Stereoselective Synthesis Of (S)-2-aminobutanoic Acid By Nitrilase

Levetiracetam is the second generation of acetylcholine agonists,developed by UCB company of Belgium,mainly for the treatment of circumscribed and generalized epilepsy.Approved by the US FDA and then listed in the United States on April 2000,it is allowed for sale and use in the Chinese market in 2006 for the first time.reaching the medicine amount of the annual increase of 60%-70%.Levetiracetam is the only anti-epilepsy drug which can prevent epileptic seizure acording to the recent reports.Because of its special effects and low medicinal properties of side effects its clinical application is increasingly wide in the global scope.Sales analysis data of the hospital market in 14 varieties of antiepileptic drugs showed levetiracetam ranked No.5 on 2009,It is predicted that by 2016,the US anti-epileptic drug market will continue to maintain sales at around 30 billion dollars.Levetiracetam has its good pharmacology,pharmacokinetics active and will be one of the strong competition in the field of medicine.Nitrilase(EC 3.5.5.1)is an important nitrile converting enzymes,directly hydrolyzing a nitrile compound to the corresponding carboxylic acid and ammonia by one reaction step.Enzymatic hydrolysis of nitrile compounds shows high efficiency and high selectivity with mild reaction conditions,light pollution,therefore conforms to the atomic economy and the developing direction of green chemistry.The regio-and stereo-specific synthesis of(S)-2-aminobutyric acid from 2-aminobutyronitrile by nitrilase and then prepare levetiracetam through hydrogenation,which exhibits the highest atom economy than other chemistry-enzymatic processes,was developed.And the difficulty is to obtain a nitrilase with high activity and selectivity towards 2-aminobutyronitrile.In this study,through the screening of nitrilase engineered bacteria from our lab,one recombinant strain E.coli BL21(DE3)/Nitbll6402/pET-28 b which had the ability of stereoselective production of(S)-2-amino-butyric acid from 2-aminobutyronitrile with nitrilase was obtained.Under the condition of 35 ℃and pH 7.2,after 12 h react with 100 mM 2-aminobutyronitrile,the e.e.value and the conversion ratio reached 100% and 11.3%,respectively.Based on Phyre server to predict three-dimensional structure of the nitrilase,then submitted to the HOTSPOT server to predict "hot spots" amino acid sites of nitrilase Nitbll6402,12 residues,including 198 Y,239P,272 P,252P,102 F,194R,196 K,195D,251 K,240E,192 L and 234 M,nearby the active site were selected as target mutation sites.The recombinant plasmid Nitbll6402 was used as the template for saturation mutagenesis using polymerase chain reaction.From the constructed mutaion clone libraries,four mutants 192L-62,195D-72,240E-91,and 251K-85 which possessed significantly higher activity towards 2-aminobutyronitrile were obtained using thin layer chromatography and High Performance Liquid Chromatography analysis.whereby the specific activity of mutant 195D-72 was 3.12-fold relative to the parental enzyme.Under 100 mM substrate,the e.e.value and the conversion reached 100% and 35.3%,respectively.The reaction conditions including temperature,pH,substrate concentrations and cell concentrations were optimized by single factor method for an improved synthesis.The optimum temperature,pH,substrate concentration and cell concentrations were 35℃,8.0,250 mM and 0.2 g/L,respectively.Under above conditions,a yield of 87.2% and an e.e.of 100% were achieved.