Dadwal Anica, Sharma Shilpa, Satyanarayana Tulasi
Department of Biological Sciences & Engineering, Netaji Subhas Institute of Technology (University of Delhi), Azad Hind Fauj Marg, Sector-3 Dwarka, New Delhi, 110078, India.
Department of Biological Sciences & Engineering, Netaji Subhas University of Technology, Azad Hind Fauj Marg, Sector-3 Dwarka, New Delhi, 110078, India.
AMB Express. 2021 Nov 4;11(1):148. doi: 10.1186/s13568-021-01311-8.
A codon optimized cellobiohydrolase (CBH) encoding synthetic gene of 1188 bp from a thermophilic mold Myceliophthora thermophila (MtCel6A) was cloned and heterologously expressed in Escherichia coli for the first time. In silico analysis suggested that MtCel6A is a GH6 CBH and belongs to CBHII family, which is structurally similar to Cel6A of Humicola insolens. The recombinant MtCel6A is expressed as active inclusion bodies, and the molecular mass of the purified enzyme is ~ 45 kDa. The rMtCel6A is active in a wide range of pH (4-12) and temperatures (40-100 °C) with optima at pH 10.0 and 60 °C. It exhibits T of 6.0 and 1.0 h at 60 and 90 °C, respectively. The rMtCel6A is an extremozyme with organic solvent, salt and alkali tolerance. The K, V, k and k/K values of the enzyme are 3.2 mg mL, 222.2 μmol mg min, 2492 s and 778.7 s mg mL, respectively. The product analysis of rMtCel6A confirmed that it is an exoenzyme that acts from the non-reducing end of cellulose. The addition of rMtCel6A to the commercial cellulase mix (Cellic CTec2) led to 1.9-fold increase in saccharification of the pre-treated sugarcane bagasse. The rMtCel6A is a potential CBH that finds utility in industrial processes such as in bioethanol, paper pulp and textile industries.
首次克隆了来自嗜热霉菌嗜热毁丝霉（Myceliophthora thermophila，MtCel6A）的1188 bp密码子优化的纤维二糖水解酶（CBH）编码合成基因，并在大肠杆菌中进行了异源表达。计算机分析表明，MtCel6A是一种GH6 CBH，属于CBHII家族，其结构与特异腐质霉的Cel6A相似。重组MtCel6A以活性包涵体形式表达，纯化酶的分子量约为45 kDa。重组MtCel6A在广泛的pH范围（4 - 12）和温度范围（40 - 100°C）内具有活性，最适pH为10.0，最适温度为60°C。在60°C和90°C时，其半衰期分别为6.0小时和1.0小时。重组MtCel6A是一种具有有机溶剂、盐和碱耐受性的极端酶。该酶的Km、Vmax、kcat和kcat/Km值分别为3.2 mg mL-1、222.2 μmol mg-1 min-1、2492 s-1和778.7 s-1 mg-1 mL。重组MtCel6A的产物分析证实它是一种从纤维素非还原端起作用的外切酶。向商业纤维素酶混合物（Cellic CTec2）中添加重组MtCel6A可使预处理甘蔗渣的糖化率提高1.9倍。重组MtCel6A是一种潜在的CBH，可用于生物乙醇、纸浆和纺织工业等工业过程。
