Preparation And Enzyme Kinetics Property Study Of A Novel GPX Mimic Basing On Hyaluronic Acid

Wild streptococcus zooepidemicus C55151 was mutated by NTG.(N-methyl-N'-nitro – N -nitrosogua – nidine). A high HA-producing strainJ18 was acquired, which can produce HA twice as much as the original strain.The optimum fermentation condition was studied by an orthogonal method,which is glucose 50g/L, yeast extract paste 30g/L, MgSO4 0.5g/L. It is foundthat SDS can improve the yield of HA obviously. Kinetics of HA fermentationprocess of the original and mutant strain was compared.Polysaccharide produced by mutated strain of Streptococcuszooepidemicus was purified by the procedure including Savage method,quaternary ammonium compound precipitation, DEAE-cellulose (DE52)column chromatograph and Sephadex G-75 gel filtration. The structure of thepurified polysaccharide has proved to be hyaluronic acid (HA) by means ofchemical composition analysis, 13C NMR, Infrared spectra, and circulardichroism. The single helix conformation of the purified HA was studied byCongo red experiment. The molecular weight of the HA was about 1.16×106D, which was measured by viscosity method.Basing on the framework of hyaluronic acid(HA) and method of chemicalmodification, novel modified hyaluronic acid compounds were synthesized tobe functioned as a mimic of glutathione peroxidase (GPX). The structure ofSeHA was characterized by means of IR and NMR with the conclusion thatthe target SeH was located at –CH2OH of the N-acetyl-D-glucosamine. Afterthe new compound was prepared and purified,SeHA was applied for DTNBmethod to identify the selenium content of 1.2%. The GPX activities of SeHA,reducing H2O2, tert-butyl hydroperoxide(t-BuOOH) and cumenylhydroperoxide (CuOOH) by glutathione, are 103.88 u/μmol 32.15 u/μmoland 152.71 u/μmol respectively, which illustrated CuOOH is the optimalsubstance of the mimic. In the steady-state kinetic studies of theSeHA-catalyzed reactions with three substrates, a ping-pong mechanism wasobserved from the data of the double reciprocal plots. All the result showedthat SeHA behaves similar to the native GPX. 2,6-di-butyk-4-methylphenol(BHT) and iodine acetic acid were applied to investigate the catalyzingmechanism of the mimic respectively. It is shown that the catalyzedmechanism is free radical and the mimic cannot be inhibited by iodine aceticacid.