The Genomic Study of Nattokinase Isolated from Soil Bacilli and its Cloning in Escherichia Coli for Medicinal Use

Nattokinase is an enzyme extracted from Japanese food produced in the fermentation process. This enzyme has significant positive effects in the treatment of cardiovascular diseases, the treatment or prevention of amyloid diseases such as Alzheimer disease and the reduction of blood cholesterol. This genomic study aimed to investigate the Nattokinase isolated from soil bacilli in areas around Tehran and its cloning in Escherichia coli bacteria for medicinal use.

In this study, 60 samples were collected from 5-10 cm depth of soil of different areas around Tehran and their Bacillus bacteria were isolated by molecular and biochemical methods. By the PCR method, the bacteria with the Nattokinase gene were identified and the amplified gene was cloned into E. coli and sequenced. The gene expression was then evaluated by Real Time PCR and its phylogenetic relationships were determined.

As a result of screening soil samples sent to the laboratory, a total of 12 suspected colonies were isolated from Gram-positive bacilli which were identified based on morphological, microscopic, and biochemical tests. Then, 11 species was determined as target genes. The cloning and expression of Nattokinase gene were successfully performed and the homology of 99-100% with other enzymes was observed.

In the present study, the Nattokinase gene from Bacillus subtilis RH5 isolated from soils around Tehran was successfully cloned into PTG19-T expression vector. The successful expression of its gene showed that it could be expanded by this method for industrial production.