Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf
Date
2020
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Teknologi Malaysia
Abstract
The lignocellulose of industrial crops consists of three main polymers: cellulose, hemicellulose, and lignin. The combination of these complex and heterogeneous polymers contributes to the recalcitrant structure of lignocellulose. Thus, it becomes a drawback for a group of hydrolytic enzymes which work synergistically to hydrolyse the lignocellulosic substrate including xylanase. Hence, this study aimed to improve the catalytic efficiency of Aspergillus fumigatus RT-1 xylanase (AfxynG1) on pretreated kenaf hydrolysis through protein engineering of amino acids that located near the substrate-binding site and at the N-terminal region. Molecular docking analysis revealed 5 subsites; -3, -2, -1, +1, and +2 and several of substrate-binding residues which distributed alongside the subsites. Two putative binding residues of Phe 146 and Phe 30 and a putative secondary binding site of residue Tyr 7 were determined. High-throughput and low-throughput screenings of 5000 clones from error-prone PCR library which acted as fine tuner and 414 clones from site-saturation mutagenesis library were successfully performed to screen out three improved mutants; c168t, Q192H, and Y7L. The site-directed mutagenesis was applied to construct double and triple mutants and this process resulted in only two improved mutants; c168t/Q192H and c168t/Q192H/Y7L. The triple mutant c168t/Q192H/Y7L was the most stable enzyme in high temperature 60 and 70 °C and acidic pH 3-6, while the double mutant c168t/Q192H showed to contribute to the most effective hydrolysis reaction with a 7.6-fold increase in catalytic efficiency. Mutant Y7L produced the highest sugar yield with 28 % increase in pretreated kenaf hydrolysis. Overall, these improved mutants are feasible to be used synergistically with cellulases for bioconversion of lignocellulose into reducing sugar.
Description
Thesis (PhD. (Bioprocess Engineering))
Keywords
Hydrolysis—Industrial applications, Molecular microbiology, Hydrolases