Design, development and production of a thermostable endoglucanase-I from Fusarium oxysporum

Abstract

Endoglucanase is one of the three enzymes required to synergistically hydrolyze cellulose to sugar monomers which can be fermented into ethanol. The purpose of this study is to computationally design and experimentally produce a thermostable endoglucanase from Fusarium oxysporum that is stable at higher temperature (above 60°C) for use in various industries. An endoglucanase, EGuia, which is same as the well-characterized endoglucanase I from Fusarium oxysporum (PDB ID: 3OVW) has been cloned into an appropriate expression vector, except for two mutations. As an attempt to improve the substrate binding characteristics of EGuia, computational molecular docking studies were carried out using Lamarkian Genetic Algorithm (LGA) with a fast, simplified potential of mean force (PMF) to understand the interactions in the active site regions and evaluate the docking efficiency. The two substrate evaluated in this study were cellobiose and cellotetraose. The active site was confirmed by comparing the docked structure with the available experimental structures. Cellotetraose was found to have stronger binding than cellobiose, and thus was used as the ligand in this study. The second part of this study was to perform molecular dynamics simulation (MDS) to identify the potential regions (residues) in EGuia that could be altered (mutated) to produce a thermostable EGuia. The temperature studied was 313K (40oC), 333K (60oC) and 353K (80oC). Simulation was done for EGuia and EGuia-cellotetraose complex to evaluate the effect of complexation. NAMD was used to perform the MDS with CHARMM36 Force field. Root mean square deviation of backbone atoms, helices, betasheets, coils, turns, buried and surface calculated and plotted. Solvent accessible surface area (SASA) and radius of gyration was also calculated. Root mean square fluctuation (RMSF) for the entire residues was calculated. Analysis at the active site reveals that the substrate (ligand) is surrounded by betasheets. Site Directed Mutagenesis (SDM) method based on Polymerase Chain Reaction (PCR) was used to produce the predicted mutation. After many failures, finally the enzyme was detected via SDS-PAGE. The rapid divergence of RMSD of enzyme-substrate complex at higher temperature compared to EGuia under same conditions suggested that the enzyme-substrate complex would easily lose its activity at higher temperatures. Among all the secondary structure analyzed, betasheets shows the least RMSD indicating stability even at higher temperature. Based on the findings of solvent accessible surface area (SASA) and radius of gyration, we conclude that the increase in temperature leads to the unfolding of the EGuia and it is more evident in the ligand-enzyme complex than the EGuia. From the root mean square fluctuation (RMSF) results, four regions with high fluctuation were identified and as expected they are in the coils secondary structure. Serine at position 230 is the most mobile residue. Based on this, a triple mutant (G229A/S230F/S231E) which would improve thermostability was proposed. This study establishes a connection between complexed enzyme structure and activity at different temperatures.