RESUMO
An exo-chitosanase was purified from the culture filtrate of Gongronella butleri NBRC105989 to homogeneity by ammonium sulfate precipitation, followed by column chromatography using CM-Sephadex C-50 and Sephadex G-100. The enzyme comprised a monomeric protein with a molecular weight of approximately 47,000 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited optimum activity at pH 4.0, and was stable between pH 5.0 and 11.0. It was most active at 45°C, but was stable at temperatures below 30°C. The enzyme hydrolyzed soluble chitosan and glucosamine (GlcN) oligomers larger than tetramers, but did not hydrolyze N-acetylglucosamine (GlcNAc) oligomers. To clarify the mode of action of the enzyme, we used thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to investigate the products resulting from the enzyme-catalyzed hydrolysis of chitosan and N1-acetylchitohexaose [(GlcN)5-GlcNAc] with a GlcNAc residue at the reducing end. The results indicated that the enzyme is a novel exo-type chitosanase, exo-chitobiohydrolase, that releases (GlcN)2 from the non-reducing ends of chitosan molecules. Analyses of the hydrolysis products of partially N-acetylated chitooligosaccharides revealed that the enzyme cleaves both GlcN-GlcNAc and GlcNAc-GlcN bonds in addition to GlcN-GlcN bonds in the substrate.
Assuntos
Cunninghamella , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Acetilglucosamina/metabolismo , Quitina/análogos & derivados , Quitina/metabolismo , Quitosana/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Cunninghamella/enzimologia , Cunninghamella/genética , Cunninghamella/metabolismo , Glucosamina/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Mucorales/enzimologia , Mucorales/genética , Oligossacarídeos , Especificidade por SubstratoRESUMO
The hydrolytic specificities of chitosanases were determined using N¹,N4-diacetylchitohexaose [(GlcN)2-GlcNAc-(GlcN)2-GlcNAc]. The results for the hydrolytic specificities of chitosanases belonging to subclasses I, II, and III toward chitohexaose and N¹,N4-diacetylchitohexaose agreed with previous results obtained by analysis of the hydrolysis products of partially N-acetylated chitosan. N¹,N4-Diacetylchitohexaose is a useful substrate to determine the hydrolytic specificity of chitosanase. On the other hand, chitosanases from Amycolatopsis sp. CsO-2 and Pseudomonas sp. A-01 showed broad cleavage specificity. They cleaved both the GlcNAc-GlcN and the GlcN-GlcNAc bonds in addition to the GlcN-GlcN bond in the substrate. Thus, both enzymes were new chitosanases. The chitosanases were divided into four subclasses according to their specificity for hydrolysis of the ß-glycosidic linkages in partially N-acetylated chitosan.
Assuntos
Glicosídeo Hidrolases/metabolismo , Oligossacarídeos/metabolismo , Hidrólise , Pseudomonas/enzimologia , Especificidade por SubstratoRESUMO
Chitosanase II was purified from the culture filtrate of Aspergillus fumigatus ATCC13073. The purified enzyme had a molecular mass of 23.5 kDa. The N-terminal amino acid sequence of chitosanase II was identical to those of other Aspergillus chitosanases belonging to glycoside hydrolase family 75. The optimum pH and temperature were pH 6.0 and 40 °C. Chitosanase II hydrolyzed 70% deacetylated chitosan faster than fully deacetylated chitosan. Analysis of the degradation products generated from partially N-acetylated chitosan showed that chitosanase II split GlcN-GlcN and GlcNAc-GlcN bonds but not GlcNAc-GlcNAc or GlcN-GlcNAc, suggesting that it is a subclass I chitosanase. It degraded (GlcN)(6) to produce (GlcN)(3) as main product and small amounts of (GlcN)(2) and (GlcN)(4). Reaction rate analyses of mono-N-acetylated chitohexaose suggested that the (+3) site of chitosanase II recognizes the GlcNAc residue rather than the GlcN residue of its substrate.