• Journal of Life Science
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• v.11 no.1
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• pp.42-46
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• 2001

Subtilisin YaB, produced by alkalophilic Bacillus strain YaB, is an extracellular alkaline serine protease having 55% homology to subtilisin BPN'. It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease. To examine the role of pro-sequence for the secretion of subtilisin YaB, we have studied the expression, in Bacillus subtilis, of a mutant preprosubtilisin YaB in which active site Ser214 is substituted with Cys. The use of a six protease-deficient strain, WB600, was required for its efficient production. The prosubtilisin YaB, thus produced, was indeed secreted into the culture medium and was processed to its mature form upon treatment with exogenously added active subtilisin YaB. From these results, we have concluded that the processing of pro-sequence is not essential for the secretion of the enzyme.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.315-318
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• 2005

The effects of inorganic salts and feather concentrations on pretense production by Bacillus megaterium F7-1 were investigated. Pretense production was dependent on the presence of phosphates in the medium. Supplementation of medium with calcium ion slightly increased protease production. The highest protease production was obtained at $1.4\%$ feather. The optimal medium contained $2.0\%$ glucose, $0.8\%$ skim milk, $0.06\%\;K_{2}HPO_{4}\%,\;0.04\%\;KH_{2}PO{4},\;0.06\%\;NaCl,\;0.03\%\;MgCl_{2}\cdot6H_{2}O,\;0.002\%\;CaCl_{2}\cdot2H_{2}O,\;and\;1.4\%$ whole feather. By using this optimized medium, increased production of the protease was achieved compared with the cases of using basal medium.

• Journal of the Korean Society of Food Science and Nutrition
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• v.20 no.4
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• pp.388-394
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• 1991

Alkaline protease producing bacteria were isolated from soil and identified as Bacillus sp. CW-1121. It was found that the production of alkaline protease reached to maximum in 5 day of fermentation at 4$0^{\circ}C$. The enzyme was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-150 and DEAE-cellulose ion-exchange chromatography. The homogeneity of the purified enzyme was verified by polyacrylamide gel electrophoresis. The enzyme was purified 5.72 fold and yield of the enzyme purification was 16.71%. When the purified enzyme was applied to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight was estimated to be 55, 000.

• Korean Journal of Food Science and Technology
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• v.32 no.1
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• pp.154-160
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• 2000

Protease production and its characteristics were investigated with Bacillus subtilis PCA20-3 which was isolated from Korean traditional meju. The optimum culture conditions of Bacillus subtilis PCA20-3 for the production of the protease were as follow: 0.2% soytone, 2% starch, 0.1% $(NH_4)_2SO_4,\;0.2%\;CaCl_2,\;0.01%\;yeast\;extract,\;0.1%\; K_2HPO_4,\;0.1%\;KH_2PO_4,\;pH\;7.0,\;30^{\circ}C$ and 20 hrs. The optimum pH and temperature for enzyme activity of protease producing Bacillus subtilis PCA20-3 were pH 8.0-10.0 and $55^{\circ}C$, respectively. The enzyme was relatively stable at pH $6.0{\sim}11.0$ and at temperature below $50^{\circ}C$. The activity of the enzyme was inhibited by $Fe^(2＋)\;and\;Cu^(2＋)$. 2 mM phenymethanesulfonyl fluoride inhibited 89.2% of enzyme activity. This indicates that the enzyme is serine protease. The $K_m$ value was $5\;{\times}\;10^{-4}\;M,\;V_{max}\;value\;was\;100\;{\mu}g/min$. This enzyme hydrolyzed casein more rapidly than bovine serum albumin.

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.26-32
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• 2002

For commercial production of Korean fermented anchovy sauce through rapid fermentation, a bacterial strain which showed the high protease activity was isolated from a commercially fermented anchovy sauce. The isolate was Bacillus amyloliquefaciens, and named as B. amyloliquefaciens HTP-8. The incubation temperature, initial pH, and cultivation time for optimal production of protease by B. amyloliquefaciens HTP-8 were $30^{\circ}C$, 7.0, and 3 days, respectively. In jar fermenter, B. amyloliquefaciens HTP-8 showed higher protease activity when grown at pH 7.0. The protease was partially purified by 80% ammonium sulfate precipitation and CM-Sephadex C-50 ion exchange chromatography. The partially purified enzyme had specific activity of 103.3 units/mg, yield of 0.4%, and purification fold of 43.0. The optimal pH and temperature for the protease activity were 10.0 and $50^{\circ}C$, respectively. The protease was relatively stable at the pH range of 7.0~12.0 and at the temperatures below 4$0^{\circ}C$. The activity of the enzyme was inhibited by $Ag^{＋}$ /, $Ba^{2＋}$ and selectively inhibited by PMSF, suggesting that it is a serine protease.

• Journal of the Korean Society of Food Science and Nutrition
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• v.20 no.1
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• pp.21-26
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• 1991

the Bacillus subtilis LY-353 which secretes the protease isolated from seafoods. The opti-mum culture condition for production of protease from B. subtilis LY-353 was as follows ; tem-perature 35$^{\circ}C$ pH 7.5 salt concentration 1.0% The purification steps involved ammonium sulfate fractionation DEAE-Sephadex A-50 column chromatography and Sephadex G-100 gel filtration A 7.33 fold purification and 6.55 yield of protease was obtained from culture broth, The optimum pH and temperature for the enzyme action were pH7.5 and 55$^{\circ}C$ respecti-bely.

• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1250-1256
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• 2011

In this study, Bacillus licheniformis cells were immobilized by entrapment in calcium alginate beads and were used for production of alkaline protease by repeated batch process. In order to increase the stability of the beads, the immobilization procedure was optimized by statistical full factorial method, by which three factors including alginate type, calcium chloride concentration, and agitation speed were studied. Optimization of the enzyme production medium, by the Taguchi method, was also studied. The obtained results showed that optimization of the cell immobilization procedure and medium constituents significantly enhanced the production of alkaline protease. In comparison with the free-cell culture in pre-optimized medium, about 7.3-fold higher productivity was resulted after optimization of the overall procedure. Repeated batch mode of operation, using optimized conditions, resulted in continuous production of the alkaline protease for 13 batches in 19 days.

• Food Science and Biotechnology
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• v.17 no.3
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• pp.519-526
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• 2008

To improve the quality of traditional kochujang, strains with high protease and amylase activity were isolated and identified from Sunchang traditional kochujang. Twenty-three strains strongly producing protease and 16 strains strongly producing $\alpha$- and $\beta$-amylase were isolated by using 1% isolated soy protein agar medium and 2% starch agar medium, respectively. Protease activities of the IA7, I5, and IA2 strain were 22.5, 21.2, and 20.6 unit/mL, respectively, and were higher than those of the other strains. Stains with high $\alpha$-amylase activity included K9 (967.8 unit/mL), K14 (828.3 unit/mL), K13 (662.5 unit/mL), K8 (601.5 unit/mL), and K11 (405.9 unit/mL). The $\beta$-amylase activity of the K11 strain was the highest, 34.3 unit/mL, among the isolated strains. Based on morphological, physiological properties, and API 50CHB-kit test for assimilation of 49 carbohydrates, 8 strains selected according to protease, $\alpha$-amylase, and $\beta$-amylase activities were tentatively identified as Bacillus megaterium (IA2), Bacillus subtilis (IA7, 15), Bacillus amyloliquefaciens (K8, K9, K11, and K13), and Bacillus stearothermophillus (K14). The IA7, 15, and K11 strains were finally identified as B. subtilis (99% ID) based on 16S rDNA sequencing.

• Applied Biological Chemistry
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• v.41 no.5
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• pp.349-354
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• 1998

The protease produced by Bacillus subtilis YG-95 was purified by precipitating with ammonium sulfate, DEAE-sepharose 6B and Sephadex G-100 column chromatogtaphies and its purified enzymological characteritics were investigated. The molecular weight of purified protease was estimated about 43kilodalton by SDS PAGE The optimum pH and temperature for the purified protease activity were pH 10.0 and $55^{\circ}C$, respectively. The enzyme was stable in broad range of pH 5.0 to 12.0. and at the below $45^{\circ}C$. The purified enzyme activty was inhibited by $Fe^{3+}$ and $Al^{3+}$. The activity was significantly inhibited more than 80% by O-Phenanthroline, PMSF and SDS. The $K_m$ value of the purified enzyme against Soy Protein Isolate as a substrate was 1.28 mg/ml.

• Journal of Environmental Health Sciences
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• v.12 no.2
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• pp.67-74
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• 1986

The study was carried out to investigate for the property of Bacillus strains, on the native growthed microflora in Korean native soybean paste, and Bacillus strains of the high enzyme producing, were selected and identificated, from the microflora, that is, identificated Bacillus strains beared resemblance to B. subtills, on the colony, appearance was pellicle, surface's spreading, color creamy-thin browned, colony elevation flated, and edge lobated, the identfficated B. subtills strain named for the B. subtilis SCF. For the protease activity of B. subtilis SCF, according to the variation with pH, the pH stability was pH 7~8, and on the its protease activity, optimum temperature was 40$\circ$C, on the other hand, temperature of the highest stability of the protease was 50$\circ$C.