• Mycobiology
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• v.32 no.2
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• pp.74-78
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• 2004

An alkaline protease produced by Aspergillus niger C-15 was purified and characterized. The enzyme was purified 19.41-fold with a specific activity of 74150 U/mg and a recovery of 34.4% by gel filtration and ion exchange chromatography. The molecular weight of the enzyme was estimated to be 34 kDa. The optimum pH and temperature for the protease activity were pH 8.0 and $60^{\circ}C$, respectively. The enzyme activity inhibited by EDTA suggests that the preparation contains a metalloprotease. The enzyme activity of the metalloprotease was completely inhibited by 5 mM $HgCl_2$ and $FeCl_3$, while partially inhibited by $CuSO_4$, and $MnCl_2$. When polyols such as glycerol, mannitol, sorbitol and xylitol, were added to the reaction medium, most polyols tested enhanced protease activity. Especially, glycerol showed the highest effect. The alkaline metalloprotease was stable at high temperature and retained more than 90% of the initial activity at $60^{\circ}C$ and 86.4% under addition of glycerol.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.5
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• pp.379-382
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• 2004

Entomopathogenic nematodes are used for insect control. Herein, an extracellular protease was partially purified from a culture supernatant of Xenorhabdus nematophilus, a symbiotic bacterium of an entomopathogenic nematode, Steinernema glaseri: using precipitation with $80\%$ v/v isopropyl alcohol followed by gel permeation chromatography with a packed Sephacryl S-300 HR media. The partially purified protease exhibited maximal activity at pH 7 in the presence of 1 mM $CaCl_2$. The protease was identified as a metallo-protease based on the inhibition of its activity by the metal chelating agent, EDTA.

• Journal of Microbiology
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• v.33 no.3
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• pp.244-251
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• 1995

An extracellular protease of Salmonella schottmulleri was purified from culture filtrate by using 0-75% ammonium sulfate precipitation, DEAE Sepharose Fast Flow ion exchange chromatography, Ultrogel HA chromatography and Sephacryl S-200 HR molecular sieve chromatography. To measure enzyme activity, synthetic dipeptide substrate (CBZ-arg-arg-AFC) with low molecular weight was employed as substrate. The molecular weight of the purified enzyme was approximately 80 kDa when determined by gel filtration on Sephacryl S-200 HR and 73 kDa when estimated by SDS-PAGE. The isoelectric point was 5.45. The activity of the purified enzyme was inhibited by metal chelating agesnts such as EDTA and 1.10-phenanthroline. The divalent cations, such as Ca$\^$2+/, Zn$\^$2+/, Fe$\^$2+/, Mg$\^$2+/ enhanced its activity. These results suggested that it was a metalloprotease. It had a narrow pH optimum of 6.5-7.5 with a maximum at pH 7.0 and a temperature optimum of 40.deg.C. It was stable at least for 1 week at 40.deg.C and maintained its activity for 24 hours at 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium 50.deg.C, but it was rapidly inactivated at 65.deg.C. This protease was shown to be sensitive to sodium dodecyl sulfate (SDS) and was inactivated in a dose-dependent manner. However, it was resistant to Triton X-100 and the activity was enhanced to 32.3% with treatment of 0.025% Triton X-100.

• 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.

• Mycobiology
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• v.31 no.4
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• pp.209-213
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• 2003

Aspergillus flavus K-03 isolated from poultry forming soil in Korea was studied for its ability to produce extracellular proteases on basal medium containing 2%(w/v) chicken feathers. The fungus was observed to be a potent producer of such enzymes. Keratinolytic enzyme secretion was the best at 15 days of incubation period at pH 9 and temperature $40^{\circ}C$. No relationship existed between the enzyme yield and increase of biomass. Enzyme production was suppressed by exogenous sugars in descending order arabinose>maltose>mannose>fructose. But glucose did not influence the enzyme activity. The keratinolytic enzyme released by the fungus demonstrated the ability to decompose keratin substrates as chicken feather when exogenous glucose was present. The keratinolytic activity was inhibited by $HgCl_2$ and serine-protease inhibitors such as phenymethylsulfonyl fluoride(100%), chymostain(88%), crystalline soybean trypsin inhibtor(80%), antipain(45%) and aprotinin(40%), and was not by cystein-protease and aspartyl-protease inhibitors. The enzyme activity is only partially inhibited by metallo-protease inhibitor. Thus, the enzyme secreted by A. flavus K-03 belongs to the alkaline serine-type protease.

• 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.

• 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)

• Korean Journal of Veterinary Research
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• v.55 no.3
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• pp.175-179
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• 2015

Proteases play important roles in parasite development and host parasite interactions. The protease of Kudoa spp. has been recognized as a key factor of severe proteolysis of fish muscle post-mortem; however, there is little information available regarding the protease of Kudoa (K.) septempunctata, which was recently identified as a cause of food poisoning in humans. The present study was conducted to isolate and characterize proteases to elucidate the type of protease contained in the parasite and determine the optimal pH for protease activity. We confirmed the cysteine protease and metalloprotease produced by K. septempunctata. While the cysteine protease showed optimal activity at pH 5 that decreased rapidly with increasing pH, the optimal activity of metalloprotease was pH 7, and it remained stable from pH 6 to pH 8. These results indicate that the pH of cysteine protease is not proper for fish muscle postmortem, and that metalloprotease can act in human intestines. Overall, the present study provides important information that improves our understanding of the role of protease physiology and the subsequent food poisoning caused by K. septempunctata.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.881-888
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• 2010

solvent-tolerant bacterium strain, MH6, was isolated by hydrophilic organic solvent DMSO enrichment in the medium and identified as Serratia marcescens. The extracellular protease with novel organic-solvent-stable properties from strain MH6 was purified and characterized. The molecular mass of the purified protease was estimated to be 52 kDa on SDS-PAGE. The open reading frame (ORF) of the MH6 protease encoded 504 amino acids with 471 amino acid residues in the mature protease. Based on the inhibitory effects of EDTA and 1,10-phenathroline, the MH6 protease was characterized as a metalloproteinase. The enzyme activity was increased in the presence of $Ni^{2+}$, $Mg^{2+}$, and $Ca^{2+}$. The protease could also be activated by the nonionic surfactants Tween 80 (1.0%) and Triton X-100 (1.0%). The protease showed remarkable solvent stability in the presence of 50% (v/v) solutions of long-chain alkanes and long-chain alcohols. It was also fairly stable in the presence of 25% solutions of hydrophilic organic solvents. Owing to its high stability in solvents and surfactants, the MH6 protease is an ideal candidate for applications in organic catalysis and other related fields.

• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.333-340
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• 2016

An alkaline protease was purified and characterized from an alkalophilic microorganism, Bacillus sp. DK1122, isolated from soil in central Korea. The optimum temperature and pH for the growth of the producer strain were 40℃ and pH 9.0, respectively. The protease was produced aerobically at 40℃ after 24 h incubation in modified Horikoshi I medium (pH 9.0) containing 0.5% (w/v) glucose, 0.8% (w/v) yeast extract, 0.5% (w/v) polypeptone, 0.1% (w/v) K₂HPO₄, 0.02% (w/v) MgSO₄·7H₂O, 1% (w/v) Na₂CO₃, and 3% (w/v) NaCl. The alkaline protease was purified by 70% ammonium sulfate precipitation of the culture supernatant of Bacillus sp. DK1122, followed by CM-Sepharose chromatography. The molecular weight of the enzyme was estimated to be 27 kDa on the basis of SDS-PAGE. The optimum temperature and pH for the protease activity were 60℃ and pH 9.0, respectively. Addition of CaCl₂ increased the thermal stability of the purified protease, where 90% of protease activity was retained at 60℃ for up to 3 h. Consequently, it is expected that the alkaline protease from this study, exhibiting stability at pH 7–9 and 60℃, may be promising for application in the food and detergent industries.