Molecular expression of a recombinant thermostable bacterial amylase from Geobacillus stearothermophilus SR74 using methanol-free Meyerozyma guilliermondii strain SO yeast system

• Published in: Bioresources Vol. 15; no. 2; pp. 3161 - 3172
• Main Authors: Nasir, Nurul S. M., Leow, Chor T., Oslan, Siti N. H., Salleh, Abu B., Oslan, Siti N.
• Format: Journal Article
• Language: English
• Published: Raleigh North Carolina State University 01-05-2020
• Subjects: Alcohol oxidase; Amylases; Cloning; E coli; Enzymes; Fermentation; Formaldehyde dehydrogenase; Geobacillus stearothermophilus; Glycerol; Industrial applications; Iodine; Methanol; Meyerozyma guilliermondii; Optimization; Plasmids; Potassium; Proteins; Starch; Yeast; Yeasts; α-Amylase; United States > US
• DOI: 10.15376/biores.15.2.3161-3172
• EISSN: 1930-2126

α-Amylase, which was isolated from Geobacillus stearothermophilus SR74, has shown its potential to be used in industrial applications. However, its expression in the Pichia pastoris expression system with the alcohol oxidase 1 promoter (PAOX1) requires high methanol consumption and is time-consuming. This study aimed to express SR74 α-amylase in an alternative yeast system, using Meyerozyma guilliermondii strain SO, which was isolated from a spoiled orange (SO) under the regulation of a formaldehyde dehydrogenase promoter (PFLD). Qualitative screening showed that strain SO possessed a native amylase grown on YPD-starch plate at 30 °C. The recombinant SR74 α-amylase was further quantified and validated using the Western blot test. It was confirmed that SR74 α-amylase was expressed by strain SO extracellularly with a size of 59 kDa. Optimization in a shake flask showed that the recombinant SR74 α-amylase, which was regulated by PFLD, was successfully produced (26 U/mL) without any external inducer in the YPT medium after 24 h of cultivation. In conclusion, strain SO was able to produce SR74 amylase without methanol in one-fifth the fermentation time of P. pastoris. Further optimization of the expression may be done to improve the yield, as this methanol-free host is still underexplored.