Mutation Breeding And Fermentation Process Optimization Of Xanthine Oxidase Producing Strain

Xanthine oxidase (XOD, EC.1232) is an important enzyme that exists in theprocess of nucleic acid metabolism as an available oxidoreductase in all kinds oforganisms. It is also an available enzyme which promotes iron absorption andtransport, detects the activity of superoxide dismutase (SOD), measures inorganicphosphorus in serum, improves the conversion rate of ribavirin and determines thefreshness of fish, etc.. Rely solely on animal sources of XOD limits its wideapplication, so since the60s of last century, microbial sources have been committed todeveloping XOD in foreign, but now for the study of XOD by bacteria is still at anearly stage, the research of XOD production by bacterial fermentation has drawnincreasing attention.Various mutating methods including ultraviolet(UV), diethyl sulfate(DES),hydroxylamine hydrochloride, nitrous acid, lithium chloride, microwave irradiationand compound mutagenesis were used to deal with a xanthine oxidase producingstrain Arthrobacter sp.X. One high production strain Arthrobacter sp.UW6withgenetic stability was screened through the substrate resistance screening method forprimary screening in the selection plate and then to shake-flask re-screening. Afterfermentation, the yield of xanthine oxidase reached7.699U/ml, increased by63.46%over the original strain.Then through the cell form in early and late phases, colony morphology,physiological and biochemical test results, and16SrDNA sequence analysis, weidentified the strain should be regarded as Arthrobacter, and named Arthrobactersp.UW6.The culture conditions of high production strain Arthrobacter sp.UW6wasoptimized by using single parameter, Plackett-Burman and Box-Benken responsesurface analysis. The optimized culture conditions was as follows: working volumeof75mL in a250mL flask, initial pH7.5, inoculum6.0%(v/v),32℃and200rpm,K2HPO43H2O4.15g/L, KH2PO40.26g/L, NaCl1.0g/L, CaCl24.5mg/L, FeSO47H2O0.5mg/L, MgSO47H2O0.2mg/L,(NH4)2SO42.217g/L, xanthine0.75g/L, lactose12.83g/L. Under the optimum condition, the enzyme peak from54hshortened to44h, the yield of xanthine oxidase reached12.67U/mL, increased65.21%by over the original strain.The studies on the enzymatic characteristic showed that the optimum pH value ofxanthine oxidase was8.0, the optimized storage buffer was Tris-HCl (pH7.0~9.0); andthe optimum temperature was45℃, poor thermal stability of the enzyme. The activitycan be stimulated by Mn2+and Ba2+, and inhibited by Pb2+, Ag+and Hg2+at aconcentration of2mM or more.