Penicillin-G acylases

Patent metadata

Field	Value
Publication number	US-10724025-B2
Application number	US-201715588378-A
Country	US
Kind code	B2
Filing date	May 5, 2017
Priority date	May 5, 2016
Publication date	Jul 28, 2020
Grant date	Jul 28, 2020

Abstract

The present invention provides engineered penicillin G acylase (PGA) enzymes having improved properties, polynucleotides encoding such enzymes, compositions including the enzymes, and methods of using the enzymes.

First claim

What is claimed is:

1. An engineered penicillin G acylase having a polypeptide sequence that is at least 95% identical to SEQ ID NO: 1220, wherein said penicillin G acylase comprises a leucine residue at position X2 as compared to the sequence of SEQ ID NO: 1220.

2. The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase comprises SEQ ID NO: 1194.

3. The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase is capable of producing phenyl acetate mono-protected or di-protected insulin.

4. The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase comprises a polypeptide sequence that is at least 95% identical to at least one sequence provided in the even-numbered sequences of SEQ ID NOS: 6-1902.

5. The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase is encoded by a polynucleotide sequence that is at least 95% identical to SEQ ID NO:1219.

6. A method for producing phenyl acetate mono-protected insulin, comprising: i) providing the engineered penicillin G acylase of claim 1 and/or a composition comprising said engineered penicillin G acylase, and insulin comprising A1/B1/B29 tri-phenyl acetate protecting groups; and ii) exposing said engineered penicillin G acylase to said insulin comprising A1/B1/B29 tri-phenyl acetate protecting groups, under conditions such that said engineered penicillin G acylase removes the A1, B1 and/or B29 tri-phenyl acetate protecting groups from said insulin thereby producing phenyl acetate mono-protected or di-protected insulin.

7. The method of claim 6 , wherein said penicillin G acylase removes the A1 tri-phenyl acetate protecting group of said insulin.

8. The method of claim 6 , wherein said penicillin G acylase removes the B1 tri-phenyl acetate protecting group of said insulin.

9. The method of claim 6 , wherein said penicillin G acylase removes the B29 tri-phenyl acetate protecting group of said insulin.

10. The method of claim 6 , wherein said penicillin G acylase removes the A1, B1, and B29 tri-phenyl acetate protecting group of said insulin.

11. The method of claim 6 , wherein said engineered penicillin G acylase produces more than 90% phenyl acetate mono-protected, as compared to the production of phenyl acetate mono-protected insulin by the polypeptide of SEQ ID NO:2.

12. The engineered penicillin G acylase of claim 1 , wherein said penicillin G acylase comprises SEQ ID NO: 1220.

Assignees

• Codexis Inc

Inventors

• Nazor Jovana
• Mitchell Vesna
• Elgart David
• Lexa Katrina W
• Dellas Nikki
• Orr Robert Kevin
• Alvizo Oscar
• Garcia Ravi David
• Viduya Judy Victoria Antonio
• Moffett Courtney Dianne

Classifications

• C12Y305/01011

  Penicillin amidase (3.5.1.11), i.e. penicillin-amidohydrolase · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N9/84 Primary

  Penicillin amidase {(3.5.1.11)} · CPC title

• C07K14/62

  Insulins · CPC title