一株β-葡萄糖苷酶产生菌株的分离鉴定及酶学性质研究

【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。

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

Objective] To isolate a novel β-glucosidase-producing strain from soil,to clarify the taxonomic status,and to study the enzymatic characteristics of the β-glucosidase.Me-thods] 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 β-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 °C.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.