dc.contributor.author	Ayob, Siti Norbaidurah
dc.date.accessioned	2024-07-14T01:29:10Z
dc.date.available	2024-07-14T01:29:10Z
dc.date.issued	2017
dc.description	Thesis (PhD. (Bioprocess Engineering))
dc.description.abstract	Kenaf (Hibiscus cannabinus) is a lignocellulosic biomass with high amount of hemicellulose composition. The hemicellulose of kenaf contains high arabinoxylan and its hydrolysis is having challenge due to a high arabinofuranosyl branches on the xylan backbone. A branching enzyme such as a-Larabinofuranosidase is important for complete arabinoxylan hydrolysis. A hemicellulose from pre-treated kenaf stem was hydrolysed with a recombinant a-Larabinofuranosidase from Aspergillus niger ATCC120120 (AnabfA). Substrate specificity of AnabfA towards hemicellulose was successfully improved by reaction optimization and protein function approach. In the first approach, the reaction conditions were optimised using rotatable central composite design (CCD). The optimised reaction conditions obtained for AnabfA loading, substrate concentration and reaction time were identified as 88 U, 0.9% (w/v) and 48 h, respectively. The addition of AnabfA as a bio-catalyst reduced the activation energy (17.58 kJ/mol) for hydrolysis of hemicellulose in kenaf. In structure-guided protein engineering approach, molecular modeling and substrate docking were performed to study the structure function relationship for rational design. The study showed that a substrate binding site, interaction between enzyme-substrate and the residues involved in steric hindrance, affected the hydrolysis of hemicellulose. This information was used to design variants that have side chain with lower steric hindrance and higher flexibility. Variant N246D increased the nucleophilicity of water, resulting 32% higher hydrolysis activity towards ρ-nitrophenyl a-L-arabinofuranoside (ρNPA). This variant also showed higher preference towards hemicellulose by increasing the flexibility of enzyme through weakening its hydrogen bond interaction with substrate. Variants W453Y and L371 exhibited preference towards hemicellulose when compared to ρNPA. The mutation to less hydrophobic residue decreased hydrolytic activity against hydrophobic residue. The decrease in hydrophobicity increased enzyme flexibility and allowed larger substrate to complement the active site. Variant E449D, W453Y and L371V reduced steric interference and allowed larger substrate to penetrate into the active site easier. Variant E449D/W453Y induced the highest hydrolysis by producing up to 62% arabinose. However, this alteration did not cause significant effect on small substrate such as ρNPA. This finding can be studied further to produce more arabinose as a source for advanced biofuel
dc.description.sponsorship	Faculty of Chemical Engineering
dc.identifier.uri	http://openscience.utm.my/handle/123456789/1231
dc.language.iso	en
dc.publisher	Universiti Teknologi Malaysia
dc.subject	Protein engineering
dc.subject	Hydrolysis
dc.subject	Kenaf
dc.title	Characterisation and protein engineering of A. niger a- L arabinofuranosidase to improve substrate binding and hydrolysis activity
dc.type	Thesis
dc.type	Dataset

Characterisation and protein engineering of A. niger a- L arabinofuranosidase to improve substrate binding and hydrolysis activity

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