Cloning, Expression And Characterization Of Chondroitin Sulfate From Bacteroides Polymorpha

Chondroitinase(Ch Sase)is a bacterial extracellular cleavage enzyme that dissociate the ?-1,4 glycoside between the disaccharide repeating units of chondroitin sulfate to produce low molecular weight chondroitin sulfate which has high biological activity because of the low viscosity,good solubility and absorbability.In this study,various bacteria containing the Ch Sase gene were screened to obtain highly expressed and viable Ch Sase.Chondroitin sulfate was degraded by Ch Sase and the content of hydrolyzed product was determined by UV spectrophotometry and the product of chondroitin sulfate was verified by mass spectrometry.The Bacteroides thetaiotaomicron CAG:40 strain containing identified three Ch Sase genes was obtained from screening.The identified Chsase enzymes were cloned and the characterization of chondroitin sulfate was also performed.The main research contents of this research are as follows:(1)Three Ch Sases genes in the B.thetaiotaomicron CAG:40 strain named Ch Sase-ABC I(Gene ID: 524777996),Ch Sase-ABC II(Gene ID: 524777977),Ch Sase-AC(Gene ID: 524780449),and encoding 1025,892,719 amino acids were identified,respectively.(2)Computational analysis showed that the three Ch Sases contained signal peptide sequences encoded 19,14 and 24 amino acids,respectively.The chondroitinase ABC(PDB ID: 2Q1F)from B.thetaiotaomicron wal2926 was selected as the template,the sequence identities to the template of the three Ch Sases were 30%,98%,and 34%,respectively.The homology modeling of Ch Sase was built using Swiss Model online sever.The PROCHECK results proved the three models were reasonable.Thus,the genomic DNA of B.thetaiotaomicron CAG:40 was extracted as the template for cloning.Three Ch Sase genses were successfully cloned and ligated into the expression vectors p Czn1 and p ET-30 a.(3)Expression of recombinant Ch Sase-ABC I was optimized and the characterization of the expressed enzyme was performed.The optimal conditions for expression of recombinant Ch Sase-ABC I wereas follows: 0.75 m M of IPTG,temperature 25 oC,and the induction time was 6 h.After purification,a single band was observed by SDS-PAGE and the molecular weight was found approximately 108 k Da.Relatively high enzyme activity of Ch Sase-ABC I in a 70 m M p H 8.0 phosphate buffer solution was detected.The enzyme displayed the highest activity of 541.3 U mg-1 at 37 oC.And it remained about 50% relative activity after 120 minutes incubation at the optimal temperature.Enzymatic reaction rate of Ch Sase-ABC I could be enhanced in the presence of 1 m M magnesian.The Km and Vmax of Ch Sase-ABC I were calculated as 0.54 mg/m L and 541.3 U·mg-protein-1 based on the double reciprocal plots,respectively.(4)The final product of reaction catalyzed by Ch Sase-ABC I is monosulfated unsaturated disaccharide with mass-to-charge ratio of 458.30 when chondroitin sulfate A was used as the substrate,which suggestted that Ch Sase-ABC I degraded chondroitin sulfate through the ?elimination mechanism.(5)The expression of the previous cloned Ch Sase-AC was investigated.The optimal expression was oberserved at the following conditions: 10 ?M of IPTG,temperature 15 oC,the induction time 20 h.Most of Ch Sase-AC was expressed in soluble form under these conditions.A single band around 80 k Da was found with SDS-PAGE after Ni-IDA purification.The optimum p H of the enzyme is 7.5 and the optimum temperature is 35 oC.More than 80% relative activity was left after incubation 4 h at 37 oC.And enzymatic reaction rate of Ch Sase-AC could be enhanced in the presence of 1 m M calcium.