Mutant penicillin G acylases
Inventors
Van Der Laan, Jan Metske • Moody, Harold Monro • Kerkman, Richard • van der Does, Thomas
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-8541199-B2
Publication Date
2013-09-24
Expiration Date
Abstract
The present invention relates to a mutant prokaryotic penicillin G acylase derived from a wild-type penicillin G acylase characterized in that the mutant is having an amino acid substitution at one or more amino acid positions selected from the group consisting of amino acid positions A3, A77, A90, A144 A192, B24, B109, B148, B313, B460 and B488 according to the amino acid numbering of the Escherichia coli penicillin G acylase having the amino acid sequence depicted in SEQ ID No: 1.
Core Innovation
The invention relates to mutant prokaryotic penicillin G acylase derived from a wild-type penicillin G acylase. The mutant comprises an amino acid substitution at least at position B460 and at least one additional amino acid position selected from A3, A77, A90, A144, A192, B24, B109, B148, B313, B460 and B488 according to the amino acid numbering of the Escherichia coli penicillin G acylase with the amino acid sequence depicted in SEQ ID NO:1. The wild type penicillin G acylase is having an amino acid sequence which is 80-100% homologous to the amino acid sequence depicted in SEQ ID NO:1, with defined signal peptide, α-subunit, connecting peptide, and β-subunit regions.
The core technical problem addressed is improving enzymatic acylation performance by increasing synthetic activity and improving the synthesis/hydrolysis (S/H) ratio for enzymatic acylation of activated side chains to β-lactam nuclei. The described mutant prokaryotic penicillin G acylases emphasize particular combinations of amino acid substitutions that maintain or increase synthetic activity while improving the synthesis/hydrolysis (S/H) ratio. The document further characterizes improved behavior in terms of higher productivity, improved synthetic activity, and/or improved S/H ratios versus controls and/or wild type.
The mutant acylases are used for enzymatic coupling to produce semi-synthetic β-lactam antibiotics. The coupling is described for activated side chain amide or ester derivatives of phenylglycine/hydroxyphenylglycine/dihydrophenylglycine to β-lactam nuclei, including 6-APA, 7-ADCA, and 7-ACA. Documented embodiments include producing amoxicillin and ampicillin from 6-APA, and cefadroxil, cefalexin, and cefradine from 7-ADCA, with examples describing improved yields and reduced hydrolysis of activated side chain precursors.
Claims Coverage
The independent claims define a mutant prokaryotic penicillin G acylase characterized by specific amino-acid substitutions relative to SEQ ID NO:1 numbering and use of the mutant acylase for enzymatic coupling to produce semi-synthetic β-lactam antibiotics. The inventive features described are centered on the mutation pattern including B460 and, in some embodiments, additional specified positions, and on using the mutant acylase for enzymatic coupling to β-lactam nuclei with stated constraints in process embodiments.
Mutant prokaryotic penicillin G acylase with B460 and additional specified substitutions
A mutant prokaryotic penicillin G acylase derived from a wild-type penicillin G acylase, wherein the mutant comprises an amino acid substitution at least at position B460 and of at least one amino acid position selected from A3, A77, A90, A144, A192, B24, B109, B148, B313, B460 and B488 according to the amino acid numbering of the Escherichia coli penicillin G acylase with the amino acid sequence depicted in SEQ ID NO:1; wherein the Escherichia coli penicillin G acylase consists of a signal peptide, an α-subunit, a connecting peptide, and a β-subunit and the wild type penicillin G acylase is having an amino acid sequence which is 80-100% homologous to the amino acid sequence depicted in SEQ ID NO:1.
Enzymatic coupling of activated side chains to β-lactam nuclei using the mutant acylase
A process to produce a semi-synthetic β-lactam antibiotic by enzymatically coupling an activated side chain, where the activated side chain is an amide derivative or an ester of HPG, PG, or DHPG, to a β-lactam nucleus using a mutant prokaryotic penicillin G acylase.
Second mutation position together with B460
A mutant prokaryotic penicillin G acylase comprising amino acid substitutions at positions B460 and B24.
Expression vector encoding the mutant acylase
An expression vector that includes the nucleic acid sequence recited in claim 5.
Overall, the claim coverage centers on a mutant prokaryotic penicillin G acylase defined by an amino acid substitution at least at B460 with additional specified substitutions, and its use in enzymatic coupling of activated side-chain amide/ester derivatives to β-lactam nuclei to produce semi-synthetic β-lactam antibiotics. Additional inventive embodiments include specific additional mutation positioning (B24 with B460) and molecular embodiments in the form of an expression vector encoding the mutant acylase.
Stated Advantages
Maintains or increases synthetic activity while improving the synthesis/hydrolysis (S/H) ratio for enzymatic acylation of activated side chains to β-lactam nuclei.
Improves productivity for the production described in the document.
Increases product yields for the semi-synthetic β-lactam antibiotics described.
Reduces hydrolysis of activated side chain precursors.
Reduces activated side chain/nucleus ratios.
Documented Applications
Enzymatic production of semi-synthetic β-lactam antibiotics by enzymatic acylation using the mutant prokaryotic penicillin G acylase, including amoxicillin and ampicillin production from 6-APA and cefadroxil, cefalexin, and cefradine production from 7-ADCA.
Amoxicillin production embodiment described in connection with a Test A setup and monitoring criteria with residual 6-APA and amoxicillin yield targets.
Interested in licensing this patent?