細菌Proteaseに関する研究 : I. Protease生産菌の分離と酵素の精製

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In the course of experiments on bacteria in milk, a strain of Bacillus cereus which produced a protease was isolated. Some experiments have been performed to isolate and purify the protease. The enzyme activity was measured by Casein-Folin calorimetric method. The results were obtained as follows;1) The bacillus is gram-positive, spore-forming and motile with peritrichous flagella. The organism is aerobic and grows well at 30°C, but does not at 55°C. The size of a cell is about 1.5×6μ. No acid or gas formed in medium containing xylose or arabinose, and acetylmethylcarbinol is produced. From these properties, the organism is regarded as a strain of B. cereus.2) The bacillus was cultured on agar slant containing glucose and skim milk for 24 hours. When the culture grown on this medium was inoculated in a synthetic medium (pH 7.0) containing 1% glucose, it excreted the protease in the medium after 7 day-incubation, and then the culture showed the highest degree of enzyme production.3) Concentrated cell-free culture was fractionated by means of methanol-fractionation. About 80% of the total enzyme activity was contained in 66-90% methanol fraction. This fraction was further purified through Sephadex G-50 equilibrated with 0.02M calcium acetate, and a fraction which showed simple peak by ultracentrifugation and single spot by electrophoresis was obtained. The sedimentation coefficient of purified enzyme was 20, ws=2.34S.4) Digestive ability against casein of the purified fraction was several times higher than some other commercial protease.5) The optimum temperature of this enzyme was 40°C and the optimum pH was 8.0.6) Loss of the enzyme activity by heat treatment and by stock at 5°C was remarkably protected in the presence of 0.02M calcium acetate.
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細菌Proteaseに関する研究 : I. Protease生産菌の分離と酵素の精製

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