Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

Shirasaka, Norifumi; Naitou, Masao; Okamura, Kazuki; Kusuda, Mizuho; Fukuta, Yasuhisa; Terashita, Takao

doi:10.1007/s10267-011-0179-3

Cited by 52 publications

(30 citation statements)

References 41 publications

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“…A similar study conducted by Sharkova et al (2015) with fungal species with fibrinolytic and collagenase activities revealed that, although eighteen micromycetes exhibited proteolytic activity, species belonging to the Mucor genus showed specific collagenase activity but no appreciable proteolytic activity towards fibrin. Another study by Shirasaka et al (2012) about protease production by Aspergillus oryzae KSK-3 isolated from commercial rice-koji for miso brewing, showed a fibrinolytic activity of 21.8 U/mL ,less activity than that found in our work. Kim (2003) observed that fourteen species of fungi associated with feather belonging to ten genera, including Mucor, showed keratinase activity in submerged fermentation, but the species that provided the best results were belonging to the genus Aspergillus with activity in the range 10-15 U/mL, well below that of the crude extract produced in this work by Mucor subtilissimus (39.6 U/mL).…”

Section: Resultscontrasting

confidence: 95%

Production and Purification of Protease by Fungus Isolated From the Brazilian Dry Tropical Forest(caatinga)

Nascimento¹,

Costa²,

Converti³

et al. 2017

Anais Do 5º Encontro Brasileiro Para Inovação Terapêutica

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A demanda industrial por enzimas proteolíticas tem estimulando a busca por novas fontes de enzimas. As enzimas fúngicas são preferidas sobre as enzimas bacterianas, por serem mais eficazes e fáceis de extrair. O objetivo deste trabalho foi à purificação de proteases produzidas na fermentação em estado sólido por Mucor subtilissimus UCP 1262. O extrato bruto foi analisado utilizando 3 atividades proteolíticas diferentes, atividade colagenolítica (161,4 U/ml), queratinolítica (39,6 U/ ml) e fibrinolítica (26,1 U/ml). Após precipitação com sulfato de amônio, as frações ativas com atividade fibrinolítica foram dialisadas em tampão Tris-HCl 15 mM, pH 8, carregadas na coluna de troca iônica DEAE-Sephadex A50 e gel filtração (Superdex 75 HR10/300). A enzima purificada mostrou atividade frente a um substrato cromogênico de quimotripsina, a SDS-PAGE mostrou uma protease fibrinolítica com massa molecular de aproximadamente 70 kDa, e uma atividade específica de 25,93 U/mg. Essas características sugerem que a enzima pode ser produzida de forma rentável e eficiente para uso na terapia trombolítica.

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Section: Resultscontrasting

confidence: 95%

Production and Purification of Protease by Fungus Isolated From the Brazilian Dry Tropical Forest(caatinga)

Nascimento¹,

Costa²,

Converti³

et al. 2017

Anais Do 5º Encontro Brasileiro Para Inovação Terapêutica

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“…In recent years, several studies have been made to search for new sources of thrombolytic agents such as bacteria, algae, plants, worms, snake venom, insects, and fungi [6][7][8][9][10]. Filamentous fungi have been shown to be a good choice for the production of fibrinolytic enzymes, since they produce them in large amounts mostly extracellularly, which makes their extraction/purification easier [11].…”

Section: Introductionmentioning

confidence: 99%

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

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Porto

et al. 2016

Journal of Chromatography B

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A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.

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“…(Shankar et al, 2011), and Trichoderma harzianum (Savitha et al, 2011). Additionally, the protease purified from submerged medium of Aspergillus oryzae KSK-3 had 182.7-fold purification, but a recovery of only 0.005% (Shirasaka et al, 2012). A metalloprotease isolated from Penicillium spp.…”

Section: Discussionmentioning

confidence: 99%

“…exhibits increased activity after pre-incubation with cobalt (Shankar et al, 2011). In contrast, the presence of aluminum decreases the activity of proteases from P. waksmanii (Graminho et al, 2013) and Aspergillus oryzae KSK-3 (Shirasaka et al, 2012). Finally, inhibitors, such as EDTA, can be used to classify the protease according to it catalytic site.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated from Eupenicillium javanicum Using Fluorescence Resonance Energy Transfer Peptides

Neto

Oliveira

et al. 2017

Front. Microbiol.

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Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60°C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The “primed” side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S′1 subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P′1. These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.

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Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

Cited by 52 publications

References 41 publications

Production and Purification of Protease by Fungus Isolated From the Brazilian Dry Tropical Forest(caatinga)

Production and Purification of Protease by Fungus Isolated From the Brazilian Dry Tropical Forest(caatinga)

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate)

Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated from Eupenicillium javanicum Using Fluorescence Resonance Energy Transfer Peptides

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