Cloning and over expression of alpha-amylase gene isolated from Bacillus licheniformis and determination of biochemical characterizations of this recombinant enzyme
Subject Areas : Bacteriology
Ashraf Kariminik
1
,
Kobra Saeedi
2
1 - PhD Student, Department of Microbiology, Kerman Branch, Islamic Azad University, Kerman, Iran
2 - PhD Student, Department of Microbiology, Kerman Branch, Islamic Azad University, Kerman, Iran
Keywords: Recombinant protein, Alpha amylase, pET-26b (+), Bl21 (DE3),
Abstract :
Background & Objectives: α-amylase, is an endoamylase to hydrolyses amylase, amylopectin and glycogen by randomly cleavage of internal alpha 1, 4 linkages. Genus Bacillus is one of the most common microbial sources of α-amylase production. The aim of the present study is identification, overexpression and activity analysis of Bacillus licheniformis α-amylase (amyS). Materials & Methods: A polymerase chain reaction was used to identify and isolate α-amylase gene in Bacillus sp. The fragments were introduced into expression vector pET-26 (+). After sequencing, the recombinant vectors were introduced into E. coli BL21 (DE3) for expression. The recombinant enzyme were purified using amicon filter and dialysis bags. α-amylase activity was measured using di-nitro salicylic acid technique. Results: Based on the results, the α-amylase gene was over-expressed in an expression system beyond the native host (Bacillus sp.). Based on the SDS-PAGE electrophoresis, the molecular weight of this enzyme was predicted 54 KDa. The α-amylase showed an enzyme activity about 4.77 U/ml. Conclusion: These results indicated a high expression of this enzyme in an expression system beyond the host, which was due to existence of a strong promoter used in this study, T7promoter. As a result, employment of this system in an industrial scale is recommended due to the secretion of the target enzyme, a proper folding of the enzyme and maintenance of its activity.
