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.     

Utilization and transport of l-arabinose by non-Saccharomyces yeasts

l-Arabinose is one of the sugars found in hemicellulose, a major component of plant cell walls. The ability to convert l-arabinose to ethanol would improve the economics of biomass to ethanol fermentations. One of the limitations for l-arabinose fermentation in the current engineered Saccharomyces cerevisiae strains is poor transport of the sugar. To better understand l-arabinose transport and use in yeasts and to identify a source for efficient l-arabinose transporters, 165 non-Saccharomyces yeast strains were studied. These yeast strains were arranged into six groups based on the minimum time required to utilize 20 g/L of l-arabinose. Initial transport rates of l-arabinose were determined for several species and a more comprehensive transport study was done in four selected species. Detailed transport kinetics in Arxula adeninivorans suggested both low and high affinity components while Debaryomyces hansenii var. fabryii, Kluyveromyces marxianus and Pichia guilliermondii possessed a single component, high affinity active transport systems.     

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.     

Enantiomeric differentiation of amino acids by a chiral crown ether derived fromd-mannose studied by the liquid membrane technique

Chiral 18-crown-6 incorporating ad-mannopyranoside unit displays noticeable enantioselectivity in the recognition of amino acids and their sodium and potassium salts in transport experiments across a liquid membrane containing the carrier.d-phenylalanine andd-phenylglycine were transported faster than their correspondingl-enantiomers, whereas the enantioselectivity was reversed with tryptophan.     

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.     

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.     

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.     

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.     

Substrate selectivity of Gluconobacter oxydans for production of 2,5-diketo-D-gluconic acid and synthesis of 2-keto-L-gulonic acid in a multienzyme system

Substrate selectivity of Gluconobacter oxydans (ATCC 9937) for 2,5-diketo-d-gluconic acid (2,5-DKG) production was investigated with glucose, gluconic acid, and gluconolactone in different concentrations using a resting-cell system. The results show that gluconic acid was utilized favorably by G. oxydans as substrate to produce 2,5-DKG. The strain was coupled with glucose dehydrogenase (GDH) and 2,5-DKG reductase for synthesis of 2-keto-l-gulonic acid (2-KLG), a direct precursor of l-ascorbic acid, from glucose. NADP and NADPH were regenerated between GDH and 2,5-DKG reductase. The mole yield of 2-KLG of this multienzyme system was 16.8%. There are three advantages for using the resting cells of G. oxydans to connect GDH with 2,5-DKG reductase for production of 2-KLG: gluconate produced by GDH may immediately be transformed into 2,5-DKG so that a series of problems generally caused by the accumulation of gluconate would be avoided; 2,5-DKG is supplied directly and continuously for 2,5-DKG reductase, so it is unnecessary to take special measures to deal with this unstable substrate as it was in Sonoyama’s tandem fermentation process; and NADP(H) was regenerated within the system without any other components or systems.     

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.     

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.

     

Macroligandd-alanyl-d-alanine-dextran for vancomycin purification

Water soluble macroligands for vancomycin purification in affinity ultrafiltration have been prepared by coupling the ligandd-alanyl-d-alanine to dextran activated by tosyl chloride, carbonyldiimidazole, and chloroformate, respectively. Centrifugal ultrafiltration has been used to study the equilibrium binding of vancomycin for the macroligands. The affinity binding can be described as Langmuir type adsorption and is strongly affected by temperature. The binding between vancomycin and macroligand is an unusual endothermic process that binding capacity of macroligand increases with temperature. Vancomycin has also been successfully purified from fermentation liquor using the macroligand in a centrifugal ultrafiltration device.     

Purification and characterization of an extracellular α-l-arabinofuranosidase from Fusarium oxysporum

An α-l-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-l-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the α-l-arabinofuranosidase and an endo-(1 →4)-β-d-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.     

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.     

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.     

Electrophoretic extraction and analysis of DNA from chitosan or poly-l-lysine-coated alginate beads

Alginate beads containing entrapped DNA were produced using both external and internal calcium sources, and coated with chitosan or poly-l-lysine membranes. The beads were assayed with DNase nuclease to determine formulation conditions offering the highest level of DNA protection fromnucleic acid hydrolysis, simulating gastrointestinal exposure. A method was developed to extract and assay intracapsular DNA through a modified agarose electrophoresis system. Both external and internally gelled beads were permeable to DNase (M_w=31 kDa), indicated by the absence of DNA after nuclease exposure. At low levels of DNase exposure, coated high guluronic content alginate beads offered a higher level of DNA protection compared with coated beads with low guluronic alginate. No apparent correlation was found with chitosan membrane molecular weight and degree of deacetylation; however, increasing poly-l-lysine molecular weight appeared to increase DNase exclusion from beads. At elevated levels of DNase exposure, DNA hydrolysis was evident within all coated beads with the exception of those coated with the highest molecular weight poly-l-lysine (M_w=197.1 kDa), which provided almost total nuclease protection. Optimal combination then for DNA protection from nucleases is a high guluronic alginate core, coated with high molecular weight poly-l-lysine.     

Poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactide) networks with tailored water sorption

A poly(l-lactide) diol was obtained through ring opening polymerization of l-lactide, using 1,6 hexanediol and tin(II) 2 ethylhexanoate as a catalyst. In the second step, the poly(l-lactide) macromer (mLA) was obtained by the reaction of poly(l-lactide) diol with methacrylic anhydride. The effective incorporation of the polymerizable end groups was assessed by Fourier transform infrared spectroscopy and nuclear magnetic resonance (¹H NMR). Besides, poly(l-lactide) networks (pmLA) were prepared by photopolymerization of mLA. Further, the macromer was copolymerized with 2-hydroxyethyl acrylate seeking to tailor the hydrophilicity of the system. A set of hydrophilic copolymer networks were obtained. The phase microstructure of the new system and the network architecture was investigated by differential scanning calorimetry, infrared spectroscopy, dynamic mechanical spectroscopy, thermogravimetry, and water sorption studies.     

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).     

Production of l-DOPA by tyrosinase immobilized on modified polystyrene

Mushroom tyrosinase was immobilized on modified polystyrene—polyaminostyrene (PSNH) and polymethylchloridestyrene (PSCL)—to produce l-DOPA from l-tyrosine. Glutaraldehyde was used as an activating agent for the PSNH to immobilize the tyrosinase and 10% (w/v) glutaraldehyde was optimal in conferring the highest specific activity (11.96 U/g) to the PSNH. Methylchloride on the PSCL was directly linked with the tyrosinase, and 1.5 mmol of Cl/g was optimal in attaining the specific activity of 17.0 U/g. The temperature and optimal acidity were, respectively, 60°C and pH 5.5 for the PSNH, and 70°C and pH 3.0 for the PSCL. In a 50-mL batch reactor working over 36 h, the l-DOPA production rate at 30°C was 1.44 mg/(L·h) for the PSNH and 2.33 mg/(L·h) for the PSCL. The production rate over 36 h was 3.86 mg/(L·h) for the PSNH at 60°C and 5.54 mg/(L·h) for the PSCL at 70°C. Both of the immobilized enzymes showed a remarkable stability with almost no change in activity after being stored wet. The operational stability study indicated a 22.4% reduction in l-DOPA production for the PSNH and an 8.63% reduction for the PSCL over seven runs (each run was for 144h at 30°C) when the immobilized enzymes were used under turnover conditions. The immobilized tyrosinase was more stable on the PSCL than on the PSNH.