Evaluation of penicillin expandase mutants and complex substrate inhibition characteristics at high concentrations of penicillin G.
- Author:
Linjun WU
;
Keqiang FAN
;
Junjie JI
;
Keqian YANG
- Publication Type:Journal Article
- MeSH:
Catalysis;
Intramolecular Transferases;
genetics;
Mutation;
Penicillin G;
pharmacology;
Penicillin-Binding Proteins;
genetics;
Streptomyces;
enzymology;
genetics
- From:
Chinese Journal of Biotechnology
2015;31(12):1690-1699
- CountryChina
- Language:Chinese
-
Abstract:
Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application.
