黑曲霉α-葡萄糖苷酶cDNA的克隆及表达

Cloning and Expression of An α-glucosidase cDNA from Aspergillus niger in E.coli

研究黑曲霉(Aspergillus niger)M-1菌株的α-葡萄糖苷酶基因在大肠杆菌中的克隆及表达。以M-1菌株的总RNA为模板,利用RT-PCR扩增α-葡萄糖转苷酶的cDNA,重组到Trc启动子控制下的表达载体pSE380中,构建重组质粒pSE-αtg,转入大肠杆菌BL21(DE3)进行IPTG诱导表达。初步研究表明:重组蛋白具有葡萄糖苷酶活性,最适pH为6.0,最适温度为45℃,金属离子Cu2+和Mn2+对酶活力有明显的促进作用。添加1.6mmol/L IPTG对重组菌的诱导作用最大,在培养中添加麦芽糖,对重组菌产酶有显著的促进作用。

The paper reports the research about cloning and expression of an α - glucosidase cDNA from Aspergillus niger in E. coli. cDNA was amplified by RT-PCR from total RNA of Aspergillus niger, and cloned into expression vector pSE380 which is controlled by Tre promoter, resultant pSE-atg was transformed to E. coli BL21（DE3 ） .The positive transformants were fermented in flasks and induced by IPTC. The recombinant α-transglucosidase activity amounted to 485.40U/g, and showed the optimal activity at pH 6.0 and 45℃. The research also found that the recombinant enzyme was obviously activated by Cu2^＋ and Mn2^＋ ion. Adding maltose in the substrate can induce the production of α - glucosidase by E. coil transformant.