Study On The Characterization And Synergism Of The Chitinolytic Enzymes From Aeromonas Veronii B565

Chitin is the second most abundant polysaccharide, merely after cellulose in nature（¹011tons），which is widely distributed in the cell walls of bacteria and fungi, the exoskeleton of insects andcrustacean （shrimp, crab, etc.），the microfilariae sheath of parasites the intestinal peritrophic membraneof insects. Chitin is a kind of gray, tasteless, solid sheet and as a natural biological extract, it hasexcellent biological compatibility and adaptability. Thus chitin is broadly utilized in many aspects,including medicine, food, agriculture, environmental protection and other fields.Liu and collegues in our laboratory isolated a bacteria-strain from the sediment of an aquaculturepond myxosporidia once broke out and recovered by itself in Wuqing, Tianjin, China. This bacterium isidentified as Aeromonas veronii, and nominal strain for B565. The bacterium is gram-negative bacilli,subordinate Vibrionaceae Aeromonas.The whole-genome sequence of A. veronii strain B565（GenBank accession number CP002607）was reported in our earlier study. Strain B565encodes three chitinases from GH18, namely Chi92（GenBank number AEB48885.1）, ChiA （AEB48887.1）, ChiB565（AEB48892.1）, one chitin bindingprotein （CBP）（YP₀04394209.1） and two chitinases from GH19, namely ChiC （AEB50059.1） andChiP （AEB48889.1）. Our previous work have cloned, expressed, and characterized the ChiB565andβ-N-glucosaminidase, as well as their application to be feed additives.In this study we cloned, sequenced and expressed two exo-chitinases genes, Chi92and ChiC. Oneexhibits both endo-and exo-chitinase activity ChiA and one acts as a chitin binding protein（CBP） inPichia pastoris GS115. Chi92had optimal activity at pH6.0and40℃. Chi92retained more than58.1%of optimal activity over the pH range4.0–9.0at40℃and>48.8%activity among temperature range20–40℃. Under the optimized conditions, Chi92had the highest activity,809.2U/mg （colloidal chitinas substrate）. Moreover, it showed certain resistance to chemical reagent, mental ions, digestiveproteinases, and Tilapia intestinal juice. Futhermore, Forage safety evaluation experiment showed thatChi92had low specific activity when mucin （the protective barrier of intestinal epithelium mucosa）（149.2U/mL） was used as substrate （¹8%of colloidal chitin as substrate）. The recombinant ChiA hadhigh yeild （442.1mg/L）. The optimum pH of ChiC and ChiA was8.0and6.0, respectively, while theywere stablized between pH5.0-10.0; The optimal temperature of ChiC and ChiA was40℃and stayedstable in low temperature range. CBP showed the ability of binding the shrimp shell chitin and colloidalchitin.Chi92were characterized for its high activity, ability to digest β-glucanase and weak damage to thefish intestinal protective barrier （mucin）, instead ChiA and ChiC showed rather higher recombinantexpression levels. Although all three chitinases showed pH and temperature stability, under aquaculturecircumstance, suggesting its potential as an enzymatic feed addictive in warm-water while thethermostability of Chi92and the chitinase activity of ChiA and ChiC need to be improved. In addition, we studied the transcription levels of chitinolytic enzymes in A.veronii B565and foundchitinases reached its peak levels at12h after induction. At this timepoint, colloidal chitin induced highexpression of five chitinolytic enzymes. Similarly, ChiA, ChiC, Chi92also showed high expression byshrimp shell chitin induction of12h. Additionally, after induced24h by colloidal chitin, ChiA and CBPcommon high expression. These data provide theoretical basis for investigating the synergismmechanism.Preliminary research on synergism showed CBP significantly promoted the chitinase activity ofChiA （colloidal chitin as the substrate, shrimp shell chitin as substrate） and ChiB565enzyme activity（shrimp shell chitin as substrate）.Our research have not only filled the unexplored area of glycoside hydrolase19family bacteriachitinase oringinating not from actinomycetes and purple bacteria, enriched the resources of availablechitinases, but also provided new methods of exploitation and utilization of yeast resources, which canbe finally employed as an ideal model to study the catalytic mechanism of chitinase.