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
The effect of middle and higher boiling sulfur compounds on wine flavor were studied. Volatile sulfur compounds were extracted from wine with chloroform and analyzed by FPD-GC and GC-MS. By these methods following seven sulfur compounds were identified and determined : 2-methylmercapto-ethanol (MME), dime thyl sulfone (DM), 3-methylthio-propanol (MTP), Dihydro-2-methyl-3 (2 H)-thiophene (DMTP), 2-methylthiophan-3-ol (MTO), 3-methylthio-prpionic acid (MTPA) and ethyl 3-methythio-propionate (EMTP). MME and DM were found in wine for the first time in this study.These compounds had unpleasant odor which were similar to onion, rubber and fermented vegetables, but they they were not so serious Off-flavor than the odor of lower boiling sulfur compounds.Their flavor units were calculated from the odor-threshold. Sensory evaluation was carried out on wine after the addition of each compund.Plus images were shown when these compunds were added to wine in amounts below three times of the flavor units, for example, fruity, powerful, or full of flavor. When these compounds were added to wine over five-fold of their flavor units, they brought minus images to wine, for ecample, "flat", "lacking of fruity", "onion-like" and "rubberlike".Apparently, these sulfur compunds made an important contribution to the wine flavor.
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