嗜热子囊菌光孢变种Thermoascus aurantiacus var.levisporusβ-葡萄糖苷酶的分离纯化及特性研究  

Purification and Properties of β-glucosidase from Thermoascus aurantiacus var.levisporus

作　　者：王冬梅[1] 李多川[1] 孟军 

机构地区：[1]山东农业大学环境生物系,山东泰安271018 [2]山东省莱芜市畜牧局

出　　处：《农业环境科学学报》2005年第5期1007-1012,共6页Journal of Agro-Environment Science

基　　金：国家863计划资助项目(2003AA241161);国家自然科学基金资助项目(3017001330270013)

摘　　要：采用室内培养、测定方法,对嗜热子囊菌光孢变种Thermoascus aurantiacus var.levisporus产生的β-葡萄糖苷酶进行了分离纯化及特性研究。粗酶液经硫酸铵沉淀、DEAE-SepharoseFastFlow阴离子层析、Phenyl-Sepharose疏水层析等步骤获得了凝胶电泳均一的β-葡萄糖苷酶。结果表明,经12%SDS-PAGE测得酶的单亚基分子量约为118kDa,凝胶过滤层析测得酶的分子量约为350kDa。该酶反应的最适温度和最适pH分别为80℃和4.5￣5.0,在pH5.0条件下,该酶在70℃条件下基本稳定,80℃保温30min,剩余酶活为15%。金属离子对β-葡萄糖苷酶活性影响较大,其中Ca2+、Ba2+对酶有激活作用;Ag+、Fe3+、Cu2+对酶有显著的抑制作用。该酶对水杨苷具有很强的底物特异性。Cellulose is the most abundant and renewable source of energy on Earth. It is an insoluble polysaccharide composed of long, linear chains of β-1,4-1inked glucose units. Cellulase system is a series of enzymes that can break down cellulose in the natural circumstance and make little pollution to the environment. The enzymes can be divided into three types： endo-β-1,4-glucanase （EC3.2.1.4）, exo-β-1,4-glucanase （EC3.2.1.91） and β-glucosidase （EC3.2.1.21）. β-Glucosidase completes the hydrolysis by converting cellobiose and cellooligosaccharides into glucose. It also stimulates the rate and extent of cellulose hydrolysis by relieving cellobiose-induced inhibition of endo-and exo-glucanases. Thermoascus aurantiacus var. levisporus Upadhyay, Farmelo, Goetz ＆ Melan is a new record species of thermophilic fungi isolated from Yunnan Province, grows well at 45℃-50℃, and the cellulase system produced by it keep higher activities. By using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography, Phenyl-Sepharose Fast Flow chromatography and SephacrylS-100 chromatography, an extracellular β-glucosidase from culture supematant of Thermoascus aurantiacus var. levisporus was purified, and its properties and substrate specificities were studied. The single subunit molecular weight and molecular weight were about 118000 and 350000, which was identified by 12% SDS-PAGE and gel filtration respectively.. The β-glucosidase was a homotrimer and optimally active at pH 4.5-5.0 and 80℃, showed almost thermostable at 70℃, and kept 15% of its optimal activity after 30 min at 80℃.It was stable at pH5.0, but the stability decreased at pH values above and below 5.0. Different metal ions had different effects on the activity of β-glucosidase, Ca^2＋ and Ba^2＋ enhanced the activity, whereas Ag^＋, Fe^3＋ and Cu^2＋ inhibited it. The enzyme acted on salicin specially.

关 键 词：嗜热子囊菌光孢变种 Thermoascus aurantiacus var.levisporus β-区区糖苷酶 纯化 特性 

分 类 号：Q936[生物学—微生物学]