1. Effects of halophilic peptide fusion on solubility, stability, and catalytic performance of D-phenylglycine aminotransferase
Hossein Javid, Juntratip Jomrit, Aiya Chantarasiri, Duangnate Isarangkul, Vithaya Meevootisom, Suthep Wiyakrutta J Microbiol Biotechnol. 2014 May;24(5):597-604. doi: 10.4014/jmb.1312.12040.
D-Phenylglycine aminotransferase (D-PhgAT) from Pseudomonas stutzeri ST-201 is useful for enzymatic synthesis of enantiomerically pure D-phenylglycine. However, its low protein solubility prevents its application at high substrate concentration. With an aim to increase the protein solubility, the N-terminus of D-PhgAT was genetically fused with short peptides (A1 α- helix, A2 α-helix, and ALAL, which is a hybrid of A1 and A2) from a ferredoxin enzyme of a halophilic archaeon, Halobacterium salinarum. The fused enzymes A1-D-PhgAT, A2-D-PhgAT, and ALAL-D-PhgAT displayed a reduced pI and increased in solubility by 6.1-, 5.3-, and 8.1- fold in TEMP (pH 7.6) storage, respectively, and 5-, 4.5-, and 5.9-fold in CAPSO (pH 9.5) reaction buffers, respectively, compared with the wild-type enzyme (WT-D-PhgAT). In addition, all the fused D-PhgAT displayed higher enzymatic reaction rates than the WT-DPhgAT at all concentrations of L-glutamate monosodium salt used. The highest rate, 23.82 ± 1.47 mM/h, was that obtained from having ALAL-D-PhgAT reacted with 1,500 mM of the substrate. Moreover, the halophilic fusion significantly increased the tolerance of D-PhgAT in the presence of NaCl and KCl, being slightly in favor of KCl, where under the same condition at 3.5 M NaCl or KCl all halophilic-fused variants showed higher activity than WT-D-PhgAT.
2. Enantioselective N-acetylation of 2-phenylglycine by an unusual N-acetyltransferase from Chryseobacterium sp
Shinji Takenaka, Yuuta Honma, Kenji Yoshida, Ken-ichi Yoshida Biotechnol Lett. 2013 Jul;35(7):1053-9. doi: 10.1007/s10529-013-1172-z. Epub 2013 Mar 12.
The demand for D-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the L-form of racemic D,L-2-phenylglycine to (2S)-2-acetylamide-2-phenylacetic acid with a molar yield of 50% and an enantiomer excess of >99.5% under optimal culture conditions, consequently resulting in 99% pure D-2-phenylglycine remaining in the culture. The enantioselective N-acetylation was catalyzed by an acetyl-CoA-dependent N-acetyltransferase whose synthesis was induced by L-2-phenylglycine. The enzyme differed from previously reported bacterial arylamine N-acetyltransferases in molecular mass and substrate specificity. The relative activity ratio of the enzyme with the substrates L-2-phenylglycine, D-2-phenylglycine, 2-(2-chlorophenyl)glycine, and 5-aminosalicylic acid (a good substrate of arylamine N-acetyltransferase) was 100:0:56.9:5.49, respectively. The biotransformation by the N-acetyltransferase-producing bacterium reported here could constitute a new preparative route for the enzymatic resolution of D,L-2-phenylglycine.
3. Production of D-phenylglycine-related amino acids by immobilized microbial cells
H Yamada, S Shimizu, H Shimada, Y Tani, S Takahashi, T Ohashi Biochimie. 1980;62(5-6):395-9. doi: 10.1016/s0300-9084(80)80171-4.
Bacterial dihydropyrimidinase was shown to catalyze the hydrolytic cleavage of various 5-substituted hydantoins to the corresponding N-carbamyl-D-amino acids under alkaline conditions. Therefore, an enzymatic method for preparing the D-forms of phenylglycine-related amino acids was developed using immobilized bacterial cells with high enzyme activity. Alkalophilic bacteria were a good enzyme source for this process. The process is simple and economical for use in the production of various amino acids with the D-configuration.
