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

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Aspergillus fumigatus基因组中含有一个 bgl基因 (1 752 bp)，编码的蛋白约65 kDa，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Escherichia coli BL21 (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Km值为17.7 mmol/L。该酶在pH 4~7范围内稳定；70 ℃保温2 h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，Ca2+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。

Abstract:

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-1-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 β-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.