Stereoselective Biocatalytic Synthesis Of (S)-2-aminobutanamide By D-aminopeptidase

Levetiracetam,（S）-α-ethyl-2-oxo-1-pyrrolidineacetamide,is a next generation anti-epileptic drug developed by Belgian UCB company.Levetiracetam has unique mechanisms of antiepileptic activity and unparalled excellent features such as high therapeutic index,high efficiency,less side effects,good tolerance and pharmacokinetic properties.It was used to prevent epilepsy.（S）-2-aminobutyramide is the key intermediate of Levetiracetam.In recent years,it became a research hotspot to find out new strategy for efficient synthesis of（S）-2-aminobutanamide.In this study,（S）-2-aminobutanamide was stereoselectively synthesized by D-aminopeptidase,which laid the groundwork for the industrial production of chiral intermediates of Levetiracetam.In our study,we cloned the D-aminopeptidase genes from Gluconobacter oxydans and Gluconobacter thailandicus.The target genes were transformed into E.coli BL21（DE3）and expressed successfully.Two recombinants E.coli BL21（DE3）/pET28a-Go-Dap and E.coli BL21（DE3）/pET28a-Gt-Dap were constructed.Another D-aminopeptidase from Brucella sp.F23/97 was screened by homologous sequence alignment.After codon optimization,it was expressed in E.coli BL21 to construct the recombinant E.coli BL21（DE3）/pET28a-Bs-Dap.E.coli BL21（DE3）/pET28a-Bs-Dap was chosen for further study in our research.The nickel column affinity chromatography was used to separate and purify D-aminopeptidase and its catalytic properties was investigated.The results showed that the optimum temperature and pH of D-aminopeptidase was 35?C and 8.0 respectively.EDTA and metal ions would improve little or inhibite the activity of D-aminopeptidase.The K_m and V_maxax of D-aminopeptidase toward 2-aminobutanamide was 15.1 mM and 476.2 U/mg,respectively.Finally,stereoselective synthesis of（S）-2-aminobutanamide was carried out by the whole cells of E.coli BL21（DE3）/pET28a-Bs-Dap.The results indicated that the optimum temperature,pH,substrate concentration and cell concentration were 35?C,pH 7.5（Tris-HCl）,300g/L,and 0.8g DCW/L,respectively.Under above optimized reaction condition,48.2%yield and 99.9%ee of（S）-2-aminobutanamide were achieved after2 h.The study indicated that the recombinant E.coli BL21（DE3）/pET28a-Bs-Dap could stereoselectively catalyze 2-aminobutanamide under high substrate concentration.It demonstrated the potential of BL21（DE3）/pET28a-Bs-Dap for the large scale production of（S）-2-aminobutanamide.