Processivity and enzymatic mechanism of a non-modular family 5 endoglucanase from Sporocytophaga sp. CX11 with potential applications in cellulose saccharification

IF 3.4 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Enzyme and Microbial Technology Pub Date : 2025-02-15 DOI:10.1016/j.enzmictec.2025.110609
Xiaoyi Chen , Zilong Gao , Shang Wang, Fan Yang
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Abstract

In this study, a novel GH5 processive endoglucanase SmCel5A from Sporocytophaga sp. CX11 was functionally expressed in E. coli. It could rapidly decrease the viscosity of carboxymethyl cellulose (CMC-Na) solution by nearly 50 % within the initial 8 min of incubation and exhibited a significantly high specific activity towards CMC-Na of 9940 U/µmol. SmCel5A could also hydrolyze other cellulosic substrates such as RAC, Avicel, filter paper, β-glucan and the chromogenic substrate pNPC. When hydrolyzing filter paper, about 89.1 % of soluble reducing sugars were generated after 180 min of incubation, and the main products were cellobiose followed by cellotriose and glucose. The processive ratio of SmCel5A increased from 2.32 to 11.22 as the reaction time was extended from 5 min to 180 min, which is significantly higher than those of other known processive endoglucanases. Moreover, SmCel5A could hydrolyze cellodextrins with the degree of polymerization (DP) ≥ 3, but it was not active on cellobiose. In combination reaction with β-glucosidase, the maximum substrate conversion rate reached 73.2 %, showing that the synergistic reaction of the two enzymes could efficiently reduce the accumulation of cellobiose and greatly improve the hydrolysis efficiency of cellulose. The three-dimensional structure of SmCel5A was predicted by AlphaFold2 and showed to feature a classic GH5 family (β/α)8-barrel structure, with a deep substrate-binding cleft formed by the amino acids at the C-terminus. Molecular docking results indicated that when hydrolyzing cellulosic substrates, SmCel5A exhibits a preference for the exo-type mechanism of action over the endo-type mechanism of action.
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Enzyme and Microbial Technology
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.
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