Development And Optimization Of L-phenylglycine Biosynthesis Process

L-Phenylglycine is an important non-naturalα-amino acid,which is widely used in the field of medicine as an important pharmaceutical intermediate,and it is of great significance to explore its green synthesis process.This research focuses on exploring the green synthesis process of L-phenylglycine,and adopts two strategies to biosynthesize L-phenylglycine.Firstly,the free enzyme is used to catalyze D-mandelate under the condition of adding coenzyme to realize the biosynthesis of L-phenylglycine.Secondly,by constructing recombinant E.coli,microbial cells are used as a catalyst,and use the coenzyme circulation system in the cells to realize the biosynthesis of D,L-mandelate to L-phenylglycine without the addition of coenzymes.Finally,the optimal process conditions for whole-cell catalyzed production of L-phenylglycine were optimized by response surface methodology,to realize the economical and efficient synthesis of L-phenylglycine.The main contents and results of this research are as follows:1.“One-pot”method to achieve high-efficiency biosynthesis of L-phenylglycine from D-mandelate.Coupling a new of highly active D-mandelate dehydrogenase（Lh DMDH）and L-leucine dehydrogenase（Es Leu DH）,using D-mandelate as a substrate,adding a lower concentration of Nicotinamide adenine dinucleotide（NAD⁺）realizes biocatalytic synthesis of L-phenylglycine from D-mandelate.After optimizing the catalytic conditions such as the amount of enzymes added to Lh DMDH and Es Leu DH,the amount of coenzymes,the concentration of NH₄⁺,and the concentration of substrates,the optimal reaction conditions for the biosynthesis of L-phenylglycine were obtained in this method:under the conditions of 200 m M D-mandelate,6.5 k U/L enzyme addition,0.1 m M NAD⁺,500 m M NH₄⁺,30℃for 12 h,the yield of the product and the enantiomeric excess value（e.e.）,it can reach 98%and 99%respectively.2.Recombinant E.coli whole cells catalyze the enantioselective production of L-phenylglycine from D,L-mandelate.With the aid of the p ACYCDuet-1 and p ET28a two-plasmid co-expression system,a recombination carrying the encoding genes for mandelate racemase（Ar MR）,D-mandelate dehydrogenase（Lh DMDH）and L-leucine dehydrogenase（Es Leu DH）was constructed recombinant E.coli,named E.coli BL21（DE3）/p ACYCDuet-1-Es Leu DH-Lh DMDH:p ET28a-Ar MR.Under the induction of low temperature and low concentration of inducer,the recombinant E.coli successfully expressed three recombinant enzymes with their respective catalytic activities.The fermentation broth contains D-mandelate dehydrogenase,L-leucine dehydrogenase and mandelate racemase.The activities were 195.8,56.2 and 174.5 U/m L,respectively.With the induced whole cells as the catalyst and D,L-mandelate as the substrate,the initial concentration of D,L-mandelate is 50 m M,p H 9.5,and 500 m M NH₄C1-NH₃·H₂O buffer Under the system,after reacting at 180 rpm and 30℃for 48 h,the yield of L-phenylglycine can reach 77.48%,and the e.e.value is greater than 99%.3.Response surface methodology to optimize the process conditions of whole-cell catalytic production of L-phenylglycine.By response surface methodology,after optimization of the conditions for the catalytic synthesis of L-phenylglycine by recombinant E.coli,under the condition of the initial concentration of D,L-mandelate was 300 m M,p H10.5,reaction temperature 25℃and 500 m M NH₄C1-NH₃·H₂O buffer for 12 h,the yield and space-time yield of L-phenylglycine were significantly increased to 87.89%and 79.70 g·L^-1·d^-1,respectively.The results obtained in this research have great potential for industrialization,laying a solid foundation for the large-scale biosynthesis of L-phenylglycine.