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Improved catalytic activity and stability of cellobiohydrolase (Cel6A) from the Aspergillus fumigatus by rational design.
Protein Engineering, Design and Selection ( IF 2.4 ) Pub Date : 2020-09-14 , DOI: 10.1093/protein/gzaa020
Subba Reddy Dodda 1 , Nibedita Sarkar 1 , Piyush Jain 1 , Kaustav Aikat 1 , Sudit S Mukhopadhyay 1
Affiliation  

Cheap production of glucose is the current challenge for the production of cheap bioethanol. Ideal protein engineering approaches are required for improving the efficiency of the members of the cellulase, the enzyme complex involved in the saccharification process of cellulose. An attempt was made to improve the efficiency of the cellobiohydrolase (Cel6A), the important member of the cellulase isolated from Aspergillus fumigatus (AfCel6A). Structure-based variants of AfCel6A were designed. Amino acids surrounding the catalytic site and conserved residues in the cellulose-binding domain were targeted (N449V, N168G, Y50W and W24YW32Y). I mutant 3 server was used to identify the potential variants based on the free energy values (∆∆G). In silico structural analyses and molecular dynamics simulations evaluated the potentiality of the variants for increasing thermostability and catalytic activity of Cel6A. Further enzyme studies with purified protein identified the N449V is highly thermo stable (60°C) and pH tolerant (pH 5–7). Kinetic studies with Avicel determined that substrate affinity of N449V (Km =0.90 ± 0.02) is higher than the wild type (1.17 ± 0.04) and the catalytic efficiency (Kcat/Km) of N449V is ~2-fold higher than wild type. All these results suggested that our strategy for the development of recombinant enzyme is a right approach for protein engineering.

中文翻译:

通过合理设计提高了烟曲霉纤维二糖水解酶(Cel6A)的催化活性和稳定性。

廉价生产葡萄糖是生产廉价生物乙醇的当前挑战。需要理想的蛋白质工程方法来提高纤维素酶成员的效率,纤维素酶是参与纤维素糖化过程的酶复合物。试图提高纤维二糖水解酶(Cel6A)的效率,纤维二糖水解酶(Cel6A)是从烟曲霉Af Cel6A)分离的纤维素酶的重要成员。设计了Af Cel6A的基于结构的变体。靶向催化位点周围的氨基酸和纤维素结合域中的保守残基(N449V,N168G,Y50W和W24YW32Y)。我使用了变体3服务器根据自由能值(∆∆G)识别潜在的变体。电脑结构分析和分子动力学模拟评估了变异体提高Cel6A热稳定性和催化活性的潜力。对纯化蛋白进行的进一步酶研究表明,N449V具有高度的热稳定性(60°C)和耐pH值(pH 5-7)。用Avicel进行的动力学研究确定N449V的底物亲和力(Km = 0.90±0.02)高于野生型(1.17±0.04),N449V的催化效率(Kcat / Km)比野生型高约2倍。所有这些结果表明,我们开发重组酶的策略是蛋白质工程的正确方法。
更新日期:2020-09-15
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