Abstract ▼ AI-Helper

Proteases are involved in every aspect of life, and are the most important kind of enzymes from an industrial point of view. Microbial proteases account for approximately 60% of the total enzyme sales in the world. Because of various application of enzyme in the industrial production, microorganisms...

Proteases are involved in every aspect of life, and are the most important kind of enzymes from an industrial point of view. Microbial proteases account for approximately 60% of the total enzyme sales in the world. Because of various application of enzyme in the industrial production, microorganisms producing protease were screened from the coastal sea water, seaweed and sea soil in Tong-yeong. The bacterial strain TS 0611, which showed the highest activity among isolated 5 strains producing proteolytic enzyme, was finally selected for further studies. The strain TS 0611 was a gram-positive rod, 1.2 um of cell length, catalase positive, motility-positive, melanin-negative and grew at 15～50℃, and hydrolyzed gelatin and casein. According to results of the API system(API 50 CHB), the strain was identified as Bacillus thuringiensis and Bacillus cereus. Also, isolated strain TS 0611 was identified as B. thuringiensis and B. cereus by MIDI with fatty acid composition of cell wall. 16s rRNA gene sequence of strain TS 0611 had identities of 99% compared with 16S rRNA sequence of Bacillus thuringiensis WS 2614. Strain TS 0611 was finally identified as Bacillus sp. TS 0611. The identified strain, Bacillus sp. TS 0611, was cultured at 27℃, 180 rpm with shaking on a medium containing 1.5% starch soluble, 0.5% peptone, 0.1% yeast extract, 0.0016 g ammonium nitrate, and 0.008 g disodium phosphate in 1 L of artificial sea water. After culture for 18 hours, the culture broth was centrifuged at 10,000 × g for 20 min and filtrated with glass microfiber filter. Ammonium sulfate was added slowly to the crude enzyme solution with gentle stirring from 60 to 80%, recovered proteins by centrifuge(10,000 × g for 20 min) was dissolved in 10 mM CaCl2, 20 mM Tris HCl(pH 7.0) buffer and dialyzed with same buffer. The dialysate was applied to HiLoad Q-Sepharose column(2.6 × 10 cm) equilibrated with same buffer, and active fraction from Q-Sepharose column was applied to Superdex-200 gel filtration column(1.6 × 60 cm) equilibrated with 0.1 M NaCl, 20 mM CaCl2, 20 mM Tris-HCl(pH 7.0). The purified protease showed a single band in native and SDS-polyacrylamide gel electrophoresis. The finally purified protease had specific activity of 133.7 units/mL/min, yield of 2.4% and purification fold of 90.7. Properties of the purified protease were investigated. As a result, molecular weight of the purified protease was 23 kDa and 20 kDa by Superdex-200 chromatography and SDS-PAGE, respectively. Optimum pH and temperature were 7.0(50 mM Tris-HCl, pH 7.0) and 40℃ for 1% casein, respectively. In case of thermal stability, remaining activity of the purified protease was 50% at 55℃. Km and Vmax values of protease against casein were 0.58% and 120 units(uM tyrosine/min/mL), respectively. The activity of the purified protease was increased by Ba2+, Ca2+, Li+, Mg2+ and Na+ ions compared with control, and greatly increased by Ca2+. The protease was inhibited by EDTA and 1,10- phenanthroline. Amino acid sequences of major peak did not show significant homology to any motifs of known protein. There were not protein corresponded with peptides generated by tryptic digestion based on NCBI database.

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