Purification and characterization of chitinase and chitobiase produced by Aeromonas hydrophila subsp. anaerogenes A52.

Abstract

Chitinase (EC 3.2.1.14) and chitobiase (EC 3.2.1.30) from culture broths of our isolate Aeromonas hydrophila subsp. anaerogenes A52 were concentrated by 80% saturation with ammonium sulfate into a crude enzyme preparation. The crude enzyme preparation was purified by separating the chitinase from the chitobiase by affinity adsorption of the chitinase on chitin. The chitinase was further purified by ion-exchange chromatography with C M-Sephadex C-50. The chitobiase was purified by successive ion-exchange chromatography with CM-cellulose and DEAE-Sephadex A-50. The homogeneity of the purified enzyme preparations was evaluated by polyacrylamide gel disc electrophoresis. The isoelectric points of the chitinase and chitobiase were determined to be 4.60 and 5.35, respectively. The chitinase showed a molecular weight of approximately 110,000, slightly higher than the chitobiase at 105,000. These values are the highest ever reported among chitinolytic enzymes from microbial origins. In addition, both the chitinase and the chitobiase showed a glycoprotein nature on polyacrylamide gel disc electrophoretogram when stained with Schiff's reagent. On examination of the effects of chemical reagents upon the activities of the enzymes, sulfhydryl groups appeared to be involved in the expression of the activities, even though they behaved differently in detail with the different reagents tested. The Km value of the chitinase was 2.8 mg chitin ml -1 and of the chitobiase was 1 x 10 -3 M of p-nitrophenyl-/3-N-acetyl-D-glucosamine. The effect of pH and temperature on the reaction rate or stability of the enzymes are also presented. By following the decrease in turbidity of chitin suspension and the formation of N-acetylglucosamine caused by the action of chitinase, the enzyme was found to degrade the substrate in an endo-splitting manner.25

Purification, Cloning, and Sequence Analysis of β-N-Acetylglucosaminidase from the Chitinolytic BacteriumAeromonas hydrophilaStrain SUWA-9

“…anaerogenes A52. 13) Although most of the kinetic behaviors of these two enzymes are similar to those of NAG20A, some characteristics are clearly different. For example, NAG of Aeromonas sp.…”

“…Species of the genus Aeromonas occur widely in water, sludge, and sewage. Chitin-degrading enzymes are apparently widely distributed in this genus, and have been reported so far from A. hydrophila, [13][14][15][16] A. caviae, 17,18) and Aeromonas sp. 19,20) Cells of A. hydrophila SUWA-9 grew well in M9 synthetic-colloidal chitin medium, but most of the chitinase activity was not detected in the culture supernatant, but was associated with cells during all growth phases (Fig.…”

“…Some bacterial NAGs were extracellular enzymes and are presumed to work in a degradation of chitooligosaccharides produced from polymeric chitin by the action of endochitinase. 13,[24][25][26] On the other hand, NAGs have been reported to be located in periplasmic space or in cytoplasm in the case of marine bacteria. 22,23,27) This intracellular localization of enzymes appears to be advantageous for an efficient hydrolysis of chitin in sea water, since hydrolyzed products can easily be taken into cells.…”

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

“…Preparation of colloidal chitin. Suspension of colloidal chitin was routinely prepared in the manner described previously (24), according to JEI NIAUx (25).…”

Chemical modification studies of the active centre of Candida albicans chitinase and its inhibition by allosamidin

“…In any case, high concentrations of thiol reagents necessary for the partial inactivation of chitinase exclude the participation of any cysteine residue in the catalytic action of the enzyme. It should be added that some other chitinases have been reported to be inhibited by Hg2+ ions (Somers et al, 1987) and iodoacetic acid (Yabuki et al, 1986). Both these agents are known as thiol-blocking compounds.…”