Understanding and Overcoming the Limitations of Bacillus badius and Caldalkalibacillus thermarum Amine Dehydrogenases for Biocatalytic Reductive Amination

Abstract

The direct asymmetric reductive amination of ketones using ammonia as the sole amino donor is a growing field of research in both chemocatalysis and biocatalysis. Recent research has focused on the enzyme engineering of amino acid dehydrogenases (to obtain amine dehydrogenases), and this technology promises to be a potentially exploitable route for chiral amine synthesis. However, the use of these enzymes in industrial biocatalysis has not yet been demonstrated with substrate loadings above 80 mM, because of the enzymes’ generally low turnover numbers (k cat < 0.1 s–1) and variable stability under reaction conditions. In this work, a newly engineered amine dehydrogenase from a phenylalanine dehydrogenase (PheDH) from Caldalkalibacillus thermarum was recruited and compared against an existing amine dehydrogenase (AmDH) from Bacillus badius for both kinetic and thermostability parameters, with the former exhibiting an increased thermostability (melting temperature, T m) of 83.5 °C, compared to 56.5 °C for the latter. The recruited enzyme was further used in the reductive amination of up to 400 mM of phenoxy-2-propanone (c = 96%, ee (R) < 99%) in a biphasic reaction system utilizing a lyophilized whole-cell preparation. Finally, we performed computational docking simulations to rationalize the generally lower turnover numbers of AmDHs, compared to their PheDH counterparts.

The Kinetic Resolution of Racemic Amines Using a Whole-Cell Biocatalyst Co-Expressing Amine Dehydrogenase and NADH Oxidase

“…The results suggest that the activities of AmDH and Nox were affected by the substrate and/or product. Several amino acid dehydrogenases have been observed to suffer from strong product inhibition, often by their native Lamino acid product [20]. In addition, in the asymmetric synthesis of amines from ketones by AmDH, product inhibition has been previously suggested by the use of a biphasic reaction system to ostensibly overcome inhibition in AmDH-catalyzed reactions [27].…”

“…These results clearly showed that the use of whole-cell was much more efficient than the reaction using purified enzymes that gave 26% ee (Figure 6b). The Shell group reported that in the asymmetric synthesis of chiral amine by AmDH, the lyophilized whole-cell gave better conversions both in the absence and presence of co-solvent heptane than lyophilized lysate [20]. The use of whole-cell apparently reduces the substrate/product inhibition, and/or it increases the stability of the enzymes.…”

“…Since then, different AmDHs have been developed using L-amino acid dehydrogenases from Rhodococcus sp. M4, Exiguobacterium sibiricum, and Caldalkalibacillus thermarum [14,19,20]. A systematic investigation of substrate acceptance, optimal reaction conditions, and the chemo-and stereoselectivity of AmDHs has been carried out [13,20].…”

“…M4, Exiguobacterium sibiricum, and Caldalkalibacillus thermarum [14,19,20]. A systematic investigation of substrate acceptance, optimal reaction conditions, and the chemo-and stereoselectivity of AmDHs has been carried out [13,20]. In addition, AmDHs have been used in elegant hydrogen borrowing dual-enzyme cascade reactions for the synthesis of chiral amines from alcohols with "closed-loop" recycling of the cofactor [19,21].…”

Mechanistic Insight into the Catalytic Promiscuity of Amine Dehydrogenases: Asymmetric Synthesis of Secondary and Primary Amines

“…All known AmDHs, to date, were obtained mainly by protein engineering, starting from α‐amino acid dehydrogenases, such as leucine dehydrogenases from Bacillus stereothermophilus , Exiguobacterium sibiricum , Lysinibacillus fusiformis and Bacillus sphaericus , as well as phenylalanine dehydrogenases from Bacillus badius , Rhodococcus sp. M4 and Caldalkalibacillus thermarum . In all of these cases, mutations of the highly conserved lysine and asparagine residues in the active site, which interact with the two oxygen atoms of the carboxylic moiety of the natural substrate, were essential for switching the substrate specificity from α‐keto carboxylic acids to ketones .…”