一个外切菊粉酶基因的克隆表达及酶学性质

从马克斯克鲁维酵母（Kluyveromycesmarxianus）DSM5418中克隆出外切菊粉酶（INU）的成熟肽编码区域，在毕赤酵母（Pichiapastoris）GS115中实现了高效分泌表达，体积酶活力达到15．27U／mL，进一步对重组酶进行了纯化与表征。经过（NH4）2SO4沉淀、透析和分子筛过滤后，得到了纯度大于95％的纯化重组酶，SDS-PAGE分析发现INU的表观相对分子质量为9．0×10^4，大于理论预测值6．0×10^4。纯化酶液的表征结果表明，INU的最适温度和最适pH分别为55℃和5．0，在此条件下INU对菊粉的K。值和比酶活分别为1．90mmol／L和433．86U／mg，对蔗糖的K。值和比酶活分别为27．81mmol/L和1249．49U／mg，I/S值为0．34；HPLC分析表明，INU酶解菊粉的产物由果糖和葡萄糖组成；金属离子Mn2＋、Fe3｜、K｜和Co2＋对酶有促进作用，而Zn2＋、Cu2＋、Ni2＋、SDS和EDTA对酶活力有不同程度的抑制作用。

Cloning, expression and characterization of exo-inulinase from Kluyveromyces marxianus DSM 5418

The mature peptide gene of exo-inulinase from Kluyveromyces marxianus DSM 5418 was cloned into the plasmid pPIC9K ,which was further expressed efficiently, by secretory expressed in Pichia pastoris GS115 and maximum activity reached 15.27 U/mL. The recombinant inulinase was purified to be more than 95% purity by ammonium sulfate precipitation, dialysis and sieve chromatography. The apparent molecular weight of INU was 9.0 ×10^4 by SDS-PAGE ,and was bigger than the theoretical value of 6. 0 ×10^4. The enzymatic characteristics of INU were as follows：the optimum temperature was 55 ℃ and the optimum pH was pH 5.0, the apparent Km and specific activity values for inulin were 1.90 mmol/L and433.86 U/mg,and 27.81 mmol/L and 1 249.49 U/rag for sucrose,the metal ions including Mn2＋ ,Fe3＋ , K ＋ , and Co2 ＋ activated the inulinase activity, but it was inhibited by Zn2 ＋ , Cu2 ＋ , Ni2 ＋ , SDS, and EDTA. The enzymatic hydrolysis product of inulin by INU consisted of glucose and fructose, which was consistent with the I/S value of 0. 34 of INU.