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

Abstract

For the production of enantiopure β-amino acids, enantioselective resolution of N-acyl β-amino acids using acylases, especially those recognizing N-acetyl-β-amino acids, is one of the most attractive methods. Burkholderia sp. AJ110349 had been reported to exhibit either (R)- or (S)-enantiomer selective N-acetyl-β-Phe amidohydrolyzing activity, and in this study, both (R)- and (S)-enantioselective N-acetyl-β-Phe acylases were purified to be electrophoretically pure and determined the sequences, respectively. They were quite different in terms of enantioselectivities and in their amino acids sequences and molecular weights. Although both the purified acylases were confirmed to catalyze N-acetyl hydrolyzing activities, neither of them show sequence similarities to the N-acetyl-α-amino acid acylases reported thus far. Both (R)- and (S)-enantioselective N-acetyl-β-Phe acylase were expressed in Escherichia coli. Using these recombinant strains, enantiomerically pure (R)-β-Phe (>99% ee) and (S)-β-Phe (>99% ee) were obtained from the racemic substrate.

Expression, purification and crystallization of N-acetyl-(R)-β-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 and structure determination of the Burkholderia enzyme

“…In a previous study, native -FAA produced by Burkholderia sp. AJ110349 was purified and the molecular weight of the purified -FAA in solution was analyzed by size-exclusion chromatography and estimated to be 206 kDa from the elution volume (Imabayashi et al, 2016). Since the molecular masses of native -FAA monomers, homodimers and homotetramers were calculated to be 81.4, 162.8 and 244.2 kDa, respectively, the oligomeric state of -FAA in solution remained unclear.…”

“…In our previous study, the native -FAA produced by Burkholderia sp. AJ110349 was purified and characterized, showing that it had strict (R)-enantiomer specificity (Imabayashi et al, 2016). The -FAA gene was cloned from the Burkholderia sp.…”

“…AJ110349 genome, and recombinant expression in Eschericia coli and purification of this enzyme were attempted for further investigations, but failed because the expression level was insufficient due to the expression system used in the study. In spite of its low expression in E. coli, the recombinantly expressed protein exhibited -FAA activity (Imabayashi et al, 2016).…”

“…On the other hand, B--FAA expressed in E. coli exhibited activity without requiring another subunit, showing that it has a different quaternary structure to the DMFases. Since the molecular mass deduced from the 760 amino-acid residues of B--FAA was calculated to be 81.4 kDa and activity of the native enzyme was detected in size-exclusion chromatography at positions corresponding to the estimated molecular mass of 206 kDa, B--FAA was thought to have a homodimeric or homotrimeric structure (Imabayashi et al, 2016).…”