Determination of serum <Emphasis Type="SmallCaps">d</Emphasis>-lactic and <Emphasis Type="SmallCaps">l</Emphasis>-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor () of 1.32 and a resolution (R_s) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5–101.2 and 89.0–100.7%, and the intra-day and inter-day precisions were 0.3–1.2 and 0.4–4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients (n=31) as compared with normal subjects (n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.     

Determination of the Enantiomeric Purity of l-Carnitine and Acetyl-l-Carnitine by Chiral Liquid Chromatography

A chiral liquid chromatographic method for determination of the enantiomeric purity of both l-carnitine and acetyl-l-carnitine is described. Separation of the enantiomers of dl-carnitine and acetyl-dl-carnitine was achieved on a commercial chiral column (Chiralcel OD-R) after derivatization with (alpha-bromo)methyl phenyl ketone. Introduction of this lipophilic UV chromophoric group to the carnitine and acetylcarnitine molecules improved their retention, resolution, and UV detection. The mobile phase was 74:26 (v/v) 0.5 mol L^-1 sodium perchlorate–acetonitrile, pH 3.8, and the flow rate was 0.4 mL min^-1. Detection was performed at 235 nm. The method is selective and reliable for determination of the enantiomeric purity of bulk drug substances l-carnitine and acetyl-l-carnitine.     

Characterization and complementation of a Pichia stipitis mutant unable to grow on d-xylose or l-arabinose

Pichia stipitis CBS 6054 will grow on d-xylose, d-arabinose, and l-arabinose. d-Xylose and l-arabinose are abundant in seed hulls of maize, and their utilization is important in processing grain residues. To elucidate the degradation pathway for l-arabinose, we obtained a mutant, FPL-MY30, that was unable to grow on d-xylose and l-arabinose but that could grow on d-arabinitol. Activity assays of oxidoreductase and pentulokinase enzymes involved in d-xylose, d-arabinose, and l-arabinose pathways indicated that FPL-MY30 is deficient in d-xylitol dehydrogenase (D-XDH), d- and l-arabinitol dehydrogenases, and d-ribitol dehydrogenase. Transforming FPL-MY30 with a gene for xylitol dehydrogenase (PsXYL2), which was cloned from CBS 6054 (Gen Bank AF127801), restored the D-XDH activity and the capacity for FPL-MY30 to grow on l-arabinose. This suggested that FPL-MY30 is critically deficient in XYL2 and that the d-xylose and l-arabinose metabolic pathways have xylitolas a common intermediate. The capacity for FPL-MY30 to grow on d-arabinitol could proceed through d-ribulose.     

Preparation of optical active aliphatic <Emphasis Type="Italic">α</Emphasis>-amino acid from fatty acid: synthesis of <Emphasis Type="SmallCaps">l</Emphasis>-norvaline and <Emphasis Type="SmallCaps">d</Emphasis>-norvaline

A new synthesis of l-norvaline is described. Using valeric acid as the raw material, α-brominevaleryl chloride was achieved by acyl chlorination and α-position bromination in one-pot with the yield of 84.7%. The yields of the following ammoniation of α-brominevaleryl chloride and resolution of dl-norvaline were 88.7% and 26.7%, respectively. d-Norvaline was also obtained in a similar method from the waste resolution solution. Other optical active amino acids, valine, α-aminobutyric acid and alanine were also synthesized in similar ways.     

Quantitative determination of protein of bacterial origin on the basis ofd-aspartic acid andd-glutamic acid content

Summary In recent decades several methods have been developed for determination of the proportion of nitrogen-containing substances passed from the rumen into the abomasum, or small intestine, which are of microbial origin. Recently, when examining thed-amino acid content of foodstuffs, particularly milk and milk products, it was observed that, in addition tod-alanine (d-Ala,d-glutamic acid (d-Glu) andd-aspartic acid (d-Asp) can also be detected in similar quantities, primarily in products which have links with bacterial activity. This gave rise to the idea of examining the diaminopimelic acid (DAPA),d-Glu, andd-Asp content of bacteria extracted from the rumen of cattle, and that of chyme from the same cattle, to establish whetherd-Asp andd-Glu can be used to estimate protein of bacterial origin. The investigations performed have provided evidence that bothd-Asp andd-Glu might be appropriate for determination of protein of bacterial origin. The results obtained using these two bacterial markers (d-Asp andd-Glu) proved to the approximately 10% lower than those obtained using DAPA; this was not because of to error attributable to the new markers but rather to the unreliability of determination using DAPA Analyses performed on samples of known bacterial protein content indicate thatd-Asp andd-Glu gave almost identical results for bacterial protein content which were very close to the theoretical (calculated) values. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001.     

Steady-state measurements of lactic acid production in a wild-type and a putative d-lactic acid dehydrogenase-negative mutant of zymomonas mobilis

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of d- and l-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of dl-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, d-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. d-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.     

The electrochemical polymerization of <Emphasis Type="Italic">o</Emphasis>-phenylenediamine on <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine functionalized glassy carbon electrode and its application

The polymerization of o-phenylenediamine (OPD) on l-tyrosine (Tyr) functionalized glassy carbon electrode (GCE) and its electro-catalytic oxidation towards ascorbic acid (AA) had been studied in this report. l-Tyrosine was first covalently grafted on GCE surface via electrochemical oxidation, which was followed by the electrochemical polymerization of OPD on the l-tyrosine functionalized GCE. Then, the poly(o-phenylenediamine)/l-tyrosine composite film modified GCE (POPD-Tyr/GCE) was obtained. X-ray photo-electron spectroscopy (XPS), field emission scanning electron microscope (SEM), and electrochemical techniques have been used to characterize the grafting of l-tyrosine and the polymerization and morphology of OPD film on GCE surface. Due to the doping of the carboxylic functionalities in l-tyrosine molecules, the POPD film showed good redox activity in neutral medium, and thus, the POPD-Tyr/GCE exhibited excellent electrocatalytic response to AA in 0.1 mol l⁻¹ phosphate buffer solution (PBS, pH 6.8). The anode peak potential of AA shifted from 0.58 V at GCE to 0.35 V at POPD-Tyr/GCE with a greatly enhanced current response. A linear calibration graph was obtained over the AA concentration range of 2.5 × 10⁻⁴–1.5 × 10^–3 mol l⁻¹ with a correlation coefficient of 0.9998. The detection limit (3δ) for AA was 9.2 × 10⁻⁵ mol l⁻¹. The modified electrode showed good stability and reproducibility and had been used for the determination of AA content in vitamin C tablet with satisfactory results.     

Fabrication of modified TiO2 nanoparticle carbon paste electrode for simultaneous determination of dopamine, uric acid, and l-cysteine

A carbon paste electrode, modified with 2, 2′-[1,7-hepthandiylbis(nitriloethylidyne)]-bis-hydroquinone and TiO₂ nanoparticles, was used for the simultaneous determination of dopamine (DA), uric acid (UA), and l-cysteine. The study was carried out by using cyclic voltammetry, chronoamperometry, and square wave voltammetry (SWV) techniques. Some kinetic parameters such as the electron transfer coefficient (α) and heterogeneous rate constant (k_s) were also determined for the DA oxidation. A dynamic range of 8.0–1400 μM, with the detection limit of 8.4 × 10⁻⁷ M for DA, was obtained using SWV (pH = 7.0). The prepared electrode was successfully applied for the determination of DA, UA, and l-cysteine in real samples.     

Metabolic engineering of Saccharomyces cerevisiae for efficient production of pure l ?(+)?lactic acid

We developed a metabolically engineered Saccharomyces cerevisiae, which produces optically pure l-lactic acid efficiently using cane juice-based medium. In this recombinant, the coding region of pyruvate decarboxylase (PDC)1 was completely deleted, and six copies of the bovine l-lactate dehydrogenase (l-LDH) genes were introduced on the genome under the control of the PDC1 promoter. To confirm optically pure lactate production in lowcost medium, cane juice-based medium was used in fermentation with neutralizing conditions. l-lactate production reached 122 g/L, with 61% of sugar being transformed into l-lactate finally. The optical purity of this l-lactate, that affects the physical characteristics of poly-l-lactic acid, was extremely high, 99.9% or over. These two authors contributed equally to this work.     

l-DOPA production by immobilized tyrosinase

The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U _C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U _IMO). The optimal conditions were t=24 h, G=2% (v/v), and U _C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U _IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h).     

Presence and origin of large amounts of d-proline in the urine of mutant mice lacking d-amino acid oxidase activity

Using a column-switching HPLC system combining a micro-ODS column and a chiral column, the amounts of d-proline (d-Pro) have been determined in 18 tissues, plasma and urine of mice. To avoid the enzymatic degradation of d-amino acids in vivo, a mutant mouse strain lacking d-amino acid oxidase activity (ddY/DAO⁻ mouse) was used. In the brain, relatively large amounts of d-Pro were observed in the anterior pituitary, posterior pituitary and pineal glands. In the peripheral tissues, the amounts of d-Pro were high in the pancreas and kidney. Above all, it is surprising that the ddY/DAO⁻ mice excreted large amounts of d-Pro in their urine (433 nmol/mL, 20 times that of l-Pro). The origin of d-Pro has also been investigated. By comparing germ-free mice and gnotobiotic mice, intestinal bacteria were shown to have no effect on the urinary d-Pro amount. Concerning the dietary origin, a notable amount of d-Pro was still excreted in the urine after starvation for 4 days, suggesting that some of the d-Pro is produced in the mice. Age-dependent changes in the urinary d-Pro amount have also been investigated from the postnatal 1st month up to 12 months, and ddY/DAO⁻ mice were found to excrete large amounts of d-Pro in the urine constantly throughout their lives. Kenji Hamase is Associate Professor in the Department of Bioanalytical Chemistry, Graduate School of Pharmaceutical Sciences at Kyushu University. His current research interests focus on the development of analytical methods for d-amino acids and the study of their physiological functions and diagnostic values. He received the Japanese Society for Analytical Chemistry Award for Young Scientists in 2003, and the PSJ Award for Young Scientists in 2006.     

Inexpensive isolation of beta-D-glucopyranosidase from alpha-L-arabinofuranosidase,alpha-L-rhamnopyranosidase,and o-acetylesterase

β-d-Glucopyranosidase (βG, EC 3.2.1.21) has been isolated from some collateral activities, α-l-arabinofuranosidase (Ara, EC3.2.1.55), α-l-rhamnopyranosidase (Rha, EC 3.2.1.40), and o-acetylesterase (Est, EC 3.1.1.53), using a commercial enzyme preparation and a simple method economically sustainable for the food industry. The procedure comprises precipitation of extraneous substances by adding ethanol and CaCl₂, ultrafiltration, and adsorption, first on bentonite and then on chitosan. The results obtained were the complete isolation of βG from the above-mentioned activities, a drastic reduction in extraneous compounds, such as brown substances and polysaccharides, and a slight increase in purification.     

Fluorescent sensing layer for the determination of<Emphasis Type="SmallCaps"> L</Emphasis>-malic acid in wine

An enzymatic method for determining L-malic acid in wine based on an L-malate sensing layer with nicotinamide adenine dinucleotide (NAD⁺), L-malate dehydrogenase (L-MDH) and diaphorase (DI), immobilized by sol-gel technology, was constructed and evaluated. The sol-gel glass was prepared with tetramethoxysilane (TMOS), water and HCl. L-MDH catalyzes the reaction between L-malate and NAD⁺, producing NADH, whose fluorescence (λ _exc = 340 nm, λ _em = 430 nm) could be directly related to the amount of L-malate. NADH is converted to NAD⁺ by applying hexacyanoferrate(III) as oxidant in the presence of DI. Some parameters affecting sol-gel encapsulation and the pH of the enzymatic reaction were studied. The sensing layer has a dynamic range of 0.1–1.0 g/L of L-malate and a long-term storage stability of 25 days. It exhibits acceptable reproducibility [s _r(%)≈10] and allows six regenerations. The content of L-malic acid was determined for different types of wine, and polyvinylpolypyrrolidone (PVPP) was used as a bleaching agent with red wine. The results obtained for the wine samples using the sensing layer are comparable to those obtained from a reference method based on UV-vis molecular absorption spectrometry, if the matrix effect is corrected for.     

Production of optically pure d-lactic acid by Nannochlorum sp. 26A4

Microalgae were screened from seawater for greenhouse gas CO₂ fixation and d-lactic acid production by self-fermentation and tested for their growth rate, starch content, and conversion rate from starch into d-lactic acid. More than 300 strains were isolated, and some of them were found to have suitable properties for this purpose. One of the best strains, Nannochlorum, sp. 26A4, which was isolated from Sakito Island, had a starch content of 40% (dry weight), and a conversion rate from consumed starch into d-lactic acid of 70% in the dark under anaerobic conditions. The produced d-lactic acid showed a high optical purity compared with the conventional one. The proposed new d-lactic acid production system using Nannochlorum sp. 26A4 should also be an effective technology for greenhouse gas CO₂ fixation and/or conversion into industrial raw materials.     

Efficient production of poly-γ-glutamic acid by bacillus subtilis ZJU-7

A strain with high poly-γ-glutamic acid (γ-PGA) production was isolated from fermented bean curd, a traditional Chinese food. The strain was named Bacillus subtilis ZJU-7 according to 16s rDNA sequencing and its taxonomic characters. The culture conditions for γ-PGA production were evaluated. The most suitable carbon and nitrogen sources were sucrose and tryptone, respectively. Exogenous l-glutamic acid was necessary for γ-PGA production, and the production of γ-PGA increased on the addition of l-glutamic acid to the medium. In the medium containing 60 g/L of sucrose, 60 g/L of tryptone, 80 g/L of l-glutamic acid, and 10 g/L of NaCl, the yield of γ-PGA reached 54.4 g/L after cultivation at 37°C for 24h, which was the highest γ-PGA production compared with values reported in the literature. The average molecular mass of γ-PGA produced was about 1.24×10⁶ Daltons. B. subtilis ZJU-7 is genetically stable and can synthesize levan instead of γ-PGA without the addition of l-glutamic acid to the medium.     

Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation

This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Glycosylation of betulin acetates with glycals

Betulin 2-deoxy-α-d-, 2-deoxy-α-l-, and 2,6-dideoxy-α-l-arabino-hexopyranosides were synthesized by acid-catalyzed glycosylation (cationite in the H⁺ form, LiBr) of betulin 3- and 28-monoacetates with glycal acetates. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 531–534, March, 1998.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

l-Lactat-Durchflu?elektrode mit immobilisierter Lactat-Oxidase

Zusammenfassung Eine leicht zu handhabende l-Lactat-Durchflußelektrode mit immobilisierter Lactat-Oxidase wird beschrieben. Sie ist Bestandteil eines dreikanaligen O₂-sensitiv-enzymatischen Meßsystems für l-Lactat, Pyruvat und -d-Glucose. Der l-Lactat-Sensor weist eine schnelle Einstellzeit, geringe Streuung um die errechnete Regressionsgerade und einen für die klinisch-chemische Analytik adäquaten linearen Anzeigebereich auf. Meßkurven werden demonstriert. Auf den Chemismus anderer üblicher elektrochemisch-enzymatischer Meßverfahren von l-Lactat wird hingewiesen und auf die klinische Bedeutung der l-Lactat-Bestimmung Bezug genommen.

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l-lactate flow-through electrode with immobilized lactate oxidase

Summary An l-lactate flow-through electrode with immobilized lactate oxidase is described that is easy to handle. It is part of a three-channel O₂-sensitive enzymatical measuring system for l-lactate, pyruvate and -d glucose. The l-lactate sensor features a short response time, little variation about the regression line computed and a linear indicating range adequate for clinical-chemical analysis. Measurement diagrams are demonstrated. The chemism of other conventional electrochemical-enzymatical measuring methods for l-lactate is mentioned, and reference is made to the clinical importance of l-lactate determination.

     

Effect of a nonmetabolizable analog of fructose-1,6-bisphosphate on glycolysis and ethanol production in strains ofSaccharomyces cerevisiae andEscherichia coli

Fructose-1,6-bisphosphate (F-1,6-P2) is an allosteric activator of two key enzymes of glycolysis: phosphofructokinase and pyruvate kinase. Regulation of glycolysis in a wild-typeSaccharomyces cerevisiae and a recombinantEscherichia coli by a dead-end structural analog of F-1,6-P2 was studied. 2,5-Anhydromannitol (2,5-AM), a structural analog of β-d-fructose, was used. On being taken up by the cells, 2,5-AM was converted into its monophosphate and diphosphate by the enzymes of the glycolytic pathway. The final product, 2,5-anhydromannitol-1,6-bisphosphate, could not be metabolized further and, therefore, accumulated inside the cells. Glucose and fructose were used as substrates. It was found that 2,5-AM at concentrations of 1 mM or less did not have any effect on either substrate consumption or ethanol production. At concentrations of 2,5-AM of 2.5 mM or greater, significant inhibition of both glucose and fructose was observed, with fructose inhibition much more severe. We discuss the possible mechanisms of glycolysis inhibition by 2,5-AM at high concentrations and the regulation of glycolysis by this compound.