# US 10,246,690 B2 · Mutant hydrolase proteins with enhanced kinetics and functional expression

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- Number: US 10,246,690 B2
- Document ID: US10246690B2
- Status: Granted
- Kind: B2
- Application number: 15/877,980
- Filed: 2018-01-23
- Published: 2018-08-09
- Granted: 2019-04-02
- Earliest claimed: 2006-10-30
- Assignees: Promega Corporation
- Applicants: Promega Corporation
- Inventors: Aldis Darzins; Lance P. Encell; Rachel Friedman Ohana; Paul Otto; Michael R. Slater; Gediminas Vidugiris; Keith V. Wood; Monika G. Wood; Kate Qin Zhao; Kris Zimmerman
- Web page: https://patentgrep.com/patent/US10246690B2

## CPC

- C12N 9/14: Hydrolases (3)
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N 11/00: Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12Q 1/34: involving hydrolase
- G01N 33/581: {with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)}
- C12Y 308/01: in C-halide substances (3.8.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Y: ENZYMES

## Abstract

The invention provides a mutant hydrolase protein with enhanced kinetics and functional expression, as well as polynucleotides encoding the mutant proteins and methods of using the polynucleotides and mutant proteins.

## Claims

### Claim 1

A polypeptide comprising a mutant dehalogenase that is capable of catalyzing hydrolytic dehalogenation of an alkylhalide substrate and forming a covalent ester intermediate with the substrate, said mutant dehalogenase having at least 85% sequence identity with the polypeptide of SEQ ID NO: 1; wherein said mutant dehalogenase comprises substitutions at:
(a) one or more positions corresponding to positions in SEQ ID NO: 1 selected from the group consisting of positions 106 and 272; and
(b) positions corresponding to positions 58, 78, 155, 167, 172, 224, and 291 of SEQ ID NO: 1.

### Claim 2 (depends on 1)

A fusion polypeptide comprising the polypeptide of claim 1 and a polypeptide of interest.

### Claim 3 (depends on 2)

The fusion polypeptide of claim 2, wherein the polypeptide of interest is at the C-terminus of the fusion polypeptide.

### Claim 4 (depends on 2)

The fusion polypeptide of claim 2, wherein the polypeptide of interest is at the N-terminus of the fusion polypeptide.

### Claim 5 (depends on 1, 2)

The fusion polypeptide of claim 2, further comprising a polypeptide linker linking the polypeptide of claim 1 and the polypeptide of interest.

### Claim 6 (depends on 5)

The fusion polypeptide of claim 5, wherein the linker comprises a protease recognition sequence.