Directed Evolution And Expression Optimization Of The Key Enzyme D-amino Acid Oxidase And Cephalosporin C Acylase In Enzymatic Methods Of7-ACA Production

The industrial production of most cephalosporin derivatives is based on the modification of7-aminocephalosporanic acid (7-ACA) as starting material. Cephalosporin C can be hydrolyzed to7-ACA in two ways, one-step enzyme process and two-step enzyme process. D-amino acid oxidase is the key enzyme in two-step process, and cephalosporin C aeylase is for one-step process. In this study, directed evolution and expression optimization were conducted on these two enzymes. Results are as follows:1. Molecular directed evolution to Rhodotorula gracilis D-amino acid oxidase gene RgDAAO was conducted by error-prone PCR. First a random mutation library of RgDAAO was constructed and optimized. Through48-well-plate based high-throughput screening method, a mutant named M3217was obtained, whose Vmax was16.8%higher than the wild type. The sequence of M3217showed that five nucleotides substitution have occurred, and three of them caused amino acid changes, D242V/Q253R/D304V. According to the three-dimension structure of M3217simulated by Swiss-Model, none of the mutations is located in the vicinity of the active site. In particular, V304is located on the loop connecting β-strands F5and F6and appears to play an important role in monomer-monomer interaction. We speculated that this substitution improves the stability or FAD binding ability of RgDAAO protein. The volumetric productivity of M3217was increased up to1.8fold after expression optimization, achieving790.6U/L.2. A true Cephalosporin C aeylase named VACs has been over-expressed in E.coli BL21(DE3)pLysS and Pichia pasloris GS115cells. By optimization of medium, alternating screenings of medium components and simplified factorial experimental designs, an improved microbial process was set up at shake-flask level giving a60fold increase in volumetric enzyme productivity. Under the best expression conditions, nearly2000U/L of VACs were produced. Notably, this expression level is very promising in the7-ACA industrial production. VACs was also expressed in Pichia pastoris system, and the volumetric productivity achieves378.2U/L, laying a good groundwork for industrial application.