Study On The Biosynthesis Of L-DOPA Catalyzed By Recombinant Tyrosine Phenol-Lyase

Parkinson's disease is a common degenerative disease of the nervous system.As the nigral-striatum nerve cells in the brain of the patient are damaged,the production of tyrosine hydroxylase is significantly reduced,resulting in a serious deficiency in the production of dopamine.Levodopa is currently the most effective drug for the treatment of Parkinson's disease clinically.With the development of an aging society,people will pay more and more attention to the enzymatic synthesis of levodopa.This method is very efficient and has important production value.Enzymatic synthesis is very efficient and has important production value.This thesis focuses on the synthesis of levodopa by recombinant tyrosine phenololytic enzyme enzymatic method.Firstly,the process of producing tyrosine phenololytic enzyme from recombinant Escherichia coli was optimized,and the optimal fermentation medium was determined as:Na2HPO4 6 g/L,KH2PO4 3 g/L,NH4Cl 1 g/L,NaCl 0.5 g/L,1 mM MgSO4 solution,1 mM CaCl2 solution,50 mM MOPS solution(pH 7.4),10 g/L glycerol,12 g/L tryptone,24 g/L yeast extract.Subsequently,the enzyme production conditions were optimized.The optimal IPTG concentration was determined to be 0.1 mM,the induction temperature was 30 0 C,and the induction time was 14 h.Finally,the recombinant tyrosine phenololytic enzyme was produced by fed-batch on a 5 L fermenter with a cell concentration of 130 g/L and a recombinant tyrosine phenololytic enzyme activity of 31 U/mLThen,the enzyme catalytic process was studied.The tyrosine phenololytic enzyme was catalyzed at 30? C,the initial pH was 7.0,the substrate catechol concentration was 1.2%,the pyruvate concentration was 1%,the enzyme amount was 2.5%,and the coenzyme PLP was 0.01%.Under the above conditions,the tyrosine phenololytic enzyme has the highest enzyme activity and has a yield of 87.8±1.1 g/L of levodopa.Finally,the post-treatment process of the enzyme catalytic product was studied.When the concentration of levodopa was 80 g/L,60? and the holding time was 1 h,the product had the best decolorization effect and the best product quality.After examination of the crystallization temperature,it was found that the crystallization was best at 15?,and the product was a regular crystal with a pure color.In the amplification study of the catalytic production of levodopa by the enzymes of 650 L and 6500 L,the production yields were all above 85%,the accelerated stability results were good and the product quality met the standard.