• Proceedings of the PSK Conference
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• 2002.10a
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• pp.332.2-333
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• 2002

Three alkaline proteases. designated JB-1. JB-2, and JB-3, are extracellular enzymes produced by Bacillus spp. JB411 which was isolated korean soil. They were separated by DEAE-sepharose CL-6B gel. and further purified using ammonium sulfate precipitation. ultra membrane filtration. and Ultrogel AcA gel filtration. The optimun pH values of proteases IB-1. JB-2. and JB-3. were shown to be 9.5. 9.5 and 7.5. respectively. All three proteases were stable in the pH range of 5-11. (omitted)

• Journal of Applied Biological Chemistry
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• v.49 no.4
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• pp.135-139
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• 2006

A alkalophilic strain, Bacillus licheniformis MH31 producing an alkaline protease was isolated from mine soil of Boryeong in Korea. Production of a high level of alkaline protease was achieved 42 h after incubation when the bacterium was grown at pH 9.0 and $35^{\circ}C$ in Horikoshi medium supplemented with 0.5%(w/v) starch and 1%(w/v) skim milk as carbon and nitrogen source, respectively. The molecular weight of partially purified enzyme was estimated to be 30 kDa by SDS-PAGE and its optimum pH was pH 10. The enzyme showed optimum temperature at $50^{\circ}C$, and was stable up to $60^{\circ}C$ after 1 h incubation. The protease was strongly inhibited by 1 mM of PMSF which was known well as strong inhibitor of serine proteases, but almost not inhibited by 5 mM of EDTA and 1,10-phenanthroline. When the protein hydrolysis products of 1% skim milk by partially purified protease was compared with available commercial proteases using HPLC analysis, most of hydrolysis products were detected below molecular weight of 10,000 and the hydrolysis ratio of purified enzyme was 24.8% lower than those(above 32%) of commercial proteases.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.103-106
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• 2000

Bacillus cereus KCTC 3674 excretes at least two kinds of extracellular proteases into the growth medium. Two major bands of the protease activity with molecular weights of approximately 100 and 38 kDa were obtained after gelatin-SDS-PAGE. The protease with a molecular weight of 38kDa was identified as an extracellular neutral (metallo-) protease. The neutral protease was quite thermostabile but labile to alkaline pH. On the contrary, the 100-kDa protease was thermolabile but stable to alkaline pH. The production of 38-kDa neutral protease was strongly affected by temperature, oxygen, carbonylcyanied m-chlorophenylhydrazone(CCCP) that was defined as a protonophofre, and cerulenin which inhibited lipid synthesis and caused changes in the membrane composition. On the other hand, the production of the 100-kDa protease was strongly affected by only temperature and cerulenin. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing proteases of Bacillus licheniformis, had no effect, whatsoever, on the production of extracellular proteases in B.cereus KCTC 3674.

• CELLMED
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• v.4 no.1
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• pp.6.1-6.12
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• 2014

Anthrax is the deadly disease for human being caused by Bacillus anthracis. Instantaneous research work on the mode of infection of the organism revealed that different proteases are involved in different steps of pathogenesis. Present study reports the in silico characterization and the detection of pathogenic proteases involved in anthrax infection through protein-protein interaction. A total of 13 acid, 9 neutral, and 1 alkaline protease of Bacillus anthracis were selected for analysing the physicochemical parameter, the protein superfamily and family search, multiple sequence alignment, phylogenetic tree construction, protein-protein interactions and motif finding. Among the 13 acid proteases, 10 were found as extracellular enzymes that interact with immune inhibitor A (InhA) and help the organism to cross the blood brain barrier during the process of infection. Multiple sequence alignment of above acid proteases revealed the position 368, 489, and 498-contained 100% conserved amino acids which could be used to deactivate the protease. Among the groups analyzed, only acid protease were found to interact with InhA, which indicated that metalloproteases of acid protease group have the capability to develop pathogenesis during B. anthracis infection. Deactivation of conserved amino acid position of germination protease can stop the sporulation and germination of B anthracis cell. The detailed interaction study of neutral and alkaline proteases could also be helpful to design the interaction network for the better understanding of anthrax disease.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.667-667
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enganced by the increase of agitation speed. Two formas of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0 and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both protease were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were 50℃ and 45℃, respectively. About 56% of the original protease BK7-2 activity remained after being treated at 50℃ for 30 min but protease BK7-1 was rapidly inactivated at above 25℃. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.677-684
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• 2000

Pseudomonas sp. BK7, an alkalophile, displayed the highest growth and protease activity when grown in a fermenter which was controlled at a pH level of 9.0, and the enzyme production was significantly enhanced by the increase of agitation speed. Two forms of alkaline proteases (BK7-1 and BK7-2) were fractionated and purified to near homogeneity. Protease BK7-1 was purified through CM-Sepharose CL-6B and Sephadex G-75 column chromatographies, and Protease BK7-2 was purified through CM-Sepharose CL-6B, DEAE-Sepharose, and Sephadex G-75 column chromatographies. The molecular weights of proteases BK7-1 and BK7-2 determined by gel filtration chromatography were 20,700 and 40,800, respectively. The $K_m$ value, isoelectric point, and optimum pH of protease BK7-1 were 2.55 mg/ml, 11.0, and 11.0, respectively, whereas those of protease BK7-2 were 1.57 mg/ml, 7.2, and 10.0, respectively. Both proteases were practically stable in the pH range of 5-11. The optimum temperatures for the activities of both protease BK7-1 and BK7-2 were $50^{\circ}C$ and $45^{\circ}C$, respectively. About 56% of the original protease BK7-2 activity remained after being treated at $50^{\circ}C$ for 30 min but protease BK7-1 was rapidly inactivated at above $25^{\circ}C$. Both proteases were completely inhibited by phenylmethane sulfonyl fluoride, a serine protease inhibitor. Protease BK7-2 was stable against EDTA, EGTA, STP, and detergents such as SDS and LAS, whereas protease BK7-1 was found to be unstable.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.501-506
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• 1992

An alkalophilic Vibrio sp. RH530 showing high proteolytic activity was isolated form soil samples by enrichment culture. The activity staining using gelatin SDS- polyacrylamide gel electrophoresis (PAGE ) revealed that the strain produced an alkaline major protease (Apr B) with a size of 27 kDa, and at least six minor proteases. The apparent sizes of four of the minor proteases were approximately 45, 28, 22 and 19 kDa. Apr B and five of the minor proteases were inhibited by serine protease inhibitors including PMSF and DFP, suggesting that they are serine proteases. One of the minor proteases was inhibited by metalloprotease inhibitors, not by serine protease inhibitors, indicating it to be a metalloprotease. Furthermore, the activities of Apr B and Prt 3 were not inhibited by SDS in the reaction mixture. The production of Apr B and some of the minor proteases was specifically affected by culture temperature (30 to 37.deg.C) and pH (7 to 10). The production of Apr B. Prt 2, Prt 5 and Prt 6 was mainly affected by culture temperature, while Prt 4 by culture pH. Prt 1 and Prt 3 were not affected by neither of these factors.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.181-186
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• 2000

MAPI (microbial alkaline protease inhibitor) was isolated from cultrue broth of Streptomyces chromofuscus SMF28. The Ki values of MAPI for the representative serine proteases such as chymotrypsin and proteinase K were 0.28 and $0.63{\;}\mu\textrm{M}$, respectively, and for the cysteine proteases cathepsin B and papain were 0.66 and $0.28{\;}\mu\textrm{M}$, respectively. These data indicate that MAPI is not a potent selective inhibitor of serine or cysteine proteases. Progress curves for the inhibition of three proteases by MAPI exhibithe characteristic patterns; MAPI exhibited slow-binding inhibition of cathepsin B. It was rapidly associated with chymotrypsin before the addition of substrate and then reactivation of MAPI-inhibited enzyme was investigated in the presence of substrate. On the other hand, MAPI-proteinase K interaction was typical for those classical inhibitors. When MAPI was incubated with trypsin, there was an extensive reduction in the ingibitory activities of MAPI corresponding to 66.5% inactivation of MAPI, indicating that trypsin-like protease may play a role in the decrease of the inhibitory activity during cultivation.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.331.1-331.1
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• 2002

Actinomycetes CS0703 has been isolated in soil sample from location in the Jeju province. Korea. and produces alkaline extracellular proteases. To maximize protease production, initial pH of the culture medium was adjusted to 12.0 with NaOH and incubated at $48^{\circ}C$ on a rotary shaking incubator(180rpm). Actinomycetes CS0703 produced high level of protease at late exponential phase when grown in OSYM medium (oatmeal 2.0%. soybean meal 1%. dried yeast 1%. mannitol 1%). (omitted)

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.639-644
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• 1998

Extracellular serine proteases were isolated from a soil bacterium, alkalophilic coryneform bacterium TU-19, which have been grown in a liquid medium optimized at 3$0^{\circ}C$ and pH 10.0. Three different sizes, 120 kDa (protease I), 80 kDa (protease II), and 45 kDa (protease III), of serine pro teases were purified using Sephadex G-150 and QAE-Sephadex chromatography (Kang et al. 1995. Agric. Chem Biotech. 38: 534-540). SDS-PAGE showed that the 120 kDa protease was degraded into the 80 kDa protease in 20 mM Tris-HCI (pH 8.0) buffer solution. This degradation was enhanced in the presence of 0.5 M NaCl and 5 mM EDTA, but was inhibited in the presence of 5 mM $CaCl_2$. These results indicated that the $Ca^{2＋}$ ion seems to stabilize the 120 kDa protease like other proteases derived from Bacillus species. The $NH_2$-terminal amino acid sequences of the 10 residues of both proteases were completely identical: Met-Asn-Thr-Gln-Asn-Ser-Phe-Leu-Ile-Lys. In contrast to this, the 80 kDa protease has 1.5 times higher specific activity than the 120 kDa protease does (Kang et al. 1995. Agric. Chern. Biotech. 38: 534-540). Therefore the C-terminal of the 120 kDa protease seems to be autolyzed to the 80 kDa protease but this autolysis did not decrease the protease activity. Optimum pH and temperature of both 80 kDa and 120 kDa proteases were pH 10.5 and $45^{\circ}C$, respectively, and pH and thermal stability were almost identical. Several divalent ions except the $Fe^{2＋}$ ion showed similar effects on activities of both proteases, which are similarly resistant to three different detergents.