Regulation of lactate production and utilization in rat tumors in vivo

These experiments were performed to determine the factor(s) that regulate lactic acid production and utilization by rat tumors in vivo. Arteriovenous differences for glucose and lactic, pyruvic, 3-OH-butyric, and acetoacetic acids were measured across "tissue-isolated" Walker 256 sarcocarcinomas and Morris 5123C hepatomas in fasted rats anesthetized with sodium pentobarbital. Twenty-six per cent of the sarcocarcinomas (n = 53) and 48% of the hepatomas (n = 29) utilized blood lactic acid. The remainder released lactic acid into the venous blood. The steady-state rate of glucose consumption was similar in both lactate-producing and lactate-utilizing tumors. The range of lactate concentrations in the blood leaving the tumors was narrower than the range of lactate concentrations in the blood entering the tumors. This difference was caused by tumor lactic acid production at low arterial lactate concentrations and tumor lactic acid utilization at high arterial lactate concentrations. Individual tumors changed from lactic acid production to lactic acid utilization in a matter of minutes in response to an increase in the arterial lactic acid concentration. Mean lactic plus pyruvic acid concentrations and lactic/pyruvic acid ratios in the tumor venous blood were 2.15 +/- 0.22 and 23.4 +/- 3.7 mM, respectively, for Walker sarcocarcinoma 256 (n = 18) and 1.28 +/- 0.13 and 48.1 +/- 5.1 mM, respectively, for hepatoma 5123C (n = 11). The results suggest: that a steady-state lactic plus pyruvic acid concentration and lactic/pyruvic acid ratio are maintained in the tumor cell cytoplasm by the active glycolytic pathway and by lactic acid dehydrogenase; that the tumor intracellular concentrations equilibrate with the arterial blood and that the tumor steady state is expressed in the tumor venous blood; and that tumor lactic acid production or utilization results from the equilibration between the variable arterial lactic acid concentration and the more constant tumor intracellular steady-state lactic acid concentration. Since the arterial lactate concentration may be less than, greater than, or equal to the intracellular steady-state concentration, an individual tumor may produce, utilize or neither produce nor utilize lactic acid.     

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H₂) are also produced. The effect of hydrogenase inhibitors (H₂, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N₂ to H₂, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H₂ or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD⁺ from NADH by H₂, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H₂ production for the conversion of synthetic gases to chemicals.     

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.     

生物基乳酸生物转化研究进展

乳酸的发酵生产技术已取得了长足的进步,作为一种重要生物基化学品,乳酸除了可用于食品工业及生产聚乳酸外,亦可作为一种重要的平台化合物,用于生产丙烯酸、丙酮酸、1,2-丙二醇、乳酸酯等。文中重点综述了以生物基乳酸为原料经脱水、脱氢、还原及酯化反应生产乳酸衍生物的生物转化工艺,对该领域的发展趋势进行了展望。     

Structure of Cyl-2, a Novel Cyclotetrapeptide from Cylindrocladium scoparium

Fermentative production of pyruvic acid by yeasts was studied using extracts from citrus natsudaidai peel as a carbon source. Many yeasts showed good growth. Of these yeasts, Debaryomyces coudertii IFO 1381 produced pyruvic acid at high yield. Pretreatment of the peel extract with Amberlite IR-120B (Na⁺) led to increased production of pyruvic acid. Under optimum conditions, the accumulation of pyruvic acid reached a maximum of 970 mg/100ml at 48 hr-fermentation. The pyruvic acid from the fermentation broth was identified with lactic acid dehydrogenase and by comparisons of properties of its 2,4-dinitrophenylhydrazone with those of authentic pyruvic acid in paper chromatography, IR spectrometry and elemental analysis.     

Immobilization and kinetics of lactate dehydrogenase at a rotating nylon disk

Lactate dehydrogenase (LDH) was covalently attached to an impervious nylon surface by an improved technique. The procedure allowed the kinetics of the rotating enzyme disk reactor to be successfully explored. This enzyme-disk configuration has potential applications in assays for lactic acid or pyruvic acid in fluids of biological importance (e.g., urine). In order to evaluate and understand the physics and chemistry underlying the kinetics of the heterogeneous biocatalyst, a mathematical model based on the von Karman-Levich theories of rotating electrodes, was developed. It applied well to LDH attached to a disk, under variable NADH concentrations and fixed pyruvic acid. The new theory, leads to the conclusion that the apparent Michaelis constant K(m)(app), varies linearly with f(-1/2), where f is the speed of rotation of the disk. Extrapolation of f(-1/2) to zero gives the Michaelis-Menten constant, K(m), corresponding to the diffusion-free behavior. With immobilized LDH, the diffusion-free K(m) for NADH obtained at 25 degrees C, in phosphate buffer (pH 7.5) using the extrapolation method was 84 muM. This value was in good agreement with the previously published value of 87 muM, obtained with LDH attached to the inner surface of a nylon tubing. However, when compared to the K(m) for a free enzyme system, the 84 muM was about nine times larger, indicating an inherent reduction in the activity of the bound LDH. Since, at extrapolated infinite rotation speeds, diffusion effects were assumed eliminated, the drop in the activity was thought to be due to sterric hinderances imposed on the substrate NADH as a result of having LDH bound to another polymer.     

Effect of oxygenation and sulfate concentrations on pyruvate and lactate formation inKlebsiella oxytoca ZS growing in chemostat cultures

Klebsiella oxytoca ZS fermented glucose to ethanol and lactic, formic, and acetic acids, but, in contrast to many strains, accumulates pyruvic and acetic acids as the principal end products in aerobic growth conditions. This strain was grown in sulfate-limited chemostat at a fixed low dilution rate (D=0.033 h^–1) with glucose present in excess. When oxygen was supplied at a high level, pyruvate and acetate were produced, and the ratio NADH/NAD⁺ was low (0.04) while the internal pyruvate concentration increased to 100 mol (g dry wt)^–1. A shortage of oxygen supply was accompanied by lactate production, an increase of the ratio NADH/NAD⁺ (0.53), and an undetectable level in internal pyruvate concentration. The observed changes in LDH activity found in vitro in extracts of the cells are not strictly related to those found in vivo. In fact, the specific activity of LDH was essentially stable at 30% of dissolved oxygen tension (d.o.t.) and decreased slightly at 60% of d.o.t., whereas specific lactic acid production decreased rapidly. The in vitro LDH activity was strongly affected by the NADH/NAD⁺ ratio.     

Efficient production of L-Lactic acid by metabolically engineered Saccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene

We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2microm plasmid-based vectors and two genome-integrated strains.     

Recovery of pyruvic acid from biotransformation solutions

The aim of this investigation was to separate pyruvic acid of biotransformation solutions from lactic acid through complex extraction. For this purpose, complex extraction was investigated from model solutions. Tri-n-octanylamine (TOA) was used as the extractant. The effects of various diluents, the stoichiometry of pyruvic acid to TOA, and the initial pH of the aqueous phase on the extraction process were investigated in this study. The effects of sodium hydroxide (NaOH) and trimethylamine (TMA) on the back extraction process were also studied, respectively. The optimal conditions attained from the model solutions proved efficient on the biotransformation solutions of different concentrations. A total recovery of 71–82% of pyruvic acid was obtained, whereas 89–92% of lactic acid was removed. The purity of pyruvic acid reached 97% after the removal of TMA by a simple distillation.     

Lactic acid production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing a <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactate dehydrogenase gene

This work demonstrates the first example of a fungal lactate dehydrogenase (LDH) expressed in yeast. A L(+)-LDH gene, ldhA, from the filamentous fungus Rhizopus oryzae was modified to be expressed under control of the Saccharomyces cerevisiae adh1 promoter and terminator and then placed in a 2μ-containing yeast-replicating plasmid. The resulting construct, pLdhA68X, was transformed and tested by fermentation analyses in haploid and diploid yeast containing similar genetic backgrounds. Both recombinant strains utilized 92 g glucose/l in approximately 30 h. The diploid isolate accumulated approximately 40% more lactic acid with a final concentration of 38 g lactic acid/l and a yield of 0.44 g lactic acid/g glucose. The optimal pH for lactic acid production by the diploid strain was pH 5. LDH activity in this strain remained relatively constant at 1.5 units/mg protein throughout the fermentation. The majority of carbon was still diverted to the ethanol fermentation pathway, as indicated by ethanol yields between 0.25–0.33 g/g glucose. S. cerevisiae mutants impaired in ethanol production were transformed with pLdhA68X in an attempt to increase the lactic acid yield by minimizing the conversion of pyruvate to ethanol. Mutants with diminished pyruvate decarboxylase activity and mutants with disrupted alcohol dehydrogenase activity did result in transformants with diminished ethanol production. However, the efficiency of lactic acid production also decreased. Electronic Publication     

Cloning,characterization and insertional inactivation of theLactobacillus helveticus D(−) lactate dehydrogenase gene

A plasmid, designated pSUW100, encoding the D(-)lactate dehydrogenase [D(-)-LDH; NAD⁺ oxidoreductase, EC 1.1.1.28] fromLactobacillus helveticus CNRZ32 was identified from a genomic library by complementation ofEscherichia coli FMJ39. The D(-)LDH gene was localized by Tn5 mutagenesis and subcloning to a 1.4-kb region of pSUW100. A 2-kbDraI fragment of pSUW100 encoding D(-)LDH activity was subcloned and its nucleotide sequence determined. Analysis of this sequence identified a putative 1,014-bp D(-) LDH open reading frame that encodes a polypeptide of 337 amino acid residues with a deduced molecular mass of 38 kDa. The distribution of homology to the CNRZ32 D(-)LDH gene in several lactic acid bacteria was determined by Southern hybridization using an internal fragment of the D(-)LDH gene as a probe. Hybridization was detected in leuconostocs and pediococci but not in lactococci orLactobacillus casei. An integration plasmid was constructed from pSA3 and a 0.60-kb internal fragment of the D(-)LDH gene. This plasmid was used to construct a D(-)LDH-negative derivative ofL. helveticus CNRZ 32 by gene disruption; this derivative was determined as producing only L(+)lactic acid. No significant difference in growth or total lactic acid production was observed between CNRZ32 and its D(-)LDH mutant.     

Encapsulation of lactate dehydrogenase in carbon nanotube doped alginate–chitosan capsules

Alginate–chitosan shell–core (AC) capsules doped with carbon nanotubes (CNTs) were prepared for lactate dehydrogenase (LDH, EC 1.1.1.27) encapsulation to convert pyruvic acid to lactic acid coupling with the oxidation of NADH to NAD⁺. LDH was entrapped within the liquid core of the capsules and the CNTs were incorporated in the alginate or chitosan matrices or both. The physical properties of the capsules and the immobilized LDH activity were investigated. The AC capsules doped with CNTs showed better mechanical strength than that without CNTs. The LDH loading efficiency of the AC capsules with CNTs (10 mg/mL) doped in both the shell and the core was 30.7% higher than that without CNTs. The optimal pH value for the bioconversion catalyzed by immobilized LDH was 7.0, lower than that by free LDH (7.5). The optimal temperature was 35 °C for both immobilized and free LDH. Operational stability of the immobilized LDH was greatly improved by doping CNTs in AC capsules. The results showed that this method was efficient for enzyme encapsulation in the biotechnology applications.     

Lactic Acid Utilization by the Cutaneous Micrococcaceae

Human cutaneous staphylococci and micrococci utilized lactic acid as an energy source on a minimal medium. Propionic acid was not utilized, but l(+)-lactic acid and pyruvic acid could replace ld-lactic acid as a substrate. Selected strains of cocci were inhibited more by the l(+) and d(-) forms of lactic acid than the balanced ld form, particularly at pH 5.6. With proper dilution of substrate, lactic acid was utilized by selected strains in the presence of 10 mug of oleic and palmitic acids per ml.     

Eperythrozoon ovis: the difference in carbohydrate metabolism between infected and uninfected sheep erythrocytes.

The glucose utilization lactic and pyruvic acid production and oxygen uptake of normal and Eperythrozoon ovis infected sheep erythrocytes were measured under aerobic conditions. Infected cells showed marded increases in both glucose utilization and acid production as compared with controls. Uninfected erythocyte samples which included a percentage of reticulocytes comparable to that found in E. ovis infection showed no apparent difference in glucose utilization and lactic acid production form the normal control erythrocytes, although considerable increases in the oxygen uptake were recorded.     

Biotechnological routes based on lactic acid production from biomass

Lactic acid, the most important hydroxycarboxylic acid, is now commercially produced by the fermentation of sugars present in biomass. In addition to its use in the synthesis of biodegradable polymers, lactic acid can be regarded as a feedstock for the green chemistry of the future. Different potentially useful chemicals such as pyruvic acid, acrylic acid, 1,2-propanediol, and lactate ester can be produced from lactic acid via chemical and biotechnological routes. Here, we reviewed the current status of the production of potentially valuable chemicals from lactic acid via biotechnological routes. Although some of the reactions described in this review article are still not applicable at current stage, due to their “greener” properties, biotechnological processes for the production of lactic acid derivatives might replace the chemical routes in the future.     

Conversion of levulinic acid to 2-butanone by acetoacetate decarboxylase from Clostridium acetobutylicum

In this study, a novel system for synthesis of 2-butanone from levulinic acid (γ-keto-acid) via an enzymatic reaction was developed. Acetoacetate decarboxylase (AADC; E.C. 4.1.1.4) from Clostridium acetobutylicum was selected as a biocatalyst for decarboxylation of levulinic acid. The purified recombinant AADC from Escherichia coli successfully converted levulinic acid to 2-butanone with a conversion yield of 8.4–90.3 % depending on the amount of AADC under optimum conditions (30 °C and pH 5.0) despite that acetoacetate, a β-keto-acid, is a natural substrate of AADC. In order to improve the catalytic efficiency, an AADC-mediator system was tested using methyl viologen, methylene blue, azure B, zinc ion, and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as mediators. Among them, methyl viologen showed the best performance, increasing the conversion yield up to 6.7-fold in comparison to that without methyl viologen. The results in this study are significant in the development of a renewable method for the synthesis of 2-butanone from biomass-derived chemical, levulinic acid, through enzymatic decarboxylation.     

Effects of methyl [5 [[4-(2-pyridinyl)-1-piperazinyl]carbonyl]-1H-benzimidazol-2-yl] carbamate on energy metabolism of Ancylostoma ceylanicum and Nippostrongylus brasiliensis

Effects of methyl [5[[4-(2-pyridinyl)-1-piperazinyl] carbonyl] 1H-benzimidazol-2-yl] carbamate (CDRI Comp. 81-470) and mebendazole on the energy metabolism of A. ceylanicum and N. brasiliensis were compared. At 10 and 50 microM concentration both compounds inhibited glucose uptake and its conversion into metabolic endproducts. The shift towards the increased production of lactic acid appeared to be the result of inhibition of PEP carboxykinase and increase in LDH activity. The compounds also caused significant inhibition of ATP production in mitochondria.