CHITINOLYSIS BY THE MARINE ASCOMYCETE COROLLOSPORA-MARITIMA WERDERMANN - PURIFICATION AND PROPERTIES OF A CHITOBIOSIDASE

Out of six marine fungi examined, two, Corollospora maritima Werderman n, strain 198 and Lindra obtusa Nakagiri et Tubaki, strain 4937, grew on chitin and produced chitinolytic enzymes. Corollospora maritima sho wed higher growth rates and enzyme activity and was studied further. C rude, purified and colloidal chitin, as well as N-acetylglucosamine (G lcNAc) and the chitin di- and trisaccharides (GlcNAc)(2) and (GlcNAc)( 3), produced fungal growth comparable to that from carbon sources unre lated to chitin. Only chitin-related substrates induced chitinolytic a ctivity. Growth in 0.2% (w/v) purified chitin and 0.2% (w/v) GlcNAc wa s maximal after 12-14 days. Chitin-induced mycelial and extracellular (culture fluid) chitinolytic activities peaked at 10 and 14 days respe ctively, whereas the activities in both fractions increased in paralle l in GlcNAc-induced cultures with twice as much activity in the cultur e fluid as in the mycelium throughout. Chitin and GlcNAc both induced chitinolytic activity in washed, glucose-grown mycelia but no activity appeal-ed if glucose was present during the induction period. The cul ture fluid of GlcNAc-induced mycelia contained N-acetyl-beta-glucosami nidase activity (EC 3.2.1.30) and a chitin 1,4-chitobiosidase as defin ed previously [Tronsmo, A. and G. E. Harman (1993) Anal. Biochem. 208: 74-79]. The latter enzyme was purified by adsorption to colloidal chi tin and desorption by enzymic digestion of the substrate, followed by chromatofocusing. One peak of chitinolytic activity eluted from the ch romatofocusing column at pH 7.55 and corresponded to a single polypept ide of relative molecular mass (M(r)) 40 000 identified by SDS polyacr ylamide gel electrophoresis. High performance liquid chromatographic a nalysis showed that the major hydrolysis product from colloidal chitin , purified chitin and the chitin tetrasaccharide (GlcNAc)(4) was (GlcN Ac)(2) (> 90% of the reducing sugars), the remainder being GlcNAc and (GlcNAc)(3). The substrate (GlcNAc)(3) also yielded (GlcNAc)(2), indic ating that the chitobiosidase catalyses a transglycosylation reaction. This 'exochitinase' depolymerized crude chitin demonstrating that Cor ollospora maritima utilises a chitinolytic enzyme system more akin to that of chitinolytic bacteria than to terrestrial chitinolytic fungi.