Lipase Variants

Abstract

The present invention relates to a newly identified lipase belonging to the Ustilaginaceae family of Basidomycetes and variants thereof. The invention also relates to polynucleotides encoding the lipase and the variants thereof. The invention further relates to procedures for producing the lipase, and variants thereof, polypeptides and polynucleotides. The invention further relates to lipase variants with an increased trans-selectivity. The invention further relates to lipase variants with a preference for long chain fatty acid esters or carboxylic acid moieties with a chain length greater than C 12 . The invention further relates to a method of reducing, or eliminating, trans-fatty acids from liquid oil stable enough for frying systems.

1 . An isolated lipase, or a variant thereof, that comprises:
 (a) an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6, or to a complement thereof;   (b) one or more amino acid substitutions corresponding to residues 269, 338, 341, 342, 357, 421, or 425, wherein each amino acid position is numbered by correspondence to a position in the amino acid sequence of the lipase as set forth in SEQ ID NO:1; or   (c) at least a first amino acid substitution in one or more amino acid residues of the polypeptide sequence of any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6, wherein the at least a first amino acid substitution confers to the polypeptide a preference for catalysis of long-chain fatty acids, or a trans-selective lipolytic activity, or a combination thereof.   
     
     
         2 . The isolated lipase, or variant thereof, of  claim 1 , wherein (a) the amino acid sequence is at least about 98% identical to the amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6, or to a complement thereof. 
     
     
         3 . The isolated lipase, or variant thereof, of  claim 2 , wherein (a) the amino acid sequence is at least about 99% identical to the amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6, or to a complement thereof. 
     
     
         4 . The isolated lipase, or variant thereof, of  claim 1 , wherein (b) the one or more amino acid substitutions is a phenylalanine-to-aspartic acid substitution, an alanine-to-asparagine substitution, a threonine-to-histidine substitution, a phenylalanine-to-serine substitution, a glycine-to-alanine substitution, a glycine-to-tyrosine substitution, a glycine-to-leucine substitution, a valine-to-histidine substitution, a valine-to-isoleucine substitution, or a leucine-to-asparagine substitution, or any combination thereof. 
     
     
         5 . The isolated lipase, or variant thereof, of  claim 4 , wherein (b) the one or more amino acid substitutions is a phenylalanine-to-aspartic acid substitution at residue 269 (F269D), an alanine-to-asparagine substitution at residue 338 (A338N), a threonine-to-histidine substitution at residue 341 (T341H), a phenylalanine-to-serine substitution at residue 342 (F342S), a glycine-to-alanine (G357A), a glycine-to-tyrosine (G357Y), or a glycine-to-leucine (G357L) substitution at residue 357, a valine-to-histidine (V421H) or a valine-to-isoleucine (V421I) substitution at residue 421, or a leucine-to-asparagine substitution at amino acid residue 425 (L425N), or any combination thereof. 
     
     
         6 . The isolated lipase, or variant thereof, of  claim 1 , wherein (c) the at least a first amino acid substitution confers to the polypeptide a preference for catalysis of long-chain fatty acids, when compared to a non-selective or un-substituted lipase. 
     
     
         7 . The isolated lipase, or variant thereof, of  claim 6 , wherein (c) the at least a first amino acid substitution confers to the polypeptide an increase in hydrolytic or ethanolytic activity in the presence of at least a first trans-fatty acid moiety, when compared to a non-selective or un-substituted lipase. 
     
     
         8 . The isolated lipase, or variant thereof, of  claim 7 , wherein (c) the at least a first amino acid substitution confers to the polypeptide an about 1.5-fold increase in hydrolytic or ethanolytic activity in the presence of at least a first trans-fatty acid moiety, when compared to a non-selective or un-substituted lipase. 
     
     
         9 . The isolated lipase, or variant thereof, of  claim 7 , (c) the at least a first amino acid substitution confers to the polypeptide an about 2-fold increase in hydrolytic or ethanolytic activity in the presence of at least a first trans-fatty acid moiety, when compared to a non-selective or un-substituted lipase. 
     
     
         10 . The isolated lipase, or variant thereof, of  claim 7 , wherein (c) the at least a first amino acid substitution confers to the polypeptide an about 2.5-fold increase in hydrolytic or ethanolytic activity in the presence of at least a first trans-fatty acid moiety, when compared to a non-selective or un-substituted lipase. 
     
     
         11 . The isolated lipase, or variant thereof, of  claim 1 , wherein (c) the at least a first amino acid substitution confers to the polypeptide an increased preference for catalyzing fatty acid moieties having a carbon chain length greater than or equal to twelve (≧C 12 ), when compared to the wild-type or parent lipase. 
     
     
         12 . The isolated lipase, or variant thereof, of  claim 1 , wherein (c) the at least a first amino acid substitution confers to the polypeptide an increased preference for catalyzing fatty acid moieties having a carbon chain length greater than or equal to fourteen (≧C 14 ), when compared to the wild-type or parent lipase. 
     
     
         13 . The isolated lipase, or variant thereof, of  claim 1 , wherein (c) the at least a first amino acid substitution confers to the polypeptide an increased preference for catalyzing fatty acid moieties having a carbon chain length greater than or equal to sixteen (≧C 16 ), when compared to the wild-type or parent lipase. 
     
     
         14 . The isolated lipase, or variant thereof, of  claim 1 , wherein (c) the at least a first amino acid substitution confers to the polypeptide an increased preference for catalyzing fatty acid moieties having a carbon chain length greater than or equal to twelve (≧C 12 ), and an about 1.5-fold increase in hydrolytic or ethanolytic activity in the presence of at least a first trans-fatty acid moiety, when compared to the wild-type or parent lipase. 
     
     
         15 . The isolated lipase, or variant thereof, of  claim 1 , comprising:
 (a) an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6, or to a complement thereof; and   (b) one or more amino acid substitutions corresponding to residues 269, 338, 341, 342, 357, 421, or 425, wherein each amino acid position is numbered by correspondence to a position in the amino acid sequence of the lipase as set forth in SEQ ID NO:1; and   further wherein the one or more amino acid substitutions confers to the polypeptide an increased preference for catalyzing fatty acid moieties having a carbon chain length greater than or equal to twelve (≧C 12 ), or an about 1.5-fold increase in hydrolytic or ethanolytic activity in the presence of at least a first trans-fatty acid moiety, when compared to the wild-type or parent lipase, or a combination thereof.   
     
     
         16 . The isolated lipase, or variant thereof, of  claim 1 , that (a) is about 425 to about 470 amino acids in length; (b) is at least about 95% identical to the amino acid sequence of SEQ ID NO:4, or SEQ ID NO:6, or to a complement thereof and (c) includes one or more amino acid substitutions corresponding to residues 269, 338, 341, 342, 357, 421, or 425, wherein each amino acid position is numbered by correspondence to a position in the amino acid sequence of the lipase as set forth in SEQ ID NO:1, and
 further wherein the one or more amino acid substitutions confers to the polypeptide an increased preference for catalysis of long-chain fatty acids, or an increased trans-selective lipolytic activity, or a combination thereof.   
     
     
         17 . The isolated lipase, or variant thereof, of  claim 16 , that (a) is about 430 to about 460 amino acids in length; and (b) is at least about 98% identical to the amino acid sequence of SEQ ID NO:4, or SEQ ID NO:6, or to a complement thereof. 
     
     
         18 . The isolated lipase, or variant thereof, of  claim 16 , that (a) is about 425 to about 470 amino acids in length; (b) is at least about 95% identical to the amino acid sequence of SEQ ID NO:4, or SEQ ID NO:6, or to a complement thereof; (c) includes one or more amino acid substitutions corresponding to residues 269, 338, 341, 342, 357, 421, or 425, wherein each amino acid position is numbered by correspondence to a position in the amino acid sequence of the lipase as set forth in SEQ ID NO:1, and (d) has an about 1.5-fold increase in hydrolytic or ethanolytic activity in the presence of at least a first trans-fatty acid moiety, or an increased preference for catalyzing fatty acid moieties having a carbon chain length greater than or equal to twelve (≧C 12 ), when compared to the wild-type, un-substituted, or parent lipase. 
     
     
         19 . A composition, comprising the isolated lipase, or variant thereof, of  claim 1 . 
     
     
         20 . The composition of  claim 19 , further comprising a lipid, a fatty acid, a sterol, a wax, an oil, a triglyceride, an ester, or a carboxylic acid moiety. 
     
     
         21 . The composition of  claim 19 , further comprising a binding medium to which the lipase, or variant thereof, binds or cross-links. 
     
     
         22 . The composition of  claim 19 , wherein the lipase, or variant thereof is substantially bound or substantially chemically cross-linked to a matrix, a column, a fiber, a filter, a resin, a gel, a bead, or any combination thereof. 
     
     
         23 . The composition of  claim 19 , further comprising one or more additional enzymes. 
     
     
         24 . The composition of  claim 23 , wherein the one or more additional enzymes comprise a second, distinct, lipase in accordance with  claim 1 , or a variant thereof. 
     
     
         25 . The composition of  claim 23 , wherein the one or more additional enzymes include one or more lipases, esterases, lyases, deproteinases, phosphatases, dehydrogenases, transglutaminases, oxidases, or any combination thereof. 
     
     
         26 . An isolated polynucleotide that encodes the lipase, or the variant thereof, of  claim 1 . 
     
     
         27 . An expression vector comprising an isolated polynucleotide that encodes the lipase, or variant thereof, of  claim 1 . 
     
     
         28 . The expression vector of  claim 27 , comprising an isolated polynucleotide that is codon-optimized for expression in a bacterial or non-basidiomycetous yeast cell. 
     
     
         29 . The expression vector of  claim 28 , wherein the bacterial cell is  E. coli,  or the non-basidiomycetous yeast cell is  P. pastoris.    
     
     
         30 . The expression vector of  claim 28 , defined as pET22-lipUMsophis, or pET22-lipUMfophis. 
     
     
         31 . A microbial host cell transformed with the isolated polynucleotide of  claim 26 , or the expression vector of  claim 27 . 
     
     
         32 . The microbial host cell of  claim 31 , defined as an  E. coli  or a  P. pastoris  host cell. 
     
     
         33 . The microbial host cell of  claim 31 , wherein the expression vector encodes, in a non- Ustilago maydis  cell, the lipase, or the variant thereof, according to  claim 1 . 
     
     
         34 . A  Pichia pastoris  host cell deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the accession number 24702. 
     
     
         35 . A method for producing a lipase variant, comprising (a) culturing a microbial host cell that encodes the lipase, or variant thereof, of  claim 1 , under conditions conducive to the expression and secretion of the lipase, or the variant thereof, and (b) recovering the expressed lipase, or variant thereof, from the culture. 
     
     
         36 . A variant of a parent lipolytic enzyme, which comprises:
 (a) one or more amino acid substitutions corresponding to residues 269, 338, 341, 342, 357, 421, or 425; wherein each amino acid position is numbered by correspondence to a position in the amino acid sequence of the lipase as set forth in SEQ ID NO:1, or   (b) one or more amino acid substitutions corresponding to residues 145, 214, 217, 218, 233, 297, or 301; wherein each amino acid position is numbered by correspondence to a position in the amino acid sequence of the lipase as set forth in SEQ ID NO:4; wherein the one or more amino acid substitutions confer to the polypeptide an increased lipolytic activity toward trans-fatty acid moieties, or a preference for catalysis of one or more long-chain fatty acids.   
     
     
         37 . The variant of  claim 36 , which comprises a total of no more than 5 amino acid substitutions as compared to SEQ ID NO:1. 
     
     
         38 . The variant of  claim 36 , wherein the parent lipolytic enzyme is a  Ustilago maydis  lipase. 
     
     
         39 . The variant of  claim 36 , comprising a phenylalanine-to-aspartic acid substitution, an alanine-to-asparagine substitution, a threonine-to-histidine substitution, a phenylalanine-to-serine substitution, a glycine-to-alanine substitution, a glycine-to-tyrosine substitution, a glycine-to-leucine substitution, a valine-to-histidine substitution, a valine-to-isoleucine substitution, or a leucine-to-asparagine substitution, or any combination thereof. 
     
     
         40 . The variant of  claim 39 , comprising a phenylalanine-to-aspartic acid substitution at amino acid residue 145 (F145D); an alanine-to-asparagine substitution at amino acid residue 214 (A214N); a threonine-to-histidine substitution at amino acid residue 217 (T217H); phenylalanine-to-serine substitution at amino acid residue 218 (F218S); a glycine-to-alanine (G233A), a glycine-to-tyrosine (G233Y), or a glycine-to-leucine (G233L) substitution at amino acid residue 233; a valine-to-histidine (V297H) or a valine-to-isoleucine (V297I) substitution at amino acid residue 297; or a leucine-to-asparagine substitution at amino acid residue 301 (L301N) of SEQ ID NO:4. 
     
     
         41 . A method of obtaining an isolated  Ustilago maydis  lipase variant, comprising:
 (a) mutagenizing a polynucleotide that encodes the polypeptide of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:6 under conditions effective to generate at least a first lipase variant that comprises an amino acid substitution in at least a first amino acid residue therein that confers to the polypeptide an increased trans-selectivity, or an increased preference for catalyzing long-chain fatty acid moieties (≧C 12 ) over that of medium or short-chain fatty acid moieties (<C 12 );   (b) transforming a suitable microbial host cell with the mutagenized polynucleotide;   (c) culturing the microbial host cell under conditions effective to express the first lipase variant; and   (d) recovering the first expressed lipase variant from the culture.   
     
     
         42 . A method of reducing or eliminating one or more trans-unsaturated fatty acid compounds or one or more long-chain (≧C 12 ) fatty acid moieties from a substrate, comprising contacting the substrate with an effective amount of a composition comprising the lipase, or variant thereof, of  claim 1 , for a time sufficient to hydrolyze or esterify at least a portion of the substrate thereby reducing or eliminating the one or more trans-unsaturated fatty acid compounds or the one or more long-chain (≧C 12 ) fatty acid moieties from the substrate. 
     
     
         43 . The method of  claim 42 , further comprising removing the lipase, or variant thereof, from the composition after the one or more trans-unsaturated fatty acid compounds or the one or more long-chain (≧C 12 ) fatty acid moieties has been substantially reduced or eliminated from the substrate. 
     
     
         44 . The method of  claim 42 , wherein the substrate comprises an edible lipid, an edible fat, an edible fatty acid, an edible sterol, an edible wax, an edible oil, or an edible triglyceride, or any combination thereof. 
     
     
         45 . A fat-containing product having reduced trans-fatty acid moieties, or essentially no trans-fatty acid moieties, produced by the method of  claim 42 . 
     
     
         46 . The fat-containing product of  claim 45 , suitable for human consumption. 
     
     
         47 . The fat-containing product of  claim 45 , characterized as a cooking ingredient, or a frying oil.