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 |
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Uncontrolled Keywords: | aspergillus niger, heterologous expression, pichia pastoris, ß-glucosidase |
Subjects: | Q Science > Q Science (General) |
Divisions: | Biosciences and Medical Engineering |
ID Code: | 58742 |
Deposited By: | Haliza Zainal |
Deposited On: | 04 Dec 2016 04:07 |
Last Modified: | 08 Aug 2021 06:42 |
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