Improvement of the enantioselectivity and activity of lipase from Pseudomonas sp. via adsorption on a hydrophobic support: kinetic resolution of 2-octanol

Pseudomonas sp. lipase (PSL) was successfully immobilized on a novel hydrophobic polymer support through physical adsorption and the immobilized PSL was used for resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed from the immobilized PSL compared with free PSL. The effects of reaction conditions such as temperature, water activity, substrate molar ratio and the amount of immobilized lipase were investigated. Under optimum conditions, the residual (S)-2-octanol was recovered with 99.5% enantiomeric excess at 52.9% conversion. The results also indicated that the immobilized PSL could maintain 94% of its initial activity even after reusing it five times.     

Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

Histamine was immobilized on Sepharose CL‐6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine‐Sepharose (HA‐S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA‐S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (ΔH_ads, ΔS_ads, and ΔG_ads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt‐buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA‐S was characterized by a less favorable ΔG_ads and an unfavorable ΔS_ads because histamine was covalently attached to Sepharose via a three‐carbon‐chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ΔS_ads was offset by a favorable ΔH_ads, thus exhibiting typical enthalpy‐entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA‐S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Immobilization of lipase from Candida rugosa on a polymer support

Lipase from Candida rugosa was immobilized by adsorption onto a macroporous copolymer support. Under optimum conditions the maximum amount of protein bound was 15.4 mg/g and the immobilization efficiency was 62%. The kinetics of lipase binding to the selected polymer carrier was assessed by using a general model of topochemical reactions. The effect of temperature on adsorption was thoroughly investigated, as was the adsorption mechanism itself. Analysis of the proposed kinetic model and the specific kinetic parameters measured suggest that surface kinetics control the adsorption process. According to the activation energy (E _a) and the rate constant, k, the enzyme has rather a high affinity for the support's active sites. The immobilized enzyme was used to catalyse the hydrolysis of palm oil in a lecithin/isooctane reaction system, in which the enzyme's activity was 70% that of the free enzyme. Kinetic parameters such as maximum velocity (V _max) and the Michaelis constant (K _m) were determined for the free and the immobilized lipase. Following repeated use, the immobilized lipase retained 56% of its initial activity after the fifth hydrolysis cycle. Received: 3 April 1998 / Received revision: 28 July 1998 / Accepted: 29 July 1998     

Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications

Abstract

The recovery of activity of lipases immobilized onto a photo-crosslinked polymer network was 76.0% and 41.0% for entrapment and adsorption methods, respectively. Both entrapped and adsorbed immobilized enzymes were very stable, retaining more than 60% of their activity over the range of temperatures studied. Immobilization by either method protected their relative activities nearly 96% at 70°C. The optimum pH was 8.0 for immobilized enzymes and 6.0 for the free enzyme at 40°C, while the relative activities after storage at 0–4°C for 30 days were 98% and 75% using entrapment and adsorption methods, respectively. These results indicated that lipase immobilized by entrapment and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Also, it can be safely stated that, photo-crosslinked polymer network can be used as alternative supports for immobilization of lipase for enzymatic polymerization reactions. In the ring-opening polymerization of ?-caprolactone, polymerization rates were clearly affected as monomer conversions were 58% and 49% and the highest molecular weights (M_n) obtained were 7890 and 5600 gmol^{? 1} for entrapment and adsorption methods, respectively.     

Comparison of physical and covalent immobilization of lipase from Candida antarctica on polyamine microspheres of alkylamine matrix

Abstract

Polyamine microspheres (PA-M) prepared using polyethyleneimine as matrix were used for the immobilization of Candida antarctica lipase. The isoelectric point of PA-M is 10.6, and the hydrophobicity of PA-M was indicated using naphthalene. Optimization of conditions showed that the maximal loading of lipase on PA-M reached 230.2 mg g^{? 1} at pH 9.0 and 35°C. An increased buffer concentration had no effect on the activity of lipase but decreased the amount of lipase adsorbed. Simulation with Langmuir and Freundlich isotherms demonstrated that the adsorption of lipase on PA-M was thermodynamically favorable. Covalent crosslinking of the lipase adsorbed extended the pH range and increased the optimal temperature of the lipase activity. The physically adsorbed lipase (P-lipase) and the covalently immobilized derivative (C-lipase) retained more than 75% and 85% of their initial activity, respectively, after 10 cycles of usage. The half-lives of P-lipase and C-lipase at 50°C were 15.70 and 27.67 times higher than that of the free enzyme, respectively. Compared to P-lipase, covalent immobilization obviously reduced the catalytic efficiency and activation energy of the enzyme.     

Stereoselective enzymatic esterification of 3-benzoylthio-2-methylpropanoic acid

Summary A key intermediate, S-(–)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(–) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(–) compound 1 was obtained in 35 mol% yield and 95% optical purity. Offsprint requests to: R. N. Patel     

Immobilization of Candida rugosa lipase on sporopollenin from Lycopodium clavatum

Sporopollenin is a natural polymer obtained from Lycopodium clavatum, which is highly stable with constant chemical structure and has high resistant capacity to chemical attack. In this study, immobilization of lipase from Candida rugosa (CRL) on sporopollenin by adsorption method is reported for the first time. Besides this, the enzyme adsorption capacity, activity and thermal stability of immobilized enzyme have also been investigated. It has been observed that under the optimum conditions (Spo-E_(0.3)), the specific activity of the immobilized lipase on the sporopollenin by adsorption was 16.3 U/mg protein, which is 0.46 times less than that of the free lipase (35.6 U/mg protein). The pH and temperature of immobilized enzyme were optimized, which were 6.0 and 40 °C respectively. Kinetic parameters V_max and K_m were also determined for the immobilized lipase. It was observed that there is an increase of the K_m value (7.54 mM) and a decrease of the V_max value (145.0 U/mg-protein) comparing with that of the free lipase.     

Immobilized copper-ion affinity adsorbent based on a cross-linked β-cyclodextrin polymer for adsorption of Candida rugosa lipase

Lipase from Candida rugosa was immobilized on a β-cyclodextrin-based polymer by adsorption and subsequent cross-linking with epichlorohydrin (EP-CD). The ligand iminodiacetic acid (IDA) was then bonded with the cross-linked β-cyclodextrin (EP-CD-IDA). This affinity adsorbent was further chelated with Cu^{2 +} for the purpose of binding affinity and stability. The properties of the immobilized lipase were assayed and compared with those of the free enzyme. Results showed that 266 µg protein with an activity of 17.85 U was bound per gram of matrix, giving 188% of the specific activity of the free enzyme and a total recovered activity of 79.7% under the optimum conditions. The pH and thermal stabilities of lipase were improved after immobilization on the β-cyclodextrin-based polymer (EP-CD-IDA-Cu^{2 +}). In addition, experimental results indicated that the residual activity of the immobilized lipase was 50% after eight cycles of reuse.     

A novel support for the immobilization of lipase and the effects of the details of its preparation on the hydrolysis of triacylglycerides

Summary The lipase from Candida cylindracea was immobilized by its adsorption on the internal surface of hydrophobic microporous poly(styrene-divinylbenzene) supports prepared by the concentrated emulsion polymerization method. The prepared supports have a surface area of the order of 200 m²/g. The immobilized enzyme catalyst is used for the hydrolysis of triacylglycerides. The effects of the amounts of surfactant and divinylbenzene used in the preparation of the hydrophobic support on the adsorption capacity for lipase and on the activity of the immobilized lipase have been investigated. The activity of the immobilized enzyme per enzyme molecule can be higher than that of the free lipase.     

Poly(styrene-divinylbenzene) beads surface functionalized with di-block polymer grafting and multi-modal ligand attachment: performance of reversibly immobilized lipase in ester synthesis

Fibrous poly(styrene-b-glycidylmethacrylate) brushes were grafted on poly(styrene–divinylbenzene) (P(S–DVB)) beads using surface-initiated atom transfer radical polymerization. Tetraethyldiethylenetriamine (TEDETA) ligand was incorporated on P(GMA) block. The ligand attached beads were used for reversible immobilization of lipase. The influences of pH, ionic strength, and initial lipase concentration on the immobilization capacities of the beads have been investigated. Lipase adsorption capacity of the beads was about 78.1 mg/g beads at pH 6.0. The K _m value for immobilized lipase was about 2.1-fold higher than that of free enzyme. The thermal, and storage stability of the immobilized lipase also was increased compared to the native lipase. It was observed that the same support enzyme could be repeatedly used for immobilization of lipase after regeneration without significant loss in adsorption capacity or enzyme activity. A lipase from Mucor miehei immobilized on styrene–divinylbenzene copolymer was used to catalyze the direct esterification of butyl alcohol and butyric acid.     

Immobilization of Candida antarctica lipase B (CALB) on surface-modified rice husk ashes (RHA) via physical adsorption and cross-linking methods

In the present study, the recovery of activity of Candida antarctica lipase B (CALB) immobilized onto surface-modified rice husk ash (RHA) was 90% for both cross-linking and adsorption methods. Both cross-linked and adsorbed immobilized preparations were very stable, retaining more than 48% of their activity over the range of temperatures studied. The optimum temperature and optimum pH values were 37?°C and 7.0, respectively for both immobilized preparations, while the relative activities after storage at 4.0?°C for 60 days were 55% and 65% using cross-linking and adsorption methods, respectively. Also, the activity of the immobilized lipase began to decrease after 10 cycles, more than 58% of the initial activities were still retained after 10 cycles for both immobilization methods. These results indicated that lipase immobilized by cross-linking and adsorption not only effected activity recovery, but also remarkably effected stability, reusability and application adaptability. It can be concluded that, surface-modified RHA can be used as alternative supports for immobilization of CALB for polymerization reactions.     

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 Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

Temperature-dependence of enantioselectivity and desymmetrization in the acetylation of 2-mono- and 2,2-di-substituted 1,3-propanediols by a novel lipase isolated from the yeast Cryptococcus spp. S-2

The effect of temperature on enantioselectivity and desymmetrization in the acetylation of 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c) and 2-benzyl-2-methyl-1,3-propanediol (1d) by a novel lipase (CSL) isolated from the yeast Cryptococcus spp. S-2 was studied. Desymmetrization of 1a, 1c and 1d by CSL-catalyzed acetylation was observed in the temperature range of ?20°C to 40°C, while diacetylation of 1b occurred considerably even at 0°C. The kinetic parameters of the selectivity indicated that the acetylation of 1a is an entropy controlled process whereas the reaction of 1c and 1d is mainly controlled by the enthalpy term. In the monoacetylation of the diol 1d, the preferred configuration in the enantiomeric induction by CSL was opposite to that of immobilized porcine pancreatic lipase (PPL).     

Enhancing performance of lipase immobilized on methyl-modified silica aerogels at the adsorption and catalysis processes: Effect of cosolvents

Hydrophobic silica aerogels modified with methyl group were applied as support to immobilize Candida rugosa lipase (CRL). At the adsorption process, different alcohols were used to intensify the immobilization of CRL. The results showed that n-butanol wetting the hydrophobic support prior to contacting with enzyme solution could promote lipase activity, but the adsorption quantity onto the support decreased. Based on this, a novel immobilization method was proposed: the support contacted with enzyme solution without any alcohols, and then the immobilized enzymes were activated by 90% (V) n-butanol solution. The experimental results showed that this method could keep high adsorption quantity (413.0 mg protein/g support) and increase the lipase specific activity by more than 50%. To improve the stability of immobilized lipase, the support after adsorption was contacted with n-octane to form an oil layer covering the immobilized lipases, thus the leakage can be decreased from over 30–4% within 24 h. By utilizing proper cosolvents, a high enzyme activity and loading capacity as well as little loss of lipase was achieved without covalent linkage between the lipase and the support. This is known to be an excellent result for immobilization achieved by physical adsorption only.     

Meetings & Symposia

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.     

Improved activity of a lipase by vacuum drying on to a hydrophobic microporous support

Summary Lipase from Rhizopus arrhizus was immobilized by physical adsorption on hydrophobic microporous polypropylene supports. The immobilized enzyme catalyst was employed for the hydrolysis of palm kernel olein in the presence of n-hexane. The initial rate of lipolysis for vacuum dried immobilized lipase is nearly double that of air dried. The initial rate of lipolysis declines with increase of drying time. Immobilized lipase clearly reveals a relatively high initial rate after 30 days of storage at 4 °C. Stability of the immobilized lipase in buffer could be enhanced up to three-fold that of the free lipase.     

Asymmetric ammonolysis of (R/S)-mandelic acid by immobilized lipases via direct amidation of mandelic acid in biphasic media

Abstract

We have investigated the direct enantioselective amidation of mandelic acid with ammonia, catalyzed by a variety of commercial lipases including those from Candida rugosa, Mucor miehei, Pseudomonas sp., Rhizomucor miehei, and Thermomyces lanuginosus covalently immobilized onto Florisil^® support via glutaraldehyde and polysuccinimide spacer arms. All the immobilized lipase preparations tested preferentially amidated the R isomer of mandelic acid. The highest amide yields were obtained for immobilized Pseudomonas sp. lipase preparations under the optimized reaction conditions. After 24 h of amidation, the reaction had proceeded with an excellent yield (50%) and enantiopurity (> 99%). The immobilized Pseudomonas sp. lipase preparations catalyzed the amidation reaction with the same yield and enantioselectivity. The enzyme immobilized via a glutaraldehyde spacer arm showed better reusability than that immobilized via a polysuccinimide spacer arm.

In view of these results, it is revealed that the direct amidation of mandelic acid catalyzed by the immobilized Pseudomonas sp. lipases is a facile and effective methodology for obtaining (S)-mandelic acid and (R)-mandelamide.     

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.     

Lipase immobilized by modification-coupled and adsorption–cross-linking methods: A comparative study

Candida antarctica lipase was immobilized by an adsorption and cross-linking method with NW-ZT2 and by modification-coupled method with a silica–PEG gel. The final product silica–PEG-lipase was confirmed by IR spectra. The optimum pH value, the optimum temperature, the thermo-stabilities and operational stabilities for two kinds of immobilized lipase were also determined. Results show that the silica–PEG-lipase gel was superior to the lipase immobilized by adsorption and cross-linking, however both are viable for use in transesterification reactions.