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Exploitation of cold‐active cephalosporin C acylase by computer‐aided directed evolution and its potential application in low‐temperature biosynthesis of 7‐aminocephalosporanic acid
Author(s) -
Tang CunDuo,
Shi HongLing,
Jiao ZhuJin,
Shi HongFei,
Yao LunGuang,
Xu JianHe,
Kan YunChao
Publication year - 2018
Publication title -
journal of chemical technology and biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 117
eISSN - 1097-4660
pISSN - 0268-2575
DOI - 10.1002/jctb.5647
Subject(s) - cephalosporin c , mutant , adaptability , cephalosporin , chemistry , mutagenesis , combinatorial chemistry , yield (engineering) , directed evolution , enzyme , flexibility (engineering) , biochemistry , computational biology , biology , physics , gene , thermodynamics , mathematics , ecology , antibiotics , statistics
BACKGROUND Cephalosporin C acylase (CA) plays a key role in the one‐step enzymatic catalysis of 7‐aminocephalosporanic acid (7‐ACA) from Cephalosporin C (CPC). However, the synthesis of 7‐ACA needs low‐temperature conditions: unless catalyzed at 13 °C, it will produce large amounts of undesired products. Unfortunately, cephalosporin CA catalyzes CPC to 7‐ACA directly at a very low efficiency at 13 °C or below. RESULTS In this work, multiple techniques including molecular docking, multi‐site random‐directed mutagenesis and high‐throughput screening were adopted and combined into an engineering strategy to improve the cold adaptability of CA . Thus, the best‐hit mutant 9H9 containing mutations of P238G, P449G and P582G was screened revealing a specific activity of 1.94 U mg −1 at 13 °C, which is 1.3‐fold higher than that of the parent. Compared with the parent, the mutant 9H9 shows a slight decline in the K m value and significant improvement in the V max value. In addition, the yield of 7‐ACA catalyzed by 9H9 was obviously higher than that of the parent at low temperature. CONCLUSION A new and viable strategy was established for improving the cold adaptability of CA and investigations were made into enhancing its conformational flexibility. This might lay a solid foundation for future study and industrial application of CAs. © 2018 Society of Chemical Industry