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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| Format: | Thesis |
| Language: | English |
| Published: |
2010
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| Subjects: | |
| Online Access: | http://eprints.utm.my/id/eprint/78295/1/NoorIzawatiAliasMFKK20101.pdf |
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| Summary: | 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. |
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