AU714570B2

AU714570B2 – Process for stabilizing proteins in an acidic environment with a high-ester pectin
– Google Patents

AU714570B2 – Process for stabilizing proteins in an acidic environment with a high-ester pectin
– Google Patents
Process for stabilizing proteins in an acidic environment with a high-ester pectin

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Publication number
AU714570B2

AU714570B2
AU66144/96A
AU6614496A
AU714570B2
AU 714570 B2
AU714570 B2
AU 714570B2
AU 66144/96 A
AU66144/96 A
AU 66144/96A
AU 6614496 A
AU6614496 A
AU 6614496A
AU 714570 B2
AU714570 B2
AU 714570B2
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AU
Australia
Prior art keywords
pectin
recombinant
pme
enzyme
seq
Prior art date
1995-07-14
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AU66144/96A
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AU6614496A
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Inventor
Hans Christian Buchholt
Tove Martel Ida Elsa Christensen
Jette Dina Kreiberg
John Nielsen
Preben Rasmussen
Hanne Thorsoe
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DuPont Nutrition Biosciences ApS

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Danisco AS
Danisco US Inc
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1995-07-14
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1996-07-12
Publication date
2000-01-06
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1996-07-12
Application filed by Danisco AS, Danisco US Inc
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1997-02-18
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2000-01-06
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2012-07-12
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Classifications

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12Y—ENZYMES

C12Y301/00—Hydrolases acting on ester bonds (3.1)

C12Y301/01—Carboxylic ester hydrolases (3.1.1)

C12Y301/01011—Pectinesterase (3.1.1.11)

A—HUMAN NECESSITIES

A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES

A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF

A23C21/00—Whey; Whey preparations

A23C21/08—Whey; Whey preparations containing other organic additives, e.g. vegetable or animal products

A—HUMAN NECESSITIES

A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES

A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF

A23C9/00—Milk preparations; Milk powder or milk powder preparations

A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes

A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives

A23C9/137—Thickening substances

A—HUMAN NECESSITIES

A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES

A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF

A23C9/00—Milk preparations; Milk powder or milk powder preparations

A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives

A23C9/154—Milk preparations; Milk powder or milk powder preparations containing additives containing thickening substances, eggs or cereal preparations; Milk gels

A23C9/1542—Acidified milk products containing thickening agents or acidified milk gels, e.g. acidified by fruit juices

A—HUMAN NECESSITIES

A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES

A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL

A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof

A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents

A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin

A23L29/231—Pectin; Derivatives thereof

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

C12N9/14—Hydrolases (3)

C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

C12N9/18—Carboxylic ester hydrolases (3.1.1)

Abstract

A process is described wherein there is added to an acidic environment, which contains at least one protein, a block-wise enzymatically de-esterified pectin, and wherein the pectin is a high ester pectin. Also described is a recombinant pectin methyl esterase.

Description

WO 97/03574 PCT/EP96/03051 1 PROCESS FOR STABILIZING PROTEINS IN AN ACIDIC ENVIRONMENT WITH A PECTIN HIGH-ESTER The present invention relates to a process and an enzyme for use in such a process.
In particular, the present invention relates to a process for preparing and using an enzymatically modified pectin.
Pectin is an important commodity in today’s industry. For example, it can be used in the food industry as a thickening or gelling agent, such as in the preparation of jams.
Pectin is a structural polysaccharide commonly found in the form of protopectin in plant cell walls. The backbone of pectin comprises a-1-4 linked galacturonic acid residues which are interrupted with a small number of 1,2 linked a-L-rhamnose units.
In addition, pectin comprises highly branched regions with an almost alternating rhamno-galacturonan chain. These highly branched regions also contain other sugar units (such as D-galactose, L-arabinose and xylose) attached by glycosidic linkages to the C3 or C4 atoms of the rhamnose units or the C2 or C3 atoms of the galacturonic acid units. The long chains of a-1-4 linked galacturonic acid residues are commonly referred to as «smooth» regions, whereas the highly branched regions are commonly referred to as the «hairy regions».
Some of the carboxyl groups of the galacturonic residues are esterified the carboxyl groups are methylated). Typically esterification of the carboxyl groups occurs after polymerisation of the galacturonic acid residues. However, it is extremely rare for all of the carboxyl groups to be esterified methylated).
Usually, the degree of esterification will vary from 0-90%. If 50% or more of the carboxyl groups are esterified then the resultant pectin is referred to as a «high ester pectin» («HE pectin» for short) or a «high methoxyl pectin». If less than 50% of the carboxyl groups are esterified then the resultant pectin is referred to as a «low ester pectin» («LE pectin» for short) or a «low methoxyl pectin». If the pectin does not contain any or only a few esterified groups it is usually referred to as pectic acid.
i WO 97/03574 PCT/EP96/03051 2 The structure of the pectin, in particular the degree of esterification (e.g.
methylation), dictates many of the resultant physical and/or chemical properties of the pectin. For example, pectin gelation depends on the chemical nature of the pectin, especially the degree of esterification. In addition, however, pectin gelation also depends on the soluble-solids content, the pH and calcium ion concentration. With respect to the latter, it is believed that the calcium ions form complexes with free carboxyl groups, particularly those on a LE pectin.
Pectic enzymes are classified according to their mode of attack on the galacturonan part of the pectin molecule. A review of some pectic enzymes has been prepared by Pilnik and Voragen (Food Enzymology, Ed.: P.F.Fox; Elsevier; (1991); pp: 303- 337). In particular, pectin methylesterases (EC 3.1.1.11), otherwise referred to as PMEs, de-esterify HE pectins to LE pectins or pectic acids. In contrast, and by way of example, pectin depolymerases split the glycosidic linkages between galacturonosyl methylester residues.
In more detail, PME activity produces free carboxyl groups and free methanol. The increase in free carboxyl groups can be easily monitored by automatic titration. In this regard, earlier studies have shown that some PMEs de-esterify pectins in a random manner, in the sense that they de-esterify any of the esterified (e.g.
methylated) galacturonic acid residues on more than one of the pectin chains.
Examples of PMEs that randomly de-esterify pectins may be obtained from fungal sources such as Aspergillus aculeatus (see WO 94/25575) and Aspergillus japonicus (Ishii et al J Food Sci 44 pp 611-14). Baron et al (Lebensm. Wiss. M-Technol 13 pp 330-333) apparently have isolated a fungal PME from Aspergillus niger. This fungal PME is reported to have a molecular weight of 39000 D, an isoelectric point of 3.9, an optimum pH of 4.5 and a Km value (mg/ml) of 3.
In contrast, some PMEs are known to de-esterify pectins in a block-wise manner, in the sense that it is believed they attack pectins either at non-reducing ends or next to free carboxyl groups and then proceed along the pectin molecules by a single-chain mechanism, thereby creating blocks of unesterified galacturonic acid units which are j WO 97/03574 PCT/EP96/03051 3 very calcium sensitive. Examples of such enzymes that block-wise enzymatically deesterify pectin are plant PMEs. Up to 12 isoforms of PME have been suggested to exist in citrus (Pilnik W. and Voragen A.G.J. (Food Enzymology P.F.Fox); Elsevier; (1991); pp: 303-337). These isoforms have different properties.
Versteeg et al (J Food Sci 45 pp 969-971) apparently have isolated a PME from orange. This plant PME is reported to occur in multiple isoforms of differing properties. Isoform I has a molecular weight of 36000 D, an isoelectric point of 10.0, an optimum pH of 7.6 and a Km value (mg/ml) of 0.083. Isoform II has a molecular weight of 36200 D, an isoelectric point of 11.0, an optimum pH of 8.8 and a Km value (mg/ml) of 0.0046. Isoform III (HMW-PE) has a molecular weight of 54000 D, an isoelectric point of 10.2, an optimum pH of 8 and a Km value (mg/ml) of 0.041. However, to date there has been very limited sequence data for such PMEs.
According to Pilnik and Voragen (ibid), PMEs may be found in a number of other higher plants, such as apple, apricot, avocado, banana, berries, lime, grapefruit, mandarin, cherries, currants, grapes, mango, papaya, passion fruit, peach, pear, plums, beans, carrots, cauliflower, cucumber, leek, onions, pea, potato, radish and tomato. However, likewise, to date there has been very limited sequence data for such PMEs.
Random or blockwise distribution of free carboxyl groups can be distinguished by high performance ion exchange chromatography (Schols et al Food Hydrocolloids 1989 6 pp 115-121). These tests are often used to check for undesirable, residual PME activity in citrus juices after pasteurisation because residual PME can cause, what is called. «cloud loss» in orange juice in addition to a build up of methanol in the juice.
PMEs have important uses in industry. For example, they can be used in or as sequestering agents for calcium ions. In this regard, and according to Pilnik and Voragen (ibid). cattle feed can be prepared by adding a slurry of calcium hydroxide WO 97/03574 PCT/EP96/03051 4 to citrus peels after juice extraction. After the addition, the high pH and the calcium ions activate any native PME in the peel causing rapid de-esterification of the pectin and calcium pectate coagulation occurs. Bound liquid phase is released and is easily pressed out so that only a fraction of the original water content needs to be removed by expensive thermal drying. The press liquor is then used as animal feed.
Pilnik and Voragen (ibid) list uses of endogenous PMEs which include their addition to fruit juices to reduce the viscosity of the juice if it contains too much pectin derived from the fruit, their addition as pectinase solutions to the gas bubbles in the albedo of citrus fruit that has been heated to a core temperature of 20 to 40 0 C in order to facilitate removal of peel and other membrane from intact juice segments (US-A-4284651), and their use in protecting and improving the texture and firmness of several processed fruits and vegetables such as apple (Wiley Lee 1970 Food Technol 24 1168-70), canned tomatoes (Hsu et al 1965 J Food Sci 30 pp 583-588) and potatoes (Bartolome Hoff 1972 J Agric Food Chem 20 pp 262-266).
Glahn and Rolin (1994 Food Ingredients Europe, Conf Proceedings pp 252-256) report on the hypothetical application of the industrial «GENU pectin type YM-100» for interacting with sour milk beverages. No details are presented at all on how GENU pectin type YM-100 is prepared.
EP-A-0664300 discloses a chemical fractionation method for preparing calcium sensitive pectin. This calcium sensitive pectin is said to be advantageous for the food industry.
Thus, pectins and de-esterified pectins, in addition to PMEs, have an industrial importance. However, there is a continuing need to improve the known methods of stabilising proteins in an acidic environment but without adversely affecting the viscosity of that environment. In this regard, an adverse effect on the viscosity of the environment can compromise the overall appearance and/or texture and/or palatability and/or mouth feel of the resultant product.
WO 97/03574 PCT/EP96/03051 According to a first aspect of the present invention there is provided a process comprising adding to an acidic environment, which contains at least one protein, a block-wise enzymatically de-esterified pectin, wherein the pectin is a higher ester pectin and wherein the pectin is block-wise de-esterified by a PME enzyme.
According to a second aspect of the present invention there is provided a method of block-wise enzymatically de-esterifying a pectin comprising treating the pectin with a recombinant enzyme comprising any one of the amino acid sequences shown as SEQ.I.D No. 1 or SEQ.I.D. No. 2, or a variant, derivative or homologue thereof that has at least 75% homology therewith, including 10 combinations thereof.
According to a third aspect of the present invention there is provided a recombinant enzyme comprising any one of the amino acid sequences shown as SEQ.I.D. No. 1 or SEQ.I.D. No. 2, or a variant, derivative or homologue thereof that has at least 75% homology therewith, including combinations thereof.
*15 According to a fourth aspect of the present invention there is provided a nucleotide sequence coding for the recombinant enzyme according to the present invention, wherein the nucleotide sequence comprises any one of the sequences shown as SEQ.I.D. No. 3 or SEQ.I.D. No. 4, or a variant, derivative or homologue thereof that has at least 75% homologue therewith.
According to a fifth aspect of the present invention there is provided a nucleotide sequence coding for the recombinant enzyme according to the present invention wherein the nucleotide sequence is obtainable from NCIMB 40749 or NCIMB 40750, or a variant, derivative or homologue thereof that has at least homology therewith.
According to a sixth aspect of the present invention there is provided a construct expressing or comprising the recombinant enzyme according to the present invention or a nucleotide sequence according to the present invention.
I I WO 97/03574 PCT/EP96/03051 6 According to a seventh aspect of the present invention there is provided a vector expressing or comprising a construct according to the present invention or the recombinant enzyme according to the present invention or a nucleotide sequence according to the present invention.
According to an eighth aspect of the present invention there is provided a combination of constructs comprising at least a first construct expressing or comprising the recombinant enzyme according to the present invention or a nucleotide sequence according to the present invention; and a second construct comprising a gene of interest (GOI) and a promoter.
According to a ninth aspect of the present invention there is provided a cell, tissue or organ expressing or comprising a vector according to the present invention or a construct according to the present invention or the recombinant enzyme according to the present invention or a nucleotide sequence according to the present invention or a combination of constructs according to the present invention.
According to a tenth aspect of the present invention there is provided a transgenic organism expressing or comprising any of the afore-mentioned aspects of the present invention.
According to an eleventh aspect of the present invention there is provided
NCIMB
40749 or NCIMB 40750.
According to a twelfth aspect of the present invention there is provided a recombinant PME enzyme which is immunologically reactive with an antibody raised against a purified recombinant enzyme according to the third aspect of the present invention, but not a tomato PME enzyme.
According to a thirteenth aspect of the present invention there is provided the use of the recombinant enzyme according to the present invention to reduce the number of ester groups of a pectin.
1 WO 97/03574 PCT/EP96/03051 7 According to a fourteenth aspect of the present invention there is provided the use of the recombinant enzyme according to the present invention to de-esterify pectin in a block-wise manner.
According to a fifteenth aspect of the present invention there is provided the use of the recombinant enzyme according to the present invention to affect the calcium sensitivity of pectin.
According to a sixteenth aspect of the present invention there is provided the use of the recombinant enzyme according to the present invention to esterify pectin containing free carboxyl groups.
According to a seventeenth aspect of the present invention there is provided a pectin obtained by use of the recombinant enzyme according to the present invention, such as by the method according to the present invention.
According to an eighteenth aspect of the present invention there is provided a combination of enzymes comprising a recombinant enzyme according to the present invention and a fungal PME and/or an other pectin degrading enzyme pectin lyase).
Other aspects of the present invention include the use of a pectin according to the present invention to prepare a foodstuff; and the use of a pectin according to the present invention to stabilise a protein in an acidic environment without adversely affecting the viscosity of the environment; and a recombinant nucleotide sequence coding for the enzyme according to the present invention.
Thus, the present invention relates to a new use of de-esterified pectins. The present invention also relates to a new recombinant PME to prepare such pectins.
WO 97/03574 WO 97/03574 PCT/EP96/03051 8 Some of the key advantages of the present invention are that the de-esterified pectin of the present invention offers stability to proteins in an acidic environment without adversely affecting the viscosity of the environment.
A preferred embodiment of the first aspect of the present invention is a process comprising adding to an acidic environment, which contains at least one protein, a block-wise enzymatically de-esterified pectin prepared by use of recombinant
DNA
techniques, wherein the pectin is a high ester pectin.
The term «a block-wise enzymatically de-esterified pectin prepared by use of recombinant DNA techniques» means a pectin containing block-wise de-esterified groups wherein the pectin is prepared by treating contacting) a pectin containing esterified groups with an enzyme that has been prepared by use of recombinant
DNA
techniques.
Some of the key advantages of this preferred aspect of the present invention are that the de-esterified pectin can be prepared with ease and with a relative degree of consistency. In this regard, the recombinant PME itself can be prepared fairly simply and easily and, also, to a high degree of homogeneity. This in turn, and unlike the prior art PME preparations, means that the resultant PME activity is more consistent and homogeneous so enables the overall de-esterification process to be more controlled.
The use of a block-wise enzymatically de-esterified pectin which is preferably prepared by use of recombinant DNA techniques in the process of the present invention for stabilising at least one protein in an acidic environment is of benefit as it allows proteins such as whey and milk proteins (such as casein) to be stable in acidic solutions. This is of importance for the drinks market, such as skimmed milk, fruit juices and whey protein drinks, wherein before it was only possible to retain the flavour of the key proteins under fairly high acidic conditions such as pH 4.2 if high amounts of stabiliser were present.
WO 97/03574 PCT/EP96/03051 9 We have now found that for some applications small amounts of the de-esterified pectin according to the present invention can be employed. At these low levels, the de-esterified pectin according to the present invention not only acts as a stabiliser but also it does not have an adverse effect on the final product.
If desired, the use of the de-esterified pectin of the present invention would enable food manufacturers to increase the pH of foods, such as drinks. In this regard, in some cases the less acidic nature of the drinks may make them more palatable for people, especially infants. Thus, in contrast to the prior art processes, it is now possible to retain the flavour of those proteins at pH conditions higher than 4.2, such as up to pH 5.5 (such as pH 5.2) by use of the block-wise enzymatically de-esterified pectin, particularly the block-wise enzymatically de-esterified pectin prepared by use of recombinant DNA techniques.
In addition, it is believed that even under low pH conditions, such as pH 4.2 or less, the block-wise enzymatically de-esterified pectin particularly the block-wise enzymatically de-esterified pectin prepared by use of recombinant DNA techniques stabilises the protein(s) more than the prior art stabilisers that are used for those pH conditions.
A further advantage is that the recombinant PME is capable of producing a substantially homogeneous block-wise de-esterified pectin. By this we mean that substantially all of the pectin chains comprise at least two adjacent de-esterified carboxyl groups. However, for some applications it may not be necessary to prepare or use such a substantially homogeneous block-wise de-esterified pectin.
Without wishing to be bound by theory it is believed that the block-wise enzymatically de-esterified pectin particularly that prepared by use of recombinant DNA techniques stabilises the protein(s) by surrounding the protein(s) in a blanket of negative charges, thus forming a stable emulsion.
WO 97/03574 PCT/EP96/03051 The recombinant PME enzyme of the present invention is useful for blockwise deesterifying pectins when the pectins are contacted with the recombinant enzyme in a substantially aqueous medium. In some instances, de-esterifying pectins can increase the calcium ion sensitivity of a pectin which in turn may be advantageous.
Alternatively, the recombinant PME enzyme of the present invention is useful for esterifying pectins when the pectins are contacted with the recombinant enzyme in a substantially non-aqueous medium, such as in the presence of methanol or in the presence of high concentrations of ammonium sulphate. This aspect is advantageous if, for example, it is desirable to reduce the calcium sensitivity of a pectin.
This method of esterifying pectins is advantageous because it obviates the need for the high temperature and methanol esterification conditions associated with the prior art processes. Thus, the present invention also includes the use of that esterified pectin in the preparation of a foodstuff, as well as the pectin per se.
In accordance with the present invention, the de-esterified pectin of the present invention is advantageous for the preparation of a foodstuff.
Typical foodstuffs include dairy products, meat products, poultry products, fish products and bakery products. Preferably, the foodstuff is a beverage.
The de-esterified pectin of the present invention is also advantageous for use as a stabiliser and/or viscosity modifier in the preparation of pharmaceuticals, pharmaceutical appliances, cosmetics and cosmetic appliances.
Preferably the acidic environment is an aqueous solution.
Preferably the aqueous solution is a beverage.
Preferably the beverage is a drinking yoghurt, a fruit juice or a beverage comprising whey protein.
WO 97/03574 PCT/EP96/03051 11 Preferably the protein is derived or derivable from dairy products, such as milk or cheese.
Preferably the protein is casein or whey protein.
Preferably the acidic environment has a pH of from about 3.5 to about Preferably the acidic environment has a pH of from 4 to about Preferably, wherein the acidic environment has a pH of about 4.
Preferably the block-wise enzymatically de-esterified pectin, particularly that prepared by recombinant DNA techniques, is a high ester pectin containing about 80% ester groups or less a degree of esterification (DE) of 80% or less), preferably about 75% ester groups or less a DE of about 75% or less). In this regard, the ratio of free carboxyl groups to esterified carboxyl groups on the pectin is from 1:1 to 1:4, preferably from 1:2 to 1:3.
Preferably, the block-wise enzymatically de-esterified pectin contains about 76 ester groups.
In some instances, preferably the block-wise enzymatically de-esterified pectin is sensitive to Ca 2 ions. Calcium sensitivity can be determined by following the Protocol as mentioned in the Examples.
More preferably, however, the block-wise enzymatically de-esterified pectin, particularly that prepared by recombinant DNA techniques, is insensitive to Ca ions. Calcium insensitivity can be determined by following the Protocol as mentioned in the Examples.
Preferably the block-wise enzymatically de-esterified pectin has a high molecular weight.
WO 97/03574 PCT/EP96/0305 1 12 Typically, the molecular weight is between from about 50 KD to about 150 KD.
Preferably the block-wise enzymatically de-esterified pectin is prepared by treating a pectin with a recombinant pectin methyl esterase that de-esterifies two or more adjacent galacturonic acid residues of the pectin on at least substantially all of the pectin chains.
Preferably the recombinant pectin methyl esterase is derived from a PME obtainable from a plant.
The term «derived from a PME obtainable from a plant» means that the recombinant PME has a sequence similar to that of a PME that is obtainable from a plant, providing the recombinant PME can de-esterify pectin in a block-wise manner.
Preferably the plant is a fruit.
Preferably the fruit is a citrus fruit.
Preferably the citrus fruit is an orange.
Preferably the recombinant pectin methyl esterase is derived from a PME obtainable from the lamella or albedo of an orange. For reference, a cross-section of a typical citrus fruit is shown in Figure 1 where the lamella and albedo are indicated.- The term «derived from a PME obtainable from the lamella or albedo of an orange» means that the recombinant PME has a sequence similar to that of a PME that is obtainable from the lamella or albedo of an orange, providing the recombinant
PME
can de-esterify pectin in a block-wise manner.
Preferably, the block-wise enzymatically de-esterified pectin is prepared by treating a pectin with a recombinant enzyme comprising any one of the amino acid sequences shown as SEQ.I.D. No.1 or SEQ.I.D. No. 2, or a variant, derivative or homologue thereof that has at least 75% homology therewith, including combinations thereof.
0T WO 97/03574 PCT/EP96/03051 13 Preferably, the block-wise enzymatically de-esterified pectin is prepared by treating a pectin with a recombinant enzyme that is obtainable by expression of a nucleotide sequence comprising the nucleotide sequence shown as SEQ.I.D.
No. 3 or SEQ.I.D. No. 4, or a variant, derivative or homologue thereof that has at least 75% homology therewith, or combinations thereof.
Preferably, the block-wise enzymatically de-esterified pectin is prepared by treating a pectin with a recombinant enzyme that is obtainable by expression of the PME coding sequence contained in NCIMB 40749 or NCIMB 40750, or a variant, derivative or homologue thereof that has at least 75% homology 10 therewith, or combinations thereof.
Preferably, the block-wise enzymatically de-esterified pectin is prepared by treating the pectin with the recombinant pectin methyl esterase in the presence of sodium ions.
Preferably, the block-wise enzymatically de-esterified pectin prepared by 15 recombinant DNA techniques is prepared by treating the pectin with the recombinant pectin methyl esterase in the presence of NaC1, NaNO 3 or Na 2 SO4.
Preferably the recombinant enzyme comprises all of the sequence shown as SEQ.I.D. No. 1 or SEQ.I.D. No. 2.
Preferably the recombinant enzyme is expressed by a nucleotide sequence that comprises all of the sequence shown as SEQ.I.D. No. 3 or SEQ.I.D. No. 4.
The present invention also covers sequences that are complementary to the aforementioned sequences.
The term «pectin» includes pectin in its normal sense, as well as fractionates and derivatives thereof, as well as modified pectins chemically modified pectins and enzymatically modified pectins).
WO 97/03574 PCT/EP96/03051 14 Preferably, the pectin is not a pectin that has been prior treated with the enzyme polygalacturonase to substantially reduce the length of the pectin backbone.
The terms «variant», «homologue» or «fragment» in relation to the recombinant enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acid from or to the sequence providing the resultant amino acid sequence has PME activity, preferably having at least the same activity of a recombinant enzyme comprising any one or more of the sequences shown as SEQ I.D. No.s 1 and 2. In particular, the term «homologue» covers homology with respect to structure and/or function providing the resultant recombinant enzyme has PME activity. With respect to sequence homology similarity), preferably there is at least 75%, more preferably at least 85 more preferably at least 90% homology to the sequences shown in the attached sequence listings. More preferably there is at least 95%, more preferably at least 98%, homology to the sequences shown in the attached sequence listings.
The terms «variant», «homologue» or «fragment» in relation to the nucleotide sequence coding for the recombinant enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for a recombinant enzyme having PME activity, preferably having at least the same activity of a recombinant enzyme comprising any one or more of the sequences shown as SEQ I.D. No.s 1 and 2. In particular, the term «homologue» covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for a recombinant enzyme having PME activity.
With respect to sequence homology similarity), preferably there is at least 75 more preferably at least 85%, more preferably at least 90% homology. More preferably there is at least 95%, more preferably at least 98%, homology.
The above terms are synonymous with allelic variations of the sequences.
WO 97/03574 PCT/EP96/03051 The term «complementary» means that the present invention also covers nucleotide sequences that can hybridise to the nucleotide sequences of the coding sequence.
The term «nucleotide» in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA for the coding sequence of the present invention.
The term «citrus fruit» means species of the genus Citrus and includes lime, lemon, orange, grapefruit, kumquat, pomelo, and mandarin. Preferably, it means orange.
The term «construct» which is synonymous with terms such as «conjugate», «cassette» and «hybrid» includes the nucleotide sequence according to the present invention or. the case of the combination of constructs, the GOI directly or indirectly attached to a promoter. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron or the ADH intron, intermediate the promoter and the nucleotide sequence of the present invention or the GOI. The same is true for the term «fused» in relation to the present invention which includes direct or indirect attachment. In each case, the terms do not cover the natural combination of the gene coding for the enzyme ordinarily associated with the wild type gene promoter and when they are both in their natural environment.
The construct may even contain or express a marker which allows for the selection of the genetic construct in, for example, a filamentous fungus, preferably of the genus Aspergillus. such as Aspergillus niger, or plants, such as potatoes, sugar beet etc., into which it has been transferred. Various markers exist which may be used, such as for example those encoding mannose-6-phosphate isomerase (especially for plants) or those markers that provide for antibiotic resistance e.g. resistance to G418, hygromycin. bleomycin, kanamycin and gentamycin.
The term «vector» includes expression vectors and transformation vectors.
WO 97/03574 PCT/EP96/03051 16 The term «expression vector» means a construct capable of in vivo or in vitro expression.
The term «transformation vector» means a construct capable of being transferred from one species to another such as from an E.coli plasmid to a filamentous fungus, preferably of the genus Aspergillus. It may even be a construct capable of being transferred from an E. coli plasmid to an Agrobacterium to a plant.
The term «tissue» includes tissue per se and organ.
The term «organism» in relation to the present invention includes any organism that could comprise the nucleotide sequence coding for the recombinant enzyme according to the present invention and/or products obtained therefrom, wherein a promoter can allow expression of the nucleotide sequence according to the present invention when present in the organism.
Preferably the organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
The term «transgenic organism» in relation to the present invention includes any organism that comprises the nucleotide sequence coding for the recombinant enzyme according to the present invention and/or products obtained therefrom, wherein the promoter can allow expression of the nucleotide sequence according to the present invention within the organism. Preferably the nucleotide sequence is incorporated in the genome of the organism.
Preferably the transgenic organism is a filamentous fungus, preferably of the genus Aspergillus, more preferably Aspergillus niger.
Therefore, the transgenic organism of the present invention includes an organism comprising any one of, or combinations of, a promoter, the nucleotide sequence coding for the recombinant enzyme according to the present invention, constructs WO 97/03574 PCT/EP96/03051 17 according to the present invention (including combinations thereof), vectors according to the present invention, plasmids according to the present invention, cells according to the present invention, tissues according to the present invention or the products thereof.
The term «transgenic organism» does not cover the native nucleotide coding sequence according to the present invention in its natural environment when it is under the control of its native promoter which is also in its natural environment. In addition, the present invention does not cover the native enzyme according to the present invention when it is in its natural environment and when it has been expressed by its native nucleotide coding sequence which is also in its natural environment and when that nucleotide sequence is under the control of its native promoter which is also in its natural environment.
The transformed cell or organism could prepare acceptable quantities of the desired compound which would be easily retrievable from, the cell or organism.
Preferably the construct of the present invention comprises the nucleotide sequence of the present invention and a promoter.
The term «promoter» is used in the normal sense of the art, e.g. an RNA polymerase binding site in the Jacob-Monod theory of gene expression.
In one aspect, the nucleotide sequence according to the present invention is under the control of a promoter that may be a cell or tissue specific promoter. If, for example, the organism is a plant then the promoter can be one that affects expression of the nucleotide sequence in any one or more of tuber, stem, sprout, root and leaf tissues.
By way of example, the promoter for the nucleotide sequence of the present invention can be the cr-Amy 1 promoter (otherwise known as the Amy 1 promoter, the Amy 637 promoter or the a-Amy 637 promoter) as described in our co-pending UK patent application No. 9421292.5 filed 21 October 1994. Alternatively, the promoter for WO 97/03574 PCT/EP96/03051 18 the nucleotide sequence of the present invention can be the ca-Amy 3 promoter (otherwise known as the Amy 3 promoter, the Amy 351 promoter or the a-Amy 351 promoter) as described in our co-pending UK patent application No. 9421286.7 filed 21 October 1994.
The promoter could additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow Box or a TATA box. The promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the nucleotide sequence of the present invention or, in the case of the combination of constructs, the GOI. For example, suitable other sequences include the Shl-intron or an ADH intron. Other sequences include inducible elements such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present. An example of the latter element is the TMV 5′ signal sequence (see Sleat Gene 217 [1987] 217-225; and Dawson Plant Mol.
Biol. 23 [1993] 97).
In addition the present invention also encompasses combinations of promoters and/or nucleotide sequences coding for proteins or recombinant enzymes and/or elements.
The present invention also encompasses the use of promoters to express a nucleotide sequence coding for the recombinant enzyme according to the present invention or the GOI, wherein a part of the promoter is inactivated but wherein the promoter can still function as a promoter. Partial inactivation of a promoter in some instances is advantageous. In particular, with the Amy 351 promoter mentioned earlier it is possible to inactivate a part of it so that the partially inactivated promoter expresses the nucleotide of the present invention or a GOI in a more specific manner such as in just one specific tissue type or organ.
The term «inactivated» means partial inactivation in the sense that the expression pattern of the promoter is modified but wherein the partially inactivated promoter still functions as a promoter. However, as mentioned above, the modified promoter is WO 97/03574 PCT/EP96/03051 19 capable of expressing the nucleotide of the present invention or a GOI in at least one (but not all) specific tissue of the original promoter. One such promoter is the Amy 351 promoter described above. Examples of partial inactivation include altering the folding pattern of the promoter sequence, or binding species to parts of the nucleotide sequence, so that a part of the nucleotide sequence is not recognised by, for example, RNA polymerase. Another, and preferable, way of partially inactivating the promoter is to truncate it to form fragments thereof. Another way would be to mutate at least a part of the sequence so that the RNA polymerase can not bind to that part or another part. Another modification is to mutate the binding sites for regulatory proteins for example the CreA protein known from filamentous fungi to exert carbon catabolite repression, and thus abolish the catabolite repression of the native promoter.
The term «GOI» with reference to the combination of constructs according to the present invention means any gene of interest. A GOI can be any nucleotide that is either foreign or natural to the organism filamentous fungus, preferably of the genus Aspergillus, or a plant) in question. Typical examples of a GOI include genes encoding for proteins and enzymes that modify metabolic and catabolic processes.
The GOI may code for an agent for introducing or increasing pathogen resistance.
The GOI may even be an antisense construct for modifying the expression of natural transcripts present in the relevant tissues. The GOI may even code for a non-native protein of a filamentous fungus, preferably of the genus Aspergillus, or a compound that is of benefit to animals or humans.
Examples of GOIs include other pectinases, pectin depolymerases, polygalacturonases, pectate lyases, pectin lyases, rhamno-galacturonases, hemicellulases. endo–glucanases, arabinases, or acetyl esterases, or combinations thereof, as well as antisense sequences thereof.
The GOI may be a protein giving nutritional value to a food or crop. Typical examples include plant proteins that can inhibit the formation of anti-nutritive factors and plant proteins that have a more desirable amino acid composition a higher WO 97/03574 PCT/EP96/03051 lysine content than a non-transgenic plant). The GOI may even code for an enzyme that can be used in food processing such as chymosin, thaumatin and a-galactosidase.
The GOI can be a gene encoding for any one of a pest toxin, an antisense transcript such as that for patatin or a-amylase, ADP-glucose pyrophosphorylase see EP- A-0455316), a protease antisense, a glucanase or genomic PME.
The GOI may even code for an intron of a particular enzyme but wherein the intron can be in sense or antisense orientation. In the latter instance, the particular enzyme could be genomic PME. Antisense expression of genomic exon or intron sequences as the GOI would mean that the natural PME expression would be reduced or eliminated but wherein the recombinant PME expression would not be affected. This is particularly true for antisense intron or sense intron expression.
The GOI can be the nucleotide sequence coding for the a-amylase enzyme which is the subject of our co-pending UK patent application 9413439.2 filed on 4 July 1994.
The GOI can be the nucleotide sequence coding for the a-amylase enzyme which is the subject of our co-pending UK patent application 9421290.9 filed on 21 October 1994. The GOI can be any of the nucleotide sequences coding for the ADP-glucose pyrophosphorylase enzymes which are the subject of our co-pending PCT patent application PCT/EP94/01082 filed 7 April 1994. The GOI can be any of the nucleotide sequences coding for the a-glucan lyase enzyme which are described in our co-pending PCT patent application PCT/EP94/03397 filed 15 October 1994.
The host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press). If a prokaryotic host is used then the gene may need to be suitably modified before transformation such as by removal of introns.
As mentioned above, a preferred host organism is of the genus Aspergillus, such as Aspergillus niger.
WO 97/03574 PCT/EP96/03051 21 A transgenic Aspergillus according to the present invention can be prepared by following the teachings of Rambosek, J. and Leach, J. 1987 (Recombinant DNA in filamentous fungi: Progress and Prospects. CRC Crit. Rev. Biotechnol. 6:357-393), Davis R.W. 1994 (Heterologous gene expression and protein secretion in Aspergillus.
In: Martinelli Kinghorn Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp 525-560), Ballance, D.J. 1991 (Transformation systems for Filamentous Fungi and an Overview of Fungal Gene structure. In: Leong, Berka R.M. (Editors) Molecular Industrial Mycology. Systems and Applications for Filamentous Fungi. Marcel Dekker Inc.
New York 1991. pp 1-29) and Turner G. 1994 (Vectors for genetic manipulation. In: Martinelli Kinghorn Editors) Aspergillus: 50 years on. Progress in industrial microbiology vol 29. Elsevier Amsterdam 1994. pp. 641-666). However, the following commentary provides a summary of those teachings for producing transgenic Aspergillus according to the present invention.
For almost a century, filamentous fungi have been widely used in many types of industry for the production of organic compounds and enzymes. For example, traditional japanese koji and soy fermentations have used Aspergillus sp. Also, in this century Aspergillus niger has been used for production of organic acids particular citric acid and for production of various enzymes for use in industry.
There are two major reasons why filamentous fungi have been so widely used in industry. First filamentous fungi can produce high amounts of extracelluar products, for example enzymes and organic compounds such as antibiotics or organic acids.
Second filamentous fungi can grow on low cost substrates such as grains, bran, beet pulp etc. The same reasons have made filamentous fungi attractive organisms as hosts for heterologous expression according to the present invention.
In order to prepare the transgenic Aspergillus, expression constructs are prepared by inserting the nucleotide sequence according to the present invention (or even the GOI) into a construct designed for expression in filamentous fungi.
WO 97/03574 PCT/EP96/03051 22 Several types of constructs used for heterologous expression have been developed.
These constructs preferably contain a promoter which is active in fungi. Examples of promoters include a fungal promoter for a highly expressed extracelluar enzyme, such as the glucoamylase promoter or the a-amylase promoter. The nucleotide sequence according to the present invention (or even the GOI) can be fused to a signal sequence which directs the protein encoded by the nucleotide sequence according to the present invention (or even the GOI) to be secreted. Usually a signal sequence of fungal origin is used. A terminator active in fungi ends the expression system.
Another type of expression system has been developed in fungi where the nucleotide sequence according to the present invention (or even the GOI) can be fused to a smaller or a larger part of a fungal gene encoding a stable protein. This can stabilize the protein encoded by the nucleotide sequence according to the present invention (or even the GOI). In such a system a cleavage site, recognized by a specific protease, can be introduced between the fungal protein and the protein encoded by the nucleotide sequence according to the present invention (or even the GOI), so the produced fusion protein can be cleaved at this position by the specific protease thus liberating the protein encoded by the nucleotide sequence according to the present invention (or even the GOI). By way of example, one can introduce a site which is recognized by a KEX-2 like peptidase found in at least some Aspergilli. Such a fusion leads to cleavage in vivo resulting in protection of the expressed product and not a larger fusion protein.
Heterologous expression in Aspergillus has been reported for several genes coding for bacterial, fungal, vertebrate and plant proteins. The proteins can be deposited intracellularly if the nucleotide sequence according to the present invention (or even the GOI) is not fused to a signal sequence. Such proteins will accumulate in the cytoplasm and will usually not be glycosylated which can be an advantage for some bacterial proteins. If the nucleotide sequence according to the present invention (or even the GOI) is equipped with a signal sequence the protein will accumulate extracelluarly.
WO 97/03574 PCT/EP96/03051 23 With regard to product stability and host strain modifications, some heterologous proteins are not very stable when they are secreted into the culture fluid of fungi.
Most fungi produce several extracelluar proteases which degrade heterologous proteins. To avoid this problem special fungal strains with reduced protease production have been used as host for heterologous production.
For the transformation of filamentous fungi, several transformation protocols have been developed for many filamentous fungi (Ballance 1991, ibid). Many of them are based on preparation of protoplasts and introduction of DNA into the protoplasts using PEG and Ca 2 ions. The transformed protoplasts then regenerate and the transformed fungi are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as argB, trpC, niaD and pyrG, antibiotic resistance markers such as benomyl resistance, hygromycin resistance and phleomycin resistance. A commonly used transformation marker is the amdS gene of A. nidulans which in high copy number allows the fungus to grow with acrylamide as the sole nitrogen source.
In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton. eds, pp 107-133, Blackie, Glasgow).
For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as WO 97/03574 PCT/EP96/03051 24 well as large-scale fermentation characteristics of Saccharomvces cerevisiae.
A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, «Yeast as a vehicle for the expression of heterologous genes», Yeasts, Vol Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).
Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.
In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence of the present invention into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The constructs contain a promoter active in yeast fused to the nucleotide sequence of the present invention, usually a promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.
For the transformation of yeast several transformation protocols have been developed.
For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).
The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibiotic resistance markers such as aminoglycoside antibiotic markers, eg G418.
Another host organism is a plant.
WO 97/03574 PCT/EP96/03051 Even though the enzyme and the nucleotide sequence coding therefor are not disclosed in EP-B-0470145 and CA-A-2006454, those two documents do provide some useful background commentary on the types of techniques that may be employed to prepare transgenic plants according to the present invention. Some of these background teachings are now included in the following commentary.
The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material.
Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205- 225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27).
Thus, in one aspect, the present invention relates to a vector system which carries a nucleotide sequence or construct according to the present invention and which is capable of introducing the nucleotide sequence or construct into the genome of an organism, such as a plant.
The vector system may comprise one vector, but it can comprise two vectors. In the case of two vectors, the vector system is normally referred to as a binary vector system. Binary vector systems are described in further detail in Gynheung An et al.
(1980), Binary Vectors, Plant Molecular Biology Manual A3, 1-19.
One extensively employed system for transformation of plant cells with a given promoter or nucleotide sequence or construct is based on the use of a Ti plasmid from Agrobacterium tumefaciens or a Ri plasmid from Agrobacterium rhizogenes An et al.
(1986), Plant Physiol. 81, 301-305 and Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203-208.
WO 97/03574 PCT/EP96/03051 26 Several different Ti and Ri plasmids have been constructed which are suitable for the construction of the plant or plant cell constructs described above. A non-limiting example of such a Ti plasmid is pGV3850.
The nucleotide sequence or construct of the present invention should preferably be inserted into the Ti-plasmid between the terminal sequences of the T-DNA or adjacent a T-DNA sequence so as to avoid disruption of the sequences immediately surrounding the T-DNA borders, as at least one of these regions appear to be essential for insertion of modified T-DNA into the plant genome.
As will be understood from the above explanation, if the organism is a plant, then the vector system of the present invention is preferably one which contains the sequences necessary to infect the plant the vir region) and at least one border part of a T- DNA sequence, the border part being located on the same vector as the genetic construct. Preferably, the vector system is an Agrobacterium tumefaciens Ti-plasmid or an Agrobacterium rhizogenes Ri-plasmid or a derivative thereof, as these plasmids are well-known and widely employed in the construction of transgenic plants, many vector systems exist which are based on these plasmids or derivatives thereof.
In the construction of a transgenic plant the nucleotide sequence or construct of the present invention may be first constructed in a microorganism in which the vector can replicate and which is easy to manipulate before insertion into the plant. An example of a useful microorganism is E. coli., but other microorganisms having the above properties may be used. When a vector of a vector system as defined above has been constructed in E. coli. it is transferred, if necessary, into a suitable Agrobacterium strain, e.g. Agrobacterium tumefaciens. The Ti-plasmid harbouring the nucleotide sequence or construct of the invention is thus preferably transferred into a suitable Agrobacterium strain, e.g. A. tumefaciens, so as to obtain an Agrobacterium cell harbouring the nucleotide sequence or construct of the invention, which DNA is subsequently transferred into the plant cell to be modified.
WO 97/03574 PCT/EP96/03051 27 As reported in CA-A-2006454, a large amount of cloning vectors are available which contain a replication system in E. coli and a marker which allows a selection of the transformed cells. The vectors contain for example pBR 322, the pUC series, the M13 mp series, pACYC 184 etc.
In this way, the nucleotide or construct of the present invention can be introduced into a suitable restriction position in the vector. The contained plasmid is used for the transformation in E. coli. The E. coli cells are cultivated in a suitable nutrient medium and then harvested and lysed. The plasmid is then recovered. As a method of analysis there is generally used sequence analysis, restriction analysis, electrophoresis and further biochemical-molecular biological methods. After each manipulation, the used DNA sequence can be restricted and connected with the next DNA sequence.
Each sequence can be cloned in the same or different plasmid.
After each introduction method of the desired promoter or construct or nucleotide sequence according to the present invention in the plants the presence and/or insertion of further DNA sequences may be necessary. If, for example, for the transformation the Ti- or Ri-plasmid of the plant cells is used, at least the right boundary and often however the right and the left boundary of the Ti- and Ri-plasmid T-DNA, as flanking areas of the introduced genes, can be connected. The use of T-DNA for the transformation of plant cells has been intensively studied and is described in EP-A- 120516; Hoekema, in: The Binary Plant Vector System Offset-drukkerij Kanters Alblasserdam, 1985, Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46; and An et al., EMBO J. (1985) 4:277-284.
Direct infection of plant tissues by Agrobacterium is a simple technique which has been widely employed and which is described in Butcher D.N. et al. (1980), Tissue Culture Methods for Plant Pathologists, eds.: D.S. Ingrams and J.P. Helgeson, 203- 208. For further teachings on this topic see Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). With this technique, infection of a plant may be done on a certain part or tissue of the plant, i.e. on a part of a leaf, a root, a stem or another part of the WO 97/03574 PCT/EP96/03051 28 plant.
Typically, with direct infection of plant tissues by Agrobacterium carrying the promoter and/or the GOI, a plant to be infected is wounded, e.g. by cutting the plant with a razor or puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium. The inoculated plant or plant part is then grown on a suitable culture medium and allowed to develop into mature plants.
When plant cells are constructed, these cells may be grown and maintained in accordance with well-known tissue culturing methods such as by culturing the cells in a suitable culture medium supplied with the necessary growth factors such as amino acids, plant hormones, vitamins, etc. Regeneration of the transformed cells into genetically modified plants may be accomplished using known methods for the regeneration of plants from cell or tissue cultures, for example by selecting transformed shoots using an antibiotic and by subculturing the shoots on a medium containing the appropriate nutrients, plant hormones, etc.
Further teachings on plant transformation may be found in EP-A-0449375.
In summation, the present invention provides a process of stabilising at least one protein in an acidic environment comprising contacting the protein with a block-wise enzymatically de-esterified pectin, in particular a block-wise enzymatically deesterified pectin prepared by recombinant DNA techniques.
In addition the present invention provides an recombinant enzyme useful in that process.
A preferred embodiment of the present invention relates to a process of stabilising at least one protein in an acidic environment comprising contacting the protein with a block-wise enzymatically de-esterified pectin prepared by recombinant DNA techniques, wherein the block-wise enzymatically de-esterified pectin is prepared by WO 97/03574 PCT/EP96/03051 29 use of a recombinant enzyme, and wherein the recombinant enzyme comprises any one of the amino acid sequences shown as SEQ.I.D. No.1 or SEQ.I.D. No. 2 or a variant, derivative or homologue thereof, including combinations thereof, and has the following characteristics: 1.
2.
3.
4.
6.
7.
8.
a molecular weight of from about 36 kD to about 64 kD; a pH optimum of pH 7 8 when measured with 0.5% lime pectin in 0.15 M NaCl; a temperature optimum of at least a temperature stability in the range of from 10°- at least a Km value of 0.07 an activity maximum at levels of about 0.25 M NaCl; an activity maximum at levels of about 0.2 M Na 2
SO
4 and an activity maximum at levels of about 0.3 M NaNO 3 Another preferred embodiment of the present invention relates to a method of blockwise enzymatically de-esterifying a pectin comprising treating the pectin with a recombinant enzyme comprising any one of the amino acid sequences shown as SEQ.I.D. No.1 or SEQ.I.D. No. 2, or a variant, derivative or homologue thereof, including combinations thereof, wherein the recombinant enzyme has the following characteristics: 1.
2.
3.
4.
6.
7.
8.
a molecular weight of from about 36 kD to about 64 kD; a pH optimum of pH 7 8 when measured with 0.5% lime pectin in 0.15 M NaCI; a temperature optimum of at least a temperature stability in the range of from 10°- at least 40 0
C;
a Km value of 0.07 an activity maximum at levels of about 0.25 M NaCI; an activity maximum at levels of about 0.2 M Na 2
SO
4 and an activity maximum at levels of about 0.3 M NaNO 3
MM_
WO 97/03574 PCT/EP96/03051 Another preferred embodiment of the present invention relates to a recombinant enzyme comprising any one of the amino acid sequences shown as SEQ.I.D. No.1 or SEQ.I.D. No. 2, or a variant, derivative or homologue thereof, including combinations thereof, wherein the recombinant enzyme has the following characteristics: 1.
2.
3.
4.
6.
7.
8.
a molecular weight of from about 36 kD to about 64 kD; a pH optimum of pH 7 8 when measured with 0.5% lime pectin in 0.15 M NaCI; a temperature optimum of at least a temperature stability in the range of from 10°- at least a Km value of 0.07 an activity maximum at levels of about 0.25 M NaCl; an activity maximum at levels of about 0.2 M Na 2
SO
4 and an activity maximum at levels of about 0.3 M NaNO 3 More preferred embodiments of the present invention relate to the afore-mentioned preferred process, method and recombinant enzyme, and wherein the recombinant enzyme has been expressed by a nucleotide sequence comprising the sequence shown as SEQ.I.No. 3 or SEQ.I.D. No. 4, or a variant, derivative or homologue thereof.
The following sample has been deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on 6 July 1995: NCIMB 40749 (which corresponds to plasmid p034).
The following sample has been deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, AB2 1RY, United Kingdom, on 12 July: NCIMB 40750 (which corresponds to plasmid p017).
WO 97/03574 PCTIEP96/03051 31 Therefore, more preferred embodiments of the present invention relate to the aforementioned process, method and recombinant enzyme and wherein the recombinant enzyme is capable of being expressed by NCIMB 40749 or NCIMB 40750.
The present invention also provides an amino acid sequence comprising the sequence shown as SEQ.I.D. No. 5, or a variant, derivative or homologue thereof that has at least 75% homology therewith, suitable for use in imparting or increasing heat stability to a protein.
S.In addition, the present invention provides a nucleotide sequence comprising the 10 sequence shown as SEQ.I.D. No. 6, or a variant, derivative or homologue thereof that has at least 75% homology therewith, suitable for use in expressing an amino acid sequence for imparting or increasing heat stability to a protein.
With respect to these last aspects, the above commentary for variants, derivatives, homologues, constructs, vectors, transformation of host organisms is 15 equally applicable though of course the amino acid sequence and nucleotide sequence in this case affect the heat stability of proteins.
In this regard, it is believed that the amino acid sequence shown as SEQ.I.D. No.
is capable of imparting or increasing the heat stability of a protein, such as the recombinant PME of the present invention. The amino acid sequence may also increase or impart heat stability to other proteins, include PMEs from other sources.
The present invention will now be described only by way of example, in which reference is made to the following attached Figures: Figure 1, which is a schematic diagram of a citrus fruit, such as an orange; Figure 2, which is a SDS PAGE gel of the recombinant enzyme of the present invention; WO 97/03574 PCT/EP96/03051 Figure 3, which is a plot to determine pH optimum; Figure 4, which is a plot to determine temperature optimum; Figure 5, which is a plot to determine temperature stability; Figure 6, which is a plot to determine the Km value; Figure 7, which is a plot to determine activity in the presence or absence of NaCI; Figure 8, which is a plot to determine activity in the presence or absence of NaSO 4 Figure 9, which is a plot to determine activity in the presence or absence of NaNO 3 Figure 10, which is a plasmid map of p017; Figure 11, which is a plasmid map of pO 3 4 Figure 12, which is a diagrammatic representation of two genes; Figure 13, which is a plasmid map of Figure 14, which is a plasmid map of pJK11; Figure 15, which is a plasmid map of pJK12; Figure 16, which is a plasmid map of Figure 17, which is a plasmid map of pJK21; and Figure 18, which is a plasmid map of pJK22.
WO 97/03574 PCT/EP96/03051 33 Figure 1 was referred to in the above description. The other Figures are discussed in the commentary below.
EXPERIMENTAL SECTION MATERIALS AND METHODS FOR BIOCHEMISTRY Plant material: Immature Spanish oranges of the Navelina variety class I without seeds, were used for isolation of PME. The oranges were peeled manually and the peels were stored at -80 0
C.
Extraction of PME from orange peel PME was purified according to the following procedure. All operations were performed at 4°C. 600 g frozen orange peels were thawed and cut into minor pieces.
They were then homogenized in a Warring blender for 2 min in 1200 ml buffer (100 mM Na-succinate pH 6.2, 1 mM DTT). 36 g solid NaCl was added to the homogenate to reach an end-concentration of 3 in order to isolate membrane bound proteins (Versteeg et al. (1978) Lebensmittel.-Wiss. u. Technol., 11: 267-274). After 2 hours incubation with gently stirring at 4 0 C the suspension was filtered through nylon mesh and the filtrate was centrifuged at 10,000 rpm for 20 min to remove insoluble residues.
The supernatant was then fractionated using (NH 4 2
SO
4 precipitation. The supernatant was first precipitated with 30% (NH 4 2
SO
4 under slowly stirring for 30 min. After centrifugation at 20,000 rpm for 10 min the supernatant was further precipitated with
(NH
4 2
SO
4 for 30 min. The suspension was centrifuged as before and the precipitate was resuspended in 50 ml 50 mM MES, 1 mM DTT pH 6.8 and dialysed against the same buffer over night.
WO 97/03574 PCT/EP96/03051 34 Chromatography The dialysed sample was further separated by cation exchange chromatography. A 40-50 ml sample was applied to a CM-Sepharose m CL-6B (1.5 x 15 cm) in two rounds with a 30 min wash between the rounds with buffer A: 50 mM MES, 1 mM DTT pH 6.8. After washing off the unbound proteins with buffer A the bound proteins were eluted with an increasing NaCl gradient from 0 0.4 M NaCl in total 500 ml. The flow was 25 ml/h and fractions of 8.33 ml were collected. The protein absorption profile was measured at 280 nm.
All fractions were analysed for PME activity and protein. The protein content was measured spectrophotometrically with the BioRad method.
The fractions containing PME activity were pooled and concentrated by press dialysis using Amicon filter system. Buffer exchange to 50 mM Tris, 1 mM DTT, 0.1 M NaCl pH 7 was done on the same system.
9 ml concentrated PME sample was then applied to a Sephacryl» S-200 (2.6 x 70 cm) gel filtration column. The column was equilibrated with 50 mM Tris, 1 mM DTT, 0.1 M NaCl pH 7. The flow was 40 ml/h and fractions of 5.33 ml were collected.
The fractions containing PME activity were pooled and concentrated.
Enzyme activity PME catalyses the cleavage of methylester groups from pectin. During the purification steps PME was detected by a fast method using methyl red indicator test.
Due to cleavage of methyl groups from galacturonic acid residues in the pectin chain, carboxyl groups were formed and the pH drops in the assay. The pH indicator methyl red changes colour at pH drop from yellow (pH 6.2) to pink (pH The assay contained 1 ml 0.5 Grindsted- Pectin 1450 (DE 70%) (supplied by Danisco Ingredients, Danisco A/S) solubilized in 0.15 M NaCl pH 7 and 125 Al sample. The samples which showed positive methyl red test after 10 min incubation at 30°C were WO 97/03574 PCT/EP96/03051 then further measured by the titration method (Versteeg et al (1978) Lebensmittel.- Wiss. u. Technol., 11: 267-274).
With the titration method the assay contained 10 ml 0.5 lime pectin (Grindsted
T
Pectin 1450 supplied by Danisco Ingredients, Danisco A/S) solubilized in 0.15 M NaCI pH 6.8 and 10 100 pl sample. Titration was performed with 0.02 M NaOH and the reaction was measured at room temperature. An automatic titrator was used (Versteeg et al. (1978) Lebensmittel.-Wiss. u. Technol., 11: 267-274).
SDS-PAGE/Western blotting The purity of the PME fraction was investigated by SDS-PAGE using Pharmacia PhastSystem T with 10 15% SDS-gradient gels. Electrophoresis and silver staining of the proteins were done as described by the manuals from Pharmacia. For determination of pi IEF 3-9 PhastSystem- gels were used.
Immuno gel electrophoresis was used for characterisation of polyclonal antibodies raised against orange peel PME. The enzyme fractions were separated on SDS- PAGE and transferred to NC-paper by semi-dry blotting technique on a Semidry transfer unit of the PhastSystem T m The NC-paper was incubated with the primer antibody diluted 1:50 and stained with the second antibody coupled to alkaline phosphatase (Dako A/S Glsotrup, Denmark) used in a dilution of 1:1000.
Peptide mapping PME was digested with either trypsin or endo-proteinase Lys-C from Lysobacter enzymogenes (both enzyme preparations were sequencing grade purchased from Boerhinger Mannheim, Germany).
100 A/g purified PME was carboxy methylated with iodoacetamide to protect the reduced SH-groups. Then the protein was cleaved with trypsin (4 jg/20-100 1l).
The hydrolytic cleavage was performed at 40 0 C for 2 x 3 hrs. The reaction was WO 97/03574 PCT/EP96/03051 36 stopped with addition of 20 zl TFA. After centrifugation at 15,000 rpm for 5 min the peptides were purified on a reverse-phase HPLC column (Vydac 10 C18 column).
2 x 500 I1 samples were applied. The peptides were eluted and separated with an increasing acetonitrile gradient from 0.05 0.35% in 60 min in 0.1% TFA. The peptides were collected manually in Eppendorf tubes.
For digestion with endo-proteinase Lys-C, freeze dried PME (0.1 mg) was dissolved in 50 1l of 8 M urea, 0.4 M NH 4
HCO
3 pH 8.4. After overlay with N 2 and addition of 5 p l of 45 mM DTT, the protein was denatured and reduced for 15 min at under After cooling to room temperature, 5 pl of 100 mM iodoacetamide was added for the cysteines to be derivatised for 15 min at room temperature in the dark under Subsequently, 90 l1 of water and 5 Ag of endo-proteinase Lys-C in 50 pl mM tricine and 10 mM EDTA, pH 8.0, was added and the digestion was carried out for 24 hrs at 37°C under N 2 The resulting peptides were separated as described for trypsin digested peptides.
Selected peptides were further purified on a Devosil 3 C 8 RP-HPLC column 0.46×10 cm (Novo Nordisk, Denmark). The purified peptides were then applied on an amino acid sequencer, Applied Biosystems 476A, using pulsed-liquid fast cycles.
STUDY 1 During purification of PME 600 g frozen orange peels were homogenized and after precipitation with 30 60% (NH 4 2
SO
4 and dialysis the sample was applied to a cation exchange column (CM-Sepharose» CL-6B). PME binds strongly to a cation exchange column material at pH 6.8 whereas most of the proteins do not bind to the column and so elute in the wash volume. With increasing NaCl gradient PME eluted into two peaks with the major activity in PME I (fraction 49-53) at a NaCl concentration of 0.25 M. A minor PME peak eluted at a lower concentration of NaCl (fraction 32). The further purification was only performed for PME I, which contained the highest activity.
WO 97/03574 PCT/EP96/03051 37 After concentration the PME fraction was further purified using gel filtration chromatography (Sephacryl» S-200 column). Fractions containing the highest PME activity were pooled and concentrated on amicon filter dialysis.
In total approximately 4 mg PME was obtained from 600 g orange peels which corresponds to a protein yield of 12%.
SDS-PAGE showed only one protein band in the purified PME fraction with a MW of 36,000 D (Figure Isoelectric focusing of PME showed that the pi was 9.
Characterization and kinetic data Characterization of PME and optima determinations were all done with the titration method as described in Materials and Methods.
pH optimum of PME activity was measured with 0.5% lime pectin (Grindsted T Pectin 1450 supplied by Danisco Ingredients, Danisco A/S) in 0.15 M NaCl. The data are shown in Figure 3. The optimum was found around pH 7 8.
Temperature optimum was found at 50 0 C see the data shown in Figure 4.
The heat (temperature) stability of PME was determined by incubating the enzyme sample in Eppendorf tubes at different temperatures for 15 min. After incubation the enzyme activity was measured by traditional assay by the titration method. The stability of the enzyme activity was between 10°- 40 0 C see the data in Figure The affinity for lime pectin (Grindsted» Pectin 1450 supplied by Danisco Ingredients, Danisco A/S) was determined by Lineweaver Burk plot of different pectin concentration versus activity. The data are shown in Figure 6. The Km was calculated from the curve to be 0.07 WO 97/03574 PCT/EP96/03051 38 We have also found that the PME could also de-esterify sugar beet pectin. Sugar beet pectin contains 60% galacturonic acid residues some of whose carboxyl groups are methylated (an approximate DE of 60%) and, in addition, some of the galacturonic acid groups are acetylated at C-2 and/or C-3. The PME activity was measured as described in Materials and Methods except that 1% sugar beet pectin solubilized in 0.15 M NaCI was used in the assay as the sugar beet pectin contained approx. pectin. The results showed that the PME could de-esterify sugar beet pectin, even though the galacturonic acid residues are acetylated at the C-2/C-3 positions.
Enzyme activity (jumol/min/ml) lime pectin 387 (Grindsted»‘ Pectin 1450 supplied by Danisco Ingredients, Danisco A/S) 1.0 sugar beet pectin Further experiments showed that the PME of the present invention preferably requires NaCl for activity see the data in Figure 7. The activity increases with increasing NaCl concentration, with an optimum at 0.25 M NaCI. Higher concentrations are reducing the activity compared to the maximal activity.
Further experiments showed that other salts e.g. NaSO 4 or NaNO 3 can substitute NaCI for PME activity. The data are shown in Figures 8 and 9. The optimum activity was found at 0.2 M Na2SO 4 and 0.3 M NaNO 3 respectively.
WO 97/03574 PCT/EP96/03051 39 N-terminal analysis Studies showed that the N-terminal sequence of native PME was blocked. Deblocking was achieved by treating PME blotted onto a PVDF membrane with anhydrous TFA for 4 min at 45 0 C in tubes. After evaporation of most of TFA the tubes were placed at 65 0 C for 4 hrs (Wellner, D. et al. (1990) Proc. Natl. Acad. Sci.
87: 1947-1949). The sequence obtained was SSSVTPNVVVAADSSGNFK and that N-acetylserine is the N-terminal residue of PME.
Immuno histo localization For the preparation of tissue samples for immuno histo chemistry, thin slices of the middle part of mature fruit were fixed in 2% paraformaldehyde, 0.25% glutaraldehyde and 3% sucrose buffered with 0.05 M phosphate buffer pH 7. After incubation for 2 hours at 25 0 C and 63 hours at 5°C the specimens were washed 3 x min in 0.05 M phosphate buffer pH 7. Dehydration was carried out using a series of ethanol washings 70%, 80% and 96%) followed by 3 washes of 99% ethanol (30 min for each ethanol concentration). After additional treatment with 2 x 2 hrs in petroleum (ShellsolP D70k, Q7712) and 2 x 2 hrs in paraffin with 7% beeswax, the samples were embedded in paraffin. Cross-sections of 12.5 prm were made on a Supercut 2050 Reichart Jung pyramitome.
Immunology Tissue sections were pre-incubated with 20% swine serum in TBS (0.5 M Tris/HCl pH 7.6, 0.15 M NaCI, 0.1% Triton X-100) for 30 min before treatment for 1 hour with PME antibodies were diluted 1: 50 in TBS. Excess antibody was removed by washing with TBS for 5 x 5 min. After washing the sections were incubated for min with secondary antibodies coupled with alkaline phosphatase 1:20 in TBS buffer.
Surplus of secondary antibody was removed by TBS washing as described above.
Before staining the sections were treated with veronal acetate buffer pH 9.2 for 5 min and then stained with Fast Red and Naphtol AS-BI phosphate (Sigma no N4875) for WO 97/03574 PCT/EP96/03051 min. Excess reagent was removed by washing with water. Controls were run in parallel and treated with pre-immune serum.
Results Immunological localizations with the antibody raised against peel PME showed that PME is situated in large amounts in the outer cell layer of the lamella between the segments, in the core and in the outer cell layer of the juice sacs and also in the inner cell layer of the albedo (see Figure These results demonstrate that the antibody against peel PME cross reacts with PME from the flesh (the flesh consists of segments, see Figure 1) indicating a high homology between the PME located in the peel and in the flesh, respectively.
STUDY 2 PME from orange peel was purified in large amounts. In this regard, approximately mg PME was isolated from 5 kg orange peels. The purified PME was then used in the application test using milk protein i.e. a drinking yoghurt. In this test, the block-wise enzymatically de-esterified pectin improved protein stabilising properties compared to non-de-esterified pectin, presumably because of the formed block structure. The final product also had a favourable viscosity.
Enzyme Isoforms Whilst not wishing to be bound by theory, it is believed that the PME of the present invention can exist in at least two isoforms. Isoform S has a molecular weight of about 36 kD, and can be referred to as the «short PME». Isoform L has a molecular weight of about 64 kD, and can be referred to as the «long PME».
It is also believed that the Isoform L is more heat stable than Isoform S. Also, it is believed that Isoform S starts the initial de-esterification step in a block-wise manner and is then superseded by Isoform L.
WO 97/03574 PCT/EP96/03051 41 In other words, it is believed that Isoform L may have a greater affinity to partially de-esterified pectin than Isoform S.
It is believed that the heat stability of Isoform L may be due to the amino acid sequence represented as SEQ.I.D.No. Further studies have shown that the gene for Isoform L has an N-terminal extension upstream of the signal sequence. This is shown diagrammatically in Figure 12.
ISOLATION AND CHARACTERIZATION OF cDNAs ENCODING PECTIN METHYL ESTERASE OBTAINABLE FROM ORANGE MATERIALS AND METHODS FOR MOLECULAR BIOLOGY 1. MATERIALS Oranges (Citrus sinensis) var. Navel originating from Morocco were used.
2. DNA Genomic DNA was isolated as described by Dellaporta S.L.et al (1983) Plant Mol Biol Rep 1(4):19-21. Plasmid DNA was isolated as described in EP-B-0470145.
3. RNA Total RNA was isolated from mature orange fruits. The outer portion of the flesh and the inner portion of the albedo layer (see Figure 1) of a Navel orange fruit were used for the RNA isolation following the procedure described by Logemann Schell J.
and Willmitzer L. (1987) Anal. Biochem 163:16-20. «Improved Method for the isolation of RNA from Plant Tissues».
WO 97/03574 PCT/EP96/03051 42 4. PCR Total orange RNA was used to make reverse PCR with the rTth Reverse PCR Kit (Perkin Elmer) according to the suppliers instructions with the following temperature cycling: Reverse transcription: 2 min 60°C 2 min 2 min 0 C 5 min 0 C 5 min 0 C 10 min 42 0 C 10 min 2,5 min soak Amplification (PCR): 94°C 2 min 92 0 C 1 min 2 min 72°C 2 min in 40 cycles 72 0 C 5 min soak CLONING OF PCR FRAGMENTS PCR fragments were cloned into the EcoRV site of the vector pT7Blue (Novagen) following the suppliers instructions.
WO 97/03574 PCT/EP96/03051 43 6. DNA SEQUENCING Double stranded DNA was sequenced essentially according to the dideoxy method of Sanger et al. (1979) using the Auto Read Sequencing Kit (Pharmacia) and the Pharmacia LKB A.L.F. DNA sequencer (Ref: Sanger, Nicklen, and Coulson, A. R. (1979). DNA sequencing with chain-determinating inhibitors. Proc. Nat.
Acad. Sci. USA 74: 5463-5467). The primers used for sequencing are listed below (presented 5′ to UNI (M13-20 Primer) 17 Mer
GTAAACGACGGCCAGT
REV 19 MER
GGAAACAGCTATGACCATG
01 20 MER
GACAACGGCAACGAGCCTCA
02 22 MER
GCACTTGTAATAACCCTAAAT
03 20 MER
CAGGGTAGTTTCCCGACGTA
04 20 MER
TACGTCGGGAAACTACCCTG
21 MER
CTCCTGGAAGCTTCATTGCTG
06 20 MER
GAAGCTTCTTGAAGAGAACG
WO 97/03574 WO 9703574PCT/EP96/03051 07 -20 MER
CGTTCTCTTCAAGAAGAAGCTTC
08 22MER
GAGGATAGCATGATGAGGCTCG
09 22 MER
GCAGTTCACAAAGAACTGGCGG
010 22 MER
CCTCATGATCATCATGTCAGTG
011 21 MER
GCTCCTCAGGGAGGCACTAAG
012 21 MER
CAGCAATGAAGCTTCCAGGAG
013 21 MER
CTTAGTGCCTCCCTGAGGAGC
014 18 MER
GCGACCGCCTGGTGCTTT
015 18 MER
AAAGCACCAGGCGGTGGC
016 20 MER
GCGGGATGCGTTGTCAGACG
017 20 MER
GAGGCACTAAGCGGTATATT
WO 97/03574 PCT/EP96/03051 018 18 MER
GCAACTGTAGCAGACTTG
019 20 MER
CACTGACATGATGATCATGA
020 20 MER
CAATTAAGCAGTTCACAAAG
021 20 MER
AATATACCGCTTAGTGCCTC
The sequenced nucleotide sequence is shown as SEQ.I.D. No. 3 (derived from p017) and SEQ.I.D.No. 4 (derived from pO34). The N-terminal sequence is shown as SEQ.I.D.No. 6.
7. SCREENING OF THE LIBRARY A cDNA library in lambda zapII (Stratagene) which had been prepared from mRNA isolated the flesh and albedo layer of the orange fruit was screened with the appropriate radio labelled PCR probe. The screening was performed according to the suppliers instructions except that the pre-hybridization and hybridization was performed in 2 x SSC, 0.1% SDS, 10 x Denhardt’s and 100g/ml denatured salmon sperm DNA. Hybridization was overnight at 67°C. The filters were washed twice in 2 x SSC. 0.1% SDS, twice in 1 x SSC, 0.1% SDS and twice in 0.1 x SSC, 0.1%
SDS.
8. PROBE The cloned PCR fragment was isolated from the pT7 blue vector by digestion with the appropriate restriction enzymes. The fragment was separated from the vector by agarose gel electrophoresis and the fragment purified and radiolabelled using the Ready to Go» DNA labelling kit (Pharmacia).
WO 97/03574 PCT/EP96/03051 46 9. SOUTHERN ANALYSIS Genomic orange DNA or plasmid DNA were digested with appropriate restriction enzymes, transferred to HybondN'» membranes and hybridized following the suppliers (Amersham) instructions.
IN SITU HYBRIDIZATION EXPERIMENTS In situ hybridization technique is based upon the principle of hybridization of an antisense ribonucleotide sequence to the mRNA. The technique is used to visualize areas in microscopic sections where said mRNA is present. In this particular case the technique was used to localize the mRNA encoding the enzyme pectin methyl esterase in sections of C. sinensis.
Preparation of tissue samples for in situ hybridization.
Thin slices of the middle part of a mature orange fruit were fixed by fixation with FAA fixation (45% ethanol, 5% formalin (40% paraformaldehyde) and 5% acetic acid) and incubation for 2 h at 25 0 C and 63 h at 5°C. The samples were washed for 3 x 20 min in a 0.05 M phosphate buffer, pH 7. Dehydration was carried out using a series of ethanol washes 70%, 80%, 96%) ending with 3 washes in 99% ethanol. Each wash was for 30 min. The samples were then treated with petroleum (Shellsol» D70k, Q7712) in 2 x 2 h and for another 2 x 2 h in paraffin with 7% beeswax. After this the samples were embedded in paraffin. Cross-sections of 12,5m were made using a Supercut 2050 Reichart Jung pyramitome.
Preparation of «S labelled probes for in situ hybridization A 501 bp PCR fragment from a second PCR amplification was cloned into the pT7blue vector (Novagen) in both directions. The pT7 vector contains a T7 promoter. The transcription of the antisense RNA and the sense RNA were driven by the SP7 promoter after digesting the plasmids with BamHI. A Maxiscript Kit WO 97/03574 PCT/EP96/03051 47 (Ambion) was used with the following modifications. The transcripts were run on a 6% sequencing gel to remove the incorporated nucleotide and eluted with the elution buffer supplied with the T7 RNA polymerase in vitro transcription kit (Ambion). The transcripts contained 55 non-coding nucleotides at one end and also 9 non-coding nucleotides at the other end. For hybridization 10 7 cpm/ml of the 3S labelled probe was used.
In situ hybridization was performed essentially as described by Langedale J.A.(1994) in the Maize Handbook-M. Freeling and V. Walbot, eds. pp 165-180 Springer- Verlag, New York, Inc. The hybridization temperature was found to be optimal at 57°C. After washing at 57°C the sections were covered with Kodak photographic emulsion and left for 3 days at 5°C in the dark.
11. RESULTS The following sequence information was used to generate primers for the PCR reactions mentioned below and to check the amino acid sequence generated by the respective nucleotide sequences see attached SEQ.I.D.No.s 7-19.
Peptide sequences of pectin methyl esterase used for generation of primers Asn Cys Asp Met Leu Ala Tyr Gin Asp Thr Leu Tyr (PE492B) Val ILe Thr Ser Ala Thr Glu Ala Gin Ala Phe Thr Pro Gly Ser Phe Ile Ala Gly Ser Ser Trp Leu Gly Ser Thr Gly Phe (PE701) The amino acid sequence (PE492B,4-7) used to generate primer A (Met Leu Ala Tvr Gin Asp Thr) Primer A ATG(CT)T(GATC)GC(GATC)TA(TC)CA(AG)GA(TC)AC 256 mix WO 97/03574 PCT/EP96/03051 48 The amino acid sequence (PE701,7-13) used to generate primer B (Glu Ala Gin Ala Phe Thr Pro) Primer B GT(AG)AA(GATC)GC(TC)TG(GATC)GC(TC)TC 128 mix (the sequence corresponds to the complementary strand) Generation of PCR DNA fragment partially encoding pectin methyl esterase The amino acid sequences of the two non-overlapping peptides (described above) were used to generate mixed oligonucleotides. These were used as primers for reverse transcription and the following amplification (PCR) of total RNA. The resulting PCR clone was sequenced and had an insert of 501 bp. The deduced amino acid sequence from the nucleotide sequence corresponded nearly perfectly with the peptide sequences given in SEQ.I.D.No.s 7-19.
In situ hybridization analysis The in situ hybridization experiments (see Materials and Methods) with the riboprobes (produced from the appropriate PCR clones) against the mRNA of pectin methyl esterase, showed strong hybridization in the outer cell layer of the lamella between the segments (see Figure Strong signals were also obtained from the outer cell layer of the juice sacs, the inner cell layer of the albedo and the core. These results correspond very well with the immunological localizations results seen with the antibody raised against peel PME as described previously.
Southern analysis Southern analysis showed that additional copies of the isolated PME genes (represented by the cDNA clones) are present in the C. sinensis genome. These other PME genes were rather homologous with the ones represented in p017 and p034.
WO 97/03574 PCT/EP96/03051 49 In this regard, we observed in each lane one band of a strong hybridization signal, two bands of a middle hybridization signal and at least two more bands of a weaker signal compared with the rest. This pattern indicates that C. sinensis has at least between 5 and 7 copies of PME genes in the genome.
Isolation and characterization of novel PME cDNAs A cDNA library in lambda zaplI (Stratagene) which had been prepared as described in Materials and Methods was screened with the radiolabelled 501 bp insert from the PCR clone. Several hybridizing clones were identified and plasmid DNA was excised in vivo according to the suppliers instructions. The size of the cDNA inserts in the clones were determined by digestion with EcoRV and SmaI followed by agarose gel electrophoresis. One clone pO34 had an insert size of approximately 2 kb whereas another clone p017 had an insert size of approximately 1.4 kb. These clones were selected for further analysis. The nucleotide sequences were determined and are shown as SEQ.I.D. NO. 3 and SEQ.I.D. No. 4 for p017 and p034, respectively.
The open reading frame in 034 starting at nucleotide 29 and ending at nucleotide 1780 encodes a PME of 584 amino acids including a putative signal peptide. A possible cleavage site between glycine at position 46 and isoleucine at position 47 can be predicted according to the rules of von Heijne, G.(1986) Nucl Acids Res 14, 4683- 4690 «A new method for predicting signal sequence cleavage sites», thereby giving a long mature PME enzyme of 538 amino acids having a calculated molecular weight of 58386 dalton. The molecular weight of the long PME including the signal sequence can be calculated to 63502 dalton. p034 nucleotide sequence includes an untranslated 5′ region of 29 nucleotides and a untranslated 3′ region of 186 nucleotides. ending in a poly A tail.
The open reading frame in 017 starts at nucleotide 18 and terminates at nucleotide 1103. It encodes a short PME of 362 amino acids including a signal peptide of 44 amino acids. A putative cleavage site can be predicted between glutamine at position 44 and the serine at position 45 leaving a mature short PME starting with the amino WO 97/03574 PCT/EP96/03051 acid sequence Ser-Ser-Ser-Val-Thr-Pro. This N-terminal amino acid sequence is identical to the N-terminal amino acid sequencing of the purified short type of PME as described in the biochemical section. The amino acids sequences obtained from the purified enzyme were aligned with the deduced mature amino acid sequence of p017. Full identity was found for the peptide fragments.
There was nearly full identity between all the sequenced peptides and the deduced amino acid sequence in p017 and also with the amino acid sequence deduced from pO34, the long PME. In this regard, p017 differs at one amino acid position (no.
24 in the sequence encoding the mature polypeptide). The mature short PME protein has a calculated molecular weight of 33954 dalton. The calculated molecular weight of the short PME form including the signal peptide is 39088 dalton. 017 includes a 5′ untranslated region of 17 nucleotides and a 3′ untranslated region of 180 nucleotides, ending in a polyA tail.
The major difference between the long and the short PME enzyme is an N-terminal extension of 220 amino acids present in the mature enzyme derived from 034 and presented as SEQ.I.D.No. 5. The corresponding nucleotide sequence encoding this region of the long PME enzyme is shown as SEQ.I.D.No. 6.
EXPRESSION OF PME IN MICRO-ORGANISMS The DNA sequence encoding either the long or the short form of orange PME was introduced into micro-organisms to produce a recombinant enzyme in large quantities having a high specific activity to be used for enzymatic treatment of pectin.
EXPRESSION IN PICHIA PASTORIS Construction of pJK10, pJK11 and pJK12 (see Figs. 13-15) p034 plasmid DNA was used as template DNA in a PCR reaction with the following set of primers: WO 97/03574 PCT/EP96/03051 51 5′-GAATTCATTGTCGCCGGAGTGAAC-3′ with an EcoRI site at one end and 5′-AAGACCAGAGACCTATGGATCCAC-3′ with a BamHI site near the end.
Combining AmpliTaqR DNA Polymerase (Perkin Elmer), template DNA, dATP, dGTP, dCTP and dTTP and the two primers in the following buffer: mM Tris-HCI (pH 15 mM (NH 4 2 SO, and 1.5 mM MgCl 2 and using the following temperature cycle: 94 0 C 2 min 94°C 1 min 2 min 72 0 C 2 min in 35 cycles 72*C in 7 min soak produced the expected PCR product of 1690 bp which was purified and subcloned into the vector pT7Blue (from Novagen) following the suppliers instructions.
Resulting clones (called pT7-034) containing an EcoRI-BamHI fragment of the expected size were further verified by DNA sequencing (see Materials and Methods for Molecular Biology). A pT7-034 subclone containing the correct sequence was digested with EcoRI and BamHI and the fragment of 1685 bp was purified and subcloned into the Pichia pastoris vector pHIL-S 1 (from Invitrogen) digested with the same enzymes.
The sequence encoding the mature long form of PME was cloned in this way in frame with the PHO1 secretion signal in the vector. The resulting plasmid is WO 97/03574 PCT/EP96/03051 52 called pJK10 and is shown in figure 13.
To create pJK11 (see figure 14) the EcoRI-BamHI fragment of the pT7-034 clone was further subcloned into a pBSK- vector (from Stratagene) digested with the same enzymes. Then, an EcoRI-NotI fragment from one of the resulting clones (verified by DNA sequencing) was subcloned into the Pichia pastoris expressions vector pPIC9 (from Invitrogen) digested with the same enzymes. This places the reading frame encoding the mature protein, of the long form of PME, downstream and in frame with the alpha-factor secretion signal in pPIC9, see figure 14.
In addition, the EcoRI-NotI fragment from pT7-034 was also subcloned into the pPIC9K (Invitrogen) vector creating the pJK12 plasmid shown in figure 15. The only difference between pJK11 and pJK12 is a gene encoding resistance for kanamycin and this is situated in pJK12.
Both the PHO1 secretion signal in pJK10 and the alpha secretion signal in pJK11 and pJK12 can direct the secretion of the mature polypeptide encoding the long form of
PME.
Construction of pJK20. pJK21 and pJK22 p017 plasmid DNA was used as a template DNA in a PCR reaction with the following primers: 5′-GAATTCTCCTCGTCGGTGACACCG-3′ with an EcoRI site at one end and 5′-AAGACCAGAGACCTATGGATCCAC-3’ with a BamHI site near the end.
The template DNA. AmpliTaqR polymerase, dATP, dGTP, dCTP and dTTP were combined with the buffer (60 mM Tris-HC1, pH 8.5; 15 mM (NH4)2SO 4 and 1.5 mM MgCI 2 and placed in a thermoblok with the following temperature cycle: WO 97/03574 PCT/EP96/03051 53 94 0 C 2 min 94°C 1 min 2 min 72 0 C 2 min in 35 cycles 72 0 C in 7 min soak This produced an expected PCR band of 1008 bp which was purified and subcloned into pT7Blue (Novagen). The resulting clones were verified by DNA sequencing as explained above. A plasmid with the correct sequence was digested with EcoRI and BamHI and the resulting fragment further subcloned into the pHIL-S1 vector (Invitrogen) the resulting plasmid is called pJK20 (shown in figure 16) and in this, is the region encoding the mature polypeptide of the short form of PME situated in frame and downstream of the PHO1 secrection signal.
Plasmid pJK21 and pJK22 were contructed in the same manner as explaned for the pJK11 and pJK12 except that the EcoRI-NotI fragment was obtained from the pBSK- 017 clone. Figure 17 and 18 show the resulting plasmids where the region encoding the mature polypeptide is situated downstream, and in frame, of the alpha secretion signal in respectively pPIC9 and pPIC9K.
Introduction of the long or the short form of PME into Pichia pastoris by spheroplast formation and transformation The plasmids pJK10, pJK11, pJK12, pJK20, pJK21 and pJK22 were introduced in separate experiments into Pichia pastoris GS115 cells.
In this regard, spheroplasts were prepared from the GS115 cells growing in yeast extract peptone dextrose medium (YPD) at 28-30 0 C. The preparation of spheroplasts WO 97/03574 PCT/EP96/03051 54 and the transformation procedure were performed as described in the Pichia expression kit: Instruction Manual (Invitrogen). The resulting Pichia pastoris Mut or Muts transformants were then analyzed for the expression of the recombinant PME gene by assaying the supernatant for PME activity using the methods decribed in: Materials and Methods for Biochemistry.
Screening of transformants for high expression of the long or the short form of PME Putative positive transformants were further grown in minimal medium (either Minimal Dextrose Medium or Minimal Glycerol Medium as specified in the Pichia expression kits Instruction Manual, Invitrogen) and the genomic DNA was isolated and analysed by PCR as decribed in the Instruction Manual.
Positive clones found in the initial screen, which also produced a PCR band of the expected size, were selected and further grown in a flask at 28-30 0 C following the procedure described in the Pichia Instruction Manual.
At least 10 verified recombinant clones of each construct (pJK10, pJKll, pJK12, pJK21 and pJK21) were screened for secretion of PME and their relative expression level were measured over time by sampling every 2 to 6 hours. The cells in the samples were pelleted and the supernatant assayet for PME activity following the methods explained in: Materials and Methods for Biochemistry.
The clones obtained after transformation with either pJK12 or pJK22 were preselected using the integrated Kanamycin resistance gene as the selector gene as decribed by Scorer C.A. et al.(1994) Bio/Technology vol 12, ppl81-184 and Laroche Y. et al (1994) Bio/Technology vol 12, pp 1119-1124. In this way multiple copy integration transformants were obtained and further screened as described above.
Transformants representing either the long or the short form of PME and shown to express the recombinant protein in high levels were selected and further analyzed by Western Blot analysis (see Materials and Methods for Biochemistry).
WO 97/03574 PCT/EP96/03051 Purification and charaterization of recombinant PME Recombinant PME protein was purified from the cultures supernatant using the procedures described in the Materials and Methods for Biochemistry as necessary.
The presumed high temperature stability of the long form of PME was verified by testing the enzyme activity after incubation at different temperatures as explained in the section: Charaterization and kinetic data. The purified recombinant enzymes (both the long and the short PME form) were further charaterized as explained in the: Characterization and kinetic data section. These analyses showed that both types had a pH optimum around 7-8 and the temperature stability of the short recombinant
PME
was found to be between 10 and 40 0 C and up to 80 0 C for the long recombinant
PME.
EXPRESSION IN ASPERGILLUS
NIGER
In another embodiment, p034 or p017 was digested with the appropriate restriction enzymes and the coding sequence for the long or the short form of PME of the present invention was cloned into the Aspergillus expression vector pBAMTE1 (containing methyl tryptophan resistance promoter from Neurospora crassa) for expression in Aspergillus niger (Pall et al. (1993) Fungal Genet Newslett. vol 40 pp 59-62).
The protoplasts were prepared according to Daboussi et al. (Curr Genet (1989) vol 15. pp453-456) using lysing enzymes Sigma L-2773 and the lyticase Sigma L-8012.
Transformation of the protoplasts followed the protocol stated by Buxton et al. (Gene (1985) vol 37 pp 207-214) except that for plating the transformed protoplasts the protocol laid out in Punt et al. (Methods in Enzymology (1992) vol 216 pp447-457) was followed but with the use of 0.6% osmotic stabilised top agarose.
The results showed that purified orange PME activity was otainable from Aspergillus niger cultures.
WO 97/03574 PCT/EP96/03051 56
USES
EFFECT OF ORANGE PME TREATED PECTIN ON THE VISCOSITY AND STABILITY OF PROTEIN DRINKS
METHODS
Enzymatic treatment of pectin with PME derivable from orange A batch of enzymatically treated pectin was prepared as follows: 125 g pectin was dissolved in hot water under efficient stirring. 45.3 g NaCl (reagent grade) was added and the volume adjusted to 4.0 1 with water. This solution was stirred until the salt had dissolved. The pectin solution was cooled to 40°C and the pH was increased to pH 7.0, using 1 N NaOH (reagent grade) and efficient stirring.
An appropriate sample of orange PME was added and the enzymatic reaction continued until the desired degree of estrification was achieved. The pH was kept constant at pH 7 by automatic dosage of 1 N NaOH (reagent grade) during the incubation period, and the enzymatic reaction was followed by the consumption of NaOH.
When the pectin sample had reached the desired degree of de-esterification the NaOH addition was stopped, the pH of the solution lowered to about 3.0 by addition of 2 HC1. The pectin solution was then heated to 70 0 C for 5 min to completely inactivate the enzyme. The treated pectin was precipitated with 1 volume of isopropanol, washed with 60% isopropanol and pressed to about 50 dry matter.
The enzymated pectin batch was then air dried at 40 0 C and finally milled to a dry powder.
WO 97/03574 PCT/EP96/03051 57
PROTOCOL
Determination of pectin samples for calcium sensitivity index (CF) Calcium sensitivity is measured as the viscosity of a pectin dissolved in a solution with 57.6 mg calcium/g pectin divided by the viscosity of exactly the same amount of pectin in solution, but without added calcium. A non calcium sensitive pectin has a CF value of 1.
4.2 g pectin sample is dissolved in 550 ml hot water with efficient stirring. The solution is cooled to about 20 0 C and the pH adjusted to 1.5 with 1N HC1. The pectin solution is adjusted to 700 ml with water and stirred. 145 g of this solution is measured individually into 4 viscosity glasses. 10 ml water is added to two of the glasses (double determinations) and 10 ml of a 250 mM CaC1, solution is added to the other two glasses under stirring.
ml of an acetate buffer (0.5 M, pH about 4.6) is added to all four viscosity glasses under efficient magnetic stirring, thereby bringing the pH of the pectin solution up over pH 4.0. The magnets are removed and the glasses left overnight at 20°C. The viscosities are measured the next day with a Brookfield viscometer. The calcium sensitivity index is calculated as follows: Viscosity of a solution with 57.6 mg Ca2/ g pectin CF Viscosity of a solution with 0.0 mg Ca 2 g pectin WO 97/03574 PCT/EP96/03051 58 Determination of pectin samples degree of esterification (%DE) To 50 ml of a 60 isopropanol and a 5 HCI solution is added 2.5 g pectin sample and stirred for 10 min. The pectin solution is filtered through a glass filter and washed with 15 ml 60 isopropanol/5 HCI solution 6 times followed by further washes with 60% isopropanol until the filtrate is free of chlorides. The filtrate is dried overnight at 80 0
C.
20.0 ml 0.5 N NaOH and 20.0 ml 0.5 N HC1 is combined in a conical flask and 2 drops of phenolphtalein is added. This is titrated with 0.1 N NaOH until a permanent colour change is obtained. The 0.5 N HCI should be slightly stronger than the NaOH. The added volume of 0.1 N NaOH is noted as V 0 g of the dried pectin sample (the filtrate) is measured into a conical flask and the sample is moistened with 96% ethanol. 100 ml of recently boiled and cooled destilled water is added and the resulting solution stirred until the pectin is completely dissolved. Then 5 drops of phenolphtalein are added and the solution titrated with 0.1 N NaOH (until a change in colour and pH is The amount of 0.1 N NaOH used here is noted as V 1 20.0 ml of 0.5 N NaOH is added and the flask shaken vigously, and then allowed to stand for 15 min. 20.0 ml of 0.5 N HC1 is added and the flask is shaken until the pink colour disappears. 3 drops of phenolphtalein are then added and then the resultant solution is titrated with 0.1 N NaOH. The volume 0.1 N NaOH used is noted as V 2 The degree of esterification DE: of total carboxy groups) is calculated as follows:
V
2 -Vo DE V, (V 2 Vo) WO 97/03574 PCT/EP96/03051 59 Yoghurt production Standardised skimmed milk (prepared by mixing powdered milk with an adequate volume of water) was heated at 90 0 C in 5 min and then homogenized at 200 kp/cm 2 and the milk cooled to 31 C. Yoghurt culture was added and the milk fermented to about pH 4.0. The yoghurt was cooled to about 20 0 C and the pectin sample was added as a saturated sugar solution (about 65 sugar) and stirred for 15 min. The pH was adjusted to pH 4.0 with lactic acid. The yoghurt was pasteurized at 88 0 C for seconds and homogenized at 150 kp/cm 2 then cooled to 20°C and filled into sterile 250 ml blue cap bottles (200 ml/bottle).
The composition of the final product was: 7.6 MSNF (milk solid content), 9.15 sugar and 0.25 or 0.35% pectin sample giving total solids of 17.0 or 17.10 respectively.
Viscosity determination of yoghurt drink The viscosity of a yoghurt sample was determined (double determinations) using either a Bohlin Rheometer» (supplied by Bohlin Instruments) with shear rates 18.5- 46.0 or a using a StressTech (Rheologica instruments AB) with the same shear rates.
Protein stability measured by a centrifugation test g of a sample drinking yoghurt) is centrifuged at 10°C, 2300xg for 20 min.
The supernatant is discarded and the centrifuge glass placed upside down for 30 min.
The glass is weighed and the sedimentation was calculated as follows: Wgt of glass after centri Wgt of glass Sedimentation X 100 Wgt of sample Where Wgt= weight and centri= centrifugation WO 97/03574 PCT/EP96/03051 Protein stability as judged by particle size distribution in the sample Particle size distribution of a yoghurt sample was determined by the use of a Malvern 2600 Easy» sizer. The particle size is determined by laser light scattering by this method. 1 ml yoghurt sample was added to 9 ml de-areated buffer solution (30.7 0.1 M citric acid, 19.3 0.2 M Na 2
HPO
4 and 50.0 water) and mixed. The deareated buffer is added to the measuring glass and the mixture of sample/buffer is added droplet by droplet until the optimum concentration is obtained. The average particle size is calculated from the measurements.
A yoghurt with the average particle size below about 3 pm is considered rather stabile while a yoghurt with an average particle size over about 10 pm and higher is considered not to be stabile for long term storage.
Determination of long-term stability Samples were stored at 4°C or at ambient temperature and the whey separation was measured (in mm of whey at the top of the sample, in the bottle). The sample was filled in 250 ml blue cap bottles (double determinations). The depth of the sample in each case was approximately 70 mm which corresponded to the 200 ml mark for each bottle.
EXAMPLE 1 SOUR MILK DRINK The purpose of adding pectin to a sour milk drink a yoghurt drink) is to produce a drink that remains physically homogenous during the bacteriological and organoleptic shelf life of the drink. In addition the treatment of the yoghurt drink for long term storage destabilizes the protein in the drink giving rise to a drink with a sandy mouthfeel and showing rather quickly syneresis, if pectin is not added.
WO 97/03574 PCT/EP96/03051 61 Treatment of the pectin A commercially available high ester pectin; Grindsted T M Pectin URS (Iltra Rapid Set pectin type) was chosen as the mother pectin, due to its high ester level DE of 82, see figure 19). The treatment with the orange PME enzyme of this mother pectin is explained in the method section.
The enzymatic reaction was stopped and the resulting experimental pectin (called pectin no 1944-96-2) was investigated for its degree of esterification, by the method described in the method section. In addition, to compare the two pectin types, the mother pectin and the treated pectin, with a known good commercial drinking yoghurt pectin type, the GrindstedT Pectin AM453 was included in the experiments.
The relative calcium sensitivity of the three chosen pectins was determined as explained in the method section: Determination of pectin samples calcium sensitivity index and the results are shown in the Table below.
Pectin-type a CF DE GrindstedTM Pectin URS 1.1 82 Pectin 1944-96-2 1.4 76 Grindsted T M Pectin AM453 >20 72 De-esterification of the GrindstedTM Pectin URS mother pectin from 82 DE down to 76 DE gave nearly no change in the calcium sensitivity of the these two pectins (a a CF of 1 have no sensitivity). A pectin with measurable calcium sensitivity could be produced by further treatment with the orange PME enzyme on the mother pectin down to 70% DE, since this pectin had a A CF of 14.
WO 97/03574 PCTIEP96/03051 62 Results of Yoghurt/Drinking Yoghurt Analysis Yoghurt was produced as explained in the method section. The three pectins (GrindstedTM Pectin URS, Pectin 1944-96-2 and GrindstedTM Pectin AM453) were used individually in the following recipes: 7.6 MSNF (milk solid content), 9.15 sugar and 0.25 or 0.35% pectin sample giving total solids of 17.0 or 17.10 respectively in the final product.
The quality of the individual yoghurt produced was investigated by their sedimentation seen in the centrifugation test, by measuring the particle size in the yoghurt, by measuring the viscosity and by examination of possible whey separation during long time storage, as described in the method section.
The sedimentation of the yoghurt produced with the three pectin types are presented in the Table below.
SEDIMENT (IN OF THE YOGHURT Pectin-type Concentration 0.25% Concentration 0.35% GrindstedTM Pectin URS 29.40 21.02 Pectin 1944-96-2 1.53 2.95 GrindstedTM Pectin 2.20 1.85 AM453 The results are the average of from one to four individual production.
WO 97/03574 PCT/EP96/03051 63 It is clear that the mother pectin (URS type) had a high sedimentation and consequently the yoghurt produced showed whey separation and was unstable at both pectin concentrations used (0.25 and 0.35 This is not surprising since normally the URS pectin type cannot be used for stabilization of heat treated yoghurt types for long time storage.
The yoghurt produced with the Pectin 1944-96-2 showed stability at both pectin dosages used and low sedimentation as seen by the above results, and no whey separation after over 75 days of storage. In comparison, the excellent GrindstedTM Pectin AM453 normally used in yoghurt production also showed low sedimentation, as expected, and produced stabile yoghurts with no whey separation (see results above).
By treating the mother URS pectin with orange PME an unsuitable pectin was made suitable as a stabilizing agent in the yoghurt, and this treated pectin behave just as well as an excellent commercial stabilizer.
Futher examination of the produced yoghurts by particle size determination (see the methods section) were performed and the results are shown in the Table below.
PARTICLE SIZE (IN uM) OF THE YOGHURT Pectin-type Concentration 0.25 Concentration 0.35% GrindstedTM Pectin URS 9.32 6.41 Pectin 1944-96-2 1.33 1.55 GrindstedTM Pectin 1.40 1.53 AM453 WO 97/03574 PCT/EP96/03051 64 The average particle size (the number corresponding to the fraction using the Malvern instrument, is shown) of the yoghurt produced with the mother URS pectin is high, as expected. Again the results are an average of one to four productions.
The average particle size of the yoghurt produced from both the pectin 1944-96-2 and the GrindstedT Pectin AM453 are small at both pectin dosages used (see results above). Again showing that the yoghurt produced with these pectin types are suitable to produce stabile yoghurts.
Finally, and very importantly, in the case of drinking yoghurt production for long time storage, the viscosity was determined and the results are shown in the Table below.
VISCOSITY (IN MPa s) OF THE YOGHURT Concentration Pectin- type Concentration 0.25% Concentration 0.35% GrindstedT Pectin URS 55 Pectin 1944-96-2 12 18 GrindstedTM Pectin 24 43 AM453 Since the mother URS pectin could not stabilize the yoghurt the viscosity obtained is, as expected rather high at both pectin concentrations. The excellent GrindstedT Pectin AM453 which produced stabile yoghurts with low sedimentation and low particle size showed a viscosity of about half the viscosity seen with the Grindted T M Pectin URS pectin at the 0.25 pectin dosage and approximately the same as the URS pectin at the 0.35 pectin dosage irrespective of the fact that only the AM453 pectin produced a stabile yoghurt.
a WO 97/03574 PCT/EP96/03051 In both the URS and the AM453 cases the higher viscosity seen could partly be attributed to the amount of pectin added, especially this seems to be the situation in the AM453 pectin case since an increase in the amount of pectin added (from 0.25 to 0.35 produced nearly twice as viscous a yoghurt.
The viscosity obtained with the orange treated PME pectin 1944-96-2 shows a dramatic fall in viscosity compared with the mother URS pectin at 0.25% concentration. This is partly due to the stabilization of the yoghurt with the 1944-96- 2 pectin but not the only reason since the AM453 yoghurt has twice as high a viscosity at that pectin dosage. Indeed. adding 0.35 of the 1944-96-2 pectin to the yoghurt only increases the viscosity with 6 units (compared with the 0.25 dosage) while the viscosity increases with 19 units going from 0.25 to 0.35 in the AM453 case.
The orange PME treated pectin 1944-96-2 can stabilize yoghurt having a very low viscosity, despite the pectin dosage was 0.25 (or 0.35 which gave nealy twice as high a viscosity using other untreated pectins e.g. GrindstedTM Pectin AM453.
This is a new and very important development for the production of sour milk drinks.
By treating the GrindstedTM Pectin URS with orange PME a new pectin type is created which in contrast to the mother pectin can stabilize yoghurt and most importantly shows much lower viscosity than normally used pectins. Other high ester pectin can also be improved by treatment with the orange PME.
EXAMPLE 2 Whey Juice Drink The modified pectin according to the present invention (as prepared above) was used in a whey juice drink as follows: WO 97/03574 PCT/EP96/03051 66 Sweet or acid whey 42.00% Fruit juice 40.00% Sugar 8.00% Sodium citrate 0.20% PME modified pectin 0.20% Grindsted Flavouring Water 9.60% Dry PME modified pectin, sodium citrate and sugar were mixed and then dissolved in water at 80°C. This pectin solution was cooled to below 5°C and whey was added at 5°C. Grindsted Flavouring (supplied by Danisco Ingredients, Danisco A/S) and juice were added slowly and the pH in the sample mixture was adjusted with citric or lactic acid to pH 4.0. The sample mixture was aged for approx. 30 min under agitation. The pasteurisation was performed at 80°C/15 seconds and homogenisation at 200 bar (2900 psi). The samples were cooled to 20’C and filled aseptically in containers.
Sample tests were analysed after 24 hours, 1 months and 6 months incubation at room temperature. The investigation analysis included viscosity measurements, stability index particle size and long-term stability the protocols for which are described above.
The results showed improved stability and a long-term stability in the whey juice drink processed with PME modified pectin compared to the reference pectin employed in the control tests. In addition, the whey juice drink had a favourable viscosity which was lower than the control drinks.
The whey juice drink was also processed with plant PME modified lime and lemon pectin, respectively, i.e. modified with the PME of the present invention. The results demonstrate that PME modified pectin has improved protein stability compared to the non-modified pectin and also compared to the reference pectin.
WO 97/03574 PCT/EP96/03051 67 EXAMPLE 3 MILK/FRUIT JUICE DRINK GrindstedT URS (obtained from Danisco Ingredients, Danisco A/S) was modified with the PME as described for the yoghurt drink. The modified pectin was used in a milk/fruit juice drink which contained: Skimmed milk 45.00% Fruit juice 40.00% Sugar 5.00% PME modified pectin 0.25% Grindsted Flavouring Water 9.75% Dry PME modified pectin and sugar were mixed and then dissolved in water at The pectin solution was cooled to below 5°C and milk was added at 5 0 C. Grindsted Flavouring and juice were added slowly and the pH in the sample mixture was adjusted (if necessary) with citric or lactic acid to pH 4.0. The sample mixture was aged, pasteurised and homogenised as described for whey juice drink. The samples were cooled to 20 0 C and filled aseptically in containers.
The results showed improved stability including long-term stability in the milk/fruit juice drink processed with PME modified pectin compared to the reference pectin employed in the control tests. In addition, the milk/fruit juice drink had a favourable viscosity which was lower than the control drinks. Improved functionality was also observed.
The milk/fruit juice drink can also be processed with lime and lemon pectin modified with the PME of the present invention.
WO 97/03574 PCT/EP96/03051 68 EXAMPLE 4 Whey stability The enzymatic modified pectins were tested at pH 4.0. The pH of the resultant pectin solutions were adjusted to pH 4.0 with KOH/HCI. The pectin concentration was adjusted to The pectins were tested in concentrations of 0.1% 0.25 Pectin, Jenness Buffer (see below) and whey solution (see below) were mixed and heated at 96 C for 25 min. After cooling to room temperature the absorbance is measured at 500 nm.
Dry Blend Jenness Buffer: Dry Powder Jenness (described in Jenness, R and Koops, J Preparation and Properties of a salt solution which simulates milk ultrafiltrate, Nederlands Melk- en Zuiveltijdschrift, vol 16 nr 3, pp 153-164, 1962): 15.80 g KH 2
PO
4 5.08 g K 3 citrate 17.91 g Na 3 citrate, 2.H 2 0 1.80 g g K2SO 4 13.20 g CaCI,, 2.H,O 5.02 g Mg 3 citrate, HO 3.00 g K2CO 3 10.78 g KCI Buffer solution: Aqueous solution of 7.5900 g/1 Dry Powder Jenness with a pH of WO 97/03574 PCT/EP96/03051 69 Whey Solution A concentrate of whey protein is freeze dried and pulverized. A solution of 0.40% w/w whey protein is prepared in Jenness Buffer at pH Pectin solutions 1% w/w aqueous solutions of pectins are made with a pH of Mixture concentrations: Pectin, Jenness Buffer and Whey solution are mixed as indicated in the Table below.
The mixtures are heated at 96 0 C for 25 minutes and after cooling to room temperature the samples are measured on a spectrophotometer at 500 nm.
k1 pectin solution Al Jenness Buffer Al whey solution b1 total volume 500 2000 2500 5000 750 1750 2500 5000 1000 1500 2500 5000 10 1250 1250 2500 5000 Results The results are shown in the Table below. The quoted absorbance value at 500 nm is a mean value taken from two measurements. For comparison between the different types of pectin and the enzymatic modified pectins according to the present invention the index for non-modified pectin Grindsted T Pectin 3450 (supplied by Danisco Ingredients, Danisco A/S) is set to 100.
1 WO 97/03574 PCT/EP96/03051 An Index of 100 indicates poorer protein stability than using the sample 3450. An Index of 100 indicates similar stability to the sample 3450. An Index of 100 indicates better protein stability than using the sample 3450. An Index of 95 or indicates very good protein stability.
Pectin type 0.10% pectin 0.15% pectin 0.20% pectin 0.25% pectin GrindstedT1 100 100 100 100 pectin 3450 Grindsted™ 127 140 155 161 pectin URS min enz. 100 108 115 113 min enz. 98 106 111 111 min enz. 92 94 100 100 min enz. 90 95 100 99 As can be seen from the results the Grindsted T pectin URS pectin modified according to the present invention exhibits favourable properties and in some instances very good properties for increasing stability when compared to the reference Grindsted™ pectin 3450 and the un-modified Grindsted» pectin URS pectin.
EXAMPLE Laban Drink with long Shelf life, low pH The Laban drink is an acidified milk drink with a pH value below 4.2. The laban drink consists of Laban base mixed with pectin solution. The formulation for laban base is: 1 WO 97/03574 PCT/EP96/03051 71 Anhydrous milk fat 2.8% Skimmed milk powder 10.0% Grindsted flavouring Water 87.2% Standardised whole milk (anhydrous milk fat and skimmed milk powder) is homogenised at 75-80°C and pressure of 200 bar (2900 psi) followed by pasteurisation at 90-95 C for 5-10 min. After culturing to pH around 4.0 the pH is adjusted to 3.8 4.2 with citric or lactic acid. Pectin solution is then added. The mixture is then agitated until it is a homogeneous mixture. It is then pasteurised at 90-95 0 C for 10-15 seconds and further homogenised at 150-200 bar. After cooling to 20-25 0 C the product is filled aseptically in containers.
After a few days, non-stabilised laban drink often show syneresis.
However, addition of the enzymatically modified pectin according to the present invention prevents syneresis and improves the viscosity.
In addition, the product has a pronounced yoghurt taste and a long shelf life.
EXAMPLE 6 Orange Juice (Protein-enriched) Orange juice drink is an acidified drink (pH approx. 4) containing 2 DANPROLACT 40TM (Central Soya, Aarhus 6% sugar, 10% orange concentrate, 0.4% lemon concentrate, 0.2% pectin and 81.4% water. The product is an orange juice drink enriched with soya protein. The product is pasteurised and homogenised. After cooling to 20-25 C the product is filled aseptically in containers and can be stored for approx. 6 months at room temperature. Addition of enzymatically modified pectins according to the present invention in orange drink WO 97/03574 PCT/EP96/03051 72 (protein-enriched) showed favourable properties such as long term stability and it had a good mouth feel.
EXAMPLE 7 Antibody production Antibodies were raised against the enzyme of the present invention by injecting rabbits with the purified enzyme and isolating the immunoglobulins from antiserum according to procedures described according to N Harboe and A Ingild S 10 («Immunization, Isolation of Immunoglobulins. Estimation of Antibody Titre» In A Manual of Quantitative Immunoelectrophoresis, Methods and Applications, N H Axelsen, er al Universitetsforlaget, Oslo, 1973) and by T G Cooper («The Tools of Biochemistry», John Wiley Sons, New York, 1977).
15 Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention.
In the following pages a number of sequence listings are presented which have been consecutively numbered from SEQ.I.D. No. 1 SEQ.I.D. No. 19, which represent nucleotide sequences and amino acid sequences.
Throughout the specification, unless the context requires otherwise, the word «comprise» or variations such as «comprises» or «comprising», will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
WO 97/03574 WO 9703574PCT/EP96/03051
SEQUENCES
SEQ.I.D. NO. 1 MIRMTDTDM MIMRTSNNP.K NVVVAADGSG NFKTVAAAVA NIMFIGDGRT RTIITGSRNV GPSKHQAVAL RVGADLSAFY FIFGNAAAVL QNCDIHARKP ATSDLKPVQG SFPTYLGRPW LNTLFYGEHQ NAGAGAGTSG LGSTGFPFSL GL
LIEETSTVDG
AAPQGGTKRY
VDGSTTFKSA
NCDMLAYQDT
NSGQKNMVTA
KEYSRTVIMQ
RVKWKGFRVI
WPAWLSTGDR
I IRIKAGVYR
TVAVVGEGFL
LYVHSNRQFF
QGRADPNQNT
SS ITDVIHPA
TSATEAQAFT
RLLQSSSVTP
ENVEVTKIK!K
ARDITFQNTA
VNCLIAG7VD
GIVIQKSRIG
GWHEWDGNFA
PGSFIAGSSW
s0 100 150 200 250 300 350 362 SEQ.I.D. NO. 2
MTRIKEFFTK
VNSRKNSGDN
DVIEMSLNIT
ELHKAVEDLE
HVRDALSDGQ
DGWPAWLSTG
RYIIRIKAGV
SATVAVVGEG
DTLYVHSNRQ
TAQGRAflPNQ MQSS ITDVIH
VITSATEAQA
LSES STNQNI
GNEPHHAILK
TTAVEHNlYFG
EYPNKKSLSQ
VHV’EKMCSNA
DRRLLQS SSV
YRENVEVTKK
FLARDITFQN
FFVNCLIAGT
NTGIVIQKSR
PAGWHEWDGN
FTPGS FIAGS SNI PKKKKKL
SSCSSTRYPD
IQKLLKRTNL
HADDLKTLMS
LAMIKNMTDT
TPNVVVAADG
HKN~IMFIGDG
TAGPSKHQAV
XTDFIFGNAAA
IGATSDLKPV
FALNTLFYGE
SWLGSTGFPF
FLALFATLLV
LCFSAIAAVP
TKREKVALHD
AAMTNQGTCL
DMMIMRTSNN
SGNFKIVAAS
RTRTI ITGSR
ALRVGADLSA
VLQNCDIHA.
QGSFPTYLG.
HQNAGAGAGT
SLGL
VAAVIGIVAG
EASKKVTSQK
CLETIDETLD
DGFSHDDANK
RKL IEETSTV
VAAAPQGGTK
NVVDGSTTFK
FYNCDMLAYQ
KPNSGQKNMV
PWKEYSRTVI
SGRVKWKGFR
SEQ.I.D. NO. 3
GTAGCAATGC
ATCATGAGGA
TGATGGGTGG
CCTCGTCGGT
TTTAAGACGG
GCGGTATATT
TGACAAAGAA
ACTATCATCA
GTCTGCTACA
CATTCCAA-AA
GTGGGAGCTG
AGACACACTC
TTGCTGGCAC
AATTGTGACA
CACAGCCCAA
GCTTGCTATG
CTTCAAACAA
CCGGCGTGGC
GACACCGAAC
TGGCGGCAGC
ATTAGGATTA
GCATAAAAAT
CAGGAAGTAG
GTTGCTGTTG
CACAGCCGGC
ACCTTTCAGC
TACGTCCACT
GGTTGATTTT
TCCATGCACG
GGCAGGGCTG
ATCAAGAACA
CAGGAAGCTG
TGTCCACCG
GTGGTGGTGG
GGTGGCGGCG
AAGCCGGTGT
ATAATGTTCA
AAATGTGGTT
TTGGTGAAGG
CCCTCAAAGC
ATTTTACAAT
CGAACCGCCA
ATTTTTGGTA
AAAGCCCAAT
ACCCTAACCA
TGACTGACAC
ATAGAGGAGA
AGACAGGAGG
CAGCAGATGG
GCTCCTCAGG
TTATCGGGAA
TCGGTGACGG
GATGGAAGCA
ATTCTTGGCC
ACCAGGCGGT
TGCGATATGT
GTTCTTTGTG
ACGCTGCAGC
TCCGGCCAAA
AAACACCGGC
TGACATGATG
CCAGTACGGT
CTGTTGCAGT
CAGCGGAAAC
GAGGCACTAA
AATGTTGAGG
GAGGACTAGA
CAACTTTCAA
CGAGACATTA
GGCACTACGA
TAGCTTACCA
AACTGCTTAA
CGTGTTACAA
AAAATATGGT
ATTGTCATTC
100 150 200 250 300 350 400 450 500 550 600 650 700 750 WO 97/03574 WO 9703574PCT/EP96/03051
AAAAATCTAG
TTCCCGACGT
CATGCAGTCA
GGGATGGTAA
GCCGGAGCCG
GGTTATTACA
TTGCTGGTAG
TTGTAATATT
CATAGGTCTC
TCGTGGCTTC
TAGCATGGGA
GATTGGTGCC ACCTCCGATT ACCTCGGCAG GCCCTGGAAG TCGATTACTG ACGTGATCCA CTTCGCGTTG AACACATTGT GTGCCGGAAC TTCAGGGAGA AGTGCTACCG AGGCTCAAGC TAGCTGGCTG GGCTCCACTG CACTAGGAGT TTTAATTAAT TGGTCTTTCA ATTTGTAA.TA GATTTCACAA ATACTATTGT AGAATAATAA TTTCCGGCTT AAAAAA AAA
TAAAACCGGT
GAGTACTCGA
CCCTGCCGGG
TTTACGGAGA
GTGAAATGGA
TTTTACTCCT
GTTTCCCATT
ATGTTTTGTA
TTTGATTGAG
GTGATTAACA
CTTTAAAAAA
TCA-GGGTAGT
GGACGGTGAT
TGGCACGAGT
GCATCAGAAC
AGGGATTTAG
GGAAGCTTCA
CTCCCTTGGT
TTAGTGGATC
CGTGTCTTAT
AGAAATAAAA
800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1323 SEQ-I.D. NO. 4 CTTTTGTTCT CTCTTATCGA GAAAAAAA.AT GACCCGCATA AAAGAATTCT
TCACAAAACT
AAGAAAAAA
CGCTGCCGTA
GCGACAACGG
AGCACAAGGT
GGCCTCCAAA
ACATCACAAC
TTGAAGAGAA
TCTTGAGACG
ATCTTGAGGA
CTCAAAACCC
TGGGTTCTCT
ACGGCCAGGT
AAGAACATGA
GAAGCTGATA
CCACCGGAGA
GTGGTGGCAG
GGCGGCGGCT
CCGGTGTTTA
ATGTTCATCG
TGTGGTTGAT
GTGAAGGATT
TCAAAGCACC
TTACAATTGC
ACCGCCAGTT
TTTGGTAACG
GCCCAATTCC
CTAACCAAAA
TCCGATTTAA
TTCTGAATCT
AGAAACTATT
ATCGGCATTG
CAACGAGCCT
ACCCGGACTT
AAGGTGACAA
AACAGCCGTG
CGAATCTCA C
ATCGATGAGA
GTACCCGAAC
TAATGAGTGC
CATGATGATG
TCATGTTGAG
CTGACACTGA
GAGGAGACCA
CAGGAGGCTG
CAGATGGCAG
CCTCAGGGAG
TCGGGAAAAT
GTGACGGGAG
GGAAGCACAA
CTTGGCCCGA
AGGCGGTGGC
GATATGTTAG
CTTTGTGAAC
CTGCAGCCGT
GGCCAAAAAA
CACCGGCATT
AACCGGTTCA
TCTACCAACC
CTTAGCTCTT
TCGCCGGAGT
CATCATGCTA
ATGCTTTTCG
GCCAAAAGGA
GAACACAACT
CAAACGGGAA
CTCTTGATGA
AAGAAATCTT
CGCGATGACC
CTAATAAGCA
AAGATGTGTA
CATGATGATC
GTACGGTTGA
TTGCAGTCCT
CGGAAACTTT
GCACTAAGCG
GTTGAGGTGA
GACTAGAACT
CTTTCAAGTC
GACATTACAT
ACTACGAGTG
CTTACCAAGA
TGCTTAATTG
GTTACAAAAT
ATATGGTCAC
GTCATTCAAA
GGGTAGTTTC
AAAACATTTC
TTTGCAACGC
GAACTCAAGA
TCCTCAAATC
GCTATTGCTG
CGTTATTGAG
ACTTCGGGAT
AAGGTTGCTC
GTTACACAAA
TATCACAGCA
AATCAGGGGA
CGTGCGGGAT
GCAATGCGCT
ATGAGGACTT
TGGGTGGCCG
CGTCGGTGAC
AAGACGGTGG
GTATATTATT
CAAAGAAGCA
ATCATCACAG
TGCTACAGTT
TCCAAAACAC
GGAGCTGACC
CACACTCTAC
CTGGCACGGT
TGTGACATCC
AGCCCAAGGC
AATCTAGGAT
CCGACGTACC
CAATATTCCC
TACTCGTTGT
AAAAACTCCG
ATCATGTAGC
CCGTTCCAGA
ATGTCCTTAA
TCAGAAGCTC
TCCATGACTG
GCCGTCGAGG
TGCGGATGAT
CGTGTCTTGA
GCGTTGTCAG
TGCTATGATC
CAAACAACAG
GCGTGGCTGT
ACCGAACGTG
CGGCATCGGT
AGGATTAAAG
TAAAAATATA
GGAGTAGAAA
GCTGTTGTTG
AGCCGGCCCC
TTTCAGCATT
GTCCACTCGA
TGATTTTATT
ATGCACGAAA
AGGGCTGACC
TGGTGCCACC
TCGGCAGGCC
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 WO 97/03574 WO 9703574PCT/EP96/03051 CTGGAAGGAG TACTCGAGGA CGGTGATCAT
TGATCCACCC
ACATTGTTTT
AGGGAGAGTT
CTCAA.GCTTT
TCCACTGGTT
AATTAATATG
TGTAATATTT
CTATTGTGTG
CCGGCTTCTT
TGCCGGGTGG
ACGGAGAGCA
AAATGGAAGG
TACTCCTGGA
TCCCATTCTC
TTTTGTATTA
GATTGAGCGT
ATTAACAAGA
TAAATTAAAA
CACGAGTGGG
TCAGAACGCC
GATTTAGGGT
AGCTTCATTG
CCTTGGTTTG
GTGGATCCAT
GTCTTATTCG
AATAAAA.TAG
GCAGTCATCG
ATGGTAACTT
GGAGCCGGTG
TATTACAAGT
CTGGTAGTAG
TAATATTCAC
AGGTCTCTGG
TGGCTTCGAT
CATGGGAAGA
ATTACTGACG
CGCGTTGAAC
CCGGAACTTC
GCTACCGAGG
CTGGCTGGGC
TAGGAGTTTT
TCTTTCAATT
TTCACAAA.TA
ATAATAATTT
1550 1600 1650 1700 1750 1800 1850 1900 1950 1975 SEQ.I.D. NO.
IVAGVNSRKN
TSQKDVIEMS
ETLDELHKAV
DANKHVRDAL
TSTVDGWPAW
SGDNGNEPHH
LNITTTAVEH
EDLEEYPNKK
SDGQVHVEKM~
LSTGDRRLLQ
AILKSSCSST
NYFGIQKLLK
SLSQHADDLK
CSNALAMIKN
RYPDLCFSAI
RTNLTKREKV
TLMSAAMTNQ
MTDTDMn’IMR
AAVPEASKKV
ALHDCLETID
GTCLDGFSHD
TSNNRKLIEE
100 150 200 220 SEQ.I.D. NO. 6
ATTGTCGCCG
GCCTCATCAT
ACTTATGCTT
ACAAGCCAAA
,,CGTGGAACAC
TCACCAAACG
GAGACTCTTG
GAACAAGAAA
GTGCCGCGAT
GATGCTAATA
TGAGAAGATG
CTGACATGAT
ACCAGTACGG
GCTGTTGCAG
GAGTGAACTC
GCTATCCTCA
TTCGGCTATT
AGGACGTTAT
AACTACTTCG
GGAAAAGGTT
ATGAGTTACA
TCT-TTATCAC
GACCAATCAG
AGCACGTGCG
TGTAGCAATG
GATCATGAGG
TTgATna=CT
AAGAAAAAAC
AATCATCATG
GCTGCCGTTC
TGAGATGTCC
GGATTCAGAA
GCTCTCCATG
CAAAGCCGTC
AGCATGCGGA
GGGACGTGTC
GGATGCGTTG
CGCTTGCTAT
ACTTCAAACA
GCCGGCGTGG
TCCGGCGACA
TAGCAGCACA
CAGAGGCCTC
TTAAACATCA
GCTCTTGAAG
ACTGTCTTGA
GAGGATCTTG
TGATCTCAAA
TTGATGGGTT
TCAGACGGCC
GATCAAGAAC
ACAGGAAGCT
CTGTCCACCG
ACGGCAACGA
AGGTACCCGG
CAAAAAGGTG
CAACAACAGC
AGAACGAATC
GACGATCGAT
AGGAGTACCC
ACCCTAATGA
CTCTCATGAT
AGGTTCATGT
ATGACTGACA
GATAGAGGAG
GAGACAGGAG
100 150 200 250 300 350 400 450 500 550 600 650 660 SEQ.Z.D. NO. 7 PE511 (14 aa) SEQ.1.D. NO. 8 PE3252 (4 aa) Ser-ala-thr-val-ala-val -val -gly-glu-gly-phe- leu-ala- Tyr-ile-ile-arg WO 97/03574 PTE9/35 PCT/EP96/03051 SEQ.I.D. NO. 9 PE8 (8 aa) SEQ.Z.D. NO. PE21 (21 aa) SEQ.I.D. NO. 11 PE492D (15 aa) SEQ.I.D. NO. 12 PE492C (11 aa) SEQ.I.D. NO. 13 PE492B (12 aa) SEQ.I.D. NO. 14 PE492A (16 aa) SEQ.I.D. NO. PE701 (28 aa) SEQ.I.D. NO. 16 PE594 (13 aa) SEQ.I.D. NO. 17 PE7 (19 aa) Asn-ile-met-phe-ile-gly-asp-gly Ile-gly-ala-thr-ser-asp-leu-lys-pro-va1 -g)n-gly-serphe -pro- thr- tyr- leu-gly-arg-pro xx-e-l-h-a-l-alvlgygugypelu ala- arg xxx-ala- tyr-pro-gly-gln- ile-thr-ser-asn-met Asn-cys-asp-met-leu-ala-tyr-gln-asp-thr-leu.tyr Val-ile-thr-ser-ala-thr-glu-ala-gln-ala-phe-thr-progly- ser-phe va-l-h-e-l-h-luaagnaapetrpo gly-ser-phe-ile-ala-gly-ser-ser-trp- leu-gly-ser-thrgly-phe Ile-ala-gly-ser-ser-trn-leu-gly-ser-thr-gly-phe-pro Asn-met-val-thr-ala-gln-gly-arg-ala-asp-pro- asn-glnasn-thr-gly- ile-val-ile WO 97/03574 PTE9/35 PCT/EP96/03051 SEQ.I.D. NO. 18 PE 251 (38 aa) xxx-arg-ile-gly-ala-thr-ser-asp-leu-lys -pro-val -glngly- ser-phe -pro- thr- tyr eu -gly-arg-pro (trp) -lysthr SEQ.I.D. NO. 19 PE 201 (27 aa) gly-ser-phe-pro-thr-tvr- leu-gly-arg-pro-xxx-lys-gu.
tyr SEQ.I.D. NO. PE 22 (21 aa) aa 2 (arg) could aa 3 (ile) could aa 6 (thr) could gly-ser-phe-pro- thr- tyr-leu-gly be replaced with val be replaced with met be replaced with val WO 97/03574 WO 9703574PCT/EP96/03051 78 INDICATIONS RELAT1ING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) The National Collections of Industrial and Marine Bac:eria Limited (NCIMB) Address of depositary institution (including postal code and country) 23 St. Machar Drive A berdeen Scotland AB2 iRY United Kingdom Date of deposit .1rAccession Number 6 July 1995 k Z\Ai- 1 NCJMB 40749; NCIMB 40750 C. ADDITIONAL :DICATIONS ((cave blank ifnot applicabh) This information is continued on an additional sheet E7 In respect of those designations in which a European patent is sought, and any other designated state having equivalent legislation, a sample Of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or w-ithdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4)
EFC).
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MIADE (I/ife indicadons are nzot for all designated States) E. SEPARATE FURN-ISaNG OF INDICATIONS (14avc blank if not applicabic) The indications listed beiowwilI be submtted to the International Bureau later (sci ttg~riaur fhidctosa. ‘Accession Numberof Deposit’) For receiving Office use only For Inter-national Bureau use only r’7This sheet was received with the international application ElTishewarevdbyteneraoalBeuo: IAuthorized officer Form PCIROII34 (July 1992)
ASH
Authorized officer WO 97/03574 PCT/EP96/03051 79 RECEIPT IN THE CASE OF AN ORIGINAL DEPOS:T issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY: Escherichia coli K12 ISLR-p034 NCDB 40749 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above was accompanied by: a scientific description a proposed taxonomic designation (Mark with a cross where applicable) I1. RECEIPT AND ACCEPTANCE This Internatlonal Depositary Authority accepts the microorganism Identified under I aoove, which was received by it on 6 July 1995 (date of the original deposit) 1 IV. RECEIPT OF REQUEST FOR CONVERSION The microorganism identified under I above was received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) V. INTERNATIONAL DEPOSITARY AUTHORITY Name: ).rd Signature(s) of person(s) having the power PA to represent the International Depositary 23 St Macha Authority or of authorized official(s): Aberdeen Scotland Dae:11 July 1995..- Address UK A82 1 RY Dae- Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.
Form BP/4 (sole page) WO 97/03574 PCT/EP96/03051 BUDAPEST TREATY ON THE :=NT-NAT=:::AL RECOG~ITION OF THE DEPOSIT OF MICRCCRGANISMS FOR THE PURPOSES OF ?AT:’T ?RCCE’ RE Danisco Biotechnology, INTERNATIONAL FCR Langebrogade 1, ?0 Box 17, DK-1001 Copenhagen K, Denmark.
VIABILITY STATE.»;T issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified on the following page NA.ME AND ADDRESS OF THE PARTY TO WHOM THE VIABILITY STATEMENT IS ISSUED 7 DEPOSITOR II. IDENTIFICATION OF THE MICRCORCA!;ISM: Name: Accession number given by the INTERNATIONAL DEOSITARY AUTHO!TY: Address: NCIMB 40749 AS ABOVE Date of the deposit or of the transfer: 6 July 1995 III. VIABILITY STATEZ NT The viability of the microorganism identified under II azove was tested on 7 July 1995 2 On that date, the said mic=rorganism was 3 F Iviable 3 Sno longer viable Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).
2 In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability tc:t.
Mark with a cross the applicable box.
Form 3P/9 (first page) WO 97/03574 PCT/EP96/03051 81 V. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN ?ERFO0PL!7E:) V. INTERNATIONAL DEPOSITARY AUTHORITY are: LdSignature(s) of person(s) having the power Mpcj O Ltdto represent the International Depositary Address: 23 St Machar DriVS Authority or of authorized official(s): abrenscotiand 11 A b e rd e e nR. e p y LC e 7 UK AB IRyDate: 11 July 1995 4Fill in if the information has been requested and if the results of the test were negative.
ForM BP/9 (second an.d last zaae) WO 97/03574 PCT/EP96/03051 82 RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT issued pursuant to Rule 7.1 by the INTERNATIONAL DEPOSITARY AUTHORITY identilied at the bottom of this page I. IDENTIFICATION OF THE MICROORGANISM Identification reference given by the Accession number given by the DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY: Escherichia Coli K12 SOLR p017 NCIMB 40750 II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION The microorganism identified under I above was accompanied by: a scientific description I a proposed taxonomic designation (Mark with a cross where applicable) Ill. RECEIPT AND ACCEPTANCE This International Depositary Authority accepts the microorganism identified under I above, which was received by it on 12 July 1995 (date of the original deposit) l IV. RECEIPT OF REQUEST FOR CONVERSION The microorganism identified under I above was received by this International Depositary Authority on (date of the original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion) V. INTEnNATIONAL DEPOSITARY AUTHORITY CI Signature(s) of person(s) having the power to represent the International Depositary 23 St Machar Drive Authority or of authorized official(s): Aberdeen Scotland Date: 21Jl Address: 11K A82 iRY 21 July 1995 Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.
Form BP/4 (sole page) WO 97/03574 WO 9703574PCT/EP96/03051 Danisco Biotechnology {Langebrogade 1 .P 0 Box 17 DK-1001 Copenhagen
K
Denmark BUJDAPEST TRE~ATY ON THE ZA-oNA R-vCO o OF THE DEPCS:: Or RCANI VIABILITY STATZ:’=NT issued pursuant to Rule 10.2 by t’-e INTERNAT’IONAL DEPOS:TARY AUTHO’RITY identified on the fcllowing page NAM’E AND ADDR=SS OF THE PARTY TO WHOM THE VIABILITY
STATEM’ENT
IS ISSUED I. DEPOSITOR Name: As above Address: 11. 0:N:ICTO O h MICRCORGAN;IS,- Accession number given by the INTERNATIONAL oEpOSIT;a~y AUTHon-y NCIMB 40750 Date cf the deposit or OF the transfer: 12 July 1995 III7. VIABILITY STATEZIENT The viabilit7 Of the microorganism~ identified under 1: a–ove was tested on 19 July 1995 2* On that date, the said micrcorgair ma 3 Iviable no longer viable 1Indicate the date of the original deposit or, where a new deposit or a transfer h’as been made, the most recent relevant date (date of the new d-eoosit or date of the transfer).
2 In the cases referred to in Rule 10.2(a) (ii) and (iii; ree to the most recent viability t toot.
Mark with a cross the applicable box.
For= B?/9 (first page WO 97/03574 WO 9703574PCT/EP96/03051 I.CONDIT:ONS UNDER WHICH THE VIA.BILITY TEST HAS BEE7N PER.7ORM-ED V. INTERNAIONAL DEPOSITARY AUTHORITY Name: LdSignature(s) of person(s) having the power 14 C Ltd to represent the International Depositary Address: 23 St Machar Drive Authority or of authorized of ficial Aberdeen~ scotfi- 7 IJK A82 I RY Date: 21 July 1995 4 Fill in if the information has been recuested and if the results of the test were negative.
7c m BP/9 second and last page)

Claims (40)

1. A process for stabilising a protein in an acidic environment comprising adding to said acidic environment, which contains at least one protein, a block-wise enzymatically de-esterified pectin, wherein the pectin is a high ester pectin and wherein the pectin is blockwise de-esterified by a PME enzyme.

2. A process according to claim 1 wherein the block-wise enzymatically de- esterified pectin is prepared by recombinant DNA techniques.

3. A process according to claim 1 or claim 2, wherein the acidic environment is an aqueous solution, preferably wherein the aqueous solution is a beverage.

4. A process according to claim 3, wherein the beverage is a drinking yoghurt, a fruit juice or a beverage comprising whey protein. A process according to any one of the preceding claims, wherein the protein is derived from or is derivable from or is in a dairy product, such as milk or cheese.

6. A process according to claim 5, wherein the protein is casein or whey protein.

7. A process according to any one of the preceding claims, wherein the acidic environment has a pH of from about 3.5 to about 5.5, preferably wherein the acidic environment has a pH of from 4 to about

8. A process according to any one of the preceding claims, wherein the acidic environment has a pH of about 4.

9. A process according to any one of the preceding claims wherein the block- wise enzymatically de-esterified pectin prepared by recombinant DNA techniques contains from about 70% to about 80% ester groups, preferably about 76% ester Nroups. A process according to any one of the preceding claims, wherein the block- wise enzymatically de-esterified pectin prepared by recombinant DNA techniques is insensitive to Ca 2 ions according to the Protocol as mentioned in the Examples.

11. A process according to any one of the preceding claims, wherein the block- wise enzymatically de-esterified pectin prepared by recombinant DNA techniques has a high molecular weight from about 50 kD to about 150 kD.

12. A process according to any one of the preceding claims, wherein the block- wise enzymatically de-esterified pectin prepared by recombinant DNA techniques is prepared by treating a pectin with a recombinant pectin methyl esterase that de- esterifies two or more adjacent galacturonic acid residues of the pectin on at least substantially all of the pectin chains.

13. A process according to any one of the preceding claims, wherein the recombinant pectin methyl esterase is derived from a PME obtainable from a plant.

14. A process according to claim 13, wherein the plant is a fruit.

15. A process according to claim 14, wherein the fruit is a citrus fruit.

16. A process according to claim 15, wherein the citrus fruit is an orange.

17. A process according to any one of claims 13 to 16, wherein the recombinant pectin methyl esterase is derived from a PME obtainable from the lamella or albedo of an orange.

18. A process according to any one of the preceding claims, wherein the block- wise enzymatically de-esterified pectin is prepared by treating a pectin with a recombinant enzyme comprising any one of the amino acid sequences shown as SEQ.I.D. No.1 or SEQ.I.D. No. 2, or a variant, derivative or homologue thereof that has at least 75 homology therewith, including combinations thereof. -7C 87

19. A process according to any one of the preceding claims, wherein the block- wise enzymatically degraded pectin is prepared by treating a pectin with an recombinant enzyme that is obtainable by expression of the PME coding sequence contained in NCIMB 40749 or NCIMB 40750, or a variant, derivative or homologue thereof that has at least 75% homology therewith, or combinations thereof; or by expression of a nucleotide sequence comprising the nucleotide sequence shown as SEQ.I.D. No. 3 or SEQ.I.D. No. 4, or a variant, derivative or homologue thereof that has at least 75% homology therewith, or combinations thereof. A process according to any one of claims 12 to 19 wherein the block-wise enzymatically de-esterified pectin is prepared by treating the pectin with the recombinant pectin methyl esterase in the presence of sodium ions. *09o

21. A process according to claim 20 wherein the block-wise enzymatically de- esterified pectin is prepared by treating the pectin with the recombinant pectin methyl esterase in the presence of NaCI, NaN0 3 or Na 2 SO 4

22. A method of block-wise enzymatically de-esterifying a pectin comprising treating the pectin with a recombinant enzyme comprising any one of the amino acid sequences shown as SEQ.I.D. No.1 or SEQ.I.D. No. 2, or a variant, derivative or homologue thereof that has at least 75% homology therewith, including combinations **0o0o thereof.

23. A method according claim 22, wherein the block-wise enzymatically degraded pectin is prepared by treating a pectin with a recombinant enzyme that is obtainable by expression of the PME coding sequence contained in NCIMB 40749 or NCIMB 40750, or a variant, derivative or homologue thereof that has at least 75% homology therewith.

24. A method according to claims 22 to 23 wherein the block-wise enzymatically de-esterified pectin is prepared by treating the pectin with the recombinant pectin methyl esterase in the presence of sodium ions. _3 fp 88 A method according to claim 24 wherein the block-wise enzymatically de- esterified pectin is prepared by treating the pectin with the recombinant pectin methyl esterase in the presence of NaC1, NaN0 3 or Na 2 SO 4

26. An recombinant enzyme capable of de-esterifying pectins in a block-wise manner comprising any one of the amino acid sequences shown as SEQ.I.D. No.1 or SEQ.I.D. No. 2, or a variant, derivative or homologue thereof that has at least homology therewith, including combinations thereof.

27. An isolated nucleotide sequence coding for the recombinant enzyme according to claim 26, wherein the nucleotide sequence comprises any one of the sequences shown as SEQ. I.D. No. 3 or SEQ. I.D. No.4, or a variant, derivative or homologue thereof that has at least 75% homology therewith.

28. An isolated nucleotide sequence coding for the recombinant enzyme according to claim 26 wherein the nucleotide sequence is obtainable from NCIMB 40749 or NCIMB 40750, or a variant, derivative or homologue thereof. or a variant, derivative or homologue thereof that has at least 75 homology therewith. S

29. A construct expressing or comprising the recombinant enzyme according to claim 26 or a nucleotide sequence according to claim 27 or claim 28. A vector expressing or comprising a construct according to claim 29 or the recombinant enzyme according to claim 26 or a nucleotide sequence according to claim 27 or claim 28.

31. A combination of constructs comprising at least a first construct expressing or comprising the recombinant enzyme according to claim 26 or a nucleotide sequence according to claim 27 or claim 28; and a second construct comprising a gene of interest (GOI) and a promoter.

32. A cell, tissue or organ expressing or comprising a vector according to claim or a construct according to claim 29 or the recombinant enzyme according to claim 26 or a nucleotide sequence according to claim 27 or claim 28 or a combination of constructs according to claim 31.

33. A transgenic organism expressing or comprising the invention according to any one of claims 26 to 32.

34. NCIMB 40749 or NCIMB 40750. A recombinant PME enzyme adapted to de-esterify pectin in a block-wise manner and which is immunologically reactive with an antibody raised against a purified recombinant enzyme according to claim 26, but not a tomato PME enzyme.

36. Use of the recombinant enzyme according to claim 26 or claim 35 to reduce the number of ester groups in a pectin.

37. Use of the recombinant enzyme according to claim 26 or claim 35 to de- o: esterify pectin in a block-wise manner.

38. Use of the recombinant enzyme according to claim 26 or claim 35 to affect the calcium sensitivity of pectin.

39. Use of the recombinant enzyme according to claim 26 or claim 35 to esterify pectin containing free carboxyl groups. A pectin obtained by use of the recombinant enzyme according to claim 26 or claim

41. Use of a pectin according to claim 40 to prepare a foodstuff.

42. Use of a pectin according to claim 40 to stabilise a protein in an acidic environment without adversely affecting the viscosity of the environment.

43. A combination of enzymes comprising a recombinant enzyme according to claim 26 or claim 35 and a fungal PME or other pectin degrading enzyme.

44. An isolated amino acid sequence comprising the sequence shown as SEQ.I.D. No. 5, or a variant, derivative or homologue thereof that has at least 75% homology therewith, suitable for use in imparting or increasing heat stability to a protein. An isolated nucleotide sequence comprising the sequence shown as SEQ.I.D. No. 6, or a variant, derivative or homologue thereof that has at least 75% homology therewith, suitable for use in expressing an amino acid sequence for imparting or increasing heat stability to a protein. a

46. An isolated recombinant nucleotide sequence coding for the enzyme according to claim 26.

47. A recombinant PME enzyme substantially as described herein.

48. A pectin prepared by use of a recombinant PME enzyme substantially as described herein.

AU66144/96A
1995-07-14
1996-07-12
Process for stabilizing proteins in an acidic environment with a high-ester pectin

Ceased

AU714570B2
(en)

Applications Claiming Priority (3)

Application Number
Priority Date
Filing Date
Title

GB9514438.2

1995-07-14

GBGB9514438.2A

GB9514438D0
(en)

1995-07-14
1995-07-14
Stabilisation process and an enzyme for use in such a process

PCT/EP1996/003051

WO1997003574A1
(en)

1995-07-14
1996-07-12
Process for stabilizing proteins in an acidic environment with a high-ester pectin

Publications (2)

Publication Number
Publication Date

AU6614496A

AU6614496A
(en)

1997-02-18

AU714570B2
true

AU714570B2
(en)

2000-01-06

Family
ID=10777675
Family Applications (1)

Application Number
Title
Priority Date
Filing Date

AU66144/96A
Ceased

AU714570B2
(en)

1995-07-14
1996-07-12
Process for stabilizing proteins in an acidic environment with a high-ester pectin

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Country
Link

US
(4)

US6083540A
(en)

EP
(5)

EP1625796A1
(en)

JP
(3)

JPH11509102A
(en)

CN
(1)

CN1112855C
(en)

AT
(2)

ATE313270T1
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AT00204485T
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DK
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DE
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1996-07-12
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Kabushiki Kaisha Yakult Honsha
Method for producing calcium enriched fermented milk and fermented milk drink and the products

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US20030157230A1
(en)

2003-08-21

DK0839006T4
(en)

2010-12-06

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2006-06-01

EP1621082A2
(en)

2006-02-01

CN1112855C
(en)

2003-07-02

DE69635625T2
(en)

2006-09-07

DE69615199T2
(en)

2002-05-02

JP2005124579A
(en)

2005-05-19

US20020009790A1
(en)

2002-01-24

EP0976822A2
(en)

2000-02-02

GB9514438D0
(en)

1995-09-13

US6083540A
(en)

2000-07-04

DK1101412T3
(en)

2006-04-24

ES2163644T5
(en)

2010-10-29

WO1997003574A1
(en)

1997-02-06

ATE313270T1
(en)

2006-01-15

AU6614496A
(en)

1997-02-18

HK1035846A1
(en)

2001-12-14

ES2163644T3
(en)

2002-02-01

JPH11509102A
(en)

1999-08-17

CN1195970A
(en)

1998-10-14

DE69635625D1
(en)

2006-01-26

JP2006262905A
(en)

2006-10-05

PL324503A1
(en)

1998-05-25

PT839006E
(en)

2001-12-28

PL185172B1
(en)

2003-03-31

EP1101412A3
(en)

2001-08-08

MX9800422A
(en)

1998-04-30

BR9609791A
(en)

1999-03-16

US6268195B1
(en)

2001-07-31

EP1101412B1
(en)

2005-12-21

DE69615199T3
(en)

2011-02-10

EP0839006A1
(en)

1998-05-06

US7166312B2
(en)

2007-01-23

CA2225481A1
(en)

1997-02-06

ATE205366T1
(en)

2001-09-15

EP1621082A3
(en)

2006-02-08

EP0839006B1
(en)

2001-09-12

EP1101412A2
(en)

2001-05-23

NZ313625A
(en)

1999-08-30

RU2207761C2
(en)

2003-07-10

EP0839006B2
(en)

2010-08-11

EP0976822A3
(en)

2000-03-15

CZ11798A3
(en)

1998-06-17

EP1625796A1
(en)

2006-02-15

DK0839006T3
(en)

2001-11-19

DE69615199D1
(en)

2001-10-18

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