Mycol. Res. 95 (2):217-219 (1991) Printed in Greaf Britain 217 Degradation of chitosan in the autolysis of Mucorales C. ALFONSO, M. J. MARTINEZ AND F. REYES Centro de Investigaciones Bioldgicas, C.S.I.C., Vekiquez 144, 28006 Madrid, Spain The degradation of chitosan by enzymes present in the culture medium of seven fungi of the order Mucorales was studied during their autolysis. Constant levels of endochitosanase activity were detected during incubation of most of these fungi. Exochitosanase activity was detected in Mucor hiemalis, Mucor rourii, Rhiwpw stolonifer and Syncephalastrurn racernosurn. No exochitinase activity was found but endochitinase activity was detected in 40 day autolysed cultures of all the fungi and chitin deacetylase activity was indirectly detected in most of the fungi. Natural autolysis of filamentous fungi is mediated by lytic enzymes that catalyse the degradation of cell wall poly- saccharides (Reyes & Lahoz, 1977) and cytoplasmic material (Foster, 1949). Generally the cell wall degrading enzymes increase their activity in the culture fluid with increasing incubation time (KO & Lockwood, 1970; Lahoz ef al., 1979; Reyes ef al., 1981). The biosynthesis of these enzymes (Reese, 1972; St Leger ef al., 1986) is considered an inducible phenomenon triggered by either monomers or oligomers maintained at low levels in the culture fluid by the turnover of substances during autolysis (Reyes ef al., 1988). The occurrence of chitosan in the cell walls of members of the Mucorales (Bartnicki-Garcia, 1968; Allan ef al., 1987) led to this study into the possible presence of chitosanases (EC 3.2.1.99) in the culture fluid during autolysis of these fungi. and the filtrate made up to 20 ml with distilled water. The mycelium was washed with distilled water and dried at 60' to constant weight. The degree of autolysis was defined as the loss in mycelial dry weight as a percentage of the maximum at the onset of autolysis. Chitin was obtained from prawn shell as described by Jeuniaux (1966), and carboxymethylchitin by the method of Trujillo (1968) with a substitution degree of 43% (Eyler ef al., 1947). Chitosan with an acetylation degree of 20% (Hayes & Davies, 1978) was obtained by deacetylation of chitin as described by Horton & Lineback (1965). Carboxymethylchitosan with a substitution degree of 23% and acetylation degree of 30% was obtained by the method of Nud'ga ef al. (1973). Total reducing substances in culture filtrate were determined by combining the methods of Somogyi (1945) and Nelson (1944) using glucose as standard. Soluble proteins were determined by the method of Lowry ef al. (1951) using bovine MATERIALS AND METHODS serum albumin as a standard. Glucosamine and N-acetyl- glucosamine were detennined as described by Tracey (1955). The seven fungi studied were Absidia californica Ellis & This method does not distinguish between mono- and Hesseltine IJFM 7459 (Institute Jaime Ferrin de Microbiologia, Madrid, Spain); Mortierella vinacea Dixon, Stewart IJFM 7443; Mucor hiemnlis Wehmer, IJFM 7408; Mucor rouxii (Calmette) Wehmer, CECT 2654 (Coleccion Espafiola ce Cultivos Tipo, Valencia, Spain); Phycomyces blakesleeanw (Burgeff), CMI 118496 (Commonwealth Mycological Institute, Kew, England); Rhizopus stolonifer (Ehrenberg ex Friex) Lind, IJFM 7425; Syncephalastmm racernosum (Cohn) Schroter CMI 77601. Each was maintained on potato-agar slants (3.9% potato-dextrose-agar) and transferred to malt-agar slants (2% malt, 0-1 % peptone and 2% agar) to provide an inoculum. Fungi were grown at 25 OC without shaking in 100 ml conical flasks containing 20 ml of mineral liquid medium (Santamaria & Reyes, 1988). Samples were taken daily until autolysis began, at which time mycelial dry weight was maximal, and then at ten days intervals. The culture medium was filtered (Whatman No. 1) ~li~osaccharides. Exochitosanase activity was assayed by estimating the glucosamine liberated when hydrolysing a colloidal chitosan suspension. One unit of chitosanase activity was defined as the amount causing the liberation of 1.0 pmol glucosamine h-' from colloidal chitosan (1 mg ml-') at 37'. P- N-acetylglucosaminidase activity (EC 3.2.1.30) was assayed by estimating 4-nitrophenol liberated when hyrolysing 4-nitrophenyl-2-acetamido-2-deoxy-~-~-glucopyranoside (Woollen ef al., 1961). Endochitosanase and endochitinase activities were assayed by measuring the percentage viscosity change of solutions of carboxymethylchitosan or carboxy- methylchitin (1 mg ml-') respectively using a Cannon-Fenske viscometer (535412) at 37'. One unit of activity was defined as that amount causing a 1 % decrease in viscosity per min.