Abstract
Utilizing plant-based materials as a biofuel source is an increasingly popular attempt to redesign the global energy cycle. This endeavour underlines the potential of cellulase enzymes for green energy production and requires the structural and functional engineering of natural enzymes to enhance their utilization. In this work, we aimed to engineer enzymatic and functional properties of Endoglucanase I (EGI) by swapping the Ala43-Gly83 region of Cellobiohydrolase I (CBHI) from Trichoderma reesei. Herein, we report the enhanced enzymatic activity and improved thermal stability of the engineered enzyme, called EGI_swapped, compared to EGI. The difference in the enzymatic activity profile of EGI_swapped and the EGI enzymes became more pronounced upon increasing metal-ion concentrations in the reaction media. Notably, the engineered enzyme retained a considerable level of enzymatic activity after thermal incubation for 90 min at 70 °C while EGI completely lost its enzymatic activity. Circular Dichroism spectroscopy studies revealed distinctive conformational and thermal susceptibility differences between EGI_swapped and EGI enzymes, confirming the improved structural integrity of the swapped enzyme. This study highlights the importance of swapping the metal-ion coordination region in the engineering of EGI enzyme for enhanced structural and thermal stability.
Highlights
Lignocellulose is considered the most abundant and sustainable energy resource in the world [1,2].Due to the growing demand on green alternative energy sources, a substantial global effort is being exerted to redesign the global carbon cycle [3] in an effort to provide sustainabile fuels from plant-based cellulosic materials [4]
We aim to alter the existing structural features of Endoglucanase I (EGI) enzyme via domain swapping from Cellobiohydrolase I (CBHI) enzyme
We find that choosing the Ala43-Gly83 region (CBHI numbering) is promising due to its closeness to the Co2+ metal ion in the crystal structure of CBHI, which does not exist in the crystal structure of EGI [24]
Summary
Due to the growing demand on green alternative energy sources, a substantial global effort is being exerted to redesign the global carbon cycle [3] in an effort to provide sustainabile fuels from plant-based cellulosic materials [4]. Plant-based cellulosic materials as a biofuel resource offer significant advantages over fossil fuel sources, e.g., decreasing global emission, providing a long-term rural development [5,6], the particular challenges still have to be surmounted in the deconstruction processes of cellulose to cellulosic biomass. Apart from efforts in different pre-treatment techniques for a more efficient breaking down of cellulose, researchers focused on the development of new cellulase enzymes with improved efficiency via protein engineering [9,10].
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