Muhammad Nauman Aftab
Government College University, Pakistan
Title: Cloning, expression and characterization of β-xylanase gene from Thermotoga naphthophila
Biography
Biography: Muhammad Nauman Aftab
Abstract
More than 90% energy requirements depend on fossil fuels that include crude oil, natural gas, coal, etc. The burning of these energy sources emit greenhouse gases that are harmful for our environment and are major cause of many diseases like lung cancer, asthma etc. The requirement of fossil fuel is increasing day by day due to rapid industrialization and increased number of vehicles. In the middle of this century, world’s fossil fuels reserves will be significantly reduced. In this scenario, the alternative fuel, bioethanol, can serve as an ideal candidate in reducing greenhouse emissions and can also help in fulfilling the partial energy needs globally. To achieve bioethanol, highly thermostable β-xylanase gene Thermotoga naphthophila was cloned and expressed in E. coli BL21 utilizing pET-21a(+) expression vector. To obtain maximum expression of recombinant xylanase, growth conditions i.e., temperature, pH, inducer and induction time of E. coli were optimized and found to be 7.0, 37ºC, 0.5 mM and 4 hours, respectively. Heat treatment was used for the partial purification of recombinant enzyme. Further purification was carried out by ammonium sulphate precipitation followed by the anion exchange chromatography. Molecular weight of pure recombinant β-xylanase enzyme was calculated to be 150 kDa by SDS-PAGE. Considerable stability was exhibited by the purified enzyme at pH value 8.0 and at temperature 90ºC. The activity of the enzyme was decreased considerably in the presence of EDTA and significantly increased in the presence of Cu+2. The effect of different organic solvents (methanol, ethanol, n-butanol, acetone and isopropanol) was also explored but no considerable effect on activity of xylanase enzyme was observed. Similarly, inhibitors (urea, triton X-100, Tween-20, Tween-80 and β-mercaptoehanol) did not have any considerable effect on the enzyme activity, however, addition of SDS significantly reduced the activity of β-xylanase activity. The β-xylanase enzyme obtained can be utilized for the saccharification of lignocellulosic mass which can in turn be used in the production of bioethanol.