Lipase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-2019390182-A1
Application number	US-201916540180-A
Country	US
Kind code	A1
Filing date	Aug 14, 2019
Priority date	Jul 6, 2015
Publication date	Dec 26, 2019
Grant date	—

Abstract

The present invention relates to a lipase variant of a parent lipase, which variant has lipase activity, at least 75% but less than 100% sequence identity to SEQ ID NO: 3 and comprises a substitution at one or more positions corresponding to positions 1; 2; 3; 4; 5; 6; 7; 9; 10; 11; 12; 13; 14; 15; 17; 18; 29; 30; 33; 34; 36; 37; 38; 40; 41; 43; 44; 45; 46; 47; 49; 50; 51; 52; 54; 55; 57; 58; 59; 60; 69; 70; 71; 72; 81; 82; 85; 86; 87; 89; 90; 91; 92; 94; 95; 96; 97; 99; 100; 101; 102; 103; 105; 108; 111; 115; 116; 118; 119; 120; 122; 123; 124; 126; 127; 128; 129; 130; 131; 132; 133; 134; 136; 144; 152; 155; 157; 158; 159; 161; 162; 163; 177; 178; 181; 182; 184; 185; 187; 189; 191; 196; 197; 199; 203; 205; 206; 207; 208; 209; 211; 215; 220; 221; 222; 223; 224; 226; 229; 230; 232; 235; 236; 240; 242; 243; 244; 247; 248; 252; 254; 255; 257; 260; 261; 262 of SEQ ID NO: 3. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

First claim

1 . A lipase variant of a parent lipase, which variant has lipase activity, has at least 75% but less than 100% sequence identity to SEQ ID NO: 3 and comprises at least one of the substitutions selected from the group consisting of:
a) N97D+T176N b) Q49K+D255G c) N97D+D255G d) S152A+D255G e) H158Q+D255G f) S152A+H158Q+K221S g) Q49K+T176N+D255G h) N97D+T176N+D255G i) S152A+T176N+D255G j) K108A+H158Q+K221S k) N97D+H158Q+K221S l) N97D+R131A+D255G m) N97D+V247A+D255G n) T176N+K221S+V247A+D255G o) S152A+T176N+K221S+V247G+D255G p) S152A+T176N+V247N+D255G q) S152A+T176N+I219N+M220D+V247P r) N97D+S152A+T176N+V247P+D255G s) R131A+S152A+T176N+V247P+D255G t) N97D+S152A+T176N+V247F+D255G u) K108A+S152A+T176N+V247F+D255G v) R131A+S152A+T176N+V247F+D255G w) R131A+S152A+T176N+L225V+P229S+V247F+D255G aa) R131A+S152A+I174V+V247F+D255G ab) K70E+R131A+S152A+T176N+V247F+D255G ac) R131A+S152A+T176N+G206P+V247F+D255G ad) L111S+R131A+S152A+T176N+V247F+D255G ae) L111T+R131A+S152A+T176N+V247F+D255G af) V101R+R131A+S152A+T176N+V247F+D255G ag) D130R+S152A+T176N+V247F+D255G ah) N97L+R131A+S152A+T176N+V247F+D255G ai) R131A+S152A+T176N+V247F aj) R131A+S152A+T176N+S223R+V247F ak) R131A+S152A+T176N+I232E+V247F+D255G al) R131A+S152A+T176N+I219N+V247F+D255G am) R131A+S152A+T176N+I219E+V247F+D255G an) N97L+V101R+R131A+S152A+T176N+V247F+D255G ao) L111T+D130R+S152A+T176N+V247F+D255G ap) V101R+R131A+S152A+T176N+V247F+D255G aq) V101R+D130R+S152A+T176N+V247F+D255G ar) R131A+S152A+T176N+A207E+V247F+D255G as) K108V+R131A+S152A+T176N+V247F+D255G at) K70E+D130R+R131A+S152A+T176N+A207E+V247F+D255G au) D130R+R131A+S152A+T176N+S223R+V247F+D255G av) V101R+R131A+S152A+T176N+S223R+L251F aw) V101R+R131A+L144I+S152A+T176N+S223R+V247F+D255G ba) V101R+D130R+R131A+S152A+T176N+S223R+V247F+D255G bb) V101R+R131A+S152A+T176N+S223R+V247F+D255G bc) V101R+R131A+S152A+T176N+G206V+S223R+V247F+D255G bd) V101R+R131A+S152A+T176N+G206S+S223R+V247F+D255G be) K70E+V101R+R131A+S152A+T176N+S223R+V247F+D255G bf) D4C+V101R+R131A+S152A+T176N+S223R+N236C+V247F+D255G bg) V101R+D130R+R131A+S152A+T176N+G206V+S223R+V247F+D255G bh) V101R+R131A+L144I+S152A+T176N+G206V+S223R+V247F+D255G bi) V101R+R131A+S152A+T176N+G206V+S223R+V247F+D255G bj) V101R+R131A+S152A+T176N+G206V+S223R+L251F+D255G bk) D4C+V101R+D130R+R131A+S152A+T176N+G206P+S223R+N236C+V247F+D255G bl) D4C+A45V+V101R+D130R+R131A+S152A+T176N+G206V+S223R+N236C+V247F+D255G bm) D4C+Q49V+V101R+D130R+R131A+S152A+T176N+G206V+S223R+N236C+V247F+D255G bn) D4C+N97D+D130R+R131A+S152A+T176N+G206V+S223R+N236C+V247F+D255G bo) D4C+V101R+R131A+S152A+T176N+G206V+S223R+N236C+V247F+D255G bp) D4C+V101R+D130R+R131A+S152A+T176N+S223R+N236C+V247F+D255G and bq) D4C+V101R+R131A+S152A+T176N+S223R+N236C+V247F+D255G.

2 . The lipase variant of claim 1 , comprising a polypeptide having at least 80%, but less than 100% sequence identity to SEQ ID NO: 3.

3 . The lipase variant of claim 1 , wherein the variant has one or more improved properties relative to the parent, measured as an Improvement Factor (IF) that is greater than 1.0, or a Tm than is higher than the Tm for the parent lipase, wherein the improved property is selected from the group consisting of: Thermostability; Thermostability in Detergent; Hydrolytic Activity in Detergent; and Stability in Detergent.

4 . The lipase variant of claim 3 , wherein the Improvement Factor (IF) is greater than 1.0 in at least one of the assays selected from the group consisting of: Thermostability Assay; Thermostability in Detergent Assay; Performance Screening Assay; Stability in Detergent Assay; DSC assay; and Thermal Shift Assay;
wherein Tm of the variant is higher than Tm for the parent lipase.

5 . The lipase variant of claim 4 , wherein the Improvement Factor (IF) is greater than 1.0 in at least two, in at least three, or in at least four of the assays.

6 . The lipase variant of claim 3 , wherein the Improvement Factor (IF) is at least 1.1.

7 . A method of hydrolyzing a lipase substrate, comprising contacting said substrate with the lipase variant of claim 1 .

8 . A composition comprising the lipase variant of claim 1 .

9 . A method of producing a composition of a lipase, comprising adding the variant of claim 1 to the composition.

10 . A method of cleaning, comprising contacting a surface or an item with the composition of claim 8 .

11 . A polynucleotide encoding the lipase variant of claim 1 .

12 . A nucleic acid construct comprising the polynucleotide of claim 11 .

13 . An expression vector comprising the polynucleotide of claim 11 .

14 . A host cell comprising the polynucleotide of claim 11 .

15 . A method of producing a lipase variant, comprising:
(a) cultivating the host cell of claim 14 under conditions suitable for expression of the variant; and (b) recovering the variant.

Assignees

• Novozymes As

Inventors

• Saikia Rakhi
• Mendez Geetha Hiremath
• Dalal Sohel
• Balumuri Padmavathi
• Mepadam Santhosh Vasu
• Baunsgaard Lone
• Vind Jesper
• Gippert Garry Paul
• Borch Kim

Classifications

• C11D3/38681

  Chemically modified or immobilised enzymes · CPC title

• C12Y301/01004

  Phospholipase A2 (3.1.1.4) · CPC title

• C12N9/20 Primary

  Triglyceride splitting, e.g. by means of lipase · CPC title

• C11D3/38627

  containing lipase · CPC title