Protective effect of l-ascorbic acid on nickel induced pulmonary nitrosative stress in male albino rats

Nickel sulfate stimulates inducible nitric oxide synthase (i-NOS) and increases serum nitric oxide concentration by overproduction of reactive nitrogen species due to nitrosative stress. The present study was undertaken to assess possible protective role of l-ascorbic acid as an antioxidant against nickel induced pulmonary nitrosative stress in male albino rats. We studied the effect of the simultaneous treatment with l-ascorbic acid (50 mg/100 g b. wt.; orally) and nickel sulfate (2.0 mg/100 g b. wt.; i.p.) on nitric oxide synthesis by quantitative evaluation of serum i-NOS activities, serum and lung nitric oxide, l-ascorbic acid and protein concentrations of Wister strain male albino rats. We have further studied histopathological changes in lung tissue after nickel sulfate treatment along with simultaneous exposure of l-ascorbic acid. Nickel sulfate treatment significantly increased the serum i-NOS activity, serum and pulmonary nitric oxide concentration and decreased body weight, pulmonary somatic index, serum and lung l-ascorbic acid and protein concentration as compared to their respective controls. Histopathological changes induced by nickel sulfate showed loss of normal alveolar architecture, inflammation of bronchioles, infiltration of inflammatory cells and patchy congestion of alveolar blood vessels. The simultaneous administration of l-ascorbic acid and nickel sulfate significantly improved all the above biochemical parameters along with histopathology of lung tissues of rats receiving nickel sulfate alone. The study clearly showed a protective role of l-ascorbic acid against nickel induced nitrosative stress in lung tissues.     

Functional cardiovascular action of l-cysteine microinjected into pressor sites of the rostral ventrolateral medulla of the rat

The endogenous sulfur-containing amino acid l-cysteine injected into the cerebrospinal fluid space of the cisterna magna increases arterial blood pressure (ABP) and heart rate (HR) in the freely moving rat. The present study examined (1) cardiovascular responses to l-cysteine microinjected into the rostral ventrolateral medulla (RVLM), where a group of neurons regulate activities of cardiovascular sympathetic neurons and (2) involvement of ionotropic excitatory amino acid (iEAA) receptors in response. In the RVLM of urethane-anesthetized rats accessed ventrally and identified with pressor responses to l-glutamate (10 mM, 34 nl), microinjections of l-cysteine increased ABP and HR dose dependently (3–100 mM, 34 nl). The cardiovascular responses to l-cysteine (30 mM) were not attenuated by a prior injection of either antagonist alone, MK801 (20 mM, 68 nl) for the NMDA type of iEAA receptors, or CNQX (2 mM) for the non-NMDA type. However, inhibition of both NMDA and non-NMDA receptors with additional prior injection of either antagonist completely blocked those responses to l-cysteine. The results indicate that l-cysteine has functional cardiovascular action in the RVLM of the anesthetized rat, and the responses to l-cysteine involve both NMDA and non-NMDA receptors albeit in a mutually exclusive parallel fashion. The findings may suggest endogenous roles of l-cysteine indirectly via iEAA receptors in the neuronal network of the RVLM for cardiovascular regulation in physiological and pathological situations.     

Polarized spectroscopic elucidation of N-acetyl-l-cysteine, l-cysteine, l-cystine, l-ascorbic acid and a tool for their determination in solid mixtures

Method of linear polarized vibrational (both IR- and Raman) spectroscopy of oriented colloids in nematic host is applied on N-acetyl-l-cysteine, l-cysteine, l-cystine and l-ascorbic acid with a view to obtain experimental bands assignment and local structural elucidation in solid-state. Structural results are compared with available crystallographic data for all of the systems studied. Scopes and limitations of the polarized method are shown. Discussion on the correlation between polarized spectroscopic data and the space group type as well as the number of the molecules in the unit cell (Z) is performed. Compounds with monoclinic space group P2₁, containing Z = 1 (N-acetyl-l-cysteine) and 2 (l-cysteine and l-ascorbic acid) are elucidated. One of the rare for organic molecules, hexagonal P6₁22 space group and Z = 6 (l-cystine) is also elucidated. Experimental assignment of the characteristics frequencies is obtained, explaining the typical for the crystals Fermi-resonance, Fermy–Davydov and Davydov splitting effects. For first time in the literature we are reported the orientation of the solid-mixture in nematic host, using the trade product ACC (Hexal, Germany), containing mainly N-acetyl-l-cysteine and l-ascorbic acid. Quantitative IR-spectroscopic approach for determination of solid mixtures is presented as well. The intensity ratio between 1,716 cm^?1 (characteristic for N-acetyl-l-cysteine) and 990 cm^?1, (attributed N-acethyl-cysteine and vitamin C) is used. Linear regression analysis between content and the peak ratio data for ten solid-binary mixtures, leads to straight-line plot y = 1.08₂ (±0.04₉) + (?0.11₄ ± 0.01₁)x, where x = 1/X _i . Factor r of 0.9641 and a reliability of 98.85% are obtained. The analysis of ACC 200 (Hexal, Germany) show that the IR measurements leads to standard deviation of 0.010 and 0.011 at P about 0.0500 for the systems and a confidence of >98.77₁%.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

Regulation of leucine catabolism by metabolic fuels in mammary epithelial cells

Lactation is associated with elevated catabolism of branched-chain amino acids (BCAA) in mammary glands to produce glutamate, glutamine, alanine, aspartate, and asparagine. This study determined effects of metabolic fuels on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 0.5 mM l-leucine and either l-[1-¹⁴C]leucine or l-[U-¹⁴C]leucine. The medium also contained 0–5 mM d-glucose, 0–2 mM l-glutamine, 0–4 mM dl-β-hydroxybutyrate, or 0–2 mM oleic acid. Rates of leucine decarboxylation were 60 % lower, but rates of α-ketoisocaproate production were 34 % higher, in the presence of 2 mM glucose than in its absence. All variables of leucine catabolism did not differ between 2 and 5 mM glucose or between 0 and 4 mM dl-β-hydroxybutyrate. Compared with 0–0.25 mM glutamine, 0.5 and 2 mM l-glutamine reduced leucine transport, transamination, and decarboxylation. In contrast, increasing the concentration of oleic acid from 0 to 2 mM dose-dependently stimulated leucine transamination, decarboxylation, and oxidation of carbons 2–6. Oleic acid also enhanced the abundance of cytosolic BCAA transaminase, while reducing the phosphorylated level (inactive state) of the E1α subunit of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. Thus, hypoglycemia or ketosis in early lactation does not likely affect BCAA metabolism in mammary epithelial cells. Increasing circulating levels of BCAA and oleic acid may have great potential to increase the syntheses of glutamate, glutamine, aspartate, alanine, and asparagine by lactating mammary glands, thereby leading to enhanced production of milk for suckling neonates.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

l-Carnitine Exposure and Mitochondrial Function in Human Neuronal Cells

l-Carnitine is a naturally occurring substance required in mammalian energy metabolism that functions by facilitating long-chain fatty acid entry into cellular mitochondria, thereby delivering substrate for oxidation and subsequent energy production. It has been purposed that l-carnitine may improve and preserve cognitive performance, and may lead to better cognitive aging through the life span, and several controlled human clinical trials with l-carnitine support the hypothesis that this substance has the ability to improve cognitive function. We further hypothesized that, since l-carnitine is an important co-factor of mammalian mitochondrial energy metabolism, acute administration of l-carnitine to human tissue culture cells should result in detectable increases in mitochondrial function. Cultures of SH-SY-5Y human neuroblastoma and 1321N1 human astrocytoma cells grown in 96-well cell culture plates were acutely administered l-carnitine hydrochloride, and then, mitochondrial function was assayed using the colorimetric 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt cell assay kit in a VERSA_max tunable microplate reader. Significant increases in mitochondrial function were observed when human neuroblastoma or human astrocytoma cells were exposed to 100 nM (20 μg l-carnitine hydrochloride/L) to 100 μM (20 mg l-carnitine hydrochloride/L) concentrations of l-carnitine hydrochloride in comparison to unexposed cells, whereas no significant positive effects were observed at lower or higher concentrations of l-carnitine hydrochloride. The results of the present study provide insights for how l-carnitine therapy may significantly improve human neuronal function, but we recommend that future studies further explore different derivatives of l-carnitine compounds in different in vitro cell-based systems using different markers of mitochondrial function.     

l-leucine, l-methionine, and l-phenylalanine share a Na+/K+-dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of ³H-l-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. ³H-l-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na⁺- and K⁺-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. ³H-l-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, l-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM ³H-l-leucine in both Na⁺- and K⁺-containing incubation media. The residual ³H-l-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an l-methionine- and cation-independent transport system. ³H-l-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [l-leucine], following the carrier-mediated Michaelis–Menten equation. In NaCl, ³H-l-leucine influx displayed a low apparent K _M (high affinity) and low apparent J _max, while in KCl the transport exhibited a high apparent K _M (low affinity) and high apparent J _max. l-methionine or l-phenylalanine (7 and 20 mM) were competitive inhibitors of ³H-l-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in ³H-l-leucine influx K _M, but no significant response in ³H-l-leucine influx J _max. Potassium was a competitive inhibitor of sodium co-transport with ³H-l-leucine, significantly (p < 0.01) increasing ³H-l-leucine influx K _M in the presence of sodium, but having negligible effect on ³H-l-leucine influx J _max in the same medium. These results suggest that shrimp BBMV transport ³H-l-leucine by a single l-methionine- and l-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na⁺ or K⁺ acting as co-transport drivers binding to shared activator sites.     

Gender-related effects on urine l-cystine metastability

Cystinuria is an autosomal recessive disease that causes l-cystine precipitation in urine and nephrolithiasis. Disease severity is highly variable; it is known, however, that cystinuria has a more severe course in males. The aim of this study was to compare l-cystine metastability in first-morning urine collected from 24 normal female and 24 normal male subjects. Samples were buffered at pH 5 and loaded with l-cystine (0.4 and 4 mM final concentration) to calculate the amount remaining in solution after overnight incubation at 4 °C; results were expressed as Z scores reflecting the l-cystine solubility in each sample. In addition, metabolomic analyses were performed to identify candidate compounds that influence l-cystine solubility. l-cystine solubility Z score was +0.44 ± 1.1 and ?0.44 ± 0.70 in female and male samples, respectively (p < 0.001). Further analyses showed that the l-cystine solubility was independent from urine concentration but was significantly associated with low urinary excretion of inosine (p = 0.010), vanillylmandelic acid (VMA) (p = 0.015), adenosine (p = 0.029), and guanosine (p = 0.032). In vitro l-cystine precipitation assays confirmed that these molecules induce higher rates of l-cystine precipitation in comparison with their corresponding dideoxy molecules, used as controls. In silico computational and modeling analyses confirmed higher binding energy of these compounds. These data indicate that urinary excretion of nucleosides and VMA may represent important factors that modulate l-cystine solubility and may represent new targets for therapy in cystinuria.     

The lipophilic vitamin C derivative, 6-o-palmitoylascorbate,protects human lymphocytes,preferentially over ascorbate,against X-ray-induced DNA damage,lipid peroxidation,and protein carbonylation

The aim of this study was to investigate protective effects of the lipophilic vitamin C derivative, 6-o-palmitoylascorbate (PlmtVC), against X-ray radiation-induced damages including cell death, DNA double-strand breaks (DSBs), lipid peroxidation, and protein carbonylation in human lymphocytes HEV0082, and the stability of PlmtVC under cell-cultured or cell-free condition. Irradiation with X-ray (1.5 Gy) diminished the cell viability and induced apoptosis, both of which were protected by pre-irradiational administration with PlmtVC. Gamma-H2A.X foci as a hallmark of DSBs were markedly enhanced in the irradiated cells. PlmtVC prevented X-ray-induced DSBs more appreciably than l-ascorbic acid (l-AA). Intracellular ROS production, lipid peroxidation, and protein carbonylation in HEV0082 cells were increased by X-ray at 1.5 Gy, all of which were significantly repressed by PlmtVC. PlmtVC also elevated endogenous reduced glutathione (GSH) in HEV0082 cells, and prevented X-ray-induced GSH depletion that are more appreciably over l-AA. Thus, PlmtVC prevents X-ray-induced cell death through its antioxidative activity. Stability tests showed that after being kept under physiological conditions (pH 7.4, 37 °C) for 14 days, vitamin C residual rates in PlmtVC solutions (62.2–82.0 %) were significantly higher than those in l-AA solutions (20.5–28.7 %). When PlmtVC or l-AA was added to HEV0082 lymphocytes, intracellular vitamin C in l-AA-treated cells was not detectable after 24 h, whereas PlmtVC-treated cells could keep a high level of intracellular vitamin C, suggesting an excellent stability of PlmtVC. Thus, X-ray-induced diverse harmful effects could be prevented by PlmtVC, which was suggested to ensue intrinsically from the persistent enrichment of intracellular vitamin C, resulting in relief to X-ray-caused oxidative stress.     

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.     

Effects of l-lysine and d-lysine on ɛ-Poly-l-lysine biosynthesis and their metabolites by Streptomyces ahygroscopicus GIM8

?-Poly-l-lysine (?-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of l-lysine, which is used as a safe food preservative. In this study, the effects of l-lysine and its isomer, d-lysine, on ?-PL biosynthesis and their metabolites by the ?-PL-producing strain Streptomyces ahygroscopicus GIM8 were determined. The results indicated that l-lysine added into the fermentation medium in the production phase mainly served as a precursor for ?-PL biosynthesis during the flask culture phase, leading to greater ?-PL production. At an optimum level of 3 mM l-lysine, a ?-PL yield of 1.16 g/L was attained, with a 41.4% increment relative to the control of 0.78 g/L. Regarding d-lysine, the production of ?-PL increased by increasing its concentrations up to 6 mM in the initial fermentation medium. Interestingly, ?-PL production (1.20 g/L) with the addition of 3 mM d-lysine into the initial fermentation medium in flasks was higher than that of the initial addition of 3 mM L-lysine (1.06 g/L). The mechanism by which d-lysine improves ?-PL biosynthesis involves its utilization that leads to greater biomass. After S. ahygroscopicus GIM8 was cultivated in the defined medium with L-lysine, several key metabolites, including 5-aminovalerate, pipecolate, and l-2-aminoadipate formed in the cells, whereas only l-2-aminoadipate was observed after d-lysine metabolism. This result indicates that l-lysine and d-lysine undergo different metabolic pathways in the cells. Undoubtedly, the results of this study are expected to aid the understanding of ?-PL biosynthesis and serve as reference for the formulation of an alternative approach to improve ?-PL productivity using l-lysine as an additional substrate in the fermentation medium.     

Enhancement of biocatalytic efficiency by increasing substrate loading: enzymatic preparation of l-homophenylalanine

Enantiomerically pure l-homophenylalanine (l-HPA) is a key building block for the synthesis of angiotensin-converting enzyme inhibitors and other chiral pharmaceuticals. Among the processes developed for the l-HPA production, biocatalytic synthesis employing phenylalanine dehydrogenase has been proven as the most promising route. However, similar to other dehydrogenase-catalyzed reactions, the viability of this process is markedly affected by insufficient substrate loading and high costs of the indispensable cofactors. In the present work, a highly efficient and economic biocatalytic process for l-HPA was established by coupling genetically modified phenylalanine dehydrogenase and formate dehydrogenase. Combination of fed-batch substrate addition and a continuous product removal greatly increased substrate loading and cofactor utilization. After systemic optimization, 40 g (0.22 mol) of keto acid substrate was transformed to l-HPA within 24 h and a total of 0.2 mM NAD⁺ was reused effectively in eight cycles of fed-batch operation, consequently giving an average substrate concentration of 510 mM and a productivity of 84.1 g l^?1 day^?1 for l-HPA. The present study provides an efficient and feasible enzymatic process for the production of l-HPA and a general solution for the increase of substrate loading.     

l-Ascorbic acid metabolism in parthenocarpic and seeded cherry tomatoes

The auxin treatment in tomato plants during anthesis has been extensively used for setting fruits in adverse climatic conditions (e.g., low temperatures and inadequate light), which is well known that reduces pollen availability and fertility. Since auxin application may affect fruit composition and quality, we examined l-ascorbic acid metabolism in seeded fruit (set by natural pollination) and parthenocarpic fruit (set by auxin) in cherry tomato cv. Conchita. Specifically, we studied the oxidized and total ascorbic acid contents, the expression of all characterized genes of l-ascorbic acid metabolism, the activity of ascorbate peroxidase and dehydroascorbate reductase and the immunolocalization of ascorbate peroxidase. Differences were detected between seeded and parthenocarpic fruits, in the expression of some of the genes of ascorbic acid metabolism. However, strong presence of l-ascorbic acid peroxidase protein was detected on the developing seeds. Our data indicate that induced parthenocarpy in auxin treated plants has a significant influence in ascorbic acid metabolism comparing to seeded tomato fruits.     

Detection of d-amino acids in purified proteins synthesized in Escherichia coli

It has long been believed that amino acids comprising proteins of all living organisms are only of the l-configuration, except for Gly. However, peptidyl d-amino acids were observed in hydrolysates of soluble high molecular weight fractions extracted from cells or tissues of various organisms. This strongly suggests that significant amounts of d-amino acids are naturally present in usual proteins. Thus we analyzed the d-amino acid contents of His-tag-purified β-galactosidase and human urocortin, which were synthesized by Escherichia coli grown in controlled synthetic media. After acidic hydrolysis for various times at 110°C, samples were derivatized with 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole (NBD-F) and separated on a reverse-phase column followed by a chiral column into d- and l-enantiomers. The contents of d-enantiomers of Ala, Leu, Phe, Val, Asp, and Glu were determined by plotting index d/(d + l) against the incubation time for hydrolysis and extrapolating the linear regression line to 0 h to eliminate the effect of racemization of amino acids during the incubation. Significant contents of d-amino acids were reproducibly detected, the d-amino acid profile being specific to an individual protein. This finding indicated the likelihood that d-amino acids are in fact present in the purified proteins. On the other hand, the d-amino acid contents of proteins were hardly influenced by the addition of d- or l-amino acids to the cultivation medium, whereas intracellular free d-amino acids sensitively varied according to the extracellular conditions. The origin of these d-amino acids detected in proteins was discussed.     

Optimization of Biotransformation of l-Tyrosine to l-DOPA by Yarrowia lipolytica-NCIM 3472 Using Response Surface Methodology

l-DOPA (3,4-dihydroxyphenyl-l-alanine) is the most widely used drug for treatment of Parkinson’s disease. In this study Yarrowia lipolytica-NCIM 3472 biomass was used for transformation of l-tyrosine to l-DOPA. The process parameters were optimized using response surface methodology (RSM). The optimum values of the tested variables for the production of l-DOPA were: pH 7.31, temperature 42.9 °C, 2.31 g l^?1 cell mass and 1.488 g l^?1 l-tyrosine. The highest yield obtained with these optimum parameters along with recycling of the cells was 4.091 g l^?1. This optimization of process parameters using RSM resulted in 4.609-fold increase in the l-DOPA production. The statistical analysis showed that the model was significant. Also coefficient of determination (R²) was 0.9758, indicating a good agreement between the experimental and predicted values of l-DOPA production. The highest tyrosinase activity observed was 7,028 U mg^?1 tyrosine. l-DOPA production was confirmed by HPTLC and HPLC analysis. Thus, RSM approach effectively enhanced the potential of Y. lipolytica-NCIM 3472 as an alternative source to produce l-DOPA.     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

Improved Protease Stability of the Antimicrobial Peptide Pin2 Substituted with d-Amino Acids

Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a d-diastereomer of Pandinin 2, d-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of l- and d-Pin2 were to some extent similar, an important reduction in d-Pin2 hemolytic activity (30–40 %) was achieved compared to that of l-Pin2 over human erythrocytes. Furthermore, d-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of l-Pin2. Nevertheless, the antimicrobial activity of d-Pin2 was equally effective as that of l-Pin2 in microdilution assays. Yet, when d- and l-Pin2 were incubated with trypsin, elastase and whole human serum, only d-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, l- and d-Pin2 maintained similar peptide stability. Finally, when l- and d-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, d-Pin2 kept its antibiotic activity while l-Pin2 was not effective.     

d-Lactic acid biosynthesis from biomass-derived sugars via Lactobacillus delbrueckii fermentation

Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly l-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-l and d-lactic acid and has a higher melting temperature. To date, several studies have explored the production of l-lactic acid, but information on biosynthesis of d-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of d-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to d-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L^?1 of d-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g^?1 and 1.01 g L^?1 h^?1, respectively. Luedeking–Piret model described the mixed growth-associated production of d-lactic acid with a maximum specific growth rate 0.2 h^?1 and product formation rate 0.026 h^?1, obtained for this strain. The efficient synthesis of d-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.