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.

Bacillus velezensis Identification and Recombinant Expression, Purification, and Characterization of Its Alpha-Amylase

“…In Thermotoga petrophila and Geobacillus sp. GS33, 1% (v/v) of Tween 20 decreased the α-amylase activity by 13% and 34%, respectively [48,50]. In WangLB bacterium, both 2% (w/v) and 5% (w/v) of Tween 20 decreased the α-amylase activity by about 14% and 32%, respectively [11].…”

“…The optimum temperature of purified BvAmylase activity was explored by measuring the α-amylase activity at different temperatures (25,30,40,50,55,60,65,70,75,80,90, 100 • C) with the DNS method. Furthermore, the thermal stability of the purified amylase was obtained by preincubating the BvAmylase at different temperatures (40,50,60,70, 80 • C) for 0-60 min, and then the activity was tested by the DNS method at optimum temperature. The results were calculated as the percentages of the highest activity reaction (100%).…”

Heterologous expression, molecular studies and biochemical characterization of a novel alkaline esterase gene fromBacillus thuringiensisfor detergent industry

“…33 Previous reports are present on the use of pET expression systems for the successful expression of recombinant proteins. [34][35][36][37][38] Expression of cloned esterase gene as well as molecular weight determination of recombinant protein was evaluated by SDS-PAGE as shown in Fig. 3.…”

A Novel Raw Starch Hydrolyzing Thermostable α-Amylase Produced by Newly Isolated Bacillus mojavensis SO-10: Purification, Characterization and Usage in Starch Industries

“…Michelin et al 25 demonstrated amylase as a single band of about 75 kDa by SDS-PAGE. Another αamylase with molecular weight of 70 kDa was indicated by Zafar et al 26 . Different molecular masses of the α-amylases from various Bacillus sp.…”