# US 10,870,838 B2 · Methods for producing lipases

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- Number: US 10,870,838 B2
- Document ID: US10870838B2
- Status: Granted
- Kind: B2
- Application number: 15/778,330
- Filed: 2016-11-30
- Published: 2018-11-29
- Granted: 2020-12-22
- Earliest claimed: 2015-12-01
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Lone Baunsgaard; Allan Svendsen; Kim Borch; Rakhi Saikia; Jakob Skjold-Jørgensen
- Web page: https://patentgrep.com/patent/US10870838B2

## CPC

- C12N 9/20: Triglyceride splitting, e.g. by means of lipase
- C11D 3/38627: {containing lipase}
- C12Y 301/01003: Triacylglycerol lipase (3.1.1.3)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C12Y: ENZYMES

## Abstract

The present invention relates to a method for generating a preparation of a lipase variant of a parent lipase comprising a step of altering one or more nucleotides in a polynucleotide encoding the parent lipase, wherein the alteration provides at least one disulfide bond and a hydrolytic activity of said lipase variant which is reduced as compared with the hydrolytic activity of the parent lipase. The invention also relates to a polynucleotide encoding the lipase variant, the lipase variant and its use.

## Claims

### Claim 1

A method for obtaining a lipase variant of a parent lipase, comprising introducing into a parent lipase an alteration at one or more positions of the parent lipase, wherein the alteration at the one or more positions provides at least one disulfide bond and wherein hydrolytic activity of the lipase variant is reduced as compared to hydrolytic activity of the parent lipase,
wherein the alteration is selected from one or more amino acid substitutions selected from the group consisting of L57C/N120C, L54C/P101C, A47C/V102C, S24C/G104C, P46C/N186C, S43C/N186C, T40C/P182C, T40C/L216C, T40C/L239C, D38C/A212C, P37C/E240C, P37C/N246C, E36C/N246C, E36C/V249C, A33C/V249C and S31C/V249C, wherein the position corresponds to the positions of SEQ ID NO: 2
and wherein the amino acid sequence of the lipase variant has at least 90% sequence identity with SEQ ID NO: 2.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the lipase variant has a propeptide and a mature polypeptide.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the parent lipase has at least 95% sequence identity with SEQ ID NO: 2.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the parent lipase is a zygomycete lipase having an amino acid sequence of SEQ ID NO: 2.

### Claim 5

A lipase variant of a parent lipase, comprising an alteration at one or more positions compared to the parent lipase, wherein the alteration provides at least one disulfide bond and wherein hydrolytic activity of said lipase variant is reduced as compared with hydrolytic activity of the parent lipase,
wherein the variant comprises one or more amino acid substitutions selected from the group consisting of L57C/N120C, L54C/P101C, A47C/V102C, S24C/G104C, P46C/N186C, S43C/N186C, T40C/P182C, T40C/L216C, T40C/L239C, D38C/A212C, P37C/E240C, P37C/N246C, E36C/N246C, E36C/V249C, A33C/V249C and S31C/V249C, wherein the positions correspond to the positions of SEQ ID NO: 2,
and wherein the amino acid sequence of the lipase variant has at least 90% sequence identity with SEQ ID NO: 2.

### Claim 6 (depends on 5)

A composition comprising the lipase variant of claim 5.

### Claim 7 (depends on 6)

The composition according to claim 6, further comprising an activator, wherein the activator is a protease or a reducing agent.

### Claim 8 (depends on 5)

A method for increasing hydrolytic activity of the lipase variant of claim 5, comprising a step of bringing said lipase variant into contact with an activator.

### Claim 9 (depends on 5)

The lipase variant of claim 5, comprising one or more amino acid substitutions selected from the group consisting of P-46C/N186C, T-40C/P182C, L-54C/P101C, A-24C/G104C, and D-38C/A212C wherein the positions correspond to the positions of SEQ ID NO: 2.

### Claim 10 (depends on 5)

The lipase variant of claim 5, having at least 95% sequence identity with SEQ ID NO: 2.

### Claim 11 (depends on 5)

The lipase variant of claim 5, having at least 97% sequence identity with SEQ ID NO: 2.

### Claim 12 (depends on 5)

The lipase variant of claim 5, having at least 98% sequence identity with SEQ ID NO: 2.

### Claim 13 (depends on 5)

The lipase variant of claim 5, having at least 99% sequence identity with SEQ ID NO: 2.

### Claim 14 (depends on 6)

The composition of claim 6, formulated as a regular, compact or concentrated liquid; a gel; a paste; a soap bar; a regular or a compacted powder; a granulated solid; a homogenous or a multilayer tablet with two or more layers (same or different phases); a pouch having one or more compartments; a single or a multi-compartment unit dose form; or any combination thereof.

### Claim 15 (depends on 6)

A method of cleaning comprising contacting a surface or an item with a composition according to claim 6.