Miniemulsion as efficient system for enzymatic synthesis of acid alkyl esters

The aim of this work is to devise an efficient enzymatic process for the production of linear alkyl esters in aqueous miniemulsion systems. The esterification reactions of linear alcohols and carboxylic acids were performed with three different enzymes, commercial Amano lipase PS from Pseudomonas cepacia, Lipase type VII from Candida rugosa, and lyophilized Fusarium solani pisi cutinase expressed in Saccharomyces cerevisiae SU50. The miniemulsion system shows a high potential for the synthesis of linear alkyl esters, for example, hexyl octanoate, which could be synthesized with an ester yield of 94% using Amano lipase PS. Even with hydrophilic alcohols as ethanol, ethyl decanoate could be obtained with a concentration of 0.45 M and a yield of 62% using F. s. pisi cutinase as catalyst. High esterification rates for ethyl‐ and hexyloleate in miniemulsion showed a significant shift in cutinase selectivity towards longer chain length carboxylic acids. The stepwise addition of the alcohol led to an increase of the esterification yield. Moreover, increasing the amount of dispersed organic phase, mainly consisting of the substrates, led to a significant increase of the final ester concentration (e.g., concentration of 1.4 M for ethyl decanoate for the esterification with Amano Lipase PS). Biotechnol. Bioeng. 2010;106: 507–515. © 2010 Wiley Periodicals, Inc.     

Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent

The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by Fusarium solani pisi cutinase in an organic solvent media (iso-octane), and to assess the influence of different parameters on the reaction yield.Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15 μmol min⁻¹ mg⁻¹ for ethyl butyrate (C₄ acid chain) and 1.06 μmol min⁻¹ mg⁻¹ for ethyl valerate (C₅ acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6 h of reaction, for an initial equimolar concentration of substrates (0.1 M).The highest product yield (97%) was observed for ethyl caproate (C₆) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2 M ethanol concentration).Cutinase affinity for short chain length carboxylic acids (C₄–C₆) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.     

Effect of irradiance and temperature on the photosynthesis of a cultivated red alga,Pyropia tenera (= Porphyra tenera), at the southern limit of distribution in Japan

The effect of irradiance and temperature on the photosynthesis of the red alga, Pyropia tenera, was determined for maricultured gametophytes and sporophytes collected from a region that is known as one of the southern limits of its distribution in Japan. Macroscopic gametophytes were examined using both pulse‐amplitude modulated fluorometry and/or dissolved oxygen sensors. A model of the net photosynthesis–irradiance (P‐E) relationship of the gametophytes at 12°C revealed that the net photosynthetic rate quickly increased at irradiances below the estimated saturation irradiance of 46 μmol photons m^?2 s^?1, and the compensation irradiance was 9 μmol photons m^?2 s^?1. Gross photosynthesis and dark respiration for the gametophytes were also determined over a range of temperatures (8–34°C), revealing that the gross photosynthetic rates of 46.3 μmol O₂ mg_chl‐a^?1 min^?1 was highest at 9.3 (95% Bayesian credible interval (BCI): 2.3–14.5)°C, and the dark respiration rate increased at a rate of 0.93 μmol O₂ mg_chl‐a^?1 min^?1°C^?1. The measured dark respiration rates ranged from ?0.06 μmol O₂ mg_chl‐a^?1 min^?1 at 6°C to ?25.2 μmol O₂ mg_chl‐a^?1 min^?1 at 34°C. The highest value of the maximum quantum yield (Fv/Fm) for the gametophytes occurred at 22.4 (BCI: 21.5–23.3) °C and was 0.48 (BCI: 0.475–0.486), although those of the sporophyte occurred at 12.9 (BCI: 7.4–15.1) °C and was 0.52 (BCI: 0.506–0.544). This species may be considered well‐adapted to the current range of seawater temperatures in this region. However, since the gametophytes have such a low temperature requirement, they are most likely close to their tolerable temperatures in the natural environment.     

Chemical modification of alpha-chymotrypsin for obtaining high transesterification activity in low water organic media

Alpha-chymotrypsin was made more hydrophilic by modifying 11 (out of 16) ε-amino groups with pyromellitic dianhydride. The hydrophilic preparation was precipitated with n-propanol. This preparation gave significantly higher initial rates at the optimum a_w (127.51 nmol mg^?1 min^?1 in n-octane and 21.30 nmol mg^?1 min^?1 in acetonitrile at a_w=0.33) compared with the lyophilized preparation (53.50 nmol mg^?1 min^?1 in n-octane and 0.26 nmol mg^?1 min^?1 in acetonitrile at a_w=0.97). FT-IR showed that the precipitate of modified alpha-chymotrypsin has a higher content of alpha-helices and beta-sheets compared to the lyophilized powder.     

ACTIVITY AND ISOENZYME PATTERN OF LACTATE DEHYDROGENASE IN ASTROBLASTS CULTURED FROM BRAINS OF NEWBORN MICE

Lactate dehydrogenase activity and isoenzyme distribution was determined in primary cultures of astroblasts as a function of the culture period. The specific activity increased during this period with a peak value (1.91 ± 0.18μmol x min^-1 x mg^-1 cell protein) after 2 weeks in culture. The isoenzyme pattern changed during 3 weeks in culture towards a higher proportion of the H₄ (LDH-1) isoenzyme which is analogous to the in vivo pattern. Omission of serum with or without dBcAMP (0.5 mM) in the culture medium during the third week of culture further enhanced this prominence of the H₄ isoenzyme. The specific activity (1.58 × 0.06 μmol x min^-1 x mg^-1 cell protein) of cultures grown in the presence of 0.5 mM-dBcAMP and absence of serum was close to the activity in the adult brain.     

Phosphatidate phosphatase from avocado (Persea americana) – purification, substrate specificity and possible metabolic implications for the Kennedy pathway and cell signalling in plants

Phosphatidate phosphatase (PAP; EC 3.1.3.4) is a pivotal enzyme in plant lipid metabolism positioned at a major branchpoint between the biosyntheses of phospholipids and triacylglycerols. The enzyme has been purified 7000-fold from the microsomes of maturing Avocado fruit. The enzyme has a subunit molecular mass, as determined on SDS-PAGE, of 49 kDa, and gel filtration studies revealed that it is monomeric. The enzyme was examined for the ability to hydrolyse sn-1,2-dioleoylglycerol-3-phosphate (PA), sn-1-oleoylglycerol-3-phosphate (LPA), sn-2-oleoylglycerol-3-phosphate (sn-2-LPA), and ceramide-1-phosphate. All substrates were used, but the apparent V_max values for ceramide-1-phosphate and sn-2-LPA were considerably lower than for PA or LPA. A kinetic analysis of the purified enzyme was conducted using PA and LPA in Triton X-100 mixed micelles according to the surface dilution kinetic model. The V_max and interfacial Michaelis constant (Km^B) were calculated for PA as 47 μmol min^–1 mg^–1 and 1.49 mol%, respectively. LPA was found to be a better substrate with a V_max and Km^B of 100 μmol min^–1 mg^–1 and 1.06 mol%, respectively. A detailed kinetic analysis of the effect of LPA on PA hydrolysis revealed LPA to be a potent competitive inhibitor of the reaction, with a K_i of 1.4 mol%. This possibly indicates that strict metabolic channelling is in operation in the Kennedy Pathway, with very low steady state concentrations of lyso-phosphatidate with respect to phosphatidate in vivo.     

The Inhibition of Clostridium chauvoei (Jakari strain) Neuraminidase Activity by Methanolic Extracts of the Stem Barks of Tamarindus indicus and Combretum fragrans

The inhibition of neuraminidase from Clostridium chauvoei (jakari strain) with partially purified methanolic extracts of some plants used in Ethnopharmacological practice was evaluated. Extracts of two medicinal plants, Tamarindus indicus and Combretum fragrans at 100–1000 μg/ml, both significantly reduced the activity of the enzyme in a dose-dependent fashion (P < 0.001).

The estimated IC₅₀ values for Tamarindus indicus and Combretum fragrans were 100 and 150 μ/ml respectively. Initial velocity studies conducted, using fetuin as substrate revealed a non-competitive inhibition with the V_max significantly altered from 500 μmole min^?1 mg^?1 to 240μmole min^?1 mg^?1 and 340 μmole min^?1 mg^?1 in the presence of Tamarindus indicus and Combretum fragrans respectively. The K_M remained unchanged at 0.42 mM. The computed Index of physiological efficiency was reduced from 1.19 min^?1 to 0.57 min^?1 and 0.75 min^?1 with Tamarindus indicus and Combretum fragrans as inhibitors respectively.     

Photosynthetic studies with mesophyll protoplasts from Notonia grandiflora, a crassulacean acid metabolism plant

A method is described for rapid enzymatic isolation of mesophyll protoplasts and cells from the crassulacean acid metabolism (CAM) plant Notonia grandiflora DC. The mesophyll protoplasts exhibited high rates of ¹⁴CO₂ fixation both in the light (45 μmol of CO₂ fixed mg^?1 Chl h^?1) and in the dark (20 μmol of CO₂ fixed mg^?1 Chl h^?1). The protoplasts also showed O₂ evolution (40 μmol of O₂ evolved mg^?1 Chl h^?1) without added bicarbonate. Exogenously added bicarbonate had no stimulating effect on the O₂ evolution. Analyses of early photosynthetic products in the light showed the formation of both C₃ and C₄ acids. Aspartate was found to be a predominant photosynthate.     

Collagenase of a new streptomycete: Its induction and purification

Rifampin and chloramphenicol inhibited the synthesis of collagenase of Streptomyces sp. A₈, suggesting de novo synthesis. The collagenase was induced by insoluble collagen, its macromolecular fragments, gelatin, peptone, hide powder and yeast extract. Growth as well as collagenase synthesis were dependent on substrate availability. Purification of collagenase by DEAE-cellulose chromatography resulted in approximately 25-fold increase in activity (268.6 μmol glycine equivalents min^?1 mg^?1 protein) relative to the activity of the culture filtrate (10.5 μmol glycine equivalents min^?1 mg^?1 protein).     

Immunodetection of a 90 000-Mr polypeptide related to yeast plasma membrane ATPase in plasma membrane from maize shoots

Maize shoot plasma membranes were prepared using either polyethyleneglycol (PEG)-dextran phase partition or centrifugation through a 30% sucrose cushion. The ATPase specific activity of membranes obtained with the phase partition method (1.4 μmol P_i · min⁻¹ · mg⁻¹ protein) was twice that of those prepared with the sucrose cushion method. After solubilization by lysolecithin and precipitation by ammonium sulfate, ATPase activities of the order of 3.0–3.5 μmol P_i · min⁻¹ · mg⁻¹ were obtained. A polypeptide of M_r = 90 000 was enriched during ATPase purification.Antibodies against pure plasma membrane ATPase from Saccharomyces cerevisiae inhibited the plant ATPase activity. Immunodetection during purification of the plant enzyme strongly supported the conclusion that the polypeptide of M_r = 90 000 belongs to plant plasma membrane ATPase.     

High throughput covalent immobilization process for improvement of shelf-life,operational cycles,relative activity in organic media and enzymatic kinetics of urease and its application for urea removal from water samples

High throughput covalent urease immobilization was performed through the amide bond formation between the urease and the amino-functional MNPs. The enzyme’s performances, including shelf-life, reusability, enzymatic kinetics, and the enzyme relative activity in organic media was improved. At optimal conditions, the immobilization efficiency was calculated about 95.0% with keeping 94.7% of the urease initial specific activity. The optimal pH for maximum activity of the free and immobilized urease was calculated as 7.0 at 37.0 °C and 8.0 at 60.0 °C, respectively. The kinetics studies showed the K_m of 26.0 mM and 8.0 mM and the V_max of 5.31 μmol mg⁻¹ min⁻¹ and 3.93 μmol mg⁻¹ min⁻¹ for the free and immobilized urease, respectively. The ratio K_cat/K_m as a measure of catalytic efficiency and enzyme specificity was calculated as 0.09 mg mL⁻¹ min⁻¹ and 0.22 mg mL⁻¹ min⁻¹ for the free and immobilized urease, respectively, indicating an improvement in the enzymatic kinetics. The shelf-life and operational studies of immobilized urease indicated that approximately 97.7% and 88.5% of its initial activity was retained after 40 days and 17 operational cycles, respectively. The immobilized urease was utilized to urea removal from water samples with an efficiency between 91.5–95.0%.     

Enzymatic removal of a cephalosporin methyl ester blocking group

Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C₄ methyl ester blocking group on 7-aminodesacetoxycephalosporanic acid (7-ADCA). Only one culture, Streptomyces capillispira Mertz and Higgens nov. sp., produced an enzyme that catalysed the reaction. Enzyme synthesis in a defined mineral salts medium was repressed by NH₃ and amino acids. Under optimum fermentation conditions, the maximum rate of substrate hydrolysis was 6 × 10^?10 mol min^?1 mg^?1 cell. The enzyme was recovered from the mycelia and partially purified by gel filtration. Kinetic studies by pH-stat titration indicated that the pH optimum was 7.5–8.5, the temperature optimum was 25–30°C, and the substrate K_m value was 2.3 mg ml^?1. The reaction products, 7-ADCA and methanol, were weak competitive inhibitors of the enzyme with K₁ values of 6.63 and 0.188 mg ml^?1, respectively. The enzyme also hydrolysed cefaclor and cephalexin methyl esters but did not hydrolyse cephalosporin ethyl esters. With further improvements in enzyme yields and stability, enzymatic deblocking of cephalosporins could provide an alternative to chemical deblocking processes.     

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ⁵3β hydroxy steroids into the active Δ⁴3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ⁵3β HSD we have analysed the initial velocity rates (V_o) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ⁵3β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/V_o versus 1/S plot, the x intercept being positive. Using a 1/V_o versus 1/S² plot the V_max was determined to be 1·0 ± 0·08 μmol min^?1 mg^?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the V_max for DHEA transformation was unchanged (0·95 ± 0·04 μmol min^?1 mg^?1, n = 3) whilst the S_0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD⁺ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.¹ Extending the analysis of NAD⁺ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent V_max and K_m being 0·84 ± 0·04 μmol min^?1 mg^?1 CL (n = 3) and 27 ± 7 μmol 1^?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding.     

Improving operational stability of thermostable Pythium myriotylum secretory serine protease by preparation of cross-linked enzyme aggregates (CLEAs)

Abstract

Present study was undertaken to develop cross-linked enzyme aggregate (CLEA)of alkaline serine proteases (sp) from Pythium myriotylum (Pm), a necrotrophic oomycete reported to considerably secrete serine proteases. Among various precipitants screened for spPm1-CLEA preparation, ammonium sulfate at 80% saturation (w/v) yielded 100% activity recovery and retention of spherical morphology as observed by SEM analysis. Addition of glutaraldehyde as cross-linker at 1% (v/v) concentration with optimized ammonium sulfate concentration for 1?hour at 100?rpm yielded 100% activity recovery of spPm1-CLEA from 8-day old P. myriotylum culture filtrate. Addition of BSA (10?mg/ml) to CLEA cross-linking reaction mix reduced CLEA size from the range of 1.82–1.19?µm to 394–647?nm. spPm1-CLEA preparations retained 100% activity at temperature of 80?°C and pH 12.0 signifying their potential commercial applications. In terms of kinetic parameters, present process enhanced kinetic parameters as revealed by 1.67?U.mg^?1 specific activity, K_m of 0.062?mM and V_max of 0.145?µmol.min^?1.mg^?1 for the spPm1-CLEA compared to 0.288?U.mg^?1 specific activity, K_m of 0.060?mM and V_max of 0.20?µmol.min^?1.mg^?1 determined for the free spPm1 enzyme. Study has successfully demonstrated the concept of CLEA in enhancing spPm1 stability and the results so generated can be translated in future towards development of robust biocatalysts.     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans oxidizes acetate to CO₂ with sulfate. This organism metabolizes acetate via a pathway in which C₁ units rather than tri- and dicarboxylic acids are intermediates. We report here that cell extracts of D. acetoxidans catalyzed an exchange between CO₂ and the carboxyl group of acetate at a rate of 90 nmol · min^-1 · mg^-1 protein which is sufficient to account for the in vivo acetate oxidation rate of 250 nmol · min^-1 · mg^-1 protein. The reaction was strictly dependent on both ATP and coenzyme A. The extracts contain high activities of acetate kinase (6.3 U · mg^-1 protein) and phosphotransacetylase (60 U · mg^-1 protein). These findings indicate that acetyl-CoA rather than acetyl-phosphate or acetate is the substrate of the carbon-carbon cleavage activity. Exchange was only observed in the presence of strong reducing agents such as Ti³⁺. Interestingly, the cell extracts also catalyzed the reduction of CO₂ to CO with Ti³⁺ as electron donor (120 nmol · min^-1 · mg^-1 protein). Carbon monoxide dehydrogenase and other oxidoreductases involved in acetate oxidation were found to be partially associated with the membrane fraction suggesting a membrane localization of these enzymes.Abbreviations MOPS Morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine - DTT d,l-1,4-Dithiothreitol - DMN 2,3-Dimethyl-1,4-naphthoquinone - MV_OX Methyl viologen, oxidized - APS Adenosinephosphosulfate - SRB Sulfate reducing bacteria - U mol product formed per min     

Decolorization of the azo dye Acid Red 18 by crude manganese peroxidase: Effect of system parameters and kinetic study

Abstract

To optimize operating conditions for the decolorization of the azo dye Acid Red 18 (AR18) by crude manganese peroxidase (MnP), some important factors affecting enzymatic decolorization were systematically investigated. Under the optimal enzyme reaction conditions, a decolorization efficiency of more than 82.3% was achieved after 60 min treatment. Furthermore, the manganese chelators, malate, tartrate, and lactate were found to be more favorable for the decolorization of AR18 than malonate, acetate, succinate, maleate, oxalate, and citrate. However, the presence of NaCl or Na₂SO₄ had a negative impact on the decolorization of AR18. The K_m and V_max values of MnP for AR18 were 169.66 μmol L^{? 1} and 20.63 μmol L^{? 1} min^{? 1}, respectively. The decolorization of AR18 by MnP followed second-order reaction kinetics with respect to the dye concentration. The decolorization rate constant increased with increasing temperature from 20°C to 35°C, which indicated an activation energy (E_a) of 15.87 kcal mol^{? 1} and frequency factor (k₀) of 1.36 × 10⁸ mg^{? 1} L min^{? 1} according to the Arrhenius equation. The results obtained provide experimental data for the application of crude MnP for the decolorization of AR18, and help to elucidate the biochemical mechanism of dye decolorization by the enzyme.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

Association of β-amyloid peptide fragments with neuronal nitric oxide synthase: Implications in the etiology of Alzheimers disease

Neuronal nitric oxide synthase (nNOS) was purified on DEAE-Sepharose anion-exchange in a 38% yield, with 3-fold recovery and specific activity of 5 µmol.min^?1.mg^?1. The enzyme was a heterogeneous dimer of molecular mass 225?kDa having a temperature and pH optima of 40°C and 6.5, K_m and V_max of 2.6 μM and 996 nmol.min^?1.ml^?1, respectively and was relatively stable at the optimum conditions (t_½?=?3?h). β-Amyloid peptide fragments Aβ_17–28 was the better inhibitor for nNOS (K_i?=?0.81 µM). After extended incubation of nNOS (96?h) with each of the peptide fragments, Congo Red, turbidity and thioflavin-T assays detected the presence of soluble and insoluble fibrils that had formed at a rate of 5?nM.min^?1. A hydrophobic fragment Aβ_17–21 [Leu₁₇ – Val₁₈ – Phe₁₉ – Phe₂₀ – Ala₂₁] and glycine zipper motifs within the peptide fragment Aβ_17–35 were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst.     

Lactate dehydrogenase in abdominal muscle of crayfishOrconectes limosus and shrimpcrangon crangon (Decapoda: Crustacea): Properties and evolutionary relationship

In crayfishOrconectes limosus and shrimpCrangon crangon abdominal muscle, lactate dehydrogenase (LDH, EC 1.1.1.27) is encoded by one locus. No polymorphism was detected. The enzymes were purified to homogeneity. The specific activities for purified crayfish and shrimp LDHs were 472 and 414 μmol NADH min⁻¹ mg⁻¹, respectively, at 30°C. Their physicochemical and kinetic properties did not resemble fish (Gadus morhua) LDH-A₄ isoenzyme. Their amino acid composition indicated greater similarity to fish LDH-C₄ isoenzymes.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate