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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Penerbit Universiti Kebangsaan Malaysia
2015
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| Online Access: | http://journalarticle.ukm.my/8690/1/44_1_02.pdf |
| _version_ | 1796930829426884608 |
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| author | Kamaruddin, S. Abu Bakar, F.D. Illias, R.M. Said, M. Hassan, O. Murad, A.M.A. |
| author_facet | Kamaruddin, S. Abu Bakar, F.D. Illias, R.M. Said, M. Hassan, O. Murad, A.M.A. |
| author_sort | Kamaruddin, S. |
| collection | UKM |
| 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. |
| first_indexed | 2024-03-06T04:08:59Z |
| format | Article |
| id | ukm.eprints-8690 |
| institution | Universiti Kebangsaan Malaysia |
| language | English |
| last_indexed | 2024-03-06T04:08:59Z |
| publishDate | 2015 |
| publisher | Penerbit Universiti Kebangsaan Malaysia |
| record_format | dspace |
| spelling | ukm.eprints-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 |
| 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 |
| title | 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_short | 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 |
| url | http://journalarticle.ukm.my/8690/1/44_1_02.pdf |
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