Optimization Of 3-phenoxyzoic Acid Degrading-enzyme Production By Sphingomonas Sp. SC-1 And Its Enzyme Immobilization

The pyrethroid pesticides is wildly used in the world with the disabled of high toxic and high-residual pesticides, and its residual and damage problems have increasingly becoming the hot issue of focuses and researches.3-Phenoxybenzoic acid (3-PBA) is an intermediate product of most pyrethroid pesticides. This product can not only caused secondary pollution to agricultural produces but also inhibit the growth of microbial metabolism, and moreover it can retard the biomineralization of the parent pesticides. As is well known microbial degradation is an efficient and green method to degrade toxic compounds, and enzyme degradation has a higher efficiency and security than microbe for it is a protein. In this experiment we study the 3-PBA-degradation enzyme on the source of Sphingomonas sp. SC-1 which has 3-PBA-degradation effect. Localization the 3-PBA-degradation enzyme in the microbial cell, optimized the conditions of the enzyme production, and immobilized the crude enzyme, study the properties of the immobilized enzyme.The localization the 3-PBA-degradation enzyme results shows that extracellular enzymes, intracellular enzyme and the microbial cell debris all have activities, and the activities is low in extracellular and intracellular compare with the microbial cell debris. Separate the microbial cell membrane and it has 3-PBA-degradation activity, so the 3-PBA-degradation enzyme located in the microbial membrane of Sphingomonas sp.SC-1.Crush the membrane samples and separate it by Native-PAGE, recycle the protein bands in the Gel and find the band c has 3-PBA-degradation activity. Separate the band c by SDS-PAGE and get two protein bands, the 3-PBA-degradation enzyme protein molecular weight about 90 Ku and 50 Ku, respectively.Take the MM medium for the initial medium to optimize the conditions of the enzyme production of SC-1. The optimum conditions contained initial pH 8.33, incubation time 37.75 h, spawn age 40.0 h,3-PBA 100 ?g/mL, peptone 0.5%, yeast extract 0.5%, glucose 0.8%, MgSO4 0.01%, rotary shaker 180 r/min, temperature 30?, inoculums volume 4% (SC-1 concentration 108cfu/mL), loading volume 30 mL(250 mL conical flask). The validation test result was 0.3871U /mL.Get the optimal immobilized conditions of 3-PBA-degradation crude enzyme embedding immobilized by single factor experiment:a ratio of enzyme liquid and sodium alginate 1:5 (v/v), sodium alginate and calcium chloride concentration at 2% and 1% respectively. Under the immobilize conditions of this article, immobilization time is not obvious to enzyme activity, but immobilized enzyme particle diameter have great influenceThe immobilized enzyme optimal reaction temperature is 30?, and it has a poor thermal stability, when temperature higher than 40? the enzyme activity is less than 50%. The optimal reaction pH is 7.0, between pH5.5-8.5 the immobilized enzyme can keep more than 60% of the enzyme activity and the stability of immobilized enzyme in alkaline conditions slightly higher than the partial acid conditions. Mg2+?Mn2+ and high concentrations of Ba+, low concentration of Cu2+promote the immobilized enzyme activity; Pb2+?Zn2+ and high concentration of Cu2+, Fe3+ has strong inhibitory effect. And EDTA?0.01%BSA?10-6 mol/L phenanthroline?0.1%Vc have promoting effect on enzyme activity,0.1%SDS?10-2mol/L phenanthroline have strong inhibitory effect on enzyme activity. The michaelis constant (Km) of immobilized enzyme is 46.63 ×106 nmol/L, maximum reaction rate (Vmax) is 3.70×103 U/g. The application of immobilized enzyme preliminary simulation experiment shows that:under the conditions of this article, the cell debris immobilized enzyme has high degradation activity, after 900 min continuous reaction in the optimal reaction conditions.The results of this article can not only lay a theoretical foundation for the study of 3-PBA-degradation enzyme in Sphingomonas sp.SC-1, but also provide a reference to eliminate the 3-PBA in environmental and agricultural products, thus possessing important theoretical significance and practical value.