【摘要】 目的:克隆解淀粉芽孢杆菌β-1,3-1,4-葡聚糖酶基因(bglA)使其在解淀粉芽孢杆菌CICIM B4081中高效表达,并对重组酶进行酶学性质研究。方法:以解淀粉芽孢杆菌(CICIM B4801)染色体DNA为模板,经过PCR扩增得到了大小约为0.8kb的β-1,3-1,4-葡聚糖酶基因(bglA),构建了重组表达质粒pQ-bglA,通过电转化的方法将其转化入解淀粉芽孢杆菌(CICIM B4801)中。结果:得到了能高效表达β-1,3-1,4-葡聚糖酶的重组解淀粉芽孢杆菌。在250mL摇瓶条件下,重组菌分解地衣多糖的胞外最高酶活达到了1 515.7U/mL,重组酶的最适作用温度为55℃,最适反应pH值为6.5。结论:重组菌的β-1,3-1,4-葡聚糖酶的酶活为原始菌株的11.84倍,实现了bglA基因在解淀粉芽孢杆菌中的高效表达。更多还原

【Abstract】 Objective: The bglA gene was cloned from Bacillus amyloliquefaciens and overexpressed in Bacillus amyloliquefaciens CICICM B4081,and the characterization of recombinant β-1,3-1,4-glucanase was studied.Method: The 0.8kb fragment of β-1,3-1,4-glucanase gene(bglA) was amplified by PCR using genomic DNA of Bacillus amyloliquefaciens(CICIM B4801) as template.The recombinant plasmid PQ-bglA containing the strong promoter PQ and bglA gene was constructed and transformed into Bacillus amyloliquefaciens(CICIM B4801) by the method of electroporation.Result: The bglA was overexpressed in the recombinant strain and the maximum enzyme activity for was 1 515.7 U/mL,the optimal pH and temperature of the enzyme was 6.5 and 55℃,respectively.Conclusion: The results show that the β-1,3-1,4-glucanase was efficiently overexpressed in Bacillus amyloliquefaciens and the enzyme activity of recombinant strain was 11.84 times higher than the original strain.更多还原