Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris

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 i...

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Main Authors:	Kamaruddin, S., Abu Bakar, F.D., Illias, R.M., Said, M., Hassan, O., Murad, A.M.A.
Format:	Article
Language:	English
Published:	Penerbit Universiti Kebangsaan Malaysia 2015
Online Access:	http://journalarticle.ukm.my/8690/ http://journalarticle.ukm.my/8690/ http://journalarticle.ukm.my/8690/1/44_1_02.pdf

id	ukm-8690
recordtype	eprints
spelling	ukm-86902016-12-14T06:47:54Z http://journalarticle.ukm.my/8690/ Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris Kamaruddin, S. Abu Bakar, F.D. Illias, R.M. Said, M. Hassan, O. Murad, A.M.A. 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 (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-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. Penerbit Universiti Kebangsaan Malaysia 2015-04 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/8690/1/44_1_02.pdf Kamaruddin, S. and Abu Bakar, F.D. and Illias, R.M. and Said, M. and Hassan, O. and Murad, A.M.A. (2015) Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris. Malaysian Applied Biology, 44 (1). pp. 7-11. ISSN 0126-8643 http://www.mabjournal.com/index.php?option=com_content&view=article&id=505&catid=59:current-view&Itemid=56
repository_type	Digital Repository
institution_category	Local University
institution	Universiti Kebangasaan Malaysia
building	UKM Institutional Repository
collection	Online Access
language	English
description	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 (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-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.
format	Article
author	Kamaruddin, S. Abu Bakar, F.D. Illias, R.M. Said, M. Hassan, O. Murad, A.M.A.
spellingShingle	Kamaruddin, S. Abu Bakar, F.D. Illias, R.M. Said, M. Hassan, O. Murad, A.M.A. Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris
author_facet	Kamaruddin, S. Abu Bakar, F.D. Illias, R.M. Said, M. Hassan, O. Murad, A.M.A.
author_sort	Kamaruddin, S.
title	Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris
title_short	Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris
title_full	Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris
title_fullStr	Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris
title_full_unstemmed	Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris
title_sort	overexpression, purification and characterization of aspergillus niger beta-glucosidase in pichia pastoris
publisher	Penerbit Universiti Kebangsaan Malaysia
publishDate	2015
url	http://journalarticle.ukm.my/8690/ http://journalarticle.ukm.my/8690/ http://journalarticle.ukm.my/8690/1/44_1_02.pdf
first_indexed	2023-09-18T19:53:01Z
last_indexed	2023-09-18T19:53:01Z
_version_	1777406345139453952

Overexpression, purification and characterization of Aspergillus niger beta-glucosidase in Pichia pastoris

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