Cloning, Expression And Properties Of Chondroitin Sulfate AC Enzyme

Chondroitinase AC(ChonAC)is a family of enzymes that depolymerize chondroitin sulfate(CS)and hyaluronic acid(HA)through a?-elimination,cleaving the?-1,4 glycosidic linkage between N-acetylgalactosamine and glucuronic acid in glycosaminoglycans to produce oligosaccharides,mainly unsaturated disaccharides.It can be used for the preparation of low molecular weight chondroitin sulfate,remove chondroitin sulfate in crude heparin,the structural-functional analyses of CS oligosaccharides,and treatment of spinal cord injury.In this thesis,three encoding novel ChonAC genes from different bacteria were cloned and successfully heterologously expressed in Escherichia coli BL21.The expressed ChonACs with6×His tag fusion were purified via a metal chelating affinity column.Biochemical characterization of the purified ChonACs was performed and molecular docking was carried out to analyze the interactions between ChonACs and the substrates.The main research contents are as follows:1)Three ChonACs from Pedobacter sp.ok626?Pedobacter rhizosphaerae DSM18610 and Pedobacter xixiisoli named PschonAC,PrchonAC and PxchonAC encode 702,701,and 699 amino acids,respectively.The BLAST analysis of the PschonAC,PrchonAC and PxchonAC sequence displayed the 43.5%,63.48%and 52.65%sequence identity with ChonAC from F.heparinum(Fhchon AC).All the gene were cloned and ligated to construct the pET-28a-chon AC expression vector and then transformed into E.coli BL21(DE3)for the expression.2)The conditions for inducing the expression of three ChonACs were optimized.The optimal conditions for recombinant PschonAC were 30?M ITPG,at 15 ^oC and the induction time was 30 h,the activity of the crude PschonAC was 150 U L^-1.The crude enzyme was then purified with a metal chelating affinity column;the purified PschonAC displayed a specific activity of 35 U mg^-1 toward CS and with a molecular weight of approximately 76.08 k Da was showed via 12%SDS-PAGE. The optimal expression of PrchonAC at the following conditions:0.1 m M IPTG, temperature 20 ^oC and the induction time was 12 h,the activity of crude PrchonAC was 1500 U L^-1.The crude enzyme was then purified with Ni-NTA affinity chromatography.The over-expressed PschonAC was clearly displayed a protein band,which is estimated to be 77 k Da.This result was consistent with the predicted molecular weight of the hypothesized PrchonAC,and the purified PrchonAC howed a specific activity of 150 U mg^-1 with CS-A.PxchonAC could achieve a ighest activity of 5082 U L^-1 under the expression conditions(0.1 m M IPTG, emperature 25 ^oC and the induction time was 15 h).The crude enzyme was then urified with Ni-NTA column to obtain the purified PxchonAC.It showed a single rotein band in SDS-PAGE gel with a molecular weight of 76.8 k Da.The specific ctivity toward CS was determined as 428.77 U mg^-1.3)The biochemical properties of three purified ChonACs were investigated. schon AC could achieve a highest enzyme activity at p H 7.5 in 50 m M PBS buffer. he optimum temperature for the catalytic reaction of PschonAC was 25 ^oC,and he half-life in this temperature was 200 min.Most metal ions have no obvious ffect on PschonAC,while the enzyme was strongly inhibited by Co²⁺.PschonAC ould utilize chondroitin sulfate as the substrate with the K_m and V_max of 0.37 mg L^-1 and 39.2 U mg^-1,respectively.The relative activity of PschonAC remained 64% fter 18 days of storage.The optimal reaction temperature was 35 ^oC,and the ptimal p H for the hydrolysis of chondroitin sulfate was 7.5.The K_m values of rchon AC towards chondroitin sulfate A,chondroitin sulfate C,and hyaluronic cid were 1.68,0.14,0.34 mg m L^-1.PrchonAC could utilize CS-A,CS-C,HA as he substrates with the V_max of 479.39,24.46 124.36 U mg^-1,respectively.The half- ife of PrchonAC at 25 ^oC was 20 min,and PrchonAC could remain more than 90% esidual activity after 10 days store at 4 ^oC.The optimum temperature and p H were 0 ^oC and p H 8.0 using chondroitin sulfate A as the substrate.The K_m and V_maxof xchon AC were 0.61 mg m L^-1and 670.18 U·mg^-1 using chondroitin sulfate A as he substrate.The enzyme activity was significantly improved by Ca²⁺,and xchon AC retained higher residual activities and exhibited a half-life 6.3 times onger than PxchonAC without Ca²⁺.While PxchonAC was strongly inhibited by n²⁺with the enzyme activity decrease 94.3%.PxchonAC exhibited good stability t physiological conditions and showed a half-life of approximately 660 minutes ith 20 m M Ca²⁺.PxchonAC possessed a half-life with 3,300 minutes at 25 ^oC pproximately.PxchonAC displayed a half-life of 27 days at 4 ^oC.4)Homology modeling and molecular docking were performed to validate the nteractions between enzyme and substrate.The theoretical structures of PschonAC, rchon AC and PxchonAC were then constructed based on the crystal structure of hchon AC(1CB8).The Ramachandran map displayed that 99.8%residues of three rotein models located at the favored regions,demonstrating the protein structures ere reasonable.The docking model revealed that Asn125,Trp126,Asn175, er233,His225,Tyr234,Arg288,Arg292,and Asn374 were the key catalytic esidues of PschonAC.These amino acids were strictly conserved.More hydrogen onds were formed in the active pocket among PrchonAC-CS-C,which limited the xtension of the substrate in the active center,resulting in lower enzyme activity; hile hyaluronic acid lacks sulfate moieties at galactosamine,result in the ecreased ability on the recognition of substrate,which making the enzyme activity lightly lower than that using CS-A as the substrate.PxchonAC exhibited higher tability than PrchonAC.The docking model revealed that five hydrogen bonds ormed between the Ca²⁺and the four amino acid residues of PxchonAC in the loop, nd there were four hydrogen bonds for PrchonAC.These hydrogen bonds robably improve the structural rigidity of the enzymes,and stabilize the tertiary tructures,and improving the protein's stability.