Characterization And Purification Of Cellulases From Mangrove Macrobenthos In Dongzhaigang Mangrove Reserve, Hainan

Numerous studies have focused on the impact of biotic factors under the role of organic matter in the dynamics of mangrove ecosystems during the last decades, but an overall view about the role of macrobenthic fauna in organic matter processing has not yet been established. Cellulase and hemicellulase (xylanase and laminiarinase) activities were measured in the extracts from eight native mangrove macrobenthic species (two gastropods, three crabs and three bivalves) of southern China with plate assay and colorimetric assay. All enzyme activities were detected in the eight species except for negligible exoglucanase activity of the crabs and the bivalves. The crabs generated the largest halo zones in the plate assays, but produced the lowest amount of reducing sugar in the colorimetric assays. The highest endoglucanase, β-1,4-glucosidase and xylanase activities were detected in Crassostrea rivularis with colorimetric assays. For each mangrove macrobenthic species, the laminarinase activity was highest among the enzyme activities that were detected. The results suggest that mangrove macrobenthos participates in the carbon cycling of the mangrove ecosystem via the consumption and conversion of mangrove primary production at different ecological niches and they prefer algae as a major food source rather than mangrove leaf litter from September to October. Furthermore, five macrobenthic species were chosen to analyze the effect of NaCl on the stability of their digestive enzymes. Cellulases and xylanases from mangrove macrobenthos all showed high levels of salt-tolerance.A novel endo-P-1,4-glucanase was purified40.6fold from the digestive gland of the oyster Crassostrea rivularis living in mangrove forests by anion exchange chromatography, gel filtration chromatography and gel extraction. The27kDa cellulase named CrCel showed the specific activity of23.4U/mg against CMC. CrCel preferably hydrolyzed (3-1,4glucosidic bonds in the amorphous parts of cellulose materials and displayed degradation activity toward xylan. The optimal pH and temperature of CrCel were pH5.5and40℃, respectively. The enzyme was stable over a wide range of pH, retaining over60%activity after incubation for80min in the pH range of3.0-9.0. Besides, CrCel showed remarkable tolerance for salt, which not only remained active at high NaCl concentrations, but retained over70%activity after incubation in0.5-2M NaCl for up to24h. CrCel is the first reported cellulase isolated from mangrove invertebrates, which participates in the assimilation of mangrove leaf litter and contributes to the carbon cycling of mangrove ecosystem. The unique properties of this enzyme make it a potential candidate for further industrial application.The tissue localization of CrCel in the body of C. rivularis was investigated by Western blot. CrCel was determined to distribute in the digestive gland of C. rivularis, but not in the intestine where symbiotic microorganisms are harbored with cellulase zymogenicity. The results suggest that C. rivularis probably synthesizes CrCel endogenously in the digestive gland.