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

Kamaruddin, Shazilah and Abu Bakar, Farah Diba and Illias, Rosli Md. and Said, Mamot and Hassan, Osman and Abdul Murad, Abdul Munir (2015) Overexpression, purification and characterization of aspergillus niger beta-glucosidase in pichia pastoris. Malaysian Applied Biology, 44 (1). pp. 7-11. ISSN 0126-8643

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Abstract

This study describes the expression of ß-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-ß-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.

Item Type:Article
Uncontrolled Keywords:aspergillus niger, heterologous expression, pichia pastoris, ß-glucosidase
Subjects:A General Works
ID Code:58742
Deposited By: Haliza Zainal
Deposited On:04 Dec 2016 12:07
Last Modified:27 Apr 2017 14:40

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