Lipase Improvement: Goals and strategies

Lipases have received great attention as industrial biocatalysts in areas like oils and fats processing, detergents, baking, cheese making, surface cleaning, or fine chemistry . They can catalyse reactions of insoluble substrates at the lipid-water interface, preserving their catalytic activity in organic solvents. This makes of lipases powerful tools for catalysing not only hydrolysis, but also various reverse reactions such as esterification, transesterification, aminolysis, or thiotransesterifications in anhydrous organic solvents. Moreover, lipases catalyse reactions with high specificity, regio and enantioselectivity, becoming the most used enzymes in synthetic organic chemistry. Therefore, they display important advantages over classical catalysts, as they can catalyse reactions with reduced side products, lowered waste treatment costs, and under mild temperature and pressure conditions. Accordingly, the use of lipases holds a great promise for green and economical process chemistry.

Lipase Improvement: Goals and strategies

Lipases have received great attention as industrial biocatalysts in areas like oils and fats processing, detergents, baking, cheese making, surface cleaning, or fine chemistry . They can catalyse reactions of insoluble substrates at the lipid-water interface, preserving their catalytic activity in organic solvents. This makes of lipases powerful tools for catalysing not only hydrolysis, but also various reverse reactions such as esterification, transesterification, aminolysis, or thiotransesterifications in anhydrous organic solvents. Moreover, lipases catalyse reactions with high specificity, regio and enantioselectivity, becoming the most used enzymes in synthetic organic chemistry. Therefore, they display important advantages over classical catalysts, as they can catalyse reactions with reduced side products, lowered waste treatment costs, and under mild temperature and pressure conditions. Accordingly, the use of lipases holds a great promise for green and economical process chemistry.

Changes in lipoprotein lipase modulate tissular energy supply during stress

We studied the variations caused by stress in lipoprotein lipase (LPL) activity, LPL-mRNA, and local blood flow in LPL-rich tissues in the rat. Stress was produced by body immobilization (Immo): the rat's limbs were taped to metal mounts, and its head was placed in a plastic tube. Chronic stress (2 h daily of Immo) decreased total LPL activity in mesenteric and epididymal white adipose tissue (WAT) and was accompanied by a weight reduction of these tissues. In limb muscle, heart, and adrenals, total LPL activity and mRNA levels increased, and, in plasma, LPL activity and mass also increased. Acute stress (30-min Immo) caused a decrease in total LPL activity only in retroperitoneal WAT and an increase in preheparin plasma active LPL, but the overall weight of this tissue did not vary significantly. We propose an early release of the enzyme from this tissue into the bloodstream by some unknown extracellular pathways or other local mechanisms. These changes in this key energy-regulating enzyme are probably induced by catecholamines. They modify the flow of energy substrates between tissues, switching the WAT from importer to exporter of free fatty acids and favoring the uptake by muscle of circulating triacylglycerides for energy supply. Moreover, we found that acute stress almost doubled blood flow in all WAT studied, favoring the export of free fatty acids.

Nitric oxide and the release of lipoprotein lipase from white adipose tissue

Background/Aim: Lipoprotein lipase (LPL) is the main enzyme responsible for the distribution of circulating triacylglycerides in tissues. Its regulation via release from active sites in the vascular endothelium is poorly understood. In a previous study we reported that in response to acute immobilization (IMMO), LPL activity rapidly increases in plasma and decreases in white adipose tissue (WAT) in rats. In other stress situations IMMO triggers a generalized increase in nitric oxide (NO) production. Methods/Results: Here we demonstrate that in rats: 1) in vivo acute IMMO rapidly increases NO concentrations in plasma 2) during acute IMMO the WAT probably produces NO via the endothelial isoform of nitric oxide synthase (eNOS) from vessels, and 3) epididymal WAT perfused in situ with an NO donor rapidly releases LPL from the endothelium. Conclusion: We propose the following chain of events: stress stimulus / rapid increase of NO production in WAT (by eNOS) / release of LPL from the endothelium in WAT vessels. This chain of events could be a new mechanism that promotes the rapid decrease of WAT LPL activity in response to a physiological stimulus.

Combining phospholipases and a liquid lipase for one-step biodiesel production using crude oils

Background Enzymatic biodiesel is becoming an increasingly popular topic in bioenergy literature because of its potential to overcome the problems posed by chemical processes. However, the high cost of the enzymatic process still remains the main drawback for its industrial application, mostly because of the high price of refined oils. Unfortunately, low cost substrates, such as crude soybean oil, often release a product that hardly accomplishes the final required biodiesel specifications and need an additional pretreatment for gums removal. In order to reduce costs and to make the enzymatic process more efficient, we developed an innovative system for enzymatic biodiesel production involving a combination of a lipase and two phospholipases. This allows performing the enzymatic degumming and transesterification in a single step, using crude soybean oil as feedstock, and converting part of the phospholipids into biodiesel. Since the two processes have never been studied together, an accurate analysis of the different reaction components and conditions was carried out. Results Crude soybean oil, used as low cost feedstock, is characterized by a high content of phospholipids (900 ppm of phosphorus). However, after the combined activity of different phospholipases and liquid lipase Callera Trans L, a complete transformation into fatty acid methyl esters (FAMEs >95%) and a good reduction of phosphorus (P <5 ppm) was achieved. The combination of enzymes allowed avoidance of the acid treatment required for gums removal, the consequent caustic neutralization, and the high temperature commonly used in degumming systems, making the overall process more eco-friendly and with higher yield. Once the conditions were established, the process was also tested with different vegetable oils with variable phosphorus contents. Conclusions Use of liquid lipase Callera Trans L in biodiesel production can provide numerous and sustainable benefits. Besides reducing the costs derived from enzyme immobilization, the lipase can be used in combination with other enzymes such as phospholipases for gums removal, thus allowing the use of much cheaper, non-refined oils. The possibility to perform degumming and transesterification in a single tank involves a great efficiency increase in the new era of enzymatic biodiesel production at industrial scale.

Spontaneous Emergence of Chirality in the Limited Enantioselectivity Model: Autocatalytic Cycle Driven by an External Reagent

The emergence of chirality in enantioselective autocatalysis for compounds unable to transform according to the Frank-like reaction network is discussed with respect to the controversial limited enantioselectivity (LES) model composed of coupled enantioselective and non-enantioselective autocatalyses. The LES model cannot lead to spontaneous mirror symmetry breaking (SMSB) either in closed systems with a homogeneous temperature distribution or in closed systems with a stationary non-uniform temperature distribution. However, simulations of chemical kinetics in a two-compartment model demonstrate that SMSB may occur if both autocatalytic reactions are spatially separated at different temperatures in different compartments but coupled under the action of a continuous internal flow. In such conditions, the system can evolve, for certain reaction and system parameters, toward a chiral stationary state; that is, the system is able to reach a bifurcation point leading to SMSB. Numerical simulations in which reasonable chemical parameters have been used suggest that an adequate scenario for such a SMSB would be that of abyssal hydrothermal vents, by virtue of the typical temperature gradients found there and the role of inorganic solids mediating chemical reactions in an enzyme-like role.

Effects of heparin on the uptake of lipoprotein lipase in rat liver

BACKGROUND: Lipoprotein lipase (LPL) is anchored at the vascular endothelium through interaction with heparan sulfate. It is not known how this enzyme is turned over but it has been suggested that it is slowly released into blood and then taken up and degraded in the liver. Heparin releases the enzyme into the circulating blood. Several lines of evidence indicate that this leads to accelerated flux of LPL to the liver and a temporary depletion of the enzyme in peripheral tissues. RESULTS: Rat livers were found to contain substantial amounts of LPL, most of which was catalytically inactive. After injection of heparin, LPL mass in liver increased for at least an hour. LPL activity also increased, but not in proportion to mass, indicating that the lipase soon lost its activity after being bound/taken up in the liver. To further study the uptake, bovine LPL was labeled with 125I and injected. Already two min after injection about 33 % of the injected lipase was in the liver where it initially located along sinusoids. With time the immunostaining shifted to the hepatocytes, became granular and then faded, indicating internalization and degradation. When heparin was injected before the lipase, the initial immunostaining along sinusoids was weaker, whereas staining over Kupffer cells was enhanced. When the lipase was converted to inactive before injection, the fraction taken up in the liver increased and the lipase located mainly to the Kupffer cells. CONCLUSIONS: This study shows that there are heparin-insensitive binding sites for LPL on both hepatocytes and Kupffer cells. The latter may be the same sites as those that mediate uptake of inactive LPL. The results support the hypothesis that turnover of endothelial LPL occurs in part by transport to and degradation in the liver, and that this transport is accelerated after injection of heparin.

Bioequivalence of meat products treated with high pressure processing

The effects of high pressure on the composition of food products have not been evaluated extensively. Since, it is necessary to take in consideration the possible effects in basis to the changes induced in the bio molecules by the application of high pressures. The main effect on protein is the denaturation, because the covalent bonds are not affected; however hydrogen bonding, hydrophobic and intermolecular interactions are modified or destroyed. 1 High pressure can modify the activity of some enzymes. If this is done the proteolysis and lipolysis could be more or less intense and the content of free amino acids and fatty acids will be different. This could be related to the bioavailability of these compounds. Low pressures (100 MPa) have been shown to activate some enzymes (monomeric enzymes). Higher pressures induce loss of the enzyme activity. However some enzymes are very stable (ex. Lipase ~ 600 - 1000 MPa). Lipoxygenase is less stable, and there is little information about the effects on antioxidant enzymes. Other important issue is the influence of high pressure on oxidation susceptibility. This could modify the composition of lipids if the degree of the oxidation would have been higher or lower than in the traditional product. Pressure produces the damage of cell membranes favouring the contact between substrates and enzymes, exposure to oxidation of membrane fatty acids and loos of the efficiency of vitamin E. These effects can also affect to protein oxidation. In this study different compounds were analysed to establish the differences between non-treated and high-pressure treated products.

Paper de la PAP en la resposta inflamatoria durant la pancreatitis aguda

Estudi elaborat a partir d’una estada al Institut national de la santé et de la recherche médicale, França, entre setembre i octubre del 2006. La PAP va ser identificada inicialment com una proteïna de secreció, que apareixia al suc pancreàtic després de la inducció d’una pancreatitis aguda experimental. Per la PAP s’ha suggerit diferents funcions, algunes no relacionades en aparença, però les més interessants en el camp de la pancreatitis són les activitats antiapoptótiques, mitogéniques i, especialment, antiinflamatóries. Per aprofundir en aquests aspectes de la PAP, s’ha emprat un model de ratolí PAP-/- per observar els efectes de la deleció del gen de la PAP durant la pancreatitis aguda.Per induir la pancreatitis es va fer servir el model de administració de ceruleina a dosi supra-màximes. En aquestes condicions es va observar que en els animals PAP-/-, la severitat del procés era menor. Els marcadors de necrosi pancreàtica, lipasa i amilasa, van presentar nivells menors en els animals PAP-/- que en els corresponents wild type. Per contra, el nombre de PMN infiltrats i la producció de citoquines pro-inflamatories va ser major en el pàncreas dels animals PAP-/-. La intensitat de la resposta inflamatòria observada suggereix que en condicions fisiològiques, el paper anti-inflamatori de la PAP es prou rellevant. Això ja s’havia suggerit en estudis in vitro, en els que es va demostrar que l’activitat antiinflamatòria de la PAP depenia de la via de transducció de senyal Jak/STAT/SOCS3. Aquesta hipòtesi s’ha comprovat in vivo, monitoritzant el nivell d’activació de STAT3 en els pàncreas dels animals després de la inducció de la pancreatitis.Tot plegat confirma que les funcions antiinflamatòries descrites in vitro per la PAP també es poden observar in vivo, de manera que la PAP sembla ser un agent important en la resposta de les cèl•lules pancreàtiques durant la pancreatitis aguda.

Preparació, estudi estructural i aplicabilitat de derivats de Poli-(4R)-hidroxi-L-prolina a la separació d’enantiòmers per tècniques cromatogràfiques o relacionades

La separació d’enantiòmers (isòmers òptics) és molt important en molts diversos camps, com les síntesis quirals, biologia, i en el camp de la farmacologia especialment. És per això, que es fa necessari de disposar de tècniques i mètodes analítics ràpids, fiables i sensibles per a la separació d’enantiòmers. La present tesi s’emmarca en el camp de la separació d’enantiòmers, concretament en la preparació de fases estacionàries quirals per al seu ús en cromatografia liquida. En aquest sentit, s’ha desenvolupat la síntesi i caracterització de molècules polimèriques quirals derivades de l’aminoàcid L-prolina que incorporades en matrius de gel de sílice poden constituïr columnes quirals per a la separació d’enantiòmers per cromatografia liquida. S’han estudiat les característiques enantioselectives d’aquests nous materials en la separació de molècules quirals, trobant-se ésser satisfactòriament enantioselectius. L’interès que suscita l’obtenció d’enantiòmers a gran escala fa que la recerca s’orienti a la recerca de materials amb elevada capacitat de càrrega, és a dir, que puguin donar lloc a la separació d’elevades quantitats d’enantiòmers. Amb aquesta finalitat s’han dut a terme assaigs de capacitat de càrrega, que han posat de manifest la possible aplicació d’aquests materials a la separació preparativa d’enantiòmers. També s’ha parat especial atenció a l’estudi de les característiques de la matriu de gel de sílice, assajant-se altres materials de sílice més porosos i que permeten així treballar amb fluxos més elevats tot reduint-ne el temps d’anàlisi i els costos associats a la separació preparativa d’enantiòmers. L'estudi conformacional d'aquests nous selectors també ha estat contemplat per tal d'explicar l'enantioselectivitat específica que s'observa en certs dissolvents orgànics en els qual es duu a terme la separació dels enantiòmers.

Teleost fish larvae adapt to dietary arachidonic acid supply through modulation of the expression of lipid metabolism and stress response genes

Dietary fatty acid supply can affect stress response in fish during early development. Although knowledge on the mechanisms involved in fatty acid regulation of stress tolerance is scarce, it has often been hypothesised that eicosanoid profiles can influence cortisol production. Genomic cortisol actions are mediated by cytosolic receptors which may respond to cellular fatty acid signalling. An experiment was designed to test the effects of feeding gilthead sea-bream larvae with four microdiets, containing graded arachidonic acid (ARA) levels (0·4, 0·8, 1·5 and 3·0 %), on the expression of genes involved in stress response (steroidogenic acute regulatory protein, glucocorticoid receptor and phosphoenolpyruvate carboxykinase), lipid and, particularly, eicosanoid metabolism (hormone-sensitive lipase, PPARα, phospholipase A2, cyclo-oxygenase-2 and 5-lipoxygenase), as determined by real-time quantitative PCR. Fish fatty acid phenotypes reflected dietary fatty acid profiles. Growth performance, survival after acute stress and similar whole-body basal cortisol levels suggested that sea-bream larvae could tolerate a wide range of dietary ARA levels. Transcription of all genes analysed was significantly reduced at dietary ARA levels above 0·4 %. Nonetheless, despite practical suppression of phospholipase A2 transcription, higher leukotriene B4 levels were detected in larvae fed 3·0 % ARA, whereas a similar trend was observed regarding PGE2 production. The present study demonstrates that adaptation to a wide range of dietary ARA levels in gilthead sea-bream larvae involves the modulation of the expression of genes related to eicosanoid synthesis, lipid metabolism and stress response. The roles of ARA, other polyunsaturates and eicosanoids as signals in this process are discussed.

Short-term oleoyl-estrone treatment affects capacity to manage lipids in rat adipose tissue

Background: Short-term OE (oleoyl-estrone) treatment causes significant decreases in rat weight mainly due to adipose tissue loss. The aim of this work was to determine if OE treatment affects the expression of genes that regulate lipid metabolism in white adipose tissue. Results: Gene expression in adipose tissue from female treated rats (48 hours) was analysed by hybridization to cDNA arrays and levels of specific mRNAs were determined by real-time PCR. Treatment with OE decreased the expression of 232 genes and up-regulated 75 other genes in mesenteric white adipose tissue. The use of real-time PCR validate that, in mesenteric white adipose tissue, mRNA levels for Lipoprotein Lipase (LPL) were decreased by 52%, those of Fatty Acid Synthase (FAS) by 95%, those of Hormone Sensible Lipase (HSL) by 32%, those of Acetyl CoA Carboxylase (ACC) by 92%, those of Carnitine Palmitoyltransferase 1b (CPT1b) by 45%, and those of Fatty Acid Transport Protein 1 (FATP1) and Adipocyte Fatty Acid Binding Protein (FABP4) by 52% and 49%, respectively. Conversely, Tumour Necrosis Factor (TNF¿) values showed overexpression (198%). Conclusion: Short-term treatment with OE affects adipose tissue capacity to extract fatty acids from lipoproteins and to deal with fatty acid transport and metabolism.

Spontaneous mirror symmetry breaking in the limited enantioselective autocatalysis model: abyssal hydrotermal vents as scenario for the emergence of chirality in prebiotic chemistry

The emergence of chirality in enantioselective autocatalysis for compounds unable to transform according to the Frank-like reaction network is discussed with respect to the controversial limited enantioselectivity (LES) model composed of coupled enantioselective and non-enantioselective autocatalyses. The LES model cannot lead to spontaneous mirror symmetry breaking (SMSB) either in closed systems with a homogeneous temperature distribution or in closed systems with a stationary non-uniform temperature distribution. However, simulations of chemical kinetics in a two-compartment model demonstrate that SMSB may occur if both autocatalytic reactions are spatially separated at different temperatures in different compartments but coupled under the action of a continuous internal flow. In such conditions, the system can evolve, for certain reaction and system parameters, toward a chiral stationary state; that is, the system is able to reach a bifurcation point leading to SMSB. Numerical simulations in which reasonable chemical parameters have been used suggest that an ade- quate scenario for such a SMSB would be that of abyssal hydrothermal vents, by virtue of the typical temper- ature gradients found there and the role of inorganic solids mediating chemical reactions in an enzyme-like role. Key Words: Homochirality Prebiotic chemistry.

Synthetic applications of chiral unsaturated epoxy alcohols prepared by sharpless asymmetric epoxidation

An overview of the synthesis and applications of chiral 2,3-epoxy alcohols containing unsaturated chains is presented. One of the fundamental synthetic routes to these compounds is Sharpless asymmetric epoxidation, which is reliable, highly chemoselective and enables easy prediction of the product enantioselectivity. Thus, unsaturated epoxy alcohols are readily obtained by selective oxidation of the allylic double bond in the presence of other carbon-carbon double or triple bonds. The wide availability of epoxy alcohols with unsaturated chains, the versatility of the epoxy alcohol functionality (e.g. regio- and stereo-selective ring opening; oxidation; and reduction), and the arsenal of established alkene chemistries, make unsaturated epoxy alcohols powerful starting materials for the synthesis of complex targets such as biologically active molecules. The popularization of ring-closing metathesis has further increased their value, making them excellent precursors to cyclic compounds.

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.