Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3

Cheng, Guangyan; He, Lin; Sun, Zhibin; Cui, Zhongli; Du, Yingxiang; Kong, Yi

doi:10.4014/jmb.1503.03015

Cited by 20 publications

(19 citation statements)

References 52 publications

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“…Earlier, an extracellular alkaline protease from Streptomyces clavuligerus, Streptomyces pseudogrisiolus and Streptomyces sp. P3, has shown the same optimal temperature of our study (Moreira et al, 2001;Awad et al, 2013;Cheng et al, 2015), as well fibrinolytic protease from Streptomyces sp. (Simkhada et al, 2012;Cheng et al, 2015).…”

Section: Discussionsupporting

confidence: 79%

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2016

Int. J. Adv. Biol. Biomed. Res.

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Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding CASRP΄s archiving and manuscript policies encouraged to visit: http://www.casrp.co.uk/journals Abstract Actinomycetes are an uncommon agent of human infections and its pathogenic factors are not known. The present study reports a rare case isolation of an actinomycete from a woman pleural fluid; the strain was identified by 16S rRNA gene sequence analysis. This strain was tested to produce an extracellular protease that hydrolysis gelatin, casein and hemoglobin on agar mediums. The purification of the enzyme was carried by ammonium sulfate precipitation, gel filtration and ion exchange chromatographies. The activity of protease was studied at different pH values and temperatures and in the presence of metallic ions and inhibitors. The molecular weight of the enzyme was determined by 12% Tricine SDS-polyacrylamide gel electrophoresis. The strain was identified as Streptomyces cinereoruber ssp. cinereoruber. Extracellular proteolytic enzyme was purified at 19.67 fold and a 3.0% recovery. The enzyme was characterized as having optimal activities at pH 11.0 and 50°C, it keeps more than 50% of activity at pH between 4.0 to 12.0 and it is thermostable at 30 and 40°C. Enzymatic activity is enhanced in the presence of metal ions and inhibited by EDTA and 1,10-phenanthroline. The molecular weight was 53 kDa. This study reports the first case isolation of Streptomyces cinereoruber ssp. cinereoruber from pleural fluid, the extracellular zinc-metalloprotease was proposed as candidate virulence factor.

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Section: Discussionsupporting

confidence: 79%

“…P3, has shown the same optimal temperature of our study (Moreira et al, 2001;Awad et al, 2013;Cheng et al, 2015), as well fibrinolytic protease from Streptomyces sp. (Simkhada et al, 2012;Cheng et al, 2015). Characteristics of zinc-metalloproteases from some human pathogenic bacteria are summarized in table 3.…”

Section: Discussionsupporting

confidence: 79%

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2016

Int. J. Adv. Biol. Biomed. Res.

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“…From the purification table, the specific activity of marine Streptomyces radiopugnans VITSD8 is 3891 U/mg, which is higher than the specific activity of 2563.6 U/mg of terrestrial Streptomyces sp. P3 strain isolated from soil [28].…”

Section: Resultsmentioning

confidence: 99%

Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges

Dhamodharan

Naine

et al. 2019

Marine Drugs

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Fibrinolytic enzymes have received more attention due to their medicinal potential for thrombolytic diseases. The aim of this study is to characterize the in vitro fibrinolytic nature of purified protease producing Streptomyces radiopugnans VITSD8 from marine brown tube sponges Agelas conifera. Three varieties of sponge were collected from the Rameshwaram Sea coast, Tamil Nadu, India. The fibrinolytic activity of Streptomyces sp. was screened and determined by casein plasminogen plate and fibrin plate methods respectively. The crude caseinolytic protease was purified using ammonium sulfate fractionation, affinity and ion-exchange chromatography. Based on the morphological, biochemical, and molecular characterization, the isolate VITSD8 was confirmed as Streptomyces radiopugnans. Maltose and peptone were found to be the best carbon and nitrogen sources for the production of fibrinolytic protease. The carbon and nitrogen source peptone showed (781 U/mL) enzyme activity. The optimum pH and temperature for fibrinolytic protease production was found to be 7.0 and 33 °C respectively. The purified enzyme showed a maximum specific activity of 3891 U. The blood clot lysis activity was compared with the standard, and it was concluded that a minimum of 0.18 U (10 µL) of purified protease was required to dissolve the blood clot. This is the first report which exploits the fibrinolytic protease activity of Streptomyces radiopugnans VITSD8 extracted from a marine sponge. Hence the investigation suggests a potential benefit of purified fibrinolytic protease which will serve as an excellent clot buster alternative.

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“…CS684 (Simkhada et al 2010), but higher than Streptomyces sp. P3 (Cheng et al 2015). The fibrinolytic enzyme from Streptomyces DPUA1576 was stable at pH 6.0-9.5, and the activity was greatly decreased below pH 6.0, exhibiting a pH stability comparably wider than that of Streptomyces sp.…”

Section: Discussionmentioning

confidence: 86%

“…The fibrinolytic enzyme from Streptomyces DPUA1576 was stable at pH 6.0-9.5, and the activity was greatly decreased below pH 6.0, exhibiting a pH stability comparably wider than that of Streptomyces sp. P3 (Cheng et al 2015) and similar to Streptomyces sp. CS684 (Simkhada et al 2010).…”

Section: Discussionmentioning

confidence: 99%

Screening, production and biochemical characterization of a new fibrinolytic enzyme produced by Streptomyces sp. (Streptomycetaceae) isolated from Amazonian lichens

et al. 2016

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Thrombosis is a pathophysiological disorder caused by accumulation of fibrin in the blood. Fibrinolytic proteases with potent thrombolytic activity have been produced by diverse microbial sources. Considering the microbial biodiversity of the Amazon region, this study aimed at the screening, production and biochemical characterization of a fibrinolytic enzyme produced by Streptomyces sp. isolated from Amazonian lichens. The strain Streptomyces DPUA1576 showed the highest fibrinolytic activity, which was 283 mm 2 . Three variables at two levels were used to assess their effects on the fibrinolytic production. The parameters studied were agitation (0.28 -1.12 g), temperature (28 -36 ºC) and pH (6.0 -8.0); all of them had significant effects on the fibrinolytic production. The maximum fibrinolytic activity (304 mm 2 ) was observed at 1.12 g, 28 ºC, and pH of 8.0. The crude extract of the fermentation broth was used to assess the biochemical properties of the enzyme. Protease and fibrinolytic activities were stable during 6 h, at a pH ranging from 6.8 to 8.4 and 5.8 to 9.2, respectively. Optimum temperature for protease activity ranged between 35 and 55 °C, while the highest fibrinolytic activity was observed at 45 ºC. Proteolytic activity was inhibited by Cu 2+ and Co 2+ ions, phenylmethylsulfonyl fluoride (PMSF) and pepstatin A, which suggests that the enzyme is a serine protease. Enzymatic extract cleaved fibrinogen at the subunits Aα-chain, Aβ-chain, and γ-chain. The results indicated that Streptomyces sp. DPUA 1576 produces enzymes with fibrinolytic and fibrinogenolytic activity, enzymes with an important application in the pharmaceutical industry. KEYWORDS: Actinomycetes, fibrinolytic protease, inhibitor protease, fibrinogenolytic activity.Seleção, produção e caracterização bioquímica de uma nova enzima fibrinolítica produzida por Streptomyces sp. (Streptomycetaceae) isolada de liquens da Amazônia RESUMO A trombose é uma doença patofisiológica causada pelo acúmulo de fibrina no sangue. Proteases fibrinolíticas com potente atividade trombolítica são produzidas por diversas fontes microbianas. Considerando a biodiversidade microbiana da região amazônica, o presente estudo teve como objetivo a seleção, produção e caracterização bioquímica da enzima fibrinolítica de Streptomyces sp. isolado de líquens da Amazônia. Streptomyces DPUA1576 foi a melhor produtora com atividade fibrinolítica de 283 mm 2 . Três variáveis em dois níveis foram utilizadas para determinar as variáveis mais relevantes na produção da enzima fibrinolítica (FA). Os parâmetros estudados foram agitação (0.28 -1.12 g), temperatura (28 -36 ºC) e pH (6.0 -8.0) e todos obtiveram efeitos significativos na produção fibrinolítica. A maior atividade fibrinolítica (304 mm 2 ) foi obtida a 1.12 g, 28 ºC e pH 8.0. O extrato bruto da fermentação foi usado para determinar as propriedades bioquímicas da enzima. Atividades proteásica e fibrinolítica foram estáveis durante 6 horas no intervalo de pH entre 6.8 -8.4 e 5.8 -9.2, respectivamente. Temperatura ...

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Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3

Cited by 20 publications

References 52 publications

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Novel Fibrinolytic Protease Producing Streptomyces radiopugnans VITSD8 from Marine Sponges

Screening, production and biochemical characterization of a new fibrinolytic enzyme produced by Streptomyces sp. (Streptomycetaceae) isolated from Amazonian lichens

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