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.

Preparation of (S)-1-halo-2-octanols using ionic liquids and biocatalysts

Preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol was carried out by the enzymatic hydrolysis of halohydrin palmitates using biocatalysts. Halohydrin palmitates were prepared by various methods from palmitic acid and 1,2-octanediol. A tandem hydrolysis was carried out using lipases from Candida antarctica (Novozym® 435), Rhizomucor miehei (Lipozyme IM), and “resting cells” from a Rhizopus oryzae strain that was not mycotoxigenic. The influence of the enzyme and the reaction medium on the selective hydrolysis of isomeric mixtures of halohydrin esters is described. Novozym® 435 allowed preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol after 1–3 h ofreaction at 40 °C in [BMIM][PF6].

Esterification of oleic acid catalayzed by an Aspergillus niger strain. Influence of water activity and alcohol concentration

Effects of water activity and 1-propanol concentration on synthesis of propyl oleate from oleic acid using Aspergillus niger cell-bound lipases in isooctane are described. A. niger produces lipases (EC 3.1.1.3) which partly bind to the mycelium during growth. Ester production was monitored for 72 hours at different 1-propanol concentrations and water activities. Aliquots were sequentially withdrawn and propyl esters were quantified using GC and methyl palmitate as an internal standard. In all assayed conditions A. niger cell-bound lipases catalysed propyl oleate synthesis, but at different degrees.

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.

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.

Localización de la lipoproteína lipasa en el cuerpo ciliar de ojos humanos: Un estudio inmunocitoquímico

La Lipoproteína lipasa (LPL, E.C. 3.1.1.34) es una glucoproteína sintetizada por diferentes tipos celulares, principalmente en adipocitos, células musculares y marcófagos.

Localización de la lipoproteína lipasa en el cuerpo ciliar de ojos humanos: Un estudio inmunocitoquímico

La Lipoproteína lipasa (LPL, E.C. 3.1.1.34) es una glucoproteína sintetizada por diferentes tipos celulares, principalmente en adipocitos, células musculares y marcófagos.

Optimisation of the lipase-catalysed preparation of a nucleoside prodrug model using an experimental design methodology

The preparation of 2', 3'-di-O-hexanoyluridine (2) by a Candida antarctica B lipase-catalysed alcoholysis of 2', 3', 5'-tri-O-hexanoyluridine (1) was optimised using an experimental design. At 25 ºC better experimental conditions allowed an increase in the yield of 2 from 80% to 96%. In addition to the yield improvement, the volume reaction could be diminished in a factor of 5 and the reaction time significantly shortened.

Estudo da influência do solvente, carboidrato e ácido graxo na síntese enzimática de ésteres de açúcares

The aim of this work was to gain knowledge of enzymatic processes for the synthesis fatty acid esters of sugar, with the objective to develop an enzymatic process for the preparation of non-toxic biodegradable surface-active agents derived entirely from renewable resources. A wide range of data were collected for reaction conditions involving different sugars (glucose, fructose and sucrose), fatty acids (oleic, palmitic, lauric), solvents (hexane, heptane and t-butanol) and different sources of lipases in both free and immobilized forms. As a solvent t-butanol provided the best conditions to create a catalytic liquid phase in which the reaction occurs. Sugars were preferentially esterified in the following order: fructose > glucose > sucrose, depending on the enzyme preparation. For fructose no influence was found concerning de acyl donor and similar rates were achieved for all tested fatty acids. Ester synthesis was maximized for substrates containing fructose, lauric or oleic acids, t-butanol and lipase from porcine pancreas immobilized on polysiloxane-polyvinyl alcohol particles. Under such conditions molar conversions were higher than 50%.

Comportamento de cristalização de lipídios estruturados obtidos a partir de gordura de palmiste e óleo de peixe

The aim of this study is to evaluate the crystal structure of binary mixtures of palm kernel fat and fish oil, before and after chemical and enzymatic interesterification. The crystal structure was analyzed by polarized light microscopy. The addition of fish oil didn't change the palm kernel fat crystallization characteristics, spherullites of types A and B being observed. However, due to chemical and enzymatic interesterification, smaller crystals were obtained. There was no difference between chemical and enzymatic interesterification, probably as a function of acyl migration in discontinuous processes catalyzed by lipases.

Co-produção de lipase e biossurfactante em estado sólido para utilização em biorremediação de óleos vegetais e hidrocarbonetos

Recently lipases have been increasing in prominence due to its wide industrial application. The lipase production can be influenced by different variables such as the producing microorganism, carbon sources, aeration and agitation conditions, inductor type and the geometry of the reactor. Biosurfactants are composites of surface active produced by microbial cells which reduce superficial and interfacial tensions. The objective of this study was to verify the influence of different process variables in the lipase production during a fermentative process. The results showed that the concomitant production of lipases and biosurfactant was possible in different cultivation conditions.

Cutinases fúngicas: propriedades e aplicações industriais

Cutinases (EC 3.1.1.74) are also known as cutin hidrolases. These enzymes share catalytic properties of lipases and esterases, presenting a unique feature of being active regardless the presence of an oil-water interface, making them interesting as biocatalysts in several industrial processes involving hydrolysis, esterification and trans-esterification reactions. They are also active in different reaction media, allowing their applications in different areas such as food industry, cosmetics, fine chemicals, pesticide and insecticide degradation, treatment and laundry of fiber textiles and polymer chemistry. The present review describes the characteristics, potential applications and new perspectives for these enzymes.