Lipolytic enzyme variants and method for their production

Abstract

The inventors have developed a method using protein engineering to produce lipolytic enzymes having a relatively high activity for one ester bond in an amphiphilic substrate with two lipophilic groups) and a relatively low activity for the ester bond in an amphiphilic substrate with one lipophilic group, e.g. a relatively high phospholipase activity and a relatively low lysophospholipase activity.

1 . A method of producing a lipolytic enzyme variant comprising: 
 a) selecting a parent fungal lipolytic enzyme,    b) selecting at least one amino acid residue which comprises an atom (excluding H atoms) which in a three-dimensional model lies within 10 Å of an atom (excluding H atoms) of a phospholipid docked with the parent lipolytic enzyme, or which is the C-terminal amino acid,    c) altering the selected amino acid,    d) optionally, altering one or more amino acids other than those selected,    e) preparing the variant resulting from the preceding steps,    f) testing hydrolytic activities of the variant towards the bond B-C of a first substrate having the general formula A-B-C and towards the bond B′-C of a second substrate having the general formula A′-B′-(A″-B″-) C where: 
 i) A and A′ are fatty acyl groups,  
 ii) B and B′ are oxygen or sulfur  
 iii) C is a polyol with B, B′ and B″ attached to OH groups, optionally having other functional groups, and optionally having a hydrophilic group attached to an OH group,  
   g) selecting a variant having a ratio of activity on the first substrate to activity on the second substrate which is lower than the parent lipolytic enzyme, and    h) producing the selected variant.    
     
     
         2 . A method of producing a lipolytic enzyme variant comprising: 
 i) selecting a parent fungal lipolytic enzyme,    j) in the parent lipolytic enzyme selecting at least one amino acid residue corresponding to any of residues 17-18, 20-23, 26, 37, 39, 62, 64, 80-96, 110-113, 144-151, 171-177, 200-211, 213, 215, 227, 253-261 and 263-269 of the  T. lanuginosus  lipase (SEQ ID NO: 14),    k) altering the selected amino acid,    l) optionally, altering one or more amino acids other than those selected,    m) preparing the variant resulting from the preceding steps,    n) testing hydrolytic activities of the variant towards the bond B-C of a first substrate having the general formula A-B-C and towards the bond B′-C of a second substrate having the general formula A′-B′-(A″-B″-) C where: 
 i) A and A′ are fatty acyl groups,  
 ii) B and B′ are oxygen or sulfur  
 iii) C is a polyol with B, B′ and B″ attached to OH groups, optionally having other functional groups, and optionally having a hydrophilic group attached to an OH group,  
   o) selecting a variant having a ratio of activity on the first substrate to activity on the second substrate which is lower than the parent lipolytic enzyme, and    p) producing the selected variant.    
     
     
         3 . The method of  claim 1  wherein the first substrate is a lyso-phospholipid and the second substrate is a phospholipid.  
     
     
         4 . The method of  claim 3  wherein the parent lipolytic enzyme has phospholipase activity, particularly phospholipase A1 activity.  
     
     
         5 . The method of  claim 4  wherein the parent lipolytic enzyme also has digalactosyl diglyceride hydrolyzing activity and optionally triacylglycerol lipase activity.  
     
     
         6 . The method of  claim 1  wherein the parent lipolytic enzyme has an amino acid sequence which is at least 50% (particularly at least 90%) identical to that of the  Thermomyces lanuginosus  lipase (SEQ ID NO: 14).  
     
     
         7 . The method of  claim 6  wherein the alterations comprise substitution of at least one of L93, F95, V203 and L206 with an amino acid residue which is smaller and/or more hydrophilic.  
     
     
         8 . The method of  claim 7  wherein the alterations comprise at least one of the substitutions L206V/S/T/A, V203T/S/A, F95I/L/Y and L93V/I/A/T.  
     
     
         9 . The method of  claim 5  wherein the alterations comprise substitution of at least one of P253, I255 and P256 with an amino acid which is larger.  
     
     
         10 . The method of  claim 5  wherein the alterations comprise an amino acid alteration in the region 247-260 or 265-269.  
     
     
         11 . The method of  claim 10  wherein the alterations comprise substitution of at least one of P253 and P256 with a different amino acid residue.  
     
     
         12 . The method of  claim 5  wherein the alterations comprise a substitution L227D, P253T/G/L, D254S/L, I255, P256L/A, A257D/A, L259 or W260H.  
     
     
         13 . The method of  claim 5  wherein the alterations comprise an amino acid alteration in the region 247-260 and an alteration of amino acid L93, F95, V203 and L206.  
     
     
         14 . The method of  claim 5  wherein the parent lipolytic enzyme compared to the  T. lanuginosus  lipase (SEQ ID NO: 14) comprises an amino acid alteration at a position corresponding to R81, R84, S85, G263, L264, 1265, G266, T267 or L269 and/or a peptide extension of 1-10 amino acids art the C-terminal.  
     
     
         15 . A method of preparing a dough or a baked product made from dough, comprising preparing a lipolytic enzyme variant by the method of any preceding  claim 1  and adding the lipolytic enzyme variant to the dough.  
     
     
         16 . A variant of a parent fungal lipolytic enzyme which variant comprises an amino acid alteration corresponding to R84G/A/Y/S or L206F of SEQ ID NO: 14, has phospholipase activity and has a lysophospholipase to phospholipase ratio corresponding to PLARN below 500 or RLPLA below 1.0.  
     
     
         17 - 19 . (canceled)