一株β-葡萄糖苷酶产生菌株的分离鉴定及酶学性质研究  

Isolation and identification of a β-glucosidase-producing strain and enzymatic characteristics of the β-glucosidase

作　　者：郑芳[1] 曹小芳[1] 张亚玲[1] 白芳[1] 白钢[1] 

机构地区：[1]南开大学药学院天津市分子药物研究重点实验室,天津300071

出　　处：《微生物学通报》2012年第8期1059-1068,共10页Microbiology China

基　　金：国家自然科学基金项目(No.21002052);天津市应用基础及前沿技术研究计划项目(No.10JCYBJC14300)

摘　　要：【目的】分离获得β-葡萄糖苷酶高产菌株,确定该菌分类地位,并对其所产β-葡萄糖苷酶的酶学性质进行初步研究。【方法】采用七叶灵显色法从土壤样品中筛选β-葡萄糖苷酶产生菌,再用对硝基苯基-β-D-吡喃葡萄糖苷(PNPG)显色法进行复筛;通过形态特征、生理生化特征及16S rDNA序列相似性分析等方法确定其分类学地位;利用超滤、疏水层析、阴离子层析、分子筛层析法对β-葡萄糖苷酶进行分离纯化;以PNPG为底物,测定β-葡萄糖苷酶的最适反应pH及最适反应温度,通过双倒数作图法确定β-葡萄糖苷酶催化不同底物水解的米氏常数Km值。【结果】从土壤样品中筛选得到一株β-葡萄糖苷酶高产菌株ZF-6C,初步鉴定为Bacillus korlensis;芽胞杆菌ZF-6C所产β-葡萄糖苷酶的分子量约为90 kD,最适反应pH和温度分别为7.0和40°C,该酶具有水解β(1,4)糖苷键的活性,最适底物为邻硝基苯-β-D-吡喃葡萄糖苷,Km值为0.73 mmol/L。金属离子Ca2+、Pb2+增强酶活,而Cu2+、Fe2+抑制酶活。【结论】首次报道从Bacillus korlensis中分离得到β-葡萄糖苷酶,Bacillus korlensis ZF-6C所产β-葡萄糖苷酶在分子量、最适反应条件及底物特异性等方面均不同于已知酶,可能为一结构新颖且催化效率较高的β-葡萄糖苷酶。[Objective] To isolate a novel β-glucosidase-producing strain from soil, to clarify the taxonomic status, and to study the enzymatic characteristics of the 13-glucosidase. [Meth- ods] A β-glucosidase-producing strain was isolated from soil sample using esculin and 4-nitrophenyl-β-D-glucopyranoside （PNPG） coloration methods; the taxonomic status of strain ZF-6C was clarified by morphological, physiological, chemotaxonomic characteristics and 16S rDNA analysis; the [3-glucosidase was isolated and purified by ultra-filtration, hydrophobic interaction chromatography, anion chromatography, molecular sieve chromatography; with PNPG as the substrate, the optimal reaction pH and temperature of β-glucosidase were determined, the Michaelis constant Km of β-glucosidase against different substrates were determined by Lineweaver-Burk plot. [Results] A strain, which produced β-glucosidase at high level, was isolated from soil and identified as Bacillus korlensis, the molecular weight of β-glucosidase isolated from Bacillus ZF-6C was 90 kD, the optimum reaction conditions for this enzyme were pH 7.0 and 40℃. The enzyme was active against a wide range of β （1,4） linked disaccharides, the optimal substrate was o-nitrophenyl-β-D-glucopyranoside, the Km value was 0.73 mmol/L. Metal ions Ca2＋ and Pb2＋ enhanced enzyme activity, Cu2＋ and Fe2＋ inhibit the enzyme activity. [Conclusion] This is the first report of isolation and identification of β-glucosidase from Bacillus korlensis. The β-glucosidase from Bacillus korlensis is widely different to known enzymes at molecular weight, optimum reaction conditions and substrate specificity aspects. This β-glucosidase may have novel structure and high catalytic efficiency.

关 键 词：芽胞杆菌 分离鉴定 Β-葡萄糖苷酶 酶学性质 

分 类 号：R587.1[医药卫生—内分泌]