Enzyme and Microbial Technology 36 (2005) 455–460 In vitro degradation of porcine skin epidermis by a fungal keratinase of Doratomyces microsporus J. Friedrich a,∗ , H. Gradiˇ sar a , M. Vrecl b , A. Pogaˇ cnik b a Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia b Institute of Anatomy, Histology and Embryology, Veterinary Faculty, University of Ljubljana, Gerbiˇ ceva 60, SI-1000 Ljubljana, Slovenia Received 27 February 2004; accepted 27 September 2004 Abstract A keratinase, produced biotechnologically by Doratomycesmicrosporus, was used to treat porcine skin in vitro under different experimental conditions. The amount of soluble proteins, their electrophoretic profile and the histological appearance of the skin cuttings were followed. The amount of soluble protein released by the keratinase was maximal after 6 h of incubation in neutral or alkaline environments up to pH 9. With prolonged incubation, the amount of protein released due to the enzyme decreased on account of further hydrolysis of soluble proteins into smaller units. As shown by SDS-PAGE, the proteins of 45–70kDa detected in controls were hydrolysed by the enzyme to fragments of 14 kDa or less after 3 h incubation in the optimal pH range. Histological examination of the skin cuttings suggested that the enzyme was active throughout the incubation period, since the effect on epidermis progressed with time. Stratum corneum was detached from the underlying layers of epidermis that were severely disintegrated and separated from the unaffected dermis at the epidermal–dermal zone. Keratinase also hydrolysed the outer epithelial sheath of hair roots provoking depilation. These data suggest the potential of this enzyme for application in ecologically-friendly leather processing. © 2004 Elsevier Inc. All rights reserved. Keywords: Fungal keratinase; Proteolysis; Porcine skin; In vitro enzymatic degradation; Doratomyces microsporus 1. Introduction Keratins are structural proteins located in skin and its ap- pendages such as hair, nail, feather and also in epithelial tis- sues within the body. They are a complex group of proteins with molecular weights (M w ) of 40–67 kDa, assembled into intermediate filaments (IFs) from pairs of type-I and type- II subclass molecules [1,2]. Most of them are insoluble in water and resistant to biodegradation. However, certain mi- croorganisms and insects synthesise keratinases that are able to degrade keratins. These enzymes enable keratinolytic bac- teria and fungi to degrade waste keratin in nature [3] and, among others, they play a role in skin invasion by dermato- phytic fungi [4]. Keratinases have several potential applica- tions: in detergent formulations for eliminating horny epithe- ∗ Corresponding author. Tel.: +386 1 476 03 33; fax: +386 1 476 03 00. E-mail address: jozica.friedrich@ki.si (J. Friedrich). lial cells adhered to textile fibres [5], ecologically friendly leather processing [6,7], waste chicken feather degradation [8–10], nutritional improvement of waste feather for live- stock feed [11] and production of protein hydrolysates from keratinous waste materials [12]. Keratinolytic activity is quite widespread among com- mon non-pathogenic fungi, especially among hyphomycetes which can be used for production of the enzymes. The amount of keratinase synthesized is dependent on the cultivation con- ditions and the source of keratin used as an inducer [13]. Among the most potent species identified in our screening process was a ubiquitous fungus Doratomyces microsporus. It can be cultivated in submerged aerobic conditions to syn- thesize an active keratinase. The enzyme is a serine proteinase with M w of 30 kDa that displays optimal activity at 50 ◦ C [14]. The isolated keratinase is able to hydrolyse keratinous and non-keratinous proteins. Among keratinous substrates it preferentially hydrolyses keratin powder prepared from hu- 0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2004.09.015