title: Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli.
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.
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Asma
authors.initials: A
authors.lastname: Zafar
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan. nauman535@yahoo.com.
authors.forename: Muhammad Nauman
authors.initials: MN
authors.lastname: Aftab
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Zia
authors.initials: Z
authors.lastname: ud Din
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Saima
authors.initials: S
authors.lastname: Aftab
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Irfana
authors.initials: I
authors.lastname: Iqbal
authors.affiliation: Institute of Industrial Biotechnology, Government College University, Lahore, 54000, Pakistan.
authors.forename: Ikram
authors.initials: I
authors.lastname: ul Haq
mesh_terms: D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D008958:Models, Molecular; D008970:Molecular Weight; D010802:Phylogeny; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D000516:alpha-Amylases
keywords: Amylase; Cloning; Expression; Thermostable; Thermotoga petrophila