| Previous studies have found that two strains of Proteobacteria:Thiomnias intermedia BCRC17547 and Xinfangfangia sp.DLY26 both contain novel nitrile pollutant degrading enzyme genes.Therefore,in this thesis,three novel nitrilase enzymes derived from BCRC17547 and DLY26 were recombinantly expressed,their enzymatic properties were characterized,and a highly efficient re comb inant strain was optimized and immobilized.Name the three enzymes BN,DN1 and DN2 respectively,through the analysis of amino acid sequence similarity,it is found that the conserved catalytic site Glu-Lys-Cys(E-K-C)in nitrilase can be found in all three enzymes.The amino acid sequence similarity comparison revealed that the similarities between BN,DN1 and DN2 were 24.24%-40.31%.Both BN and DN1 have the highest similarity with the nitrilase of R.eutropha H16,with 62.96%and 44.90%,respectively.DN2 has similar sequences to the nitrilase of P.abyssi.The highest is 31.16%.It shows that the three nitrilase enzymes are relatively novel nitrilase enzymes.The PCR technology was used to successfully amplify the three nitrilase genes from BCRC17547 and DLY26,and the recombinant strain E.coli BL21-pDE1-BN/DN1/DN2 was constructed at the same time.And the three enzymes have achieved soluble expression in the recombinant engineering bacteria.The molecular weights of the three recombinant nitrilase enzymes,BN,DN1 and DN2 are approximately 57.0 kDa,59.2 kDa and 50.7 kDa,respectively.Each of the three enzymes is composed of two single subunits that are identical to each other.At 30℃ C and pH 7.0,with 10 mM 3-cyanopyridine as the substrate,the activities of the three nitrilase enzymes BN,DN1 and DN2 measured for 60 min are:11836.83 U·L-1,9597.98 U·L-1 and 9100.47 U·L-1.The optimum pH and optimum temperature of BN,DN1 and DN2 are 8.0 and 75℃,7.0 and 55℃,7.0 and 60℃,respectively,and they are all thermostable nitrilase enzymes.The catalytic activity of BN,DN1 and DN2 is relatively stable at pH 5.0-8.0 and 65℃,pH 6.0-8.0 and 50-55℃,pH 6.0-8.0 and 50-55℃,respectively.The three enzymes all have a broad substrate spectrum.Among them,BN has the best degradation effect on lauric nitrile,3-phenylpropionitrile,sebaconitrile and 3-indoleacetonitrile,DN1 has the best degradation effect on 4-chlorobutyronitrile,2-Methylglutaronitrile and 3-phenylpropionitrile have the best degradation effects,DN2 has better degradation effects on most nitriles.In the investigated metal ions and chemical reagents,except Ba2+has almost no effect on BN,all other metal ions have a certain inhibitory effect on BN,except for Ni2+ which has almost no effect on DN1,the other metal ions have almost no effect onm DN1.A certain inhibitory effect,Fe2+ and Ni2+ have a strong promoting effect on DN2,and Mg2+,Fe3+,Mn2+and Ba2+all have a certain inhibitory effect on DN2.The metal chelator EDTA and the reducing agent DTT have no great influence on the activities of the three enzymes,L-cysteine and L-glutathione have little effect on the activities of the three enzymes.Among the organic solvents and surfactants,SDS hardly affects the activities of the three enzymes.Span-80 has a certain inhibitory effect on BN,but has a certain promotion effect on DN1,and has little effect on DN2.Triton X-100 has a certain inhibitory effect on BN,a slight promotion effect on DN2,and basically no effect on DN1.After adding organic solvents,methanol,glycerol,DMSO and acetone have a certain inhibitory effect on BN,DMSO has a certain inhibitory effect on DN1,while methanol,ethanol,glycerol,isopropanol and ether have a certain promotion effect on DN1,Propanol and DMSO have a certain inhibitory effect on DN2.The fermentation process of recombinant strain BL21-pDEl-BN to produce nitrilase was optimized:yeast extract was used as carbon source,peptone was used as nitrogen source,the concentration of inducer IPTG was 0.5 mM,and the induction time was 8 h.The induction temperature is 25℃,the immobilization conditions of recombinant strain BL21-pDEl-BN are optimized:sodium alginate concentration is 3.0%,polyvinyl alcohol concentration is 12%,CaCl2 concentration is 1.0%,immobilization time for 3 hours.The optimal reaction pH of SA-PVA immobilized cells is 8.0,and the optimal reaction temperature is 45℃.After 4 weeks of storage in a refrigerator at 4℃,it can still retain high catalytic activity,the half-life of SA-PVA immobilized cells is greatly increased,When the substrate 3-cyanopyridine concentration is 50 mM,the SA-PVA immobilized cells can be reused in 8 batches,and the conversion rate of the first 5 batches can reach more than 90%,in the eighth batch In the transformation experiment,the remaining catalytic activity of the immobilized cells retained at 45.3%of the initial catalytic activity. |
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