A rapid and specific method to screen environmental microorganisms for cephalosporin acylase activity.
Medically useful semisynthetic cephalosporin antibiotics are made from precursor 7-aminocephalosporanic acid (7-ACA). Cephalosporin acylase (CA), which catalyzes hydrolysis of both glutaryl-7-aminocephalosporanic acid (GL-7ACA) and cephalosporin C (CPC) to 7-ACA, is thus a very important enzyme for producing semisynthetic beta-lactam antibiotics. To facilitate the attempts of obtaining the microorganisms with higher CA activity from natural environments, a new and specific method for screening environmental microorganisms with cephalosporin acylase activity was developed. The core part of cephalosporin was replaced by 6-amino penicillinic acid (6-APA) to generate new substrates glutaryl-6-APA and adipoyl-6-APA for screening. Serratia marcescens that is sensitive to 6-APA and resistant to penicillin G, glutaryl-6-APA and adipoyl-6-APA was used as an indicator strain in an overlaid-agar screening system. A strain capable of producing cephalosporin acylase was selected from thousands of candidates by this method. Because of its specificity, simplicity and sensitivity, the method could be easily installed into a high-throughout system.
Laboratory of Molecular Microbiology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, PR China.
Songcheng
S
Zhu
Yunliu
Y
Yang
Guoping
G
Zhao
Weihong
W
Jiang
D001419:Bacteria / Q000201:enzymology*; D002511:Cephalosporins / Q000096:biosynthesis* / Q000378:metabolism; D003470:Culture Media; D004783:Environmental Microbiology; D010399:Penicillin Amidase / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013997:Time Factors
D002511:Cephalosporins; D003470:Culture Media; C081063:glutaryl-7-aminocephalosporanic acid; C025163:cephalosporin C; D010399:Penicillin Amidase