A novel β-glucosidase gene was cloned from genomic DNA of the filamentous fungus Rhizopus stolonifer var.reflexus TP-02 and expressed in Escherichia coli BL21.Structure and function of the gene product β-glucosidase (abbreviated as BGLⅢ) were analyzed by a series of bioinformatics software.Trp127 was considered to be the key amino acid in the enzymatic process.Results from structure analysis indicated that the conformation of Trp127 had a significant impact on the structure and function of the active center of BGLⅢ.Leu could maintain the original conformation of active centre,meanwhile,increase hydrophobic interaction with glucose,thus it could increase the efficiency of the enzyme and substrate binding.The fermentation characteristics of recombinants showed that W127A without side chain greatly decreased the activity,and W127F with benzene ring side chain did not change the activity greatly,while W127L with long chain improved the activity greatly.%从匍枝根霉TP-02(Rhizopus stolonifer)基因组DNA中扩增得到β-葡萄糖苷酶bgl3全长基因,并在大肠杆菌BL21中实现表达.通过对β-葡萄糖苷酶的生物信息学分析,确定其与底物作用过程中的关键氨基酸为Trp127.对该位点进行突变研究,结果显示:突变为不带侧链的Ala时,酶活降低20.8％;突变为具有相同苯环侧链的Phe时,酶活相差不大;突变为带有非苯环长链的Leu时,酶活提高34.07％.Leu的长链在维持原有活性中心构象的基础上,与酶解产物葡萄糖的疏水作用增强,从而提高酶与底物结合效率.即127位氨基酸的空间构象对BGLⅢ活性中心的结构功能有显著影响.
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