title: Expression and Characterization of Geobacillus stearothermophilus SR74 Recombinant α-Amylase in Pichia pastoris.
abstract: 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.
authors.affiliation: Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Sivasangkary
authors.initials: S
authors.lastname: Gandhi
authors.affiliation: Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia ; Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Abu Bakar
authors.initials: AB
authors.lastname: Salleh
authors.affiliation: Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Raja Noor Zaliha Raja Abd
authors.initials: RN
authors.lastname: Rahman
authors.affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Thean
authors.initials: T
authors.lastname: Chor Leow
authors.affiliation: Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
authors.forename: Siti Nurbaya
authors.initials: SN
authors.lastname: Oslan
mesh_terms: D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D000516:alpha-Amylases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D000516:alpha-Amylases