Study Of Fermentation And Purification Of L-glutamate Oxidase

L-Glutamate oxidase（GLOD）is a relatively kind of new enzyme which wasdiscovered at the beginning of80s. GLOD which have been isolated from varioussources，including snake venom，rat kidney，invertebrates and microorganiaims. It is aflavin protease with FAD for cofactor, and specifically catalyzes the reaction that onemole of L-glutamic acid was converted to one mole of hydrogen peroxide,α-ketoglutaric acid and ammonia. It has been widely used in the area of food, medicine,fermentation, etc. and became a very useful tool enzyme. At present, the domesticresearch on the enzyme is restricted to the optimum conditions of fermentation andGLOD crude products preparation, while the preparation of the pure GLOD is rarelyreported. the pure GLOD with high price and not timely supply we currently usedlargely depend on several foreign companies, therefore the localization of the pureGLOD is imperative.Streptomyces splendens.4.666, which was preserved in Biology Institute ofShandong Academy of Sciences was studied. After renovationofvarieties，activation，astrain which has higher GLOD production ability than other strains was selected，and itsmorphology characteristic and the physiological and biochemical characteristics weredetected. The strain was used as the GLOD producing strain in the followingexperiments.In order to further improve use value of the strain biological activity，the growth ofthe strain and the enzyme production condition are optimized. Then the the culturemedium composition optimization were studied by response surface and the effects ofthe fermentation conditions on the basis of one factor test. The result showed that theoptimum medium was sucrose3.2%，corn serum2.2%，glucose3.0%，ammoniumsulfate1.0%，sodium glutamate1.0%，MgCl₂0.05%， KCl0.05%，NaH₂PO₄0.05%，Each shake flask plus3%calcium carbonateï¼›the optimum initial was pH6.7，the liquidvolume was30mL，the inversion temperature，transition time and inoculated ratio were28℃，48h and3.0%，respectively. The largest product was increased from9.56g/L to19.1g/L，which was2times of the initial yield.The GLOD was separated and purified from the cuture solution of GLODproducing strain of Streptomyces sp.4.666and the purification line was designed. Thepurified enzyme was prepared from the fermentation culture through frozencentrifugation， ammonium sulfate fractionation，Sephadex G-25desalination，Mono Q iron exchange，Superdex G-200gel chromatography and freeze-drying. The specificactivity of GOLD was176U/mg. The purification yield was12.95%, and it had beenpurified by926times.The purified GLOD shows a single channel by SDS-PAGE eletrophoresis and，isabout140kDa single protein after SDS-PAGE and Superdex G-200gel chromatographyanalysis. it shows that most unwanted poly-saccharide was moved after FPLC fastprotein purification system in the enzyme，and98.67%GLOD was yield.Finally，The properties of GLOD and its application were studied. The enzyme wasstable below50℃. The optimal pH and temperature were7.0and50℃，respectively.The Michaelis-constant（Km）is2.1×10^-4mol/L. Most of all the metal ions had littleeffect on the activity of GLOD，trace amounts of SDS、EDTA、Cu²⁺can reduce theactivity of GLOD in varying degrees，While Hg²⁺and Ag⁺can inhibit the activity of theenzyme obviously. At last，we make a comparison between the enzyme membrane madeby puried GLOD and international commodity GLOD in the biosensor.This paper just do some research in the theory and practical for our countrycommercial GLOD production. When compared to the international commodityGLOD，there is still a large distance. It has not been able to carry on the amino acidsequence analysis because of the condition limits. moreover, this strain’s trial productionresearch also waits for exploring. It not only can open a new application field for thebiosensor but also can lower the consumables cost, saves the foreign exchange andpromote bio-sensor’s promoted application, which has the extremely high value of eachcontribution and the market potential.