Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris
Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the ther...
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Hindawi
2015
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| Online Access: | http://psasir.upm.edu.my/id/eprint/44734/ http://psasir.upm.edu.my/id/eprint/44734/1/Expression%20and%20characterization%20of%20Geobacillus%20stearothermophilus%20.pdf |
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| author | Gandhi, Sivasangkary Salleh, Abu Bakar Raja Abdul Rahman, Raja Noor Zaliha Thean, Adam Chor Leow Oslan, Siti Nurbaya |
| author_facet | Gandhi, Sivasangkary Salleh, Abu Bakar Raja Abdul Rahman, Raja Noor Zaliha Thean, Adam Chor Leow Oslan, Siti Nurbaya |
| author_sort | Gandhi, Sivasangkary |
| building | UPM Institutional Repository |
| collection | Online Access |
| description | Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL−1 at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification. |
| first_indexed | 2025-11-15T10:06:35Z |
| format | Article |
| id | upm-44734 |
| institution | Universiti Putra Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-15T10:06:35Z |
| publishDate | 2015 |
| publisher | Hindawi |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | upm-447342021-04-22T08:12:50Z http://psasir.upm.edu.my/id/eprint/44734/ Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris Gandhi, Sivasangkary Salleh, Abu Bakar Raja Abdul Rahman, Raja Noor Zaliha Thean, Adam Chor Leow Oslan, Siti Nurbaya Geobacillus stearothermophilus SR74 is a locally isolated thermophilic bacteria producing thermostable and thermoactive α-amylase. Increased production and commercialization of thermostable α-amylase strongly warrant the need of a suitable expression system. In this study, the gene encoding the thermostable α-amylase in G. stearothermophilus SR74 was amplified, sequenced, and subcloned into P. pastoris GS115 strain under the control of a methanol inducible promoter, alcohol oxidase (AOX). Methanol induced recombinant expression and secretion of the protein resulted in high levels of extracellular amylase production. YPTM medium supplemented with methanol (1% v/v) was the best medium and once optimized, the maximum recombinant α-amylase SR74 achieved in shake flask was 28.6 U mL−1 at 120 h after induction. The recombinant 59 kDa α-amylase SR74 was purified 1.9-fold using affinity chromatography with a product yield of 52.6% and a specific activity of 151.8 U mg−1. The optimum pH of α-amylase SR74 was 7.0 and the enzyme was stable between pH 6.0–8.0. The purified enzyme was thermostable and thermoactive, exhibiting maximum activity at 65°C with a half-life (t1/2) of 88 min at 60°C. In conclusion, thermostable α-amylase SR74 from G. stearothermophilus SR74 would be beneficial for industrial applications, especially in liquefying saccrification. Hindawi 2015 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/44734/1/Expression%20and%20characterization%20of%20Geobacillus%20stearothermophilus%20.pdf Gandhi, Sivasangkary and Salleh, Abu Bakar and Raja Abdul Rahman, Raja Noor Zaliha and Thean, Adam Chor Leow and Oslan, Siti Nurbaya (2015) Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris. BioMed Research International, 2015. art. no. 529059. pp. 1-9. ISSN 2314-6133; ESSN: 2314-6141 https://www.hindawi.com/journals/bmri/2015/529059/ 10.1155/2015/529059 |
| spellingShingle | Gandhi, Sivasangkary Salleh, Abu Bakar Raja Abdul Rahman, Raja Noor Zaliha Thean, Adam Chor Leow Oslan, Siti Nurbaya Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris |
| title | Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris |
| title_full | Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris |
| title_fullStr | Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris |
| title_full_unstemmed | Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris |
| title_short | Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris |
| title_sort | expression and characterization of geobacillus stearothermophilus sr74 recombinant a-amylase in pichia pastoris |
| url | http://psasir.upm.edu.my/id/eprint/44734/ http://psasir.upm.edu.my/id/eprint/44734/ http://psasir.upm.edu.my/id/eprint/44734/ http://psasir.upm.edu.my/id/eprint/44734/1/Expression%20and%20characterization%20of%20Geobacillus%20stearothermophilus%20.pdf |