Pectate lyase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-10662417-B2
Application number	US-201716308711-A
Country	US
Kind code	B2
Filing date	Jul 5, 2017
Priority date	Jul 5, 2016
Publication date	May 26, 2020
Grant date	May 26, 2020

Abstract

The present invention relates to pectate lyase variants exhibiting alterations relative to a parent enzyme exhibiting pectate lyase activity; to a method of producing such enzymes; and to methods for using such enzymes in the textile, detergent and cellulose fiber processing industries. Compared to the parent enzyme, the pectate lyase variants of the present invention exhibit improved stability in detergents.

First claim

The invention claimed is:

1. A pectate lyase variant, wherein said variant has pectate lyase activity and comprises an alteration at one or more positions selected from the group consisting of: 250, 176, 124, 325, 108, 149, 99+176+325, 49+99+176+325+356, 49+99+176+257+325+356, 49+99+176+229+257+325+356, 99+176+325+356, 99+49+176+325+356, 99+124+176+325+356, 48+99+176+325+356, 99+108+176+325+356, 99+49+108+176+325+356, 229+250, 250+257, 48+250, 250+356, 48+49+99+176+257+325+356, 48+49+99+176+257+325+356, 49+99+124+176+257+325+356, 49+99+124+176+229+250+257+325+356, 49+99+176+229+250+257+325+356, 49+99+108+124+176+229+250+257+325+356, 48+49+99+176+250+257+325+356, 49+99+176+250+325+257+356, and 229+250+356, wherein each position corresponds to a position of the amino acid sequence of the pectate lyase having the amino acid sequence of SEQ ID NO:1, and wherein the variant has at least 90%, but less than 100% identity to the amino acid sequence of SEQ ID NO:1.

2. The variant of claim 1 , which is a variant of a parent pectate lyase having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1.

3. The variant according to claim 1 , wherein said alteration is a substitution with a positively charged amino acid.

4. The variant according to claim 1 , wherein the variant comprises one or more substitutions selected from the group consisting of: E108A, E108G, E108H, E108K, E108L, E108M, E108N, E108R, E108S, E108T, E108V, E108W, D124A, D124E, D124F, D124G, D1241, D124L, D124M, D124N, D124P, D124Q, D124R, D124S, D124T, D124V, D124W, S149K, S149L, S149R, S149W, S176A, S176C, S176D, S176E, I250A, I250G, I250L, I250M, I250N, I250S, I250T, I325F, I325L, I325Y, K99D+S176D+I325F, T49R+K99D+S176D+I325F+Q356F, T49R+K99D+S176D+K257L+I325F+Q356F, 49R+K99D+S176D+S229I+K257L+I325F+Q356F, K99D+S176D+I325F+Q356F, K99D+T49W+S176D+I325F+Q356F, K99D+D124W+S176D+I325F+Q356F, P48W+K99D+S176D+I325F+Q356F, K99D+E108N+S176D+I325F+Q356F, K99D+T49W+E108N+S176D+I325F+Q356F, S229I+I250N, I250N+K257L, P48W+I250N, I250N+Q356F, P48W+T49R+K99D+S176D+K257L+I325F+Q356F, P48W+T49W+K99D+S176D+K257L+I325F+Q356F, T49R+K99D+D124W+S176D+K257L+I325F+Q356F, T49R+K99D+S176D+S229I+K257L+I325F+Q356F, S229I+I250N, T49R+K99D+D124W+S176D+S229I+I250N+K257L+I325F+Q356F, T49R+K99D+S176D+S229I+K257L+I325F+Q356F, T49R+K99D+S176D+S229I+I250N+K257L+I325F+Q356F, T49R+K99D+D124W+S176D+S229I+I250N+K257L+I325F+Q356F, T49R+K99D+E108N+D124W+S176D+S229I+I250N+K257L+I325F+Q356F, P48W+T49R+K99D+S176D+I250N+K257L+I325F+Q356F, T49R+K99D+S176D+I250N+I325F+K257L+Q356F, and S229I+I250N+Q356F.

5. A pectate lyase variant comprising an alteration at one or more positions selected from the group consisting of positions 250, 176, 124, 108, 149, and 325, wherein numbering is according to SEQ ID NO: 1, and wherein the alteration(s)
is a substitution of the amino acid which occupies the position with a different amino acid,

and wherein each position corresponds to a position of the amino acid sequence of the pectate lyase having the amino acid sequence of SEQ ID NO: 1, and wherein the variant pectate lyase has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.

6. The variant of claim 5 , which is a variant of a parent pectate lyase having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1.

7. The variant according to claim 1 , wherein the variant has improved stability in detergent or improved thermal stability compared to a parent enzyme.

8. A composition comprising the variant of claim 1 .

9. The composition of claim 8 , wherein said composition is a cleaning or detergent composition.

10. The composition according to claim 8 , further comprising:
i) one or more detergent components selected from the group consisting of: surfactants, hydrotropes, builders, co-builders, chelators, bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors, enzyme stabilizers, enzyme activators, antioxidants, and solubilizers; and/or
ii) one or more additional enzymes selected from the group consisting of: proteases, amylases, lichenases, lipases, cutinases, cellulases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases, mannanases, oxidoreductases, hemicellulases, xylanases, galactanases, arabinofuranosidases, esterases, arabinanases, pectate lyases, DNases, perhydrolases, betaglucanases, xanthan lyases, acyl transferases, phospholipases, laccases, aryl esterases, alpha-amylases, glucoamylases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenases, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, endo-beta-mannanases, exo-beta-mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, or combinations thereof.

11. A polynucleotide encoding the variant of claim 1 .

12. An isolated host cell comprising the polynucleotide of claim 11 .

13. A method for improving the detergent stability of a parent pectate lyase, comprising: introducing into said parent pectate lyase an alteration at one or more positions selected from the group consisting of:108, 124, 149, 176, 250, 325, 99+176+325, 49+99+176+325+356, 49+99+176+257+325+356, 49+99+176+229+257+325+356, 99+176+325+356, 99+49+176+325+356, 99+124+176+325+356, 48+99+176+325+356, 99+108+176+325+356, 99+49+108+176+325+356, 229+250, 50+257, 48+250, 250+356, 48+49+99+176+257+325+356, 48+49+99+176+257+325+356, 49+99+124+176+257+325+356, 49+99+176+229+257+325+356, 229+250, 229+356, 49+99+124+176+229+250+257+325+356, 49+99+176+229+257+325+356, 49+99+176+229+250+257+325+356, 49+99+124+176+229+250+257+325+356, 49+99+108+124+176+229+250+257+325+356, 48+49+99+176+250+257+325+356, 49+99+176+250+325+257+356, and 229+250+356, wherein each position corresponds to a position of the amino acid sequence of the pectate lyase having the amino acid sequence of SEQ ID NO:1, and wherein the altered pectate lyase has at least 90%, but less than 100% sequence identity to the amino acid sequence of SEQ ID NO:1, and incorporating the pectate lyase into a detergent composition.

14. The method of claim 13 , wherein said alteration at one or more positions is selected from the group consisting of: E108A, E108G, E108H, E108K, E108L, E108M, E108N, E108R, E108S, E108T, E108V, E108W, D124A, D124E, D124F, D124G, D124I, D124L, D124M, D124N, D124P, D124Q, D124R, D124S, D124T, D124V, D124W, S149K, S149L, S149R, S149W, S176A, S176C, S176D, S176E, I250A, I250G, I250L, I250M, I250N, I250S, I250T, I325F, I325L, I325Y, K99D+S176D+I325F, T49R+K99D+S176D+I325F+Q356F, T49R+K99D+S176D+K257L+I325F+Q356F, T49R+K99D+S176D+S229I+K257L+I325F+Q356F, K99D+S176D+I325F+Q356F, K99D+T49W+S176D+I325F+Q356F, K99D+D124W+S176D+I325F+Q356F, P48W+K99D+S176D+I325F+Q356F, K99D+E108N+S176D+I325F+Q356F, K99D+T49W+E108N+S176D+I325F+Q356F, S229I+I250N, I250N+K257L, P48W+I250N, I250N+Q356F, P48W+T49R+K99D+S176D+K257L+I325F+Q356F, P48W+T49W+K99D+S176D+K257L+I325F+Q356F, T49R+K99D+D124W+S176D+K257L+I325F+Q356F, T49R+K99D+S176D+S229I+K257L+I325F+Q356F, S229I+I 250N, T49R+K99D+D124W+S176D+S229I+I250N+K257L+I325F+Q356F, T49R+K99D+S176D+S229I+K257L+I325F+Q356F, T49R+K99D+S176D+S229I+I250N+K257L+I325F+Q356F, T49R+K99D+D124W+S176D+S229+I250N+K257L+I325F+Q356F, T49R+K99D+E108N+D124W+S176D+S229I+I250N+K257L+I325F+Q356F, P48W+T49R+K99D+S176D+I250N+K257L+I325F+Q356F, T49R+K99D+S176D

Assignees

• Novozymes As

Inventors

• Monrad Rune Nygaard
• Hansen Peter Kamp
• Rasmussen Frank Winther
• Due Anne Vindum
• Nielsen Jens Erik
• Gippert Garry Paul

Classifications

• C12N9/88 Primary

  Lyases (4.) · CPC title

• C12Y402/02002

  Pectate lyase (4.2.2.2) · CPC title

• C11D3/38636

  containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase · CPC title