Discrimination between chitinase and chitobiase activities in Candida albicans.

Abstract

Measurement of chitinase activity in crude homogenates of Candida albicans with the artificial substrates 4-methylumbelliferyl β-D-N, N′, N"-triacetylchitotrioside and 4-methylumbelliferyl-β-D-N, N′-diacetylchitobioside (MU-[GlcNAc]3, 2) showed that both chitobiase and chitinase were involved in the liberation of the fluorophore methylumbelliferone (MU). The substrate MU-[GlcNAc]3 appeared to be digested by means of two separate mechanisms, resulting in a lag-phase in the time curve of the MU-release, and thus making this release non-linear with time. The first mechanism is the action of an endochitinase that released MU in one step; this reaction could be inhibited by allosamidin. The second probable mechanism involves the digestion of the substrate by the stepwise release of the outer two G1cNAc residues by a chitobiase, followed either by a third chitobiase-catalyzed release of the final GlcNAc or by the action of a β-N-acetylhexosaminidase, that is also present in C. albicans. Hydrolysis of the substrate MU-[GlcNAc]2 was not inhibited by allosamidin, suggesting that the two GlcNAc residues in this substrate are probably released in two steps, either by the action of chitobiase alone, or by the successive action of chitobiase (step 1) and β-N-acetylhexosaminidase (step 2). Our results show that the activities of the individual enzymes involved in chitin degradation cannot be determined on the basis of MU release in unfractionated homogenates of C. albicans.