Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris

A recombinant gene encoding α-ʟ-arabinofuranosidase (AnabfA) amplified from the genomic deoxyribonucleic acid (DNA) of Aspergillus niger ATCC 120120 was successfully cloned and expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promoter. Molecular analysis of the nucleotide s...

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Main Author:	Alias @ Che Rashid, Noor Izawati\
Format:	Thesis
Language:	English
Published:	2010
Subjects:	TP Chemical technology
Online Access:	http://eprints.utm.my/id/eprint/78295/1/NoorIzawatiAliasMFKK20101.pdf
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spelling	my-utm-ep.782952018-08-03T08:47:13Z Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris 2010 Alias @ Che Rashid, Noor Izawati\ TP Chemical technology A recombinant gene encoding α-ʟ-arabinofuranosidase (AnabfA) amplified from the genomic deoxyribonucleic acid (DNA) of Aspergillus niger ATCC 120120 was successfully cloned and expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promoter. Molecular analysis of the nucleotide sequence showed that the AnabfA gene contain eight exons and seven introns. Introns deletion was performed via modified overlap extension-polymerase chain reaction (MOE-PCR). The sequence was found to contain an open reading frame composed of 1887 base pairs (bp) nucleotides and encode 628 amino acid (aa) residues. Amino acids sequence analysis suggested that the mature enzyme was preceded by a 25 aa signal sequences. The effect of cultural conditions on recombinant AnabfA expression was studied and the enzyme was expressed as soluble protein. The recombinant AnabfA expressed as an active enzyme at 28°C when cultured in buffered complex methanol medium (BMMY), pH 6 and induced with 2% methanol for every 24 hours. Purified recombinant AnabfA before and after treatment with PNGase F migrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a relative molecular mass of 83 kDa and 66 kDa, suggesting that the AnabfA contains N-linked oligosaccharides. The optimum temperature and pH of recombinant AnabfA were 50°C and pH 4, respectively. The enzyme was stable at pH 2 to pH 6 and retained more than 80% of its activity after pre-incubation at 40°C for 30 minutes. Recombinant AnabfA activity was stimulated by K+, Mn2+, Na2+ and Triton X-100 and significantly inhibited by Cu2+ and Fe2+, while the enzyme activity was relatively unaffected by Ca2+, CO2+, Mg2+ and ethylenediamine-tetraacetate (EDTA). The Michaelis-Menten constant (Km) and maximum reaction velocity, (Vmax) of the recombinant AnabfA activity towards ρ-nitrophenyl α-ʟ-arabinofuranoside (ρNPA) were 0.93 mM and 17.86 μmol.ml-1.min-1, respectively. 2010 Thesis http://eprints.utm.my/id/eprint/78295/ http://eprints.utm.my/id/eprint/78295/1/NoorIzawatiAliasMFKK20101.pdf application/pdf en public http://dms.library.utm.my:8080/vital/access/manager/Repository/vital:78065 masters Universiti Teknologi Malaysia, Faculty of Chemical and Energy Engineering Faculty of Chemical and Energy Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	TP Chemical technology
spellingShingle	TP Chemical technology Alias @ Che Rashid, Noor Izawati\ Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris
description	A recombinant gene encoding α-ʟ-arabinofuranosidase (AnabfA) amplified from the genomic deoxyribonucleic acid (DNA) of Aspergillus niger ATCC 120120 was successfully cloned and expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promoter. Molecular analysis of the nucleotide sequence showed that the AnabfA gene contain eight exons and seven introns. Introns deletion was performed via modified overlap extension-polymerase chain reaction (MOE-PCR). The sequence was found to contain an open reading frame composed of 1887 base pairs (bp) nucleotides and encode 628 amino acid (aa) residues. Amino acids sequence analysis suggested that the mature enzyme was preceded by a 25 aa signal sequences. The effect of cultural conditions on recombinant AnabfA expression was studied and the enzyme was expressed as soluble protein. The recombinant AnabfA expressed as an active enzyme at 28°C when cultured in buffered complex methanol medium (BMMY), pH 6 and induced with 2% methanol for every 24 hours. Purified recombinant AnabfA before and after treatment with PNGase F migrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a relative molecular mass of 83 kDa and 66 kDa, suggesting that the AnabfA contains N-linked oligosaccharides. The optimum temperature and pH of recombinant AnabfA were 50°C and pH 4, respectively. The enzyme was stable at pH 2 to pH 6 and retained more than 80% of its activity after pre-incubation at 40°C for 30 minutes. Recombinant AnabfA activity was stimulated by K+, Mn2+, Na2+ and Triton X-100 and significantly inhibited by Cu2+ and Fe2+, while the enzyme activity was relatively unaffected by Ca2+, CO2+, Mg2+ and ethylenediamine-tetraacetate (EDTA). The Michaelis-Menten constant (Km) and maximum reaction velocity, (Vmax) of the recombinant AnabfA activity towards ρ-nitrophenyl α-ʟ-arabinofuranoside (ρNPA) were 0.93 mM and 17.86 μmol.ml-1.min-1, respectively.
format	Thesis
qualification_level	Master's degree
author	Alias @ Che Rashid, Noor Izawati\
author_facet	Alias @ Che Rashid, Noor Izawati\
author_sort	Alias @ Che Rashid, Noor Izawati\
title	Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris
title_short	Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris
title_full	Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris
title_fullStr	Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris
title_full_unstemmed	Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris
title_sort	isolation, expression and characterization of recombinant α-l-arabinofuranosidase from aspergillus niger atcc 120120 in pichia pastoris
granting_institution	Universiti Teknologi Malaysia, Faculty of Chemical and Energy Engineering
granting_department	Faculty of Chemical and Energy Engineering
publishDate	2010
url	http://eprints.utm.my/id/eprint/78295/1/NoorIzawatiAliasMFKK20101.pdf
_version_	1747817954599239680

Isolation, expression and characterization of recombinant α-L-arabinofuranosidase from Aspergillus niger ATCC 120120 IN Pichia pastoris

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