产乙醇酸氧化酶的重组毕赤酵母的构建及催化合成乙醛酸的研究

乙醇酸氧化酶（Go）是植物光呼吸途径中的一种关键酶,可以催化乙醇酸生产乙醛酸。从新鲜菠菜叶中提取总RNA,利用RT-PCR技术获得编码GO基因的cDNA片断。通过基因重组将GO基因克隆到载体pA0815中,构建了胞内表达载体pA0815／GO,重组质粒经电转整合至甲醇营养酵母GS115染色体。在混合碳源为10g/L山梨醇和0．5g/L甲醇的培养条件下,细胞的GO酶活达到474IU／g（DCW）。利用该重组毕赤酵母作为催化剂生产乙醛酸,结果表明：在乙醇酸浓度为0．25mol／L,重组酵母湿菌体为10dL,黄素单核苷酸（FMN）浓度为0．01mmol／L,pH8．0,20℃,反应18h后乙醛酸的产率达到51．8％。     

Oxalic acid metabolism and calcium oxalate formation in Lemna minor L.

Abstract Axenic Lemna minor plants, which form numerous calcium oxalate crystals, were exposed to [¹⁴C]-glycolic acid, -glyoxylic acid, -oxalic acid and -ascorbic acid and prepared for microautoradiography by a technique that preserves only insoluble label to determine specifically the pathway leading to oxalic acid used for crystal formation. Label from glycolic, glyoxylic, and oxalic acids was incorporated into crystals. Label from oxalic acid was also found in starch when exposure to label was done in the light but not dark, while plastids specialized for lipid storage were heavily labelled under both conditions. Incorporation of label from glycolic and glyoxylic acids, but not oxalic acid, was inhibited in the presence of the glycolate oxidase inhibitors, αHPMS (2-pyridylhydroxy methanesulphonic acid) and mHBA (methyl 2-hydroxy-3-butynoic acid), and inhibition of labelling was not due to an effect on uptake. These studies show that the glycolate oxidase pathway to oxalic acid is operational in L. minor and that the product is available for crystal formation. Dark-grown plants form almost four times as many crystal cells (idioblasts) as do light-grown plants, indicating crystal formation is not in response to photorespiratory glycolate production. Label from [1-¹⁴C]ascorbic acid was also incorporated into crystals and labelling was inhibited by mHBA, indicating glycolic acid and/or glyoxylic acid are possible intermediates of ascorbic acid catabolism. The effect of nitrogen source on crystal formation was also investigated. Significantly more crystal idioblasts were formed, on a surface area basis, by plants grown on ammonium than by plants grown on nitrate nitrogen. When grown with mixed ammonium and nitrate, an intermediate number of crystal idioblasts were formed.     

Recombinant Hansenula polymorpha as a biocatalyst: coexpression of the spinach glycolate oxidase (GO) and the S. cerevisiae catalase T (CTT1) gene

 The methylotrophic yeast Hansenula polymorpha has been developed as an efficient production system for heterologous proteins. The system offers the possibility to cointegrate heterologous genes in anticipated fixed copy numbers into the chromosome. As a consequence coproduction of different proteins in stoichiometric ratios can be envisaged. This provides options to design this yeast as an industrial biocatalyst in procedures where several enzymes are required for the efficient conversion of a given inexpensive compound into a valuable product. To this end recombinant strains have been engineered with multiple copies of expression cassettes containing the glycolate oxidase (GO) gene from spinach and the catalase T (CTT1) gene from S. cerevisiae. The newly created strains produce high levels of the peroxisomal glycolate oxidase and the cytosolic catalase T. The strains efficiently convert glycolate into glyoxylic acid, oxidizing the added substrate and decomposing the peroxide formed during this reaction into water and oxygen. Received: 31 October 1995/Received last revision: 23 February 1996/Accepted: 4 March 1996     

Glycolate Metabolism in Eurasian Watermilfoil (Myriophyllum spicatum)

The metabolism of glycolate by Eurasian watermilfoil (Myriophyllum spicatum L.), a submersed angiosperm, was studied by feeding radioactive glycolate and glyoxylate and by analysis of glycolate and glycolic acid oxidase. Evidence for operation of the glycolate pathway is given. Glycolic acid oxidase occurs at levels comparable to amounts in species showing photorespiration. This species has a high affinity for CO₂ and a possible mechanism for it is described.     

代谢工程改造大肠杆菌提高乙醇酸产率

乙醇酸(Glycolate)是一种在工业上有多种用途的重要化合物。本研究首先在大肠杆菌MG1655(DE3)中敲除了ldh A(乳酸脱氢酶),获得菌株Mgly1,作为出发菌株。然后通过调节乙醇酸合成途径的关键酶——异柠檬酸裂解酶(ace A)、乙醛酸还原酶(ycd W)、异柠檬酸脱氢酶激酶/磷酸化酶(ace K)的表达水平,得到乙醇酸产率为0.24 g/g葡萄糖(占理论产率的28.2%)。过量表达柠檬酸合成酶(glt A),乙醇酸产率提高到0.326 g/g葡萄糖(占理论产率的38.3%)。然后在Mgly1中敲除了glc B和ace B(苹果酸合成酶),减少了乙醇酸合成的前体乙醛酸的消耗。最终获得的工程菌株Mgly335乙醇酸产率达到0.522 g/g葡萄糖(占理论产率的61.4%)。     

Isolation and characterization of glycolic acid oxidase from human liver.

Glycolic acid oxidase has been isolated from human liver and purified over 3000-fold to a specific activity of 123 U/mg protein by a 5-step procedure. The preparation gave a single protein band on polyacrylamide gel electrophoresis, required flavin mononucleotide for catalytic activity, had a pH optimum between 8.2-8.8 depending on the substrate, and had a molecular weight of 105 000. The enzyme has a broad specificity towards alpha-hydroxy acids. Glycolate (Km = 3.3 . 10(-4) M) was the most effective substrate. The enzyme was stable for several months when stored as an (NH4)2SO4 precipitate or in 15% glycerol. Since glycolate inhibits the oxidation of glyoxylate to oxalate by glycolic acid oxidase, it is suggested that glycolic acid oxidase contributes to the synthesis of oxalate in vivo when the glyoxylate concentration is high and the glycolate concentration is low.     

The pathways of oxalate formation from phenylalanine,tyrosine, tryptophan and ascorbic acid in the rat

The metabolic pathway by which L-[¹⁴C₁]phenylalanine, L-[¹⁴C₁]tyrosine, L-[¹⁴C₁]tryptophan, and L-[¹⁴C₁]ascorbic acid are converted to [¹⁴C]oxalate have been investigated in the male rate. Only [¹⁴C]oxalate was detected in the urine of rats injected with L-[¹⁴C₁]ascorbic acid, but [¹⁴C]-labeled oxalate, glycolate, glyoxylate, glycolaldehyde, glycine, and serine were recovered from the [¹⁴C₁]-labeled aromatic amino acids. DL-Phenyllactate, an inhibitor of glycolic acid oxidase and glycolic acid dehydrogenase, reduced the amount of [¹⁴C]oxalate recovered in the urine of rats given the [¹⁴C₁]-labeled aromatic amino acids, but increased the amount of [¹⁴C]glycolate formed from L-[¹⁴C₁]-phenylalanine and L-[¹⁴C₁]tyrosine and the amount of [¹⁴C]glycolate produced from [¹⁴C₁]tryptophan. Based on the [¹⁴C]labeled intermediates identified and the relative distribution of the radioactivity, it is postulated that phenylalanine and tyrosine are converted to oxalate via glycolate which is oxidized directly to oxalate by glycolic acid dehydrogenase. Tryptophan is metabolized via glyxylate which is oxidized directly to oxalate by glycolic acid oxidase. Neither glycolate, glyoxylate, glycolic acid oxidase or glycolic acid dehydrogenase are involved in the formation of oxalate from ascorbic acid.     

Effect of banana stem juice on biochemical changes in liver of normal and hyperoxaluric rats.

Influence of stem extract of banana (family Musaceae), was studied on glycolic acid oxidase (GAO) and lactate dehydrogenase enzymes, calcium, phosphorus, oxalate and glycolic acid in liver tissues of sodium glycolate-induced hyperoxaluric rats. Activity of GAO was significantly lowered in the extract-treated rats compared to that of the glycolate-fed rats. LDH increased significantly in glycolate administered rats when compared with the extract-treated rats. The levels of calcium, phosphorus, oxalate and glycolic acid during hyperoxaluric state showed remarkable alterations in liver tissue.     

Immobilization of glycolate oxidase from Medicago falcata on magnetic nanoparticles for application in biosynthesis of glyoxylic acid

Glycolate oxidase was isolated from Medicago falcata Linn. after a screening from 13 kinds of C₃ plant leaves, with higher specific activity than the enzyme from spinach. The M. falcata glycolate oxidase (MFGO) was partially purified and then immobilized onto hydrothermally synthesized magnetic nanoparticles via physical adsorption. The magnetic nanoparticles were characterized with scanning electron microscope (SEM), transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectroscopy. The maximum load of MFGO was 56 mg/g support and the activity recovery was 45%. Immobilization of MFGO onto magnetic nanoparticles enhanced the enzyme stability, and the optimum temperature was significantly increased from 15 °C to 30 °C. The immobilized biocatalyst was successfully used in a batch reactor for repeated oxidization of glycolic acid to synthesize glyoxylic acid, retaining ca. 70% of its initial activity after 4 cycles of reaction at 30 °C for nearly 70 h, and its half-life was calculated to be 117 h.     

Involvement of hydrogen peroxide in the regulation of senescence in pear

Endogenous peroxide levels in pear fruit (Pyrus communis) were measured using a titanium assay method, and were found to increase during senescence in both Bartlett and Bosc varieties. Application of glycolic acid or xanthine, serving as substrates for the formation of H₂O₂, increased the peroxide content of the tissue and accelerated the onset of ripening, as measured by increased softening and ethylene evolution. Application of ethylene also induced increased peroxide levels. Ripening processes were similarly promoted when peroxides were conserved by inhibiting the activity of catalase with hydroxylamine or potassium cyanide. By comparison, the inhibition of glycolate oxidase with alphahydroxy-2-pyridinemethanesulfonic acid decreased the peroxide content of the tissue and delayed the onset of ripening. These results indicate that the onset of ripening correlates with the peroxide content of fruit tissues as occurring under normal conditions or as influenced by the treatments. Hydrogen peroxide may be involved in oxidative processes required in the initiation and the promotion of ripening.     

Oxidation of sarcosine and N-alkyl derivatives of glycine by D-amino-acid oxidase.

1. Sarcosine was oxidized by D-amino-acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) to yield methylamine and glyoxylic acid. A seriies of N-alkyl glycines were also oxidized by this enzyme. 2. N-Acetyl- and N-Phenylglycine inhibited the oxidase by competing with the substrate, while N-methyl-N-acetylglycine did not bind to the enzyme. This suggests the requirement of at least one unsubstituted hydrogen atom at the amino group ofglycine for binding. 3. The primary step in the reaction was the release of a proton from the substrate, indicating the formation of a substituted imino acid, which was spontaneously hydrolyzed to glyoxylic acid acid and an amine.     

A possible role for abscisic Acid in regulation of photosynthetic and photorespiratory carbon metabolism in barley leaves

The influence of abscisic acid (ABA) on carbon metabolism, rate of photorespiration, and the activity of the photorespiratory enzymes ribulose bisphosphate oxygenase and glycolate oxidase in 7-day-old barley seedlings (Hordeum vulgare L. var. Alfa) was investigated. Plants treated with ABA had enhanced incorporation of labeled carbon from ¹⁴CO₂ into glycolic acid, glycine, and serine, while ¹⁴C incorporation into 3-phosphoglyceric acid and sugarphosphate esters was depressed. Parallel with this effect, treated plants showed a rise in activity of RuBP oxygenase and glycolic acid oxidase. The rate of photorespiration was increased twofold by ABA treatment at IO⁻⁶ molar while the CO₂-compensation point increased 46% and stomatal resistance increased more than twofold over control plants.     

Oxidation-reduction properties of glycolate oxidase

This is the first report of the redox potentials of glycolate oxidase. The pH dependence of the redox behavior as well as the effects of activators and inhibitors was studied. At pH 7.1 in 10 mM imidazole-chloride, Eo1' (EF1ox/EF1-.) is -0.033 +/- 0.010 V and Eo2' (EF1-./EF1redH-) is -0.017 +/- 0.017 V vs. the standard hydrogen electrode at 10 degrees C. A maximum of 29% flavin mononucleotide (FMN) anion radical is stabilized at half-reduction at pH 7.1 and 10 degrees C. Both redox couples of glycolate oxidase are pH-dependent from pH 7 to pH 9, and the FMN anion radical is stabilized in this range. The redox potentials of glycolate oxidase are shifted markedly positive of those of unbound FMN, consistent with the enzyme's function. The midpoint potential of glycolate oxidase is more positive than that of the glyoxalate/glycolate couple, and two-electron reduction of glycolate oxidase is thermodynamically favorable. The redox behavior of glycolate oxidase markedly contrasts that of other flavoprotein oxidases. For most flavoprotein oxidases, Eo1' is independent of pH from pH 7 to pH 9 and is much more positive than Eo2', which is pH-dependent. We present a mechanism that suggests a structural basis for the positive shifts and pH dependence of both Eo1' and Eo2' of glycolate oxidase.     

Determination of glycolic acid level in higher plants during photorespiration by stable isotope dilution mass spectrometry with double-labeling experiments

Determination of glycolic acid by stable isotope dilution was applied to the measurement of the glycolic acid pool size in tomato and maize leaves during photorespiration. Detached leaves were maintained in the presence of 18O2; [13C]glycolate was added to the foliar extract as an internal standard and the mixture of biological glycolate and [13C]glycolate was analyzed by combined gas chromatography-mass spectrometry. The level of foliar glycolate pool was measured via the 13C label, and 18O incorporation was determined.     

THE EXTRACELLULAR RELEASE Of GLYCOLIC ACID BY A MARINE DIATOM1

The excretion of glycolic acid by the marine diatom Chaetoceros socialis through time was studied. Excretion in axenic cultures was linear for the time intervals used, but for nonaxenic cultures an equilibrium was created, suggesting bacterial uptake of glycolic acid. In studies with an inhibitor of glycolate dehydrogenase, the level of glycolic acid in the medium jumped 15–fold. This shows the presence of this enzyme, and implies the presence of the entire set of enzymes which convert glycolic acid to serine and release carbon dioxide. In both axenic and nonaxenic cultures a steady state was reached. All of the data suggest that at high cell densities glycolic acid is liberated from the cell by a passive mechanism. The effect of such an excretion in natural waters is discussed.     

Studies on the Residual Respiration not Inhibited by KCN Plus Hydroxamic Acid in Tobacco Callus Cultures

A residual respiration not inhibited by KCN plus hydroxamic acid had been observed in many plant organs and tissues. The relative O2 uptake of it was 20–30% of total respiration in tobacco callus cultures. However, there is no report concerning the nature of the residual respiration and its localization in cell. The object of this study is to elucidate the characteris- tics of this residual respiration and its localization in cell. The experimental results are as follows: 1. The additions of glycolate and glyoxylate cause a marked rise in residual respiration not inhibited by KCN (or NaN3) plus m-CLAM. 2. The O2 uptake induced by glycolate and the residual respiration is inhibited by the addition of α-hydroxy ethanesulfonate. 3. The mitochondrial respiration is completely inhibited by KCN plus m-CLAM, but no effect by adding of glycolate. 4. Oxidation reactions of glycolate and glyoxylate in supernatant are observed after mitochondria are removed. Based on the above results, it is suggested that the residual respiration not inhibited by KCN plus m-CLAM in tobacco callus cultures is primarily catalyzed by glycolic acid oxidase localized within microbodies.     

Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings

Vigna cutjang Endl. cv. Pusa Barsati seedlings, subjected to increasing degrees of water stress (−0.5, −1.0, −1,5 MPa), produced an approximately proportional increase in glycolate oxidase activity, hydrogen peroxide (H₂O₂) and proline content but a decrease in catalase activity, ascorbic acid and protein content. Leaf water potential (leaf ψ) and relative water content (RWC) were also lowered with increasing stress. Pretreatment with l -cysteine and reduced glutathione (10-3 M) decreased glycolate oxidase activity, H₂O₂ content, ascorbic acid oxidase activity, proline content and also slightly improved the water status of leaves stressed (−1.0 MPa) for 2 days. Pretreatment of non-stressed seedlings with these antioxidants had little or no effect. These studies indicate that treatment with antioxidants makes the plant tolerant against water stress by modulating the endogenous levels of H₂O₂ and ascorbic acid in stressed tissue.     

In situ kinetic measurements of d -amino acid oxidase in rat liver with respect to its substrate specificity

Summary d-Amino acid oxidase activity was demonstrated in peroxisomes of rat liver using unfixed cryostat sections and a histochemical technique using cerium ions as capture reagent for hydrogen peroxide and diaminobenzidine, cobalt ions and exogenous hydrogen peroxide to visualize the final reaction product for light microscopical analysis. Cytophotometric analysis of liver sections revealed similar zero-order reaction velocities of d-amino acid oxidase with activity twice as high in periportal areas as in pericentral areas of liver lobuli when using either d-proline or d,l-thiazolidine-2-carboxylic acid as substrates. On the other hand, a 4–5 times higher K _M value was found for d-proline than for d,l-thiazolidine-2-carboxylic acid. The K _M values in periportal and pericentral areas were similar for each substrate. These findings support the suggestion that the physiological substrate for d-amino acid oxidase may be d,l-thiazolidine-2-carboxylic acid, the adduct of cysteamine and glyoxylic acid. d-Amino acid oxidase may play a role in vivo in the production of oxalate which may participate in metabolic control processes as an intracellular messenger molecule.     

A new rapid method of glycolate test by diethyl ether extraction,which is applicable to a small amount of bacterial cells of less than one milligram

The glycolate test is a method to discriminate N-acyl groups of muramyl residue in peptidoglycan of bacterial cell walls by color reaction without purification of the cell walls. The glycolyl residue presents red purple color by heating with 0.02% 2,7-dihydroxynaphthalene (DON) dissolved in concentrated H(2)SO(4). Instead of the previous column methods for quantitative analysis, a qualitative method by solvent works was developed to simplify and to miniaturize the analysis. In this method, solvents played two roles, removal of interfering materials and extraction of glycolic acid from the cell hydrolysates. Of several solvent systems tested, diethyl ether was studied in detail on such properties as the efficiency of glycolic acid extraction under several conditions, the ability of removing various interfering compounds, and the advantage on evaporation procedure of the solvent from extracts. DON reaction of the second diethyl ether extract from cell hydrolysate of "Micromonospora nigra" JCM 3328 showed a clear red purple color of a strong absorbance at 530 nm, which is the same as that of authentic glycolic acid. The solvent method was applied to 20 strains of typical actinomycete species whose acyl types have already been known (Uchida and Seino, 1997). All glycolate test positive strains showed the clear red purple color mentioned above, whereas acetyl type strains revealed no apparent color by the same procedures. Additional experiments indicated that the glycolate test could be determined with less than 1 mg of actinomycete cells by using a smaller amount of DON reagent and ordinary polypropylene tubes. The new method was discussed for advantages in the identification of actinomycetes and for possible applications to other fields.