New Enzymatic Method of Chiral Amino Acid Synthesis by Dynamic Kinetic Resolution of Amino Acid Amides: Use of Stereoselective Amino Acid Amidases in the Presence of α-Amino-ε-Caprolactam Racemase

Abstract

D-and L-amino acids were produced from L-and D-amino acid amides by D-aminopeptidase from Ochrobactrum anthropi C1-38 and L-amino acid amidase from Pseudomonas azotoformans IAM 1603, respectively, in the presence of ␣-amino--caprolactam racemase from Achromobacter obae as the catalyst by dynamic kinetic resolution of amino acid amides.Several dynamic kinetic enzymatic resolutions of synthetic substrates have been developed for the industrial production of chiral amino acids. The use of L-␣-amino-ε-caprolactam (ACL)-hydrolase in the presence of ACL racemase is a typical example of L-lysine production (1, 2, 12). L-cysteine is produced by hydrolases acting on amino-⌬ 2 -thiazoline-4-carboxylic acid in the presence of a racemase (23). Used in combination with D-specific hydantoinase (22,26), either with spontaneous racemization of hydantoin or with hydantoin racemase (21,24), N-carbamoyl-D-amino acid amidohydrolase catalyzes the hydrolysis of N-carbamoyl-D-amino acids to form D-amino acids (3,(16)(17)(18)(19)(20). N-acyl-D-amino acid amidohydrolase (27) and N-acyl amino acid racemase (25) are utilized together to form D-amino acids. Fig. 1 compares several dynamic kinetic enzymatic resolutions of synthetic substrates. The optically active amino acids can be synthesized in three steps with amidases from aldehydes via aminonitrile and amino acid amide, while the D-amino acylase route requires four steps via aminonitrile, amino acid, and N-acyl amino acid.We isolated and characterized several D-stereospecific amino acid amidases, such as D-aminopeptidase (DAP), Damino acid amidase, alkaline D-peptidase, and R-amidase, and utilized them in the kinetic resolution of amino acid amides (4-9, 14). L-Amino acid amide hydrolases such as LaaA and LaaA Bd have been characterized previously (15).In this report, the effect of a high concentration of the substrate on the dynamic kinetic resolution of amino acid amide, together with the optimum pH, was investigated using DAP or LaaA in the presence of ACL racemase (10, 11).ACL racemase was purified from Escherichia coli JM109/ pACL60, and its activity was assayed as described previously (10). The expression level of ACL racemase was about 300 U/liter culture (5% of the total soluble protein). The amount of inclusion body was not measured. DAP expressed in E. coli JM109 was purified by a similar procedure, and its activity was assayed as described previously (8). About 8,000 U/liter of DAP could be produced maximally in the cell extract (about 6% of the total soluble protein). LaaA activity toward L-alanine amide was determined by measuring the amount of Lalanine formed. The reaction mixture (1.0 ml) contained 0.1 M potassium phosphate buffer (KPB; pH 7.0), 0.1 M L-alanine amide, and an appropriate amount of LaaA. The reaction was performed at 30°C for 10 min and stopped by adding 200 l 2 N HClO 4 . The amount of L-alanine formed was determined by the method used for D-ACL, and the enzyme activity was measured by the same method as described previously (10). Recombinant E. coli JM1...

Amidases: versatile enzymes in nature

“…It helps the enzymatic digestion of proteins. Yamaguchi et al (2007) used different enzymes in combinations like D-aminopeptidase from Ochrobactrum anthropi C1-38 and L-amino acid amidase from Pseudomonas azotoformans IAM 1603 as the catalyst for D-and Lamino acids synthesis from respective amides, in the presence of a-amino-e-caprolactam racemase from Achromobacter obae. Asano and coworkers isolated the D-aminopeptidases (type of amidase that hydrolysed peptides) and used in the synthesis of D-amino acid containing peptides.…”

Hydrolysis of Amides

“…More recently, Asano and Yamaguchi showed that this ACL racemase, in contrast to earlier reports [125], is also able to racemize linear a-H-a-amino acid amides [176], although with at least 35-fold lower specific activity than for ACL [177]. The A. obae ACL racemase has been combined with the D-aminopeptidase from Ochrobactrum anthropi C1-38 (Section 15.4.1.3) for the synthesis of D-alanine from Land DL-alanine amide in near stoichiometric amounts [177,178]. Similarly, complete conversion into L-alanine was obtained when the ACL racemase was combined with the L-amino acid amidase from Pseudomonas azotoformans IAM 1603 (Section 15.4.1.1) [178].…”

“…The A. obae ACL racemase has been combined with the D-aminopeptidase from Ochrobactrum anthropi C1-38 (Section 15.4.1.3) for the synthesis of D-alanine from Land DL-alanine amide in near stoichiometric amounts [177,178]. Similarly, complete conversion into L-alanine was obtained when the ACL racemase was combined with the L-amino acid amidase from Pseudomonas azotoformans IAM 1603 (Section 15.4.1.1) [178].…”

Dynamic Kinetic Resolution of α‐Aminonitriles to Form Chiral α‐Amino Acids

“…The optical purity of the isolated (R)-a-aminobutyric acid was more than 99% ee with an isolated yield of 68% (0.28 g, 2.72 mmol); Optical rotations were measured on a SEPA-300 (Horiba, Ltd., Kyoto, Japan) instrument: [ 13 C NMR, MS and IR spectra of the isolated (R)-a-aminobutyric acid are described in the Supporting Information. 10 12 > 99 97.5…”

“…We previously reported that a-amino-e-caprolactam (ACL) racemase catalyzes the racemization of aamino acid amides as new substrates and is effective for the DKR of a-amino acid amides, when used with R-or S-stereoselective amino acid amide hydrolase. [8][9][10] Our purpose in this study is to obtain optically pure a-amino acids in theoretical yield with high enantiomeric excess by the DKR of racemic a-aminonitriles. This DKR process consists of three steps: (i) hydrolysis of both enantiomer of racemic a-aminonitrile by non-stereoselective NHase, (EC 4.2.1.84), (ii) stereoselective hydrolysis of the resulting racemic aamino acid amide by stereoselective amino acid amide hydrolase, shifting the equilibrium toward the product, and at the same time, (iii) racemization of the remaining a-amino acid amide by ACL racemase (Scheme 1).…”