Biosynthesis Of α-Keto Acids And Their Derivatives Via Whole Cell Biocatalyst Expressing Membrane Bound L-amino Acid Deaminase

As a kind of compounds containing double carbonyl,a-keto acids always act as intermediates for many physiological substances,hence having a wide range of application in food,paramarceutical and chemical industry.Preparing a-keto acids from amino acids via enzyme catalysis is high-efficiency,low-cost and eco-friendly.Among the enzymes that able to convert amino acids to α-keto acids,membrane bound L-amino acid deaminase(mL-AAD)seems to be ideal since there is no hydrogen peroxide(which can oxygenize the a-keto acids)produced and no cofactors needed during the reaction.Based on mL-AAD from Proteus vulgaris,secretory express strategies and express conditions were optimized in this study.The determined mL-AAD recombinant was used to prepare phenylpyruvic acid(PPA),3,4-dihydroxyphenylpyruvic acid(DHPPA)and their corresponding derivative 3-phenyllactic(PLA),salvianic acid(SAA).Major results are listed below.1.Influence of different mL-AAD secretory paths on catalytic activity were investigated in pET-28a and pET,20b,where a wild-type mL-AAD secreted through its own twin-arginine signal peptide(twin-arginine translocation pathway,Tat)was expressed by pET-28a,and a fusion mL-AAD secreted by integrated pelB(the general secretory pathway,Sec)-tat signal peptide was expressed by pET-20b.It was showed that mL-AAD secreted through Tat path(corresponding recombinant was BL21(DE3)-pET-28a-mlaad)has higher biomass and biocatalytic activity.2.L-phenylalanine(L-Phe)was converted to PPA via BL21(DE3)-pET-28a-mlaad.Under the optimized conditions(pH = 8.0,temperature 37℃,cell concentration:0.39 g/L),150 mmol/L L-Phe was converted to 141 mmol/L PPA in 42 h.Based on this deamination reaction,recombinant coexpressing D-2-hydroxyisocaproate dehydrogenase(come from Lactobacillus delbrueckii subsp.Bulgaricus)and formate dehydrogenase(come from Mycobacterium vaccae N10)was constructed to convert PPA to PLA.Under the optimized conditions,121 mmol/L PLA was obtained,with a two-step yield of 80.7%.4.L-Dopa was converted to DHPPA via BL21(DE3)-pET-28a-mlaad.Under the optimized conditions(pH = 8.5,temperature 37 ℃,cell concentration 0.37 g/L),100 mmol/L L-Dopa was converted to 28.5 mmol/L DHPPA in 60 min.Based on this deamination reaction,recombinant coexpressing D-lactate dehydrogenase(come from Pediococcus acidilactici DSM 20284)and formate dehydrogenase(come from M.vaccae N10)was constructed to convert DHPPA to SAA.Under the optimized conditions,11.66 mmol/L SAA was obtained.