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Applied and Environmental Microbiology, July 2005, p. 3420-3426, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3420-3426.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Helena Gradisar,1* Jozica Friedrich,1 Igor Krizaj,2 and Roman Jerala1

Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia,1 Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, Ljubljana 1000, Slovenia2

Received 3 January 2005/ Accepted 6 January 2005

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 Km 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.

* Corresponding author. Mailing address: Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia. Phone: 386 1 4760 331. Fax: 386 1 4760 300. E-mail: helena.gradisar{at}ki.si.

Applied and Environmental Microbiology, July 2005, p. 3420-3426, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3420-3426.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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