Zafar Asma, Aftab Muhammad Nauman, Manzoor Asma, Iqbal Irfana, Uddin Zia, Kaleem Afshan, Khooharo Ateeque Rahman, Saleem Mushtaq Ahmad
Faculty of Life Sciences, University of Central Punjab, Lahore, Pakistan.
Institute of Industrial Biotechnology, GC University, Lahore, Pakistan.
Pak J Pharm Sci. 2018 Nov;31(6 (Supplementary):2755-2762.
Present research work is aimed to purify and characterize a recombinant β-xylosidase enzyme which was previously cloned from Bacillus licheniformis ATCC 14580 in to Escherichia coli BL21. Purification of recombinant enzyme was carried out by using ammonium sulphate precipitation method followed by single step immobilized metal ion affinity chromatography. Specific activity of purified recombinant β-xylosidase enzyme was 20.78 Umg-1 with 2.58 purification fold and 33.75% recovery. SDS-PAGE was used to determine the molecular weight of recombinant purified β-xylosidase and it was recorded as 52 kDa. Purified enzyme showed stability upto 90°C within a pH range of 3-8 with and optimal temperature and pH, 55ºC and 7.0, respectively. The enzyme activity was not considerably affected in the presence of EDTA. An increase in the enzyme activity was found in the manifestation of Mg+2. Enzyme activity was also increased by 6%, 18% and 22% in the presence of 1% Tween 80, β-mercaptoethanol and DTT, respectively. Higher concentrations (10 - 40%) of organic solvents did not show any effect upon activity of enzyme. All these characteristics of the recombinant enzyme endorsed it as a potential candidate for biofuel industry.
目前的研究工作旨在纯化和表征一种重组β-木糖苷酶，该酶先前已从地衣芽孢杆菌ATCC 14580克隆到大肠杆菌BL21中。重组酶的纯化采用硫酸铵沉淀法，随后进行单步固定化金属离子亲和层析。纯化后的重组β-木糖苷酶的比活性为20.78 Umg-1，纯化倍数为2.58，回收率为33.75%。采用SDS-PAGE测定重组纯化β-木糖苷酶的分子量，记录为52 kDa。纯化后的酶在3-8的pH范围内，高达90°C时表现出稳定性，最佳温度和pH分别为55°C和7.0。在EDTA存在下，酶活性没有受到显著影响。在Mg+2存在下，酶活性增加。在1%吐温80、β-巯基乙醇和二硫苏糖醇存在下，酶活性也分别增加了6%、18%和22%。较高浓度（10 - 40%）的有机溶剂对酶活性没有任何影响。重组酶的所有这些特性表明它是生物燃料工业的潜在候选者。
