• 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.

• Microbiology and Biotechnology Letters
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• v.18 no.4
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• pp.344-351
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• 1990

An alkalophilic microoganism producing a detergent-resistant alkaline protease was isolated from soil and identified as Baeiltus sp. The alkaline protease has been partially purified by ammonium sulfate fractionation, DEAE-Cellulose, CM-Cellulose and Sephdex G-100 column chromatography. The purified alkaline protease was highly active at pH 12-13 toward casein and stable at pH values from 6 to ll. The optimum temperature for the enzyme reaction was $55^{\circ}C$. The enzyme was completely inactivated by diisopropyl fluorophosphate (DFP) indicating that the enzyme was serine protease, but considerabiy stable in the presence of surface active agents.

• The Korean Journal of Mycology
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• v.33 no.2
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• pp.69-74
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• 2005

Fibrinolytic enzyme has been isolated and purified from the edible mushroom, Lepista nuda. The apparent molecular mass of purified enzyme was estimated to be 34 KDa by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the enzyme was Tyr-Pro-Ser-Pro-Ser-His-Gln-Thr-Ala-Val-Asn-Ala-Ile-Ile-X. It has a pH optimum at $7.0.{\sim}9.5$, suggesting that the purified enzyme is an alkaline protease. It shows the maximum fibrinolytic activity at $55^{\circ}C$. The fibrinolytic activity was inhibited by phenylmethylsulfonyl fluoride, indicating that the purified enzyme is a serine protease. The activity of the purified enzyme was totally inhibited by $Hg^{2+}$.

• 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.

• BMB Reports
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• v.33 no.2
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• pp.112-119
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• 2000

Three kinds of serine protease inhibitors, members of the Bowman-Birk trypsin inhibitor, were purified from Dolichos lablab seeds and named Dolichos protease inhibitor 1, 2 and 3 (DI-1, DI-2 and DI-3), respectively. Each inhibitor showed a single band with gel mobility at around 15.9, 12.1 and 14.6 kDa on 20% SDS-PAGE under reducing conditions. To characterize inhibitory specificity, the inhibition constant (Ki) for these inhibitors was measured against several known serine proteases. All three Dolichos protease inhibitors (DI-1, DI-2 and DI-3) inhibited the activity of trypsin and plasmin, but had no effect on thrombin and kallikrein (either for human plasma kallikrein or for porcine pancreas kallikrein). DI-1 inhibited chymotrypsin most effectively (Ki = $3.6{\times}10^{-9}\;M$), while DI-2 displayed inhibitory activity for porcine pancreatic elastase (Ki = $6.2{\times}10^{-8}\;M$). Pre-treatment of the 33 mg/kg of DI-mixture (active fractions from $C_{18}$ open column chromatography that included DI-1, DI-2 and DI-3) inhibited the induction of pseudomonal elastase-induced septic hypotension and prevented an increase in bradykinin generation in pseudomonal elastase-treated guinea pig plasma. Also, the increase of kallikrein activity, by injection of pseudomonal elastase, was inhibited by the pretreatment of the DI-mixture in a guinea pig. Since the DI-mixture had no inhibitory effect on kallikrein activity when Z-Phe-Arg-MCA was used as a substrate in vitro, its inhibitory activity in the pseudomonal elastase-induced septic hypotension model might not be due to a direct inhibition of plasma kallikrein in the activation cascade of the Hageman factor and prekallikrein system. These results suggest that the Dolichos DI-mixture might be used as an inhibitor in pathogenic bacterial protease-induced septic shock.

• Journal of Microbiology and Biotechnology
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• v.32 no.1
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• pp.99-109
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• 2022

This study is the first report on production and characterization of the enzyme from an Ornithinibacillus species. A 4.2-fold increase in the extracellular protease (called L9^T) production from Ornithinibacillus caprae L9^T was achieved through the one-factor-at-a-time approach and response surface methodological optimization. L9^T protease exhibited a unique protein band with a mass of 25.9 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This novel protease was active over a range of pH (4-13), temperatures (30-80℃) and salt concentrations (0-220 g/l), with the maximal activity observed at pH 7, 70℃ and 20 g/l NaCl. Proteolytic activity was upgraded in the presence of Ag⁺, Ca²⁺ and Sr²⁺, but was totally suppressed by 5 mM phenylmethylsulfonyl fluoride, which suggests that this enzyme belongs to the serine protease family. L9^T protease was resistant to certain common organic solvents and surfactants; particularly, 5 mM Tween 20 and Tween 80 improved the activity by 63 and 15%, respectively. More importantly, L9^T protease was found to be effective in dehairing of goatskins, cowhides and rabbit-skins without damaging the collagen fibers. These properties confirm the feasibility of L9^T protease in industrial applications, especially in leather processing.

• Microbiology and Biotechnology Letters
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• v.22 no.5
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• pp.515-521
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• 1994

A bacterial strain, which showed the high protease activity at low temperature and the high tolerance for the surfactant, was isolated from soil and identified as Xanthomonas sp. YL-37. The optimal temperature, initial pH, and cultivation time for the production of the alkaline protease by Xanthomonas sp. YL-37 were 20$\circC , 11.0, and 84 hours, respectively. In the jar fermenter culture of Xanthomonas sp. YL-37, the alkaline protease activity was about 15,000 DU/ml/-broth after cultivating for 108 hours. The optimal pH and temperature for the protease activity were 70$\circC and 11.0, respectively. The protease was relatively stable at the pH range of 7.0~12.0 and at the temperatures below 50$\circC . The protease activity at 20$\circC was about the level of 40% of its activity at 70$\circC . The enzyme was suggested as a serine protease because the enzyme activity was inhibited by phenylmethane sulfonyl fluoride, a serine modifier.

• Parasites, Hosts and Diseases
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• v.27 no.3
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• pp.161-170
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• 1989

The proteases of Toxoplasma gcndii were purified partially and characterisrd for some biochemical properties including various chromatographic patterns, major catalytic classes, and conditions to promote the activity of these enzymes. When Toxoplasma extract was incubated with 3H-casein at various pH, peak hydrolysis of casein was observed at pH 6.0 and at pH 8.5. Proteasfs working at pH 6.0 and at pH 8.5 were purified partially by conventional methods of chromatographies of DE52 anion rxchange, Sephadex G-200 gel permeation, and hydroxylapatite chromatography. Partially purified enzymes were tested by site-specific inhibitors and promotorf. The protease working at pH 6.0 was inactivated by iodoacetamide with LDso of 10-5 M and promoted by dithiothreitol, while the protease working at pH 8.5 was inhibited by phenylmethylsulfonyl fluoride with LD50 of 10-5 M and was Promoted by ATP (excess ATP beyond 2 mM inhibited the activity reversely). The protease of pH 8.5 had the activity of ATPase which might exert the energy to its action. Therefore the former was referred to as a cysteinyl acid protease and the latter, ATP-dependent neutral serine protease.

• BMB Reports
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• v.40 no.4
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• pp.604-609
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• 2007

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.

• International Journal of Industrial Entomology and Biomaterials
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• v.9 no.2
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• pp.167-171
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• 2004

We constructed an oligo-d(T) primed directional cDNA library from the Bombyx mandarina whole larvae. In an effort to isolate genes expressed in the B. mandarina, 227 expressed sequence tags (ESTs) were generated by single-pass sequencing from the cDNA library. Sequence analysis showed that 107 clones (47.1%) were classified into known genes and 120 clones (52.9%) were novel transcripts, which are unknown for their function. Of the 107 known genes, the most abundant gene was found to be actin and followed by serine protease in the expression profile. Among these clones, a serine protease homolog (BmSP) which is a class of proteolytic enzymes isolated. Full-length sequence of the BmSP cDNA clone was 922 bp in length and has an open reading frame of 276 amino acids. The conserved histidine, aspatic acid and serine residues forming the catalytic center as well as cysteine residues contributing to three disulphide bonds also were found in Bmsp gene. mRNA expression analysis revealed a high and specific expression of the gene only in midgut tissue, suggesting that BmSP gene is closely associated with the expression of digestive enzyme.