Hydrolases, nucleic acids encoding them and methods for making and using them

Abstract

Provided are hydrolases, including lipases, saturases, palmitases and/or stearatases, and polynucleotides encoding them, and methods of making and using these polynucleotides and polypeptides. Further provided are polypeptides, e.g., enzymes, having a hydrolase activity, e.g., lipases, saturases, palmitases and/or stearatases and methods for preparing low saturate or low trans fat oils, such as low saturate or low trans fat animal or vegetable oils, e.g., soy or canola oils.

1 . An isolated, synthetic or recombinant polynucleotide comprising a nucleic acid sequence that encodes the polypeptide of  claim 3 . 
     
     
         2 . An expression cassette, a vector, or a cloning vehicle comprising the nucleic acid sequence as set forth in  claim 1 . 
     
     
         3 . An isolated, synthetic or recombinant variant polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:2, and at least one amino acid residue substitution to the amino acid sequence of SEQ ID NO:2, wherein the amino acid residue substitution is selected from the group consisting of: Y7R, D16M, G27Q, G27S, L43V, G45A, G45L, A48T, S54H, D61A, D61E, D61S, V62E, V62A, V62G, V62M, V62N, V62Q, V62S, V62T, R72E, R72K, R72P, R72S, R72T, R72Y, F741, F74L, F74P, F74R, G77P, G80P, V83C, V83M, D84V, R89S, A96C, A961, A96S, G98A, G98L, Y113F, E116A, E116F, E116G, E116H, E116L, E116P, E116Q, E116R, E1165, E116T, E116V, K1201, K120L, K120F, K120M, K1205, K146A, I147F, I147L, I151A, I151G, I151H, I151P, I151S, I151T, T155C, D157S, D157G, D268T, N158A, L159M, P160T, I167R, I167S, R172P, R172Q, R172S, D197K, A210V, A211T, A211S, A211I, S212C, S212A, S212E, S212G, S212H, S212L, S212P, S212Q, S212R, S212T, S212V, S212W, S212Y, K213I, K213G, K213T, T214V, T214P, T214N, T214R, T214Y, G215A, G215H, G215S, G215M, G215V, G215P, G215C, G215W, A216T, A216R, A216Y, A216V, A216C, A216C, A216S, A216L, E217Q, E217R, E217S, E217A, E217G, E217P, A218M, A218H, A218Q, A218R, A218W, A218W, A218S, A218T, A218T, A218K, V223A, V223M, V223R, V223T, V223T, V223F, A224F, A224G, A224I, A224Q, A224Y, A225G, A225L, A225M, A225Q, and A225T, and the variant polypeptide has palmitase activity. 
     
     
         4 . A protein preparation comprising a polypeptide as set forth in  claim 3 , wherein the protein preparation comprises a liquid, a solid or a gel. 
     
     
         5 . A food, feed, food supplement, feed supplement, dietary aid or dietary composition comprising a polypeptide as set forth in  claim 3 . 
     
     
         6 . An edible enzyme delivery matrix comprising a polypeptide as set forth in  claim 3 . 
     
     
         7 . A method of producing the polypeptide of  claim 3 , comprising:
 (a) providing the nucleic acid of  claim 1  operably linked to a promoter; and   (b) expressing the nucleic acid of step (a) under conditions that allow expression of the polypeptide, thereby producing the polypeptide.   
     
     
         8 . A detergent composition comprising a polypeptide as set forth in  claim 3 . 
     
     
         9 . A cosmetic or a cream comprising the polypeptide as set forth in  claim 3 . 
     
     
         10 . An isolated, synthetic, or recombinant variant polypeptide comprising the amino acid sequence selected from the group consisting of:
 (a) a variant polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence as set forth in SEQ ID NO:2 and the combination of amino acid substitutions of  FIG. 8 ;   (b) a variant polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence as set forth in SEQ ID NO:2 and the combination of amino acid substitutions of Table 5;   (c) a variant polypeptide comprising an amino acid sequence that is at least 85% identical to the amino acid sequence as set forth in SEQ ID NO:2 and the combination of amino acid substitutions of Table 6;   
       wherein the variant polypeptide has palmitase activity. 
     
     
         11 . An isolated, synthetic or recombinant polynucleotide comprising a nucleic acid sequence that encodes the polypeptide of  claim 10 . 
     
     
         12 . An expression cassette, a vector, or a cloning vehicle comprising the nucleic acid sequence of  claim 11 . 
     
     
         13 . A protein preparation comprising a polypeptide of  claim 10 , wherein the protein preparation comprises a liquid, a solid, or a gel. 
     
     
         14 . A food, feed, food supplement, feed supplement, dietary aid, or dietary composition comprising the polypeptide of  claim 10 . 
     
     
         15 . An edible enzyme delivery matrix comprising a polypeptide of  claim 10 . 
     
     
         16 . A detergent composition comprising a polypeptide of  claim 10 . 
     
     
         17 . A cosmetic or a cream comprising the polypeptide of  claim 10 .