β-葡萄糖苷酶的分离纯化和性质研究  

Purification and Properties of β glucosidase from Aspergillus niger L 22

作　　者：孙迎庆[1] 曹淑桂[1] 韩四平[1] 

机构地区：[1]吉林大学酶工程国家重点实验室

出　　处：《中国生物化学与分子生物学报》1998年第1期82-82,共1页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：β－葡萄糖苷酶是纤维素酶的重要组分之一，它不仅可水解纤维二糖和寡糖，更可解除纤维二糖对β－１，４－内切葡聚糖酶和外切葡聚糖酶的抑制，提高水解速率和程度．利用ＳｅｐｈａｄｅｘＧ－１５０和ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０层析法从黑曲霉变异株Ｌ－２２中分离提纯了β－葡萄糖苷酶，该酶是由两个分子量相同的亚基组成的二聚体，每个亚基分子量为２０３ｋＤ．该酶最适ｐＨ为４．８，ｐＨ稳定范围在３．６～６．４；最适温度是６０℃，温度稳定范围为４～６０℃；酶分子含糖量为８．３５％．它是一个酸性β－葡萄糖苷水解酶，专一性地水解β－糖苷键．而不水解α－糖苷键，对短链底物表现了相对高的活力．用动力学分析和共价化学修饰方法探讨了与该酶活力有关的必需基团．由ｐＨ对ｌｇＶｍ和ｌｇＶｍ／Ｋｍ的影响，推测出酶活性部位至少有两个可解离基团为酶活性所必需，它们在酶－底物复合物中的ｐＫｅｓ１和ｐＫｅｓ２的值分别为４．０和５．６，在游离酶中的ｐＫ值分别为４．２和５．９．由此可初步判断这两个可解离基团可能为组氨酸和含羧基的氨基酸，它们与酶的催化和底物结合可能有关．β glucosidase is an important component of the cellulase complex.It not only hydrolyzes oligosaccharides to glucose cellobiose,but also removes the inhibitory effect of cellobiose on the β 1,4 endoglucanase and exoglucanase,thereby increaing the overall rate of cellulose biodegradation.a β glucosidase from Aspergillus niger L 22 was purified by Sephadex G 150 and DEAE Sephadex A 50 chromatography,and their properties and substrate specificites were studied.The enzyme is composed of two duplicate subunits,and with the same molecular mass of 203 kD;it is glycoprotein with 8.53% carbohydrates.Its optimum pH value is 4.8,and it is stable at pH3.6～6.4.Its optimum temperature is 60℃.and it is stable at 4～60℃.The enzyme is an acidic β glucosyl hydrolase,and it specifically hydrolyzes β glucosidic bonds rather than α glucosidic bonds.The ability to hydrolyze shorter oligosaccharide is relatively higher than that to hydrolyze longer oligosaccharides.Both kinetic and chemical modification studies provided experimental evidence for the essential groups of the enzyme.The pH dependence of V max and V max / K m with salicin as substrate indicated that the catalytic activity depended on the ionization of at least two essential groups at the active site of the enzyme.The pH profile showed apparent p K values of 4.0 and 5.6 for the enzyme substrate complex and 4.2 and 5.9 for the free enzyme.The two ionazable may be histidine and amino acid residue containing the carboxyl group.

关 键 词：Β-葡萄糖苷酶 分离 纯化 动力学 

分 类 号：Q556.2[生物学—生物化学]