Zafar Asma, Hamid Attia, Peng Liangcai, Wang Yanting, Aftab Muhammad Nauman
Faculty of Life Sciences, University of Central Punjab Lahore Pakistan.
Institute of Industrial Biotechnology, Government College University Lahore 54000 Pakistan
RSC Adv. 2022 May 18;12(23):14917-14931. doi: 10.1039/d2ra00304j. eCollection 2022 May 12.
The present study describes the cloning, expression, purification and characterization of the xylosidase gene (1650 bp) from a thermophilic bacterium into BL21 (DE3) using the expression vector pET-21a(+) for utilization in biofuel production. The recombinant xylosidase enzyme was purified to homogeneity by heat treatment and immobilized metal ion affinity chromatography. SDS-PAGE determined that the molecular weight of purified xylosidase was 60 kDa. This purified recombinant xylosidase showed its maximum activity at a temperature of 37 °C and pH 6.0. The purified recombinant xylosidase enzyme remains stable up to 90 °C for 4 h and retained 54.6% relative activity as compared to the control. The presence of metal ions such as Ca and Mg showed a positive impact on xylosidase enzyme activity whereas Cu and Hg inhibit its activity. Organic solvents did not considerably affect the stability of the purified xylosidase enzyme while DMSO and SDS cause the inhibition of enzyme activity. Pretreatment experiments were run in triplicate for 72 h at 30 °C using 10% NaOH. Saccharification experiment was performed by using 1% substrate (pretreated plant biomass) in citrate phosphate buffer of pH 6.5 loaded with 150 U mL of purified recombinant xylosidase enzyme along with ampicillin (10 μg mL). Subsequent incubation was carried out at 50 °C and 100 rpm in a shaking incubator for 24 h. Saccharification potential of the recombinant xylosidase enzyme was calculated against both pretreated and untreated sugarcane bagasse and wheat straw as 9.63% and 8.91% respectively. All these characteristics of the recombinant thermotolerant xylosidase enzyme recommended it as a potential candidate for biofuel industry.
本研究描述了利用表达载体pET-21a(+)将嗜热细菌的木糖苷酶基因（1650 bp）克隆、表达、纯化并鉴定到BL21(DE3)中，以用于生物燃料生产。重组木糖苷酶通过热处理和固定化金属离子亲和色谱法纯化至均一。SDS-PAGE测定纯化的木糖苷酶分子量为60 kDa。这种纯化的重组木糖苷酶在37°C和pH 6.0时表现出最大活性。纯化的重组木糖苷酶在高达90°C的温度下保持4小时稳定，与对照相比保留了54.6%的相对活性。Ca和Mg等金属离子的存在对木糖苷酶活性有积极影响，而Cu和Hg则抑制其活性。有机溶剂对纯化的木糖苷酶稳定性没有显著影响，而DMSO和SDS会导致酶活性受到抑制。预处理实验在30°C下使用10% NaOH进行一式三份，持续72小时。糖化实验是在pH 6.5的柠檬酸盐磷酸盐缓冲液中使用1%底物（预处理的植物生物质），加入150 U/mL的纯化重组木糖苷酶以及氨苄青霉素（10 μg/mL）进行的。随后在50°C和100 rpm的振荡培养箱中孵育24小时。计算重组木糖苷酶对预处理和未处理的甘蔗渣和小麦秸秆的糖化潜力分别为9.63%和8.91%。重组耐热木糖苷酶的所有这些特性使其成为生物燃料行业的潜在候选者。
