Lipases with high specificity towards short chain fatty acids and uses thereof

Abstract

The present invention relates to novel polynucleotide sequences comprising genes that encode novel lipolytic enzymes, as well as functional equivalents of the gene of the acid sequences with high homology thereto. The invention also relates to methods of using these lipolytic enzymes in industrial processes, for example in the diary or baking industry.

1 . An isolated polynucleotide which comprises:
 (a) the nucleotide sequence as set out in SEQ ID NO: 1 or a functional equivalent thereof having at least 90% homology to the nucleotide sequence of SEQ ID NO: 1;   (b) a nucleotide sequence which hybridizes with a polynucleotide being the complement of SEQ ID NO: 1 and wherein said nucleotide sequence is at least 90% homologous to the nucleotide sequence of SEQ ID NO: 1;   (c) a nucleotide sequence encoding the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 or a functional equivalent thereof having at least 90% homology to the mature polypeptide in the amino acid sequence of SEQ ID NO: 2;   (d) a nucleotide sequence encoding an isolated polypeptide having lipolytic activity which is a functional equivalent of the mature polypeptide in the amino acid sequence of SEQ ID NO:2, which is at least 60% homologous to said mature polypeptide and which isolated polypeptide has a degree of specificity towards triglycerides R spec  which is at least 0.7;   (e) a sequence which is degenerate as a result of the degeneracy of the genetic code to a sequence as defined in any one of (a), (b), (c), (d);   (f) a nucleotide sequence which is the complement of a nucleotide sequence as defined in (a), (b), (c), (d), (e).   
     
     
         2 . An isolated polynucleotide according to  claim 1  encoding for a lipolytic enzyme. 
     
     
         3 . An isolated polynucleotide according to  claim 1  which is produced synthetically. 
     
     
         4 . An isolated polynucleotide according to  claim 1  which is a functional equivalent of the polynucleotide according to SEQ ID NO: 1 having at least 90% homology thereto and which is a polynucleotide having a nucleotide sequence according to SEQ ID NO: 3 or according to SEQ ID NO: 5 or according to SEQ ID NO: 7. 
     
     
         5 . An isolated polynucleotide according to  claim 1  which hybridises under high stringency conditions with a nucleotide sequence being the complement of SEQ ID NO: 1. 
     
     
         6 . A vector comprising a polynucleotide sequence according to  claim 1 . 
     
     
         7 . A vector according to  claim 6  which is an expression vector wherein the polynucleotide sequence is operably linked with at least one regulatory sequence allowing for expression of the polynucleotide sequence in a suitable host cell. 
     
     
         8 . A vector according to  claim 7  wherein the suitable host cell is a filamentous fungus. 
     
     
         9 . A recombinant host cell comprising a polynucleotide according to  claim 1 . 
     
     
         10 . A recombinant host cell according to  claim 9  capable of expressing or over-expressing said polynucleotide or vector. 
     
     
         11 . A method for manufacturing a polynucleotide according to  claim 1  comprising the steps of culturing a host cell transformed with said polynucleotide or said vector and isolating said polynucleotide or said vector from said host cell. 
     
     
         12 . An isolated polypeptide having lipolitic activity comprising:
 (a) an amino acid sequence according to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 or a functional equivalent thereof having an amino acid sequence at least 90% homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2;   (b) a polypeptide which is a functional equivalent of the mature polypeptide in the amino acid sequence of SEQ ID NO: 2, which is at least 60% homologous to said mature polypeptide and which polypeptide has a degree of specificity towards triglycerides R spec  which is at least 0.7;   (c) an amino acid sequence encoded by a polynucleotide according to  claim 1 .   
     
     
         13 . An isolated polypeptide according to  claim 12  which is a functional equivalent of the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 having at least 90% homology thereto and is a polypeptide having an amino acid sequence according to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 4 or is a polypeptide having an amino acid sequence according to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 6 or is a polypeptide having an amino acid sequence according to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 8. 
     
     
         14 . The isolated polypeptide of  claim 12 , wherein said polypeptide consists of an amino acid sequence selected from the group consisting of:
 amino acid residues 34-304 of SEQ ID NO: 4;   amino acid residues 34-303 of SEQ ID NO: 4;   amino acid residues 31-304 of SEQ ID NO: 4;   amino acid residues 31-303 of SEQ ID NO: 4;   amino acid residues 34-307 of SEQ ID NO: 4;   amino acid residues 34-302 of SEQ ID NO: 4; and   amino acid residues 31-307 of SEQ ID NO: 4.   
     
     
         15 . The isolated polypeptide of  claim 12 , wherein said polypeptide consists of an amino acid sequence selected from the group consisting of:
 amino acid residues 34-304 of SEQ ID NO: 6;   amino acid residues 34-303 of SEQ ID NO: 6;   amino acid residues 31-304 of SEQ ID NO: 6;   amino acid residues 31-303 of SEQ ID NO: 6;   amino acid residues 34-307 of SEQ ID NO: 6;   amino acid residues 34-302 of SEQ ID NO: 6; and   amino acid residues 31-307 of SEQ ID NO: 6.   
     
     
         16 . The isolated polypeptide of  claim 12 , wherein said polypeptide consists of an amino acid sequence selected from the group consisting of:
 amino acid residues 34-304 of SEQ ID NO: 8;   amino acid residues 34-303 of SEQ ID NO: 8;   amino acid residues 31-304 of SEQ ID NO: 8;   amino acid residues 31-303 of SEQ ID NO: 8;   amino acid residues 34-307 of SEQ ID NO: 8;   amino acid residues 34-302 of SEQ ID NO: 8; and   amino acid residues 31-307 of SEQ ID NO: 8.   
     
     
         17 . An isolated polypeptide according to  claim 12  obtainable by expressing a polynucleotide. 
     
     
         18 . A method for manufacturing a polypeptide according to  claim 12  comprising cultivating a recombinant host cell under condition which allow for expression of a polynucleotide and optionally recovering the encoded polypeptide from the cell or culture medium. 
     
     
         19 . Use of an isolated polypeptide according to  claim 12  in food manufacturing. 
     
     
         20 . Use according to  claim 19  in the manufacture of a dairy product, preferably in the manufacture of cheese, cheese-like product, enzyme modified cheese (EMC) or in the manufacture of free fatty acid mixtures obtainable by the lypolisis of butter fat or cream. 
     
     
         21 . A method for preparing a dairy product wherein an isolated polypeptide according to  claim 12 , is added to a dairy composition used in the production of a dairy product under conditions sufficient for the enzyme to react. 
     
     
         22 . The use according to  claim 19  wherein the ΣRelative C4-C10 content/ΣRelative C12-C18 content is at least 0.7, wherein “ΣRelative C4-C10 content” is the sum of the relative content of C4-containing, C6-containing, C8-containing and C10-containing free fatty acids present in the composition which has been treated with the polypeptide having lipolytic activity and wherein “ΣRelative C12-C18 content” is the sum of the relative content of C, 12-containing, C14-containing, C, 16-containing and C, 18-containing free fatty acids present in the composition which has been treated with the polypeptide having lipolytic activity. 
     
     
         23 . The use according to  claim 19 , wherein the polypeptide is used in the development of flavour. 
     
     
         24 . The use or method according to  claim 22  wherein the sharp, tangy, spicy notes are higher than the soapy notes in the flavour profile of the dairy product. 
     
     
         25 . A dairy product obtainable by the method of  claim 21 . 
     
     
         26 . The use according to  claim 19  in the manufacture of a baked product. 
     
     
         27 . Baking enzyme composition comprising an isolated polypeptide having lipolytic activity according to  claim 12  and one or more additional enzymes. 
     
     
         28 . Baking composition comprising DATEM and an isolated polypeptide having lipolytic activity according to  claim 12 . 
     
     
         29 . Method to prepare a dough comprising the steps of adding the polypeptide according to  claim 12 . 
     
     
         30 . A dough comprising the polypeptide according to any  claim 12 . 
     
     
         31 . A dough according to  claim 30 , having improved dough stability. 
     
     
         32 . A dough according to  claim 30  having at least one of the improved properties selected from the group consisting of increased strength, increased stability, increased elasticity, reduced stickiness, and improved extensibility of the dough. 
     
     
         33 . Method to prepare a baked product comprising the step of baking the dough according to  claim 30 . 
     
     
         34 . Baked product obtainable by the method according to  claim 33 . 
     
     
         35 . Baked product according to  claim 34  having at least one improved property selected from the group consisting of increased volume, improved flavour, improved crumb structure, improved crumb softness, improved crispiness, reduced blistering and improved anti-staling. 
     
     
         36 . Baked products according to  claim 35 , wherein the product is a bread, a cake or baked product prepared from a laminated dough. 
     
     
         37 . Baked product according to  claim 36 , wherein the cake is an emulsifier-free cake or an egg and/or fat-reduced cake. 
     
     
         38 . Use of a composition comprising a lipolytic enzyme according  claim 12  and a phospholipase A in the production of cake to improve at least one of the properties selected from the group consisting of: (i) batter viscosity, (ii) specific density, (iii) initial crumb softness, (iv) crumb pore homogeneity, (v) crumb pore diameter, (vi) crumb softness upon storage, (vii) shelf life and/or (viii) cake volume or the use of said composition in the production of cake to enable reduction of the amount of eggs and/or fat used in the cake recipe, preferably whilst at least maintaining at least one of the properties selected from the group consisting of: (i) batter viscosity, (ii) specific density, (iii) initial crumb softness, (iv) crumb pore homogeneity, (v) crumb pore diameter, (vi) crumb softness upon storage, (vii) shelf life and/or (viii) cake volume. 
     
     
         39 . A method to prepare a batter comprising the steps of:
 a. preparing a batter of a cake by adding at least:
 i. sugar 
 ii. flour 
 iii. phospholipase A, the lipolytic enzyme according to  claim 12  and eggs or 
 a method to prepare a cake derived from said batter which further comprises the step of 
   b. baking the batter to yield a cake
 wherein the cake comprises a reduced amount of eggs and/or fat or wherein the cake is a regular cake. 
   
     
     
         40 . The use of  claim 38  wherein one or more other ingredients are present in the composition selected from protein sources, hydrocolloids, yeast extract, modified starch, calcium. 
     
     
         41 . Method to produce a cake or a batter from which a cake can be derived wherein a lipolytic enzyme according to  claim 12  is used. 
     
     
         42 . A cake batter obtainable by the method of  claim 41 .