Overexpression, purification and characterization of aspergillus niger beta-glucosidase in pichia pastoris

S. Kamaruddin, Farah Diba Abu Bakar, R. M. Illias, M. Said, Osman Hassan, Abdul Munir Abd. Murad

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 languageEnglish
Pages (from-to)7-11
Number of pages5
JournalMalaysian Applied Biology
Volume44
Issue number1
Publication statusPublished - 2015

Fingerprint

Pichia pastoris
beta-Glucosidase
Pichia
Aspergillus niger
beta-glucosidase
alcohol oxidase
enzymes
Glucosidases
Cellobiose
p-nitrophenol
Sodium Acetate
sodium acetate
cellobiose
glucosidases
Enzyme Assays
Enzymes
thermal stability
Recombinant Proteins
recombinant proteins
Methanol

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 journalArticle

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