Production of (R)-3-Amino-3-phenylpropionic Acid and (S)-3-Amino-3-phenylpropionic Acid from (R,S)-N-Acetyl-3-amino-3-phenylpropionic Acid Using Microorganisms Having Enantiomer-Specific Amidohydrolyzing Activity

Abstract

(R)-3-Amino-3-phenylpropionic acid ((R)--Phe) and (S)-3-amino-3-phenylpropionic acid ((S)--Phe) are key compounds on account of their use as intermediates in synthesizing pharmaceuticals. Enantiomerically pure non-natural amino acids are generally prepared by enzymatic resolution of the racemic N-acetyl form, but despite the intense efforts this method could not be used for preparing enantiomerically pure -Phe, because the effective enzyme had not been found. Therefore, screening for microorganisms capable of amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid ((R,S)-N-Ac--Phe) in an enantiomer-specific manner was performed.A microorganism having (R)-enantiomer-specific amidohydrolyzing activity and another having both (R)-enantiomer-and (S)-enantiomer-specific amidohydrolyzing activities were obtained from soil samples. Using 16S rDNA analysis, the former organism was identified as Variovorax sp., and the latter as Burkholderia sp. Using these organisms, enantiomerically pure (R)--Phe (> 99:5% ee) and (S)--Phe (> 99:5% ee) with a high molar conversion yield (67%-96%) were obtained from the racemic substrate.

Purification and characterization of enantioselective N-acetyl-β-Phe acylases from Burkholderia sp. AJ110349

“…11) The pH profile and thermal stability showed the same trend, but the thermal profile was slightly different. This could be attributed to the reaction conditions or the enzyme source with not purified.…”

“…20) The characterization data for N-acetyl-(R)-β-Phe acylase were compared with data for the corresponding wild-type strain-derived activity. 11) The pH and thermal profiles showed an almost identical trend; there was no activity under pH 5 and high activity at 60°C. About the effects of metal ions, the addition of Fe ions increased the enzyme activities but EDTA showed no effect.…”

“…In our previous report, 11) we discovered two microorganisms that enantioselectively amidohydrolyze N-acetyl-(R,S)-β-Phe. In one of the microorganisms, Burkholderia sp.…”

“…AJ110349 has been reported previously. 11) For the purification of acylases, AJ110349 was grown in enzyme-producing medium comprising 10 g/L D-glucose, 10 g/L (NH 4 ) 2 SO 4 , 10 g/L (R,S)-β-Phe, 2 g/L casamino acid, 1 g/L KH 2 PO 4 , 0.4 g/L MgSO 4 ·7H 2 O, 1 g/L NaCl, 19.5 g/L 2-(N-morpholino)ethanesulfonic acid (MES), 5 mg/L nicotinamide, 0.2 mg/L thiamin, 10 mg/L FeSO 4 ·7H 2 O, and 10 mg/L MnSO 4 ·4-5H 2 O. Cultivation was carried out with shaking at 120 rpm in a 500-mL Sakaguchi flask, for 66 h at 30°C.…”

“…In our previous report, 11) we newly discovered two microorganisms capable of enantioselectively amidohydrolyzing (R,S)-N-acetyl-3-amino-3-phenylpropionic acid [(R,S)-N-acetyl β-Phe] and identified them to be Burkholderia sp. AJ110349 and Variovorax sp.…”