为了获得耐热的β-1,3-1,4-葡聚糖酶,利用PCR技术从解淀粉芽孢杆菌(Bacillus amyloliquefaciens)中扩增β-1,3-1,4-葡聚糖酶基因(bgl),构建重组表达质粒pET30a-bgl转化至大肠杆菌BL21( DE3),经异丙基硫代-β-D-半乳糖苷酸(IPTG)诱导表达获得重组β-1,3-1,4-葡聚糖酶,并对纯化后的β-1,3-1,4-葡聚糖酶进行酶学特性研究.结果表明,摇瓶培养的重组基因工程菌的酶活力为80 U/ml,纯化后酶的比活力为4 969 U/mg,是纯化前的54.6倍；酶的分子量为2.6×104左右,最适温度为50℃,最适pH为6.0,在pH 5.0和pH 8.0之间有很好的稳定性,90℃下酶的半衰期(t1/2)为21 min；该酶对燕麦葡聚糖和地衣多糖有很好的专一性.重组β-1,3-1,4-葡聚糖酶有较好的耐酸、耐热性,在酿造和饲料加工过程中能够保持较高的活性,具有很好的工业应用价值.%In order to obtain β-l ,3-1,4-glucanase with improved thermostability, β-l ,3-1,4-glucanase gene from Bacillus amyloliquefaciens was amplified by PCR. The recombinant expression plasmid pET30a-bgl was constructed and transformed into E. Coli BL21 (DE3). The (3-1,3-1,4-glucanase was expressed efficiently after induction with isopropyl-thio-β-D-galactoside (IPTC). The enzymatic characteristics of β-1 ,3-1,4-glucanase was also studied after purification. The results show that the activity of the recombinant enzyme was 80U/ml in the shaker flask culture. The specific activity of the purified enzyme was 4 969 U/mg, which was purified 54.6-fold. The enzyme molecular weight was about 2.6xl04. Enzyme activity assay showed that the optimum temperature and pH of the recombinant enzyme were 50℃ and 6. 0, respectively. The recombinant enzyme was stable between pH 5.0 and 8.0, which was thermostable with the half life value (t1/2) of 21 min at 90℃ and exhibited strict preference for oat β-glucan and lichenan. The thermostable and acidic-tolerant recombinant β-1,3-1,4-glucanase can retain high activity in the process of brewing and animal feed additive industry.These biochemical properties make it ideal for industrial applications.
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