A Bioreactor-Crystallizer for L-Malic Acid Production

A bioreactor with associated crystallizer for the accumulation of a highly concentrated slurry product has been developed and investigated. The transformation of Ca-fumarate to Ca-L-malate by the action of the fumarase of immobilized Brevibacterium flavum cells focussed on the performance of this newly-devised bioreactor-crystallizer system.

The following results were obtained

(1) The fumarase reaction in the bioreactor proceeded at a rate that was first-order in apparent substrate concentration.

(2) The reaction rate increased with the addition of Na₂-fumarate to the substrate solution.

(3) The reaction rate was independent of the substrate circulation rate and the initial substrate concentration in the crystallizer.

(4) Fumarase activity of immobilized B. flavum cells was stable after 10 repeated uses over a period of 10 days.

(5) Maximum concentration of the product, final conversion ratio of the substrate and the productivity of the bioreactor-crystallizer system were much higher than those for a conventional bioreactor using solubilized Ca-fumarate as a substrate.     

Development of 1-O-sinapoyl-β-D-glucose: L-malate sinapoyltransferase activity in cotyledons of red radish (Raphanus sativus L. var. sativus)

Protein preparations from cotyledons of red radish (Raphanus sativus L. var. sativus) catalyzed the the formation of depsides between cinnamic acids and L-malate, using 1-O-acyl glucose conjugates as the donors. This activity showed an absolute acceptor specificity towards L-malate and a pronounced donor specificity with 1-sinapoylglucose (1-O-sinapoyl--D-glucose). Maximal rate of sinapoyl-L-malate formation was found to be at pH 6.3, and there was no requirement for metal ions or sulfhydryl group reagents. The K _m values were found to be 0.46 mM for 1-sinapoylglucose and 54 mM for L-malate. Protein extracts obtained from seedlings at different stages of seedling development did not significantly differ with respect to the properties of the enzymatic activity. Appearance and development of extractable activities correlated well with the in vivo transacylation kinetics of 1-sinapoylglucose to sinapoyl-L-malate during seedling growth. Maximal activity was extracted from 10–14-d-old seedlings and found to be at 67 pkat pair^-1 of cotyledons. This new enzymatic activity in phenylpropanoid metabolism refers to an enzyme which can be classified as 1-sinapoylglucose: L-malate sinapoyltransferase (SMT) (EC 2.3.1.-).Abbreviations DTE dithioerythriol - HPLC high performance liquid chromatography - IAA indoleacetic acid - ME 2-mercaptoethanol - Mes 2-(N-morpholino)ethanesulfonic acid - Mops 3-(N-morpholino)propanesulfonic acid - SMT 1-O-Sinapoyl--D-glucose: L-malate sinapoyltransferase     

Inhibition of two mitochondrial enzymes by gold(III) halo complexes. Evidence for a binding mechanism

Fumarase (EC 4.2.1.2) and mitochondrial L-malate dehydrogenase (EC 1.1.1.37) were both inhibited by NaAuCl₄ and KAuBr₄. The inhibition for both was measured as a function of gold complex concentration and aquation time, and the NaAuCl₄ inhibition was also measured in the presence of 0.15 M NaCl. Regeneration of the enzyme activity after NaAuCl₄ inhibition using L-cysteine, L-methionine and NaCN was also investigated. Sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis and amino acid analysis was performed on the NaAuCl₄ inhibited enzymes as well as on ribonuclease A (EC 3.1.26.2), lysozyme (EC 3.2.1.17) and liver alcohol dehydrogenase (EC 1.1.1.1). It was observed that the inhibition was proportional to the gold complex concentration but decreased markedly after aquation of the complex. In the presence of NaCl the initial rate of inactivation is essentially unaffected unless the complex has been aquated and then the initial rate is increased. Gel electrophoresis on gold complex-enzyme mixtures show polymerization for ribonuclease and lysozyme and amino acid analysis indicates that no oxidation has taken place. From these results, a binding mechanism is postulated for the inhibition of the dehydrogenases by direct displacement of a halide ligand, probably by two groups on the enzyme, at least one of which may be a sulfur containing acid.     

Purification and Stability during Storage of Phoshoenolpyruvate Carboxylase from Leaves of Amaranthus hypochondriacus,a NAD-ME Type C4 Plant

A traditional method is reported for purification of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) from leaves of Amaranthus hypochondriacus L. with a high yield of 50 %, 135-fold purification, and specific activity of 900 mmol kg^–1(protein) s^–1. PEPC was purified from light-adapted leaves of A. hypochondriacus, involving 40–60 % ammonium sulphate fractionation, followed by chromatography on columns of DEAE-Sepharose, hydroxylapatite (HAP), and Seralose 6-B. The enzyme appeared as a single band on 10 % SDS-PAGE, with a molecular mass of about 100 kDa. Kinetic studies with purified enzyme confirmed the PEPC to be the light-form of the enzyme. Glycerol generally increased the stability of PEPC. The stability and storage of the purified enzyme was studied at temperatures of 4 °C, –20 °C, and liquid nitrogen. PEPC maintained its activity for up to 3 months upon storage with 50 % (v/v) glycerol in liquid nitrogen.     

Vacuolar localization of the enzymatic synthesis of hydroxycinnamic acid esters of malic acid in protoplasts from Raphanus sativus leaves

Protoplasts from leaves of radish ( Raphanus sativus L. var. sativus ) were examined for the subcellular localization of p -coumaric, caffeic, ferulic and sinapic acid esters of malic acid and the enzyme(s) involved in their syntheses. Vacuoles isolated from leaf protoplasts contained all the hydroxycinnamic acid esters as well as all the dependent enzyme activities. Protein from leaf vacuoles was shown to form the hydroxycinnamoylmalic acids, using the corresponding hydroxycinnamic acid glucose esters (1-O-acyl glucosides) as acyl donors. It is proposed that the vacuole is the cell compartment for synthesis and deposition of the hydroxycinnamoylmalic acids.     

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter

Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).     

Specific features of changes in levels of endogenous respiration substrates in Saccharomyces cerevisiae cells at low temperature

The rate of endogenous respiration of Saccharomyces cerevisiae cells incubated at 0 degrees C under aerobic conditions in the absence of exogenous substrates decreased exponentially with a half-period of about 5 h when measured at 30 degrees C. This was associated with an indirectly shown decrease in the level of oxaloacetate in the mitochondria in situ. The initial concentration of oxaloacetate significantly decreased the activity of succinate dehydrogenase. The rate of cell respiration in the presence of acetate and other exogenous substrates producing acetyl-CoA in mitochondria also decreased, whereas the respiration rate on succinate increased. These changes were accompanied by an at least threefold increase in the L-malate concentration in the cells within 24 h. It is suggested that the increase in the L-malate level in the cells and the concurrent decrease in the oxaloacetate level in the mitochondria should be associated with a deceleration at 0 degrees C of the transport of endogenous respiration substrates from the cytosol into the mitochondria. This deceleration is likely to be caused by a high Arrhenius activation energy specific for transporters. The physiological significance of L-malate in regulation of the S. cerevisiae cell respiration is discussed.     

Effects of phosphate on the activity, stability and regulatory properties of phosphoenolpyruvate carboxylase from the C4 plant Cynodon dactylon

The extraction of phosphoenolpyruvate carboxylase, PEPC (EC 4.1.1.31) from leaves of Cynodon dactylon (L.) Pers. with phosphate buffer (pH 7.4, 105 mM) was advantageous in comparison to the usual extraction with Tris-HCl buffer (pH 7.4, 100 mM); a higher activity was obtained, which was most evident at low substrate (phosphoenolpyruvate) concentrations. The PEPC activity was stable under dilution or in storage for at least 48 h at room temperature. The effects of phosphate buffer were not due to inhibition of phosphatase(s) action during the extraction, since they were also observed when the phosphates were added after the extraction with Tris-HCl. The phosphate-extracted enzyme was less responsive to both L-malate inhibition and activation by glucose-6-phosphate. The effects of phosphates might be due to preferential exclusion from the enzymic protein domain and, therefore, to a confinement of the enzyme to a fraction of the total volume. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phosphate and Sulfate Activate the Phosphoenolpyruvate Carboxylase from the C4 Plant Cynodon dactylon L.

The effect of phosphate, sulfate and other inorganic ions on the activity of phosphoenolpyruvate carboxylase (PEPC) from the C₄ plant Cynodon dactylon were investigated for the first time, as well as their interaction with Clc-6-P, AMP and ma-late. Activation of PEPC by phosphate and sulfate ions was demonstrated and it was not dependent on the accompanying cations, something that was not clarified for PEPCs from other plant sources. No activation of this enzyme was observed by nitrate. PEPC activation was found to be competitive with glucoses-phosphate (Clc-6-P) and AMP stimulation and less sensitive to malate inhibition. This work showed that PEPC from C₄plants could exhibit similar activation properties with the enzyme from CAM plants and different activation properties in plants of the same type, rendering the study of this enzyme from different plant sources necessary.     

Modulation in vivo by Nitrate Salts of the Activity and Properties of Phosphoenolpyruvate Carboxylase in Leaves of Alternanthera pungens (C4 plant) and A. sessilis (C3 species)

Feeding K⁺ or Na⁺ nitrate salts in vivo enhanced the activity of phosphoenolpyruvate carboxylase (PEPC) in the leaf extracts of Alternanthera pungens (C₄ plant) and A. sessilis (C₃ species). The increase was more pronounced in A. pungens than in A. sessilis. Chloride salts increased the PEPC activity only marginally. However, the sulfate salts were either not effective or inhibitory. Feeding nitrate modulated the regulatory properties of PEPC in A. pungens, resulting in increased K_I (malate) and decreased K_A (glucose-6-P). The sensitivity of PEPC to malate, which gives a measure of phosphorylation status of the enzyme, indicated that feeding leaves with NO₃ ^– enhanced the phosphorylation status of the enzyme. The reduction in PEPC activity due to cycloheximide treatment suggested that increased synthesis of PEPC protein kinase may be one of the reasons for the enhancement in PEPC activity, after the nitrate feeding. We suggest that nitrate salts could be used as a tool to modulate and analyze the properties of PEPC in C₃ and C₄ plants.