Gene Mining,Directed Engineering Of Amino Acid Dehydrogenases And Its Application In Synthesis Of Chiral Amino Acid

Optical pure chiral amines,including amino acids,amino alcohols and amino nitrile,are the key intermediates for the synthesis of various compounds,such as active pharmaceutical ingredients,fine chemicals,pesticides and dyestuffs etc.Chiral amines could be synthesized through chemical or enzymatic enantioselective acylation,oxiditive resolution and asymmetric hydrogenation.Among them,biocatalytic preparation of chiral amines through amino acid dehydrogenase(AADH)is one of the most promising approaches,due to its high enantioselectivity and efficiency,mild reaction and environmental friendliness.In this study,six AADHs were identified through gene mining.Among them,BcAADH from Bacilllus clausii and BaAADH from Bacilllus amyloliquefaciens were much higher activing in the asymmetric reductive amination of aromatic ketoacid.(1)Enzymatic properties of BcAADH and BaAADH were characterized.The optimal pHs for reduction and oxidation activity of BcAADH were 9.5 and 10.5 respectively.BcAADH displayed the highest activity at 30°C.Half-lives at 30,40 and 50°C were 315,86.6 and 4.49 h,indicating BcAADH is stable.Furthermore,BcAADH exhibited high activity toward aromatic ketoacids.The specific activity and kcat/Km in the reduction of benzoylformic acid were 17.7 U·mg–1 and 5.05 s–1·mmol·L–1,respectively.This BcAADH was coexpressed with glucose dehydrogenase for cofactor regeneration.The reaction condition for the asymmetric preparation of chiral L-phenylglycine using this coexpressed E.coli was optimized.Under the optimized conditons,as much as 400 mmol·L–1 benzoylformic acid could be fully reduced into L-phenylglycine within 6 h at >99.9% ee and 91% isolation yield with merely 0.5 g DCW·L?1.In this reaction,the substrate to biocatalyst ratio of 60 g·g?1 was achieved.Enzyme consumption number(e.c.n.)was calculated to be 0.018 and environmental factor was 4.7.(2)Recombinant BaAADH was also characterized.BaAADH exhibited the highest reduction and oxidation activity at pH 8.5 and 10.5.The optimum temperation was at 45°C.This Ba AADH shows higher thermos-stability than BcAADH,with half-lives of 462,117.6 and 6.17 h at 30,40 and 50°C repectively.The specific activity and kcat/Km of BaAADH toward benzoylformic acid were 44.5 U·mg–1 and 9.8 s–1·mmol·L–1.To expending the substrate spectrum and conferring the asymmetric reductive amination activity towards prochiral ketones,double mutation of K68 S and N261 L on all the five AADHs mined in this study were performed,and only BaAADHK68S/N261 L exhibited activity toward aliphatic ketones.Futhermore,homology modeling,molecular docking and directed evolution of this BaAADHK68S/N261 L was carried out in order to achieve the reductive amination of aromatic ketones.Saturation mutagenesis libraries on the substrate binding pocket of BaAADHK68S/N261 L were constructed,and high-throughput screening was performed using DNPH-based methods.Two variants,Ba AADHK68S/N261L/L74 N and BaAADHK68S/N261L/L76 N,were obtained with detectable reductive activity towards acetophenone.The specivity activity were 75.6 and 46.8 mU·mg–1,respectively.In summary,chiral non-natural amino acids could be synthesized employing this newly identified BcAADH.And two residues,L74 and L76,were identified with vital role reductive amination activity of AADHs.This study provides an efficient biocatalyst for the synthesis of chiral amino acids,and hot-spots for further engineering and application of AADH for the synthesis of chiral amines.