Compositions and Methods Comprising a Lipolytic Enzyme Variant

Abstract

The present invention provides lipolytic enzyme variants. Specifically, the present invention provides lipolytic enzyme variants having two, three, or more modifications as compared to a parent lipolytic enzyme and having at least one improved property. In addition, the present invention provides compositions comprising a lipolytic enzyme variant of the invention. The present invention also provides methods of cleaning using compositions comprising a lipolytic enzyme variant of the invention.

1 . A lipolytic enzyme variant or an active fragment thereof comprising at least two, three, four, five, six, seven, eight, nine or ten or more amino acid modifications to a parent lipolytic enzyme, wherein a first amino acid modification is at a position of the lipolytic enzyme variant selected from the group consisting of 4, 11, 18, 23, 24, 27, 29, 33, 45, 48, 49, 51, 56, 58, 73, 75, 77, 90, 91, 94, 111, 130, 137, 154, 156, 163, 187, 189, 227, 233, 252, 256, and 264, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of  Thermomyces lanuginosa  lipase set forth in SEQ ID NO: 1. 
     
     
         2 . The lipolytic enzyme variant or active fragment thereof of  claim 1 , wherein the at least two, three, four, five, six, seven, eight, nine or ten or more amino acid modifications to a parent lipolytic enzyme are at a position of the lipolytic enzyme variant selected from the group consisting of 4, 11, 18, 23, 24, 27, 29, 33, 45, 48, 49, 51, 56, 58, 73, 75, 77, 90, 91, 94, 111, 130, 137, 154, 156, 163, 187, 189, 227, 233, 252, 256, and 264, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of  Thermomyces lanuginosa  lipase set forth in SEQ ID NO: 1. 
     
     
         3 . (canceled) 
     
     
         4 . The lipolytic enzyme variant or active fragment thereof of  claim 1 , wherein the variant or active fragment thereof comprises amino acid modifications of Tables 2-1, 3-1, 3-7, 3-13, 3-25, and/or 3-31, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of  Thermomyces lanuginosa  lipase set forth in SEQ ID NO: 1. 
     
     
         5 . The lipolytic enzyme variant or active fragment thereof of  claim 1 , wherein the variant or active fragment has lipolytic activity. 
     
     
         6 . The lipolytic enzyme variant or active fragment thereof of  claim 1 , wherein the variant or active fragment has a performance index (pI) relative to the parent lipolytic enzyme for hydrolysis of p-nitrophenyl butyrate, p-nitrophenyl caprylate, or p-nitrophenyl palmitate that is greater than 1.0. 
     
     
         7 . The lipolytic enzyme variant or active fragment thereof of  claim 6 , wherein the performance index is measured using the p-nitrophenyl butyrate, p-nitrophenyl caprylate, or p-nitrophenyl palmitate assay of Example 1, and, optionally, at a pH of 8. 
     
     
         8 - 9 . (canceled) 
     
     
         10 . The lipolytic enzyme variant or active fragment thereof of  claim 4 , wherein the variant or active fragment thereof comprising: (i) the amino acid modifications of Table 3-1 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for hydrolysis of p-nitrophenyl butyrate that is greater than 1.0; (ii) the amino acid modifications of Table 3-7 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for hydrolysis of p-nitrophenyl caprylate that is greater than 1.0; and/or (iii) the amino acid modifications of Table 3-13 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for hydrolysis of p-nitrophenyl palmitate that is greater than 1.0. 
     
     
         11 . The lipolytic enzyme variant or active fragment thereof of  claim 10 , wherein the performance index for (i) hydrolysis of p-nitrophenyl butyrate is measured using the p-nitrophenyl butyrate assay of Example 1, (ii) hydrolysis of p-nitrophenyl caprylate is measured using the p-nitrophenyl caprylate assay of Example 1, and/or (iii) hydrolysis of p-nitrophenyl palmitate is measured using the p-nitrophenyl palmitate assay of Example 1. 
     
     
         12 . The lipolytic enzyme variant or active fragment thereof of  claim 11 , wherein the performance index is measured at a pH of 8. 
     
     
         13 - 21 . (canceled) 
     
     
         22 . The lipolytic enzyme variant or active fragment thereof of  claim 4 , wherein the variant or active fragment comprising the amino acid modifications of Table 3-1 has an increased pNPP/pNPB or pNPO/pNPB or pNPB/pNPP specific activity ratio compared to SEQ ID NO:1. 
     
     
         23 - 24 . (canceled) 
     
     
         25 . The lipolytic enzyme variant or active fragment thereof of  claim 1 , wherein the variant comprises amino acid modifications as shown in Tables 4-1, 5-1, 6-1, 7-1, 7-7, 8-1 and/or 8-7, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of  Thermomyces lanuginosa  lipase set forth in SEQ ID NO: 1. 
     
     
         26 . The lipolytic enzyme variant or active fragment thereof of  claim 25 , wherein the variant or active fragment thereof comprising: (i) the amino acid modifications of Table 4-1 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for thermostability that is greater than 1.0; (ii) the amino acid modifications of Table 5-1 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for detergent stability that is greater than 1.0; (iii) the amino acid modifications of Table 6-1 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for LAS stability that is greater than 1.0; (iv) the amino acid modifications of Table 7-1 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for fabric adhesion that is greater than 1.0; (v) the amino acid modifications of Table 7-7 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for fabric adhesion that is less than 1.0; and/or (vi) the amino acid modifications of Tables 8-1 and 8-7 has a performance index (pI) relative to a reference lipolytic enzyme having the sequence of SEQ ID NO:1 for cleaning performance that is greater than 1.0. 
     
     
         27 - 32 . (canceled) 
     
     
         33 . The lipolytic enzyme variant or active fragment thereof of  claim 26 , wherein the performance index for: (i) fabric adhesion is measured using the fabric adhesion assay of Example 1; (ii) cleaning performance of said variant or active fragment thereof comprising the amino acid modifications of Table 8-1 is measured using the CS-61 microswatch assay using Tide® half dose+adjuvant of Example 1; and/or (iii) cleaning performance of said variant or active fragment thereof comprising the amino acid modifications of Table 8-7 is measured using the CS-61 microswatch assay using Tide® full dose of Example 1. 
     
     
         34 - 42 . (canceled) 
     
     
         43 . The lipolytic enzyme variant or active fragment thereof of  claim 1 , wherein said variant has at least 50% identity to a lipolytic enzyme TLL homolog. 
     
     
         44 . The lipolytic enzyme variant of  claim 43 , wherein the lipolytic enzyme TLL homolog is from a genus selected from the group consisting of  Thermomyces, Verrucosispora, Saccharomonospora, Streptomyces, Micromonospora, Streptosporangium, Amycolatopsis, Cellulomonas, Actinosynnema, Kribbella, Thermomonospora, Deinococcus, Kineococcus, Nocardiopsis, Frankia, Jonesia, Pseudomonas, Acidovorax  and Nocardioidaceae. 
     
     
         45 - 46 . (canceled) 
     
     
         47 . A composition comprising at least one lipolytic enzyme variant of  claim 1 . 
     
     
         48 . (canceled) 
     
     
         49 . The composition of  claim 47 , wherein said composition is a granular, powder, solid, bar, liquid, tablet, gel, unit dose, or paste composition. 
     
     
         50 - 51 . (canceled) 
     
     
         52 . The composition of  claim 47 , wherein said composition is a laundry detergent composition, a dish detergent composition, or a hard surface cleaning composition. 
     
     
         53 - 55 . (canceled) 
     
     
         56 . The composition of  claim 47 , further comprising at least one bleaching agent and/or at least one additional enzyme selected from the group consisting of protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, and amylase. 
     
     
         57 - 61 . (canceled) 
     
     
         62 . A method of cleaning, comprising contacting a surface or an item with (i) a composition comprising at least one lipolytic enzyme variant of  claim 1 , or (ii) the composition of  claim 47 . 
     
     
         63 - 64 . (canceled) 
     
     
         65 . The method of  claim 62 , wherein said item is dishware or fabric. 
     
     
         66 - 71 . (canceled)