| L-norvaline is a non-protein amino acid that was first identified as a natural component of antimicrobial peptides in Bacillus subtilis.L-norvaline is mainly used as a vital intermediate for the synthesis of perindopril.Perindopril is safe ACE inhibitors and used to treat hypertension and congestive heart failure.The synthesis of L-norvaline is mainly depends on chemical method at present.But direct chemical synthesis of chiral pure L-Nor not only involve highly toxic raw material cyanide but also low yield,which is not conducive to environmental protection.Because of the advantage of biocatalyst,including stereo-selective,mild reaction conditions and high catalytic efficiency,it has been paid much attention in the area of synthesis of chiral compounds.In this paper,a multi-enzyme cascade system was developed for the synthesis of chiral pure L-norvaline.The chiral pure products were obtained by converting D-norvaline in the racemate into its enantiomer L-norvaline through enzyme specific oxidation and asymmetric reduction.By constructing engineering strains with multi-enzyme cascade expression,carrying out molecule modification of the key enzymes,optimizing the multi-enzyme cascade system by mathematical model simulation and on this basis further controlling the expression levels of enzymes,thus through the fermentation of a single engineering bacteria,the best multi-enzyme cascade system used to efficiently produce L-norvaline was obtained,and there was no need to try the different proportion of the enzymes in the system before the conversion.The main content was as follows:?1?Recombinant strain E.coli BL21/pXMJ19-RtdaaoS-Bpcat-Cbfdh-Bcldh used for the synthesis of L-norvaline was constructed for co-expression enzymes of the multi-enzyme cascade system,including D-amino acid oxidase?DAAO?,catalase?CAT?,formate dehydrogenase?FDH?and leucine dehydrogenase?LDH?.Main transformation processes were as follows:The D-norvaline in mixed substrate DL-norvaline was specifically oxidized to 2-oxovaleric acid by DAAO which was then asymmetrically reduced to enantiomer L-norvaline by LDH.In the process CAT was used to eliminate the by-product H2O2 to prevent decarboxylation of the intermediate product 2-oxyvalonic acid,and the combination of FDH and LDH was used for regeneration coenzyme NADH.The crude enzymes and whole-cell cascade catalyst prepared by recombinant bacteria can be directly used for the synthesis of chiral pure L-norvaline.Conditions for the transformation of the two systems were optimized,in which the best conversion temperature and pH were 30°C and 7.5,respectively,for crude enzyme system,and for whole-cell system they were 35°C and 8,respectively.Comparing the two transformation systems about the time spent for complete conversion of 40 mM substrate,crude enzyme system was 1 h shorter than that of whole-cell system.The operational stability of formate dehydrogenase was affected by the protein oxidant hydrogen peroxide generated during the conversion process,and the supply of cofactor NADH was insufficient at the later conversion stage,and these reasons resulted in the conversion time of both systems being extended by about 1 h.?2?Through rational design of disulfide bond,free-cysteine residues in formate dehydrogenase were eliminated by disulfide bond pairing to overcome its deactivation by protein oxidant.The mutant enzymes A10C,I239C and A10C/I239C were constructed by site-directed mutation,and the formation of disulfide bonds in these enzymes was verified.The study of the enzymatic properties found that the characteristic of mutant enzymes A10C and A10C/I239C on thermal stability and antioxidant were improved evidently,of which t1/2value was increased 6.7 and 7.8 times at 60°C,respectively,and the tolerance to strong protein oxidant copper ions was improved from 5 mM to 15 mM.In particular,catalytic efficiency and specific activity of mutant enzyme A10C was 1.3 and 1.4 fold higher than that of wild-type,respectively.Therefore,FDH in the multi-enzyme cascade system was replaced by A10C.The results of batch transformation tests verified that the problem of intermediate accumulation caused by the inactivation of FDH due to the oxidation of protein oxidant in the mid to late stage of transformation was effectively solved,and the conversion time by the multi-enzyme cascade system was shortened.?3?In order to further improve the conversion efficiency and shorten the conversion time of multi-enzyme cascade system,a series of modifications were made to the N terminal amino acid sequence of DAAO to improve its soluble expression in E.coli,so as to enhance the enzyme activity of it and shorten the time of complete oxidation of D-norvaline in the substrate.The study of the second amino acid at the N terminal showed that histidine at the N terminal had an important effect on the soluble expression of DAAO,and the addition of glycine could further improve the activity of DAAO,but the biomass of recombinant bacteria was decreased compared with the wild type.Furthermore,the activity of DAAO and the corresponding biomass of recombinant bacteria were adjusted by the number of histidine at N-terminal.It was found that when there were 3 histidine at N-terminal?i.e.,mutant enzyme3-HG?,the final enzyme activity per volume of recombinant bacteria reached the highest,while the cell biomass was not significantly reduced.Furthermore,the recombinant bacteria with the random coil region?sequence MHSQK?in the N-terminal of the wild type DAAO substituted by the optimized sequence MHHHG was obtained,and fermented in 5 L tank.The final enzyme activity per volume of the fermentation was 80.7 U/mL,and the yield per volume reached 4.48 kU·L-1·h-1,which was 3.4 fold higher than that of wild type.The time of complete oxidation of substrate was shortened from 5 h to 2 h when the mutant enzyme was replaced into the multi-enzyme cascade system.However,the activity ratio of FDH and DAAO was not appropriate,so it was necessary to establish a mathematical model of the transformation system to accurately regulate the proportion of enzymes in it.?4?The transformation process of L-norvaline referred to the reaction rate equation and related substrates and products was analyzed to fully understand various factors in the process.Michaelis-Menten equation with multiple substrates used for describing the reaction rate of enzymes in the system was constructed,and mass conservation equation about substances in the process was also established.After the preparation work mentioned above,the mathematical model describing the whole transformation system was obtained.The kinetic parameters of enzymes involved in the system under optimal temperature and pH conditions were determined by initial rate method,then the data were plugged to the model for simulating.The data obtained from simulating were almost consistent with the experiment data of transformation process of L-norvaline under the same conditions,and it proved that the mathematical model can accurately describe the transformation process of L-norvaline.Furthermore,the model was used to predict the conversion efficiency of different proportion of DAAO and FDH as well as LDH and FDH,to achieve a maximum conversion rate of over98%and conversion ratio of over 99.8%with the minimum requirements for enzymatic activity in the systems.?5?The enzyme activity of FDH was improved 4.7 fold after the optimization of the strength of RBS in the Cbfdh A10C gene,while the activity of the other three enzymes remained above 96%.The multi-enzyme cascade catalyst prepared by recombinant bacteria E.coli BL21/L-nor3-r4 can still meet the requirements of the optimal transformation system after being diluted by 2.4 times and used to transform for 24 hours,which greatly improved the utilization rate of the cascade system.Moreover,step-wise feed strategy was adopted to carry out the transformation on 5 L fermentation tanks.The final conversion concentration of L-norvaline by crude enzyme system was 498.2 mM?58.4 g/L?,and that of whole-cell system was 398.6 mM?46.7 g/L?,and the conversion ratio and ee value of both systems were over99%.Considering the cost and production line,the multi-enzyme cascade system was prepared by fermentation in a 5 tons tank,and then the diluted crude enzyme was used in a 20tons fermentation tank for the production of L-norvaline.The final consequences of the amplification test were as follows:The concentration of L-norvaline was 429.6 mM?50.3g/L?,the conversion ratio was over 95%,the chiral purity ee value was over 99%,and the yield was over 90%. |
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