[Evaluation of penicillin expandase mutants and complex substrate inhibition characteristics at high concentrations of penicillin G].
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.
Linjun
L
Wu
Keqiang
K
Fan
Junjie
J
Ji
Keqian
K
Yang
D002384:Catalysis; D019751:Intramolecular Transferases / Q000235:genetics*; D009154:Mutation; D010400:Penicillin G / Q000494:pharmacology*; D046915:Penicillin-Binding Proteins / Q000235:genetics*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D046915:Penicillin-Binding Proteins; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; D010400:Penicillin G