In-silico designing of Bi-functional enzyme constructs (Cellulase and βGlucosidase) for enhanced hydrolysis of Lignicellulosic biomass
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Date
2023-09-12
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UMT Lahore
Abstract
The rapid depletion of fossil fuels and the escalating environmental concerns have fueled the exploration of renewable energy sources, such as lignocellulosic biomass, for biofuel production. The hydrolysis of lignocellulosic biomass into fermentable sugars is a critical step in this process, and enzymatic approaches using cellulase and beta-glucosidase enzymes have shown promise. However, the suboptimal catalytic efficiency and synergistic interactions between these enzymes limit the hydrolysis yield. This thesis presents an innovative approach to enhance the hydrolysis of lignocellulosic biomass by employing in silico design of bifunctional enzyme constructs. The genetic sequences responsible for cellulase from Bacillus altitudinis and β-glucosidase from Periconia sp. were combined through fusion and subsequently introduced for expression within E. coli K12 bacterial strain. This fusion enzyme was stable at 60 °C for 2 h. Molecular docking simulations were utilized to predict potential binding sites and interactions between the enzymes, enabling the identification of suitable fusion points. Optimum pH for both cellulase and β-glucosidase activities was found to be 6.0. Optimum temperature for cellulase and β-glucosidase activities was found to be 60-65°C, respectively. In future, the docked complexes of stable fusion constructs would be evaluated using molecular dynamic simulations to assess conformational stability in dynamic environment. Combining cellulase and β-glucosidase makes it easier to channel one enzyme's output as a substrate to the other, improving the total efficiency of biomass hydrolysis. Therefore, these fusion enzymes might be useful in industrial applications.