Protein engineering of Acidovorax facilis 72W nitrilase for bioprocess development

Hydroxycarboxylic acid monomers can be used to prepare industrially important polymers. Enzymatic production of such hydroxycarboxylic acids is often preferred to chemical production since the reactions are run at ambient temperature, do not require strongly acidic or basic reaction conditions, and produce the desired product with high selectivity at high conversion. However, native enzymes often do not perform desired reactions with the efficiency required for commercial applications. Protein engineering was used to significantly increase the specific activity of nitrilase from Acidovorax facilis 72W for the conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid. Overexpression of engineered nitrilase enzymes in Escherichia coli, combined with immobilization of whole cells in alginate beads that can be recycled many times has facilitated the development of a commercially viable bioprocess for production of 3-hydroxyvaleric acid.     

Which factors determine plant invasions in man-made habitats in the Czech Republic?

Factors determining the invasibility of different types of anthropogenic vegetation were studied in the Czech Republic. A data set of 3420 vegetation plots recorded between 1945 and 2005, containing 913 species, was used. A set of climatic variables (mean annual temperature and precipitation, together with elevation), propagule pressure (substituted by human population density) and local habitat conditions (substituted by values of CSR life strategies and Ellenberg indicator values of native species) was obtained for each plot. All species were classified as native, archaeophytes (i.e. alien species introduced before 1500), and neophytes (i.e. aliens introduced after 1500) and their relative proportion was calculated for each plot. Regression tree models were used to determine the ecological characteristics of the most invasible man-made habitats in the Czech Republic. The plots contained on average 31.9% archaeophytes and 7.3% neophytes. Correlation between the proportions of archaeophytes and neophytes was positive and significant. Both archaeophytes and neophytes were found predominantly in strongly disturbed habitats with a high nutrient supply located at low elevations in warmer climatic areas of the Czech Republic. Archaeophytes are more influenced by local habitat conditions and preferentially colonize sunny and dry man-made habitats with higher soil reaction. Neophytes have no special preferences for local habitat conditions and their highest proportion was found mainly in disturbed habitats at low elevations. Our results show that for anthropogenic vegetation in the Czech Republic, ecological and habitat characteristics are more important factors for plant invasions than different land use in the surrounding area.     

Calcium efflux parallel to total phosphate retention in rat liver mitochondria

Phosphate efflux from uncoupled rat liver mitochondria was completely inhibited when mersalyl plus butylmalonate and ATP were added to a sucrose suspending medium. Despite the total retention of phosphate a calcium efflux was observed even in presence of ruthenium red. Under the above conditions no phosphate is transported in association with the ADP/ATP carrier. While mersalyl completely blocked the phosphate release induced by ruthenium red or EGTA from coupled mitochondria it only partially inhibited the CA2+-efflux. The inhibition of Ca2+ efflux was almost completely abolished in the presence of acetate. The existence of a co-transport of Ca2+ associated with phosphate is discussed.     

Platelet responses promoted by the activation of protein kinase C or the increase of cytosolic Ca2+ are potentiated by adrenaline. Effects of cAMP and staurosporine.

We studied the action of the alpha 2 adrenergic agonist adrenaline on the platelet responses evoked by the activation of protein kinase C or by the ionophore induced increase of cytosolic Ca2+. Both the phorbol ester and ionomycin-induced aggregation are strongly potentiated by adrenaline which per se does not behave as an activating agonist. The potentiation by adrenaline is observed both when added before and after the aggregating agent; in the latter case the effect increases on increasing the delay of adrenaline addition. Adrenaline also reverses the inhibition by cAMP of the PMA (or ionomycin) induced aggregation. It also has a strong potentiating effect (over 100%) on the phorbol ester induced ATP secretion and a weaker effect on the secretion induced by ionomycin. The effect on secretion is visible only when adrenaline is added prior to the stimulus. The inhibition by cAMP of the PMA or ionomycin induced secretion is also counteracted by adrenaline. In no case adrenaline modifies the pattern of platelet phosphoproteins. Ionomycin induces some platelet aggregation also in the presence of the protein kinase inhibitor staurosporine; also this phosphoprotein independent aggregation is strongly stimulated by adrenaline.     

Formation of acetoacetate from 3-hydroxy-3-methylglutarate by rat liver and isolation of a mitochondrial coenzyme A-transferase activity involved

1. Formation of acetoacetate from 3-hydroxy-3-methylglutarate was observed in the perfused rat liver. Production of 3.5mumol of acetoacetate/h per g of tissue was obtained. 2. Formation of acetoacetate was catalysed mainly by the mitochondrial fraction of the homogenized liver, at a rate of 62nmol/h per mg of protein. 3. Experiments with hydroxy-[3-(14)C]methylglutarate demonstrated that the acetoacetate formed was derived mainly from this compound. 4. A mitochondrial transferase activity catalysing the transfer of a CoA molecule from succinyl-CoA (3-carboxypropionyl-CoA) to hydroxymethylglutarate was shown. The K(m) value for hydroxymethylglutarate was 5x10(-3)m.     

The localization of enzymatic activities involved in uridine nucleotides reactions in human erythrocytes

Enzymatic activities involved in the transformations of uridine nucleotides by intact erythrocytes and their subfractions have been studied and the following enzymatic activities have been identified: UTPase, UDPase, UMPase and uridylate kinase. UTPase activity was present exclusively in the stromal fraction while UMPase and uridylate kinase activities were specific in the soluble fraction. UDPase was present in both the stromal and soluble fractions. This compartmentation in erythrocytes differs from that reported for the enzymes involved in adenine nucleotides transformations. Due to its sensitivity to Zn²⁺, uridylate kinase could be differentiated from adenylate kinase.     

Pharmacological evaluation of selected arylpiperazines with atypical antipsychotic potential

Six active compounds, among previously synthesized and screened arylpiperazines, were selected and evaluated for the binding affinity to rat dopamine, serotonin and alpha(1) receptors. Two compounds with benztriazole group had a 5-HT(2A)/D(2) binding ratio characteristic for atypical neuroleptics (>1, pK(i) values). Compound 2, 5-[2-[4-(2,3-dimethyl-phenyl)-piperazin-1-yl]ethyl]1H-benzotriazole, expressed clozapine-like in vitro binding profile at D(2), 5-HT(2A) and alpha 1 receptors and a higher affinity for 5-HT(1A) receptors than clozapine. Also, it exhibited the noncataleptic behavioural pattern of atypical antipsychotics and antagonized d-amphetamine-induced hyperlocomotion in rats.     

Biocatalysis: applications and potentials for the chemical industry

The chemical industry is exploring the use of renewable feed stocks to improve sustainability, prompting the exploration of bioprocesses for the production of chemicals. Attractive features of biological systems include versatility, substrate selectivity, regioselectivity, chemoselectivity, enantioselectivity and catalysis at ambient temperatures and pressures. However, a challenge facing bioprocesses is cost competitiveness with chemical processes because capital assets associated with the existing commercial processes are high. The chemical industry will probably use biotechnology with existing feed stocks and processes to extract higher values from feed stocks, process by-products and waste streams. In this decade, bioprocesses that offer either a process or a product advantage over traditional chemical routes will become more widely used.