Synthesis And Expression Of The Cephalosporin C Acylase

Cephalosporin C acylase can catalyze cephalosporin C(CPC) or Glutaryl-7-aminocephalosporanic(GL-7-ACA) to 7-aminocephalosporanic(7-ACA), plays an important role in the industrial production of cephalosporins. As a part of antibacterial construction,7-ACA is an important intermediate for all kinds of semi-synthetic cephalosporin antibiotics. In the production of 7-ACA, chemical method are gradually eliminated,because of the multiple steps, harsh conditions, toxic liquid waste generated in the process, CPC acylase must be used in the process of enzymatic preparation.The two-step enzymatic conversion isn`t the best way,because oxidative conditions is difficult to control, enzymatic reaction need a long chain of step, the equipment costs too much, The method of one-step enzymatic conversion has a positive application prospect.Because the reaction process is simple.It is an ideal method for the production of 7-ACA. However, the activity of CPC acylase is not high enough and the product has a greater inhibitory effect on the enzyme, as a result the enzyme is difficult to be applied to industrial production. Getting a CPC acylase with high activity and transformating into engineering bacteria become the key to solve this problem.In this assay,A gene has been synthetized according to the gene sequence of CPC acylase from Pseudomonas sp.strain SE83.The mutant site(Val122Ala-Gly140Ser-Phe58βAsn-Ile75βThr-Ile176βVal-Ala436βGly-Ser471βCys) has been introduced into the gene, The content of GC is reduced and the gene sequence has been optimized before the full sequence synthesis according to the preference codon of E. coli. Fragment length 2325 bp,the DNA squence is same as expected after sequencing.The recombinant plasmid p ET28-CPC with T7 promoter has been constructed and expressed in four kinds of carriers: E.coil BL21(DE3), E.coil BL21 star(DE3), E.coil ROSETTA(DE3), E.coil JM109(DE3). Analysis of SDS-PAGE and mass spectrometry showed that the gene was transferred into Escherichia coli.Enzyme activity is as follows: E.coil BL21(DE3) 2.04 U/m L, E.coil BL21 star(DE3) 1.2 U/m L, E.coil BL21 star(DE3) 0.48 U/m L, E.coil JM109(DE3) 1.36 U/m L.BL21(DE3) is the best host bacteria to express protein, The single factor experiment has been used to determine the best composition in culture medium volume respectively: glycerol 12 g/L, yeast extract 27 g/L, beef extract 15 g/L. Optimization of the fermentation medium by response surface method, the best medium composition is as follows : glycerol 11.99 g/L, yeast extract 26.83 g/L, beef extract 15.23 g/L. the highest enzyme activity is 3.68U/m L, which is 1.8 times than the initiative activity before optimization. In 2008, Ming [31] et al who come from Peking University synthesize the acylase gene sequence from Pseudomonas strain SE83 and express it in E.coli.After the optimization of the fermentation medium and culture conditions, the activity of CPC acylase was 2.96 U /m L.This study amplified pyruvate decarboxylase(PDC) promoter(Spdc)and terminator(Tpdc) sequence from Trichoderma reesei QM9414, CPCacy,Spdc, Tpdc is connected to the plasmid p PICZαA by double enzyme digestion method, constructs the expression vector which is named p PIC-CPCacy. The expression vector p PIC-CPC and the plasmid p AN7-1 containing hygromycin resistance selection marker was transferred into Trichoderma QM9414 at the same time using protoplast transformation technology.There are 10 strains of Trichoderma reesei contain targer gene.But enzyme activity can not be detected in fermentation cultureA new CPC acylase gene was expressed in Escherichia coli, and the fermentation medium has been optimized by the method of surface response, the enzyme activity increased significantly. As a result,We obtain a CPC acylase engineering bacteria with a high activity, CPC acylase has a wide application in the producing of 7-ACA, Using CPC acylase to produc 7-ACA is the future direction, The application of CPC acylated enzyme in preparation of 7-ACA can optimize the process of production,reduce the production cost, and has good economic benefit and social benefits. The gene transformate and integrate into the genome of Trichoderma reesei. And we try to express the gene in fungal systems, In the future,it will provide the theoretical basis for the expression of CPC acylase in fungi.