Expression of gene encoding raw starch digesting α -amylase (Amyl III) in Escherichia coli

International Symposium on Bioremediation, Biomaterial, Revegetation, and Conservation

生物科学;自然科学（总论）

Anindyawati, T.^1

Research Center for Biotechnology, Indonesian Institute of Sciences, Jl. Raya Bogor Km. 46, Cibinong

16911, Indonesia^1

关键词: Alcohol fermentation;    Aspergillus awamori;    Expression vectors;    Glycosidic linkages;    Induction temperatures;    Industrial enzymes;    Protein expressions;    Waxy maize starch;   

Others  :  https://iopscience.iop.org/article/10.1088/1755-1315/308/1/012031/pdf
DOI  :  10.1088/1755-1315/308/1/012031

来源: IOP

【 摘 要 】

Amylase generally known as an industrial enzyme that hydrolyzes starch and glycogen. The α-amylase hydrolyzes α-1,4 glycosidic linkage that acts on raw starch. This reaction is very important for direct starch processing for alcohol fermentation and functional oligosaccharide or food additives development. Amyl III gene was isolated from Aspergillus awamori KT-11 and inserted into pDONR 201 as entry clone. The recombinant construct was subcloned into pDEST 17 plasmid as the expression vector and into E. Coli BL21AI cells. The objective of this study was to optimize induction temperatures in the expression process and to know the ability of recombinant Amyl III to hydrolyze raw starch. The expression study of Amyl III was performed using LB medium at 27°C and 37°C. Purification was done using affinity chromatography (His Tag) with 100 mM imidazole. Protein analysis was done using 7.5% of SDS-PAGE gel. Digestion on soluble starch and raw waxy maize starch by Amyl III was performed using TLC plate. The results show that the best protein expression was obtained by incubation at 27°C. Digestion on those starch by using recombinant Amyl III resulted in the production of malto-tetraose, -pentaose, -hexaose and -heptaose.