Purification and Mode of Action of a Chitosanase from Penicillium islandicum

Abstract

Penicillium islandicum produced an inducible extracellular chitosanase when grown on chitosan. Large-scale production of the enzyme was obtained using R hizopus rhizopodijorrnis hyphae as substrate. Chitosanase was purified 3 8-fold to homogeneity by ammonium sulphate fractionation and sequential chromatography on DEAE-Biogel A, Biogel P,, and hydroxylapatite. Crude enzyme was unstable at 37 OC, 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 b~ro [~H]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.

Purification and properties of chitosanase from Bacillus circulans MH-K1.

“…The molecular weights of the enzymes are distributed within a range of 26,000 to 41,000. Three of them, determined by two different methods, showed more or less different values (11,13,15,17). But the mean value of the molecular weight is probably about 30,000.…”

“…Since mucoraceous fungi are widely distributed in the soil and chitosan is produced by those organisms, it can be expected that the chitosan may attract soil microorganisms producing chitosanase (10)(11)(12)(13)(14)(15)(16)(17). In this respect, we have tried to isolate microorganisms producing chitosanase, and recently we obtained one, a strain of Bacillus circulars MH-Kl (18).…”

Purification and characterization of a chitinase from Trichoderma viride.

“…According to Fenton and Eveleigh (1981), GlcN within the oligosaccharides is converted to 2,5-anhydromannose, whereas glucosaminitol at the reducing end residue is converted to 2-deoxyglucose. 2-Deoxyglucose in the reaction products of nitrous acid deamination therefore implies the 58 OMUMASABA, YOSHIDA, and OGAWA Vol.…”

“…Nitrous acid degradation of oligosaccharides was carried out by the method described by Fenton and Eveleigh (1981).…”

Immobilization of β-Glucosidase from Exiguobacterium sp. DAU5 on Chitosan Bead for Improved Enzymatic Properties

“…Chitosan, a partly acetylated or nonacetylated counterpart (4-linked-2-amino-2 deoxy-β-D-glucopyranan) of chitin, is present in the mycelial and sporangiophore walls of fungi and the exoskeletons of insects and crustacean [6,13]. It is usually obtained by the artificial deacetylation of chitin in the presence of alkali.…”