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

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<0.0...

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

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.