重组蔗糖磷酸化酶表达培养基的优化及化学试剂对酶活的影响

张蕙; 周晓梅; 张彪; 孙晓; 李文杰; 李拖平

doi:10.13386/j.issn1002-0306.2020110137

重组蔗糖磷酸化酶表达培养基的优化及化学试剂对酶活的影响

Optimization of Medium Composition for Expression of Recombinant Sucrose Phosphorylase and Effect of Chemical Reagents on Enzyme Activity

摘要

摘要: 为了提高重组蔗糖磷酸化酶的表达水平，本研究通过单因素实验，研究了不同的碳源、氮源、稳定剂和金属离子对重组蔗糖磷酸化酶在大肠杆菌中表达的影响，并通过正交试验确定了最优的重组蔗糖磷酸化酶表达培养基的成分。另外，本研究也考察了不同的化学试剂和金属离子对重组蔗糖磷酸化酶的活性影响。结果表明，维生素C和FeCl₂显著（P<0.01）抑制了蛋白的表达，Cu²⁺和Zn²⁺完全抑制了菌体的生长，而淀粉、K⁺和Mg²⁺对重组蔗糖磷酸化酶的表达具有一定的促进作用，且三者的协同作用可以极大的提高重组蔗糖磷酸化酶的表达效率。其中在50 mL LB培养基中加入0.5%淀粉、0.1% K⁺和0.05% Mg²⁺的组合，总酶活达1207.83 U，比优化前提高了3.6倍。，Fe²⁺（10 mmol/L）、Mg²⁺（10 mmol/L）、牛血清蛋白（1%）和土温80（0.1%）均可在一定程度上提高该酶的活性。

Abstract: In order to improve the expression level of recombinant sucrose phosphorylase, the effects of different carbon sources, nitrogen sources, protectants and metal ions on the expression of recombinant sucrose phosphorylase in Escherichia coli was studied by single factor test, then the optimal composition of recombinant sucrose phosphorylase expression medium was determined by orthogonal test. In addition, the effects of different chemical reagents and metal ions on the stability of recombinant sucrose phosphorylase were investigated.The results showed that vitamin C and FeCl₂ significantly (P<0.01) inhibited the protein expression, and Cu²⁺ and Zn²⁺ completely killed the microorganisms. On the contrary, starch, K⁺ and Mg²⁺ could promote the expression of recombinant sucrose phosphorylase, and the synergistic effect could greatly improve the expression efficiency of recombinant sucrose phosphorylase. The total enzyme activity of optimal combination, with 0.5% starch, 0.1% K⁺ and 0.05% Mg²⁺ in 50 mL LB medium, was 1207.83 U, which was increased by 3.6 -fold. The results showed that 10 mmol/L Fe²⁺, 10 mmol/L Mg²⁺, 1% bovine serum protein and 0.1% Tween 80 could increase the enzyme activity and stability of sucrose phosphorylase in a certain extent.