title: Engineering of a CPC acylase using a facile pH indicator assay.
abstract: Cephalosporin C (CPC) acylase is important for the one-step production of 7-aminocephalosporanic acid (7-ACA), a key intermediate for cephalosporin antibiotics. However, its application is hampered by the low activity, substrate inhibition, and product inhibition. In this study, two rounds of combinatorial active-site saturation testing (CASTing) were carried out on the CPC acylase acyII from Pseudomonas SE83, and one mutant H57βA/H70βY with no substrate inhibition was obtained. For further engineering to reduce the product inhibition, a quick pH indicator assay was developed, allowing for real-time monitoring of the product inhibition in the presence of added 7-ACA. The utility of the assay was demonstrated by screening six libraries of site-directed saturation mutagenesis libraries of H57βA/H70βY. A new mutant H57βA/H70βY/I176βN was obtained, which showed a k cat 3.26-fold and a K IP 3.08-fold that of the wild type, respectively. Given the commercial value of the enzyme, both this pH indicator assay and the triple mutant should be useful for further engineering of the enzyme to increase the specific activity and to decrease the product inhibition.
authors.affiliation: Department of Chemical Engineering, National Engineering Laboratory for Industrial Enzymes, Tsinghua University, One Tsinghua Garden Road, Beijing, 100084, China.
authors.forename: Yingzhou
authors.initials: Y
authors.lastname: Xiao
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Huo
authors.forename: Yu
authors.initials: Y
authors.lastname: Qian
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Chen
authors.forename: Pingkai
authors.initials: P
authors.lastname: Ouyang
authors.forename: Zhanglin
authors.initials: Z
authors.lastname: Lin
mesh_terms: D020134:Catalytic Domain; D002511:Cephalosporins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D057075:Enzyme Assays; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010399:Penicillin Amidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D011549:Pseudomonas / Q000201:enzymology
chemical_list: D002511:Cephalosporins; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; D010399:Penicillin Amidase