A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.

A method of chemical phosphorylation was developed to modify soy protein so as to improve its functional properties. The reaction was carried out by incubating soy protein isolate and cyclic sodium trimetaphosphate in an aqueous solution at pH 11.5 and 35'C for about 3 hours. The reactions ensued were the phosphoesterification of serine residues and the phosphoramidation of lysine residues in soy protein. The phosphorylated soy protein isolate prepared therefrom exhibited much improved functional properties in terms of aqueous solubility, water-holding capacity, emulsifiability and whippability. The nutritive bioavailability of soy protein isolate was not impaired by phosphorylation.

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