化学抑制剂Woodward's Reagent K对来源于Erwinia rhapontici NX-5蔗糖异构酶的抑制动力学

Inhibition kinetics of sucrose isomerase from Erwinia rhapontici NX-5 by Woodward's Reagent K

从重组大肠杆菌E.coli BL21(p ET22b-palⅠ)中纯化得到来源于Erwinia rhapontici NX-5的蔗糖异构酶(sucrose isomerase,SIase,EC 5.4.99.11),以纯酶为对象考察其酶活力抑制动力学。结果表明:SIase纯比酶活1 512.77 U/mg,动力学常数Km=260 mmol/L,Vmax=39.41μmol/(L·s)。以化学抑制剂Woodward's Reagent K(WRK)对重组蔗糖异构酶进行抑制反应,反应体系中随着WRK浓度的升高,SIase与底物蔗糖的亲和力常数Km增大,最大反应速度Vmax在一定范围内保持稳定。通过对SIase的抑制动力学分析可得到,WRK对SIase的抑制类型为可逆的竞争性抑制,这可能与WRK与蔗糖的结构类似,与可竞争性的结合SIase的活性中心有关。

Sucrose isomerase （ SIase,EC5.4.99.11） from Erwinia rhapontici NX-5 was purified from the extract of recombinant E-coli BL21 （ pET22b-palⅠ） culture,and the inhibition kinetics of the pure SIase was studied with chemical inhibitor Woodward′s Reagent K（ WRK）-Results show that SIase had the high specific activity of 1 512-77 U/mg,as well as the Michaelis-Menten constants of Km=260 mmol/L and Vmax=39-41 μmol/（ L＆#183;s）-Km increased as the concentration of inhibitor increased,but Vmax kept stable within limits-The inhibition of SIase by WRK was reversible and competitive, probably caused by the similar structure of WRK and sucrose.