克鲁维酵母Y-85菊粉酶的纯化和性质

克鲁维酵母(Kluyveromyces sp.)Y-85产生的胞内菊粉酶(endocellular inulinase)和胞外菊粉酶(exocellular inulinase)粗酶液分别经PEG6000-磷酸盐缓冲液双水相抽提得部分纯化酶液。前者进一步用硫酸铵分级沉淀、Protein-PAK DEAE离子交换、Protein-PAK200SW凝胶过滤后得到两个菊粉酶组分EⅠ和EⅡ;后者采用DEAE-Sephacel离子交换、Sephadex G150凝胶过滤后得到菊粉酶Eexo。经Waters 650E蛋白纯化系统鉴定,三者均呈单一的对称峰;EⅠ和EⅡ达聚丙烯酰胺盘状凝胶电泳纯。EⅠ、EⅡ和Eexo的分子量分别为42kD、65kD和57kD;三者均为糖蛋白,多糖含量分别为30％、35％和25％;I/S(Inulinaseactivity/Sucrase activity)比值分别为0.086、0.078和0.072;三者均属外切菊粉酶。EⅠ、EⅡ和Eexo酶反应最适pH分别为4.6、4.5和4.6,最适温度分别为52℃、52℃和55℃;Ag^+、Hg^(2+)和PCMB对酶活性有强烈的抑制作用;三者水解菊芋粉糖液的产物均为果糖(86.5％)和葡萄糖(13.5％)。

PURIFICATION AND PROPERTIES OF INULINASE FROM KLUYVEROMYCES SP. Y-85

The crude endocellular inulinase from Kluyveromyces sp. Y-85 was purified to two components, designated as EI and EII, using PEG6000-phosphate buffer extraction, (NH4)2SO4 fractionation, DEAE chromatography and gel filtration (Protein-PAK); The crude exocellular inulinase from this strain was purified to Eexo by means of PEG6000-phosphate buffer extraction, double DEAE-Sephace chromatography, Sephadex G-150 gel filtration. EI, EII and Eexo were demonstrated to be homogeneous by Waters 650E protein purification system. Their molecular weights are 42 kD, 65 kD and 57 kD, respectively. All the inulinases were glycoproteins containing a saccharide (from 25% to 35%) and belonged to the endo-inulinase. In addition, EI, EII, Eexo were optimally reactive at pH4.6, 4.5, 4.6 and at 52 degrees C, 52 degrees C, 55 degrees C, respectively. Ag+, Hg2+ and PCMB inhibited these enzymes' activity strongly. The products of raw inulin extracted from Helianthus tuberosus hydrolyzed by these three enzymes were fructose (86.5%) and glycose (13.5%).