Catalytic tunnel engineering of thermostable endoglucanase of GH7 family (W356C) from Aspergillus fumigatus gains catalytic rate

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology Pub Date : 2025-03-21 DOI:10.1016/j.enzmictec.2025.110632

Musaddique Hossain, Subba Reddy Dodda, Shalini Das, Kaustav Aikat, Sudit S. Mukhopadhyay

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引用次数: 0

Abstract

Tunnel engineering targets the access tunnels in enzymes, which is crucial for substrate binding and product release. Modifying the tunnels can lead to better biomass-degrading abilities of the lignocellulolytic enzymes. In this report, we have engineered the thermostable GH7 family endoglucanase from Aspergillus fumigatus (AfEgl7). The residues in the open tunnel having the highest bottleneck radius are mutated. Mutations are created (T229F, W356C) in the non-conserved region. The mutant W356C showed a 2-fold increase in product release rate (V_max = 375.8 µM/min) and 2.5-fold higher catalytic activity (K_cat = 75.1 min⁻¹) compared to wild-type (V_max= 232 µM/min; K_cat = 30.9 min⁻¹) using CM cellulose as substrate. The mutant T229F lost both catalytic activity and thermostability. Molecular dynamic simulations and docking studies of W356C revealed a change in structure near the product exit region, which may facilitate faster product release and account for the increased catalytic efficiency of the mutant. This study showed how redesigning the access pathways can be a promising strategy for protein engineering and de novo protein design by tailoring the open tunnel geometry to a ligand-specific manner.

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来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.