PURIFICATION AND CHARACTERIZATION OF AN EXO-N,N'-DIACETYLCHITOBIOHYDROLASE-LIKE ENZYME FROM CELLULOMONAS-FLAVIGENA NTOU-1

Cellulomonas flavigena NTOU 1 could grow well and show higher chitinas e activity in the modified salts-pectin broth additionally containing 1% (w/v) shrimp shell powder and 0.5% yeast extract. When crude enzyme solution was concentrated and purified through ion-exchange chromatog raphy, hydrophobic interaction chromatography, and gel filtration on f ast protein liquid chromatography, a chitinase was isolated. The enzym e displayed a specific activity of 232.9 U mg(-1) protein and gave a s ingle band on SDS-PAGE. The molecular weight of the purified chitinase was estimated at around 34.2 kDa by gelfiltration and 32.5 kDa by SDS -PAGE. The optimum pH and temperature were 10.0 and 50 degrees C, resp ectively. The protein was stable in the pH range from 6-10 and up to 4 5 degrees C. Fe3+, Hg2+, N-ethylmaleimide, monoiodoacetate, and beta-m ercaptoethanol inhibited enzyme activity while Na+, Zn2+, and Mg2+ cau sed activation. The chitinase showed a K-m value of 0.15 mM against 4- methylumbelliferyldiacetylchitobiose. The chitinase degraded substrate s in an exo-splitting manner as an exo-N,N'-diacetylchitobiohydrolase. (C) 1997 by Elsevier Science Inc.