| Chitin is a long-chain biotic macromolecular polymer of a N-acetyl-D-glucosamine linked withβ-1,4glycosidic bonds. It is reported chitin accounted for18-60%of dry weight of the shells of shrimpand crab. These shells comprise75%total body weights of the shrimp and crab, but unfortunately haveusually been wasted. These materials can be partially digested by chitinolytic enzymes tochitooligosaccharides with diverse biologic activities, which represents the high value-added products inpresent chitin industry. The chemical hydrolysis for chitin in industry is confronted with high cost,imcomplete reaction and heavy pollutions. Compared with it, enzymatic digestion can overcome theseshortages, and also increase chitin utilization and bring economic value.In our laboratory, an Aeromonas veronii Strain B565was isolated from farmed pond sediment inTianjin. The whole genome sequencing has been fulfilled. Five chitinase genes and oneN-acetylaminoglycosidase gene are confirmed, which are probably related with chitin hydrolysis.A chitinase gene chiB565was cloned and expressed in Pichia pastoris, with chitinase activity of112.9U/mL and specific activity of557U/mg. The enzymatic characterization of purified chitinase wascarried out. The substrate specificity analysis indicated that the enzyme had the highest activity oncolloid chitin. Besides, it also can hydrolyze shrimp shell powder, chitin powder and glucan. It showedoptimal activity at50°C and maintained more than90%activity left after120min preincubation underthe same temperature. Its highest activity reached at pH5.0and keeps above80%relative activity at pH5.0-9.0. In addition, the enzyme showed resistance to various mental ions and neutral proteinases. Dueto its activity at actual aquiculture conditions, the in vitro hydrolysis research of shrimp shell powderwas also performed. The results showed that the best reaction time were3hours and30U of enzymeswere required to hydrolyze0.1g shrimp shell power. The combination of the enzymes with trypsincould significantly increase the release of tyrosine from shrimp shell powder, which is regarded as oneof necessary amino acids. Based on the results above, a35-day Tilapia feeding experiment withdifferent dosages of ChiB565additive was carried out. Interestedly, low content addition (16.2U/kgfeed) increased the weight of Tilapia at10.89%ratio, which decrease the feed transformation coefficientand increase the feed usage. The mRNA levels of HSP70and IL-1β declined after the low contentenzyme injection, suggested ChiB565addition did not cause any stress responses to Tilapia anddecreased basal inflammation. A challenge test by Aeromonas hydrophila, a disease-producing bacterialstrain, showed that ChiB565addition feeding for35days significantly improve the immune protectioneffects of Tilapia.ChiB565is firstly reported chitinase with shrimp shell powder degradation ability, so it is thoughtto be the potential feed enzymatic additive in aquaculture. It is also the first research that chitinase asfeed additive for animal feed products, which provide new thoughts and new methods in chitinaseapplication.In order to increase the hydrolytic effects, a β-N-acetylaminoglycosidase (chitobiase) gene nagB565 was cloned and expressed in E.coli. Purified NagB565showed activity of2257U/mL and specificactivity of7328U/mg presented the second highest specific activity chitinase reported presently. Itsproperty studies showed that its optimal pH was7.0and stable at pH5.0-9.0(maintained70%above oftop activity). Its optimal temperature was37°C, the activity declined as the temperature increased from37-70°C, and pretreatment below30°C showed no effects on enzyme activity. NagB565showedresistance to various metal ions and neutral proteinases. It can hydrolyse several chitoligosaccharides,with the highest activtity on chitobiose.In view of enzyme characterization, NagB565can be used in aquaculture conditions, and representsa potential aquacultural feed additive, facilitating the complete degradation of chitin. However, extraeffects assess should be carried out to ensure its safety in aquaculture. |
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