Sadaqat Beenish, Sha Chong, Rupani Parveen Fatemeh, Wang Hongcheng, Zuo Wanbing, Shao Weilan
Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China.
Front Bioeng Biotechnol. 2021 Mar 16;9:637649. doi: 10.3389/fbioe.2021.637649. eCollection 2021.
() contains genes encoding various hyperthermophilic enzymes with great potential for industrial applications. The gene TM1752 in genome has been annotated as cellulase gene encoding protein Cel5B. In this work, the gene TM1752 was cloned and expressed in , and the recombinant enzyme was purified and characterized. Interestingly, the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxy methyl cellulose, which is the highest among thermophilic mannanases. However, the putative enzyme did not show sequence homology with any of the previously reported mannanases; therefore, the enzyme Cel5B was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B. Man/Cel5B exhibited maximum activity at 85°C and pH 5.5. This enzyme retained more than 50% activity after 5 h of incubation at 85°C, and retained up to 80% activity after incubated for 1 h at pH 5-8. The K and V of Man/Cel5B were observed to be 4.5 mg/mL galactomannan and 769 U/mg, respectively. Thin layer chromatography depicted that locust bean gum could be efficiently degraded to mannobiose, mannotriose, and mannooligosaccharides by Man/Cel5B. These characteristics suggest that Man/Cel5B has attractive applications for future food, feed, and biofuel industries.
（）包含编码各种具有巨大工业应用潜力的嗜热酶的基因。基因组中的基因TM1752已被注释为编码蛋白质Cel5B的纤维素酶基因。在这项工作中，基因TM1752被克隆并在 中表达，重组酶被纯化并进行了表征。有趣的是，纯化后的酶对底物半乳甘露聚糖和羧甲基纤维素的比活性分别为416和215 U/mg，这在嗜热甘露聚糖酶中是最高的。然而，推测的这种酶与之前报道的任何甘露聚糖酶都没有序列同源性；因此，该酶Cel5B被鉴定为双功能甘露聚糖酶和纤维素酶，并重新命名为Man/Cel5B。Man/Cel5B在85°C和pH 5.5时表现出最大活性。该酶在85°C孵育5小时后仍保留超过50%的活性，在pH 5 - 8孵育1小时后仍保留高达80%的活性。观察到Man/Cel5B的Km和Vmax分别为4.5 mg/mL半乳甘露聚糖和769 U/mg。薄层色谱显示，刺槐豆胶可被Man/Cel5B有效降解为甘露二糖、甘露三糖和甘露寡糖。这些特性表明Man/Cel5B在未来的食品、饲料和生物燃料行业具有诱人的应用前景。
