面包酵母麦芽糖酶的异源表达及产酶条件优化

Cloning and Expression of Maltase Encoding Gene from Baker's Yeast and Optimization of the Expression Conditions

通过设计特异性引物从快速发酵面包酵母BY-14中PCR克隆获得麦芽糖酶编码基因。选择Bam H I和Hind III分别为上下游引物的酶切位点,将麦芽糖酶基因克隆到p ET28a载体上。表达质粒经PCR扩增、双酶切和测序进行验证。正确的表达质粒p ET28a-Mal62导入大肠杆菌宿主BL(21),经IPTG诱导表达,结果显示,重组蛋白能够可溶性表达。随后对大肠杆菌产酶条件进行优化,结果表明,当IPTG终浓度为0.4 mmol/L,菌浓OD600达到1.0时加入诱导剂,在26℃下振荡培养12 h可获得最高的产酶量39.9 U/L,比优化前提高了4倍。

The maltase encoding gene was amplified via PCR from baker’s yeast. Then it was digested by BamH I and Hind III, and inserted in-to plasmid pET28a. The expression plasmid was identified via PCR, restriction endonuclease digestion and sequencing. The valid expression plasmid （pET28a-Mal62） was transformed into competence Escherichia coli BL（21） for heterologous expression. The experimental results showed that, the target protein was able to be expressed and most of them were soluble. Afterwards, the expression conditions for the target pro-tein was optimized as follows：as IPTG concentration was 0.4 mmol/L, the induced timing OD600 for 1.0, oscillation induced culture for 12 h at 26℃, it was feasible to express the recombinant fusion protein in the supernatant with the highest production of 39.9 U/L, which increased by 4 fold compared with unoptimized conditions.