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José Manuel Guisán - One of the best experts on this subject based on the ideXlab platform.
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Ethyl esters production catalyzed by immobilized Lipases is influenced by n-hexane and ter-amyl alcohol as organic solvents
Bioprocess and Biosystems Engineering, 2020Co-Authors: Janaina Pires Borges, José Carlos Quilles Junior, Sônia Moreno-perez, Glória Fernandez-lorente, Mauricio Boscolo, Eleni Gomes, Roberto Silva, Daniela Alonso Bocchini, José Manuel GuisánAbstract:Lipase stability in organic solvent is crucial for its application in many biotechnological processes as biocatalyst. One way to improve lipase’s activity and stability in unusual reaction medium is its immobilization on inert supports. Here, Lipases from different sources and immobilized through weak chemical interactions on hydrophobic and ionic supports had their transesterification ability dramatically dependent on the support and also on the solvent that had been used. The ethanolysis of sardine oil was carried out at the presence of cyclohexane and tert-amyl alcohol, in which Duolite A568- Thermomyces lanuginosa lipase derivative achieved 49% of ethyl esters production after 24 h in cyclohexane. The selectivity of immobilized Lipases was also studied and, after 3 h of synthesis, the reaction with Duolite A568- Thermomyces lanuginosa derivative in cyclohexane produced 24% ethyl ester of eicosapentaenoic acid and 1.2% ethyl ester of docosahexaenoic acid, displaying a selectivity index of 20 times the ethyl ester of eicosapentaenoic acid. Different derivatives of Candida antarctica Lipases fraction B (CALB) and phospholipase Lecitase ^® Ultra (Lecitase) were also investigated. Along these lines, a combination between these factors may be applied to improve the activity and selectivity of immobilized Lipases, decreasing the total cost of the process.
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single step purification of different Lipases from staphylococcus warneri
Journal of Chromatography A, 2010Co-Authors: Giandra Volpato, Marco Filice, Marco Antonio Zachia Ayub, José Manuel Guisán, José Maria PalomoAbstract:Abstract Three different Lipases from the extract crude of Staphylococcus warneri have been purified by specific lipase–lipase interactions using different Lipases (TLL, RML, PFL, BTL2) covalently attached to a solid support as adsorption matrix. BTL2 immobilized on glyoxyl-DTT adsorbed selectivity only a 30 kDa lipase from the crude, which was desorbed by adding 0.1% triton X-100. Using glyoxyl-PFL as matrix, two new Lipases (28 and 40 kDa) were adsorbed, and completely pure 40 kDa lipase was obtained after desorption using 0.01% triton, whereas 28 kDa lipase was desorbed after the incubation of the lipase matrix with 3% detergent. When using other matrixes as glyoxyl-TLL or glyoxyl-RML, different Lipases were adsorbed. This methodology could be a very efficient and useful method to purify several Lipases from crude extracts from different sources.
Frédéric Carrière - One of the best experts on this subject based on the ideXlab platform.
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An ultraviolet spectrophotometric assay for the screening of sn-2-specific Lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate.
Journal of Lipid Research, 2012Co-Authors: Lilia D Mendoza, Frédéric Carrière, Jorge A Rodriguez, Julien Leclaire, Gerard Buono, Frédéric Fotiadu, Abdelkarim AbousalhamAbstract:In the present study, we propose a continuous assay for the screening of sn-2 Lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective Lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the Lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of Lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 Lipases.
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comparative study on digestive lipase activities on the self emulsifying excipient labrasol medium chain glycerides and peg esters
Biochimica et Biophysica Acta, 2007Co-Authors: Sylvie Fernandez, Vincent Jannin, Jeandavid Rodier, Nicolas Ritter, Bruno Mahler, Frédéric CarrièreAbstract:Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive Lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive Lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main Lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These Lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.
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Evidence for a pancreatic lipase subfamily with new kinetic properties.
Biochemistry, 1994Co-Authors: Kenneth Thirstrup, Robert Verger, Frédéric CarrièreAbstract:Several new members of the pancreatic lipase family have been reported recently, and amino acid sequence comparison reveals that this family can now be divided into three subgroups: (1) "classical" pancreatic Lipases, (2) related proteins 1 (RP1), and (3) related proteins 2 (RP2) (Giller, T., et al. (1992) J. Biol. Chem. 267(23), 16509-16516). Whereas "classical" pancreatic Lipases are well characterized with respect to kinetic properties, i.e., interfacial activation and dependence on colipase in the presence of bile salts, the two latter subfamilies have been poorly investigated so far. The kinetic behavior of a lipase from guinea pig pancreas differs, however, from that of "classical" Lipases (Hjorth, A., et al. (1993) Biochemistry 32, 4702-4707). This enzyme is highly homologous to RP2 Lipases with the exception of a deletion in the so-called lid domain that regulates access to the active center of pancreatic Lipases. We have now characterized a novel lipase from coypu (Myocastor coypus) pancreas. This enzyme, also belonging to the RP2 subfamily, possesses a full-length lid domain, but its kinetic properties are very similar to those of the guinea pig enzyme: (1) a high phospholipase activity, (2) the absence of interfacial activation, and (3) the absence of a colipase effect at high bile salt concentrations. Since both guinea pig and coypu pancreas produce a classical pancreatic lipase and no measurable phospholipase A2 activity, it is suggested that RP2 enzymes act as real phosphoLipases under physiological conditions. In fact, all RP2 Lipases from other species might share phospholipase activity and fulfill new biological functions.
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Structure-function relationships in naturally occurring mutants of pancreatic lipase
Protein engineering, 1994Co-Authors: Frédéric Carrière, Esper Boel, Robert Verger, Kenneth Thirstrup, Lars ThimAbstract:From primary structure comparison, the pancreatic lipase family is now divided into three subgroups: classical pancreatic Lipases, pancreatic lipase-related proteins 1 (RPI) and pancreatic lipase-related proteins 2 (RP2). Among the RP2 subfamily, the guinea-pig and coypu enzymes share kinetic properties which differ from those of classical pancreatic Lipases. Both enzymes display a high phospholipase activity and are not interfacially activated using a short chain triglyceride as substrate. Their activity towards insoluble triglycerides is inhibited by micellar concentrations of bile salts and is not restored by addition of colipase. These atypical kinetic properties are discussed in the light of amino acid sequence comparison between RP2 and classical pancreatic Lipases, based on the closed and open conformations of the 3-D structure of human pancreatic lipase.
Aran Hkittikun - One of the best experts on this subject based on the ideXlab platform.
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mixed Lipases for efficient enzymatic synthesis of biodiesel from used palm oil and ethanol in a solvent free system
Journal of Molecular Catalysis B-enzymatic, 2010Co-Authors: Ketsara Tongboriboon, Benjamas Cheirsilp, Aran HkittikunAbstract:The enzymatic synthesis of biodiesel from used palm oil and ethanol using immobilized Lipases in a solvent-free system was attempted. Five immobilized Lipases, Lipase AK from Pseudomonas fluorescens, Lipase PS from Pseudomonas cepacia, Lipase AY from Candida rugosa, Lipozyme TL IM from Thermomyces lanuginosa and Novozym 435 from Candida antarctica, were screened based on their catalytic activities on reactions involved in biodiesel synthesis. The combined use of Lipase AY and Lipase AK gave a higher yield of biodiesel than using Lipase AK alone. The optimal conditions for biodiesel synthesis using mixed Lipases in a batch system were: 2% water content, 10% enzyme dosage and 3:1 molar ratio of ethanol to oil. The mixed Lipases could be used in 15 replicates with retained relative activity higher than 50%. In a continuous system using mixed Lipases packed in packed-bed reactor, >67% of biodiesel was achieved.
Abdelkarim Abousalham - One of the best experts on this subject based on the ideXlab platform.
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An ultraviolet spectrophotometric assay for the screening of sn-2-specific Lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate.
Journal of Lipid Research, 2012Co-Authors: Lilia D Mendoza, Frédéric Carrière, Jorge A Rodriguez, Julien Leclaire, Gerard Buono, Frédéric Fotiadu, Abdelkarim AbousalhamAbstract:In the present study, we propose a continuous assay for the screening of sn-2 Lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective Lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the Lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of Lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 Lipases.
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Egg yolk lipoproteins as substrates for Lipases
Biochimica et Biophysica Acta, 2000Co-Authors: Abdelkarim Abousalham, Robert VergerAbstract:Egg yolk emulsions containing phospholipids (about 31%, w/w) are classically used as substrates for measuring phospholipase A2 activity using the pH-stat method. Here we investigated the susceptibility of egg yolk lipoproteins to lipolysis by various highly purified Lipases of animal or microbial origin. Egg yolk lipoproteins, which contain up to 65% triacylglycerols, were found to be effective substrates for all the Lipases tested. The specific activities measured on egg yolk lipoproteins using the pH-stat technique were found to be 8000, 1000, 1250 and 1700 U/mg in the case of human pancreatic lipase, horse pancreatic lipase, porcine pancreatic lipase and Humicola lanuginosa lipase, respectively. No activity was detected in the absence of colipase with any of the pancreatic Lipases tested. Consequently, the classical egg yolk assay cannot be considered as a specific phospholipase A2 assay.
José Maria Palomo - One of the best experts on this subject based on the ideXlab platform.
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single step purification of different Lipases from staphylococcus warneri
Journal of Chromatography A, 2010Co-Authors: Giandra Volpato, Marco Filice, Marco Antonio Zachia Ayub, José Manuel Guisán, José Maria PalomoAbstract:Abstract Three different Lipases from the extract crude of Staphylococcus warneri have been purified by specific lipase–lipase interactions using different Lipases (TLL, RML, PFL, BTL2) covalently attached to a solid support as adsorption matrix. BTL2 immobilized on glyoxyl-DTT adsorbed selectivity only a 30 kDa lipase from the crude, which was desorbed by adding 0.1% triton X-100. Using glyoxyl-PFL as matrix, two new Lipases (28 and 40 kDa) were adsorbed, and completely pure 40 kDa lipase was obtained after desorption using 0.01% triton, whereas 28 kDa lipase was desorbed after the incubation of the lipase matrix with 3% detergent. When using other matrixes as glyoxyl-TLL or glyoxyl-RML, different Lipases were adsorbed. This methodology could be a very efficient and useful method to purify several Lipases from crude extracts from different sources.