Penicillin-G acylases
The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes.
1. An engineered penicillin G acylase capable of acylating insulin, wherein the polypeptide sequence of said penicillin G acylase is at least 85% identical to SEQ ID NO: 100, and wherein said penicillin G acylase comprises a substitution at position 436 as compared to SEQ ID NO: 100.
2. The engineered penicillin G acylase of claim 1 , wherein said engineered penicillin G acylase comprises a polypeptide sequence selected from SEQ ID NOS:100, 110 and 142.
3. A method for producing acylated insulin, comprising: providing the engineered penicillin G acylase of claim 1 and insulin; exposing said engineered penicillin G acylase and said insulin under conditions such that said engineered penicillin G acylase acylates said insulin, thereby producing acylated insulin.
4. The method of claim 3 , wherein said acylation is conducted in the presence of methyl phenylacetate.
5. The method of claim 3 , wherein said acylation occurs at any of positions A1, B1, and/or B29 of said insulin.
6. The method of claim 4 , wherein said acylation occurs at any of positions A1, B1, and/or B29 of said insulin.
7. The method of claim 3 , wherein said acylation occurs at position A1 of said insulin.
8. The method of claim 3 , wherein said acylation occurs at position B1 of said insulin.
9. The method of claim 3 , wherein said acylation occurs at position B29 of said insulin.
10. The method of claim 3 , wherein said acylation occurs at positions A1, B1, and B29 of said insulin.