Mari Yasuda

N-Methyl-L-amino acid dehydrogenase from Pseudomonas putida: A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily

We found N‐methyl‐l‐amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH‐dependent formation of N‐alkyl‐l‐amino acids from the corresponding α‐oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)‐dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)‐dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)‐dependent amino acid dehydrogenases and opine dehydrogenases.

A practical synthesis of (R)-3-chlorostyrene oxide starting from 3-chloroethylbenzene

Abstract A novel and practical synthesis of ( R )-3-chlorostyrene oxide (−)- 1 was achieved starting from commercially available 3-chloroethylbenzene 3 . Enantiopure (−)-3-chlorostyrene bromohydrin (−)- 5 was obtained by the treatment of racemic (±)- 5 with lipase QL in the presence of acylating reagents. 3-Chloro-α,β-dibromoethylbenzene 4 , a precursor of (±)- 5 , was synthesized via the expeditious bromination of 3 which was developed by these authors.

Enzymatic Synthesis of L-Pipecolic Acid by Δ1-Piperideine-2-carboxylate Reductase from Pseudomonas putida

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine α-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Δ1-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.

Separation of therapeutic peptides with cyclofructan and glycopeptide based columns in hydrophilic interaction liquid chromatography.

Three cyclofructan-based, two glycopeptide-based, and one zwitterionic column used in the HILIC mode were assessed within a graphical framework based on different functional characteristics contributing to selectivity. The characteristics of these six HILIC columns are put in the perspective of 33 columns evaluated previously. The isopropyl carbamate modified cyclofructan 6 (CF6) stationary phase, Larihc P, showed reduced component contributions for hydrophilicity and hydrogen bonding relative to the native cyclofructan 6 column (Frulic N). Both Frulic N and Larihc P exhibited cation exchange attributed primarily to deprotonation of residual unsubstituted silica with the greater exchange ascribed to the reduced loading of CF6 observed for Larihc P. The cyclofructan 6 column with a polymeric styrene divinylbenzene support (MCI GEL™ CRS100) showed distinct selectivities consistent with its decreased cation exchange attributable to its nonionic core. The Chirobiotic T, Chirobiotic V, and ZI-DPPS columns displayed hydrophilicity and ion exchange selectivities similar to other zwitterionic stationary phases. All of the more hydrophilic columns showed excellent separation for the four classes of therapeutic peptides investigated: microbial secondary metabolites used as immune suppressants, synthetic gonadotropin hormones, synthetic cyclic disulfide-linked hormone-regulating hormones, and non-ribosomally derived polycyclic antibiotics. Resolution provided by these columns and ZIC-HILIC is compared for each class of peptide. Frulic N is primarily suitable for use in the HILIC mode whereas Chirobiotic T, because of its increased efficiency and selectivity, can be useful in both HILIC and reverse phase modes. In some Chirobiotic T applications, addition of low levels of a strong additive (trifluoroacetic acid, formic acid, etc.) to the mobile phase can be beneficial. In these peptide analyses, a relative weakening of the often-dominant ionic interaction between analyte and residual charge on the stationary phase improved resolution and selectivity.

High level production of 3-cyano-6-hydroxypyridine from 3-cyanopyridine by Comamonas testosteroni MCI2848

Summary We optimized culture and reaction conditions for the production of 3-cyano-6-hydroxypyridine (6-CHP) from 3-cyanopyridine (3-CP) by resting cells of Comamonas testosteroni MCI2848. High reaction activity was induced by the addition of 6-halo-nicotinic acid, as inducer, to the culture medium. The productivity by the intact cells was increased by addition of nicotinic acid or 6-hydroxy nicotinic acid to the reaction mixture. Intact cells of this strain incubated at 30°C in an optimal reaction mixture containing 5 mM sodium nicotinate produced 57.2 g/l of 6-CHP with 100% reaction-selectivity for 40 h.