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For the selection of detergent resistant and alkaline protease producing microorganisms, the alkalophilic microorganisms were isolated from various samples such as soils, composts, animal dungs, muds, waste water by growing in high alkaline media containing 1% of sodium carbonate and 0.5% of detergent. Among the 600 colonies which showed protease activity by forming a clear zone on the alkaline media containing 1% skim milk, an alkalophilich bacterium producing a quantitatuve amount of protease was selected. The characteristic point of this bacterium was especially a good growth in alkaline media, but no or scant growth was observed in neutral media, such as nutrient broth. The isolated and selected strain No. TG-423 was an aerobic, aporeforming, Gram positive, motile, rod-shaped bacterium with peritrichous flagella. From these points of view, it is clear that this bacterium belongs to the genus Bacillus. From the microbiological properties including the morphological, cultural, physiological, biochemical, and taxonomical characteristics, the strain No. TG-423 was very resemble to Bacillus alkalophilus. However, the strain was distinguished from the reference strain of B. alkalophilus in some characteristics such as nitrate and methylene blue reduction tests. Finally, the bacterium was identified as Bacillus sp. TG-423. The optimum pH for growth and the alkaline protease production of the bacterium was 10. and the bacterium grew at range of pH 7.5~12. The optimum temperature for growth and the alkaline protease production of the atrain was 40℃. Dextrine and soytone were found to be the most effective carbon and nitrogen sources for the formation of alkaline protease. The alkaline protease of Bacillus sp. TG-423 was purified by (NH₄)₂SO₄ Fractionation and dialysis. DEAE-Cellulose, CM-Sephadex column chromatography, and Sephades G-150 gel filtration. The enzyme was purified to homogeneity with specific activity of 9,850 unit/㎎ protein which means about 14 fold higher than that of the culture filtrate. The molecular weight of the enzyme was estimated as 25,000 by gel filtration method and 26,000 by SDS-polyacrylamide gel electrophoresis. The isoelectrie point of the enzyme was determined as 9.90 by isoelectric focusing method. The enzyme was the most active at pH 11.5 towards casein and stable at pH values from 6 to 12 for 10min, incubation at 50℃. Calcuum ion was effective to stabilize the enzyme especially at higher temperatures. The optimum temperature for the enzyme action of the purified enzyme was 55℃ in the presence of calcium ion. The enzyme was completely inactivated by urea and diisopropyl fluorophosphate, suggesting that the active site of the enzyme consisted of serine residue, but not affected by sulfhydryl reagent, ethylenediamine tetra acetic acid, sodium lauryl sulfate, and sodium dodecyl benzene sulfonate. Michaelis constant of the enzyme for casein was calculated to be 0.47% which indicated a excellent interaction between the enzyme and substrate.
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