Characterization of three chitosanase isozymes isolated from a commercial crude porcine pepsin preparation.
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 degrees C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were approximately 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.
Fu
Ju-Yueh
JY
Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan 106, R.O.C.
Wu
Sheng-Ming
SM
Chang
Chen-Tien
CT
Sung
Hsien-Yi
HY
D000595:Amino Acid Sequence; D000818:Animals; D002686:Chitin / Q000031:analogs & derivatives* / Q000378:metabolism; D048271:Chitosan; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010434:Pepsin A / Q000737:chemistry*; D013552:Swine; D013696:Temperature
D007527:Isoenzymes; D002686:Chitin; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase; D010434:Pepsin A