In-silico designing of bi-functional enzyme constructs (cellulase and xylanase) for enhanced hydrolysis of lignocellulosic biomass
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Date
2023
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UMT Lahore
Abstract
Current biological techniques for breaking down lignocellulosic biomass face challenges due to slow enzymatic degradation. A particular challenge in cellulose degradation is the presence of hemicellulose chains intertwined with cellulose fibrils, hindering overall cellulose hydrolysis. Moreover, hemicellulose is a polymer of various sugars valuable for product synthesis. Efficient hydrolysis of lignocellulosic biomass necessitates breaking down nearly all its components into constituent monosaccharides. This study focuses on computationally designing bifunctional fusion enzymes combining cellulase and xylanase, connected by flexible and rigid linkers while preserving enzyme functionality. Four constructs were designed, and their 3D structures, folding patterns, and molecular interactions with respective substrates were evaluated. The structural characteristics and molecular interactions of individual, unbound enzymes were also analyzed for comparison. Among the four fusion constructs, (Cell-Xyl)flx, with cellulase at the N-terminus and xylanase at the C-terminus, linked by the flexible linker (GGGS)3, emerged as the optimal choice. It exhibited proper folding, ideal substrate binding to enzymatic domains, maximal molecular interactions with substrates, and higher binding affinity values with both glucan (-7.9kcal/mol) and xylan (-9.2kcal/mol) substrates. Additionally, the nucleotide sequence of the fusion construct was underwent the codon adaptation by JCat tool and in silico cloning of this fusion construct into the pET28a(+) vector resulted in stable expression without altering the amino acid sequence. These findings highlight the effectiveness of computational strategies in designing and molecularly examining various fusion constructs, considering structural parameters, substrate binding, and molecular interactions. Once validated through wet-lab experiments, these ideal constructs hold significant potential for enhancing lignocellulosic biomass hydrolysis into fermentable sugars.