f Purification and Mode of Action of a Chitosanase from Penicillium islandicum
- Authors: DENNIS M. FENTON†, DOUGLAS E. EVELEIGH
- First Published Online: 01 September 1981, Microbiology 126: 151-165, doi: 10.1099/00221287-126-1-151
- Subject: Biochemistry
- Issue Published:
Penicillium islandicum produced an inducible extracellular chitosanase when grown on chitosan. Large-scale production of the enzyme was obtained using Rhizopus rhizopodiformis hyphae as substrate. Chitosanase was purified 38-fold to homogeneity by ammonium sulphate fractionation and sequential chromatography on DEAE-Biogel A, Biogel P60 and hydroxyl-apatite. Crude enzyme was unstable at 370C, but was stabilized by 1·0 mm-Ca2+. The pH optimum for activity was broad and dependent on the solubility of the chitosan substrate. Various physical and chemical properties of the purified enzyme were determined.
Penicillium islandicum chitosanase cleaved chitosan in an endo-splitting manner with maximal activity on polymers of 30 to 60% acetylation. No activity was found on chitin (100% acetylated chitosan) or trimers and tetramers of N-acetylglucosamine. The latter two oligomers and all small oligomers of glucosamine inhibited the activity of chitosanase on 30% acetylated chitosan. The pentamer of N-acetylglucosamine and glucosamine oligomers were slowly cleaved by the enzyme. Analysis of the reaction products from 30% acetylated chitosan indicated that the major oligomeric product was a trimer; with 60% acetylated chitosan as substrate a dimer was also found. The new terminal reducing groups produced by chitosanase hydrolysis of 30% acetylated chitosan were reduced by sodium boro[3H]hydride. The new end residues were found to be N-acetylglucosamine. The analyses strongly indicated that P. islandicum chitosanase cleaved chitosan between N-acetylglucosamine and glucosamine. Both residues were needed for cleavage, and polymers containing equal proportions of acetylated and non-acetylated sugars were optimal for chitosanase activity. The products of reaction depended on the degree of acetylation of the polymer.
Present address: Pfizer Central Research, Eastern Point Road, Groton, Connecticut 06340, U.S.A.
© Society for General Microbiology 1981 | Published by the Microbiology Society
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