Malaysian Applied Biology Journal

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44_1_02

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Malays. Appl. Biol. (2015) 44(1): 07-11

OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF Aspergillus niger BETA-GLUCOSIDASE IN Pichia pastoris

KAMARUDDIN, S.1, ABU BAKAR, F.D.1, ILLIAS, R.M.2, SAID, M.3, HASSAN, O.3 and MURAD, A.M.A.1*

1School of Biosciences & Biotechnology, Faculty Science & Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

2Department of Bioprocess Engineering, Faculty of Chemical Engineering & Natural Resources Engineering, Universiti Teknologi Malaysia,

81300 Skudai, Johor, Malaysia

3School of Chemical Sciences & Food Technology, Faculty Science & Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

*Email: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

ABSTRACT

This study describes the expression of ?-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 (OD600) 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-?-D-glucopyranoside 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.

Key words: Aspergillus niger, ?-glucosidase, Pichia pastoris, heterologous expression


 

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