1.A novel D-mandelate dehydrogenase used in three-enzyme cascade reaction for highly efficient synthesis of non-natural chiral amino acids.
Fan CW1, Xu GC1, Ma BD1, Bai YP1, Zhang J1, Xu JH2. J Biotechnol. 2015 Feb 10;195:67-71. doi: 10.1016/j.jbiotec.2014.10.026. Epub 2014 Oct 27.
A novel NAD(+)-dependent D-mandelate dehydrogenase was identified from Lactobacillus brevis (LbDMDH). After purified to homogeneity, the optimum pH and temperature for oxidation of D-mandelate were pH 10.0 and 40 °C, and the Km and kcat were 1.1 mM and 355 s(-1) respectively. Employing the LbDMDH together with a mandelate racemase from Pseudomonas putida and a leucine dehydrogenase (EsLeuDH) from Exiguobacterium sibiricum, we established a three-step one-pot domino reaction system for preparing chiral L-phenylglycine from racemic mandelic acid with internal cofactor recycling. Under the optimum conditions, 30.4 g rac-mandelic acid (0.2 M) at 1L scale had been converted into chiral L-phenylglycine, with 96.4% conversion, 86.5% isolation yield, >99% eep and 50.4 gL(-1)d(-1) space-time yield.
2.Enantioselective N-acetylation of 2-phenylglycine by an unusual N-acetyltransferase from Chryseobacterium sp.
Takenaka S1, Honma Y, Yoshida K, Yoshida K. 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.