Protease variants and uses thereof

Patent metadata

Field	Value
Publication number	US-2023257728-A1
Application number	US-202318166707-A
Country	US
Kind code	A1
Filing date	Feb 9, 2023
Priority date	Dec 21, 2016
Publication date	Aug 17, 2023
Grant date	—

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 and/or soil removal compared to one or more reference subtilisin.

First claim

1 . A subtilisin variant comprising an amino acid sequence comprising an amino acid substitution at position 76 selected from 76A/C/D/E/F/G/H/I/K/L/M/P/Q/S/T/V/W/Y, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 17, and wherein said variant comprises an amino acid sequence with 85% sequence identity to the amino acid sequence of SEQ ID NO: 11, wherein said variant has one or more improved property when compared to a parent or reference subtilisin of SEQ ID NO:11, wherein the improved property is selected from improved cleaning performance in detergent, improved stability and combinations thereof.

2 . (canceled)

3 . The subtilisin variant according to claim 1 wherein said variant
(i) is a member of the Bacillus gibsonii-clade;
(ii) is isolated;
(iii) has proteolytic activity; or
(iv) comprises a combination of (i) to (iii).

4 . The subtilisin variant according to claim 1 , wherein said variant is derived from a parent or reference polypeptide with
(i) 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:11 ; or (ii) 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:11.

5 . The subtilisin variant of claim 1 , wherein said variant comprises an amino acid sequence with
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:11 .

6 . The subtilisin variant of claim 1 , wherein said variant has one or more improved property when compared to a parent or reference subtilisin having the amino acid sequence of SEQ ID NO: 11; wherein the improved property is selected from improved cleaning performance in detergent, improved stability; and combinations thereof.

7 . (canceled)

8 . The subtilisin variant of claim 6 , wherein the improved property is
(i) improved cleaning performance in detergent, wherein said variant has a blood/milk/ink (BMI), crème brûlée and/or egg stain cleaning performance index (PI )>1 compared to a parent subtilisin having the amino acid sequence of SEQ ID NO: 11; and/or (ii) improved stability, wherein said variant has a stability PI > 1 or >50% residual activity compared to a parent subtilisin having the amino acid sequence of SEQ ID NO: 11 .

9 . (canceled)

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

11 . The composition according to claim 10 , wherein said composition is selected from an enzyme composition and a detergent composition.

12 . The composition according to claim 11 , wherein said detergent composition is selected from a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning detergent.

13 . The composition according to claim 10 , further comprising (i) one or more other enzymes selected acyl transferases, amylases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinases, arabinosidases, aryl esterases, beta-galactosidases, beta-glucanases, carrageenases, catalases, chondroitinases, cutinases, endo-beta-mannanases, exo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipolytic enzymes, lipoxygenases, mannanases, metalloproteases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonases, cellulases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, and xylosidases; (ii) one or more surfactants; (iii) one or more ions selected from calcium and zinc; (iv) one or more adjunct material; (v) one or more stabilizers; (vi) from about 0.001% to about 1.0 weight % of the variant; (vii) one or more bleaching agents; and/or (viii) combinations thereof.

14 . The composition of claim 10 , wherein said composition contains phosphate or contains boron.

15 . The composition of claim 10 , wherein said composition is a granular, powder, solid, bar, liquid, tablet, gel, paste and/or unit dose composition.

16 - 18 . (canceled)

19 . A polynucleotide comprising a nucleic acid sequence encoding a variant of claim 1 , wherein said polynucleotide is, optionally, isolated.

20 . The polynucleotide of claim 19 , wherein the nucleic acid sequence is operably linked to a promoter.

21 . An expression vector or cassette comprising the polynucleotide of claim 19 .

22 . A recombinant host cell comprising the polynucleotide of claim 19 .

23 . A composition comprising a variant of claim 1 , wherein said composition is a disinfectant, medical instrument cleaning, animal feed, contact lens cleaning, wound cleaning, or textile, leather or feather processing composition.

24 - 32 . (canceled)

33 . A method of improving the stability of a subtilisin variant, comprising introducing an amino acid substitution at position 76 selected from 76A/C/D/E/F/G/H/I/K/L/M/P/Q/S/T/V/W/Y,
(iii) wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 17, and wherein the parent subtilisin has an amino acid sequence with at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 11.

Assignees

• Danisco Us Inc

Inventors

• Babe Lilia Maria
• Dankmeyer Lydia
• Estell David A
• Goedegebuur Frits
• Kaper Thijs
• Mulder Harm
• Pricelius Sina

Classifications

• C12N9/54 Primary

  bacteria being Bacillus · CPC title

• C11D3/386

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

• C12Y304/21062

  Subtilisin (3.4.21.62) · CPC title

• C12N9/64

  derived from animal tissue · CPC title