Hydrolysis Of Olive Oil Research Articles

Immobilization of lipase onto cellulose ultrafine fiber membrane for oil hydrolysis in high performance bioreactor

Practical application of biphasic enzyme-immobilized membrane bioreactors (EMBR) requires efficient loading of the enzyme with retention of enzymatic activity. Here, we report a method to fabricate an ultrafine fiber membrane conjugated to lipase with high levels of enzyme loading and activity retention. A cellulose acetate (CA) non-woven ultrafine fiber membrane was prepared with 200 nm nominal fiber diameter by electrospinning, followed by alkaline hydrolysis to obtain regenerated cellulose (RC). The RC ultrafine fiber membrane was oxidized by exposure to NaIO4, simultaneously generating aldehyde groups to couple with pentaethylenehexamine (PEHA) as a spacer for lipase immobilization. A biphasic EMBR was assembled with the PEHA-modified and lipase-immobilized membranes. The effect of operation variables, namely aqueous-phase system, reaction pH, accelerant (sodium taurocholate) content, reaction temperature, and membrane usage on the performance of this bioreactor was investigated with the hydrolysis of olive oil. A bioreactor activity as high as 9.83 × 104 U/m2 was obtained under optimum operational conditions.

Hen egg white as a feeder protein for lipase immobilization

Abstract Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K m (2.08 mM) and apparent V max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K m (8.0 mM) and V max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.

Immobilization and stabilization of Pseudomonas aeruginosa SRT9 lipase on tri(4-formyl phenoxy) cyanurate

Lipase was extracted and purified from Pseudomonas aeruginosa SRT9. Culture conditions were optimized and highest lipase production amounting to 147.36 U/ml was obtained after 20 h incubation. The extracellular lipase was purified on Mono QHR5/5 column, resulting in a purification factor of 98-fold with specific activity of 12307.81 U/mg. Lipase was immobilized on tri (4-formyl phenoxy) cyanurate to form Schiff’s base. An immobilization yield of 85% was obtained. The native and immobilized lipases were used for catalyzing the hydrolysis of olive oil in aqueous medium. Comparative study revealed that immobilized lipase exhibited a shift in optimal pH from 6.9 (free lipase) to 7.5 and shift in optimal temperature from 55 °C to 70 °C. The immobilized lipase showed 20–25% increase in thermal stability and retained 75% of its initial activity after 7 cycles. It showed good stability in organic solvents especially in 30% acetone and methanol. Enzyme activity was decreased by ∼60% when incubated with 30% butanol. The kinetic studies revealed increase in KM value from 0.043 mM (native) to 0.10 mM for immobilized lipase. It showed decrease in the Vmax of immobilized enzyme (142.8 μmol min−1 mg−1), suggesting enzyme activity decrease in the course of covalent binding. The immobilized lipase retained its initial activity for more than 30 days when stored at 4 °C in Tris-HCl buffer pH 7.0 without any significant loss in enzyme activity.

Characterization of Crude Lipase from Rhizopus Arrhizus and Purification of Multiplicity Forms of the Enzyme

ABSTRACTSome of the biochemical properties of a crude lipase enzyme from Rhizopus arrhizus were determined. The enzyme displayed maximal catalytic activity at pH 9.0 and temperature of 35°C. It was stable at basic values of pH and 40°C and in the presence of detergents. Lipase showed substrate specificity against different plant oils. Three multiplicity forms of the enzyme (Lip I, Lip II and Lip III) were isolated by gel chromatography. They differed in their molecular weight (Lip I- 80 000 Da, Lip II- 39 700 Da and Lip III- 6 900 Da) and degree of glycosylation (51.61% for Lip I, 74.17% for Lip II and 55.60% for Lip III). Some of the specific biochemical properties of the multiplicity forms were established. No synergistic effect of the three multiplicity forms of lipase in hydrolysis of olive oil was observed.

Desempenho da matriz híbrida SiO2-quitosana na imobilização da lipase microbiana de Candida rugosa

Lipase from Candida rugosa was immobilized by covalent attachment on hybrid SiO2-chitosan obtained by sol-gel technique. A comparative study between free and immobilized lipase was provided in terms of pH, temperature, kinetic parameters and thermal stability on the olive oil hydrolysis. The pH and temperature for maximum activity shifted from 7.0 and 45 oC for the free lipase to 7.5 and wide range of temperature (40-50 oC) after immobilization. Kinetics parameters were found to obey Michaelis-Menten equation and KM values indicated that immobilization process reduced the affinity of enzyme-substrate; however Kd values revealed an increase of thermal stability of lipase.

Isolation, Optimisation and Purification of Lipase Production by Pseudomonas Aeruginosa

Six isolates of lipase producing (Ps1, Ps2, Ps3, Ps4, Ps5 and Ps6) were secreened from wastewater on a selective medium agar that contained tween 80 or olive oil as the only source of carbon. The isolate showed highest lipase activity was Ps5 which later was identified as Pseudomonas aeruginosa. The effect of media composition was analysed to maximize production of lipase. The maximum extracellular lipase present in the broth was purified 11 folds with an overall yield of 65.51 % through purification procedure of ammonium sulphate precipitation and DEAE cellulose chromatography. The purified lipase had maximal activity within the pH range of 6-8, with an optimum PH of 7, and within the temperature range of 25 – 35°C, with optimum temperature for the hydrolysis of olive oil at 35°C. The lipase activity of the enzyme was enchanced by Ca+2 and Mg+2 but strongly inhibited by heavy metals Zn+2, Cu+2and Mn+2.

Using silk woven fabric as support for lipase immobilization: The effect of surface hydrophilicity/hydrophobicity on enzymatic activity and stability

Silk fibers in the form of woven fabric were used as a novel and inexpensive carrier for the immobilization of lipase from Candida sp.99–125. In this study, the activity and stability of lipases adsorbed on two silk fabrics with different hydrophilic/hydrophobic properties were compared. Hydrophobic silk fibers functionalized with methyl groups were prepared by treatment with amino-functional polydimethylsiloxane (PDMS). The lipase immobilized on PDMS-treated fiber exhibited an over 2-fold increase in both hydrolysis and esterification activity due to the interfacial activation as compared to its immobilization on a hydrophilic support (native fiber). To characterize the properties of different immobilized derivatives, the effects of pH and temperature were investigated in the hydrolysis of olive oil. The esterification behavior in organic media with variable water contents and operational stability of immobilized derivatives were also compared. The lipase immobilized on the hydrophobic fibers could maintain a constantly high activity at a water content range from 1 to 10% (v/v), while the activity of lipase immobilized on native fibers showed a clear dependence on water content in organic media and decreased rapidly at high water content (> 2%). Furthermore, lipase immobilization on the hydrophobic support exhibited a significantly improved operational stability in esterification reaction system. After 27 batches were recycled, a high esterification yield (97%) was maintained. The results in this work indicate that a hydrophobic surface of fabric fiber promotes the interfacial activation of lipase and that woven silk could be a potential material as an immobilization matrix for industrial process.

Immobilization of Candida rugosa lipase on glutaraldehyde-activated polyester fiber and its application for hydrolysis of some vegetable oils

Candida rugosa lipase was effectively immobilized on the poly(ethylene terephthalate) grafted acrylamide (PET-g-AAm) fiber which was prepared through Hofmann reaction. The activities of the immobilized enzyme and the free enzyme were investigated in the hydrolysis reaction of olive oil in isooctane–water biphasic medium. The activities of the free and the immobilized lipases were measured at different pH values, and temperatures. The thermal stability and storage stability of them were also determined. The kinetic parameters of the free and the immobilized lipases, Km and Vmax were calculated, as well. Moreover, the application of immobilized lipase to the hydrolysis of different vegetable oils was realized. The effect of organic solvents on olive oil hydrolysis was examined and time course of the oil hydrolysis was studied. The optimum pH was shifted from 6 to 7 by immobilization of the enzyme. The maximum activity of the free and the immobilized enzymes occurred at 40 °C. It was found that the immobilized lipase stored at 4 °C retained 90% of its original activity after 60 days, whereas the free lipase stored at 4 °C retained 75% of its activity after the same period. In addition, the immobilized lipase exhibited as 0.06 U of the remaining activity even after 10 times reuses. Kinetics studies show that the corresponding values of Km and Vmax were 47.2 mg ml−1 and 48.1 U mg−1 protein for free lipase and 151.6 mg ml−1 and 10.9 U mg−1 protein for immobilized lipase. The immobilized and free enzymes showed similar behavior with respect to the different oil hydrolysis. The time course of the immobilized lipase on canola and olive oils hydrolysis used as substrate was much better than that of the free lipase. A high rate of oil hydrolysis was obtained when isooctane was used as solvent.

Immobilisation of Pseudomonas gessardii acidic lipase derived from beef tallow onto mesoporous activated carbon and its application on hydrolysis of olive oil

The study was carried out to immobilise the acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon (MAC 400) for the application of hydrolysis of olive oil. MAC 400 was prepared from rice husk by the two stages process. P. gessardii was isolated from the beef tallow acclimatised soil. The acidic lipase (ALIP) was produced from a slaughterhouse waste, namely beef tallow as a substrate and immobilised onto MAC 400. The maximum immobilisation capacity of the MAC 400 was 3570 U/g at optimum immobilisation conditions; time (180 min), pH (5.0) and temperature (30 °C). The immobilised lipase had better thermal stability and reusability than the free lipase. The immobilisation of ALIP onto MAC 400 (MAC 400–ALIP) followed pseudo-first-order rate kinetics with rate constant 0.012/min. The Michaelis–Menten constant of MAC 400–ALIP was lower than that of the ALIP, which confirmed the higher affinity between enzyme and substrate. The immobilization of acidic lipase onto MAC 400 was confirmed by Fourier transform-infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) pattern analysis. Reusability study of MAC 400–ALIP on olive oil hydrolysis showed 82% of hydrolysis up to 13 runs and 50% of hydrolysis up to 35 cycles of reuse. This work concludes that the acidic lipase immobilised mesoporous activated carbon matrix can be considered as a potential biocatalyst for the hydrolysis of olive oil. Thus, the enzyme immobilised matrix has potential industrial applications.”

Lipase immobilization on Polysulfone globules and their performances in olive oil hydrolysis

We prepared Polysulfone (PS) globules by pouring the PS solution droplets in to the non-solvent water. PS solutions (10, 15, and 20% (w/v), concentrations) of different viscosities resulted different physical appearances in globules. The lipase immobilizing amount (4.32 mg/g) was maximum for PS 20% globules as it possessed maximum BET pore surface area. The olive oil hydrolytic reaction parameters were fitted into the Lineweaver-Burk plot. The K(m) (apparent) values of all the immobilized globules were higher (83.3mM) with respect to free lipases (62.5mM) where as V(max) (apparent) values followed the reverse trend (129.8 U/mg for 20% and 120.5 U/mg for both 15% and 10% PS immobilized globules and 153.8 U/mg for the free lipase respectively). The hydrolytic activity to olive oil was decreased ( approximately 16-20%) after five cycles.

Maté Tea Inhibits In Vitro Pancreatic Lipase Activity and Has Hypolipidemic Effect on High‐fat Diet‐induced Obese Mice

The inhibitory effects of maté tea (MT), a beverage produced with leaves from Ilex paraguariensis, in vitro lipase activity and on obesity in obese mice models were examined. For the in vitro experiment, porcine and human pancreatic lipase (PL) activities were determined by measuring the rate of release of oleic acid from hydrolysis of olive oil emulsified with taurocholate, phospholipids, gum arabic, or polyvinyl alcohol. For the in vivo experiments, animals were fed with a standard diet (SD, n = 10) or high-fat diet (HFD, n = 30) for 16 weeks. After the first 8 weeks on the HFD, the animals were treated with 1 and 2 g/kg of body weight of MT. The time course of the body weight and obesity-related biochemical parameters were evaluated. The results showed that MT inhibited both porcine and human PL (half-maximal inhibitory concentration = 1.5 mg MT/ml) and induced a strong inhibition of the porcine lipase activity in the hydrolysis of substrate emulsified with taurocholate + phosphatidylcholine (PC) (83 +/- 3.8%) or PC alone (62 +/- 4.3%). MT suppressed the increases in body weight (P < 0.05) and decreased the serum triglycerides and low-density lipoprotein (LDL)-cholesterol concentrations at both doses (from 190.3 +/- 5.7 to 135.0 +/- 8.9 mg/dl, from 189.1 +/- 7.3 to 129.3 +/- 17.6 mg/dl; P < 0.05, respectively) after they had been increased by the HFD. The liver lipid content was also decreased by the diet containing MT (from 132.6 +/- 3.9 to 95.6 +/- 6.1 mg/g of tissue; P < 0.05). These results suggest that MT could be a potentially therapeutic alternative in the treatment of obesity caused by a HFD.

Immobilization of Candida antarctica lipase B by adsorption in organic medium

Candida antarctica lipase B (CALB) was immobilized on the macroporous resin by physical adsorption in organic medium. The immobilization was performed in 5 mL isooctane, and the immobilization conditions were optimized. The results were achieved with the mass ratio of lipase to support 1:80, the buffer of pH 6.0, initial addition of PBS 75 microL, and immobilization time of two hours at 30 degrees C. Under the optimal conditions, the activity recovery was 83.3%. IM-CALB presented enhanced pH and thermal stability compared to the free lipase, and showed comparable stability with the commercial Novozym 435, after 7 times repeated use for catalyzing the synthesis of ethyl lactate, 56.9% of its initial activity was retained, and only 24.7% was retained when used for catalyzing the hydrolysis of olive oil.

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10 3, 98 × 10 3, 147 × 10 3 and 196 × 10 3 U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters ( K m and V max ) were found using Michaelis–Menten enzyme kinetics. The K m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.

Isolation and purification of Mucor circinelloides intracellular chitosanolytic enzymes

This study aimed at isolation, purification and characterization of a chitosanase from Mucor circinelloides mycelium. The latter contains also a mycelium-bound lipase and lipids. The chitosanase and lipase were extracted from defatted M. circinelloides mycelium with a detergent and purified through a two-step procedure comprising chromatography on bacitracin–CNBr-Sepharose 4B and gel filtration on Sephadex G-100. Purification degree of the chitosanase (endo-type enzyme) and lipase was 23 and 12, respectively. These enzymes were optimally active at pH of 5.5–6.0 (chitosanase) and 7.2 (lipase in olive oil hydrolysis) and at 37 °C. Both purified enzymes were activated by Ca 2+ and Mg 2+ ions. The preferred substrates of chitosanase were chitosan preparations with a high degree of deacetylation. This enzyme showed no activity for colloidal chitin, Na-CMC and starch. SDS–PAGE of both purified enzymes showed two bands with molecular masses of 42 and 43 kDa. Our results suggest that M. circinelloides synthesizes an oligomeric (bifunctional) lipase which also efficiently depolymerizes chitosan.

Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for immobilization. Using this approach, a 1,3 regiospecific, alkaline and thermostable lipase from Thermomyces lanuginosa was immobilized. Efficient cross-linking was observed when ammonium sulphate was used as precipitant along with a two fold increase in activity in presence of SDS. The TEM and SEM microphotographs of the CLEAs formed reveal that the enzyme aggregates are larger in size as compared to the free lipase due to the cross-linking of enzyme aggregates with glutaraldehyde. The stability and reusability of the CLEA with respect to olive oil hydrolysis was evaluated. The CLEA showed more than 90% residual activity even after 10 cycles of repeated use.

Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome

To search for new cold-active lipases, a metagenomic library was constructed using cold-sea sediment samples at Edison Seamount and was screened for lipolytic activities by plating on a tricaprylin medium. Subsequently, a fosmid clone was selected, and the whole sequence of 36 kb insert of the fosmid clone was determined by shotgun sequencing. The sequence analysis revealed the presence of 25 open reading frames (ORF), and ORF20 (EML1) showed similarities to lipases. Phylogenetic analysis of EML1 suggested that the protein belonged to a new family of esterase/lipase together with LipG. The EML1 gene was expressed in Escherichia coli, and purified by metal-chelating chromatography. The optimum activity of the purified EML1 (rEML1) occurred at pH 8.0 and 25 degrees C, respectively, and rEML1 displayed more than 50% activity at 5 degrees C. The activation energy for the hydrolysis of olive oil was determined to be 3.28 kcal/mol, indicating that EML1 is a cold-active lipase. rEML1 preferentially hydrolyzed triacylglycerols acyl-group chains with long chain lengths of > or = 8 carbon atoms and displayed hydrolyzing activities toward various natural oil substrates. rEML1 was resistant to various detergents such as Triton X-100 and Tween 80. This study represents an example which developed a new cold-active lipase from a deep-sea sediment metagenome.

Immobilization of lipase on surface modified magnetic nanoparticles using alkyl benzenesulfonate

The surface of nano-sized magnetite (NSM) particles synthesized by coprecipitation method was modified by alkyl benzenesulfonate (ABS) as a coating material. ABS on the NSM was expected to form a spacer between the surface of the NSM particles and the enzyme adsorbed and to play a role of strong enzyme adsorption onto a hydrophobic surface. Transmission electron microscopy showed that the NSM particles had an average size of 10 nm. Magnetic measurement revealed that the nanoparticles were superparamagnetic and the saturation magnetization was about 68 emu/g. Porcine pancreas lipase (PPL) was immobilized onto the ABS-NSM, which was to catalyze hydrolysis of olive oil and showed enhanced durability in the reuse after being recovered by magnetic separations.

Comparative performance of microbial lipases immobilized on magnetic polysiloxane polyvinyl alcohol particles

Microbial lipase from Mucor miehei and Candida rugosa were immobilized by covalent binding onto magnetized polysiloxane polyvinyl alcohol particles (POS-PVA). The resulting immobilized derivatives were evaluated in aqueous solution (olive oil hydrolysis) and organic solvent (butyl butyrate synthesis). Higher catalytic activities were found when the coupling procedure was made with M. miehei lipase. Immobilized M. miehei lipase also showed a better operational stability and a higher half-life than C. rugosa lipase after the successive batches of esterification. The performance of C. rugosa immobilized derivative was constrained by the low lipase loading used in the immobilizing step. Further information regarding the both immobilized derivatives was also obtained through chemical composition (FTIR).

Modeling of the Mass Transfer Effect in Biphasic Enzyme Membrane Reactor for Hydrolysis of Olive Oil

This article presents a model to explain the mass transfer phenomenon of the ions (OH-, Na+ and RCOO-) formed during the hydrolysis reaction on the aqueous side of the reaction sites in the biphasic enzyme membrane reactor (EMR) using lipase immobilized on modified poly (methyl methacrylate-ethylene glycol dimethacrylate) (PMMA-EGDM) clay composite membrane. Our model divides the aqueous side of the system into two regions, an unstirred film (aqueous side) adjacent to the pore end (region I) and charged capillaries of the membrane (region II). The process involves transport of Na+, OH- and RCOO- ions in region I whereas the film is considered as an electro-neutral region. Space charge model (consisting of Nernst-Planck equation for ion transport and nonlinear Poisson-Boltzmann (PB) equation for charge distribution) is used to describe transport of ions inside charged capillaries (pores) in region II. The solution of the nonlinear PB equation is computationally intensive and to overcome this difficulty, a series solution of the PB equation for the charge distribution inside the capillaries is developed. The suggested solution is used to determine the rate of formation of oleic acid using the information on pore size of the membrane (determined from molecular weight cut-off experiment) and diffusivity data from the literature. The result shows that the concentration of RCOO- ions at the boundary of region I of the aqueous phase increases with increasing the duration of reaction. The pore wall charge determined using the model is found to be in the range of 0.715-0.735 mV.

Lipase Immobilization on Oleic Acid−Pluronic (L-64) Block Copolymer Coated Magnetic Nanoparticles, for Hydrolysis at the Oil/Water Interface

Here, we have reported a new approach for utilizing oleic acid-Pluronic L-64 block copolymer coated iron oxide nanoparticles as supports for enzyme immobilization. Iron oxide nanoparticles were prepared by a coprecipitation method and were coated with oleic acid and Pluronic to achieve higher stability and dispersibility. The surface morphology and size of the particle, as determined by transmission electron microscopy (TEM), was +/- 10 nm. X-ray diffraction (XRD) patterns were taken over a range from 10 degrees to 90 degrees 20, using Cu K alpha radiation. Saturation magnetization values, measured at 300 K, varied from 34.6 emu/g to 60.8 emu/g. The possible interaction behavior of oleic acid and Pluronic was observed by Fourier transform infrared (FTIR) analysis and nuclear magnetic resonance (NMR) studies. Further potential of this material as a support for lipase immobilization and enzymatic hydrolysis at the oil/water interface was also investigated. The features of the surface-coated magnetic particles enable the adsorption of lipase from Candida cylindraces via strong hydrophobic interactions, which enhances the stability of the adsorbed enzyme molecules. The stability of the catalyst and, hence, its industrial applicability was tested by performing subsequent reaction cycles for the hydrolysis of olive oil. The activity of the immobilized lipase, pretreated with its substrate, was 510 U/g-matrix and was observed to be maintained at levels as high as 90% of its original activity for up to at least seven reuses.