# US 9,040,280 B2 · Protease variants

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- Number: US 9,040,280 B2
- Document ID: US9040280B2
- Status: Granted
- Kind: B2
- Application number: 13/510,076
- Filed: 2010-12-10
- Published: 2012-12-06
- Granted: 2015-05-26
- Earliest claimed: 2009-12-11
- Assignees: Novozymes A/S
- Applicants: Tomoko Matsui; Allan Noergaard; Thomas Agersten Poulsen; John Matthews
- Inventors: Tomoko Matsui; Allan Noergaard; Thomas Agersten Poulsen; John Matthews
- Web page: https://patentgrep.com/patent/US9040280B2

## CPC

- C12N 9/58: derived from fungi
- C12P 7/06: Ethanol, i.e. non-beverage
- C12Y 304/24: Metalloendopeptidases (3.4.24)
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention relates to proteases having at least 75% identity to a protease derived from Thermoascus aurantiacus and comprises at least one modification in the amino acid sequence thereof. These protease variants have improved thermostability. The invention also relates to DNA encoding these proteases, methods of their production, as well as the use thereof.

## Claims

### Claim 1

An isolated polypeptide comprising an amino acid sequence having at least 95% sequence identity to residues 1-177 of SEQ ID NO: 2 and a substitution at position 142 of SEQ ID NO: 2, wherein the amino acid sequence has the protease specificity of the amino acid sequence of 1-177 of SEQ ID NO: 2 and has, at 80° C.-95° C., improved thermostability compared to the amino acids sequence of SEQ ID NO: 1-177 of SEQ ID NO: 2.

### Claim 2 (depends on 1)

The isolated polypeptide of claim 1, wherein the substitution at position 142 is D142L.

### Claim 3 (depends on 1)

The isolated polypeptide of claim 1, wherein the amino acid sequence has at least 99% identity to residues 1-177 of SEQ ID NO: 2.

### Claim 4 (depends on 3)

The isolated polypeptide of claim 3, wherein the substitution at position 142 is D142L.