# US 10,246,696 B2 · Pectinases with improved thermostability

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- Number: US 10,246,696 B2
- Document ID: US10246696B2
- Status: Granted
- Kind: B2
- Application number: 16/062,595
- Filed: 2016-12-08
- Published: 2019-01-03
- Granted: 2019-04-02
- Earliest claimed: 2015-12-15
- Assignees: Metgen Oy
- Applicants: Metgen OY
- Inventors: Klara Birikh; Anu Minna Maaret Suonpaa
- Web page: https://patentgrep.com/patent/US10246696B2

## CPC

- C12N 9/88: Lyases (4.)
- C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12Y 402/02002: Pectate lyase (4.2.2.2)
- 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 invention is in the field of protein chemistry, in particular in the field of enzymology. It provides pectinases, i.e. polypeptides with pectin-degrading properties. In particular the invention provides polypeptides with pectate lyase activity (EC 4.2.2.2). Enzymes according to the invention have improved properties, such as improved thermostability and decreased calcium dependence.

## Claims

### Claim 1

A polypeptide with pectate lyase activity, the polypeptide comprising:
an amino acid sequence that is at least 70% identical to the amino acid according to SEQ ID NO: 1, and a small, polar, non-charged amino acid residue at an amino acid position corresponding to position 235 in SEQ ID NO: 1, and wherein the polypeptide has an improved thermostability compared to an identical polypeptide not having said substitution.

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein the small, polar, non-charged amino acid residue is selected from the group consisting of amino acids Threonine, Serine, Asparagine and Cysteine.

### Claim 3 (depends on 1)

The polypeptide of claim 1, wherein the small, polar, non-charged amino acid residue is Threonine.

### Claim 4 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 75% identical to the amino acid according to SEQ ID NO: 1.

### Claim 5 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises a leucine residue at an amino acid position corresponding to position 231 in SEQ ID NO: 1.

### Claim 6 (depends on 1)

A composition comprising the polypeptide of claim 1.

### Claim 7 (depends on 1)

A nucleic acid encoding the polypeptide of claim 1.

### Claim 8 (depends on 7)

A vector comprising the nucleic acid of claim 7.

### Claim 9 (depends on 7)

A composition comprising the nucleic acid of claim 7.

### Claim 10 (depends on 7)

A recombinant host cell comprising the nucleic acid of claim 7.

### Claim 11 (depends on 10)

The recombinant host cell of claim 10, wherein the host cell is selected from the group consisting of Escherichia coli, Bacillus, Corynebacterium, Pseudomonas, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, filamentous fungi, yeast and insect cells.

### Claim 12 (depends on 1, 10)

A method for producing the polypeptide of claim 1, the method comprising:
culturing the recombinant host cell of claim 10 under conditions suitable for the production of the polypeptide, and
recovering the polypeptide obtained, and
optionally purifying said polypeptide.

### Claim 13

A method for improving the thermostability of a polypeptide with pectate lyase activity comprising an amino acid sequence that is at least 70% identical to the amino acid according to SEQ ID NO: 1, the method comprising:
altering the amino acid at a position corresponding to position 235 in SEQ ID NO: 1 to a small, polar, non-charged amino acid residue, and
optionally altering the amino acid at a position corresponding to position 231 in SEQ ID NO: 1 to a leucine residue.

### Claim 14 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid according to SEQ ID NO: 1.

### Claim 15 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 85% identical to the amino acid according to SEQ ID NO: 1.

### Claim 16 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid according to SEQ ID NO: 1.

### Claim 17 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid according to SEQ ID NO: 1.

### Claim 18 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is identical to the amino acid according to SEQ ID NO: 1.

### Claim 19 (depends on 8)

A composition comprising the vector of claim 8.

### Claim 20 (depends on 8)

A recombinant host cell comprising the vector of claim 8.