Zafar Asma, Aftab Muhammad Nauman, ud Din Zia, Aftab Saima, Iqbal Irfana, ul Haq Ikram
Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
Appl Biochem Biotechnol. 2016 Feb;178(4):831-48. doi: 10.1007/s12010-015-1912-8. Epub 2015 Nov 2.
A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and β-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose.
利用pET-21a(+)作为表达载体，克隆嗜热栖热袍菌假定的α-淀粉酶基因并在大肠杆菌BL21(DE3)中进行表达。使用各种参数（如pH、温度、诱导时间和诱导剂添加量）对生长条件进行优化，以实现α-淀粉酶的最大表达。α-淀粉酶最大表达的最适温度和pH分别为22℃和7.0个pH单位。通过热处理方法对重组酶进行纯化，随后进行离子交换色谱，纯化倍数为34.6倍，比活性为126.31 U mg(-1)，回收率为56.25%。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)测定纯化的α-淀粉酶分子量为70 kDa。该酶在100℃温度和pH 7.0条件下稳定。在金属离子尤其是Ca(+2)存在下酶活性增加，而在EDTA存在下酶活性降低，这表明α-淀粉酶是一种金属酶。然而，添加1%吐温20、吐温80和β-巯基乙醇后，酶活性分别被抑制至87%、96%和89%。未观察到有机溶剂（乙醇、甲醇、异丙醇、丙酮和正丁醇）对酶活性有显著影响。以可溶性淀粉为底物，优化条件下的酶活性为73.8 U mg(-1)。该α-淀粉酶能够水解淀粉形成麦芽糖。
