Design Of Artificial Chitinosome To Degrade Chitin

Chitin is an N-acetyl-D-glucosamine polymer formed by the linkage of ?-(1,4)glycosidic bonds.It is the second most abundant renewable resource on the earth only after cellulose.N-acetyl-D-glucosamine,a complete degradation product of chitin,has beenwidely applied in medicine,food,fertilizer,cosmetics and biofuels fields.In the current traditional production,the degradation of chitin is mainly used chemical method with harsh acid-base reagents,causing the low yield,impure product and environmental pollution during the degradation process On the contrary,enzymatic convertion is superior in the adventage of lower cost,high yield and product purity under the mild condition.Therefore,it is worth of developing a bioconvertion method to produce N-acetyl-D-glucosamine from chitin with the great potential social and economic value.The recalcitrant property of chitin hampered the efficiency of enzymatic degradation.In this study,we designed an artificial chitinosome by assembly of the recombinant three different chitiolytic enzymes with protein scaffold together,mimicking the structure of the cellulosome.,Then,we assessed the convertion efficiency of this chitinosome.This study has mainly achieved the following results:1.The chitinosome was designed according to the mode of cellulosome.A chitin-binding module CBD was linked on the C-terminal of the protein scaffold Scaf consisted of three different binding module Cohesins.These fusion enzymes with dockerins can anchor to protein scaffold automatically to form the chitinosome.2.The fragments encoding chitolytic enzymes and dockerins were amplified by polymer chain reaction(PCR).These fragments and plasmid pET30a(+)were ligased by seamless cloning to constructed the recombinant LPMO plasmid,the recombinant SaHex plasmid,and the recombinant SaChi19 B plasmid were obtained.The positive clones were confirmed by enzyme digestion and sequencing.3.The plasmids carrying the recombinase gene and the synthetic scaffold protein Scaf were transferred into Escherichia coli BL21(DE3)respectively,and achieved the soluble expression.The recombinant enzymes and scaffold protein were purified to apparent homogeneity by SDS-PAGE analysis.The equimolar ratio of purified recombinant enzymes and scaffold protein were assemblied togetherd by Native-PAGE gel detection.Thus,the artificial chitinosome t was successful obtained.4.The enzymatic properties of the assembled chitinosome were determine by DNS method.The optimum pH is 8,and the optimum reaction temperature is 30?.40 mM of chitinosome is enough to hydrolyze 1% crystalline chitin from shrimp shell completely in 8hrs,.Compared to the combination system of free enzymes,the degradation efficiency of chitinosome increased 2.53 folds under the optimal reaction condition.