Penicillin-G acylases
The present disclosure relates to engineered penicillin G acylase (PGA) enzymes having improved properties, polynucleotides encoding such enzymes, compositions including the enzymes, and methods of using the enzymes.
1. An engineered penicillin G acylase (PGA) capable of cleaving substrate 6-nitro-3-(phenylacetamido)benzoic acid (NIPAB) to product 5-amino-2-nitrobenzoic acid, wherein the PGA comprises an α-chain sequence having at least 85% identity to SEQ ID NO:179 and an β-chain sequence having at least 85% identity to SEQ ID NO:180, wherein the polypeptide has improved activity as compared to the naturally occurring PGA of Kluyvera citrophila , wherein said engineered penicillin G acylase comprises SEQ ID NO:8.
2. A polynucleotide encoding the engineered PGA of claim 1 , wherein said polynucleotide sequence comprises SEQ ID NO:7.
3. An expression vector comprising the polynucleotide of claim 2 .
4. A host cell comprising the vector of claim 3 .
5. A method for cleaving penicillin G of structural formula (I) (“the substrate”) to 6-amino penicillanic acid of structural formula (II) and phenylacetic acid of structural formula (III) (“the products”):
which method comprises:
contacting the penicillin G with an engineered PGA of claim 1 , under reaction conditions suitable for cleaving the substrate to the products.
6. A composition comprising: (i) a penicillin G of structural formula (I), a 6-amino penicillanic acid of structural formula (II), and/or a phenylacetic acid of structural formula (III); and (ii) an engineered PGA comprising SEQ ID NO: 8.