• 생명과학회지
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• 제23권2호
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• pp.273-281
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• 2013

본 연구는 부산 인근의 해수욕장에서 얻은 해수에서 protease를 생산하는 균주를 분리하여 동정하고 균주의 배양학적인 특성과 protease의 효소학적 특성을 확인하였다. 해수에서 분리한 protease를 생산하는 미생물은 16S rDNA sequencing을 통해 Micrococcus sp. PS-1으로 동정하였다. Protease 생산의 최적조건은 2% skim milk와 1% NaCl이 포함된 pH 7.0의 LB배지에 48시간 배양이었다. 효소의 부분정제를 위해 ultrafiltration과 acetone 침전법을 사용하였고, zymography를 통해 분자량이 35.0 kDa과 37.5 kDa인 protease를 확인하였다. 또한 효소의 최적 활성은 pH 9.0와 $37^{\circ}C$에서 나타났고, 효소는 pH 8.0에서 11.0까지, $25^{\circ}C$에서 $37^{\circ}C$까지 80% 이상의 효소활성이 유지되어 안정한 것으로 확인되었으며 PMSF, EDTA 처리시 protease가 저해되는 것을 통해 alkaline metallo-serine protease로 확인되었다.

• Applied Biological Chemistry
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• 제35권4호
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• pp.237-241
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• 1992

Halomonas sp. ES 10이 생산하는 protease를 methanol 침전, Sephadex G-150, G-200 및 DEAE-Sephadex A-50으로 여과하여 비활성이 1,014 units/mg protein, 수율이 7%로 정제하였다. 이 효소의 작용 최적온도 및 pH는 $35^{\circ}C$ 와 pH 11.0 이었고, $50^{\circ}C$ 에서 40분에 70%의 잔존활성을 보였으며 $pH\;7.5{\sim}11.0$ 범위에서 안정하였다. 정제효소의 우유 casein에 대한 Km값은 7.4 mg/ml 이었다. $Li^+$, $Ca^{2+}$, SDS, Tween 80 등은 효소 활성을 다소 증가시키고 $Hg^{2+}$과 EDTA는 심히 저해하였다. DFP와 PMSF에 의해서는 각각 63%, 107%의 상대활성을 보여 이 효소는 serine protease가 아님을 시사하였다. 0.5 M과 1 M의 NaCl 농도에서 각각 95%와 65%의 상대활성을 보여 일반 미생물의 protease 보다 각각 20%, 15%씩 상대활성이 높았다.

• BMB Reports
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• 제37권5호
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• pp.574-581
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• 2004

The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro- and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat' venous was reduced by approximately 60% versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro- and mature regions.

• International Journal of Industrial Entomology and Biomaterials
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• 제25권2호
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• pp.181-185
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• 2012

Chymotrypsin serine protease is one of the main digestive proteases in the midgut of and is involved in various essential processes. In a previous study, a gene encoding a chymotrypsin-like protease, Hi-SP1, was cloned from the larvae of Hermetia illucens and characterized. In this study, we produced the recombinant chymotrypsin-like protease Hi-SP1 in Escherichia coli cells. The molecular weight of the recombinant Hi-SP1 was estimated to be approximately 26 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western-blotting. Chymotrypsin activity was detected when AAPF was used as the substrate. Examination of the effects of temperature and pH revealed that the proteolytic activity of recombinant Hi-SP1 decreased markedly at temperatures above $30^{\circ}C$, and the optimum pH was found to be 10.0.

• 한국미생물·생명공학회지
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• 제25권1호
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• pp.56-61
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• 1997

An alkaline protease producing microorganism was isolated from korean traditional Meju and identified as Scopulariopsis brevicaulis. The optimum culture condition of Scopulariopsis brevicaulis for the production of alkaline protease was as follow: 2% soluble starch, 0.2$, tryptophan, 0.1% (NH$_{4}$) $_{2}$S$_{2}$O$_{8}$ 0.2% NaHPO$_{4}$, pH 7.5, 35$\CIRC $C. The optimum pH and temperature for the enzyme activity of alkaline protease producing Scopulariopsis brevicaulis were pH 9.0 and 50$\circ $C, respectively. The enzyme was relatively stable at pH 6.0~11.0 and at temperature below 40$\circ $C. The activity of the enzyme was inhibited by Hg$^{2+}$ whereas Cu$^{2+}$ gave rather activating effects on the enzyme activity. Phenylmethanesulfonyl fluoride inhibited the enzyme activity. This result indicates that serine is very important role in this enzyme. Km value for casein was 1.2410$^{4}$ M/L, V$_{max}$ value for casein was 25.99 $\mu $g/min. This enzyme hydrolyzed casein more rapidly than the hemoglobin.

• Journal of Microbiology
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• 제41권4호
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• pp.289-294
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• 2003

Gelatinase is a proteolytic enzyme that hydrolyzes gelatin. Gelatinolytic activity was detected from culture broths of Streptomyces griseus IFO13350 and HH1 by paper disc assays on 0.5% agar plates containing 1% gelatin. The concentrated extracellular protein from the S. griseus was analyzed by SDS polyacrylamide gel, and two proteins, with molecular weights of 30 and 28 kDa, respectively, were identified to have gelatinase activity by gelatin zymography. The protein with a molecular weight of 28 kDa was confirmed to be S. griseus trypsin (SGT). The effects of metal ions and metal chelators on the protease activity of the SGT were studied. Of the metal ions tested, only manganese was found to enhance the protease activity, 2.6 times, however, $Co^{2+},\;Cu^{2+},\;and\;Zn^{2+}$, and metal chelators, such as EDTA and EGTA, inhibited the SGT activity. When the protease activity of the SGT was measured at various pHs, in the presence of 5 mM $MnCl_2$, its highest activity was at pH 11.0, whereas only 60% of the maximum activity was observed between pHs 4.0 and pH 6.0, and almost 80% activity between pHs 7.0 to pH 10.0. The protease activity was measured at various temperatures in the presence of 5 mM $MnCl_2$. The SGT was found to be stable up to $60^{\circ}C$ for 30 min, while only 16% of the enzyme activity remained at $60^{\circ}C$, and at $80^{\circ}C$ almost all the activity was lost. The optimal temperature for the protease activity was $50^{\circ}C$.

• 생명과학회지
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• 제17권5호
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• pp.606-612
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• 2007

혈전(fibrin clot)은 심혈관계 질환을 일으키는 주요 인자로서 전신의 미세동맥이나 모세혈관 내에서 형성되어 주의 조직이나 장기에 혈류의 공급방해가 생겨 허혈, 경색, 나아가 괴사까지도 발생시킨다. 혈전이 원인이 되어 발생한 심혈관계 질환을 예방 혹은 치료할 목적으로 기존으로 사용되고 있는 항혈전제(antithrombolytic drug)나 혈전용해제(thrombolytic drug)의 개발을 위해 많은 연구들이 진행되고 있다. 본 연구에서는 치료목적으로 사용할 혈전용해제를 분리하고자 Protaetia brevitarsis의 maggot로부터 ammonium sulfate 분획과 desalting column을 이용하여 fibrinolytic protease를 분리하고 생화학적 특성을 조사하였다. 활성의 최적 pH는 9.0였고 최적온도는 $50^{\circ}C$였다. pH 7.0-9.0 사이와 온도 $60^{\circ}C$이하에서는 비교적 활성이 안정성을 나타냈다. 효소활성이 phenylmethanesulfonyl fluoride에 의해 강하게 저해되고 있는 것으로 보아 serine protease로 추정되며 금속이온에 의한 영향을 조사해 본 결과 $Ca^{2+}$과 $Zn^{2+}$에 의해서는 저해되지만 $Mg^{2+}$와 $Fe^{2+}$에 의해서는 저해를 받지 않았다.

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.135-140
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• 1998

Protease nexin-1 (PN-1)은 활성화 자리에 serine기를 갖는 단백질 분해 효소 즉, 트롬빈, 트립신, 플라스미노겐 활성화 효소 등의 작용을 억제한다. 본 연구에서는 흰쥐의 Sprague-Dawley계통을 이용하여 조직별 mRNA발현여부 및 정도를 조사하였다. PN-1의 발현이 나타난 조직은 뇌 (전뇌, 후뇌), 심장, 간, 폐, 난소, 난관 등이다. 이들 중 유전자 발현이 가장 높은 조직은 암컷의 전뇌(forebrain) 였다. 특히, 생식기관들 중에서는 암컷의 난소와 난관에서만 발현이 관찰되는 등 PN-1 유전자는 성별에 따라 서로 다르게 발현됨이 확인되었다 이러한 결과들로 미루어 PN-1은 여포 형성과정과 초기배 형성과정 등의 생식 및 발생작용과 관련이 있을 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제17권4호
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• pp.594-599
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• 2007

It is well known that the Escherichia coli inner membrane-bound protease DegS is a periplasmic stress sensor for unfolded outer membrane proteins (OMPs). Previous studies have also shown that the outer membrane protease OmpT activates plasminogen in vitro and this may be exploited by bacteria in the course of pathogenesis. However, there has been no research on the plasminogen activation ability of the important periplasmic protein DegS. Accordingly, in this study, the whole-length and truncated degS genes were separately overexpressed in Escherichia coli, the recombinant proteins purified by affinity chromatography, and their plasminogen activator role tested in vitro. The results suggested that the whole-length DegS was able to activate plasminogen on a plasma plate. The truncated form of DegS (residues 80-345), designated ${\Delta}DegS$, also acted as a plasminogen activator, as confirmed by different assays. The serine protease property of ${\Delta}DegS$ was verified based on the complete inhibition of its enzyme activity by PMSF (phenylmethanesulfonyl fluoride). Therefore, the present results indicate that DegS is a plasminogen activator in vitro.

• Journal of Microbiology
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• 제38권3호
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• pp.160-168
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• 2000

A-factor I a microbial hormone that can positively control cell differentiation leading to spore formation and secondary metabolite formation in Streptomyces griseus. to identify a protease that is deeply involved in the morphological and physiological differentiation of Streptomyces, the proteases produced by Streptomyces griseus IFO 13350 and its A-factor deficient mutant strain, Streptomyces griseus HH1, as well as Streptomyces griseus HH1 transformed with the afsA gene were sturdied. In general Streptomyces griseus showed a higher degree of cell growth and protease activity in proportion to its ability to produce a higher amount of A-factor. In particular, the specific activity of the trypsin of Streptomyces griseus IFO 13350 was greatly enhanced more than twice compared with that of Streptomyces griseus HH1 in the later stage of growth. The specific activity of the metalloprotease of Streptomyces griseus HH1 was greatly enhanced more than twice compared with that of Streptomyces griseus IFO 13350, and this observation was reversed in the presence of thiostreptione, However, Streptomyces griseus HH1 transformed with the afsA gene showed a significantly decreased level of trypsin and metalloprotease activity compared with that of the HH1 strain. There was no significant difference between Streptomyces griseus IFO 13350 and HH1 strain in their chymotrypsin and thiol protease activity, yet the level of leu-amionpeptidase activity was 2 times higher in Streptomyces griseus HH1 than in strain IFO 13350 . Streptomyces griseus HH1 harboring afsA showed a similar level of enzyme activity , however, all the three protease activities sharply increased and the thiol protease activity was critically increased at the end of the fermentation. When a serine protease inhibitor, pefabloc SC, and metalloprotease inhibitor, EDTA, were applied to strain IFO 13350 to examine the in vivo effects of the protease inhibitors on the morpholofical differentiation, the formation of aerial meycelium and spores was delayed by two or three days.