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Abstract

Porcine pancreatic lipase (PPL) and Candida cylindracea lipase (CCL) were immobilized on Celite and Amberlite IRA 938 by deposition from the aqueous solution by the addition of hexane. The influence of the immobilization on the activities of the immobilized lipase derivatives has been studied. The immobilized lipases were used in synthesis of pentyl isovalerates. Various reaction parameters affecting the synthesis of pentyl isovalerates were investigated. The reaction rates were compared with the rates of esterification with free lipases. The immobilized lipases were found to be very effective in the esterification reaction. The lipases immobilized on Celite 545 exhibited better operational stabilities than that of immobilized on Amberlite IRA‐938.     

Studies of reaction parameters on synthesis of Citronellyl laurate ester via immobilized Candida rugosa lipase in organic media

Immobilized Candida rugosa lipase was used for the synthesis of citronellyl laurate from citronellol and lauric acid. Screening of different types of support (Amberlite MB-1 and Celite) for immobilization of lipase and solvent (n-hexane, n-heptane, and iso-octane) and optimization of reaction conditions, such as catalyst loading, effect of substrates molar ratio and temperature, have been studied. The maximum enzyme activity was obtained at 310 K. The immobilized C. rugosa lipase onto Amberlite MB-1 support was found to be the best support with a conversion of 89% of citronellyl laurate ester in iso-octane compared to Celite 545. Deactivation of C. rugosa lipase at 313, 318 and 323 K were observed. Ordered bi bi mechanism with dead end complex of lauric acid was found to fit the initial rate data and the kinetic parameters were obtained by non-linear regression analysis.     

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(?) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)).     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

Summary Lipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.Dedicated to Professor H. Holzer, Freiburg University, on his 60th birthday (June 13, 1981)     

Enantioselective behavior of lipases from <Emphasis Type="Italic">Aspergillus niger</Emphasis> immobilized in different supports

Considering the extraordinary microbial diversity and importance of fungi as enzyme producers, the search for new biocatalysts with special characteristics and possible applications in biocatalysis is of great interest. Here, we report the performance in the resolution of racemic ibuprofen of a native enantioselective lipase from Aspergillus niger, free and immobilized in five types of support (Accurel EP-100, Amberlite MB-1, Celite, Montmorillonite K10 and Silica gel). Amberlite MB-1 was found to be the best support, with a conversion of 38.2%, enantiomeric excess of 50.7% and enantiomeric ratio (E value) of 19 in 72 h of reaction. After a thorough optimization of several parameters, the E value of the immobilized Aspergillus niger lipase was increased (E = 23) in a shorter reaction period (48 h) at 35°C. Moreover, the immobilized Aspergillus niger lipase maintained an esterification activity of at least 80% after 8 months of storage at 4°C and could be reused at least six times.     

Esterification of phenolic acids catalyzed by lipases immobilized in organogels

Lipases from Rhizomucor miehei and Candida antarctica B were immobilized in hydroxypropylmethyl cellulose organogels based on surfactant-free microemulsions consisting of n-hexane, 1-propanol and water. Both lipases kept their catalytic activity, catalyzing the esterification reactions of various phenolic acids including cinnamic acid derivatives. High reaction rates and yields (up to 94%) were obtained when lipase from C. antarctica was used. Kinetic studies have been performed and apparent kinetic constants were determined showing that ester synthesis catalyzed by immobilized lipases occurs via the Michaelis–Menten mechanism.     

Production of diacylglycerols from glycerol monooleate and ethyl oleate through free and immobilized lipase-catalyzed consecutive reactions

The ability of free and immobilized lipase on the production of diacylglycerols (DAG) by transesterification of glycerol monooleate (GMO) and ethyl oleate was investigated. Among three free lipases such as lipase G (Penicillium cyclopium), lipase AK (Pseudomonas fluorescens) and lipase PS (Pseudomonas cepacia), lipase PS exhibited the highest DAG productivity, and the DAG content gradually increased up to 24 hours reaction and then remained steady. The comparative result for DAG productivity between free lipase PS and immobilized lipases (lipase PS-D and Lipozyme RM IM) during nine times of 24 hours reaction indicated that total DAG production was higher in immobilized lipase PS-D (183.5mM) and Lipozyme RM IM (309.5mM) than free lipase PS (122.0mM) at the first reaction, and that the DAG production rate was reduced by consecutive reactions, in which more sn-1,3-DAG was synthesized than sn-1,2-DAG. During the consecutive reactions, the activity of lipase PS was relatively steady by showing similar DAG content, whereas DAG production of lipase PS-D and Lipozyme RM IM was gradually decreased to 69.9 and 167.1mM at 9th reaction, respectively, resulting in 62% and 46% reduced production when compared with 1st reaction. Interestingly, from 7th reaction lipase PS produced more DAG than immobilized lipase PS-D, and exhibited a stable activity for DAG production. Therefore, the present study suggested that DAG productivity between GMO and ethyl oleate was higher in immobilized lipases than free lipases, but the activity was reduced with repeated uses.     

Immobilization of Lipases on Clay, Celite 545, Diethylaminoethyl-, and Carboxymethyl-Sephadex and their Interesterification Activity

Lipases AK, PS (both from Pseudomonas sp), and AY (from Candida rugosa) were immobilized on clay, Celite 545, DEAE-Sephadex, or CM-Sephadex and used to synthesize structured lipids from tricaprylin and trilinolein. Lipases AK and AY immobilized on Celite 545 had the highest interesterification activity (0.11 and 0.35 units, respectively) while lipase PS had the highest activity (0.18 units) when immobilized on CM-Sephadex. During 72 h reaction, the highest product yields were obtained in Celite AK (224 µmol), and CM PS (290 µmol) after 48 h and Celite AY (501 µmol) after 24 h. Lipase AK immobilized on DEAE-Sephadex was the most stable for repeated (5 times) operation.     

Recent Advances in the use of Immobilized Lipases Directed Toward the Asymmetric Syntheses of Complex Molecules

Water-insoluble compounds can be substrates for enzymatic reactions when lipases are immobilized properly and suitable organic solvents are used. In this review, three type of lipase immobilization method and their application to the asymmetric syntheses of complex molecules are described. Lipases immobilized with Celite or synthetic prepolymers such as urethane prepolymer and photo-crosslinkable resin prepolymer have been applied for the kinetic resolution of many kinds of water-insoluble substrate.

Phospholipid-lipase aggregates with ether linkages are novel and have been found to function effectively as immobilized lipases in asymmetric hydrolysis or esterification reactions in water-saturated organic solvent. The phospholipid-lipase aggregates are considered to have a stacked bilayer based on X-ray diffraction analysis structure of the lipid in the crystalline phase.     

Production of Ricinoleic Acid from Castor Oil by Immobilised Lipases

Abstract

Porcine pancreatic lipase (PPL), Candida rugosa lipase (CRL), and Castor bean lipase (CBL) were immobilized on celite by deposition from aqueous solution by the addition of hexane. Lipolytic performance of free and immobilized lipases were compared and optimizations of lipolytic enzymatic reactions conditions were performed by free and immobilized derivatives using olive oil as substrate. Afterwards, the influence on lipolysis of castor oil of free lipases and immobilized lipase derivatives have been studied in the case of production of ricinoleic acid. All of the lipases performances were compared and enzyme derivative was selected to be very effective on the production of ricinoleic acid by lipolysis reaction. Various reaction parameters affecting the production of ricinoleic acid were investigated with selected the enzyme derivative.

The maximum ricinoleic acid yield was observed at pH 7–8, 50°C, for 3 hours of reaction period with immobilized 1,3-specific PPL on celite. The kinetic constants K_m and V_max were calculated as 1.6 × 10^?4 mM and 22.2 mM from a Lineweaver–Burk plot with the same enzyme derivative. To investigate the operational stability of the lipase, the three step lipolysis process was repeated by transferring the immobilized lipase to a substrate mixture. As a result, the percentange of conversion after usage decreased markedly.     

Immobilization of lipases for non-aqueous synthesis

Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n-butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.     

Immobilization of lipase within carbon nanotube–silica composites for non-aqueous reaction systems

The immobilization of lipases within sol–gel derived silica, using multi-walled carbon nanotubes (MWNTs) as additives in order to protect the inactivation of lipase during sol–gel process and to enhance the stability of lipase, was investigated. Three sol–gel immobilized lipases (Candida rugosa, Candida antarctica type B, Thermomyces lanuginosus) with 0.33% (w/w) MWNT showed much higher activities than lipase immobilized without MWNT. The influence of MWNT content and MWNT shortened by acid treatment in the sol–gel process on the activity and stability of immobilized C. rugosa lipase was also studied. In hydrolysis reaction, immobilized lipase containing 1.1% pristine MWNT showed 7 times higher activity than lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT showed 10 times higher activity in esterification reaction, compared with lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT retained 96% of initial activity after 5 times reuse, while the lipase immobilized without MWNT was fully inactivated under the same condition.     

The effect of substrate polarity on the lipase-catalyzed synthesis of aroma esters in solvent-free systems

The aim of this study was to compare activities of commercial lipases in synthesis of various esters in solvent-free system and in isooctane. Moreover, the effect of substrate polarity (expressed as log P) on solvent-free synthesis was investigated. The decrease of yields of esters of butanoic acid in absence of organic solvent was observed, while similarly high yields were noticed in synthesis of esters of octanoic acid in both systems (solvent-free and organic solvent). The kinetic analysis has shown that ester synthesis can be described with Ping-pong bi-bi kinetics. In a case of esterification of butanoic acid in solvent-free system additional term, which represents enzyme inactivation by acid substrate, must be included. It was found out that log P of initial substrate mixture was in linear correlation with kcat of ester synthesis, while final yields depend only on type of acid substrate. Each of the examined lipases showed similar properties, although immobilized lipase from Rhizomucor miehei was slightly more resistant to harmful influence of butanoic acid. Finally, it was also shown that detrimental influence of butanoic acid could be circumvented by two-step addition of acid substrate in reaction catalyzed with immobilized lipase from R. miehei.     

Modification of Phospholipids with Lipases and Phospholipases

Different commercial lipases and phosphoiipases were studied in the hydrolysis and transesterification of synthetic phosphatidylcholine and soybean lecithin. Wide variations in the lipase and phospholipase activities and in the protein contents of the preparations were observed. The substrate specificity varied between different enzymes. A high degree of hydrolysis of synthetic and soybean phospholipids was achieved with both types of enzymes.

Enzymes immobilized on Celite were used in the transesterification of dimyristoyl phosphatidylcholine and oleic acid. The conversions were carried out both without solvent and in the presence of toluene. The amount of modified phosphatidylcholine was measured using HPLC. The highest amount of modified phosphatidylcholine was obtained in solvent-free transesterification. The best results were obtained with Aspergillus niyer lipase.     

Morphological,biochemical and kinetic properties of lipase from Candida rugosa immobilized in zirconium phosphate

Zirconium phosphate (ZrP), a low-cost inorganic material with well-defined physicochemical properties, was successfully used as support for immobilizing Candida rugosa lipase by covalent bonding. The immobilized derivative showed high catalytic activity in both aqueous and non-aqueous media. Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy measurements demonstrated that the ZrP fulfilled the morphological requirements for use as a matrix for immobilizing lipases. The free and immobilized lipases were compared in terms of pH, temperature and thermal stability. The immobilized lipase had a higher pH optimum (7.5) and higher optimum temperature (50°C) than the free lipase. Immobilization also increased the thermal stability. The hydrolysis of p-nitrophenyl palmitate (pNPP) by immobilized lipase, examined at 37°C, followed Michaelis–Menten kinetics. Values for K_m=1.18 µM and V_max=325Umg^?1 indicated that the immobilized system was subject to mass transfer limitations. The immobilized derivative was also tested under repetitive reaction batches in both ester hydrolysis and synthesis.     

Triglyceride interesterification by lipases. 3. Alcoholysis of pure triglycerides

Lipase-catalyzed alcoholysis of triolein dissolved in ethanol or isopropanol for the formation of ethyl and isopropyl esters was investigated. Of 16 lipases screened, Amano lipase from P. fluorescens was selected for investigation of the effects of basic reaction conditions on alcoholysis yields. Ethanolysis yields were only slightly affected by water additions to immobilized lipase preparations. Isopropyl ester yields decreased with water addition. Good operational stability was observed over 17 days. Changes in initial triolein concentration in the range 5–50 mM had very little effect on ester yields. The ionic strength of the phosphate buffer used in lipase immobilization affected ethanolysis and isopropanolysis yields in opposite ways. The highest ethanolysis yields were obtained with lipases immobilized from 250 mM buffer, while isopropyl ester yields were highest with lipases immobilized from water. In addition, the quantities and isomers of monoglyceride intermediates in ethanolysis were affected by the immobilization buffer strength. Larger quantities of 2-monoglycerides were formed in ethanolysis reactions with lipase preparations immobilized from water.     

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.     

Modification of Phospholipids with Lipases and Phospholipases

Different commercial lipases and phosphoiipases were studied in the hydrolysis and transesterification of synthetic phosphatidylcholine and soybean lecithin. Wide variations in the lipase and phospholipase activities and in the protein contents of the preparations were observed. The substrate specificity varied between different enzymes. A high degree of hydrolysis of synthetic and soybean phospholipids was achieved with both types of enzymes.

Enzymes immobilized on Celite were used in the transesterification of dimyristoyl phosphatidylcholine and oleic acid. The conversions were carried out both without solvent and in the presence of toluene. The amount of modified phosphatidylcholine was measured using HPLC. The highest amount of modified phosphatidylcholine was obtained in solvent-free transesterification. The best results were obtained with Aspergillus niyer lipase.