Promoter engineering-mediated Tuning of esterase and transaminase expression for the chemoenzymatic synthesis of sitagliptin phosphate at the kilogram-scale.

Here, we report a bienzymatic cascade to produce ß-amino acids as an intermediate for the synthesis of the leading oral antidiabetic drug, sitagliptin. A whole-cell biotransformation using recombinant Escherichia coli coexpressing a esterase and transaminase were developed, wherein the desired expression level of each enzyme was achieved by promotor engineering. The small-scale reactions (30 ml) performed under optimized conditions at varying amounts of substrate (100-300 mM) resulted in excellent conversions of 82%-95% for the desired product. Finally, a kilogram-scale enzymatic reaction (250 mM substrate, 220 L) was carried out to produce ß-amino acid (229 mM). Sitagliptin phosphate was chemically synthesized from ß-amino acids with 82% yield and > 99% purity.

Kidney proteome responses in the teleost fish Paralichthys olivaceus indicate a putative immune response against Streptococcus parauberis.

The proteomic response to bacterial infection in a teleost fish (Paralichthys olivaceus) infected with Streptococcus parauberis was analyzed using label-free protein quantitation coupled with LC-MS(E) tandem mass spectrometry. A total of 82 proteins from whole kidney, a major lymphoid organ in this fish, were found to be differentially expressed between healthy and diseased fish analyzed 6, 24, 72 and 120 h post-infection. Among the differentially expressed proteins, those involved in mediating immune responses (e.g., heat shock proteins, cathepsins, goose-type lysozyme and complement components) were most significantly up-regulated by infection. In addition, cell division cycle 48 (CDC48) and calreticulin, which are associated with cellular recovery and glycoprotein synthesis, were up-regulated in the universal protein group, whereas the other proteins in that group were down-regulated. There was continuous activation of expression of immune-associated proteins during infection, but there was also loss of expression of proteins not involved in immune function. We expect that our findings regarding immune response at the protein level would offer new insight into the systemic response to bacterial infection of a major immune organ in teleost fish.

In-depth analysis of cysteine oxidation by the RBC proteome: advantage of peroxiredoxin II knockout mice.

Peroxiredoxin II (Prdx II, a typical 2-Cys Prdx) has been originally isolated from erythrocytes, and its structure and peroxidase activity have been adequately studied. Mice lacking Prdx II proteins had heinz bodies in their peripheral blood, and morphologically abnormal cells were detected in the dense red blood cell (RBC) fractions, which contained markedly higher levels of reactive oxygen species (ROS). In this study, a labeling experiment with the thiol-modifying reagent biotinylated iodoacetamide (BIAM) in Prdx II-/- mice revealed that a variety of RBC proteins were highly oxidized. To identify oxidation-sensitive proteins in Prdx II-/- mice, we performed RBC comparative proteome analysis in membrane and cytosolic fractions by nano-UPLC-MSE shotgun proteomics. We found oxidation-sensitive 54 proteins from 61 peptides containing cysteine oxidation, and analyzed comparative expression pattern in healthy RBCs of Prdx II+/+ mice, healthy RBCs of Prdx II-/- mice, and abnormal RBCs of Prdx II-/- mice. These proteins belonged to cellular functions related with RBC lifespan maintain, such as cytoskeleton, stress-induced proteins, metabolic enzymes, signal transduction, and transporters. Furthermore, protein networks among identified oxidation-sensitive proteins were analyzed to associate with various diseases. Consequently, we expected that RBC proteome might provide clues to understand redox-imbalanced diseases.

Hydroxycinnamoylmalic acids and their methyl esters from pear (Pyrus pyrifolia Nakai) fruit peel.

Two novel caffeoylmalic acid methyl esters, 2-O-(trans-caffeoyl)malic acid 1-methyl ester (6) and 2-O-(trans-caffeoyl)malic acid 4-methyl ester (7), were isolated from pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) fruit peels. In addition, 5 known hydroxycinnamoylmalic acids and their methyl esters were identified: 2-O-(trans-coumaroyl)malic acid (1), 2-O-(cis-coumaroyl)malic acid (2), 2-O-(cis-coumaroyl)malic acid 1-methyl ester (3), 2-O-(trans-coumaroyl)malic acid 1-methyl ester (4), and 2-O-(trans-caffeoyl)malic acid (phaselic acid, 5). The chemical structures of these compounds were determined by spectroscopic data from ESI MS and NMR. Of all the isolated compounds, five hydroxycinnamoylmalic acids and their methyl esters (2-4, 6, 7) were identified in the pear for the first time.

Metabolite profiling of Angelica gigas from different geographical origins using 1H NMR and UPLC-MS analyses.

Angelica gigas obtained from different geographical regions was characterized using (1)H nuclear magnetic resonance (NMR) spectroscopy and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) followed by multivariate data analyses. Principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) score plots from (1)H NMR and UPLC-MS data sets showed a clear distinction among A. gigas from three different regions in Korea. The major metabolites that contributed to the discrimination factor were primary metabolites including acetate, choline, citrate, 1,3-dimethylurate, fumarate, glucose, histamine, lactose, malate, N-acetylglutamate, succinate, and valine and secondary metabolites including decursin, decursinol, nodakenin, marmesin, 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-ethyl)coumarin in A. gigas roots. The results demonstrate that (1)H NMR and UPLC-MS-based metabolic profiling coupled with chemometric analysis can be used to discriminate the geographical origins of various herbal medicines and to identify primary and secondary metabolites responsible for discrimination.