Engineering of the critical residues at the stereochemistry-gate loops of Brevibacillus agri dihydropyrimidinase for the production of l-homophenylalanine

Rational re-design of the “double-racemase hydantoinase process” for optically pure production of natural and non-natural l-amino acids

“…Although most hydantoinases have been described as d-enantioselective [16], the above-mentioned systems were able to produce l-amino acids, as they included hydantoinases with unusual enantiomeric properties (see introduction section for details). Additionally, these two hydantoinases were improved by directed evolution (hydantoinase from Arthobacter aurescens DSM3745, AaHYD) and by site-directed mutagenesis (d-hydantoinase from Brevibacillus agri NCHU1002, BaDHP), increasing the l-amino acid productivity, but leaving scope for further enhancement of the process [23,24]. Our group has achieved d-amino acid production from racemic mixtures of 5-monosubstituted hydantoins by the "hydantoinase process" [19,20].…”

“…One variant of BaDHP (L159 V) was used to convert d,l-HPAH to l-HPA using the "hydantoinase process". As compared with that of the wild-type enzyme, the conversion yield of l-HPA increased from 39% to 61% for this variant [24]. The l-selectivity of these designed enzymes was not impressive and left room for further improvements [23].…”

“…However, despite this scant l-hydantoinase activity, together with an l-Ncarbamoylase from Bacillus kaustophilus CCRC11223, a conversion yield of 49% for l-homophenylalanine (l-HPA) was reached in 16 h at pH 7.0 from 10 mM d,l-HPAH. Both hydantoinase enzymes have been improved by directed evolution (AaHYD) and by sitedirected mutagenesis (BaDHP), and subsequently integrated in the "hydantoinase process" to produce l-methionine from d,l-MTEH [23] or l-HPA from d,l-HPAH [24], respectively. The evolved lhydantoinase from AaHYD together with an l-N-carbamoylase and a hydantoin racemase produced 91 mM l-methionine from 100 mM d,l-MTEH in less than 2 h, whereas the whole-cell catalyst with the wild-type pathway produced about 66 mM of the l-amino acid in the same time [23] .…”

Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids

“…However, this system still depends on the L-residual enantioselective activity of D-hydantoinase for those substrates whose chemical racemization is not favored. Different reports on the hydantoinase/NSAR/L-carbamoylase system have been reported (Bommarius et al, 2002;Lo et al, 2009). Engineered dihydropyrimidinase from Brevibacillus agri (L159V mutant), Bacillus kaustophilus L-carbamoylase and Deinococcus radiodurans NSAR were purified, and allowed the production of L-homophenylalanine (90% yield, 5 h, Lo et al, 2009).…”

“…Different reports on the hydantoinase/NSAR/L-carbamoylase system have been reported ( Bommarius et al, 2002 ; Lo et al, 2009 ). Engineered dihydropyrimidinase from Brevibacillus agri (L159V mutant), Bacillus kaustophilus L -carbamoylase and Deinococcus radiodurans NSAR were purified, and allowed the production of L -homophenylalanine (90% yield, 5 h, Lo et al, 2009 ). This L -enantiospecific MEC system has further been expanded by inclusion of a hydantoin racemase, speeding up the process by racemization of the remaining L -5-monosubstituted hydantoin for substrates whose chemical racemization is not favored ( Rodríguez-Alonso et al, 2015 , 2016 , 2017 ; Figure 1D ).…”