A vitamin-K2-binding factor secreted from Bacillus subtilis

The synthesis of vitamin K2 by Bacillus subtilis was found to increase in parallel with the number of cells. In the exponential growth phase, most vitamin K2 was found in the bacterial cells. At the end of the growth phase, however, the soluble form of vitamin K2 appeared in the incubation media and amounted to 40% of the total vitamin K2 at the stationary phase. DEAE ion-exchange chromatography of the incubation medium showed a single elution peak corresponding to vitamin K2, suggesting the secretion of vitamin K2 in the form of a soluble complex with a specific acidic binding factor. This factor was partially purified by consecutive DEAE chromatography, and examined by polyacrylamide disc gel electrophoresis. A band stainable with acridine orange, a cationic dye, was found to co-migrate with vitamin K2. This band was also visualized by periodic acid/Schiff staining. Gel-permeation chromatography showed that the binding factor in its active form has an apparent molecular mass of 100 kDa and is capable of binding exogenous menaquinone but not menadione. Chemical analysis indicated that this binding factor contained about 20% carbohydrate and 80% peptide which consisted mainly of Leu, Glu, Asp and Val. The new acidic glycoconjugate was designated as the vitamin-K2-binding factor.