Comparative Fibrinolytic Activities of Nattokinases from Bacillus subtilis var. natto

Nattokinase is a potent fibrinolytic enzyme which was first found in a traditional Japanese soybean food. Two Nattokinase genes were cloned using chromosomal DNA from Bacillus subtilis (Ehrenberg 1835) Cohn 1872. Recombinant Natto-1.3 kb (GenBank accession number KF734090) and Natto-1.1 kb were expressed using a pQE-30 expression vector, and their fibrinolytic activities were studied. Natto-1.3 kb nucleotide sequence contained an open reading frame of 1332 base pairs encoding 106 amino acids for signal peptide and 275 amino acids for mature subtilisin. It showed 100, 99.74 and 98.69 % identities with subtilisin NAT, subtilisin E and subtilisin J from Bacillus subtilis, respectively. While the new Natto-1.1 kb sequence contained 1088 base pairs encoding only 87 amino acids for signal peptide and 275 amino acids for mature subtilisin. Nattokinase was produced by Bacillus subtilis at pH 7.0 in a fermentation medium (g%): 1.0 glucose, 5.5 peptone, 0.5 CaCl2, 0.2 MgSO4. Quantitative analysis of the fibrinolytic activity was conducted by the fibrin plate method using urokinase (20000 U/mg) as the reference standard. The fibrinolytic activities of Natto-1.3 kb and Natto-1.1 kb were 1000 ± 101 and 1187.5 ± 134 U/mg, respectively, compared to 400 ± 97 U/mg of the supernatant of the fermented culture broth of B. subtilis.