Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution of Corresponding Amide and Nitrile Substrates with a Multi‐Enzyme System

Abstract

Mutant α‐amino‐ε‐caprolactam (ACL) racemase (L19V/L78T) from Achromobacter obae with improved substrate specificity toward phenylalaninamide was obtained by directed evolution. The mutant ACL racemase and thermostable mutant D‐amino acid amidase (DaaA) from Ochrobactrum anthropi SV3 co‐expressed in Escherichia coli (pACLmut/pDBFB40) were utilized for synthesis of (R)‐phenylalanine and non‐natural (R)‐phenylalanine derivatives (4‐OH, 4‐F, 3‐F, and 2‐F‐Phe) by dynamic kinetic resolution (DKR). Recombinant E. coli with DaaA and mutant ACL racemase genes catalyzed the synthesis of (R)‐phenylalanine with 84% yield and 99% ee from (RS)‐phenylalaninamide (400 mM) in 22 h. (R)‐Tyrosine and 4‐fluoro‐(R)‐phenylalanine were also efficiently synthesized from the corresponding amide compounds. We also co‐expresed two genes encoding mutant ACL racemase and L‐amino acid amidase from Brevundimonas diminuta in E. coli and performed the efficient production of various (S)‐phenylalanine derivatives. Moreover, 2‐aminophenylpropionitrile was converted to (R)‐phenylalanine by DKR using a combination of the non‐stereoselective nitrile hydratase from recombinamt E. coli and mutant ACL racemase and DaaA from E. coli encoding mutant ACL racemase and DaaA genes.

Soluble and functional expression of a recombinant enantioselective amidase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization

“…Some amidases have very strict chemo-, regio-, and enantioselectivities, allowing them to be used to produce optically pure amides, carboxylic acids, and related derivatives. They have been used to produce (S)-2,2-dimethylcyclopropanecarboxamide [2,[10][11][12], piperazine-2-carboxylic acid [13], phenylalanine [14], S-naproxen [15], and (S)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide [16]. These chemicals and their derivatives have important applications in the fine chemical and pharmaceutical industries.…”

Lipases: Valuable catalysts for dynamic kinetic resolutions

“…Wang et al, 2013;Z.-Y. Wang et al, 2014;Yasukawa and Asano, 2012). Nevertheless, only few DKR reactions co-catalyzed by racemases and lipases have been reported so far, since the former usually present low stability in non-aqueous solvents Larissegger-Schnell et al, 2005;Strauss and Faber, 1999).…”

Why Leave a Job Half Done? Recent Progress in Enzymatic Deracemizations

“…Yasukawa and Asano have showed the use of these enzymes coupled to racemases in order to synthesize several phenylalanine derivatives from the corresponding racemic phenylalanine amides. [79] Hence, when a mutant from α-amino-ε-caprolactam (ACL) racemase was co-expressed with D-amino acid amidase from Ochrobactrum anthropi SV3 in E.…”