Aeromonas caviae NCHU1發酵生產幾丁酵素之純化及特性

Abstract

In this investigation, a bacterial strain with chitinase activity was isolated from soil in Tainan and identified as Aeromonas caviae NCHU1. This strain can product chitinases when it is grown in a medium containing chitin power of marine waste. A chitinase was purified from culture broth of Aeromonas caviae NCHU1 by a series of purification steps, i.e. ammonium sulfate precipitation, DEAE-Sepharose, and gel filitration with Sephadex G-75. The molecular weight of NCHU1 chitinase is 26.2kDa by sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) analysis. The optimum temperature and pH, thermal and pH stability ranges of NCHU1 chitinase are 60°C, pH6, 10-40°C, and pH5-10, respectively. To determine the oligosaccharide contents of NCHU1 chitin hydrolysates, it was found that (GlcNAc), (GlcNAc)2, (GlcNAc)5, and (GlcNAc)6 existed. This implies that Aeromonas caviae NCHU1 may produce endo-chitinase. Furthermore, the affinity chitin chromatography with fermentation- modified chitin was employed for purification of chitinase. By observing the structural changes of chitin and fermentation-modified chitin by SEM, it was found that chitin surface became porous and branching structure, which might reveal more active sites and less the steric hinderance to the adsorption of chitinase. For chitinase adsorption capacity, factors such as loading temperature, pH and elution solutions were studied. The optimal loading conditions were found at 10℃, pH7. The elution solution was 0.05M glycine buffer. Under these conditions, a purification fold of 3.97 was obtained. Then, the modified chitin can be regeneration and reused.