Gene Cloning and Recombinant Expression of Chondroitinase AC from Pedobacter heparinus and Characterization of Recombinant Fusion Enzyme

Chondroitinase AC(ChonAC) is an important enzyme to reveal the biological function,structure and mechanism of chondroitin sulfate,and it is important for low molecular weight chondroitin sulfate preparation and diseases treatment.To achieve the efficient expression of ChonAC with high activity in recombinant Escherichia coli(E.coli),the ChonAC gene(cslA) from Pedobacter heparinus was amplified by polymerase chain reaction(PCR),and the fusion expression vector for expressing maltose-binding protein(MBP) fused ChonAC(MBP-ChonAC) was constructed.The results showed that the fusion strategy using MBP was effective to enhance the solubility of the MBP-ChonAC when induced at low temperature of 15 ℃,the purity and the specific activity of MBP-ChonAC could reach 95% and 94.1 IU/mg-fusion protein(equivalent to 143.8 IU/mg ChonAC) by one-step MBPTrap HP purification,respectively.The enzyme characteristic study showed that the optimum pH value,Ca2+concentration,NaCl concentration,and reaction temperature was 7.5—8.0,20 mmol/L,50 mmol/L,and 20—35 ℃,respectively.The half-life of MBP-ChonAC could reach 8.3 h at 30 ℃.The kinetic characterization showed that the Kmwas higher while the kcat was a little bit lower than the reported native ChonAC for chondroitin sulfate A,while the biocatalysis experiment for MBP-ChonAC indicated that the fusion of ChonAC with MBP did not affect the enzyme function.Furthermore,the total activity of MBP-ChonAC by shake flask cultivation could reach 10800.5 IU/L,the highest value reported so far,through the optimizations of host cells,IPTG induction concentration,and M9-based culture medium.