Proteins

Abstract

The present invention relates to a method of producing a variant lipid acyltransferase enzyme comprising: (a) selecting a parent enzyme which is a lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S; (b) modifying one or more amino acids to produce a variant lipid acyltransferase; (c) testing the variant lipid acyltransferase for activity on a galactolipid substrate, and optionally a phospholipid substrate and/or optionally a triglyceride substrate; (d) selecting a variant enzyme with an enhanced activity towards galactolipids compared with the parent enzyme; and optionally (e) preparing a quantity of the variant enzyme. The present invention further relates to variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues when aligned to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.

1 . A method of producing a variant lipid acyltransferase enzyme comprising: (a) selecting a parent enzyme which is a lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S; (b) modifying one or more amino acids to produce a variant lipid acyltransferase; (c) testing the variant lipid acyltransferase for activity on a galactolipid substrate, and optionally a phospholipid substrate and/or optionally a triglyceride substrate; (d) selecting a variant enzyme with an enhanced activity towards galactolipids compared with the parent enzyme; and optionally (e) preparing a quantity of the variant enzyme.  
     
     
         2 . The method according to  claim 1  wherein one or more of the one or more of the following amino acid residues identified by alignment with SEQ ID No. 2 is modified compared with a parent sequence SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.  
     
     
         3 . The method according to  claim 1  wherein the parent enzyme comprises an amino acid sequence as shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45, or an amino acid sequence which has at least 70% identity therewith.  
     
     
         4 . The method according to  claim 1  wherein amino acid residue 18 of the parent sequence identified by alignment with SEQ ID No. 2 is substituted by one of the following amino acids A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N, D, T.  
     
     
         5 . The method according to  claim 1  wherein amino acid residue 30 of the parent sequence identified by alignment with SEQ ID No. 2 is by one of the following amino acids A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.  
     
     
         6 . The method according to  claim 1  wherein amino acid residue 20 of the parent sequence identified by alignment with SEQ ID No. 2 is by one of the following amino acids A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.  
     
     
         7 . The method according to  claim 1  wherein the parent enzyme is an enzyme which comprises the amino acid sequence shown as SEQ ID No. 2 and/or SEQ ID No. 28.  
     
     
         8 . The method according to  claim 1  wherein the X of the GDSX motif is L.  
     
     
         9 . The method according to  claim 1  wherein the method further comprises one or more of the following steps: structural homology mapping or sequence homology alignment.  
     
     
         10 . The method according to  claim 9  wherein the structural homology mapping comprises one or more of the following steps: 
 a) aligning a parent sequence with a structural model (1IVN.PDB) shown in  FIG. 52 ;    b) selecting one or more amino acid residue within a 10 Å sphere centred on the central carbon atom of the glycerol molecule in the active site (see  FIG. 53 ); and    c) modifying one or more amino acids selected in accordance with step (b) in said parent sequence.    
     
     
         11 . The method according to  claim 9  wherein the structural homology mapping comprises one or more of the following steps: 
 a) aligning a parent sequence with a structural model (1IVN.PDB) shown in  FIG. 52 ;    b) selecting one or more amino acids within a 10 Å sphere centred on the central carbon atom of the glycerol molecule in the active site (see  FIG. 53 );    c) determining if one or more amino acid residues selected in accordance with step (b) are highly conserved (particularly are active site residues and/or part of the GDSx motif and/or part of the GANDY motif, SEQ ID NO: 54); and    d) modifying one or more amino acids selected in accordance with step (b), excluding conserved regions identified in accordance with step (c) in said parent sequence.    
     
     
         12 . The method according to  claim 9  wherein the sequence homology alignment comprises one or more of the following steps: 
 a) selecting a first parent lipid acyltransferase;    b) identifying a second related lipid acyltransferase having a desirable activity;    c) aligning said first parent lipid acyltransferase and the second related lipid acyltransferase;    d) identifying amino acid residues that differ between the two sequences; and    e) modifying one or more of the amino acid residues identified in accordance with step (iv) in said parent lipid acyltransferase.    
     
     
         13 . The method according to  claim 9  wherein the sequence homology alignment may comprise one or more of the following steps: 
 i. selecting a first parent lipid acyltransferase;    ii. identifying a second related lipid acyltransferase having a desirable activity;    iii. aligning said first parent lipid acyltransferase and the second related lipid acyltransferase;    iv. identifying amino acid residues that differ between the two sequences;    v. determining if one or more amino acid residues selected in accordance with step (iv) are highly conserved (particularly are active site residues and/or part of the GDSx motif and/or part of the GANDY motif; SEQ ID NO: 54); and    vi. modifying one or more of the amino acid residues identified in accordance with step (iv) excluding conserved regions identified in accordance with step (v) in said parent sequence.    
     
     
         14 . The method according to  claim 1  wherein one or more of the following modifications is made to the parent enzyme: S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or Y; A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; a C-terminal addition (−318) of at least one amino acid.  
     
     
         15 . The method according to  claim 14  wherein one or more of the following modifications is made to the parent enzyme S3T, S3N, S3Q, S3K, S3R, S3P, S3M; D157 is substituted with a polar uncharged amino acid; Q182 is substituted with an aliphatic amino acid residue; A309 is substituted with an aliphatic residue; Y230 is substituted with an aliphatic amino acid or one of the following amino acid residues G, D, T, V, R or M; a C-terminal addition comprising one or more of I, L or V.  
     
     
         16 . The method according to  claim 1  wherein one or more of the following modifications is made to the parent enzyme K187D, E309A, Y230T, Y230G, S3Q.  
     
     
         17 . The method according to  claim 1  wherein one or more of the following modifications is made to the parent enzyme K187D, K187D, Y230G, Y230T, Y230R, Y230M, Y230V, D157C, E309A, G2181.  
     
     
         18 . The method according to  claim 1  wherein one or more of the following modifications is made to the parent enzyme S3K, S3R, S3Q, S3N, S3P, S3M.  
     
     
         19 . The method according to  claim 1  wherein one or more of the following modifications is made to the parent enzyme Y230T, K187D, Y230G, E309A  
     
     
         20 . A variant lipid acyltransferase enzyme characterised in that the enzyme comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T N, M or S, and wherein the variant enzyme comprises one or more amino acid modifications compared with a parent sequence at any one or more of the following amino acid residues when aligned to SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.  
     
     
         21 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the variant enzyme comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43 or SEQ ID No. 45 except for one or more amino acid modifications at any one or more of the following amino acid residues identified by sequence alignment with SEQ ID No. 2: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88, −318.  
     
     
         22 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the enzyme comprises one or more of the following amino acid modifications S18A, L, M, F, W, K, Q, E, P, I, C, Y, H, R, N, D, T; Y30A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T; Y230A, G, L, M, W, K, Q, S, E, P, V, I, C, H, R, N, D, T.  
     
     
         23 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the enzyme comprises one or more of the following amino acid modifications: S3A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y; D157A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Q182A, C, D, E, F, G, H, I, K, L, M, N, Q, P, R, S, T, V, W, or Y; A309A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; Y230A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; a C-terminal addition (−318) of at least one amino acid.  
     
     
         24 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the enzyme comprises one or more of the following amino acid modifications: S3T, S3N, S3Q, S3K, S3R, S3P, S3M; D157 is substituted with a polar uncharged amino acid; Q182 is substituted with an aliphatic amino acid residue; A309 is substituted with an aliphatic residue; Y230 is substituted with an aliphatic amino acid or one of the following amino acid residues G, D, T, V, R or M; a C-terminal addition comprising one or more of I, L or V.  
     
     
         25 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the enzyme comprises one or more of the following amino acid modifications: K187D, E309A, Y230T, Y230G, S3Q.  
     
     
         26 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the enzyme comprises one or more of the following amino acid modifications: K187D, K187D, Y230G, Y230T, Y230R, Y230M, Y230V, D157C, E309A, G218I.  
     
     
         27 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the enzyme comprises one or more of the following amino acid modifications: S3K, S3R, S3Q, S3N, S3P, S3M.  
     
     
         28 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the enzyme comprises one or more of the following amino acid modifications: Y230T, K187D, Y230G, E309A.  
     
     
         29 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the variant enzyme has an enhanced ratio of activity on galactolipids to either phospholipids and/or triglycerides when compared with the parent enzyme.  
     
     
         30 . The variant lipid acyltransferase enzyme according to  claim 20  wherein the variant enzyme is an enzyme which comprises an amino acid sequence, which amino acid sequence is shown as SEQ ID No. 2 or SEQ ID No. 28 except for one or more amino acid modifications at any one or more of the following amino acid residues: Ser3, Leu17, Ala114, Trp111, Tyr117, Leu118, Pro156, Gly159, Gln160, Asn161, Pro162, Ser163, Ala164, Arg165, Ser166, Gln167, Lys168, Val169, Val170, Glu171, Ala172, Tyr179, Gln182, Lys187, His180, Asn181, Met209, Leu210, Arg211, Asn215, Lys284, Met285, Gln289, Val290, Ala309, Ser310, Lys22, Met23, Gly40, Asn80, Pro81, Lys82, Val112, Asn87, Asn88.  
     
     
         31 . A method of using a variant lipolytic enzyme according to  claim 20  in a substrate for preparing a lyso-glycolipid, for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG)) with the variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to produce the partial hydrolysis product, i.e. the lyso-glycolipid.  
     
     
         32 . A method of using a variant lipolytic enzyme obtained by the method according to  claim 1  in a substrate for preparing a lyso-glycolipid, for example digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG) by treatment of a glycolipid (e.g. digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG)) with the variant lipolytic enzyme according to the present invention or obtained by a method according to the present invention to produce the partial hydrolysis product, i.e. the lyso-glycolipid.  
     
     
         33 . The method according to  claim 31  wherein the substrate is a foodstuff.  
     
     
         34 . The method according to  claim 32  wherein the substrate is a foodstuff.  
     
     
         35 . A method of preparing a foodstuff the method comprising adding a variant lipolytic enzyme according to  claim 20  to one or more ingredients of the foodstuff.  
     
     
         36 . A method of preparing a foodstuff the method comprising adding a variant lipolytic enzyme obtained by the method according to  claim 1  to one or more ingredients of the foodstuff.  
     
     
         37 . A method of preparing a baked product from a dough, the method comprising adding a variant lipolytic enzyme according to  claim 20  to the dough.  
     
     
         38 . A method of preparing a baked product from a dough, the method comprising adding a variant lipolytic enzyme obtained by the method according to  claim 1  to the dough.  
     
     
         39 . A method of using a variant lipolytic enzyme according to  claim 20  in a process of treating egg or egg-based products to produce lysophospholipids.  
     
     
         40 . A method of using a variant lipolytic enzyme obtained by the method according to  claim 1  in a process of treating egg or egg-based products to produce lysophospholipids.  
     
     
         41 . A process of enzymatic degumming of vegetable or edible oils, comprising treating the edible or vegetable oil with a variant lipolytic enzyme according to  claim 20  so as to hydrolyse a major part of the polar lipids (e.g. phospholipid and/or glycolipid).  
     
     
         42 . A process of enzymatic degumming of vegetable or edible oils, comprising treating the edible or vegetable oil with a variant lipolytic enzyme obtained by the method according to  claim 1  so as to hydrolyse a major part of the polar lipids (e.g. phospholipid and/or glycolipid).  
     
     
         43 . A method of using a variant lipolytic enzyme according to  claim 20  in a process for reducing the content of a phospholipid in an edible oil, comprising treating the oil with said variant lipolytic enzyme so as to hydrolyse a major part of the phospholipid, and separating an aqueous phase containing the hydrolysed phospholipid from the oil.  
     
     
         44 . A method of using a variant lipolytic enzyme obtained by the method according to  claim 1  in a process for reducing the content of a phospholipid in an edible oil, comprising treating the oil with said variant lipolytic enzyme so as to hydrolyse a major part of the phospholipid, and separating an aqueous phase containing the hydrolysed phospholipid from the oil.  
     
     
         45 . A method of using a variant lipolytic enzyme according to  claim 20  in the bioconversion of polar lipids (preferably glycolipids) to make high value products, such as carbohydrate esters and/or protein esters and/or protein subunit esters and/or a hydroxy acid ester.  
     
     
         46 . A method of using a variant lipolytic enzyme obtained by the method according to  claim 1  in the bioconversion of polar lipids (preferably glycolipids) to make high value products, such as carbohydrate esters and/or protein esters and/or protein subunit esters and/or a hydroxy acid ester.  
     
     
         47 . An immobilised variant lipolytic enzyme according to  claim 20 .  
     
     
         48 . An immobilised variant lipolytic enzyme obtained by the method according to  claim 1.