Immobilization of lipase for effective interesterification of fats and oils in organic solvent

In order to investigate quantitatively the interesterification reaction, triolein and stearic acid were used as substrates and eight commercially available lipases were tested for their suitability for the reaction. Three fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipases was tested with olive oil, and it 2was noted that there was no correlation between their hydrolytic and interesterification activities. Among the lipases tested, Mucor miehei lipase was chosen for further study because of it high protein content and its relatively high hydrolytic and interesterification activities, both of which are required for effective interesterification. The effect of water activity of the interesterification reaction was investigated. interesterification activity was shown to be maximum at the water activity of 0.25. As the water activity of the lipase increased, hydrolysis of triglyceride was accelerated. At zero water activity, high conversion was achieved, although interesterification activity was relatively lower than that at the water activity of 0.25. A new and simple immobilization method was developed in order to render hydrophobicity to the lipase and hence to improve the interesterification activity of the lipase. The lipase was immobilized covalently with glutaraldehyde or with six alkyl chains as spacers onto Florisil (magnesium silicate, a inorganic matrix). Interesterification activity of the immobilized lipase with the hydrophobic spacers were increased against that of re lipase. The increase of activity was up to 8-fold that of the original activity of free lipase when the spacer was 7-aminoheptanoic acids. Relatively high stability of the immobilized lipase was shown in a continuous packed bed column reactor with a half-life of 97 days. (c) 1993 John Wiley & Sons, Inc.     

Enzymic interesterification of fats: The effect of non-lipase material on immobilized enzyme activity

An immobilized lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) suitable for fat interesterification has been prepared by precipitation onto diatomaceous earth (Celite) with acetone of a crude lipase preparation from an Aspergillus. Non-lipase material present in the preparation which precipitated at high acetone concentrations or ovalbumin added prior to the immobilization reduced the measured interesterification activity without affecting lipolytic activity. The non-lipase material reduced the interesterification activity by as much as 50%. The interesterification activity of immobilized preparations was enhanced by the use of higher concentrations of the crude lipase or, more substantially, by admixture of purified lipase.     

Enhanced thermostability of silica-immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate

A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55 degrees C, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70 degrees C. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55 degrees C.     

Culture conditions for intracellular lipase production by Rhizopus chinensis and its immobilization within biomass support particles

Acetone-dried cells of Rhizopus chinensis (with a 1,3-positional specificity lipase) were investigated for the interestierification reaction of olive oil and methyl stearate. First, the culture conditions for intracellular lipase production were examined, and then the activities of dried cells obtained from immobilization in Biomass Support Particles (BSPs) were compared with those of freely suspended cells.It was clear from cultivation of freely suspended cells that intracellular lipase activity for the interesterification reaction was enhanced sifnificantly by the presence of oleic acid, oil, and tea oil, but that the presence of glucose reduced the activity.The specific activity of dried cells within BSPs increased 7-fold compared with that obrained from freely suspended cells.The process presented here, using immobilization within BSPs, can provide cells directly as a catalyst with high activity, where cells become immobilized simply during batch operation, and no special preparation of cells is necessary. Therefore, the reaction system using dried cells immobilized within BSPs is a promising interesterifcation process for industrial applications.     

Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor

Ethyl acetate was explored as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel from the crude oils of Jatropha curcas (jatropha), Pongamia pinnata (karanj) and Helianthus annuus (sunflower). The optimum reaction conditions for interesterification of the oils with ethyl acetate were 10% of Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, ethyl acetate to oil molar ratio of 11:1 and the reaction period of 12h at 50 degrees C. The maximum yield of ethyl esters was 91.3%, 90% and 92.7% with crude jatropha, karanj and sunflower oils, respectively under the above optimum conditions. Reusability of the lipase over repeated cycles in interesterification and ethanolysis was also investigated under standard reaction conditions. The relative activity of lipase could be well maintained over twelve repeated cycles with ethyl acetate while it reached to zero by 6th cycle when ethanol was used as an acyl acceptor.     

Immobilization in the presence of Triton X-100: modifications in activity and thermostability of <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> CCR11 lipase

A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.     

Mass transfer effects in solvent-free fat interesterification reactions: influences on catalyst design

The use of solvent-free systems in the oil and fats industry is commonplace. Initial studies on interesterification were carried out in solvent systems because the lipase was immobilized solely by adsorption onto particles of diatomaceous earth. In this study, the mass transfer characteristics associated with the continuous interesterification of olive oil in a solvent-free system have been examined, for lipase immobilized on the three ion-exchange materials: Duolite ES562, Duolite ES568, and Spheroil DEA. The process of immobilization is influenced by the internal structure of the material and this in turn influences the interesterification activity of the catalyst. Individually prepared catalysts for the three support materials have shown that external mass transfer limitations are unlikely even at low flowrates.In the case of Spherosil DEA, with a mean pore diameter of 1480 A, the wide pores would be expected to reduce internal mass transfer limitations; however, it is more likely that the reduction in activity with increased catalyst loading is due to the lipase molecules being immobilized in a tightly packed monolayer. In such a situation, some active sites of the lipase molecules would become inaccessible to substrate molecules leading to an observed reduction in activity. For Duolite ES568, the observed results are very similar to those seen for Spherosil DEA, however, the pore structure of this support material indicate that some internal mass transfer limitations may also be occurring. Yet the contribution of the individual effects cannot be determined. The results observed for the support Duolite ES562 are different than those observed for the other materials and reflect the heterogeneity of Duolite ES562. The large proportion of narrow pores in the support mean that, for the catalysts examined, immobilization is most likely to have occurred in the external pores of the particles, and as such no internal mass transfer limitation is observed.It is clear that for interesterification the material chosen for enzyme immobilization will have an important role in determining the catalyst efficiency. External mass transfer limitations are very minor and observed internal mass transfer limitations may be caused by both internal mass transfer and the manner in which the immobilization process occurs. (c) 1994 John Wiley & Sons, Inc.     

Scale-up of flat-plate reactors for enzymatic hydrolysis of fats and oils

To determine the feasibility of continuous enzymatic fat-splitting, immobilized lipase reactors were constructed from alternating layers of enzyme support material and separator screens. Partially purified lipase from Thermomyces lanuginosus was loaded onto the support material at pH 5.5 by irreversible adsorption. Melted edible tallow at 51°C was pumped through the immobilized enzyme layers and swept from the downstream separator screens by buffer recycled from a continuous oil/water separator. Results from continuous operation of 10-layer reactors were compared with data from single-layer reactors. The activity per square centimeter of 10-layer reactors was nearly as much as that of single-layer reactors at the same enzyme loading and oil feed rate. Data were fitted to an empirical mathematical model.     

Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysosporium) thermophile Apinis

Summary Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture. Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%–100%. The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium. The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads. Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium. A minimum of 8 h regeneration period was necessary for lipase synthesis. Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts. Tween 80 enhanced lipase activity of the immobilized protoplasts. Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion. Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity.     

Interesterification of fats and oils by immobilized fungus at constant water concentration

The kinetics of enzymatic interesterification of oils and fats, using acetone-dried cells of Rhizopus chinensis immobilized on biomass support particles as a lipase catalyst, were investigated in batch operations at several constant water concentrations.Even under microaqueous (i.e., low-water-content) conditions, not only interesterification but also hydrolysis occured, and the water content in the reaction system decreased. The reaction rates of interesterification and hydrolysis at constant water concentrations were determined.For the reactions between olive oil and methyl stearate at several water concentrations, the parameters involved in the reaction model were determined by a trial-and-error method so as to make the calculated results correlate with the experimental data. The relationship between the parameters obtained and water concentration were examined.The rate constants involved in the reaction model of both interesterification and hydrolysis increased or decreased monotonically with the increasing water content, while the apparent activity of the lipase catalyst for interesterification had a maximum value at a water concentration of about 50 ppm. This suggests that when the water content is excessive the hydrolysis activity of lipase is accelerated more than its interesterification activity, and that when the water content is too little lipase activity can not be activated for either hydrolysis or interesterification.     

Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization

Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1). Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg. Activity of immobilized enzyme was 107.31 IU/mg. Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively. Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively. Ca2+ (5 mM) was found to be stimulator for enzyme activity. Immobilized lipase retained 68.18% of the original activity when stored for 40 days.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system

Candida rugosa lipase immobilized by adsorption on swollen Sephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in isooctane. Kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional to the enzyme concentration of 720 mug/g wet gel. The specific enzyme activity was 217 units/mg protein at 60% (v/v) olive oil concentration. When the initial rate is plotted versus concentration of olive oil, this system did not follow Michaelis-Menten kinetics. Maximum activity was obtained at pH 7, but optimum temperature shifted towards higher one with the increase of olive oil concentration. Among the various chemical compounds tested, Hg(2+) and Fe(2+) inhibited the lipase seriously. As the concentration of olive oil increased, the rate of the hydrolysis also increased, but degree of the hydrolysis was observed to decrease. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times. Achievement of chemical equilibrium took a longer time with the addition of water and the degree of hydrolysis decreased in the second consecutive trial. After the second hydrolysis trial, the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one.     

Effect of moisture on enzymatic reaction in supercritical carbon dioxide

The continuous acidolysis of triolein and stearic acid was carried out by an immobilized lipase to elucidate the characteristics of supercritical carbon dioxide (SC-CO₂) as a reaction medium. At first, an effect of temperature and pressure on the water adsorption to the immobilized lipase in the SC-CO₂ was examined. Then, the continuous interesterification of triolein and stearic acid by the moist immobilized lipase was examined. The amount of water adsorption to the immobilized lipase in the supercritical carbon dioxide measured under the condition of a different temperature and pressure has been expressed by a correlation equation of Freundlich type by using relative water standardized with the solubility of water in each condition. Optimum operating conditions of the interesterification by immobilized lipase in the SC-CO₂ was 323 K, 16.9 MPa and adsorbed-water concentration of 2 wt%. The production rate obtained by enzymatic acidolysis in the SC-CO₂ was found to be about 0.03 mmol/h2g-immobilized enzyme, leaving 74% residual triglyceride at the optimum operating conditions.     

Covalent-bonded immobilization of lipase on poly(phenylene sulfide) dendrimers and their hydrolysis ability

Covalent-bonded immobilization of lipase from burkholderia cepacia onto two poly(phenylene sulfide) (PPS) dendrimers with different generations (two and three) was achieved using carbodiimide as a coupling reagent. The hydrolysis activity of olive oil to fatty acid was studied on enzyme-immobilized PPS dendrimers. Enzyme activity was proportional to the enzyme loading, and highest recovered activity was obtained at the medium enzyme loading for both G2 and G3 dendrimers. The immobilization improved the optimum pH and caused the temperature range to widen. Immobilization of enzyme has enhanced the thermal stability of enzyme activity in comparison with free enzyme. The immobilized enzyme as a biocatalyst for batch hydrolysis of olive oil retained 80 approximately 90% activity even after 20 times of recycling. This retention of activity after recycle is very valuable and powerful in enzyme technology. The present noteworthy and vital availability on enzyme reaction of the covalently bonded immobilized lipase on dendrimer came from the structure of dendrimer with a large number of functional terminal groups, which are easily available for immobilization of many lipases at the situation keeping reactive enzymes on the surface of dendrimer.     

Continuous interesterification of oils and fats using dried fungus immobilized in biomass support particles

The industrial feasibility of an interesterification process using acetone-dried fungus (as a lipase catalyst) immobilized in biomass support particles (BSPs) was examined by continuous interesterification between olive oil and methyl stearate, where the water content of the reaction mixture (C_w) was controlled at a given value. The C_w affected not only the inactivation rate of lipase in the cells but also the production rate of the by-product (diglycerides). The optimal C_w was determined as about 100 ppm. The half-life of lipase in the cells was about 1200 h at the optimal C_w, suggesting that the interesterification process using the immobilized fungus is industrially feasible.     

Production of high-oleic acid tallow fractions using lipase-catalyzed directed interesterification, using both batch and continuous processing

Immobilized lipases were used to catalyze batch-directed interesterification of tallow, resulting in oleins containing significantly higher levels of unsaturated fatty acids than obtained by fractionation without lipase. After 14 days, a reaction catalyzed by 2% Novozym 435 yielded 57% olein unsaturation, compared with 45% in a no-enzyme control. Free fatty acid levels increased to 2-3% during reactions. Incubation of the enzyme in multiple batches of melted fat caused a gradual loss of interesterification activity, apparently due to progressive dehydration. The activity could be restored by addition of water to the reaction medium. Immobilized lipase was also used to catalyze directed interesterification in a continuous flow reactor. Melted tallow was circulated through a packed bed enzyme reactor and a separate crystallization vessel. The temperatures of the two parts of the apparatus were controlled separately to allow crystallization to occur separately from interesterification. Operation of the reactor with conventionally dry, prefractionated tallow allowed the formation of an olein consisting of up to 60% unsaturated fatty acids. The greatest changes in olein fatty acid composition were achieved when the fractionation temperature was kept constant at a value that promoted selective crystallization of trisaturated triglycerides that were continuously produced by enzymic interesterification. The enzyme could be reused without apparent loss of activity, and its activity was apparently enhanced by preincubation in melted tallow for up to several days. Control of both the water activity of the enzyme and tallow feedstock and of the absorption of atmospheric water vapor were required to maintain enzyme activity, during multiple reuse and minimize free fatty acid formation. This method may form the basis for a process to produce highly mono-unsaturated tallow fractions for use in food applications (e.g. frying) where a "healthy" low saturated fat product is required.     

Partial purification and characterization of almond seed lipase

A lipase was partially purified from the almond (Amygdalus communis L.) seed by ammonium sulfate fractionation and dialysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with K(m) and V(max) values of 25 mM and 113.63 micromol min(-1) mg(-1) for tributyrin as substrate. All triglycerides were efficiently hydrolyzed by the enzyme. The partially purified almond seed lipase (ASL) was stable in the pH range of 6-9.5, with an optimum pH of 8.5. The enzyme was stable between 20 and 90 degrees C, beyond which it lost activity progressively, and exhibited an optimum temperature for the hydrolysis of soy bean oil at 65 degrees C. Based on the temperature activity data, the activation energy for the hydrolysis of soy bean oil was calculated as -5473.6 cal/mol. Soy bean oil served as good substrate for the enzyme and hydrolytic activity was enhanced by Ca(2+), Fe(2+), Mn(2+), Co(2+), and Ba(2+), but strongly inhibited by Mg(2+), Cu(2+), and Ni(2+). The detergents, sodiumdeoxicholate and Triton X-100 strongly stimulated enzyme activity while CTAB, DTAB, and SDS were inhibitors. Triton X-405 had no effect on lipase activity. The partially purified enzyme retained its activity for more than 6 months at -20 degrees C, beyond which it lost activity progressively.     

Monoclonal antibodies to avian lipoprotein lipase. Purification of the enzyme by immunoaffinity chromatography

Three monoclonal antibodies to avian lipoprotein lipase have been isolated by fusing spleen cells from immunized BALB/c mice with myeloma P3X-63 Ag 8. The antibodies were detected by their ability to bind immobilized lipoprotein lipase in enzyme-linked immunosorbent assay (ELISA) and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibodies. Two of these antibodies, CAL1-7 and CAL1-11, inhibited catalytic activity, whereas with CAL1-2 interaction with lipoprotein lipase could be demonstrated only in ELISA and in Western blot assays following denaturation of the enzyme with sodium dodecyl sulfate. An immunoadsorbent column was prepared by coupling immunopurified CAL1-11 to Sepharose-4B. When acetone powder extracts of adipose tissue were applied on the column, 70% of the catalytic activity bound to the matrix. Effective elution was achieved with 1.8 M NaCl, 40% glycerol, 5% acetone, 20 mM Chaps (3[(3-cholamidopropyl)dimethylammonio]propanesulfonate), 0.5 mM EDTA, 1 mM phosphate (pH 6.5). After concentration of the active fractions on a heparin-Sepharose 4B column, the purified enzyme was obtained with an overall recovery of 25%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrates that the preparation is homogeneous with a major band at Mr 60900. Thus, avian adipose lipoprotein lipase has been purified by a one-step immunoaffinity followed by a concentrating step on heparin-Sepharose 4B.