Biocatalytic Production Of Phenyllactic Acid Based On Improving The Cell Viability Of Escherichia Coli

Phenyllactic acid?PLA?,a natural organic acid widely found in the fermentation products of honey and lactic acid bacteria.The second carbon atom in the phenyl lactic acid structure is a chiral carbon atom,so it has D-PLA and L-PLA Enantiomers.Phenyllactic acid has a broad spectrum of antibacterial properties,PLA has good inhibitory effect on a series of food-borne bacteria,yeast and mildew.And the structure of phenyllactic acid is relatively stable,with good acid and alkali resistance,high temperature and other characteristics,it has non-toxic to humans and cells.In addition,phenyllactic acid has the extensive application by the researchers attention in the pharmaceutical,chemical,feed,agriculture and other aspects.In recent years,the synthesis methods of phenyllactic acid are mainly chemical synthesis,microbial fermentation,whole cell transformation et,al.The opration of chemical synthesis is complicated,and it will cause serious environmental pollution.The fermentation process has long production cycle,low substrate utilization ratio and complex product separation.The whole cell transformation method has the advantages of short synthesis time,simple operation and high optical activity.However,product inhibition effect has a significant effect on the cell viability of E.coli,resulting in a lower production of phenyllactic acid.In this study,we constructed the recombinant strain by mutant strain with the tolerance on PLA and the water/organic solvent biphasic system for the synthesis of D-and L-phenyllactic acid.Improve the cell viability of Escherichia coli by the above manner.The main research results are as follows:?1?In this paper,we investigated the inhibitory effect of D-phenyllactic acid on Escherichia coli,low concentration of phenyllactic acid inhibited the growth of Escherichia coli.The survival ratio of Escherichia coli was 2.56%at 1.0 g/L D-PLA stress.A mutant strain of E.coli BL21?DE3?by UV mutagenesis was capable of tolerating 1.5 g/L D-PLA,named E.coli Z2016,and it was stored in the Chinese Culture Collection,No.M2016332.The mutant strain grew well and was stably inherited when 1.5 g/L PLA was added in media.The original strain was completely unable to grow.In the high concentration of phenyllactate stress,the viability of resting mutant cells decreased slowly,the mutant strain could reduce the self-aggregation of the cells,reduce the hydrophobicity of the cell surface,and the mutant strain also had the ability to maintain the intracellular pH stability.These results have shown that the selected mutant strains are resistant to higher concentrations of D-PLA than the original strain.?2?The recombinant strains E.coli Z2016 pET28a-LDH-FDH and E.coli Z2016pETduet-Lp LDH were constructed by the mutant E.coli Z2016 as competent cells.After the verification of the recombinant mutant strains,these were used to synthesis of D-PLA and L-PLA.The inhibitory effect of recombinant mutant bacteria on the synthesis of phenyllactic acid was decreased and the viability of Escherichia coli was improved obviously.The yield of D-PLA was 31.43 g/L after 6 h,which was higher by19.41%than that of the original recombinant strain,L-PLA production was 32.87 g/L,increasing by 13.82%.The optimal transformation conditions were as follows:the conversion system pH 7.0,the conversion temperature was 37?,and the substrate concentration was 12 g/L.?3?The biphasic system,which included phosphate buffer and different polar organic solvents,was selected to increase PLA production.It was found that the yield of D-phenyllactic acid was increased in the water/n-octane biphasic system by whole cell transformation.In the biphasic system,the process of whole cell transformation and synthesis of D-phenyllactic acid by E.coli pET28a-LDH^Y52V was optimized and orthogonal analysis was carried out.The optimal conversion conditions were as follows:the conversion temperature 37?,the volume fraction of n-octane 40%,cell concentration 30 g/L,substrate concentration 12.5 g/L.Under the optimum conditions,the concentration of D-PLA was 20.98 g/L,the yield of D-PLA was 8.392 g/L/h,and the final conversion ratio was 85.13%.The yield of D-PLA was 32.31 g/L,and the yield was increased to 12.92 g/L/h.The results were higher than the production of D-PLA synthesized by pure water phase system.