Expression and Characterization ofGeobacillus stearothermophilusSR74 Recombinantα-Amylase inPichia 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 (t 1/2) 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.

Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus

“…Two criteria were emphasized when choosing the target proteins: thermostability and significant primary sequence heterogeneity between isoenzymes. As shown in Table 1 , the α-amylases from B. licheniformis (AmyL), B. subtilis (AmyE) and G. stearothermophilus (AmyS) selected for this study are reported to have temperature optima at 90 °C, 50 °C and 65 °C, respectively [ 77 – 79 ], while apr -encoded subtilisin from B. licheniformis has a temperature optimum at 50 °C, and the aprE -encoded protease from B. subtilis 168 has a homolog from B. subtilis A26 (91.34% identity) whose optimum temperature is at 60 °C [ 80 , 81 ]. Even though the α-amylases chosen for this study were derived from closely related organisms, their amino acid sequences differ substantially from the B. methanolicus -derived Amy, with sequence similarities of 22.47%, 27.62% and 24.86% for AmyS, AmyE and AmyL, respectively, ensuring that different signal peptides and also different model proteins were tested in B. methanolicus .…”

Simultaneous production of alpha and beta amylase enzymes using separate gene bearing recombinant vectors in the same Escherichiacoli cells

“…Molecular analysis showed that both proteins have molecular weights consistent with their monomeric native structure. In previous studies carried out by Gandhi et al (2015) and Özcan et al (2001), the molecular mass of the recombinant α amylase from Geobacillus stearothermophilus was calculated as 59 kDa and 65 kDa from Bacillus subtilis, respectively. The molecular mass of the recombinant β amylase from barley calculated as 60 kDa by Ziegler (1999).…”

Nanoparticle-Based Delivery of Anaplasma marginale Membrane Proteins; VirB9-1 and VirB10 Produced in the Pichia pastoris Expression System

“…Furthermore, expression of VirB10 protein in E. coli is difficult, with extremely low yields. Gandhi and colleagues demonstrated a two-fold increase in the yield of Geobacillus stearothermophilus derived SR74 recombinant β-Amylase using the P. pastoris expression system compared to an E. coli expression system [28]. …”