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Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli

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Abstract

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.

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    1. Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan

      Asma Zafar, Muhammad Nauman Aftab, Zia ud Din, Saima Aftab, Irfana Iqbal & Ikram ul Haq

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    Zafar, A., Aftab, M.N., ud Din, Z. et al. Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli . Appl Biochem Biotechnol 178, 831–848 (2016). https://doi.org/10.1007/s12010-015-1912-8

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