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

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 w...

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Main Author:	Damis, Siti Intan Rosdianah
Format:	Thesis
Language:	English
Published:	2020
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/id/eprint/92509/1/SitiIntanRosdinahPSChE2020_%20valet-20210309-113851.pdf.pdf
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spelling	my-utm-ep.925092021-09-30T15:11:20Z Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf 2020 Damis, Siti Intan Rosdianah TP Chemical technology 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. 2020 Thesis http://eprints.utm.my/id/eprint/92509/ http://eprints.utm.my/id/eprint/92509/1/SitiIntanRosdinahPSChE2020_%20valet-20210309-113851.pdf.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:139254 phd doctoral Universiti Teknologi Malaysia Faculty of Engineering - School of Chemical & Energy Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Damis, Siti Intan Rosdianah Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf
description	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.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Damis, Siti Intan Rosdianah
author_facet	Damis, Siti Intan Rosdianah
author_sort	Damis, Siti Intan Rosdianah
title	Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf
title_short	Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf
title_full	Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf
title_fullStr	Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf
title_full_unstemmed	Molecular engineering for catalytic efficiency of Xylanase from Aspergillus fumigatus RT-1 and its application in hydrolysis of pretreated kenaf
title_sort	molecular engineering for catalytic efficiency of xylanase from aspergillus fumigatus rt-1 and its application in hydrolysis of pretreated kenaf
granting_institution	Universiti Teknologi Malaysia
granting_department	Faculty of Engineering - School of Chemical & Energy Engineering
publishDate	2020
url	http://eprints.utm.my/id/eprint/92509/1/SitiIntanRosdinahPSChE2020_%20valet-20210309-113851.pdf.pdf
_version_	1747818599306756096

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

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