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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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Main Authors: Gandhi, Sivasangkary, Salleh, Abu Bakar, Raja Abdul Rahman, Raja Noor Zaliha, Thean, Adam Chor Leow, Oslan, Siti Nurbaya
Format: Article
Language:English
Published: Hindawi 2015
Online Access:http://psasir.upm.edu.my/id/eprint/44734/1/Expression%20and%20characterization%20of%20Geobacillus%20stearothermophilus%20.pdf
http://psasir.upm.edu.my/id/eprint/44734/
https://www.hindawi.com/journals/bmri/2015/529059/
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spelling my.upm.eprints.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
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
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.
format Article
author Gandhi, Sivasangkary
Salleh, Abu Bakar
Raja Abdul Rahman, Raja Noor Zaliha
Thean, Adam Chor Leow
Oslan, Siti Nurbaya
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
author_facet Gandhi, Sivasangkary
Salleh, Abu Bakar
Raja Abdul Rahman, Raja Noor Zaliha
Thean, Adam Chor Leow
Oslan, Siti Nurbaya
author_sort Gandhi, Sivasangkary
title 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_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_sort expression and characterization of geobacillus stearothermophilus sr74 recombinant a-amylase in pichia pastoris
publisher Hindawi
publishDate 2015
url http://psasir.upm.edu.my/id/eprint/44734/1/Expression%20and%20characterization%20of%20Geobacillus%20stearothermophilus%20.pdf
http://psasir.upm.edu.my/id/eprint/44734/
https://www.hindawi.com/journals/bmri/2015/529059/
_version_ 1698698913821753344
score 13.211869

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