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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.


The effect of temperature and pH on recombinant β-xylosidase. (A) The optimal temperature was measured in 50 mM sodium phosphate buffer (pH 6.0) in the presence of 1 mM PNPX at various temperatures for 15 minutes. (B) The optimum pH was determined by incubating at 50°C for 15 minutes in the presence of 2 mM PNPX in sodium phosphate buffer varying pH.
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Figure 2: The effect of temperature and pH on recombinant β-xylosidase. (A) The optimal temperature was measured in 50 mM sodium phosphate buffer (pH 6.0) in the presence of 1 mM PNPX at various temperatures for 15 minutes. (B) The optimum pH was determined by incubating at 50°C for 15 minutes in the presence of 2 mM PNPX in sodium phosphate buffer varying pH.

Mentions: The optimum temperature was determined by incubation of the recombinant enzyme in the presence of 50 mM sodium phosphate buffer pH 6.0 and 2 mM 4-nitrophenyl β-xylopyranoside (PNPX) for 15 min at varying temperatures between 20°C and 90°C. The enzyme displayed optimum activity at 55°C and retained more than 90% catalytic activity between the ranges of 50 – 60°C (Figure 2A).


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

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

The effect of temperature and pH on recombinant β-xylosidase. (A) The optimal temperature was measured in 50 mM sodium phosphate buffer (pH 6.0) in the presence of 1 mM PNPX at various temperatures for 15 minutes. (B) The optimum pH was determined by incubating at 50°C for 15 minutes in the presence of 2 mM PNPX in sodium phosphate buffer varying pH.
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Related In: Results  -  Collection

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

Figure 2: The effect of temperature and pH on recombinant β-xylosidase. (A) The optimal temperature was measured in 50 mM sodium phosphate buffer (pH 6.0) in the presence of 1 mM PNPX at various temperatures for 15 minutes. (B) The optimum pH was determined by incubating at 50°C for 15 minutes in the presence of 2 mM PNPX in sodium phosphate buffer varying pH.
Mentions: The optimum temperature was determined by incubation of the recombinant enzyme in the presence of 50 mM sodium phosphate buffer pH 6.0 and 2 mM 4-nitrophenyl β-xylopyranoside (PNPX) for 15 min at varying temperatures between 20°C and 90°C. The enzyme displayed optimum activity at 55°C and retained more than 90% catalytic activity between the ranges of 50 – 60°C (Figure 2A).

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.