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Characterisation of a recombinant β-xylosidase (xylA) from Aspergillus oryzae expressed in Pichia pastoris.

Kirikyali N, Wood J, Connerton IF - AMB Express (2014)

Bottom Line: Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax).The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4).Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.

ABSTRACT
β-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae β-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with Km and Vmax values of 1.0 mM and 250 μmol min(-1) mg(-1) respectively against 4-nitrophenyl β-xylopyranoside. Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4). Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length.

No MeSH data available.


SDS-PAGE of recombinant β-xylosidase. Lane M, pre-stained Novex-Invitrogen protein marker. Lane 1, recombinant β-xylosidase.
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Figure 1: SDS-PAGE of recombinant β-xylosidase. Lane M, pre-stained Novex-Invitrogen protein marker. Lane 1, recombinant β-xylosidase.

Mentions: The β-xylosidase gene is contained within an open reading frame of 2397 nucleotides with no introns, which encodes a protein of 798 amino acids. A putative signal peptide was identified by SignalP software, thus the mature protein was predicted to be 778 amino acids with a molecular mass of 84.7 kDa. The recombinant enzyme was recovered from Pichia pastoris culture supernatant at approximately 100 mg L−1. NetNGly 1.0 predicted 12 potential N-glycosylation sites for β-xylosidase, and consistent with this, the recombinant enzyme was heterogeneous with a molecular mass estimated between 153 and 165 kDa on SDS-PAGE (Figure 1). The recombinant protein was excised from SDS-PAGE and the masses and protein sequences of tryptic peptides were determined using mass spectrometry to confirm the protein product was exo-1,4 β-xylosidase originating from Aspergillus oryzae.


Characterisation of a recombinant β-xylosidase (xylA) from Aspergillus oryzae expressed in Pichia pastoris.

Kirikyali N, Wood J, Connerton IF - AMB Express (2014)

SDS-PAGE of recombinant β-xylosidase. Lane M, pre-stained Novex-Invitrogen protein marker. Lane 1, recombinant β-xylosidase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230903&req=5

Figure 1: SDS-PAGE of recombinant β-xylosidase. Lane M, pre-stained Novex-Invitrogen protein marker. Lane 1, recombinant β-xylosidase.
Mentions: The β-xylosidase gene is contained within an open reading frame of 2397 nucleotides with no introns, which encodes a protein of 798 amino acids. A putative signal peptide was identified by SignalP software, thus the mature protein was predicted to be 778 amino acids with a molecular mass of 84.7 kDa. The recombinant enzyme was recovered from Pichia pastoris culture supernatant at approximately 100 mg L−1. NetNGly 1.0 predicted 12 potential N-glycosylation sites for β-xylosidase, and consistent with this, the recombinant enzyme was heterogeneous with a molecular mass estimated between 153 and 165 kDa on SDS-PAGE (Figure 1). The recombinant protein was excised from SDS-PAGE and the masses and protein sequences of tryptic peptides were determined using mass spectrometry to confirm the protein product was exo-1,4 β-xylosidase originating from Aspergillus oryzae.

Bottom Line: Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax).The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4).Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Food Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 5RD, UK.

ABSTRACT
β-xylosidases catalyse the hydrolysis of short chain xylooligosaccharides from their non-reducing ends into xylose. In this study we report the heterologous expression of Aspergillus oryzae β-xylosidase (XylA) in Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The recombinant enzyme was optimally active at 55°C and pH 4.5 with Km and Vmax values of 1.0 mM and 250 μmol min(-1) mg(-1) respectively against 4-nitrophenyl β-xylopyranoside. Xylose was a competitive inhibitor with a Ki of 2.72 mM, whereas fructose was an uncompetitive inhibitor reducing substrate binding affinity (Km) and conversion efficiency (Vmax). The enzyme was characterised to be an exo-cutting enzyme releasing xylose from the non-reducing ends of β-1,4 linked xylooligosaccharides (X2, X3 and X4). Catalytic conversion of X2, X3 and X4 decreased (Vmax and kcat) with increasing chain length.

No MeSH data available.