Partial characterization of the extracellular keratinase from<i>Microsporum canis</i>

Takiuchi, Iwao; Sei, Yoshihiro; Takagi, Hisae; Negi, Makoto

doi:10.1080/00362178485380351

Cited by 49 publications

(20 citation statements)

References 10 publications

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“…A T. mentagrophytes 48 kDa protease (Yu et al, 1968) may be meMep4 and a 45 kDa M. canis protease (Takiuchi et al, 1982(Takiuchi et al, , 1984 is probably caMep3 or caMep4. In contrast, the T. rubrum keratinolytic protease active as a dimer with subunits of 44 kDa (Asahi et al, 1985) and the T. mentagrophytes 41 kDa protease (Tsuboi et al, 1989) are not fungalysins but serine proteases as attested by the effect of different inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum

et al. 2004

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Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55-75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72-97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19-36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.

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Section: Discussionmentioning

confidence: 99%

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum

et al. 2004

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“…Serine proteases (Chandrasekaran and Dhar 1987;Kitadokoro et al 1994;Yum et al 1994;Taguchi et al 1995) and other proteases have been purified and characterized in Streptomycetes. Some serine proteases derived from Streptomycetes (Sinha et al 1991;Moormann et al 1993;Böckle et al 1995;Suzuki et al 1997;Bressollier et al 1999) or other microorganisms (Takiuchi et al 1984;Lin et al 1992;Kulakova et al 1999) were identified as keratinolytic proteases. Keratinases could be very useful in industrial applications, e.g., hydrolysis of high molecular mass substrates such as feather, hair, and leather to produce amino acids or peptides (Papadopoulos 1989;Dalev and Neitchev 1991).…”

Section: Introductionmentioning

confidence: 97%

Gene cloning and heterologous expression of a serine protease fromStreptomyces fradiaevar.k11

Meng

Cao

et al. 2007

Can. J. Microbiol.

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The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 degrees C for its caseinolytic activity and pH 9 and 62 degrees C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.

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“…They are produced by some insects and mostly by microorganisms. The best studied are keratinases from the dermatophytic genera Microsporum (26,37) and Trichophyton (30,38) as well as from bacteria of the genera Bacillus (4,14,20,24,34,35) and Streptomyces (2,3,27). There are relatively few reports on characterization of the keratinases from nondermatophytic fungi (5,7,12,25,32,33).…”

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Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases

Gradišar

Friedrich

Križaj

et al. 2005

Appl Environ Microbiol

209

112

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Based on previous screening for keratinolytic nonpathogenic fungi, Paecilomyces marquandii and Doratomyces microsporus were selected for production of potent keratinases. The enzymes were purified and their main biochemical characteristics were determined (molecular masses, optimal temperature and pH for keratinolytic activity, N-terminal amino acid sequences). Studies of substrate specificity revealed that skin constituents, such as the stratum corneum, and appendages such as nail but not hair, feather, and wool were efficiently hydrolyzed by the P. marquandii keratinase and about 40% less by the D. microsporus keratinase. Hydrolysis of keratin could be increased by the presence of reducing agents. The catalytic properties of the keratinases were studied and compared to those of some known commercial proteases. The profile of the oxidized insulin B-chain digestion revealed that both keratinases, like proteinase K but not subtilisin, trypsin, or elastase, possess broad cleavage specificity with a preference for aromatic and nonpolar amino acid residues at the P-1 position. Kinetic studies were performed on a synthetic substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The keratinase of P. marquandii exhibited the lowest K m among microbial keratinases reported in the literature, and its catalytic efficiency was high in comparison to that of D. microsporus keratinase and proteinase K. All three keratinolytic enzymes, the keratinases of P. marquandii and D. microsporus as well as proteinase K, were significantly more active on keratin than subtilisin, trypsin, elastase, chymotrypsin, or collagenase.Keratins are the most abundant proteins in epithelial cells of vertebrates and represent the major constituents of skin and its appendages such as nail, hair, feather, and wool. The protein chains are packed tightly either in ␣-chain (␣-keratins) or in ␤-sheet (␤-keratins) structures. Keratins belong to the superfamily of intermediate filament proteins. Their high degree of cross-linking by disulfide bonds, hydrophobic interactions, and hydrogen bonds stabilizes keratin filament structure (11). Therefore, keratinous material is water insoluble and extremely resistant to degradation by proteolytic enzymes such as trypsin, pepsin, and papain.A group of proteolytic enzymes which are able to hydrolyze insoluble keratins more efficiently than other proteases are called keratinases (29). They are produced by some insects and mostly by microorganisms. The best studied are keratinases from the dermatophytic genera Microsporum (26, 37) and Trichophyton (30, 38) as well as from bacteria of the genera Bacillus (4,14,20,24,34,35) and Streptomyces (2, 3, 27). There are relatively few reports on characterization of the keratinases from nondermatophytic fungi (5,7,12,25,32,33).Most keratinases have some common characteristics despite their different origins. They belong mainly to the extracellular serine proteases, with the exception of keratinases from yeasts, which belong to the aspartic proteases (23, 28). The molecular masses of ...

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Partial characterization of the extracellular keratinase fromMicrosporum canis

Cited by 49 publications

References 10 publications

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum

Gene cloning and heterologous expression of a serine protease fromStreptomyces fradiaevar.k11

Similarities and Specificities of Fungal Keratinolytic Proteases: Comparison of Keratinases of Paecilomyces marquandii and Doratomyces microsporus to Some Known Proteases

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