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摘 要：本文是关于重组马克斯克鲁维酵母产内切葡聚糖酶的纯化和酶学性质研究。实验将来源于里氏木霉的内切葡聚糖酶基因endoglucanaseII(egII)在马克斯克鲁维酵母中实现异源表达，然后将构建完成的重组马克斯克鲁维酵母接种在含有5mmol/L的乙酰胺的培养基上在30℃、150rpm发酵培养72h，制备重组内切葡聚糖酶发酵液，粗酶发酵液纯化需经过透析浓缩、Ni柱分离以及SDS-PAGE，SDS-PAGE以及酶谱分析得到酶的分子量为51kDa，酶的比活力达到465.868U/mg，纯化倍数为2.6，回收率为36.9%。而酶学性质研究表明：该酶最适反应pH为4.8，最适反应温度为55℃,Mg2+、Fe2+、Ca2+ 和Zn2+等离子对该酶催化反应有激活作用，Hg2+、K+ 、Cu2+、Co2+、Ag+等离子可抑制该酶的活力。该酶以2%CMC为底物时其反应米氏常数为7.83mg/ml。70568
毕业论文关 键 词：重组马克斯克鲁维酵母，内切葡聚糖酶，纯化，酶学性质
Abstract: This article is about the purification and characterization of recombinant Kluyveromyces Marx which produced endoglucanase II .In this study, endoglucanaseII from Trichoderma reesei (egII) would achieve heterologous expression in Kluyveromyces marxianus ,then the constructed recombinant Kluyveromyces marxianus would be cultured in the fermentation medium(containing 5mmol/L acetamide) at 30℃ with agitation speed of 150rpm for 72h,to prepare recombinant endoglucanase fermentation broth.Next,the crude enzyme was purified by concentration of dialysis,Ni column and SDS-PAGE.SDS-PAGE and zymogram assay revealed endoglucanaseII egII with the molecular weight of 51000.A purified endoglucanaseIIwas achieved with purification fold of 2.6 and a recovery rate of 36.9%,and the specific activity of the purified enzyme was 465.868U/mg .The optimal values of pH and temperture on purified cellulase response were found to be 4.8 and 55℃,respectively.The cellulase can be activated by Mg2+、Fe2+、Ca2+ and Zn2+,while inhinbited by Hg2+、K+ 、Cu2+、Co2+、Ag+.The enzyme with 2%CMC as substrate for the reaction of Michaelis
sustained 7.83mg/ml .
Keywords：recombinant Kluyveromyces marxianus，endoglucanase，purification，enzymologlcal properties
目 录
1 前言 4
2 材料与方法 5
2.1 材料 5
2.1.1 主要仪器和设备 5
2.1.2 菌种 5
2.1.3 培养基的配制 5
2.2 方法 6
2.2 .1 重组马克斯克鲁维酵母诱导产内切葡聚糖酶 6
2.2.2 重组内切葡聚糖酶酶活力测定（粗酶活） 6
2.2.3 总蛋白含量的测定 8
2.2.4 重组内切葡聚糖酶发酵液浓缩 9
2.2.5 Ni亲和层析（Ni Sepharose 6 Fast Flow，GE，USA） 10
2.2.6 不连续SDS聚丙烯酰胺凝胶电泳（SDS-PAGE） 11
2.2.7 纯化重组内切葡聚糖酶最适反应温度和温度稳定性 12
2.2.8 重组内切葡聚糖酶最适反应pH和pH稳定性 12
2.2.9 金属离子对重组内切葡聚糖酶活性的影响 13
2.2.10 动力学常数Km和最大反应速度Vmax的测定