耐热β-1,3-1,4-葡聚糖酶基因的克隆、表达及其酶学特性

Cloning,expression and enzymatic characteristics of thermostable β-1,3-1,4-glucanase

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-1,3-1,4-glucanase with improved thermostability,β-1,3-1,4-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 β-1,3-1,4-glucanase was expressed efficiently after induction with isopropylthio-β-D-galactoside（IPTG）.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 80 U/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 2.6×104.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.