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Showing papers on "Lipase published in 1997"


Journal ArticleDOI
TL;DR: The crystal structure of a triacylglycerol lipase from Pseudomonas cepacia (PcL) in the absence of a bound inhibitor using X-ray crystallography suggests that this enzyme shares the same mechanisms of catalysis and interfacial activation as other lipases.

309 citations


Journal ArticleDOI
TL;DR: The results showed that the degradation of PCL films in phosphate buffer solution containing PP or AY was very slow: no weight loss could be found within 1 week, however, PCL film could degrade rapidly and completely within 4 days in phosphatebuffer solution containing PS lipase.

207 citations


Journal ArticleDOI
TL;DR: Hemperidin was identified as a simple and small molecular weight inhibitor in the peels of Citrus unshiu and inhibited lipase activity from porcine pancreas and that from Pseudomonas.
Abstract: In the course of our screening work for new types of lipase inhibitors, hesperidin was identified as a simple and small molecular weight inhibitor in the peels of Citrus unshiu. Hesperidin inhibited lipase activity from porcine pancreas and that from Pseudomonas, and their IC50 was 32 and 132 micrograms/ml, respectively. Hesperidin, neohesperidin, narirutin, and naringin are known as the main flavonoids in Citrus unshiu. Neohesperidin also inhibited the lipase from procine pancreas, but did not have any effect on Pseudomonas. Narirutin and naringin did not show this effect on lipases from porcine pancreas and Pseudomonas. In animal experiments, the concentration of plasma triglyceride in rats fed a diet containing 10% hesperidin were significantly lower than that fed the control diet.

195 citations


Journal ArticleDOI
TL;DR: The overexpressed BTL2 lipase shows a strong tendency to aggregate and under certain conditions, a direct relationship was found between the molecular mass of the lipase aggregates and the increase in activity upon the addition of 1% (w/v) sodium cholate.

179 citations


Journal ArticleDOI
TL;DR: Investigations with seven different lipases showed no correlation between the specific lipolytic activity of dissolved enzyme in aqueous solution and the specific activity of adsorbed enzyme in an esterification reaction in organic solvent.

177 citations


Journal ArticleDOI
TL;DR: The reaction of 1-phenyethylamine with ethylmethoxyacetate in the presence of a lipase from Burkholderia plantarii is presented and excellent yields and selectivity and minimal amount of enzyme characterize this new process.
Abstract: Optically Active Amines via Lipase-Catalyzed Methoxyacetylation Racemic amines can be efficiently resolved using ethylmethoxyacetate as acylating agent in a lipase-catalyzed reaction. The reaction of 1-phenyethylamine with ethylmethoxyacetate in the presence of a lipase from Burkholderia plantarii is presented. Excellent yields and selectivity and minimal amount of enzyme characterize this new process.

145 citations


Journal ArticleDOI
TL;DR: Kinetic and stoichiometric parameters for the utilisation of olive oil were determined and a thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp.
Abstract: A thermostable lipase was produced in continuous cultivation of a newly isolated thermophilic Bacillus sp. strain IHI-91 growing optimally at 65 degrees C. Lipase activity decreased with increasing dilution rate while lipase productivity showed a maximum of 340 U l-1 h-1 at a condition rate of 0.4 h-1. Lipase productivity was increased by 50% compared to data from batch fermentations. Up to 70% of the total lipase activity measured was associated to cells and by-products or residual substrate. Kinetic and stoichiometric parameters for the utilisation of olive oil were determined. The maximal biomass output method led to a saturation constant Ks of 0.88 g/l. Both batch growth data and a washout experiment yielded a maximal specific growth rate, mu max, of 1.0 h-1. Oxygen uptake rates of up to 2.9 g l-1 h-1 were calculated and the yield coefficient, Y X/O, was determined to be 0.29 g dry cell weight/g O2. From an overall material balance the yield coefficient, Y X/S, was estimated to be 0.60 g dry cell weight/g olive oil.

140 citations


PatentDOI
TL;DR: In this paper, the LIP1 gene was completely synthesised with an optimised nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation, and the recombinant CRL was produced at high level and purity, accounting for 90-95 % of the secreted proteins.
Abstract: The dimorphic yeast Candida rugosa has an unusual codon-usage which hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Lipases produced by this yeast are extensively used in industrial bioconversions, but commercial lipase samples contain several different isoforms encoded by the LIP genes family. In a first laborious attempt the LIP1 gene, encoding the major isoform of the C. rugosa lipases (CRLs), was systematically modified by site-directed mutagenesis to gain functional expression in S. cerevisiae. As alternative approach, the gene (1688 bp) was completely synthesised with an optimised nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally overexpressed in Pichia pastoris. The recombinant CRL was produced at high level and purity, accounting for 90-95 % of the secreted proteins. The physical-chemical and catalytic properties of the recombinant lipase were compared with those of a commercial, non-recombinant C. rugosa lipase preparation.

136 citations


Journal ArticleDOI
TL;DR: The present enzyme, named phosphatidylserine-phospholipase A1, is the first phospholipases that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.

134 citations


Journal ArticleDOI
TL;DR: The lipolytic enzymes displayed a non-parallel pattern of development, and it is suggested that this reflects the importance of these enzymes during the suckling and postweaning phases, respectively.
Abstract: The effect of age and weaning on the activities of digestive enzymes with emphasis on the lipolytic enzymes before and after weaning was investigated. The activities of amylase, chymotrypsin, trypsin, carboxyl ester hydrolase, pancreatic lipase, and colipase in pancreatic tissue and the activity of gastric lipase in the cardiac mucosa of the stomach in 45 pigs were response variables. The activity of trypsin was not affected by weaning and the rate of increase was similar during the whole experiment. The activities of chymotrypsin and amylase decreased at weaning (P < .05). After weaning the activity of chymotrypsin increased more slowly than before weaning (P < .001), whereas the rate of increase of amylase activity remained unchanged. Lipase, colipase, and carboxyl ester hydrolase activities decreased at weaning (P < .001), whereas gastric lipase activity increased at weaning (P < .01). The development of lipase, colipase, and carboxyl ester hydrolase activity decreased postweaning (P < .01), whereas gastric lipase activity increased before weaning and remained constant after weaning. Pancreatic lipase had a considerably higher capacity for hydrolyzing tributyrin, and the total activity of pancreatic lipase was up to 600 times higher than that of gastric lipase. The lipolytic enzymes displayed a non-parallel pattern of development, and we suggest that this reflects the importance of these enzymes during the suckling and postweaning phases, respectively. However, the significance of gastric lipase for the digestion of fat in pigs remains to be elucidated.

131 citations


Journal ArticleDOI
TL;DR: It is hypothesized that the reduction in efficiency at low loadings is due to a distortion of the active molecular conformation caused by the lipase maximizing its contact with the support as a result of its high affinity for the support surface.
Abstract: Rhizomucor miehei, Humicola sp., Rhizopus niveus, and Candida antarctica B lipases were immobilized by physical adsorption onto a macroporous polypropylene support. In an esterification reaction, the enzyme efficiency, and therefore cost-effectiveness, is greatly affected by enzyme loading, with an apparent suppression of efficiency at low lipase loadings for both R. miehei and Humicola sp. lipases. This results in the appearance of a pronounced maximum in the efficiency-loading relationship at approximately 100,000 lipase units (LU)/g for R. miehei lipase (10% of its saturation loading) and at approximately 200,000 LU/g for Humicola sp. lipase (50% of its saturation loading). The other lipases studied do not show similar trends. At low loadings, only a small portion of the surface area is occupied and gives the lipase the opportunity to spread; it is hypothesized that the reduction in efficiency at low loadings is due to a distortion of the active molecular conformation caused by the lipase maximizing its contact with the support as a result of its high affinityfor the support surface. The relationship between efficiency and loading was different for each of the lipases studied, which may reflect both differences in the strength of the affinity of the lipase for the support and in the ease at which the molecular conformation of the lipase can be distorted.

Journal ArticleDOI
TL;DR: The physical chemistry of lipolysis has been well documented and in recent years there have been significant advances in the understanding at the molecular level, with publication of crystal structures of the pancreatic lipase-colipase complex, both in the presence and absence of lipids as discussed by the authors.

Journal ArticleDOI
TL;DR: This review discusses the advances made in protein structure and in understanding the relationships of structure to function of pancreatic triglyceride lipase and colipase.
Abstract: Dietary fats are essential for life and good health. Efficient absorption of dietary fats is dependent on the action of pancreatic triglyceride lipase. In the last few years, large advances have been made in describing the structure and lipolytic mechanism of human pancreatic triglyceride lipase and of colipase, another pancreatic protein that interacts with pancreatic triglyceride lipase and that is required for lipase activity in the duodenum. This review discusses the advances made in protein structure and in understanding the relationships of structure to function of pancreatic triglyceride lipase and colipase.

Journal ArticleDOI
TL;DR: Lipase-catalyzed transesterification has been shown to be an excellent alternative to traditional esterification and short-path distillation for concentrating the combined PUFA-content in fish oils.
Abstract: Because of the complexity of marine lipids, polyunsaturated fatty acid (PUFA) derivatives in highly purified form are not easily prepared by any single fractionation technique. The products are usually prepared as the ethyl esters by esterification of the body oil of fat fish species and subsequent physicochemical purification processes, including short-path distillation, urea fractionation, and preparative chromatography. Lipase-catalyzed transesterification has been shown to be an excellent alternative to traditional esterification and short-path distillation for concentrating the combined PUFA-content in fish oils. At room temperature in the presence of Pseudomonas sp. lipase and a stoichiometric amount of ethanol without any solvent, efficient transesterification of fish oil was obtained. At 52% conversion, a concentrate of 46% eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) was obtained in excellent recovery as a mixture of mono-, di-, and triacylglycerols. The latter can be easily separated from the saturated and monounsaturated ethyl esters and converted into ethyl esters either by conventional chemical means or enzymatically by immobilized Candida antarctica lipase. Urea-fractionation of such an intermediary product can give an EPA+DHA content of approximately 85%.

Journal ArticleDOI
TL;DR: The effect of water activity on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes, shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase.
Abstract: Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.

Book ChapterDOI
TL;DR: A preliminary screening work selected Penicillium restrictum as a promising micro-organism for lipase production based on the physiological response of the fungus towards cell growth and enzyme production upon variable carbon and nitrogen nutrition, specific air flow rate and agitation.
Abstract: A preliminary screening work selected Penicillium restrictum as a promising micro-organism for lipase production. The physiological response of the fungus towards cell growth and enzyme production upon variable carbon and nitrogen nutrition, specific air flow rate (Qa) and agitation (N) was evaluated in a 5-L bench-scale fermenter. In optimized conditions for lipase production meat peptone at 2% (w/v) and olive oil at 1% (w/v) were used in a growth medium with a C/N ratio of 9.9. Higher C/N ratios favored cell growth in detriment of enzyme production. Low extracellular lipase activities were observed using glucose as carbon source suggesting glucose regulation. Final lipase accumulation of 13,000 U/L was obtained, using optimized specific air flow rate (Qa) of 0.5 vvm and an impeller speed (N) of 200 rpm. Agitation showed to be an important parameter to ensure nutrient availability in a growth medium having olive oil as carbon source.

Journal ArticleDOI
TL;DR: In this article, the six-membered lactide was polymerized using the ring-opening polymerization with lipase as a catalyst at a temperature between 80 and 130°C in bulk to yield the corresponding polylactide with weight-average molecular weights of up to 126000.
Abstract: The six-membered lactide was polymerized using the ring-opening polymerization with lipase as a catalyst at a temperature between 80 and 130°C in bulk to yield the corresponding polylactide with weight-average molecular weights of up to 126000. The most preferable conditions with respect to the molecular weight of the polylactide are the bulk polymerization using lipase PS at a temperature of 100°C. The D,L-lactide gave higher molecular weight compared to the D,D- and L,L-lactide.

Journal ArticleDOI
TL;DR: The catalytic activities of lyophilized powders of alpha-chymotrypsin and Candida antarctica lipase were found to increase 4- to 8-fold with increasing amounts of either buffer salts or potassium chloride in the enzyme preparation and the enhancement of hydration level and flexibility of the macromolecule upon addition of the compounds partly provides the explanation for the observed results.
Abstract: The catalytic activities of lyophilized powders of alpha-chymotrypsin and Candida antarctica lipase were found to increase 4- to 8-fold with increasing amounts of either buffer salts or potassium chloride in the enzyme preparation. Increasing amounts of sorbitol in the chymotrypsin preparation produced a modest increase in activity. The additives are basically thought to serve as immobilization matrices, the sorbitol being inferior because of its poor mechanical properties. Besides their role as supports, the buffer species were indispensable for the transesterification activity of chymotrypsin because they prevented perturbations of the pH during the course of the reaction. Hence, increasing amounts of buffer species yielded a 100-fold increase in transesterification activity. Effects of pH changes were not as predominant in the peptide synthesis and the lipase-catalyzed reactions. Immobilization of the protease on celite resulted in a remarkable improvement of transesterification activity as compared to the suspended protease, even in the absence of buffer species. Immobilization of the lipase caused a small improvement of activity. The activity of the immobilized enzymes was further enhanced 3-4 times by including increasing amounts of buffer salts in the preparation.The inclusion of increasing amounts of sodium phosphate or sorbitol to chymotrypsin rendered the catalyst more labile against thermal inactivation. The denaturation temperature decreased with 7 degrees C at the highest content of sodium phosphate, as compared to the temperature obtained for the denaturation of the pure protein. The apparent enthalpy of denaturation increased with increasing contents of the additives. The enhancement of hydration level and flexibility of the macromolecule upon addition of the compounds partly provides the explanation for the observed results.

Journal ArticleDOI
TL;DR: Results suggest that the enzyme may participate in the intracellular neutral lipid metabolism since the enzyme is located in the endoplasmic reticulum, an organelle where de novo triacylglycerol synthesis and assembly of lipoproteins take place.
Abstract: We have purified an enzyme from porcine liver microsomes which catalyzes hydrolysis of triacylglycerols. The enzyme was solubilized from the membranes by the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and was purified to apparent homogeneity by sequential chromatography on Q-Sepharose, hydroxyapatite, Affi-Gel heparin, and Mono-Q. The purified hydrolase migrated in SDS−polyacrylamide gel electrophoresis (PAGE) as a single polypeptide band of an apparent molecular mass of 60 kDa. The enzyme hydrolyzed long-, medium-, and short-chain triacylglycerols, as well as a chromogenic lipase substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. The highest specific activity was obtained with tributyroylglycerol (240 μmol·min-1·mg-1). The reaction rate was maximal at pH 8.5. Sulfhydryl-directed reagents, such as N-ethylmaleimide (NEM), 5,5‘-dithiobis(2-nitrobenzoic acid) (DTNB), and dodecyldithio-5-(2-nitrobenzoic acid) (C12-TNB) had no effect on the hydr...

Journal ArticleDOI
TL;DR: This article showed that cyclic carbonate 1,3-dioxan-2-one was readily polymerized in the presence of lipase at a temperature between 60 and 100°C for 24h.
Abstract: It was fopun dthat cyclic carbonate 1,3-dioxan-2-one was readily polymerized in the presence of lipase at a temperature between 60 and 100°C for 24h. Among the lipases tested, porcine pancreatic lipase showed the best results with respect to the monomer conversion and the molecular weight of the polymer

Journal ArticleDOI
TL;DR: The data suggest that C. albicans, in addition to phospholipases, also has lipases, and that a lipase gene(s) may be present inCandida parapsilosis, Candida tropicalis and Candida krusei, but not in Candida pseudotropicalis,candida glabrata or S. cerevisiae.
Abstract: Extracellular phospholipases are demonstrated virulence factors for a number of pathogenic microbes. The opportunistic pathogen Candida albicans is known to secrete phospholipases and these have been correlated with strain virulence. In an attempt to clone C. albicans genes encoding secreted phospholipases, Saccharomyces cerevisiae was transformed with a C. albicans genomic library and screened for lipolytic activity on egg-yolk agar plates, a traditional screen for phospholipase activity. Two identical clones were obtained which exhibited lipolytic activity. Nucleotide sequence analysis identified an ORF encoding a protein of 351 amino acid residues. Although no extensive homologies were identified, the sequence contained the Gly-X-Ser-X-Gly motif found in prokaryotic and eukaryotic lipases, suggesting a similar activity for the encoded protein. Indeed, culture supernatants from complemented yeast cells contained abundant hydrolytic activity against a triglyceride substrate and had no phospholipase activity. The data suggest that C. albicans, in addition to phospholipases, also has lipases. Southern blot analyses revealed that C. albicans may contain a lipase gene (LIP) family, and that a lipase gene(s) may be present in Candida parapsilosis, Candida tropicalis and Candida krusei, but not in Candida pseudotropicalis, Candida glabrata or S. cerevisiae. Northern blot analyses showed that expression of the LIP1 transcript, the cloned gene which encodes a lipase, was detected only when C. albicans was grown in media containing Tween 80, other Tweens or triglycerides as the sole carbon source, and not in Sabouraud Dextrose Broth or yeast/peptone/dextrose media. Additionally, carbohydrate supplementation inhibited LIP1 expression. Cloning this gene will allow the construction of LIP1-deficient null mutants which will be critical in determining the role of this gene in candidal virulence.

Journal ArticleDOI
TL;DR: Extracellular lipase production by Yarrowia lipolytica was increased by mutant selection from 28 U/ml to 1000 U/ML and this activity was also reached in a 500 l bioreactor.
Abstract: Extracellular lipase production by Yarrowia lipolytica was increased by mutant selection from 28 U/ml to 1000 U/ml. This activity was also reached in a 500 l bioreactor. The properties of the mutant lipase were the same of those of the wild type: M 38 kDa, optimum pH 7 and optimum temperature 37iC.

Journal ArticleDOI
TL;DR: Several glycopyranosides have been acetylated, showing that the regioselectivity displayed by Novozym 435 towards sugar secondary OH's is deeply influenced by the nature of the aglycone and by the stereochemistry of the glycosidic bond.
Abstract: A systematic investigation on the transesterification activity and regioselectivity of the immobilized lipase B from Candida antarctica (Novozym 435) has been performed. This enzyme has been found to be quite active in such solvents as THF, acetone, dioxane, and in mixtures of these solvents with pyridine. Several glycopyranosides have been acetylated, showing that the regioselectivity displayed by Novozym 435 towards sugar secondary OH's is deeply influenced by the nature of the aglycone and by the stereochemistry of the glycosidic bond. This information on the regioselectivity of the lipase has been exploited for the preparation of acetyl derivatives of flavonoid glycosides.

Journal ArticleDOI
TL;DR: The neutron structure of the activated lipase–colipase–micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method concludes that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.
Abstract: The concept of lipase interfacial activation stems from the finding that the catalytic activity of most lipases depends on the aggregation state of their substrates. It is thought that activation involves the unmasking and structuring of the enzyme's active site through conformational changes requiring the presence of oil-in-water droplets. Here, we present the neutron structure of the activated lipase-colipase-micelle complex as determined using the D2O/H2O contrast variation low resolution diffraction method. In the ternary complex, the disk-shaped micelle interacts extensively with the concave face of colipase and the distal tip of the C-terminal domain of lipase. Since the micelle- and substrate-binding sites concern different regions of the protein complex, we conclude that lipase activation is not interfacial but occurs in the aqueous phase and is mediated by colipase and a micelle.


Journal ArticleDOI
TL;DR: The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702, and an open reading frame, orfA, is found whose deduced protein sequence shows similarity to various membrane-localized transporters.
Abstract: Streptomyces cinnamomeus Tu89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.

Journal ArticleDOI
TL;DR: A new way of solubilizing hydrolytic enzymes so that they can be used in organic solvents is to coat them with lipid monolayers.

Journal ArticleDOI
TL;DR: Porcine pancreas lipase (triacylglycerol ester hydrolase, EC 3.1.3) was immobilized with the highest activity on polyacrylamide beads possessing carboxylic functional groups activated by a water-soluble carbodiimide to stabilized the enzyme against heat and urea treatment.

Journal ArticleDOI
TL;DR: To purify docosahexaenoic acid (DHA), the selective esterification of fatty acids originating from tuna oil with lipases was attempted, and DHA was purified to 89% with a recovery of 71% of its initial content.
Abstract: To purify docosahexaenoic acid (DHA), we attempted the selective esterification of fatty acids originating from tuna oil with lipases. Tuna oil was hydrolyzed in NaOH-ethanol solution, and the resulting fatty acid mixture [DHA, 23.2%; named tuna-free fatty acid (FFA)] was used as a starting material. Rhizopus delemar which acted lightly on DHA, was a suitable catalyst for the selective esterification of tuna-FFA, and lauryl alcohol was the best substrate. The reaction proceeded most effectively when a mixture of 2.4 g lauryl alcohol/tuna-FFA (2:1, mol/mol), 0.6 g water, and 600 U Rhizopus lipase was incubated at 30°C for 20 h with stirring at 500 rpm. Under these conditions 72% of tuna-FFA was esterified, and 84% of DHA was recovered in the unesterified fatty acid fraction. The DHA content in the fatty acid fraction rose from 23 to 73% with this reaction. To further elevate the DHA content, the unesterified fatty acids were extracted, and then esterified again under the same conditions. By this repeated esterification, DHA was purified to 89% with a recovery of 71% of its initial content.

Journal ArticleDOI
TL;DR: The purified lipase from Pseudomonas cepacia was immobilized on a commercially available microporous polypropylene support and showed that the immobilized enzyme was fully active as soon as activity was assayed on a soluble substrate rather than an insoluble one.
Abstract: The purified lipase from Pseudomonas cepacia (PS, Amano) was immobilized on a commercially available microporous polypropylene support. The enzyme was rapidly and completely adsorbed on the support. Special attention was devoted to the demonstration of the lack of diffusional limitations, either internal or external, when a soluble substrate (p-nitrophenylacetate, pNPA) was used. The activity yield was high (100%) with pNPA and very low (0.4%) with p-nitrophenylpalmitate (pNPP). These values clearly showed that the immobilized enzyme was fully active as soon as activity was assayed on a soluble substrate rather than an insoluble one. With the latter one, the low activity was due mainly to a slow rate of substrate diffusion inside the porous support. The same diffusional phenomenon could explain the complete change of fatty acid specificity of the immobilized lipase. After immobilization, the lipase was mainly specific for short chain fatty acid esters, whereas the free enzyme was mainly specific for long chain esters. The activity-versus-temperature profiles were not greatly affected by immobilization with maximal reaction rates in the range 45 degrees to 50 degrees C for both enzyme preparations. However, immobilization increased enzyme stability mainly by decreasing the sensitivity to temperature of the inactivation reaction. Half-lives at 80 degrees C were 11 and 4 min for the immobilized and free enzymes, respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 181-189, 1997.