Enhancing Enzymatic Properties of Endoglucanase I Enzyme from Trichoderma Reesei via Swapping from Cellobiohydrolase I Enzyme

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.

ROLE OF SUBSTRATE BINDING ON THE PROTEIN DYNAMICS OF AN ENDOGLUCANASE FROM FUSARIUM OXYSPORUM AT DIFFERENT TEMPERATURES

: Thermostability is an important requirement for protein function, and one goal of protein engineering is improvement of activity of the enzymes at higher temperatures, particularly for industrial applications. Computational approaches to investigate factors influencing thermostability of proteins are becoming researchers’ choice. This study investigates the influence of substrate binding on the protein dynamics by comparing the molecular dynamics simulations of substrate-enzyme complex against un-bound enzyme, using endoglucanase I from Fusarium oxysporum. Endoglucanase-substrate complex was prepared by docking and molecular dynamics simulations were carried out at three different temperatures, 313 K, 333 K and 353 K. Our finding shows that the secondary structures for substrate-enzyme complex show more fluctuations relative to un-complexed structure. The same trend was observed for solvent accessible surface area and radius of gyration. At the highest temperature studied (353 K), the substrate-enzyme complex form showed the highest fluctuations. The fluctuations around the active site regions reach a minimum at the optimum temperature, compared to the other structural regions and other temperatures. ABSTRAK: Kestabilan (ketahanan) terhadap haba merupakan keperluan yang penting untuk fungsi protin, salah satu matlamat kejuruteraan protin adalah penambahbaikan aktiviti enzim pada suhu yang tinggi khususnya untuk aplikasi industri. Kini para penyelidik memilih kaedah komputasi, bagi mengkaji faktor yang mempengaruhi kestabilan terhadap haba. Kajian ini menyelidik pengaruh ikatan substrat pada protin dengan membandingkan simulasi molekular dinamik diantara substrat-enzim kompleks dan enzim sahaja, menggunakan endoglucanase I dari Fusarium oxysporum. Kompleks endoglucanase-substrat disediakan melalui kaedah docking dan simulasi molekular dinamik dilakukan pada suhu 313 K, 333 K dan 353 K. Kajian kami menunjukkan struktur sekunder bagi substrat-enzim kompleks kurang stabil berbanding enzim sahaja. Pola yang sama bagi luas permukaan boleh dicapai pelarut (SASA) dan jejari gyrasi. Pada suhu tertinggi dikaji (353 K), substrat-enzim kompleks paling tidak stabil. Pada suhu optimum, kadar ubah-ubah sekitar amino asid aktif adalah minimum berbanding struktur dan suhu lain.

Improving the activity of endoglucanase I (EGI) from Saccharomyces cerevisiae by DNA shuffling

To enhance the endo-β-1,4-glucanase activity of three mixedTrichodermasp. (Trichoderma reesei, Trichoderma longibrachiatum, andTrichoderma pseudokoningii), we optimized the efficiency of the encoding gene using DNA shuffling andSaccharomyces cerevisiaeINVSc1 as a host.