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.

What is claimed is: 
     
         1 . 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 contains at least one amino acid residue substitution modification relative to SEQ ID NO:2, 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, F74I, F74L, F74P, F74R, G77P, G80P, V83C, V83M, D84V, R89S, A96C, A96I, A96S, G98A, G98L, Y113F, E116A, E116F, E116G, E116H, E116L, E116P, E116Q, E116R, E116S, E116T, E116V, K120I, K120L, K120F, K120M, K120S, 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. 
     
     
         2 . The polypeptide of  claim 1 , wherein the combination of amino acid residue modifications is set forth in  FIG. 8 . 
     
     
         3 . The polypeptide of  claim 1 , wherein the combination of amino acid residue modifications is set forth in Table 5. 
     
     
         4 . The polypeptide of  claim 1 , wherein the combination of amino acid residue modifications is set forth in Table 6. 
     
     
         5 . The polypeptide of  claim 1 , comprising at least one further amino acid modification selected from the group consisting of D61A, D61E, R72E, R72K, E116A, E116Q, E116R, E116T, E116V, S133A, I151A, I151G, D164R, or a combination thereof. 
     
     
         6 . A composition comprising the polypeptide of  claim 1 , wherein the composition is a detergent, a cosmetic, a cream, a pharmaceutical, a liposome, a tablet, a capsule, a formulation, a drug delivery agent, a food, feed, food supplement, feed supplement, dietary aid, dietary composition, or enzyme delivery matrix. 
     
     
         7 . A protein preparation comprising the polypeptide of  claim 1 , wherein the protein preparation comprises a liquid, a solid or a gel. 
     
     
         8 . A food, feed, food supplement, feed supplement, dietary aid or dietary composition comprising a polypeptide of  claim 1 . 
     
     
         9 . An edible enzyme delivery matrix comprising the polypeptide of  claim 1 . 
     
     
         10 . A detergent composition comprising the polypeptide of  claim 1 . 
     
     
         11 . The detergent composition of  claim 10 , wherein the polypeptide is formulated in a non-aqueous liquid composition, a cast solid, a granular form, a particulate form, a compressed tablet, a gel form, a powder, a gel, a hydrogel, a liposome, an aerosol, a paste or a slurry form. 
     
     
         12 . A cosmetic or a cream comprising the polypeptide of  claim 1 . 
     
     
         13 . A heterodimer or a homodimer comprising the polypeptide of  claim 1 . 
     
     
         14 . The polypeptide of  claim 1  wherein the polypeptide is immobilized on a cell, a metal, a resin, a polymer, a ceramic, a glass, a microelectrode, a graphitic particle, a bead, a gel, a plate, an array or a capillary tube. 
     
     
         15 . A method of producing the polypeptide of  claim 1 , comprising:
 (a) providing an isolated, synthetic or recombinant polynucleotide comprising a nucleic acid sequence that encodes the polypeptide 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.   
     
     
         16 . An isolated, synthetic or recombinant polynucleotide comprising a nucleic acid sequence that encodes the polypeptide of  claim 1 . 
     
     
         17 . An expression cassette, a vector, or a cloning vehicle comprising the nucleic acid sequence of  claim 16 .