Min-Ho Cha

Cloning and characterization of a novel beta-transaminase from Mesorhizobium sp. strain LUK: a new biocatalyst for the synthesis of enantiomerically pure beta-amino acids.

ABSTRACT A novel β-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the β-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The β-transaminase showed higher activities toward d-β-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The β-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and α-ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of β-aminocarboxylic acids in the active site is reversed relative to that of α-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the β-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic α-keto acids such as α-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the α-carboxylate group of the α-amino acids and α-keto acids. The β-transaminase was used for the asymmetric synthesis of enantiomerically pure β-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.

Simultaneous synthesis of 2‐phenylethanol and L‐homophenylalanine using aromatic transaminase with yeast Ehrlich pathway

2‐Phenylethanol is a widely used aroma compound with rose‐like fragrance and L‐homophenylalanine is a building block of angiotensin‐converting enzyme (ACE) inhibitor. 2‐phenylethanol and L‐homophenylalanine were synthesized simultaneously with high yield from 2‐oxo‐4‐phenylbutyric acid and L‐phenylalanine, respectively. A recombinant Escherichia coli harboring a coupled reaction pathway comprising of aromatic transaminase, phenylpyruvate decarboxylase, carbonyl reductase, and glucose dehydrogenase (GDH) was constructed. In the coupled reaction pathway, the transaminase reaction was coupled with the Ehrlich pathway of yeast; (1) a phenylpyruvate decarboxylase (YDR380W) as the enzyme to generate the substrate for the carbonyl reductase from phenylpyruvate (i.e., byproduct of the transaminase reaction) and to shift the reaction equilibrium of the transaminase reaction, and (2) a carbonyl reductase (YGL157W) to produce the 2‐phenylethanol. Selecting the right carbonyl reductase showing the highest activity on phenylacetaldehyde with narrow substrate specificity was the key to success of the constructing the coupling reaction. In addition, NADPH regeneration was achieved by incorporating the GDH from Bacillus subtilis in the coupled reaction pathway. Based on 40 mM of L‐phenylalanine used, about 96% final product conversion yield of 2‐phenylethanol was achieved using the recombinant E. coli. Biotechnol. Bioeng. 2009;102: 1323–1329.

Synthesis of enantiopure (S)-2-hydroxyphenylbutanoic acid using novel hydroxy acid dehydrogenase from Enterobacter sp. BK2K.

Enterobactersp. BK2K, screened from soil samples, can enantioselectively reduce 2‐oxo‐4‐phenylbutanoic acid into (S)‐2‐hydroxy‐4‐phenylbutanoic acid. α‐Hydroxy acid dehydrogenase (HADH) (specific activity 62.6 U/mg) was purified from the crude extract of Enterobactersp. BK2K, and its gene was cloned and functionally expressed in E. coli BL21. The optimal pH and temperature for the HADH activity were 6.5 and 30 °C, respectively. The purified enzyme catalyzes the reduction of various aromatic and aliphatic 2‐oxo carboxylic acids to the corresponding (S)‐2‐hydoxy carboxylic acids using NADH as cofactor. For example, the Km and kcat/Km for 2‐oxo‐4‐phenylbutaonoic acid in the presence of 2 mM NADH were 6.8 mM and 350 M−1min−1, respectively. For practical applications, a NADH recycle system employing the recombinant formate dehydrogenase from E. coli K12 was coupled with HADH in E. coli BL21. Using the recombinant HADH (110 U of 11 U/mg crude cell extract) and formate dehydrogenase (670 U of 67 U/mg crude cell extract) in 10 mL of 500 mM phosphate buffer (pH 6.5), 96 mM of (S)‐phenyllactic acid (>94% ee) and 95 mM of (S)‐2‐hydroxy‐4‐phenylbutanoic acid (>94% ee) were produced in quantitative yields from 100 mM of phenylpyruvate and 2‐oxo‐4‐phenylbutanoic acid.