Characterization of Three Chitosanase Isozymes Isolated from a Commercial Crude Porcine Pepsin Preparation

• Published in: Journal of agricultural and food chemistry Vol. 51; no. 4; pp. 1042 - 1048
• Main Authors: Fu, Ju-Yueh, Wu, Sheng-Ming, Chang, Chen-Tien, Sung, Hsien-Yi
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 12.02.2003
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chitin - analogs & derivatives; Chitin - metabolism; Chitosan; Chromatography, Affinity; Chromatography, Ion Exchange; Enzyme Stability; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Hydrogen-Ion Concentration; Hydrolases; Hydrolysis; Isoelectric Point; Isoenzymes - chemistry; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Molecular Weight; Pepsin A - chemistry; Swine; Temperature; Enzyme preparation; Purification; Enzyme; Isozyme; Pepsin A; Pig; Peptidases; Vertebrata; Mammalia; Enzymatic activity; Glycosidases; Chitosanase; Hydrolases; Aspartic endopeptidases; Artiodactyla; O-Glycosidases; Ungulata
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf020675g

Three chitosanases designated PSC-I, PSC-II, and PSC-III were purified from commercial pepsin preparation by sequentially applying pepstatin A-agarose affinity chromotography, DEAE-Sephacel ion-exchange chromatography, Mono Q column chromatography, and Mono P chromatofocusing. With respect to chitosan hydrolysis, the optimal pHs were 5.0, 5.0, and 4.0 for PSC-I, PSC-II, and PSC-III, respectively; optimal temperatures were 40, 40, and 30 °C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were ∼40 kDa, as estimated by both gel filtration and SDS−PAGE, and the isoelectric points were 4.9, 4.6, and 4.4, respectively, as estimated by isoelectrofocusing electrophoresis. All three chitosanase isozymes showed activity toward chitosan polymer and N,N‘ ‘,N‘ ‘‘-triacetylchitotriose oligomer. Most effectively hydrolyzed were chitosan polymers that were 68−88% deacetylated. Keywords: Chitosanase; purification; characterization; pepsin