BACKGROUNDCephalosporin 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 degrees 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 degrees C or below. RESULTSIn 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 1.94Umg(-1) at 13 degrees 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. CONCLUSIONA 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. (c) 2018 Society of Chemical Industry