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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.


Related in: MedlinePlus

Enzyme activity profiles in the presence of (A) 20 mM carbohydrates and (B) metal ions and chemical compounds within reaction mixtures. All reactions were performed in 50 mM sodium phosphate buffer (pH 6.0) and 2 mM PNPX at 50°C. Control reaction performed in the absence of carbohydrates and chemicals under identical conditions.
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Figure 3: Enzyme activity profiles in the presence of (A) 20 mM carbohydrates and (B) metal ions and chemical compounds within reaction mixtures. All reactions were performed in 50 mM sodium phosphate buffer (pH 6.0) and 2 mM PNPX at 50°C. Control reaction performed in the absence of carbohydrates and chemicals under identical conditions.

Mentions: The catalytic activity of recombinant β-xylosidase was measured using 1 mM pNPX as substrate in the presence of 20 mM sugar solutions to test whether the enzyme was inhibited or enhanced (Figure 3A). In the presence of 20 mM xylose and fructose the catalytic activity was reduced to 18% and 26% respectively, with none of the others (arabinose, mannose, galactose, glucose and sucrose) showing any change in catalytic activity.


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

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

Enzyme activity profiles in the presence of (A) 20 mM carbohydrates and (B) metal ions and chemical compounds within reaction mixtures. All reactions were performed in 50 mM sodium phosphate buffer (pH 6.0) and 2 mM PNPX at 50°C. Control reaction performed in the absence of carbohydrates and chemicals under identical conditions.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Enzyme activity profiles in the presence of (A) 20 mM carbohydrates and (B) metal ions and chemical compounds within reaction mixtures. All reactions were performed in 50 mM sodium phosphate buffer (pH 6.0) and 2 mM PNPX at 50°C. Control reaction performed in the absence of carbohydrates and chemicals under identical conditions.
Mentions: The catalytic activity of recombinant β-xylosidase was measured using 1 mM pNPX as substrate in the presence of 20 mM sugar solutions to test whether the enzyme was inhibited or enhanced (Figure 3A). In the presence of 20 mM xylose and fructose the catalytic activity was reduced to 18% and 26% respectively, with none of the others (arabinose, mannose, galactose, glucose and sucrose) showing any change in catalytic activity.

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.


Related in: MedlinePlus