• 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.

• Applied Biological Chemistry
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• v.41 no.5
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• pp.342-348
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• 1998

A bacteria producing protease against soy protein was isolated from various traditional Me-Ju, in order to improve utililization and characteritics of soy protein hydrolysates reduced bitterness and advanced flavors. The optimal culture conditions for protease production was investigated. The isolated bacteria was identified as Bacillus subtilis by morphological and physiological charateristics and named Bacillus subtilis YG-95. The optimal culture condition of liquid medium for protease production by Bacillus subtilis YG-95 composed of 3.5% soluble starch, 3.0% soy protein isolate, 0.25%, dextrose, 0.5% NaCl, 0.25% $K_2HPO_4$ with on initial pH of 7.6, for 60 hrs at $45^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.427-434
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• 1998

An alkaline protease was 4-fold purified, yielding 2.3% of recovery by ammonium sulfate precipitation, CM-cellulose column chromatography and Sephadex G-100 column chromatography. The purified enzyme was estimated to be monomeric with molecular weight of about 62,000 from polyacrylamide gel eletrophoresis (PAGE) and sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-FAGE). The optimal pH and temperature of the alkaline pretense activity were 11.0 and 50$^{\circ}C$, respectively, exhibiting high stability at pH value from 6.0 to 11.0 at 50$^{\circ}C$ for 30 minute. The alkaline pretense was activated by MnSO$_4$, CaCl$_2$, and was inhibited by CuSO$_4$, ZnSO$_4$, HgCl$_2$, EDTA and EGTA. Also, the enzyme was found to be a metaloenzyme requiring Mn$\^$2+/ as cofactor. The NH$_2$-terminal amino acid of alkaline protease was alanine. The Km and Vmax values of this enzyme for casein was 4.0 mg/$m\ell$ and 5,500 unit/$m\ell$, respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.3
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• pp.373-378
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• 2008

The objective of this paper was to investigate optimal culture conditions for the production of an inhibitor against subtilisin-like protease from Streptomyces thermocarboxydus (S. thermocarboxydus) C12 isolated from sediments of Gwangyang coast. The optimal temperature and initial pH for the production of subtilisin-like protease inhibitor were $40^{\circ}C$ and pH 8.0, respectively. Inhibition activities were high for galactose, glucose and fructose. The best carbon source and its concentration were galactose and 1.6% (w/v), respectively. Inhibition activities were also high in medium containing polypeptone, proteose and peptone. Optimal nitrogen source and concentration were protease peptone and 0.5% (w/v), respectively. Optimal concentrations for inhibitor production were 1% (w/v) for NaCl and 1 mM LiCl for metal salts. The subtilisin-like protease inhibitor from S. thermocarboxydus C12 showed a maximum inhibitor activity after cultivation for 84 h under the optimized medium.

• Journal of Applied Biological Chemistry
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• v.48 no.2
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• pp.75-78
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• 2005

Halotolerant protease produced by Halomonas marisflava KCCM 10457 was partially purified through ammonium sulfate precipitation and Sephacryl S-200HR gel permeation chromatography. Optimal pH and temperature of protease were 11.0 and $45^{\circ}C$, respectively. Enzyme activity was inhibited by $Cu^{2+}$, $Hg^{2+}$, $Fe^{2+}$, and $Fe^{3+}$, and selectively inhibited by p-chloromercuribenzoic acid (PCMB), suggesting this enzyme is cysteine protease. The enzyme is halotolerant, because it retained 77% of original activity in presence of 3.33 M NaCl. The protease showed broad substrate specificity to various natural proteins; BSA, casein, egg albumin, gelatin, and hemoglobin.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.4
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• pp.636-641
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• 1995

Proteolytic activities were compared using three species involving in squid jeotkal fermentation and showing positive reaction upon casein test : Pseudomonas D2, Flavovacterium odoratum and Acinetobacter calcoaceticus. Pseudomonas D2 produced highest activity of protease at 72h when incubated in our own modified medium(polypeptone, 0.5% ; tryptone, 0.5% ; NaCl, 3% ; pH, 7.5). Thus, this specie was selected for the further study. The growth pattern was coincided with the production of protease. Thus purification of protease was proceeded by ethanol precipitation, sephadex G-100 gel filtration, and DEAE sepharose ion exchange chromatography. The purified protease showed highest activity at pH 7.0 and 5$0^{\circ}C$. The enzyme was very stable over the wide ragnes of the temperature ; even with one hour heat treatment at 7$0^{\circ}C$, the enzyme showed substantial amount of the activity toward casein. In addition, the enzyme was stable over the wide range of pH. Molecular weight of the protease was determined to be 17.4 kD by SDS-PAGE.

• 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.

• Bulletin of the Korean Chemical Society
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• v.31 no.1
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• pp.87-91
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• 2010

For the discovery of new antivirals against Severe Acute Respiratory Syndrome-coronavirus (SARS-CoV), we prepared and evaluated several benzoquinoline compounds as its 3C-like protease (3CLpro) inhibitors. Based on the computer modeling study that each of the two rigid benzoquinolinone and N-phenoltetrazole moieties of the compound 1 is bound to the S1 and S2 sites, respectively, of the SARS protease by forming H-bonds and hydrophobic interactions, we designed and synthesized alkylated benzoquinolines at both the sites of the hydroxyl groups. We found that the compound 2a showed five times higher inhibiting activity against the 3CLpro compared to the compound 1.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.51 no.4
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• pp.369-375
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• 2018

This study investigated the effects of selected seaweeds on rice koji preparation (rice inoculated with Aspergillus luchuensis) and enzyme activity. Four types of rice koji were prepared using one of three seaweeds (0.5% laver, 0.5% kelp and 0.5% green laver) or a control for 72 h. The changes in the moisture content, water activity, pH, total mold cell count, amylase and protease activities were measured. During preparation, there was no significant difference in the moisture content among the four kojis, whereas the pH in the kojis made with either laver or green laver decreased rapidly compared with the control (P<0.05). This seemed to result from the seaweeds promoting the growth of mold cells. In terms of the activities of both amylase and protease, the koji with laver was superior. Subsequently, the amylase and protease activities of the koji made with laver were evaluated at various pHs (3 to 9), temperatures ($15-55^{\circ}C$) and NaCl concentrations (0-10%). The activities of both enzymes decreased notably at pH 9 and the protease activity decreased at temperatures above $45^{\circ}C$. Although the activities of both enzymes decreased at greater than 2.5% NaCl, activity was present at 10% NaCl.

• Journal of Life Science
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• v.29 no.10
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• pp.1126-1135
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• 2019

From a sample of bamboo byproduct, the protease-producing yeast strain CO-1 was newly isolated. Strain CO-1 is spherical to ovoid in shape and measures $3.1-4.0{\times}3.8-4.4{\mu}m$. For the growth of strain CO-1, the optimal temperature and initial pH were $30^{\circ}C$ and 4.0, respectively. The strain was able to grow in 0.0-15.0%(w/v) NaCl and 0.0-9.0%(v/v) ethanol. Based on a phylogenetic analysis of its 18S rDNA sequences, strain CO-1 was identified as Pichia anomala. The extracellular protease produced by P. anomala CO-1 was partially purified by ammonium sulfate precipitation, which resulted in a 14.6-fold purification and a yield of 7.2%. The molecular mass of the protease was recorded as approximately 30 kDa via zymogram. The protease activity reached its maximum when 1.0%(w/v) CMC was used as the carbon source, 1.0%(w/v) yeast extract was used as the nitrogen source, and 0.3%(w/v) $MnSO_4$ was used as the mineral source. The protease revealed the highest activity at pH 7.0 and $30^{\circ}C$. This enzyme maintained more than 75% of its stability at a pH range of 4.0-10.0. After heating at $65^{\circ}C$ for 1 hr, the neutral protease registered at 60% of its original activity. The protease production coincided with growth and attained a maximal level during the post-exponential phase.