Showing papers on "Lipase published in 2002"

Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing

Abstract: Biodiesel fuel (fatty acid methyl esters; FAMEs) can be produced by methanolysis of waste edible oil with a lipase. The degree of methanolysis was low in reaction systems so far reported, and the lipase catalyst could not be reused in spite of using immobilized enzyme. We clarified this problem was due to the irreversible inactivation of the lipase by contact with insoluble methanol (MeOH). Based on this result, we developed a stepwise methanolysis system with immobilized Candida antarctica lipase. Two-step batch methanolysis was most effective for the production of biodiesel fuel from waste oil: the first-step reaction was conducted in the presence of 1/3 molar equivalent of MeOH for the stoichiometric amount, and the second-step reaction was performed by adding 2/3 molar equivalent of MeOH. If the immobilized carrier is destroyed by agitation in a reactor with impeller, three-step flow reaction will be available: the first-step substrates were waste oil and 1/3 molar equivalent of MeOH; the second-step, the first-step eluate and 1/3 molar equivalent of MeOH; the third-step, the second-step eluate and 1/3 molar equivalent of MeOH. The conversion of waste oil to biodiesel fuel reached >90% in the two reaction systems, and the lipase catalyst could be used for >100 days without decrease of the activity. The stepwise alcoholysis could successfully be applied to ethanolysis of tuna oil.

Interfacial adsorption of lipases on very hydrophobic support (octadecyl-Sepabeads): Immobilization, hyperactivation and stabilization of the open form of lipases

Abstract: Octadecyl–Sepabeads (Mitsubishi Chemical Corporation) was used to immobilize the lipases from Candida antarctica (fraction B), from Mucor miehei and from Candida rugosa via interfacial adsorption. The activity and stability properties of the derivatives obtained using this strategy by “stabilization of the open structure of the lipase” were compared to other more conventional immobilized derivatives (e.g. those obtained by multipoint covalent attachment) where the lipases are likely to be immobilized exhibiting its closed structure. Lipases adsorbed on hydrophobic supports exhibited a clear hyper-activation compared to the soluble enzyme or other types of derivatives. Their specific activities were greatly improved after immobilization (M. miehei lipase derivative was even 20 times more active than the soluble enzyme). Furthermore, lipases adsorbed on hydrophobic supports were very stable against heat and organic solvents inactivation. For example, C. antarctica B lipase octadecyl derivatives preserved 100% of the activity after 200 h of incubation at pH 7 and 50 °C. In addition, these derivatives remained also fully active after a very long incubation (200 h) in 50% dioxane at pH 7 and 25 °C. In spite of being immobilized by simple physical adsorption these lipase derivatives were more stable than multipoint covalently immobilized derivatives and much more stable than their respective soluble enzyme. It seems that the “open structure” of lipases, adsorbed on hydrophobic supports, is much more active and much more stable than the corresponding “closed” structure even when the closed structure is undergoing a very intense multipoint covalent attachment.

Enzyme producing bacterial flora isolated from fish digestive tracts

Abstract: Isolationand enumeration of aerobic bacterial flora in the gastrointestinal tract of nineculturable freshwater teleosts, namely catla, rohu, mrigal, silver carp, grasscarp, common carp, tilapia, walking catfish and murrel have been carried outAmylolytic, cellulolytic, lipolytic and proteolytic microflora were identifiedfrom the culture plate using selective media The isolates were qualitativelyscreened on the basis of their extracellular enzyme producing ability Theselected strains were further quantitatively assayed for amylase, cellulase,lipase and protease activities Protease activity was exhibited by almost allthe bacterial isolates, while strains isolated from tilapia, grass carp andcommon carp showed considerable amylolytic and cellulolytic activities Maximumactivity of lipase was exhibited by a strain isolated from silver carp Thestudy indicates that there is a distinct microbial source of the digestiveenzymes – amylase, cellulase, lipase and protease, apart from endogenoussources in fish gut The information generated from the present investigationmight contribute towards better feed formulations for carp at low cost,incorporating the enzyme producing bacterial isolates as probiotics

Protein expression and secretion in the yeast Yarrowia lipolytica.

Abstract: Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant.

Cross-linked enzyme aggregates with enhanced activity: application to lipases

Abstract: Seven commercially available microbial lipases were immobilised as their cross-linked enzyme aggregates (CLEAs). Preparations with enhanced activity were obtained by a judicious choice of the precipitant [(NH4)2SO4, 1,2-dimethoxyethane or acetone] and by adding either a crown ether or surfactant, depending on the source of the enzyme. Thus, precipitation of the lipases from Thermomyces lanuginosus and Rhizomucor miehei with (NH4)2SO4 in the presence of SDS, followed by cross-linking with glutaraldehyde, afforded CLEAs with three and two times, respectively, the hydrolytic activity of the native enzymes. Preparations with up to ten times enhanced activity in organic medium were similarly prepared.

Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase

Abstract: Crude soybean oil did not undergo methanolysis with immobilized Candida antarctica lipase but degummed oil did. Therefore, the substance that was removed in the degumming step was estimated to inhibit the methanolysis of soybean triacylglycerols (TAGs). The main components of soybean gum are phospholipids (PLs), and soybean PLs actually inhibited the methanolysis reaction. In addition, PLs were detected in chloroform/methanol (MeOH) extracts from the immobilized lipase preparation that had been used in the methanolysis of crude soybean oil. These results showed that PLs were at least one of the inhibitory substances in methanolysis of TAGs. The inhibition may due to the interference of the interaction of the lipase molecule with substrates by PLs bound on immobilized preparation. These findings indicated that degummed oil has to be used as a substrate for enzymatic methanolysis. Indeed, three-step methanolysis successfully converted 93.8% degummed soybean oil to its corresponding methyl esters, and the lipase could be reused for 25 cycles without any loss of the activity.

Salacia reticulata and Its Polyphenolic Constituents with Lipase Inhibitory and Lipolytic Activities Have Mild Antiobesity Effects in Rats

Abstract: Salacia (S.) reticulata, a Hippocrateaceae plant distributed in Sri Lankan and Indian forests, has been used as a supplementary food in Japan to prevent obesity and diabetes. We examined the antiobesity effects of the hot water-soluble extract (SRHW) from the roots of S. reticulata using obese rat models and an in vitro study. Body weight (P = 0.07) and periuterine fat storage (P = 0.10) in female Zucker fatty rats (8-9 wk old) tended to be suppressed by oral administration of SRHW (125 mg/kg) for 27 d. Male rats fed a high fat diet were not affected by SRHW. Furthermore, SRHW inhibited porcine pancreatic lipase (PL), rat adipose tissue-derived lipoprotein lipase (LPL) and glycerophosphate dehydrogenase (GPDH) activities with 50% inhibitory concentrations (IC(50)) of 264 (95% confidence limits: 203-393) mg/L, 15 (12-18) mg/L and 54 (35-85) mg/L, respectively, but did not inhibit hormone-sensitive lipase activity in rat adipose tissue. Next, we examined the effects of polyphenols, di- and triterpenes and salacinol isolated from the roots of S. reticulata on lipid metabolizing enzymes and lipolysis. (-)-Epigallocatechin and (-)-epicatechin-(4beta-->8)-(-)-4'-O-methylepigallocatechin inhibited PL activity with IC(50) of 88 (not calculated) and 68 (26-122) mg/L, respectively. (-)-Epicatechin, 3beta, 22beta-dihydroxyolean-12-en-29-oic acid and the tannin fraction inhibited LPL activity with IC(50) of 81 (54-214), 89 (62-214) and 35 (24-62) mg/L. Only the tannin fraction inhibited GPDH activity with an IC(50) of 6.8 (3.4-10.9) mg/L. These constituents may be involved in the lipase and GPDH inhibitory activities of SRHW. On the other hand, SRHW at 100 mg/L tended to enhance lipolysis in rat adipocytes (P = 0.06). Significant lipolytic effects were exerted by mangiferin, (-)-4'-O-methylepigallocatechin and maytenfolic acid at 100 mg/L (P < 0.01). In conclusion, polyphenolic compounds may be involved in the antiobesity effects of SRHW in rats through inhibition of fat metabolizing enzymes (PL, LPL and GPDH) and enhanced lipolysis.

Lipases and lipase-catalyzed esterification reactions in nonaqueous media

Abstract: Enzymatic reactions in nonaqueous solvents offer new possibilities for the biotechnological production of many useful chemicals using reactions that are not feasible in aqueous media. In the recent years, the use of enzymes in nonaqueous media has found applications in organic synthesis, chiral synthesis or resolution, modification of fats and oils, synthesis of sugar-based polymers, etc. The use of lipases in esterification reactions to produce industrially important products such as emulsifiers, surfactants, wax esters, chiral molecules, biopolymers, modified fats and oils, structured lipids, and flavor esters is well documented. The interest in using lipases as biotechnological vectors for performing various reactions in both macro- and microaqueous systems has picked up tremendously during the last decade. This review covers important features of lipases and lipase-catalyzed esterification reactions including the kinetics and stability of lipases, and modeling aspects.

Lipase polystyrene giant amphiphiles.

Abstract: A new type of giant amphiphilic molecule has been synthesized by covalently connecting a lipase enzyme headgroup to a maleimide-functionalized polystyrene tail (40 repeat units). The resulting biohybrid forms catalytic micellar rods in water.

Phospholipase A2 activity triggers the wound-activated chemical defense in the diatom Thalassiosira rotula

Abstract: The activation of oxylipin-based chemical defense in the diatom Thalassiosira rotula is initiated by phospholipases that act immediately after cell damage. This lipase activity is responsible for the preferential release of free mono- and polyunsaturated fatty acids. Among these, eicosatetraenoic- and eicosapentaenoic acid are further converted by lipoxygenases to reactive defensive metabolites such as the antiproliferative α,β,γ,δ-unsaturated aldehydes 2,4-decadienal and 2,4,7-decatrienal. We show that mainly saturated free fatty acids are present in the intact diatom T. rotula, whereas the amount of free polyunsaturated eicosanoids is drastically increased in the first minutes after wounding. Using fluorescent probes, the main enzyme activity responsible for initiation of the aldehyde-generating lipase/lipoxygenase/hydroperoxide lyase cascade was characterized as a phospholipase A2. All enzymes involved in this specific defensive reaction are active in seawater over several minutes. Thus, the mechanism allows the unicellular algae to overcome restrictions arising out of potential dilution of defensive metabolites. Only upon predation are high local concentrations of aldehydes formed in the vicinity of the herbivores, whereas in times of low stress, cellular resources can be invested in the formation of eicosanoid-rich phospholipids. In contrast to higher plants, which use lipases acting on galactolipids to release C18 fatty acids for production of leaf-volatile aldehydes, diatoms rely on phospholipids and the transformation of C20 fatty acids to form 2,4-decadienal and 2,4,7-decatrienal as an activated defense.

Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain

Abstract: We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3' region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5' upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.

Hydrolysis of oils by using immobilized lipase enzyme: A review

Abstract: This review focuses on the use of immobilized lipase technology for the hydrolysis of oils. The importance of lipase catalyzed fat splitting process, the various immobilization procedures, kinetics, deactivation kinetics, New immobilized lipases for chiral resolution, reactor configurations, and process considerations are all reviewed and discussed.

Ionic liquid-coated enzyme for biocatalysis in organic solvent.

Abstract: Ionic liquid-coated enzyme (ILCE) is described as a useful catalyst for biocatalysis in organic solvent An ionic liquid, [PPMIM]-[PF6] (1, [PPMIM] = 1-(3'-phenylpropyl)-3-methylimidazolium), which is solid at room temperature and becomes liquid above 53 degreesC, was synthesized in two steps from N-methylimidazole The coating of enzyme was done by simply mixing commercially available enzyme with 1 in the liquid phase above 53 degreesC and then allowing the mixture to cool A representative ILCE, prepared with a lipase from Pseudomonas cepacia, showed markedly enhanced enantioselectivity without losing any significant activity

Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production

Abstract: With a view to utilizing Rhizopus oryzae cells immobilized within biomass support particles (BSPs) as a whole-cell biocatalyst for biodiesel fuel production, an investigation was made of the effect of cross-linking treatment with glutaraldehyde (GA) on the stability of lipase activity. Although the lipase activity of the BSP-immobilized cells decreased considerably in the presence of the methyl esters produced by methanolysis, the activity of cells treated with 0.1% GA solution showed no significant decrease during six batch cycles, with the methyl ester content of the reaction mixture reaching 70–83% in each cycle. In contrast, without GA treatment, activity decreased gradually with each cycle to give a methyl ester content of only 50% at the sixth batch cycle. These findings indicate that, given the simplicity of the lipase production process and the long-term stability of lipase activity, the use of whole-cell biocatalysts immobilized within BSPs and treated with GA solution offers a promising means of biodiesel fuel production for industrial application.

Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1.

Abstract: An extracellular lipase, LipA, extracted from Acinetobacter sp. RAG-1 grown on hexadecane was purified and properties of the enzyme investigated. The enzyme is released into the growth medium during the transition to stationary phase. The lipase was harvested from cells grown to stationary phase, and purified with 22% yield and > 10-fold purification. The protein demonstrates little affinity for anion exchange resins, with contaminating proteins removed by passing crude supernatants over a Mono Q column. The lipase was bound to a butyl Sepharose column and eluted in a Triton X-100 gradient. The molecular mass (33 kDa) was determined employing SDS/PAGE. LipA was found to be stable at pH 5.8-9.0, with optimal activity at 9.0. The lipase remained active at temperatures up to 70 degrees C, with maximal activity observed at 55 degrees C. LipA is active against a wide range of fatty acid esters of p-nitrophenyl, but preferentially attacks medium length acyl chains (C6, C8). The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides, as demonstrated by zymogram analysis. RAG-1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+. The lipase is strongly inhibited by EDTA, Hg2+, and Cu2+, but shows no loss in activity after incubation with other metals or inhibitors examined in this study. The protein retains more than 75% of its initial activity after exposure to organic solvents, but is rapidly deactivated by pyridine. RAG-1 lipase offers potential for use as a biocatalyst.

Enzymatic Biodiesel Production from Sunflower Oil by Candida antarctica Lipase in a Solvent-free System

Abstract: Methanolysis (transesterification with methanol) of sunflower oil by lipase from Candida antarctica (Novozym 435) in a solvent-free system has been studied. Stepwise as well as continuous methanol feeding was applied to avoid strong substrate inhibition. Glycerol was found to cause strong product inhibition on the enzymatic reaction, therefore glycerol removal by dialysis was investigated using a flat sheet membrane module.