| Estrogen pollution is one of the most serious environmental pollution problems in the world,which has great harm and becomes a hot issue to research.We studied the estradiol degrading bacteria ED4which have preserved in laboratory,and tried to explain the enzymatic degradation mechanism.We optimized its enzyme production conditions and used response surface methodology sonication strains to obtain estradiol degrading enzymes.The estradiol-degrading enzyme positioning experiment shows ED4strains estradiol-degrading enzymes are intracellular enzymes.Enzyme-producing conditions of E2-degrading strains named Sphingo.multivorum.ED4are optimized by the single factor and orthogonal experiments,then the result shows that, the enzyme production can be the highest after72hours under such conditions:taking1%glucose as carbon source supplement, the peptone as nitrogen source, and the initial pH is7.0, culture temperature is30℃, the strain age is24hours, inoculums is2%. That is1.223times of the enzyme production before optimization.Three factors were investigated, including disruption power,disruption time and cell concentration, for their effects on enzyme activity;concurrently, single-factor tests were combined with the Response Surface Analysis(RSA) method to optimize the treatment conditions. The results showed that optimum conditions were achieved using a disruption power of400W, a disruption time of29min and a cell concentration of188mg/mL. Under these conditions, crude enzyme activity could reach1.477IU/mL.Enzyme nature has been analyzed after extracting the E2-degradation enzyme with acetone. Its most suitable pH is7.0, and the most suitable reaction temperature is35℃; controlling pH between7.0to7.5and the temperature between30℃to35℃can make the enzyme maintain higher stability; Al3+and Sn2+can suppress enzyme catalytic reaction, Mn+and Ca2+can increase enzyme activity.Using GC/MS analysis the estradiol degradation products and try to speculate the degradation pathways of estradiol. |
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