Identification of D۷۵C, E۲۹۶L as the most promising thermostable mutant of the acetylesterase enzyme from Trichoderma harzianum IOC-۳۸۴۴ by an in-silico approach

سال انتشار: 1402
محل انتشار: دوازدهمین همایش ملی و سومین همایش بین المللی بیوانفورماتیک
کد COI اختصاصی: IBIS12_210
زبان مقاله: انگلیسی
تعداد مشاهده: 121

نویسندگان

Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran

چکیده

Introduction: Trichoderma harzianum IOC-۳۸۴۴ has good characteristics for biomass degradation Its acetylesterase (EC ۳.۱.۱.۶) [۲] hydrolyzes glucuronoxylan acetyl side groups and plays a role in the biodegradation of xylan [۳]. It’s a safe alternative to harmful chemicals like chlorine as a bleaching agent in the pulp and paper industries [۴]. This study aims to improve the enzyme's thermal stability using an in-silico approach, making it suitable for high-temperature industrial applications. Methods: The study used Augustus gene prediction to identify the enzyme's coding gene and created a ۳D model using the Alphafold server. FoldX۵ was used for stability measurement, and key residues were found through the NCBI CDD database. Alanine scanning identified the best enzyme-stabilizing mutations, creating single, double, triple, and quadruple mutants while keeping key residues intact. and molecular docking and MM-GBSA scoring were performed on the wild-type enzyme and over thirty mutants. Four top candidates were chosen for molecular dynamics simulation to assess mutations' effect on enzyme thermal stability over time. Results: The enzyme model's stability was found to be -۳۶.۱۷ kcal/mol. However, all the mutants picked for docking showed better stability scores. This enzyme belongs to the SGNH hydrolase family and has four key residues: Ser۲۱, Gly۵۴, Asn۱۲۱, and His۲۹۳. The wild-type enzyme had an average docking and MM-GBSA score of -۲۱.۵۴ kcal/mol, with all four top mutants performing better. After running molecular dynamics and comparing geometrical parameters, including RMSD, RMSF, H-bonds, SASA, and Rg, for the wild-type enzyme and all four mutants at temperatures of ۳۷ and ۷۰ °C, we identified a double mutant (D۷۵C, E۲۹۶L) as the most promising variant for temperature stability. Conclusion: The study identifies D۷۵C, E۲۹۶L as the most thermostable and effective mutant of the acetylesterase enzyme, making it a safer alternative to hazardous chemicals and a viable solution for industrial applications.

کلیدواژه ها

Acetyl Esterase, Enzyme Engineering, Rational Design, Thermal Stabilization, Computational Biology

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