Logical engineering of cellulase enzyme isolated from Acidothermus cellulolyticus bacteria for increasing thermal stability

Cellulases of microbial origin have shown their potential application in industrytoday.However, the search for thermostable natural cellulases remains challenging. Therefore,alternative approaches like rational engineering offer promising prospects for their production. In thisstudy, we isolated and identified a highly cellulase-producing strain from the microbial flora of Taftanvolcanic region. Molecular methods, including ۱۶srRNA gene sequencing, were employed tocharacterize the bacterial strain and the cellulase enzyme gene, which were the main focus of ourresearch . Our findings highlighted the Acidothermus cellulolyticus strain as an excellent cellulaseproducer.Subsequently, we performed homology modeling of the cellulase enzyme using AlphaFoldserver patterns and assessed the model’s quality for the next steps. The constructed ۳D model exhibitedhigh structural quality, rendering it suitable for subsequent stages such as in-silico mutagenesis.Notably, it possessed four unique mutations (G۳۶۰I, G۴۶۴F, G۴R, N۴۷۹C) that would stabilize theenzyme. These mutations, as validated by FoldX۵, amplified the enzyme’s stability, achieving a scoreof -۱۷.۵۴ kcal/mol. We employed Schrödinger software to carry out molecular docking and MM-GBSAscoring for the wild-type enzyme and the mutants variants. The wild-type enzyme yielded an averagedocking and MM-GBSA score of -۳۴.۸۱۹ kcal/mol, serving as a reference point for evaluating themutants. The research then proceeded with Alanine scanning using Discovery Studio ۲۰۱۸ to pinpointthe most stable enzyme-stabilizing mutations, leading to the creation of single, double, and triplemutants while preserving key residues. We identified G۳۶۰I, G۴R as the most potent mutant variant ofthe cellulase enzyme.To assess the behavior of the mutant cellulase under thermal stress, we plan toconduct molecular dynamics simulations that compare the mutant and native structures. In conclusion,we discovered a unique cellulase from an extreme environment and enhanced its stability through astreamlined computer-assisted protein engineering process. This modified enzyme could be beneficialfor industrial applications that require cellulase activity at high temperatures. [۱-۳]

کلیدواژه ها:

the cellulase enzyme ، thermal stability ، laboratory experiments ، computational methods