# US 2014/0154778 A1 · High Fidelity Restriction Endonucleases

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- Number: US 2014/0154778 A1
- Document ID: US20140154778A1
- Status: Published application
- Kind: A1
- Application number: 14/137,608
- Filed: 2013-12-20
- Published: 2014-06-05
- Earliest claimed: 2007-07-12
- Assignees: New England Biolabs, Inc.
- Applicants: New England Biolabs, Inc.
- Inventors: Zhenyu Zhu; Aine Quimby; Shengxi Guan; Dapeng Sun; Yishu Huang; Xuhui Lai; Siu-hong Chan; Xianghui Li; Shuang-Yong Xu; Chunhua Zhang
- Web page: https://patentgrep.com/patent/US20140154778A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/16: acting on ester bonds (3.1)
- C12Q 1/34: involving hydrolase
- C12Q 1/44: involving esterase
- C12Q 1/68: involving nucleic acids
- C12Y 301/21004: Type II site-specific deoxyribonuclease (3.1.21.4)
- G01N 2333/916: acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N 2333/922: Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
- 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
- C12Y: ENZYMES
- 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)

## Abstract

Methods and compositions are provided for engineering mutant enzymes with reduced star activity where the mutant enzymes have a fidelity index (FI) in a specified buffer that is greater than the FI of the non-mutated enzyme in the same buffer.

## Claims

### Claim at position 1

1-74. (canceled)

### Claim 75

A composition comprising a variant MluI restriction endonuclease having reduced star activity, wherein the variant MluI restriction endonuclease comprises an amino acid sequence that differs from the amino acid sequence of the parent MluI restriction endonuclease by two amino acid substitutions at positions corresponding to positions 112 and 132 of SEQ ID NO:28.

### Claim 76 (depends on 75)

A composition according to claim 75, wherein the amino acid substitutions are E112A and R132A.

### Claim 77 (depends on 75)

A DNA encoding the variant of claim 75.