• Applied Biological Chemistry
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• 제31권4호
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• pp.356-360
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• 1988

토양에서 분리된 Alkaline Protease생산균주는 Bacills subtilis로 동정되었다. 본 균주의 최적배양온도는 최적 배지조건에서 $35^{\circ}C$이며, 초기 배지 pH는 10.5였다. 효소역가는 Casein-Folin법으로 약 2,300 Unit/ml로 나타났다. 배양액에 일련의 정제과정을 실시한 결과 정제배수 9.2, 회수율 14%의 정제효소를 얻었으며, 분자량은 19,000이었다. 정제효소의 반응최적온도는 $70^{\circ}C$, 최적 PH는 12였다. $Fe^{++}$에 의해 활성이 저하되었으며, 또한 serine protease inhibitor인 PMSF에 의해 활성이 저해되었다. 이것으로 부터 본 효소는 활성부위에 serine을 갖는 serine protease의 일종임을 알 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제21권1호
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• pp.20-27
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• 2011

The purification and characterization of a $Mn^{2+}$-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be $60^{\circ}C$. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. $Mn^{2+}$ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants ($H_2O_2$, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.

• 한국미생물·생명공학회지
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• 제23권1호
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• pp.53-59
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• 1995

A protease isolated and purified 51 fold from the culture filtrate of a soil bacterium, Bacillus sp. KUN-17, which was appeared to be a monomeric protein with molecular weight of 38, 000 daltons, was suggested to be involved in the serine (-alkaline) protease (E.C 3.4.21.14) since its activity was selectively inhibited by phenylmethylsulfonyl fluoride (PMSF) and required 40$\circ$C and pH 10.5 for optimal condition. The half-life of the enzyme activity was 1 hr at 55$\circ$C, and the activity was maintained even under high concentrations of SDS or urea. The enzyme was indicated to perform random proteolysis from the fact that most of the chromogenic substrates employed were hydrolyzed by the enzyme. The affinity of the enzyme for natural proteins was approximately 10-times higher than ester compounds, and both substrates showed mutual inhibitory effect competitively for the enzyme activity.

• 한국미생물·생명공학회지
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• 제18권4호
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• pp.344-351
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• 1990

계명활성제 내성이 있으면서 호알카리성인 protease를 생산하는 미생물을 토양에서 분리하였다. 분리된 미생물을 형태적, 생리학적, 화학분류학적 및 5S RNA 분석으로 동정한 결과 Bacillus sp.인 것으로 판명되었다. 호알카리성 protease는 황산암모늄 분획, DEAE-Cellulose, CM-Cellulose, Sephadex G-100 column chromatogrphy로 분리, 정제하였다. 정제된 호알카리성 protease는 casein에 대하여 pH6.0에서 11.0 사이에서 안정성을 나타내었다. 분리된 효소의 작용 최적 온도는 $55^{\circ}C$이었다. 이 효소는 diisopropyl fluorophosphate(DFP)로 완전히 불활성화되는 것으로 보아 serine protease로 추정되며 계면활성제의 존재하에서도 안정하였다.

• Mycobiology
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• 제31권4호
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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.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.275-283
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• 2018

Ischemic stroke can result from blockage of blood vessels, forming fibrin clots in the body and causing irreparable brain damage. Remedial thrombolytic agents or anticoagulants have been studied; however, because the FDA-approved tissue plasminogen activator has low efficacy and side effects, it is necessary to develop safer and more effective treatment candidates. This study aimed at assessing the fibrinolytic and anticoagulation features of a novel serine protease extracted and purified from Diopatra sugokai, a polychaeta that inhabits tidal flats. The purified serine protease was obtained through ammonium sulfate precipitation, affinity chromatography, and ion-exchange chromatography. Its molecular size was identified via SDS-PAGE. To characterize its enzymatic activities, the protease activity at various pH and temperatures, and in the presence of various inhibitors, was measured via azocasein assay. Its fibrinolytic activity and anticoagulant effect were assessed by fibrin zymography, fibrin plate assay, and fibrinogenolytic activity assays. The novel 38 kDa serine protease had strong indirect thrombolytic activity rather than direct activity over broad pH (4-10) and temperature ($37^{\circ}C-70^{\circ}C$) ranges. In addition, the novel serine protease exhibited anticoagulant activity by degrading the ${\alpha}$-, ${\beta}$-, and ${\gamma}$-chains of fibrinogen. In addition, it did not produce cytotoxicity in endothelial cells. Therefore, this newly isolated serine protease is worthy of further investigation as a novel alkaline serine protease for thrombolytic therapy against brain ischemia.

• Applied Biological Chemistry
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• 제38권2호
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• pp.106-110
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• 1995

알칼리성 protease를 생산하는 균을 고추장에서 분리하여 Alteromonas sp. CN301로 동정하고 그 알칼리성 protease를 생산하여 정제효소의 성질을 조사한 결과, 최적 pH 12.0, 최적 온도 $35^{\circ}C$이었으며 pH 안정성은 $pH\;6.0{\sim}13.0$ 범위에서 80% 이상의 잔존효소 활성을 나타냈고 $50^{\circ}C$에서 1시간 처리로 64%의 활성을 보였다. SDS-PAGE에 의한 분자량은 31,000 dalton이었고 $Hg^{2+}$를 제외한 다른 금속이온에 대해서는 저해를 받지 않았다. 계면활성제인 Triton X-100, Tween 20과 80은 이 효소의 활성을 상승시키는 효과를 보였으며 EDTA와 PMSF에 의하여 효소활성이 저해되므로 효소분자 중에 금속이온을 가지는 serine protease로 생각되었다.

• 한국미생물·생명공학회지
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• 제22권5호
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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.

• 미생물학회지
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• 제47권3호
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• pp.194-199
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• 2011

알칼리성 protease를 생산하는 호알칼리성 균주를 분리하여 Bacillus pseudofirmus HS-54로 동정하였고, HS-54가 생산하는 알칼리성 protease를 ammonium sulfate 침전, DEAE cellulose chromatography, sephadex G-100 gel filtration을 통과시켜 정제하였는데, 정제된 protease의 분자량은 27 kDa이었다. 정제된 효소의 반응최적 pH는 10.0이었고 pH 7.0-11.0에서 비교적 안정하였다. 또한 정제된 효소의 반응최적 온도는 $50^{\circ}C$이었고 $10-55^{\circ}C$에서 안정하였다. 금속이온에 대한 영향은 $Ca^{2+}$와 $Mg^{2+}$ 등에 의해 효소활성이 촉진되었으나, $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, $Al^{3+}$ 등에 의해서 효소활성이 저해되었다. 본 효소는 PMSF에 의해 강하게 저해를 받는 것으로 보아 serine protease에 속하는 것으로 판단된다.

• Journal of Microbiology
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• 제40권1호
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• pp.26-32
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• 2002

From the culture supernatant of the psychrotrophic strain of Bacillus amyloliquefaciens an extracellular serine protease was purified to apparent homogeneity by successive purification steps using QAE-Sephadex, SP-Sephadex and Sephacryl S-100 column chromatography. The pretense is monomeric, with a relative molecular mass of 23,000. It is inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride, but not by EDTA. The enzyme is most active at pH 9-10 and at $45^{\circ}C$, although it is unstable at $60^{\circ}C$.