Subtilisin variants having improved stability

Patent metadata

Field	Value
Publication number	US-12509672-B2
Application number	US-201917295959-A
Country	US
Kind code	B2
Filing date	Nov 25, 2019
Priority date	Nov 28, 2018
Publication date	Dec 30, 2025
Grant date	Dec 30, 2025

Abstract

Disclosed herein is one or more subtilisin variant, nucleic acid encoding same, and compositions and methods related to the production and use thereof, including one or more subtilisin variant that has improved stability compared to one or more reference subtilisin.

First claim

We claim:

1 . A subtilisin variant having subtilisin protease activity and sharing at least 70% amino acid sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22, wherein the variant comprises the mutations X040E, X078N, and X166Q, wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, wherein the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

2 . The subtilisin variant of claim 1 , wherein the variant has at least one, two, or more additional mutations selected from the group consisting of X003V, X009E, X024Q, X069S, X076D, X079I, X087D, X118R, X124I, X128S, X129P, X130S, X145R, X182E, X185Q, X210I, X217L, X218S, X248D, and X259P, wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, wherein the variant does not have 100% sequence identity to a naturally-occurring amino acid sequence.

3 . The subtilisin variant of claim 1 , wherein said variant is derived from a parent or reference polypeptide sharing at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22.

4 . The subtilisin variant of claim 1 , wherein said variant comprises an amino acid sequence sharing at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with any one of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 22.

5 . The subtilisin variant of claim 1 , wherein said variant has improved stability when compared to a reference subtilisin lacking the mutations X040E, X078N, and X166Q numbered by correspondence with the amino acid sequence of SEQ ID NO: 1.

6 . The subtilisin variant of claim 5 , wherein the improved stability is measured as (i) a performance index (PI) of 1.1 or greater after 20 minutes at 30-70 degrees Celsius in a 10% detergent solution in comparison to a respective parent from which the variant is derived or (ii) an improved residual activity of at least 10% greater in comparison to the respective parent from which the variant is derived under the same assay conditions.

7 . The subtilisin variant of claim 6 , wherein the improved stability is measured as (i) a PI of 1.1 or greater after 20 minutes at 30-70 degrees Celsius in 10% detergent solution without protease-stabilizers in comparison to the respective parent from which the variant is derived or (ii) an improved residual activity of at least 10% greater in comparison to the respective parent from which the variant is derived under the same assay conditions in a 10% detergent solution without protease-stabilizers.

8 . The subtilisin variant of claim 1 , wherein the subtilisin variant has a cleaning performance index (PI) value of 1.0 or greater in comparison to a reference subtilisin.

9 . A polynucleotide comprising a nucleotide sequence that encodes the subtilisin variant of claim 1 , wherein said polynucleotide is optionally isolated.

10 . An expression vector or cassette comprising the polynucleotide of claim 9 .

11 . The expression vector or cassette of claim 10 , wherein the polynucleotide is operably linked to a promoter.

12 . A recombinant host cell comprising the expression vector or cassette of claim 10 .

13 . A composition comprising one or more subtilisin variant according to claim 1 .

14 . The composition according to claim 13 , wherein said composition is selected from the group consisting of an enzyme composition and a detergent composition.

15 . The composition according to claim 14 , wherein said detergent composition is selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning product.

16 . The composition of claim 13 , wherein said composition further comprises one or more ions selected from calcium and/or zinc; one or more enzyme stabilizer from 0.001 to 1.0 weight % of said subtilisin variant; one or more bleaching agent; one or more adjunct material; and/or one or more additional enzymes or enzyme derivatives selected from the group consisting of acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, DNase or nuclease, endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, lysozymes, mannanases, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhannogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, metalloproteases, nucleases, additional serine proteases, and combinations thereof.

17 . The composition of claim 13 , wherein said composition contains phosphate or is phosphate-free and/or contains boron or is boron-free.

18 . The composition of claim 13 , wherein said composition does not contain a protease stabilizer.

19 . The composition of claim 13 , wherein said composition is a granular, powder, solid, bar, liquid, tablet, gel, paste or unit dose composition.

20 . A method of cleaning, comprising contacting a surface or an item in need of cleaning with the subtilisin variant of claim 1 ; and optionally further comprising the step of rinsing said surface or item after contacting said surface or item with said variant, wherein, optionally, said item is dishware or fabric.

21 . The composition according to claim 14 , wherein said enzyme composition is an enzyme granule.

Assignees

• Danisco Us Inc

Inventors

• Babe Lilia Maria
• Alekseyev Viktor Yuryevich
• Basler Joshua Roy
• Engin H Billur
• Estell David A
• Ghirnikar Roopa Santosh
• Goedegebuur Frits
• Kaper Thijs
• Mulder Harm
• Pricelius Sina
• Redestig Nils Henning
• Van Stigt Thans Sander

Classifications

• C11D3/386

  Preparations containing enzymes {, e.g. protease or amylase} · CPC title

• C12Y304/21062

  Subtilisin (3.4.21.62) · CPC title

• C11D3/38636

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

• C12N9/54 Primary

  bacteria being Bacillus · CPC title