• Korean Journal of Microbiology
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• v.9 no.4
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• pp.163-168
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• 1971

Neutral protease which was obtained from a genus of Aspergilli as the crystal form were investigated for their purification and properties. The results of biochemical and enzymatic studies for their purification and properties in this enzyme were as follows. 1) On the wheat media containing 70%-water and $CaCo_{3}$, Aspergilus oryzae S.H.W. 131 is satisfactorily grown under the basic optimum conditions temperature $27^{\circ}C$- $30^{\circ}C$at relative humidity 100% for three days. 2) The enzyme solution extracted with water is successively purified through the passing on column of Asmti-177N for decolorization of it. And ion exchanger such as DEAAE Sphadex A-50 or Shepadex G-100 and fraction collector is necessary for the sepearte treatments of this enzyme. After washing it with organic solvents as aceton-EtOH, etc., it should be dried on the vacuum dryer at $40^{\circ}C$) The protease activity is determined by the amounts of amino acids, tyrosine. 4) The optimum pH of neutral protease is 6.0-8.0. 5) In effectively decomposing with this neutral protease, the optimum temperature is $35^{\circ}C$. 6) It is interesting that the amounts of metal ion affects the activity of neutral protease. For examples, if it were treated with manganic ion, its activity would be more effective than any other that.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.407-411
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• 1989

Extracellular neutral pretense of Streptomyces rimosus producing oxytetracycline was purified by ammonium sulfate fractionation, DEAE Sephadex A-50 chromatography and Sephadex G-100 gel filteration, and was showed single band on the cathodic gel electrophoresis. The optimum pH and temperature of the enzyme were pH 8.0 and 6$0^{\circ}C$, respectively. The enzyme was activated about 80% in the presence of Co$^{2+}$ ion, and strongly inhibited by Hg$^{2+}$, Fe$^{2+}$ and chelatig agent, EDTA. Molecular weight of the enzyme was estimated to be 12, 000. The Km value of the enzyme of casein as a substrate was 2.7$\times$10$^{-4}$M.

• Journal of Pharmaceutical Investigation
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• v.18 no.3
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• pp.125-131
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• 1988

This study was undertaken to investigate the proteolytic enzyme from Neungee mushroom [Sarcodon aspratus (Berk) S. Ito]. The proteolytic activity of Neungee was higher than other several edible mushrooms under various pHs. The potency of proteolytic enzyme of Neungee was same as the digestive drugs containing protease. So the proteolytic activity of the enzyme was increased in neutral or weak alkaline pH, whose characteristics would be alkaline protease. The specific activity of the purified enzyme obtained by using Tris acryl CM-cellulose ion exchange increased 20 times as compared with that of the crude extract. The proteolytic enzyme was stable at room temperature, but decomposition was fast when incubated at higher temperature more than $40^{\circ}C$. The half life of the enzyme was longest in neutral pH and rate constant was increased in acidic or alkaline solution.

• 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.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.675-678
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• 1999

Fibrinolytic protease activity was detected in the fruit body of Pleurotus sajor-caju using a fibrin plate method. Two fibrinolytic activities (FPI and Ⅱ) were found at the regions of 14.5 and 86.0 kDa by using gel-filtration column chromatography. FPⅡ was identified as an alkaline protease, whereas FPⅠ was a neutral protease. Both were inhibited by phenanthrolin and EDTA, suggesting that they are metalloprotease. Inactivated enzyme activities were restored by adding Co/sup 2+/ or Zn/sup 2+/. Iodoacetate inhibited FPⅠ, but not FPⅡ. Both enzymes cleaved B/sub β/ and γ chains of the human fibrinogen. FPⅡ showed a preference to hydrophobic and bulky residues of nitroanilidine compounds as substrates, whereas FPⅠ preferred positively charged residues.

• 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.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.526-531
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• 1988

Procedure for the purification of pretense from culture broth of Streptomyces sp. SMF301 was developed. It was evident that the strain produced two different proteases of which molecular weights were estimated to be 23, 500 and 38, 900 dalton. It was found that the optimum pH of the smaller was 9.0 and that of the larger was 1.0. The optimal temperature of the alkaline pretense was 5$0^{\circ}C$ and that of the neutral pretense was much more stable than neutral protease at extreme condition viz. high temperature, and pH.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.593-598
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• 2005

A collagenolytic enzyme, produced by Vibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.583-588
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• 1998

A fibrinolytic enzyme has been isolated from the edible honey mushroom, Armillariella mellea and purified. The apparent molecular mass of purified enzyme was estimated to be 19800Da by SDS polyacryl amide gel electrophoresis and 19900Da by gel filtration, indicating that it was a monomer. The enzyme was optimal at pH 7, suggesting that the purified enzyme was a neutral proteinase. It shows the maximum fibrinolytic activity at $55^{\circ}C$, is completely inactivated above $65^{\circ}C$, and still indicates 40% of activity at $37^{\circ}C$. The fibrinolytic activity has been decreased by the addition of EDTA. Fifteen amino acid sequence was determined by protein sequencing techniques.

• Journal of Microbiology
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• v.44 no.6
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• pp.622-631
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• 2006

In this study we purified a fibrinolytic enzyme from Cordyceps militaris using a combination of ion-exchange chromatography on a DEAE Sephadex A-50 column, gel filtration chromatography on a Sephadex G-75 column, and FPLC on a HiLoad 16/60 Superdex 75 column. This purification protocol resulted in a 191.8-fold purification of the enzyme and a final yield of 12.9 %. The molecular mass of the purified enzyme was estimated to be 52 kDa by SDS-PAGE, fibrin-zymography, and gel filtration chromatography. The first 19 amino acid residues of the N-terminal sequence were ALTTQSNV THGLATISLRQ, which is similar to the subtilisin-like serine protease PR1J from Metarhizium anisopliae var. anisopliase. This enzyme is a neutral protease with an optimal reaction pH and temperature of 7.4 and $37^{\circ}C$, respectively. Results for the fibrinolysis pattern showed that the enzyme rapidly hydrolyzed the fibrin $\alpha$-chain followed by the $\gamma$-$\gamma$ chains. It also hydrolyzed the $\beta$-chain, but more slowly. The A$\alpha$, B$\beta$, and $\gamma$ chains of fibrinogen were also cleaved very rapidly. We found that enzyme activity was inhibited by $Cu^{2+}$ and $Co^{2+}$, but enhanced by the additions of $Ca^{2+}$ and $Mg^{2+}$ ions. Furthermore, fibrinolytic enzyme activity was potently inhibited by PMSF and APMSF. This enzyme exhibited a high specificity for the chymotrypsin substrate S-2586 indicating it's a chymotrypsin-like serine protease. The data we present suggest that the fibrinolytic enzyme derived from the edible and medicinal mushroom Cordyceps militaris has fibrin binding activity, which allows for the local activation of the fibrin degradation pathway.