Expression and characterization of Geobacillus stearothermophilus SR74 recombinant a-Amylase in Pichia pastoris

• 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/
• Institution: Universiti Putra Malaysia

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.