Isolation Screening And Identification Of Strains Producing Nattokinase

Thrombus disease severely threatens the life and health of human beings and causes enormous material and mental losses to individuals, families and the society. However, thrombolytics, which is most commonly used to cure thrombus, does not work satisfactorily, due to serious side effect, short acting time, insufficient thrombolytic activity and difficulties in preparation. Thus the thrombolytics is expensive and requires a large dose taken in a long time.Natto, a kind of traditional food in Japan, is made by fermenting boiled soybean inoculated with Bacillus subtilis. Nattokinase is an alkaline Serine protease produced by Bacillus subtilis during the fermentation process, which has a high thrombolytic activity and long acting time. It can be administered orally or through intravenous injection, and causes no side effect such as bleeding. The merits above make nattokinase a prospective substitute of thrombolytics and new health product.In our study, strain N391 with high nattokinase producing activity and stable enzymatic activity, was isolated from five starting strains of Bacillus subtilis through fermentation and nattokinase activity assay of the fermentation products. We draw the following conclusions:1. After primary screening for the nattokinase producing strain and repetitious secondary screening on fibrin plate, 24 strains with high nattokinase producing activity were isolated. They are identified as Gram positive and the nattokinase activity are all above 1000U/mL, using standard curve of urokinase activity.2. Strain N391 was obtained via genetic stability test and organoleptic evaluation of natto solid fermentation. Its nattokinase activity is equivalent to 1722.4U/mL urokinase activity.3. Routine tests of N391, including morphology, physiological and biochemical characteristics, and comparison with Bacillus subtilis, preliminarily suggest that strain N391 is Bacillus subtilis.4. A 1511bp fragment of 16S ribosomal RNA was obtained from total DNA of strain N391 by PCR amplification, using the universal primer of bacterial 16S ribosome DNA. Sequence analysis was carried out by Blast on NCBI website and the phylogenetic tree was constructed by DNA MAN and MEGA3.1 software. The results show that N391 shared 99.60% 16S ribosomal RNA sequence homology with Bacillus subtilis(AB065370). Strain N391 and Bacillus subtilis were in the same branch of the phylogenetic tree and their genetic distance was the shortest. Combining the similarity of morphological and physiological characters, strain N391 was identified as Bacillus subtilis, and the position of the strain in microbiological phylogeny was set up.