嗜热古菌高温酸性淀粉酶基因合成和大肠杆菌中的表达

BD5088是来源于1种嗜热球菌Thermococcus sp.的高温酸性α-淀粉酶的人工突变体,研究中根据BD5088基因的氨基酸序列,经密码子优化,用2步PCR法合成去信号肽后的成熟肽基因。该基因全长1311bp,由436个氨基酸组成。现将其克隆到大肠杆菌的表达载体pET30a上,在大肠杆菌BL21(DE3)中表达,表达出的目的蛋白分子量约为48ku,大小与理论值一致,经过Ni+树脂纯化,得到纯化后的重组酶BD5088。重组α-淀粉酶BD5088具有α-淀粉酶的活性,最适反应温度范围为70～85°C,最适反应pH值为5.6～6.0,在100℃下酶活性半衰期约30min。活性不依赖于Ca2+。本研究为该基因在毕赤酵母中的表达和基因的定向进化改造打下了基础。

Gene Synthesis and Expression of Thermoacidophili a-amylase in Escherichia coli

α-Amylase BD5088 is a thermostable and acid-resistance α-amylase from Thermococcus sp.In this research,the BD5088 gene with optimal codon usage was synthesized by two-step synthesis method.This synthesized amylase gene encodes a protein of 436 amino acids,without the nature signal peptide sequence.It was cloned into expression vector pET30a,and the reconstructed vector was designed as pET-BD5088 and transformed into BL21(DE3).Positive transformant was cultivated and IPTG was added into culture to induce expression of the α-amylase.The recombinant enzyme BD5088 had a molecular mass of 48 ku which was analyzed by SDS-PAGE.The optimum reaction temperature and pH scope of the recombinant α-amylase were 70~85°C and 5.6~6.0 respectively.The half life of the enzymatic activity at 100°C was 30 min,and the activity of BD5088 was not depended on the addition of Ca2+.