Showing papers on "Lipase published in 2013"

Immobilisation and application of lipases in organic media.

Abstract: Different methods of preparing lipases for use in organic media are critically reviewed. Solid lipase preparations can be made by typical immobilisation methods such as adsorption, entrapment, covalent coupling or cross-linking. Immobilisation is especially attractive for lipases because, in addition to the normal benefits of enzyme immobilisation, it can also lead to a considerable increase in catalytic activity, probably caused by conformational changes in the lipase molecules. Activation can be achieved, for example, using hydrophobic support materials or surfactants during the immobilisation procedure. Surfactants can also be used to solubilise lipases in organic media via the formation of hydrophobic ion pairs, surfactant-coated lipase or reversed micelles. Lipase preparation methods are discussed with regard to potential lipase inactivation and activation effects, mass transfer limitations, lipase stability and other features important for applications. The practical applications of lipases in organic media reviewed include ester synthesis, modification of triacylglycerols and phospholipids, fatty acid enrichment, enantiomer resolution, biodiesel production and acylation of carbohydrates and bioactive compounds.

Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities

Abstract: Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C6–C18) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C8–C16) or fatty alkanes (C7–C15) was reconstituted in vitro. This concept was applied in vivo, in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L−1 was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C8–C18). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities.

Enzyme immobilisation on amino-functionalised multi-walled carbon nanotubes: structural and biocatalytic characterisation.

Abstract: Background: The aim of this work is to investigate the structure and function of enzymes immobilised on nanomaterials. This work will allow better understanding of enzyme-nanomaterial interactions, as well as designing functional proteinnanomaterial conjugates. Methodology/Principal Findings: Multiwalled carbon nanotubes (MWNTs) were functionalised with amino groups to improve solubility and biocompatibility. The pristine and functionalised forms of MWNTs were characterised with Fouriertransform infrared spectroscopy. Thermogravimetric analysis was done to examine the degree of the functionalisation process. An immobilised biocatalyst was prepared on functionalised nanomaterial by covalent binding. Thermomyces lanuginosus lipase was used as a model enzyme. The structural change of the immobilised and free lipases were characterised with transmission electron Microscopy, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy and Circular dichroism spectroscopy. Biochemical characterisation of immobilised enzyme showed broader pH and thermal optima compared to soluble form. Reusability of the immobilised enzyme for hydrolysis of long chain esters was demonstrated up to ten cycles. Conclusion/Significance: Lipase immobilised on MWNTs has exhibited significantly improved thermal stability. The exploration of advanced nanomaterial for enzyme immobilisation support using sophisticated techniques makes nanobiocatalyst of potential interest for biosensor applications.

Angptl3 Deficiency Is Associated With Increased Insulin Sensitivity, Lipoprotein Lipase Activity, and Decreased Serum Free Fatty Acids

Abstract: Objective— Angiopoietin-like 3 (Angptl3) is a regulator of lipoprotein metabolism at least by inhibiting lipoprotein lipase activity. Loss-of-function mutations in ANGPTL3 cause familial combined hypolipidemia through an unknown mechanism. Approach and Results— We compared lipolytic activities, lipoprotein composition, and other lipid-related enzyme/lipid transfer proteins in carriers of the S17X loss-of-function mutation in ANGPTL3 and in age- and sex-matched noncarrier controls. Gel filtration analysis revealed a severely disturbed lipoprotein profile and a reduction in size and triglyceride content of very low density lipoprotein in homozygotes as compared with heterozygotes and noncarriers. S17X homozygotes had significantly higher lipoprotein lipase activity and mass in postheparin plasma, whereas heterozygotes showed no difference in these parameters when compared with noncarriers. No changes in hepatic lipase, endothelial lipase, paraoxonase 1, phospholipid transfer protein, and cholesterol ester transfer protein activities were associated with the S17X mutation. Plasma free fatty acid, insulin, glucose, and homeostatic model assessment of insulin resistance were significantly lower in homozygous subjects compared with heterozygotes and noncarriers subjects. Conclusions— These results indicate that, although partial Angptl3 deficiency did not affect the activities of lipolytic enzymes, the complete absence of Angptl3 results in an increased lipoprotein lipase activity and mass and low circulating free fatty acid levels. This latter effect is probably because of decreased mobilization of free fatty acid from fat stores in human adipose tissue and may result in reduced hepatic very low density lipoprotein synthesis and secretion via attenuated hepatic free fatty acid supply. Altogether, Angptl3 may affect insulin sensitivity and play a role in modulating both lipid and glucose metabolism.

Parameters Affecting the Performance of Immobilized Enzyme

Abstract: Enzyme immobilization has been investigated to improve lipase properties over the past few decades. Different methods and various carriers have been employed to immobilize enzyme. However, the application of enzymatic technology in large scale is rarely seen during the industrial process. The main obstacles are a high cost of the immobilization and the poor performance of immobilized lipase. This review focuses on the current status of enzyme immobilization, which aims to summarize the latest research on the parameters affecting the performance of immobilized enzyme. Particularly, the effect of immobilization methods, immobilization carriers, and enzyme loading has been discussed.

Dieselzymes: development of a stable and methanol tolerant lipase for biodiesel production by directed evolution.

Abstract: Background: Biodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed transesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially. Results: We employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the halfinactivation time at 50°C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis. Conclusions: Directed evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.

Lipase applications in oil hydrolysis with a case study on castor oil: a review

Abstract: Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.

Lipase in aqueous-polar organic solvents: Activity, structure, and stability

Abstract: Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ∼20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]-HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active-site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased Km); however significantly enhanced the kcat. Melting temperature (Tm) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.

Characterization of Binding Interactions of (-)-Epigallocatechin-3-gallate from Green Tea and Lipase

Abstract: Understanding the interaction of (-)-epigallocatechin-3-gallate (EGCG) and lipase is important for understanding EGCG's inhibition of lipase In this paper, the interaction of EGCG and porcine lipase was characterized by fluorescence spectroscopy, circular dichroism (CD), isothermal titration calorimetry, and molecular docking EGCG might act as a noncompetitive pancreatic lipase inhibiter EGCG bound to lipase with affinity of K(a) = 270 × 10⁴ L mol⁻¹ Thermodynamic features suggested that the interaction process was spontaneous, with hydrogen bonds and electrostatic force perhaps primarily responsible for the interaction, with 1:1 interaction of lipase and EGCG CD studies indicated conformation change of lipase on binding to EGCG Furthermore, docking results supported experimental findings and revealed hydrogen-bonding interaction with Val21, Glu188, and Glu220 This noncovalent bonding between EGCG and lipase alters the molecular conformation of lipase, which decreases the enzyme catalytic activity This study will help further understand the antiobesity mechanisms of green tea

Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library

Abstract: A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K m and k cat of 39 μM and 25.8 s(-1), respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 A resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80-114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.

Enzyme Immobilization on Carboxyl-Functionalized Graphene Oxide for Catalysis in Organic Solvent

Abstract: Carboxyl-functionalized graphene oxide (GO–COOH) was utilized to immobilize lipase. Fourier transform infrared (FTIR), UV–visible, and X-ray photoelectron (XPS) spectra were measured to characterize lipase immobilization. At the optimal temperature of 40 °C, the immobilized lipase retains 80% of the hydrolytic activity of the native lipase. For catalyzing the enantioselective reaction in the organic solvent heptane, at 50 °C (optimal), the catalysis efficiency of the immobilized lipase is 1.6 times that of native lipase, and the immobilized lipase retains the selectivity of the native lipase. This work demonstrates that graphene oxide is a suitable support for immobilization of lipase for catalysis in organic solvent.

Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa

Abstract: As an opportunistic human pathogen Pseudomonas aeruginosa is able to cause acute and chronic infections. The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa during an infection process and mediates the pathogenicity of the bacterium. Within a biofilm mucoid strains of P. aeruginosa simultaneously produce and secrete several hydrolytic enzymes and the extracellular polysaccharide alginate. The focus of the current study was the interaction between extracellular lipase LipA and alginate, which may be physiologically relevant in biofilms of mucoid P. aeruginosa. Fluorescence microscopy of mucoid P. aeruginosa biofilms were performed using fluorogenic lipase substrates. It showed a localization of the extracellular enzyme near the cells. A microtiter plate-based binding assay revealed that the polyanion alginate is able to bind LipA. A molecular modeling approach showed that this binding is structurally based on electrostatic interactions between negatively charged residues of alginate and positively charged amino acids of the protein localized opposite of the catalytic centre. Moreover, we showed that the presence of alginate protected the lipase activity by protection from heat inactivation and from degradation by the endogenous, extracellular protease elastase LasB. This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain. We demonstrate that the extracellular lipase LipA from P. aeruginosa interacts with the polysaccharide alginate in the self-produced extracellular biofilm matrix of P. aeruginosa via electrostatic interactions suggesting a role of this interaction for enzyme immobilization and accumulation within biofilms. This represents a physiological advantage for the cells. Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed towards the substrate and is protected from denaturation and proteolytic degradation.

Immobilized Burkholderia cepacia lipase for biodiesel production from crude Jatropha curcas L. oil

Abstract: The present world is in a dilemma with fast depletion of fossil fuels. So there arises a drastic need for development of biofuels to power the earth. From an environmental point of view, biodiesel has great potential as an alternative diesel fuel. In this study, lipase from Burkholderia cepacia was first cross linked with glutaraldehyde followed by entrapment in a hybrid matrix of equal proportions of alginate and kappa-carrageenan. Later, this biocatalyst was employed for biodiesel production from crude Jatropha curcas L. oil. The optimal conditions for processing 10 g crude Jatropha oil were: 35 degrees C, 1:10 mol ratio of oil to ethanol, 1 g water, 5.25 g immobilized lipase, 6 g RCF and 24 h reaction time. At the optimal conditions, 100% yield of fatty acid ethyl esters could be achieved. The immobilized lipase was stable and retained 73% relative transesterification activity after six cycles of reuse. This shows that the immobilized lipase in alginate/kappa-carrageenan matrix is a potential environmental friendly biocatalyst for biodiesel industry. (C) 2013 Elsevier Ltd. All rights reserved.