烟曲霉β-葡萄糖苷酶的基因克隆、表达及酶学性质分析  

Cloning, expression and characterization of β-glucosidase from Aspergillus fumigatus

作　　者：谢贻[1] 欧阳浩淼[1] 黄日波[2] 陈东[2] 金城[1] 

机构地区：[1]中国科学院微生物研究所真菌学国家重点实验室,北京100101 [2]广西科学院非粮生物质酶解国家重点实验室国家非粮生物质能源工程技术研究中心广西生物炼制重点实验室,广西南宁530007

出　　处：《生物工程学报》2013年第9期1245-1253,共9页Chinese Journal of Biotechnology

基　　金：中国科学院院地合作项目资助~~

摘　　要：探索获得优良的β-葡萄糖苷酶基因，对实现其工业化生产具有重要意义。烟曲霉Aspergillusfumigatus基因组中含有一个堙，基因（1752bp），编码的蛋白约65kDa，推测为属于糖苷水解酶家族的β-葡萄糖苷酶。将bgl基因克隆并构建了重组表达载体pGEX．bgl，转化大肠杆菌EscherichiacoliBL21（DE3），经IPTG诱导获得表达。重组蛋白经亲和层析纯化后，以七叶苷为底物进行了酶学分析，结果表明该酶的最适温度是45℃，最适pH在5．5～6．0之间，对七叶苷的‰值为17．7mmol／L。该酶在pH4～7范围内稳定；70℃保温2h后仍能保持60％的活性。金属离子和化学试剂对酶活性有不同程度的影响，Ca计对重组酶有轻微的激活作用，而SDS可强烈抑制其活性。由于其相对于真菌来源的其他葡萄糖苷酶稳定性较高，为进一步的研究与应用奠定了基础。Exploring new β-glucosidase genes is of great importance to industrialize β-glucosidase. The genomes of Aspergillus fumigatus contain a bgl gene, which encodes a 65 kDa putative β-glucosidase. The bgl gene was cloned into an expression plasmid and transformed to Escherichia coli BL21 （DE3）. The bgl was expressed upon induction of Isopropyl β-D-l-thiogalactopyranoside （IPTG）. The recombinant protein was purified by GST-tag affinity chromatography. The purified recombinant Bgl was characterized using Esculin as substrate. The optimum temperature and pH were 45 ℃ and 5.0 6.0, respectively. The Km for Esculin was 17.7 mmol/L. The enzyme was stable in the range of pH 4 7. After incubation at 70 ℃ for 2 h, the recombinant Bgl remained 60% of its activity. Metal ions and chemical reagents had different influences on the activity of 13-glucosidase. Ca2＋ （1 mmol/L） could increase enzyme activity slightly. On the contrary, the enzyme activity was greatly inhibited by 5 mmol/L Sodium dodecyl sulfate （SDS）. Based on our results, the A. fumigatus Bgl was thermostable β-glucosidase.

关 键 词：烟曲霉 Β-葡萄糖苷酶 原核表达 酶学性质 

分 类 号：Q78[生物学—分子生物学]