摘要
对重组细胞色素P450 BM-3基因工程菌的培养条件进行了单因素优化,确定了较佳的P450BM-3培养和表达条件:接种量1%,装液量100 mL/500 mL,发酵培养基加入0.1 mg?L-1FeCl3,OD578达到1.0左右时升温至42℃诱导,诱导时间为5 h.考察了DEAE-Sepharose FF与Resource Q 2种阴离子介质对分离纯化P450 BM-3的影响.结果表明,Resource Q纯化效果优于DEAE-Sepharose FF,通过KTA explorer100对Resource Q分离条件进行了优化,0.3 mol?L-1、0.5 mol?L-1的两步NaCl阶跃洗脱,P450 BM-3纯度较高,活性收率为30.1%,纯化倍数为2.12.
The optimum culture conditions of P450 BM-3 expression by recombinant Escherchia coli DH 5a were studied in detail. Using inoculum quantity corresponding to 1% of the culture medium, adding of 0. lmg·L^-1 FeC13 of the culture solution and rapid temperature shift from 37 ℃ to 42 ℃ as soon as OD578 reached about 1.0 and maintained for 5 hours, were proved to be the favorable conditions for growth of the bacteria and expression of the gene coding for P450 BM-3 monooxygenase. Purification of P450 BM-3 was investigated by two anion-exchange media DEAE-Sepharose FF, Resource Q. The results indicated that purification effectiveness of DEAE-Sepharose FF was inferior to that of Resource Q. The parameter of purification by Resource Q was optimized on AKTA explorer 100 system. By optimising the elution condition of P450 BM-3 , the results showed that the purification factor was 2.12 and P450 BM-3 activity recovery was 30.1% through 0.3 mol·L^-1, 0.5 mol·L^-1 two-step NaCl elution.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2006年第1期96-100,共5页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
国家自然科学基金资助项目(30570411)
浙江省回国人员基金资助项目
浙江省科技计划资助项目
中德合作DAAD-CSC科技人员交流资助项目
