Isolation And Identification Of A New Brevibacterium Epidermidis For Asymmetric Amination Of Ketones To (S)-chiral Amines

Enantiomerically pure chiral amines are highly valuable functionalized molecules with a wide range of applications in the pharmaceutical,agrochemical and fine chemical industry.They are often used as chiral auxiliaries and building blocks of various active pharmaceutical ingredients.Compared with classical chemical methods,biocatalysis is becoming attractive method for the preparation of chiral molecules.Because it possesses the advantages of high atomic utilization rate,high selectivity,less by-products,mild reaction conditions and environmental friendliness.Recently,the amine dehydrogenase that catalyzes the asymmetric reductive amination of ketones aroused widespread interest of researchers in industrial and academic fields.Because the reaction uses unexpensive free ammonium as amine donor and generates only water as by-product,and representing a green chiral amine preparation process.Using the natural L-amino acid dehydrogenase as starting scaffold,the amine dehydrogenase has been created by active-site-targeted protein engineering for the synthesis of chiral amine from prochiral ketones and ammonia.However,these amine dehydrogenases developed are(R)-selective enzyme and only used to synthesize(R)-chiral amines.To date,no(S)-selective amine dehydrogenase has been reported.Therefore,we sought to isolate some novel strains from the soil for synthesis of the(S)-chiral amines from prochiral ketones and ammonia,and then identify natural(S)-amine dehydrogenases from these microorganisms.In this paper,two strains with asymmetric reductive amination activity were obtained by screening from the soil using(S)-?-phenylethylamine as the sole nitrogen source.Subsequently,these two strains were further examinated using(4-fluorophenyl)acetone as the substrate.Finally,the strain ES11 could converted efficiently(4-fluorophenyl)acetone into(S)-4-fluoro-?-methylphenethylamine with>99% ee.and was therefore chosen for the further study.Phylogenetic analysis based on 16S rDNA gene sequence demonstrated that ES11 belongs to the genus Brevibacterium.and was named as Brevibacterium epidermidis ECU 1015.Next,the catalytic performance of B.epidermidis ECU1015 was studied.For the asymmetric reductive amination.the optimal conditions are 1.25 M NH4Cl as amino donor.KPB(pH 7.5.0.2 M) as reaction system and 30? as reaction temperature.While for the deamination process,the reaction is carried out in Glycine/NaOH buffer solution(pH 10.5,0.2 M)and 30?.In addition,the high loading of substrate could reduce catalytic efficiency of B.epidermidis ECU 1015 in the reductive amination reaction and deamination reaction.Finally,under the optimum reaction conditions,the scaled-up kinetic resolution of the 4-fluoro-a-methylphenethylamine and asymmetric reductive amination of the(4-fluorophenyl)acetone were conducted to generate the correspending(R)-4-fluoro-a-methylphenethylamine and(S)-4-fluoro-a-methylphenethylamine with>99% ee,respectively.These results demonstrated that B.epidermidis could catalyze reductive amination of ketones with free ammonium to synthesize the(S)-chiral amines.