产β-葡萄糖苷酶真菌的筛选鉴定、纯化及酶学性质分析  

Screening and Identification of β-Glucosidase-Producing Fungi,and Purification and Enzymatic Analysis

作　　者：陈静[1] 郝伟伟 王春梅[1] 陈惠[1] 吴琦[1] 韩学易[1] 

机构地区：[1]四川农业大学生命科学与理学院,四川雅安625014 [2]四川省华派生物制药有限公司,四川简阳641401

出　　处：《食品科学》2013年第5期191-196,共6页Food Science

摘　　要：经刚果红平板染色法从土样中筛选到3株产纤维素酶的真菌,通过对其β-葡萄糖苷酶活力的测定,选择一株相对活力较高的菌株进行菌种鉴定。在形态鉴定的基础上,通过内转录间隔区(ITS)序列构建系统发育树初步鉴定为米曲霉(Aspergillus oryzae),命名为giF-10。对米曲霉giF-10进行摇瓶发酵,经硫酸铵沉淀、Sephadex G-100凝胶层析、DEAE Cellulose 52离子交换层析进行纯化。得到纯化后的β-葡萄糖苷酶,比活力为40.84U/mg,分子质量约90kD;该β-葡萄糖苷酶最适温度为55℃;在30～50℃之间热稳定性好;最适pH值为4.5;pH4.0～6.0稳定性好;金属离子对酶活力具有一定的影响,Mn2+对酶具有较强的激活作用;Fe3+和Cu2+对β-葡萄糖苷酶活力有较强的抑制作用;该酶对水杨素和纤维二糖具有较强的底物特异性;β-葡萄糖苷酶对水杨素的动力学参数:Km为0.676mmol/L;对纤维二糖的动力学参数为:Km为2.906mmol/L。Three cellulase-producing strains were screened from the soil sample and identified by Congo red method.A strain with the most powerful capability of producing β-glucosidase was also explored.The strain was identified as Aspergillus oryzae by morphological and ITS sequence analysis,and named as giF-10.Aspergillus oryzae giF-10 was fermented in shaking flask to obtain a great deal of β-glucosidase.Crude enzyme was purified by ammonium sulfate precipitation,Sephadex G-100 gel chromatography,and DEAE cellulose ion exchange chromatography.The specific activity of purified β-glucosidase was 40.84 U/mg.Molecular weight of β-glucosidase was approximately 90 kD as identified by SDS-PAGE.Its optimal temperature and pH were 55 ℃ and 4.5,respectively.It was stable at 30—50 ℃ and pH 4.0—6.0.Different metal ions revealed different effects on β-glucosidase activity.Mn2＋ could activate β-glucosidase while Fe3＋ and Cu2＋ could inhibit β-glucosidase.The enzyme showed stronger substrate specificity to salicin and cellobiose.Its Km for salicin and cellobiose were 0.676 mmol/L and 2.906 mmol/L,respectively.

关 键 词：米曲霉 Β-葡萄糖苷酶 纯化 酶学性质 

分 类 号：Q814[生物学—生物工程]