重组β-1,3-1,4-葡聚糖酶的纯化及部分酶学性质研究

Study on the Purification and Some Enzymological Properties of Recombinant β-1,3-1,4-glucanase

[目的]研究重组β-1,3-1,4-葡聚糖酶的性质。[方法]由重组大肠杆菌制备重组β-1,3-1,4-葡聚糖酶发酵液,经乙醇分级沉淀和DEAE-650M离子交换层析分离纯化后进行SDS-PAGE纯度鉴定,最后分析纯化后重组酶的酶学性质。[结果]SDS-PAGE分析表明纯化后得到了较纯的重组β-1,3-1,4-葡聚糖酶,比活力达13 437 U/mg,纯化倍数为19.85倍,回收率为20.60%,分子量约30 kDa。纯化后重组酶的最适pH值为6.0,pH值4.5～6.0较稳定;最适温度为50℃,40～50℃较稳定;Cu2＋和Fe2＋对其活力有显著的抑制作用,Mg2＋、Zn2＋和Mn2＋对其活力有抑制作用,比较适合在啤酒酿造上使用。[结论]该研究为重组β-1,3-1,4-葡聚糖酶的工业化应用奠定了基础。

[Objective] The purpose was to study the properties of recombinant β-1,3-1,4-glucanase.[Method] The ferment broth of recombinant β-1,3-1,4-glucanase was prepared from recombinant Escherichia coli.SDS-PAGE purity identification was conducted after the recombinant β-1,3-1,4-glucanase was isolated and purified by ethanol precipitation and DEAE-650M ion-exchange chromatography.Finally,the enzymological properties of purified recombinant enzyme were analyzed.[Result] SDS-PAGE analysis showed that the fairly pure recombinant β-1,3-1,4-glucanase was obtained after purification,with specific activity of 13 437 U/mg,purification fold of 19.85,recovery of 20.60% and molecular weight of 30 kDa.The optimum pH value of purified recombinant enzyme was 6.0 and it was relatively stable in pH of 4.5～6.0;its optimum temperature was 50 ℃ and it was relatively stable in 40～50 ℃.Cu2＋ and Fe2＋ had significant inhibition effect and Mg2＋,Zn2＋ and Mn2＋ had inhibition effect on its activity.It was comparatively suitable for brewing beer.[Conclusion] The study laid a foundation for the industrial application of recombinant β-1,3-1,4-glucanase.