Abstract
This study describes the expression of β-glucosidase (BglA) from Aspergillus niger in Pichia pastoris, a methylotrophic yeast strain, under the regulation of an alcohol oxidase promoter. The heterologous expression of BglA was optimized in a shake flask. Optimal conditions were achieved using an initial cell density (OD 600) of 4-5 and an inducer concentration of 2.5% methanol for 72 hours. A recombinant protein with a molecular weight of ~116 kDa was produced. This recombinant BglA has optimal activity at 60°C in sodium acetate buffer at pH 4. This enzyme is stable between pH 3.0-6.0 and retained more than 50% of its maximum activity at pH 6.0 after incubation at 60°C for 30 min. However, it lost almost 80% of its maximal activity at pH 7.0 under the same conditions. A thermostability assay of this enzyme revealed that BglA is relatively stable up to 60°C. This enzyme retained 50% of its original activity at 60°C but was completely inactive after incubation at 70°C for 30 min. BglA showed highest activity and specificity towards the synthetic substrate p-nitrophenol-β-Dglucopyranoside with a specific activity of 347.62 U mg -1 and a specificity constant of 466.19 mL mg -1s-1 . BglA had a specific activity of 6.2 U mg -1 and a specificity constant of 6.01 mL mg -1s-1 for cellobiose.
| Original language | English |
|---|---|
| Pages (from-to) | 7-11 |
| Number of pages | 5 |
| Journal | Malaysian Applied Biology |
| Volume | 44 |
| Issue number | 1 |
| Publication status | Published - 2015 |
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Keywords
- Aspergillus niger
- Heterologous expression
- Pichia pastoris
- β-glucosidase
ASJC Scopus subject areas
- Agricultural and Biological Sciences(all)
Cite this
Overexpression, purification and characterization of aspergillus niger beta-glucosidase in pichia pastoris. / Kamaruddin, S.; Abu Bakar, Farah Diba; Illias, R. M.; Said, M.; Hassan, Osman; Abd. Murad, Abdul Munir.
In: Malaysian Applied Biology, Vol. 44, No. 1, 2015, p. 7-11.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Overexpression, purification and characterization of aspergillus niger beta-glucosidase in pichia pastoris
AU - Kamaruddin, S.
AU - Abu Bakar, Farah Diba
AU - Illias, R. M.
AU - Said, M.
AU - Hassan, Osman
AU - Abd. Murad, Abdul Munir
PY - 2015
Y1 - 2015
N2 - This study describes the expression of β-glucosidase (BglA) from Aspergillus niger in Pichia pastoris, a methylotrophic yeast strain, under the regulation of an alcohol oxidase promoter. The heterologous expression of BglA was optimized in a shake flask. Optimal conditions were achieved using an initial cell density (OD 600) of 4-5 and an inducer concentration of 2.5% methanol for 72 hours. A recombinant protein with a molecular weight of ~116 kDa was produced. This recombinant BglA has optimal activity at 60°C in sodium acetate buffer at pH 4. This enzyme is stable between pH 3.0-6.0 and retained more than 50% of its maximum activity at pH 6.0 after incubation at 60°C for 30 min. However, it lost almost 80% of its maximal activity at pH 7.0 under the same conditions. A thermostability assay of this enzyme revealed that BglA is relatively stable up to 60°C. This enzyme retained 50% of its original activity at 60°C but was completely inactive after incubation at 70°C for 30 min. BglA showed highest activity and specificity towards the synthetic substrate p-nitrophenol-β-Dglucopyranoside with a specific activity of 347.62 U mg -1 and a specificity constant of 466.19 mL mg -1s-1 . BglA had a specific activity of 6.2 U mg -1 and a specificity constant of 6.01 mL mg -1s-1 for cellobiose.
AB - This study describes the expression of β-glucosidase (BglA) from Aspergillus niger in Pichia pastoris, a methylotrophic yeast strain, under the regulation of an alcohol oxidase promoter. The heterologous expression of BglA was optimized in a shake flask. Optimal conditions were achieved using an initial cell density (OD 600) of 4-5 and an inducer concentration of 2.5% methanol for 72 hours. A recombinant protein with a molecular weight of ~116 kDa was produced. This recombinant BglA has optimal activity at 60°C in sodium acetate buffer at pH 4. This enzyme is stable between pH 3.0-6.0 and retained more than 50% of its maximum activity at pH 6.0 after incubation at 60°C for 30 min. However, it lost almost 80% of its maximal activity at pH 7.0 under the same conditions. A thermostability assay of this enzyme revealed that BglA is relatively stable up to 60°C. This enzyme retained 50% of its original activity at 60°C but was completely inactive after incubation at 70°C for 30 min. BglA showed highest activity and specificity towards the synthetic substrate p-nitrophenol-β-Dglucopyranoside with a specific activity of 347.62 U mg -1 and a specificity constant of 466.19 mL mg -1s-1 . BglA had a specific activity of 6.2 U mg -1 and a specificity constant of 6.01 mL mg -1s-1 for cellobiose.
KW - Aspergillus niger
KW - Heterologous expression
KW - Pichia pastoris
KW - β-glucosidase
UR - http://www.scopus.com/inward/record.url?scp=84946184696&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84946184696&partnerID=8YFLogxK
M3 - Article
AN - SCOPUS:84946184696
VL - 44
SP - 7
EP - 11
JO - Malaysian Applied Biology
JF - Malaysian Applied Biology
SN - 0126-8643
IS - 1
ER -