Developing Amine Dehydrogenases By Engineering Phenylalanine Dehydrogenases For Chiral Amine Synthesis

Chiral amines are key intermediates for the production of a plethora of chemical compounds that have applications in the pharmaceutical,agrochemical and bulk chemical industries.Nowadays,?-chiral amines are mainly obtained in industry via the asymmetric hydrogenation of activated intermediates such as enamides,enamines or pre-formed N?substituted imines.These strategies consume a considerable amount of energy and require multiple chemical steps to afford chiral amines.Amine dehydrogenases(AmDH)could catalyze the asymmetric reductive amination of ketones in the presence of ammonia,which is of high atom efficiency and is friendly-environment.While the reported amine dehydrogenases are limited and their narrow substrate scope limits its utilization in chiral amine synthesis.At the same time,the thermostability of the biocatalysts is one of the most important parameters that affect their practical application in industry.Thus,we attempted to recruit more thermostable amine dehydrogenases by data-mining to improve the catalytic efficiency and expand the substrate spectrum via protein engineering.Firstly,the putative phenylalanine dehydrogenases were acquired by blasting in the Uniprot database with BbAmDH and RsAmDH sequences as the templates,respectively.Consequently,four phenylalanine dehydrogenases(PheDH)were cloned from strains conserved in our lab.Taking the soluble expression,activity and thermostability into consideration,the GkPheDH from the Geobacillus kaustophillus was used for further investigation.Secondly,the two residues Lys77 and Asn276 that interact with the carboxyl group of the phenylalanine were mutated simultaneously and the double mutant K77S/N276L(GkAmDH)was obtained.Then the GkAmDH was purified and characterized:The optimal pH and temperature were 9.0 and 55?,respectively;it could retain good activity in NH3·H2O/NH4COOH buffer and could efficiently catalyze the asymmetric reductive amination of ketones for chiral amine synthesis.Furthermore,it was found that GAkAmDH could afford chiral secondary amines with organic primary amino donors.At last,we attempted to improve the catalytic efficiency of GkAmDH by protein engineering.The double mutant T129L/K282N obtained via several rounds of semi-rational design and random mutagenesis displayed a 2.3-fold improvement in kcat/KM compared to the GkAmDH.To further expand the substrate spectrum,site-directed saturation mutagenesis was performed at Lys77 and Asn276,respecitively.Then the positive mutants were combined and an amine dehydrogenase tool box was developed for the chiral amino alcohol synthesis.