Parashar Deepak, Satyanarayana T
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Int J Biol Macromol. 2017 Jun;99:274-281. doi: 10.1016/j.ijbiomac.2017.02.083. Epub 2017 Feb 24.
For saccharifying starch in one step, a chimeric biocatalyst (Amy-Glu) was generated from engineered α-amylase (Ba-Gt-amy) of Bacillus acidicola and glucoamylase (Glu) gene of Aspergillus niger. In order to join two enzymes, a linker peptide of 25 amino acids was used. Chimeric Amy-Glu was expressed in E. coli. Glu is of 75kDa, while Amy-Glu is of 145kDa. Both Amy-Glu and Glu displayed similar pH profile with good activity in the acidic pH range like that of Ba-Gt-amy with optimum at pH 4.0. All three enzymes (Ba-Gt-amy, Amy-Glu and glucoamylase) exhibited activity in the temperature range between 40 and 70°C with optimum at 60°C. Amy-Glu and Glu have T of 90 and 70min at 60 and 70°C, respectively. The K, V and K values of Glu (soluble starch) are 0.34mgmL, 606μmolmgmin and 727s, while for Amy-Glu are 0.84mgmL, 13,886μmolmgmin and 4.2×10s, respectively. The end product analysis suggested that Amy-Glu retains the activity of both parental enzymes and forms maltodextrins along with glucose as the major products. Amy-Glu saccharifies wheat and corn starches more efficiently than the Ba-Gt-amy and glucoamylase.
为了一步糖化淀粉，构建了一种嵌合生物催化剂（Amy-Glu），它由嗜酸芽孢杆菌的工程化α-淀粉酶（Ba-Gt-amy）和黑曲霉的糖化酶（Glu）基因组成。为了连接这两种酶，使用了一个含25个氨基酸的接头肽。嵌合Amy-Glu在大肠杆菌中表达。Glu的分子量为75kDa，而Amy-Glu的分子量为145kDa。Amy-Glu和Glu都表现出相似的pH谱，在酸性pH范围内具有良好的活性，与Ba-Gt-amy相似，最适pH为4.0。这三种酶（Ba-Gt-amy、Amy-Glu和糖化酶）在40至70°C的温度范围内均有活性，最适温度为60°C。Amy-Glu和Glu在60°C和70°C下的半衰期分别为90分钟和70分钟。Glu（可溶性淀粉）的Km、Vmax和kcat值分别为0.34mg/mL、606μmol/(mg·min)和727/s，而Amy-Glu的相应值分别为0.84mg/mL、13886μmol/(mg·min)和4.2×10/s。终产物分析表明，Amy-Glu保留了两种亲本酶的活性，主要产物为麦芽糖糊精和葡萄糖。Amy-Glu比Ba-Gt-amy和糖化酶更有效地糖化小麦和玉米淀粉。
