Microbial metabolism of d- and l-phenylglycine by Pseudomonas putida LW-4

A strain of Pseudomonas putida capable of utilizing both stereoisomers of phenylglycine as the sole carbon and energy source was isolated from soil. No phenylglycine racemase was detected in cells grown on either stereoisomer. In an initial reaction each steroisomer of phenylglycine was transaminated yielding phenylglyoxylate which was further metabolized via benzaldehyde to benzoate. Subsequently, benzoate was further degraded via an ortho-cleavage of catechol.Abbreviation HPLC high-performance liquid chromatography     

l-Ribulose production from l-arabinose by an l-arabinose isomerase mutant from Geobacillus thermodenitrificans

Geobacillus thermodenitrificans, with a double-site mutation in L: -arabinose isomerase, produced 95 g L-: ribulose l(-1 ) from 500 g L: -arabinose l(-1) under optimum conditions of pH 8, 70 degrees C, and 10 units enzyme ml(-1) with a conversion yield of 19% over 2 h. The half-lives of the mutated enzyme at 70 and 75 degrees C were 35 and 4.5 h, respectively.     

An expedient synthesis of l-ribulose and derivatives

A significant improvement in the production of l-ribulose from inexpensive and commercially available starting materials, l-arabinose and sodium aluminate, is demonstrated. This has facilitated expeditious access to gram-scale quantities of l-ribulofuranoside derivatives.     

Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.     

Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.     

Pathways of d-fructose catabolism in species of Pseudomonas

Cell-free extracts of d-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose and contained 1-P-fructokinase activity suggesting that in these species fructuse-1-P and fructose-1,6-P₂ were intermediates of d-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P₂ aldolase, fructose-1,6-P₂ phosphatase, and P-hexose isomerase which could convert fructose-1,6-P₂ to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-¹⁴C-d-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-meyerhof pathway. An edd ^- mutant of P. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on d-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.Non-Standard Abbreviations cpm counts per min - edd ^- mutant lacking Entner-Doudoroff dehydrase (6-PGA dehydrase) - EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTs PEP: d-fructose phosphotransferase system - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

Regulation of metabolite production by precursors and elicitors in liquid cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

Four precursors (l-phenylalanine, l-tryptophan, cinnamic acid and emodin) and one signal elicitor (methyl jasmonate, MeJA) were added to liquid cultures of Hypericum perforatum L. to study their effect on production of hyperforin and hypericins (pseudohypericin and hypericin). The addition of l-phenylalanine (75 to 100 mg l⁻¹) enhanced production of hypericins, but hyperforin levels were decreased. Hypericin, pseudohypericin and hyperforin concentrations were all decreased when l-tryptophan (25 to 100 mg l⁻¹) was added to the medium. However, addition of l-tryptophan (50 mg l⁻¹) with MeJA (100 μM) stimulated hyperforin production significantly (1.81-fold) and resulted in an increased biomass. Cinnamic acid (25, 50 mg l⁻¹) and emodin (1.0 to 10.0 mg l⁻¹) each enhanced hyperforin accumulation in H. perforatum, but did not affect accumulation of hypericins.     

N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

Biochemical and genetic characterization of l-glutamate transport and utilization in Salmonella typhimurium LT-2 mutants

Two systems for l-glutamate transport were found in Salmonella typhimurium LT-2 GltU⁺ (glutamate utilization) mutants. The first one is similar to the glt system previously described in Escherichia coli; by transductional analysis the structural gene, gltS, coding for the transport protein was located at minute 80 of the chromosome as part of the operon gltC-gltS, and its regulator, the gltR gene, near minute 90; the gltS gene product transports both l-glutamate and l-aspartate, is sodium independent, and is -hydroxyaspartate sensitive. The second transport system, whose structural gene was called gltF and is located at minute 0, was l-glutamate specific, sodium independent, and -methylglutamate sensitive. Two aspartase activities occurred in S. typhimurium LT-2: the first one was present only in the GltU⁺ mutants, had a pH 6.4 optimum, was essential for both l-glutamate and l-aspartate metabolism, and mapped at minute 94, close to the ampC gene. The second one had a pH 7.2 optimum, could be induced by several amino acids, and thus may have a general role in nitrogen metabolism.     

DL-4-hydroxyphenylglycine catabolism in Pseudomonas putida MW27

Thirteen bacteria were isolated on D-4-hydroxyphenylglycine as sole carbon and energy source. Seven strains transaminated only the D-enantiomer while the other six isolates transaminated both enantiomers of 4-hydroxyphenylglycine. One of the six strains utilizing both enantiomers was characterized as a Pseudomonas putida. This strain, MW27, employed two enantioselective transaminases, to catalyze the initial step in the metabolism of DL-4-hydroxyphenylglycine. The product of the transamination, 4-hydroxyphenylglyoxylate, was further metabolized via 4-hydroxybenzaldehyde and 4-hydroxybenzoate to protocatechuate. Preliminary results indicate that both transaminases are co-ordinately synthesized together with the 4-hydroxyphenylglyoxylate decarboxylase and the NADP⁺-dependent 4-hydroxybenzaldehyde dehydrogenase.     

Intracellular l-arginine concentration does not determine NO production in endothelial cells: Implications on the “l-arginine paradox”

We examined the relative contributory roles of extracellular vs. intracellular l-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of ¹⁵N₄-ARG, ARG, or l-arginine ethyl ester (ARG-EE) for 2 h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, ¹⁵N₄-ARG, dimethylarginines, and l-citrulline by an LC–MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by¹⁵N-nitrite or estimated ¹⁵N₃-citrulline concentrations when ¹⁵N₄-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced ¹⁵N₄-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by ¹⁵N-nitrite, total nitrite and ¹⁵N₃-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the “l-arginine paradox” should not consider intracellular ARG concentration as a reference point.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Microbial production of L -glutamate and L -glutamine by recombinant Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb

Vitreoscilla hemoglobin (VHb) gene vgb equipped with a native promoter Pvgb or a tac promoter Ptac was introduced into Corynebacterium glutamicum ATCC14067, respectively. Ptac was proven to be more suitable for expressing VHb protein in higher concentration in both Escherichia coli and C. glutamicum strains compared with the native vgb promoter Pvgb. VHb-expressing C. glutamicum exhibited higher oxygen uptake rate and enhanced cell growth. Recombinant C. glutamicum harboring vgb gene equipped with Ptac promoter produced 23% more l-glutamate in shake-flask culture and grew to 30% more cell density and formed 22% more l-glutamate in fermentor studies compared with the wild-type strain. When a site-directed mutagenesis in which Tyr405 was replaced by a phenylalanine residue (Y405F) was performed on glutamine synthesis gene, recombinant C. glutamicum overexpressing the mutated gene glnA′ was able to produce l-glutamine effectively. Co-expression of vgb and glnA′ genes in C. glutamicum produced 17 g/l l-glutamine in shake flask culture, approximately 30% more than that produced by the recombinant harboring only glnA′ gene. In fermentor cultivation, the recombinant yielded 25% more cells and produced 40.5 g/l l-glutamine. In this study, it was clearly demonstrated that VHb significantly enhanced cell growth, l-glutamate, and l-glutamine production by recombinant C. glutamicum.     

Prolonged inhibition of brain nitric oxide synthase by short-term systemic administration of nitro-l-arginine methyl ester

We studied the dose-response characteristics and the temporal profile of inhibition of brain nitric oxide (NO) synthase (NOS) elicited by i.v. administration of the NOS inhibitor nitro-l-arginine methyl ester (L-NAME). L-NAME was administered i.v. in awake rats equipped with a venous cannula. L-NAME was injected in cumulative doses of 5, 10, 20 and 40 mg/kg and rats were sacrificed 30 min after the last dose. NOS catalytic activity was assayed in forebrain cytosol as the conversion of [³H]l-arginine into [³H]l-citrulline. L-NAME attenuated brain NOS activity in a dose-dependent manner but enzyme activity could not be inhibited by more than 50%. After a single 20 mg/kg injection of L-NAME the inhibition of brain NOS activity was time dependent and reached a stable level at 2 hrs (52% of vehicle). Inhibition after a single injection was still present at 96 hrs, albeit to a lower magnitude. We conclude that intravenous administration of L-NAME in rats at concentrations commonly used in physiological experiments leads to a dose and time-dependent but partial inhibition of brain NOS catalytic activity. The finding that the inhibition persists for several days after a single administration is consistent with the hypothesis that nitro-L-arginine, the active principle of L-NAME, binds to NOS irreversibly.     

Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase from <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A novel transglycosylation reaction from sucrose to l-ascorbic acid by a recombinant sucrose phosphorylase from Bifidobacterium longum was used to produce a stable l-ascorbic acid derivative. The major product was detected by HPLC, and confirmed to be 2-O-α-d-glucopyranosyl-l-ascorbic acid by LC-MS/MS analysis.     

Synthesis and application of dipeptides; current status and perspectives

The functions and applications of l-α-dipeptides (dipeptides) have been poorly studied compared with proteins or amino acids. Only a few dipeptides, such as aspartame (l-aspartyl-l-phenylalanine methyl ester) and l-alanyl-l-glutamine (Ala-Gln), are commercially used. This can be attributed to the lack of an efficient process for dipeptide production though various chemical or chemoenzymatic method have been reported. Recently, however, novel methods have arisen for dipeptide synthesis including a nonribosomal peptide-synthetase-based method and an l-amino acid α-ligase-based method, both of which enable dipeptides to be produced through fermentative processes. Since it has been revealed that some dipeptides have unique physiological functions, the progress in production methods will undoubtedly accelerate the applications of dipeptides in many fields. In this review, the functions and applications of dipeptides, mainly in commercial use, and methods for dipeptide production including already proven processes as well as newly developed ones are summarized. As aspartame and Ala-Gln are produced using different industrial processes, the manufacturing processes of these two dipeptides are compared to clarify the characteristics of each procedure.     

L-alanine:2-oxoglutarate aminotransferase isoenzymes from Arabidopsis thaliana leaves

The occurrence of four l-alanine:2-oxoglutarate aminotransferase (AOAT) isoenzymes (AOAT-like proteins): alanine aminotransferase 1 and 2 (AlaAT1 and AlaAT2, EC 2.6.1.2) and l-glutamate:glyoxylate aminotransferase 1 and 2 (GGAT1 and GGAT2, EC 2.6.1.4) was demonstrated in Arabidopsis thaliana leaves. These enzymes differed in their substrate specificity, susceptibility to pyridoxal phosphate inhibitors and behaviour during molecular sieving on Zorbax SE-250 column. A difference was observed in the electrostatic charge values at pH 9.1 between GGAT1 and GGAT2 as well as between AlaAT1 and AlaAT2, despite high levels of amino acid sequence identity (93 % and 85 %, respectively). The unprecedented evidence for the monomeric structure of both AlaAT1 and AlaAT2 is presented. The molecular mass of each enzyme estimated by molecular sieving on Sephadex G-150 and Zorbax SE-250 columns and SDS/PAGE was approximately 60 kDa. The kinetic parameters: K_m (Ala)=1.53 mM, K_m (2-oxoglutarate)=0.18 mM, k_cat=124.6 s⁻¹, k_cat/K_m=8.1 × 10⁴ M⁻¹·s⁻¹ of AlaAT1 were comparable to those determined for other AlaATs isolated from different sources. The two studied GGATs also consisted of a single subunit with molecular mass of 47.3–70 kDa. The estimated K_m values for l-glutamate (1.2 mM) and glyoxylate (0.42 mM) in the transamination catalyzed by putative GGAT1 contributed to indentification of the enzyme. Based on these results we concluded that each of four AOAT genes in Arabidopsis thaliana leaves expresses different AOAT isoenzyme, functioning in a native state as a monomer.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

Cultivation ofCandida blankii in simulated bagasse hemicellulose hydrolysate

Summary All fourCandida blankii isolates evaluated for growth in simulated bagasse hemicellulose hydrolysate utilized the sugars and acetic acid completely. The utilization ofd-xylose,l-arabinose and acetic acid were delayed by the presence ofd-glucose, but after glucose depletion the other carbon sources were utilized simultaneously. The maximum specific growth rate of 0.36 h^–1 and cell yield of 0.47 g cells/g carbon source assimilate compared with published results obtained withC. utilis. C. blankii appeared superior toC. utilis for biomass production from hemicellulose hydrolysate in that it utilizedl-arabinose and was capable of growth at higher temperatures.