Showing papers on "Lipase published in 2008"

Enzymatic production of biodiesel from canola oil using immobilized lipase

Abstract: In the present work, a novel method for immobilization of lipase within hydrophilic polyurethane foams using polyglutaraldehyde was developed for the immobilization of Thermomyces lanuginosus lipase to produce biodiesel with canola oil and methanol. The enzyme optimum conditions were not affected by immobilization and the optimum pH for free and immobilized enzyme were 6, resulting in 80% immobilization yield. Using the immobilized lipase T. lanuginosus, the effects of enzyme loading, oil/alcohol molar ratio, water concentration, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 20 g of refined canola oil were: 430 μg lipase, 1:6 oil/methanol molar ratio, 0.1 g water and 40 °C for the reactions with methanol. Maximum methyl esters yield was 90% of which enzymatic activity remained after 10 batches, when tert-butanol was adopted to remove by-product glycerol during repeated use of the lipase. The immobilized lipase proved to be stable and lost little activity when was subjected to repeated uses.

Multipoint covalent immobilization of microbial lipase on chitosan and agarose activated by different methods

Abstract: In this work Candida antarctica lipase type B (CALB) was immobilized on agarose and chitosan. The influence of activation agents (glycidol, glutaraldehyde and epichlorohydrin) and immobilization time (5, 24 and 72 h) on hydrolytic activity, thermal and alkaline stabilities of the biocatalyst was evaluated. Protein concentration and enzymatic activity in the supernatant were determined during the immobilization process. More active derivatives were attained when the enzymatic extract was first purified through dialysis. The highest activities achieved were: for agarose-glyoxyl (with glycidol), 845 U/g of gel, after 72 h of immobilization; for chitosan-glutaraldehyde and agarose-glutaraldehyde, respectively, 1209 U/g of gel and 2716 U/g of gel, after 5 h of immobilization. Thermal stability was significantly increased, when compared to the soluble enzyme: 20-fold for agarose-glyoxyl (with glycidol)-CALB, 18-fold for chitosan-glutaraldehyde-CALB and 21-fold for agarose-glutaraldehyde. The best derivative, 58-fold more stable than the soluble enzyme, was obtained when CALB was immobilized on chitosan activated in two steps, using glycidol and glutaraldehyde, 72 h immobilization time. The stabilization degree of the derivative increased with the immobilization time, an indication that a multipoint covalent attachment between enzyme and the support had really occurred.

Modeling structure and flexibility of Candida antarctica lipase B in organic solvents

Abstract: Background The structure and flexibility of Candida antarctica lipase B in water and five different organic solvent models was investigated using multiple molecular dynamics simulations to describe the effect of solvents on structure and dynamics. Interactions of the solvents with the protein and the distribution of water molecules at the protein surface were examined.

Influence of lipid physical state on the in vitro digestibility of emulsified lipids.

Abstract: The objective of this study was to investigate the influence of the physical state of emulsified lipids on their in vitro digestibility by pancreatic lipase. A 10 wt % tripalmitin oil-in-water emulsion stabilized by sodium dodecyl sulfate (0.9 wt % SDS) was prepared at a temperature (>70 degrees C) above the melting point of the lipid phase (T(m) approximately 60 degrees C). A portion of this emulsion was cooled to a temperature (0 degrees C for 15 min) well below the crystallization temperature of the emulsified lipid (T(c) approximately 22 degrees C) and then warmed to 37 degrees C so as to have completely solid lipid particles. Another portion of the emulsion was directly cooled from 70 to 37 degrees C (which is above the T(c)) to have completely liquid (supercooled) lipid particles. Pancreatic lipase (8 mg/mL) and bile extract (5.0 mg/mL) were then added to each emulsion at 37 degrees C, and the evolution of the particle charge, particle size, appearance, and free fatty acid release were measured over a period of 2 h. It was found that the rate and extent of lipid digestion were higher in the emulsion containing liquid particles but that appreciable lipid digestion still occurred in the emulsion containing solid particles (i.e., >35% lipid digestion after 2 h). These results may have important consequences for controlling the digestion rate of lipids or for developing solid lipid particle delivery systems for lipophilic functional components.

Purification and Biochemical Characterization

Abstract: hydrolyticresistancetowardtriacylglycerideswithlongerfattyacylchainlengthcontainingunsaturation 12 as evident from the lower Vmax (0.23 mM/mg/min) of the lipase toward glycerol trioleate (C18:1n9) 13 comparedwiththefattyacidtriglycerideshavingshorttomediumcarbonchainlengths(C18:0-12:0,Vmax 14 0.32-0.51 mM/mg/min). This indicates a preferential specificity of the lipase toward cleaving shorter 15 carbon chain length fatty acid triglycerides. The lipase exhibited optimum activity at 40 C and pH 8.0, 16 respectively. A combination of Ca 2+ and sorbitol induced a synergistic effect on the thermostability of 17 lipasewithasignificantlyhighresidualactivity(100%)after30minat40C,ascomparedto90.6%after 18 incubation with Ca 2+ alone. The lipase activity was inhibited by Cu 2+ and Fe 2+ (42 and 48%, 19 respectively) at 10 mM. The enzyme lost 31% of its initial activity by 0.001 mM EDTA and 42% by 20 0.1 mM EDTA. Significant reduction in lipase activity was apparent by 2-mercaptoethanol and 21 phenylmethanesulfonyl fluoride at diluted concentration (0.001 mM), thereby indicating an important 22 role of sulfhydryl groups in the catalytic mechanism. 23 24

Function of a novel GDSL-type pepper lipase gene, CaGLIP1, in disease susceptibility and abiotic stress tolerance

Abstract: GDSL-type lipase is a hydrolytic enzyme whose amino acid sequence contains a pentapeptide motif (Gly-X-Ser-X-Gly) with active serine (Ser). Pepper GDSL-type lipase (CaGLIP1) gene was isolated and functionally characterized from pepper leaf tissues infected by Xanthomonas campestris pv. vesicatoria (Xcv). The CaGLIP1 protein was located in the vascular tissues of Arabidopsis root. The CaGLIP1 gene was preferentially expressed in pepper leaves during the compatible interaction with Xcv. Treatment with salicylic acid, ethylene and methyl jasmonate induced CaGLIP1 gene expression in pepper leaves. Sodium nitroprusside, methyl viologen, high salt, mannitol-mediated dehydration and wounding also induced early and transient CaGLIP1 expression in pepper leaf tissues. Virus-induced gene silencing of CaGLIP1 in pepper conferred enhanced resistance to Xcv, accompanied by the suppressed expression of basic PR1 (CaBPR1) and defensin (CaDEF1) genes. The CaGLIP1 lipase produced in Escherichia coli hydrolyzed the substrates of short and long chain nitrophenyl esters. The CaGLIP1-overexpressing Arabidopsis exhibited enhanced hydrolytic activity toward short and long chain nitrophenyl ester, as well as enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato and the biotrophic oomycete Hyaloperonospora parasitica. SA-induced expression of AtPR1 and AtGST1, also was delayed in CaGLIP1-overexpressing plants by SA application. During seed germination and plant growth, the CaGLIP1 transgenic plants showed drought tolerance and differential expression of drought- and abscisic acid (ABA)-inducible genes AtRD29A, AtADH and AtRab18. ABA treatment differentially regulated seed germination and gene expression in wild-type and CaGLIP1 transgenic Arabidopsis. Overexpression of CaGLIP1 also regulated glucose- and oxidative stress signaling. Together, these results indicate that CaGLIP1 modulates disease susceptibility and abiotic stress tolerance.

A modeling study by response surface methodology and artificial neural network on culture parameters optimization for thermostable lipase production from a newly isolated thermophilic Geobacillus sp. strain ARM

Abstract: Thermostable bacterial lipases occupy a place of prominence among biocatalysts owing to their novel, multifold applications and resistance to high temperature and other operational conditions The capability of lipases to catalyze a variety of novel reactions in both aqueous and nonaqueous media presents a fascinating field for research, creating interest to isolate novel lipase producers and optimize lipase production The most important stages in a biological process are modeling and optimization to improve a system and increase the efficiency of the process without increasing the cost Different production media were tested for lipase production by a newly isolated thermophilic Geobacillus sp strain ARM (DSM 21496 = NCIMB 41583) The maximum production was obtained in the presence of peptone and yeast extract as organic nitrogen sources, olive oil as carbon source and lipase production inducer, sodium and calcium as metal ions, and gum arabic as emulsifier and lipase production inducer The best models for optimization of culture parameters were achieved by multilayer full feedforward incremental back propagation network and modified response surface model using backward elimination, where the optimum condition was: growth temperature (523°C), medium volume (50 ml), inoculum size (1%), agitation rate (static condition), incubation period (24 h) and initial pH (58) The experimental lipase activity was 047 Uml-1 at optimum condition (47-fold increase), which compared well to the maximum predicted values by ANN (047 Uml-1) and RSM (0476 Uml-1), whereas R2 and AAD were determined as 0989 and 0059% for ANN, and 095 and 0078% for RSM respectively Lipase production is the result of a synergistic combination of effective parameters interactions These parameters are in equilibrium and the change of one parameter can be compensated by changes of other parameters to give the same results Though both RSM and ANN models provided good quality predictions in this study, yet the ANN showed a clear superiority over RSM for both data fitting and estimation capabilities On the other hand, ANN has the disadvantage of requiring large amounts of training data in comparison with RSM This problem was solved by using statistical experimental design, to reduce the number of experiments

Comparison among immobilised lipases on macroporous polypropylene toward biodiesel synthesis

Abstract: Eight commercial lipases were characterised (protein content and specific activity) and immobilised on macroporous polypropylene via physical adsorption. The lipases showed a different level of adaptation to the support as determined by the comparison of their catalytic efficiencies (activity/loading). The immobilised lipases were compared toward methanolysis of vegetable oil to obtain biodiesel in solvent-free conditions. Immobilised Pseudomonas fluorescens lipase was the most active biocatalyst (ester yield = 58 mol% after 22 h), followed by immobilised Pseudomonas cepacia lipase (ester yield = 37 mol% after 51.5 h), whereas all the other lipases (from Rhizopus oryzae , Candida rugosa , Mucor javanicus , Penicillium roqueforti , Aspergillus niger , Penicillium camembertii ), were inactive toward biodiesel synthesis. The effect of triglyceride feedstock, reaction temperature, water content, and enzyme loading was determined. Under the optimal conditions, i.e. soybean oil, T = 30 °C, water content = 0.5 mg water/mg of biocatalyst, loading = 600 mg lipase AK/g support, an ester yield of 98 mol% after 70 h was obtained.

Influence of Surfactants on Lipase Fat Digestion in a Model Gastro-intestinal System

Abstract: In the present study, we use a model gastro-intestinal system to study the influence of different food-grade surface-active molecules (Sn-2 monopalmitin, β-lactoglobulin, or lysophosphatodylcholine) on lipase activity. The interfacial activity of lipase and surfactants are assessed with the pendant drop technique, a commonly used tensiometry instrument. A mathematical model is adopted which enables quantitative determination of the composition of the water–oil interface as a function of bulk surfactant concentration in the water–oil mixtures. Our results show a decrease in gastric lipolysis when interfacially active molecules are incorporated into a food matrix. However, only the Sn-2 monopalmitin caused a systematic decrease in triglyceride hydrolysis throughout the gastro-intestinal tract. This effect is most likely due to exclusion of both lipase and triglyceride from the water–oil interface together with a probable saturation of the solubilization capacity of bile with monoglycerides. Addition of β-lactoglobulin or lysophopholipids increased the hydrolysis of fat after the gastric phase. These results can be attributed to an increasing interfacial area with lipase and substrate present at the interface. Otherwise, β-lactoglobulin, or lysophopholipids reduced fat hydrolysis in the stomach. From the mathematical modeling of the interface composition, we can conclude that Sn-2 monopalmitin can desorb lipase from the interface, which, together with exclusion of substrate from the interface, explains the gradually decreased triglyceride hydrolysis that occurs during the digestion. Our results provide a biophysics approach on lipolysis that can bring new insights into the problem of fat uptake.

Supported Ionic Liquid Enzymatic Catalysis for the Production of Biodiesel

Abstract: Pseudomonas cepacia lipase supported in the 1-n-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ionic liquid is an alternative “green” method for the production of biodiesel from the alcoholysis of soybean oil. The transesterification reaction catalyzed by this ionic liquid-supported enzyme can be performed at room temperature, in the presence of water and without the use of organic solvents. It is also compatible with various alcohols (including isoamyl alcohol). The biodiesel is separated by simple decantation and the recovered ionic liquid/enzyme catalytic system can be re-used at least four times without loss of catalytic activity and selectivity.