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【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】 The β-glucosidase-producing strain was isolated and its taxonomic status was determined. The enzymatic properties of its β-glucosidase were also studied. 【Method】 The β-glucosidase-producing bacteria were screened from soil samples by the method of Qiuye Ling, and then screened by p-nitrophenyl-β-D-glucopyranoside (PNPG) , Physiological and biochemical characteristics and 16S rDNA sequence similarity analysis to determine its taxonomic status; using ultrafiltration, hydrophobic chromatography, anion chromatography, molecular sieve chromatography of β-glucosidase isolated and purified; PNPG as a substrate The optimal reaction pH of β-glucosidase and the optimum reaction temperature were determined. The Km value of β-glucosidase catalyzed hydrolysis of different substrates was determined by double-reciprocal mapping. 【Result】 A strain of β-glucosidase high producing strain ZF-6C was screened from soil samples and initially identified as Bacillus korlensis. The molecular weight of β-glucosidase produced by Bacillus sp. ZF-6C was about 90 kD and the optimum reaction pH and temperature were 7.0 and 40 ° C, respectively. The enzyme had the activity of hydrolyzing β (1,4) glucosidic bond. The optimum substrate was o-nitrophenyl-β-D-glucopyranoside with a Km value of 0.73 mmol / L. Metal ions Ca2 +, Pb2 + enhance enzyme activity, while Cu2 +, Fe2 + inhibit enzyme activity. 【Conclusion】 For the first time, β-glucosidase was isolated from Bacillus korlensis. The β-glucosidase produced by Bacillus korlensis ZF-6C was different from known enzymes in molecular weight, optimum reaction conditions and substrate specificity, May be a new structure and high catalytic efficiency of β-glucosidase.