文献类型：期刊文献

中文题名：头孢菌素脱乙酰酶的重组表达及固定化

英文题名：Expression and immobilization of a recombinant cephalosporin-C deacetylase

作者：王群[1];魏东芝[1];沈亚领[1];陈鑫[2];金雄熊[2];叶凤起[2]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]浙江永宁药业股份有限公司,浙江台州318020

年份：2014

卷号：44

期号：4

起止页码：66

中文期刊名：工业微生物

外文期刊名：Industrial Microbiology

收录：CSCD:【CSCD_E2013_2014】；

基金：上海市重点学科建设项目资助(B505);国家重点实验室专项资金资助(2060204)

语种：中文

中文关键词：头孢菌素脱乙酰酶;生物催化;固定化;重组大肠杆菌;7-ACA

外文关键词：CAH; biocatalysts ; immobilized enzyme; recombinant E. coli; 7-ACA

摘要：本文构建了利用trp启动子表达头孢菌素脱乙酰酶(CAH)的重组大肠杆菌DH5α-pCAH。重组菌在7L发酵罐(装液量2L)中发酵28 h,发酵液OD_(600)达到27,产酶313 kU/L发酵液,粗略估算重组蛋白占细胞总蛋白的70%。发酵生产的重组CAH粗酶液经过硫酸铵分级沉淀分离纯化和超滤除盐浓缩两步操作,纯化倍数为1.44,总酶活回收率56%,聚丙烯酰胺凝胶电泳检测纯化后蛋白没有明显杂蛋白条带出现。纯化后的CAH共价结合固定在环氧基载体LX-1000EP(c)上,通过对固定化条件的优化最终得到固定化酶比活443 U/g。该固定化酶重复催化50 mL 5%7-ACA底物100次后,酶活没有降低。
Abstract In this work, a recombinant E. coli DHSct-pCAH, with trp promoter expressing CAH, was constructed. After 28 h fermentation of 2 L medium in 7 L-fermentor at 37℃, the recombinant E. coli produced 313 kU per liter and its OD600 reaehed to 27. The amount of CAH was approximately 70% of the total cellular protein. The recombinant CAH crude extract was purified by ammonium sulphate precipitation and ultrafiltration to homogeneity in SDS-PAGE with a yield of 56% and purification factor of 1.44. The purified CAH was immobilized on the epoxy carrier LX-1000EP（c） covalently. After optimizing the immobilizing conditions, the activity of the immobilized CAH reached to 443 U/g and the activity yield was 27% of free enzyme. The immobilized CAH activity was not reduced after repeated use for 100 times in 50 mL 5% 7 -ACA. Key words CAH; biocatalysts ; immobilized enzyme; recombinant E. coli; 7-ACA