The conversion of L-phenylalanine into benzoic acid on the thylakoid membrane of higher plants.

The conversion of L-phenylalanine into benzoic acid and other aromatic carboxylic acids was investigated in Nasturtium officinale (watercress), Astilbe chinensis, and Hydrangea macrophylla in vivo and in vitro. Comparative feeding experiments with radioactively labelled L-phenylalanine and cinnamic acid administered to intact leaf discs of A. chinensis indicated a rapid formation of benzoic acid from L-phenylalanine, whereas cinnamic acid was a poor precursor. Using a pulse-chase labelling technique followed by a fractionation of the tissue into subcellular components, chloroplasts could be identified as the predominant, if not exclusive, site of benzoic acid formation in A. chinensis. Experiments in vitro with chloroplasts and thylalkoids of N. officinale, H. macrophylla, and A. chinensis demonstrate the capacity of thylakoid membranes to catalyze the degradation of L-phenylalanine to benzoic acid. The results obtained upon stimultaneous incubation with [4'-3H]L-phenylalanine and [3-14C]cinnamic acid lead to the hypothesis that the reaction of L-phenylalanine to benzoic acid proceeds via a cinnamic acid pool which is different from that of soluble cinnamic acid.     

大肠杆菌EP8—10转化苯丙酮酸生成L—苯丙氨酸的研究

E. coli EP8-10 was selected from the soil. It was able to produce the transaminase with high activity when it was cultivated on the medium containing peptone and beef extract. Optimum conditions of enzyme reaction was: phenylpyruvic acid's concentration of 0.3-0.5 mol/L, L-Asptaric acid used as amino donor, pH 8.5 37 degrees C. When phenylpyruvic acid was 0.3 mol/L, 48 g/L L-phenylalanine was produced after 6 h with 97% conversion rate.     

Efficient conversion of phenylpyruvic acid to phenyllactic acid by using whole cells of Bacillus coagulans SDM

Background

Phenyllactic acid (PLA), a novel antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi, can be produced by many microorganisms, especially lactic acid bacteria. However, the concentration and productivity of PLA have been low in previous studies. The enzymes responsible for conversion of phenylpyruvic acid (PPA) into PLA are equivocal.

Methodology/Principal Findings

A novel thermophilic strain, Bacillus coagulans SDM, was isolated for production of PLA. When the solubility and dissolution rate of PPA were enhanced at a high temperature, whole cells of B. coagulans SDM could effectively convert PPA into PLA at a high concentration (37.3 g l⁻¹) and high productivity (2.3 g l⁻¹ h⁻¹) under optimal conditions. Enzyme activity staining and kinetic studies identified NAD-dependent lactate dehydrogenases as the key enzymes that reduced PPA to PLA.

Conclusions/Significance

Taking advantage of the thermophilic character of B. coagulans SDM, a high yield and productivity of PLA were obtained. The enzymes involved in PLA production were identified and characterized, which makes possible the rational design and construction of microorganisms suitable for PLA production with metabolic engineering.     

Characterization of d-lactate dehydrogenase from Pediococcus acidilactici that converts phenylpyruvic acid into phenyllactic acid

The gene coding for D-lactate dehydrogenase (D-LDH) from Pediococcus acidilactici DSM 20284 was cloned and expressed in E. coli. The recombinant enzyme was purified by nickel-affinity chromatography. It converted phenylpyruvic acid (PPA) to 3-phenyllactic acid maximally at 30°C and pH 5.5 with a specific activity of 140 and 422 U/mg for PPA and pyruvate, respectively. The K(m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) for PPA were 2.9 mM, 305 s(-1), and 105 mM(-1) s(-1), respectively.     

Specific conversion of d-galactose into d-galacturonic acid residues in glycoproteins: a facile method for carbohydrate linkage-analysis

The terminal d-galactopyranosyl residues of asialoglycopeptides isolated from human α₁-acid glycoprotein were oxidized in nearly quantitative yield to the corresponding uronic acid residues by a two-step sequence employing d-galactose oxidase followed by treatment with Tollens reagent, Ag(NH₃)₂⁺. Mild acid hydrolysis of the oxidized glycopeptides led to the isolation of the corresponding aldobiuronic acid(s). Structural and colorimetric analysis revealed that only one aldobiuronic acid, 2-amino-2-deoxy-4-O-(β-d-galactopyranosyluronic acid)-d-glucose, was isolated from the oxidized glycopeptides of α₁-acid glycoprotein. This method can readily distinguish between the (1→3), (1→4), and (1→6) isomers of the corresponding aldobiuronic acids.     

Rapid conversion of spirostans into furostan skeletons at room temperature

We report a facile protocol to obtain 22-substituted furostans and pseudosapogenins in high yields from (25R)- and (25S)-sapogenins. This method involves the treatment of the sapogenin with acetic-trifluoroacetic mixed anhydride and BF(3)·OEt(2) at room temperature, followed by the addition of a nucleophile (H(2)O, MeOH or KSeCN). In the case of 22-hydroxyfurostans, they can be transformed to pseudosapogenins by treatment with p-toluensulfonic acid.     

Stereoselectivity in the biosynthetic conversion of xanthoxin into abscisic acid

All stereoisomers of xanthoxin (XAN) and abscisic aldehyde (ABA-aldehyde) were prepared from (R) and (S)-4-hydroxy--cyclogeraniol via asymmetric epoxidation. Their stomatal closure activities were measured on epidermal strips of Commelina communis L. Natural (S)-ABA-aldehyde showed strong activity comparable to that of (S)-abscisic acid (ABA). Natural (1S, 2R, 4S)XAN and (1S, 2R, 4R)-epi-XAN also induced stomatal closure at high concentrations. On the other hand, unnatural (1R)-enantiomers of XAN, epi-XAN, and ABA-aldehyde were not effective. To further examine the Stereoselectivity on the biosynthetic pathway to ABA, deuterium-labeled substrates were prepared and fed to Lycopersicon esculentum Mill, under non-stressed or water-stressed conditions. Substantial incorporations into ABA were observed in the cases of natural (1S, 2R, 4S)-XAN, (1S, 2R, 4R)-epi-XAN and both enantiomers of ABA-aldehyde, leading to the following conclusions. The negligible effect of unnatural (1R)-enantiomers of XAN, epi-XAN and ABA-aldehyde can be explained by their own biological inactivity and/or their conversion to inactive (R)-ABA. Even in the isolated epidermal strips, putative aldehyde oxidase activity is apparently sufficient to convert ABA-aldehyde to ABA while the activity of XAN dehydrogenase seems very weak. The stereochemistry of the 1, 2-epoxide is very important for the XAN-dehydrogenase while this enzyme is less selective regarding the 4-hydrdxyl group of XAN and converts both (1S, 2R, 4S)-XAN and (1S, 2R, 4R)-epi-XAN to (S)-ABA-aldehyde. Abscisic aldehyde oxidase can nonstereoselectively convert both (S) and (R)-ABA-aldehyde to biologically active (S) and inactive (R)-ABA, respectively.Abbreviations ABA abscisic acid - ABA-aldehyde abscisic aldehyde - DET diethyl tartrate - epi-XAN xanthoxin epimer - FCC flash column chromatography - GC-EI-MS gas chromatography-electron impact-mass spectrometry - MeABA abscisic acid methyl ester - IR infrared - NMR nuclear magnetic resonance - PCC pyridinium chlorochromate - THF tetrahydrofuran - XAN xanthoxin The authors are very grateful to Mr J.K. Heald (Department of Biological Sciences, University of Wales, Aberystwyth, UK) and Dr. R. Horgan for carrying out GC-EI-MS analyses and advice, respectively.This work was supported by the Japan Society for the Promotion of Science (Fellowship for Young Japanese Researcher No. 0040672).