1. Purification of the thiaminase I produced by B. thiaminolyticus Matsukawa et Misawa has been described. By applying the 70% ammonium sulfate fraction of crude thiaminase I to chromatographic fractionation using an ion exchange resin, Duolite A2, and zone electrophoresis, an enzyme preparation having 200 times as much specific activity as that of the original crude enzyme was obtained. Ultracentrifugal analysis indicated that the preparation contained a single component and was free from any organic bases. The purified thiaminase alone does not show any thiaminase activity, but its activity appears in the presence of an organic base (pyridine) leading to the formation of heteropyrithiamine.
2. K_m for thiamine and pyridine of the purified enzyme were 0.9×10^-3 and 1.0×10^-3M at pH 6.5 and 30°, respectively, according to Lineweaver and Burk's method. V_max was 190mg/min/mg protein-N, expressed as the amount of thiamine decomposed.
3. The sedimentation constant and other observations suggest a molecular weight of approximately 40, 000.
4. According to the results of inhibition experiments, the enzyme is considered to be an SH-enzyme.