Process for stabilizing proteins in an acidic environment with a pectin high-ester

Abstract

A process is described wherein there is added to an acidic environment, which contains 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.

1 . 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 high ester pectin.  
     
     
         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.  
     
     
         5 . 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 5.5.  
     
     
         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 groups.  
     
     
         10 . 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.  
     
     
         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, including combinations thereof.  
     
     
         19 . A process according to any one of the preceding claims, wherein the blocks 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, 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, or combinations thereof.  
     
     
         20 . 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.  
     
     
         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 NaCl, NaNO 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, including combinations 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.  
     
     
         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.  
     
     
         25 . 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 NaCl, NaNO 3  or Na 2 SO 4 .  
     
     
         26 . 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.  
     
     
         27 . A 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.  
     
     
         28 . A 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.  
     
     
         29 . A construct expressing or comprising the recombinant enzyme according to  claim 26  or a nucleotide sequence according to  claim 27  or  claim 28 .  
     
     
         30 . 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 30  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 32 .  
     
     
         33 . A transgenic organism expressing or comprising the invention according to any one of  claims 26  to  32 .  
     
     
         34 . NCIMB 40749 or NCIMB 40750.  
     
     
         35 . A recombinant PME enzyme 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-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.  
     
     
         40 . A pectin obtained by use of the recombinant enzyme according to  claim 26  or  claim 35 .  
     
     
         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 amino acid sequence comprising the sequence shown as SEQ.I.D. No. 5, or a variant, derivative or homologue thereof, suitable for use in imparting or increasing heat stability to a protein.  
     
     
         45 . A nucleotide sequence comprising the sequence shown as SEQ.I.D. No. 6, or a variant, derivative or homologue thereof, suitable for use in expressing an amino acid sequence for imparting or increasing heat stability to a protein.  
     
     
         46 . A 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.