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島根大学農学部研究報告 Volume 15
published_at 1981-12-15
土壌由来性細菌の生産するチアミナーゼIの精製とその酵素学的性質
Purification and Enzymological Properties of the Bacterial Thiaminase I Produced by a Stain Isolated from Soil
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Purification and enzymological properties of the bacterial thiaminase I produced by B. thiaminolyticus Strain, B isolated from soil has been described.
By applying the culture filtrate of the bacteria to ammonium sulfate fractionation and chromatographic fractionation using a DEAE-Sephadex A-50, Sephadex G-100(1st), Hydroxyl apatite, and Sephadex G-100(2nd), an enzyme preparation having ca. 260 times as much specific activity as that of the original crude enzyme was obtained. The enzyme was homogeneous on polyacrylamide disc gel electrophoresis and had a molecular weight, estimated by gel filtration, of 37,000 daltons. The optimum pH and the optimum temperature were 5.0 and 50℃ respectively. The metal ions such as <Cu>^<2+>, <Ag>^+, and <Hg>^<2+>, and PCMB remarkably inhibited the activity of the enzyme.
The kinetic parameters for the base-exchange reaction of the enzyme were determined at pH 5.0 and 50℃. The Km values for thiamine and pyridine were 2.7 and 12.8mM, respectively, and the V_max value was 120mg/mg protein/mm, expressed as the amount of thiamine decomposed.
These properties were compared with those of thiaminases from B. thiaminolyticus Matsukawa et Misawa and Cl. sporogenes.
By applying the culture filtrate of the bacteria to ammonium sulfate fractionation and chromatographic fractionation using a DEAE-Sephadex A-50, Sephadex G-100(1st), Hydroxyl apatite, and Sephadex G-100(2nd), an enzyme preparation having ca. 260 times as much specific activity as that of the original crude enzyme was obtained. The enzyme was homogeneous on polyacrylamide disc gel electrophoresis and had a molecular weight, estimated by gel filtration, of 37,000 daltons. The optimum pH and the optimum temperature were 5.0 and 50℃ respectively. The metal ions such as <Cu>^<2+>, <Ag>^+, and <Hg>^<2+>, and PCMB remarkably inhibited the activity of the enzyme.
The kinetic parameters for the base-exchange reaction of the enzyme were determined at pH 5.0 and 50℃. The Km values for thiamine and pyridine were 2.7 and 12.8mM, respectively, and the V_max value was 120mg/mg protein/mm, expressed as the amount of thiamine decomposed.
These properties were compared with those of thiaminases from B. thiaminolyticus Matsukawa et Misawa and Cl. sporogenes.
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