Recombinant Expression And Characterization Of Catechol 2,3-dioxygenase And Nitrilase From Thauera Sinica K11~T

Thauera sinica K11^T is a high-efficiency pollutant-degrading bacterium isolated from the activated sludge of refinery wastewater treatment plant.Previous studies have found that this strain contains a number of novel pollutant-degrading enzyme genes(clusters)such as such as the genes of catechol 2,3-dioxygenase(C23O)and nitrilase(KN).Therefore,based on the identification of new bacterium in our previous work,the catechol 2,3-dioxygenase and nitrilase derived from T.sinica K11^Twere recombinantly expressed,and the enzymatic properties of the two enzymes were studied.Firstly,recombinant strain E.coli BL21-p DE1-C23O was constructed by genetic engineering technology,and heterologous expression of catechol 2,3-dioxygenase was achieved by induction.The catechol 2,3-dioxygenase was purified by gel filtration chromatography,and its enzymatic properties were characterized.The results showed that the activity center of the enzyme was Mg²⁺,the molecular weight was 140 kDa,the optimum reaction conditions were 50?and pH 9.0,and the stability was better when the pH was 7.0-11.0 and the temperature was less than 50?.The secondary structure of the enzyme is mainly?-sheet(content is 46.69%);the K_mand V_maxof the enzyme are:0.17 m M and 0.38 m M/min·mg,respectively.In addition,Ca²⁺,Cu²⁺and Mn²⁺have a strong inhibitory effect on C23O,while Fe²⁺and Fe³⁺have no effect on enzyme activity;EDTA and Na N₃ had little effect on the activity of C23O,but the activity of C23O was zero after adding hydrogen peroxide;the enzyme had a broad substrate spectrum and had the best catalytic effect on 3-methylcatechol.Secondly,recombinant strain E.coli BL21-p DE1-KN was constructed by genetic engineering technology,and the heterologous expression of nitrilase was achieved by induction.The enzymatic properties of nitrilase showed that the molecular weight of the enzyme was 30 kDa;the optimum pH and the temperature were pH 8.0 and 70?respectively;the enzyme was relatively stable at pH 7.0-11.0 and temperature less than 70?;the K_m and V_max of the enzyme were 17.85 m M and 2.67?M/min·mg,respectively.In addition,K⁺,Zn²⁺,Fe²⁺,Ag⁺,Mg²⁺,Mn²⁺,Hg²⁺,Cr³⁺,Ca²⁺,L-cysteine and L-glutathione had little effect on the activity of KN,while Cu²⁺,Co²⁺,Ba²⁺and DTT had certain inhibitory effect on KN;trichloromethane,n-hexane,diethyl ether,isopropanol and Span-80 had no effect on the enzyme,n-heptane promoted the enzyme,and DMSO strongly inhibited the enzyme activity.The enzyme has a broad substrate spectrum and has the best catalytic effect on dodecanenitrile.Finally,in order to further increase the expression level of nitrilase,two nitrilase recombinant strains E.coli BL21-p ET28a-KN and E.coli BL21-p QE30-KN were reconstituted by using double endonuclease restriction.After comparing the expression of nitrilase by three recombinant strains,it was found that the expression of nitrilase in recombinant E.coli BL21-p QE30-KN was the highest.By constructing phylogenetic tree and substrate degradation experiments,it was found that the nitrilase from T.Sinica K11^T was a multifunctional nitrilase.