乳糖诱导纤维二糖差向异构酶的表达及热处理纯化

Expression and thermal-purification of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus induced by lactose

以Caldicellulosiruptor saccharolyticus纤维二糖差向异构酶(Cs CE)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Cs CE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Cs CE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Cs CE的热稳定性,采用热处理工艺对粗酶液进行纯化。经70℃、2 h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Cs CE的高效表达结合热处理的初步纯化工艺,为Cs CE的工业化生产提供有益的借鉴。

The feasibility of using lactose to replace IPTG as the inducer for expression of Cs CE in E. coli BL21( DE3) was investigated. The induction conditions for Cs CE 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 ℃ for another 20 h,the maximum Cs CE activity was approximately 801 U / L,the total activity and the specific activity were 1. 4 times and37% higher than those induced by IPTG,respectively. In addition,thermal-purification of Cs CE 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 Cs CE production on industrial scale.