乳糖诱导纤维二糖差向异构酶的表达及热处理纯化

摘要

The feasibility of using lactose to replace IPTG as the inducer for expression of CsCE in E.coli BL21 (DE3) was investigated.The induction conditions for CsCE expression were optimized.Final concentration of 10 g/L lactose was added into broth when the cell growth reached OD600 =0.6.After incubation at 25 ℃C for another 20 h,the maximum CsCE activity was approximately 801 U/L,the total activity and the specific activity were 1.4 times and 37％ higher than those induced by IPTG,respectively.In addition,thermal-purification of CsCE was carried out.The results showed that approximately 85.9％ of the soluble protein was removed due to denaturation.The activity recovery was 86.6％ with a specific activity of 5.46 U/mg and a purifying fold of 6.14.The experimental results presented a promising technology to attain high CsCE production on industrial scale.%以CaldiceUulosiruptor saccharolyticus纤维二糖差向异构酶(CsCE)高效表达菌株E.coli BL21(DE3)为表达载体,研究乳糖替代异丙基-β-D-硫代半乳糖苷(IPTG)诱导CsCE表达的效果.结果表明,在菌体培养至OD600=0.6时加入终浓度为10 g/L的乳糖,25℃下诱导20 h,最终发酵液中CsCE酶活达801 U/L,较最优条件下IPTG诱导的酶活力提高1.4倍,粗酶液的比酶活提高37％.然后,根据CsCE的热稳定性,采用热处理工艺对粗酶液进行纯化.经70 ℃、2h热处理,绝大部分的杂蛋白变性沉淀,粗酶液中可溶性蛋白浓度降低85.9％,比酶活由0.89 U/mg提高到5.46 U/mg,纯化倍数达到6.14倍,酶活回收率为86.6％.乳糖诱导CsCE的高效表达结合热处理的初步纯化工艺,为CsCE的工业化生产提供有益的借鉴.