Recombinant Expression,Immobilization And Characterization Of Cephalosporin-c Deacetylase

Cephalosporin-C deacetylase(CAH)[EC3.1.1.41]is an esterase that catalyzes deacetylation at the 3' position of cephalosporins.Deacetyl cephalosporins are used as starting materials for various semisynthetic cephalosporin antibiotics such as cefuroxime,cefoxitin,cefpodoxime proxetil,etc.In this work,high-level production Bacillus subtilis Cephalosporin-C deacetylase in Escherichia coli was successfully carried out with optimization of expression plasmids and fermentation conditions.To maximize the expression of the cephalosporin-C deacetylase gene,8 kinds of inducible plasmids and 16 kinds of constitutive plasmids were constructed.A constitutive expression plasmid pB1-SIL3-K,which has a replication origin derived from pUC19 and B1 sequence between the SIL3 gene and Trp promoter,was selected as the most efficient expression plasmid.CAH activity expressed by E.coli DH5?/pB1-SIL3-K was 144U/mL when cultivating at 37? for 24 h?To produce large quantities of CAH in a cost-effective manner,we further investigated the high-level fermentation,paying special attention to the conditions of the seed culture,medium composition and fermentation conditions.The optimum compositions of the fermentation medium were:2%dextrin,2%Angel Yeast Powder,2%Fish bone peptone and 1 xM9 base salt.The highest enzyme activity of CAH was 420 U/mL when cultivating in shaken flasks under the optimum conditions.Through constant pH control and 4%glycerol feeding,the highest CAH activity increased to 1340 U/mL at 24h in a 7L fermenter,which was the highest reported enzyme activity until now.CAH was immobilized on both amino-support HA and epoxy-support EP.By optimizing the immobilizing conditions,2 kinds of immobilized CAH were successfully prepared.Both of the 2 kinds of immobilized CAH exhibit higher enzyme activity than that of the commercial one.The pH and temperature stability ranges for the immobilized CAH were broader than those for free enzyme.The optimal temperature and pH of immobilized CAH were 60-65 ? and 8.0-8.5,respectively.