| Fumarase(EC 4.2.1.2)is one of the key enzymes in the tricarboxylic acid(TCA)cycle,catalyzing the reversible hydration/dehydration of fumarate to L-malate.Fumarase C(FumC)has been successfully applied in industrial production of L-malate because of its relative thermal stability and reaction independent of ferric ions.With the increasing emphasis on environmental protection and the introduction of environmental taxes,the commencial demand for energy-saving and emission reduction has also increased.Further improving the stability of FumC will not only reduce the deactivation rate of immobilized enzyme in industrial environment,prolong its service life,but also help reduce the consumption of raw materials and energy for unit products,reduce pollutant emissions,and has an important economic and social benefits.In this paper,based on the research of domestic and foreign literature,five different sources of FumC genes were selected and cloned and expressed in Escherichia coli BL21(DE3).Based on the the results of determination and comparison of the enzyme activity of the unit broth fermentation volume and specific enzyme activity of these five recombinases,studies were conducted on the conversion and stability of FumC from E.coli(EEF)and FumC from Corynebacterium glutamicum(EGF).After investigating the two immobilized recombinant strains,it was found that the substrate conversion rate of both bacteria can reach 80%,and the conversion rate of the both did not decrease significantly between the consecutive 22 reaction batches in this experiment.Therefore,both of these two kinds of FumC were selected for thermal stability modification.First of all,after the high-throughput screening method suitable for this experiment was successfully established,EGF was used as a parent and an efficient directed evolution mutant library was constructed using error-prone PCR at a appropriate Mn2+ concentration.A mutant 2G(A411V)was screened from the mutant library of EGF.Its specific activity was 684.5 U/mg,T5015 was 47.2 ℃,t1/2。50 ℃increased from 1 min to 2.2 min.At the same time,the 3D-structure model of EGF was obtained by homology modeling and semi-rational design method was applied to modified the thermostability of EGF.Functional hotspots discovered by multiple sequence alignments were subjected to simulated mutations in 3D-structural model,then eight sites were selected for site-directed mutagenesis and mutants were constructed.3G(A227V),a mutant with improved stability was obtained from eight mutants of EGF.Its specific activity was 654.9 U/mg and showed 3.3 min increase in half-life at 50 ℃ and a 4.6℃ increased in T5015 compared with those of EGF.2/3G(A227V/A411V)was obtained by combining the mutant 2G with the mutant 3G,of which specific activity was 642.0 U/mg,the t1/2.50℃ increases to more than 768 min,t1/2,52℃ was 30.4 min,T5015 was increased by about 7.6 ℃.This showed that the effect of these two mutations on the thermal stability of proteins can be superimposed.Next,ten sites of amino acid residues that may have an important influence on the stability of the enzyme were determined by the rigidity analysis of the 3D-structure model,and ten site-directed saturation mutation libraries were constructed using the 2/3G as the parent.One positive mutant,175G(A411V/A227/E175K)with enhanced thermostability was screened from the saturated mutation library.The specific activity was 697.0 U/mg,T5015 increased from 52.4 ℃ to 54.6 ℃,t1/2,52 ℃ increased from 30.4 min to 1302 min while t1/2,50 ℃ was over 2700 min.In addition,enzymatic properties study found that the optimal reaction temperature for EGF was 30 ℃,and the optimum pH was 6.6.The optimal reaction temperature for 175G was 10 ℃ higher than that of EGF,reached 40 ℃,and the optimum reaction pH for 175G increased from 6.6 to 7.0.Then,EEF was used as a parent and an efficient directional evolution mutant library was constructed using error-prone PCR.7D(P220S),a mutant with improved thermostability,was obtained from the mutant library of EEF with its specific activity of 330 U/mg comparable to the parent enzyme.T5015 of 7D(P220S)increased 1℃from that of the parent enzyme(reached 50.5 ℃);Its t1/2,50 ℃ was 36.5 min.On the basis of 7D(P220S),directed evolution was carried out again,and the mutant strain 3D-2(P220S/A280T)with further improved thermal stability was isolated from the mutated library.The specific activity of the mutant enzyme was 346.6 U/mg,the T5015 reached 51.5 ℃,t1/2,50℃.was increased from 19.4 min to 55.9 min.However,in the subsequent transformation,neither the rational design method nor the semi-rational design method has obtained EEF mutants with improved stability.Moreover,enzymatic properties analysis showed that the optimal reaction temperature for EEF was 40 ℃ and the optimal reaction pH was 7.0.The optimum reaction temperature for mutant 3D-2 was 5 ℃ higher than that of EEF.The optimum pH of 3D-2 increased from 7.0 to 7.4.Comparing the resulting mutants transformed from EGF and EEF,it was found that the thermal stability of 175G(A411V/A227/E175K)was higher than that of mutant 3D-2(P220S/A280T).The T5015 between the two kinds of FumC has a difference of 2.8 ℃.And the specific activity of 175G(A411V/A227/E175K)was also higher than that of 3D-2(P220S/A280T)at the ideal temperature for industrial production of L-malate(40-60 ℃).From a food safety point of view,enzymes derived from C.glutamicum are more easily accepted by the public than enzymes derived from E.coli,and the former has been used for industrial production of L-malate.Therefore,The thermostable mutant 175G(A411VA227E175K)without affecting the activity in this study would be a more suitable candidate for industrial applications. |
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