Fragiskos N. Kolisis

Bioorganic reactions in microemulsions: the case of lipases

Water-in-oil microemulsions, or reverse micelles, are being evaluated as a reaction medium for a variety of enzymatic reactions. These systems have many potential biotechnological applications. Important examples are the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. This review illustrates the biotechnological applications of microemulsions as media for bioorganic reactions. The principal focus is on lipase catalyzed processes.

Coactivation of GR and NFKB alters the repertoire of their binding sites and target genes

Glucocorticoid receptor (GR) exerts anti-inflammatory action in part by antagonizing proinflammatory transcription factors such as the nuclear factor kappa-b (NFKB). Here, we assess the crosstalk of activated GR and RELA (p65, major NFKB component) by global identification of their binding sites and target genes. We show that coactivation of GR and p65 alters the repertoire of regulated genes and results in their association with novel sites in a mutually dependent manner. These novel sites predominantly cluster with p65 target genes that are antagonized by activated GR and vice versa. Our data show that coactivation of GR and NFKB alters signaling pathways that are regulated by each factor separately and provide insight into the networks underlying the GR and NFKB crosstalk.

Enzymatic synthesis of hydrophilic and hydrophobic derivatives of natural phenolic acids in organic media

The enzymatic esterification of natural phenolic antioxidants such as cinnamic acid and benzoic acid derivatives, with aliphatic alcohols, monosaccharides as well as alkylglucosides, using various lipases and esterases in non-aqueous media, was investigated. Reaction rate and esterification yield seems to be linked to the structural characteristics of the substrates (aromatic acids and alcohols or sugars) used.

Enzymes in non-conventional phases

The use of enzymes for technical applications has become increasingly important in different areas of biotechnology such as the food or pharmaceutical industries. Various processes have been developed using soluble or immobilized enzymes mainly in aqueous reaction phases. However, the catalytic activity of enzymes not only in water, but also in other solvents was first investigated at the beginning of the century (Bourquelot, E. and Bridel, M., 1911). Apart from water as the exclusive solvent and reaction phase, many kinds of solvent systems for enzymatic reactions became the subject of intensive research over the last 10-15 years (Buckland, Dunnill and Lilly, 1975; Klibanov, Samokhin, Martinek and Berezin, 1977; Nakanishi and Matsuno, 1986; Chen and Sih, 1989; Klibanov, 1990; Blanch, 1992; Carrea et al., 1992).

Lipase-catalyzed esterification of rutin and naringin with fatty acids of medium carbon chain

Abstract Flavonoids rutin and naringin were acylated with fatty acids of medium carbon chain (with 8–12 carbon atoms on their molecule) in a reaction catalyzed by immobilized lipase from Candida antarctica (Novozyme) in various solvent systems. The reaction parameters affecting the acylation rate and the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity ( a w ) of the system, as well as the kinetic of the reaction have been investigated. In all cases studied, only flavonoid monoester is identified as the product, which indicates that this lipase-catalyzed esterification is regioselective. The enzymatic acylation of flavonoids seems to follow Michaelis–Menten kinetics.

Factors affecting the lipase catalyzed transesterification reactions of 3-hydroxy esters in organic solvents.

Abstract Chiral resolutions of racemic 3-hydroxy esters were performed in organic phases with lipases from Pseudomonas cepacia , Chromobacterium viscosum and Porcine pancreas. The reaction conditions have been optimized with 3-hydroxy octanoic acid methyl ester. Different organic solvents have been tested showing a tendentious correlation with the hydrophobicity of the solvents expressed as log P. The reaction time was shortened six fold by using irreversible acylating agents. We have found solvent type, lipase type and acylating agent acting as tools for changing the enantioselectivity. Lipase from Pseudomonas cepacia was lyophilized at different pH and the influence of the amount of water added was investigated, resulting in the highest activity at the pH optimum and a denaturation of the lipase above 1 % water (w/w lipase ). The water activity was measured on-line with a humidity sensor. Water activities greater than 0.4 led to a decrease in enantioselectivity and reaction rate. In the optimized system the resolutions of other 3-hydroxy esters were tested. Aliphatic compounds reacted with lower enantioselectivity, only the substrates could be isolated in high enantiomeric purity. In contrast, aromatic 3-hydroxy esters were acylated by lipases with high stereoselectivity. A model of the active site of lipase from Pseudomonas sp . explained these experimental observations.

Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Mastic oil from Pistacia lentiscus var. chia inhibits growth and survival of human K562 leukemia cells and attenuates angiogenesis.

Abstract: Mastic oil from Pistacia lentiscus var. chia, a natural plant extract traditionally used as a food additive, has been extensively studied for its antimicrobial activity attributed to the combination of its bioactive components. One of them, perillyl alcohol (POH), displays tumor chemopreventive, chemotherapeutic, and antiangiogenic properties. We investigated whether mastic oil would also suppress tumor cell growth and angiogenesis. We observed that mastic oil concentration and time dependently exerted an antiproliferative and proapoptotic effect on K562 human leukemia cells and inhibited the release of vascular endothelial growth factor (VEGF) from K562 and B16 mouse melanoma cells. Moreover, mastic oil caused a concentration-dependent inhibition of endothelial cell (EC) proliferation without affecting cell survival and a significant decrease of microvessel formation both in vitro and in vivo. Investigation of underlying mechanism(s) demonstrated that mastic oil reduced 1) in K562 cells the activation of extracellular signal–regulated kinases 1/2 (Erk1/2) known to control leukemia cell proliferation, survival, and VEGF secretion and 2) in EC the activation of RhoA, an essential regulator of neovessel organization. Overall, our results underscore that mastic oil, through its multiple effects on malignant cells and ECs, may be a useful natural dietary supplement for cancer prevention.

An in silico compartmentalized metabolic model of Brassica napus enables the systemic study of regulatory aspects of plant central metabolism

Biochemical network reconstructions represent valuable tools for the computational metabolic modeling of organisms that present a great biotechnological interest. An in silico multi‐compartmental model of the central metabolism of the plant Brassica napus (Rapeseed) was constructed, aiming to investigate the metabolic properties of the Brassicaceae family. This family comprises many plants with major importance for the energy and nutrition sector, including the model plant Arabidopsis thaliana. The model utilized as objective function to be subsequently optimized, the biomass production of rapeseed developing embryos, which are characterized by a very high, oil content, up to 60% of biomass weight. In order to study global network properties of seed metabolism, various methods were employed, like Flux Balance Analysis, Principal Component Analysis of the flux space and reaction deletion studies, which simulate the effect of gene knock‐out experiments. The model successfully simulated seed growth during the stage of oil accumulation and provided insight, regarding certain aspects of network plasticity, with the emphasis given in lipid biosynthesis regulation. Biotechnol. Bioeng. 2011; 108:1673–1682.

Gene ARMADA: an integrated multi-analysis platform for microarray data implemented in MATLAB

BackgroundThe microarray data analysis realm is ever growing through the development of various tools, open source and commercial. However there is absence of predefined rational algorithmic analysis workflows or batch standardized processing to incorporate all steps, from raw data import up to the derivation of significantly differentially expressed gene lists. This absence obfuscates the analytical procedure and obstructs the massive comparative processing of genomic microarray datasets. Moreover, the solutions provided, heavily depend on the programming skills of the user, whereas in the case of GUI embedded solutions, they do not provide direct support of various raw image analysis formats or a versatile and simultaneously flexible combination of signal processing methods.ResultsWe describe here Gene ARMADA (Automated Robust MicroArray Data Analysis), a MATLAB implemented platform with a Graphical User Interface. This suite integrates all steps of microarray data analysis including automated data import, noise correction and filtering, normalization, statistical selection of differentially expressed genes, clustering, classification and annotation. In its current version, Gene ARMADA fully supports 2 coloured cDNA and Affymetrix oligonucleotide arrays, plus custom arrays for which experimental details are given in tabular form (Excel spreadsheet, comma separated values, tab-delimited text formats). It also supports the analysis of already processed results through its versatile import editor. Besides being fully automated, Gene ARMADA incorporates numerous functionalities of the Statistics and Bioinformatics Toolboxes of MATLAB. In addition, it provides numerous visualization and exploration tools plus customizable export data formats for seamless integration by other analysis tools or MATLAB, for further processing. Gene ARMADA requires MATLAB 7.4 (R2007a) or higher and is also distributed as a stand-alone application with MATLAB Component Runtime.ConclusionGene ARMADA provides a highly adaptable, integrative, yet flexible tool which can be used for automated quality control, analysis, annotation and visualization of microarray data, constituting a starting point for further data interpretation and integration with numerous other tools.