Catechol 2,3-dioxygenase From Thauera Sinica K11: Fermentation Optimization,Purification And Characterization

Catechol 2,3-dioxygenase which catalyzes the dioxygenation of catechol to form2-hydrox-ymuconate semialdehyde,is one of the most important enzymes in many bacterial pathways for the degradation of aromatic compounds.Due to its potential application in environmental protection,C23O has been purified from a variety of organisms.Recently,a new bacterial strain,Thauera sinica K11,was isolated from an activated sludge sample from a sewage treatment plant and could degrade phenol derivatives aerobically.First of all,the single factor experiment and response surface method was used to cultivate Thauera sinica K11,in order to improve the production and activity of catechol2,3-dioxygenase determining the optimal culture medium composition as follows:phenol308.0 mg/L,ammonium sulfate 153.1 mg/L,phosphate 2.7 g/L.Undering the condition of optimal culture medium composition,the largest enzyme activity of catechol 2,3-dioxygenase was 1921 U.In addition,the method of dimension exclusion chromatography was used to isolate and extract the high purity catechol 2,3-dioxygenase from Thauera sinica K11 strain,and then characterized the structural properties systematically.The results showed that the molecular weight of the enzyme was 140 kDa,consisting of four identical subunits,each containing a Fe²⁺ion.The particle size of the enzyme in dilute solution was 14.5 nm,and its secondary structure was mainly composed of?-sheet?51.1%?.The optimum reaction conditions was45^oC,pH 8.4.The enzyme in the pH 8 to 11 and the temperature less than 50^oC have good stability.The K_m and V_max of the enzyme were 20.36?M and 105.28 mU mg^-1 respectively.In addition,Cu²⁺has the highest positive impact on the enzyme.If the dissolved oxygen is too high or too low,it can inhibit the enzyme activity.The inhibitory effect of EDTA and sodium azide on enzyme was not obvious,but H₂O₂ could inactivate enzyme.The enzyme can degrade monocyclic phenolic compounds and has a relatively wide substrate specificity.Finally,using the technology of genetic engineering successfully built recombinant strains,E.coli BL21/pET28a?+?-C93O,implementing the recombinant expression of catechol 2,3-dioxygenase by inducing expression.On that basis,using affinity chromatography separation technology obtained high purity recombinant catechol2,3-dioxygenase?C93O?,and characterized the properties of enzyme by different techniques.The results showed that the production of recombinant catechol 2,3-dioxygenase?C93O?was greatly increased,and the optimal reaction conditions to pH 8.0 and 50^oC,Mg²⁺had the highest stimulative effect on the enzyme.