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Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf

Damis, Siti Intan Rosdianah (2020) Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf. PhD thesis, Universiti Teknologi Malaysia.

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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.

Item Type:Thesis (PhD)
Uncontrolled Keywords:polymers, pretreated kenaf, hydrolysis
Subjects:T Technology > TP Chemical technology
Divisions:Chemical and Energy Engineering
ID Code:92509
Deposited By: Narimah Nawil
Deposited On:30 Sep 2021 15:11
Last Modified:30 Sep 2021 15:11

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