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.     

Immobilization of Candida rugosa lipase on poly(3-hydroxybutyrate-co-hydroxyvalerate): a new eco-friendly support

The overall objective of this study is to evaluate the morphological [scanning electron microscopy (SEM)], physicochemical [differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), chemical composition analysis, Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR)], and biochemical properties of Candida rugosa lipase (CRL) immobilized on a natural biopolymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. CRL was immobilized by physical adsorption with efficiency of 30%. Compared with free CRL enzyme, there were slight changes in immobilized CRL activity as a function of temperature (from 37°C to 45°C), but a similar optimal pH value of 7.0. Inactivation rate constants for immobilized CRL enzyme were 0.009 and 0.334 h⁻¹, and half-lives were 77 and 2 h at 40°C and 60°C, respectively. Kinetic parameters obtained for immobilized CRL include the Michaelis–Menten constant of K _m = 213.18 mM and maximum reaction velocity of V _max = 318.62 U/g. The operational stability of immobilized CRL was tested repeatedly, and after 12 cycles of reuse, the enzyme retained 50% activity. Based on our results, we propose that PHBV-immobilized CRL could serve as a promising biocatalyst in several industrial applications.     

Investigation of deactivation thermodynamics of lipase immobilized on polymeric carrier

In the present work, we have investigated biochemical thermo-kinetic stability of lipases immobilized on a biocompatible polymeric material. Immobilization of lipase Candida rugosa (CRL) was carried out on biocompatible blend of poly vinyl alcohol (PVA) and chitosan (CHY) support via entrapment and glutardehyde (Glu) cross-linking method to produce PVA:CHY:CRL and PVA:CHY:Glu:CRL as robust biocatalyst. These immobilized lipases were characterized by various physico-biochemical characterization techniques. Later on, thermal and solvent stability of polymer immobilized lipase was determined in term of half-life time (t _0.5), D values, enthalpy (ΔH°), entropy (ΔS°), and free energy (ΔG°) of deactivation at different temperatures and in various solvents. The thermodynamic deactivation stability trend was found as: cross-linked lipase CRL > entrapped lipase CRL > free lipase CRL. Moreover, kinetic parameters, such as K _m, V _max, and catalytic efficiency, were also determined to understand the kinetic features. The polymer immobilized enzyme was reused to investigate the economic viability of the developed biocatalyst.

     

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.     

A chemo-enzymatic process for sequential kinetic resolution of (R,S)-2-octanol under microwave irradiation

A combination of the enzymatic resolution and chemical racemization for the heterogeneous sequential kinetic resolution (SKR) was employed to resolve (R,S)-2-octanol under microwave irradiation. Mesoporous molecular sieves SBA-15, alumina and strong basic styrene anion exchange resin were screened and selected as the optimum supports to immobilize the lipase from Pseudomonas sp. (PSL), oxidant-Chromium trioxide (CrO₃) and reductant-Sodium borohydride (NaBH₄), respectively. The immobilized catalysts exhibited good reusability: it remained 90%, 72% and 80% of their initial activities after five reuses for the immobilized lipase, the immobilized oxidant and the immobilized reductant, respectively. Further, the E values of the immobilized PSL was increased from 23 under conventional heating to 40 under microwave irradiation in resolution of (R,S)-2-octanol. The immobilized catalysts were then used in SKR of (R,S)-2-octanol under microwave irradiation after optimizing the reaction media. Under the optimum conditions, (R)-2-octanol acetate was obtained at 99% enantiomeric excess with 84% yield in 2 h.     

Immobilized CALB Catalyzed Transesterification of Soybean Oil and Phytosterol

Candida antarctica lipase B (CALB) was immobilized on Fe₃O₄/SiO_x-g-P(GMA) polymer carrier to catalyzed the transesterification of soybean oil and phytosterol. The enzyme loading of the obtained particles was 98.7 mg/g supports and the enzyme activity was 1226.5 U/g. The average particle size was 100.5?±?1.30 nm and the magnetization was 15.80 emu/g. The immobilized enzyme showed higher activities at a wider range of pH and temperatures. Its optimum reaction temperature was up to 50 °C; increased by 5 °C compared to the free enzyme. The obtained magnetic immobilized Fe₃O₄/SiO_x-g-P(GMA) lipase was nanoscale. First-grade soybean oils were used as a substrate. System pH was adjusted to 7.0. The optimal reaction temperature was 50 °C and the reaction time was 3 h. The phytosterol concentration of 5% and immobilized CALB of 2% were obtained. The conversion rate of transesterification reaction between soybean oil and phytosterol was 86.2%. The use of magnets can quickly separate the immobilized enzymes from the substrates. The relative activity of the immobilized enzymes was 83.0% when reused seven times. The prepared immobilized CALB can improve efficiently enzyme activity and reutilization.     

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.     

Synthesis of calix[n]arene-based silica polymers for lipase immobilization

The objective of this study was to prepare new calix[n]arene-based silica polymers for immobilization of Candida rugosa lipase. The amino functionalized calix[4]arene (C4P), calix[6]arene (C6P) and calix[8]arene (C8P)-based silica polymers were used for the covalent attachment of C. rugosa lipase using glutaraldehyde as a coupling agent. The characterization of synthesized CnP polymers and immobilized lipases were made by Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and scanning electron microscope (SEM) techniques. The hydrolytic activities of immobilized lipases (CnP-L) were evaluated and compared with the free enzyme. The activity recovery of immobilized CRL (C. rugosa lipase) based on the carrier C4P, C6P and C8P reaches 74.6%, 68.5% and 51.4%, respectively. The optimal pH and temperature region of the immobilized lipases for the hydrolysis of p-NPP were 7.0 and 50 °C. Nevertheless, the immobilized lipase has good stability, adaptability and reusability in comparison with the free enzyme.     

Stereochemistry of a diastereoisomeric amphiphile and the species of the lipase influence enzyme activity in the transesterification catalyzed by a lipase-co-lyophilizate with the amphiphile in organic media

Modified Candida rugosa and Pseudomonas cepacia lipase (CRL and PCL) were co-lyophilized with two pairs of synthetic diastereoisomeric amphiphiles, d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl-propionylamino)-pentanedioic acid didodecyl ester (d- and l-BIG2C₁₂CA); d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-pentanedioic acid didodecyl ester (d- and l-2C₁₂GE). Enzyme activities of the modified lipase in the transesterification in organic solvent were evaluated. Both pairs of the diastereoisomeric amphiphiles showed enhanced enzyme activity in the transacetylation between racemic sulcatol and isopropenyl acetate in diisopropyl ether, catalyzed by the PCL-co-lyophilizate, by 19–48 fold when compared to the native lipase lyophilized from buffer alone independent of the stereochemistry of the amphiphiles, while in the case of the CRL-co-lyophilizate only the l-BIG2C₁₂CA showed enhanced enzyme activity in the transbutyrylation between racemic solketal and vinyl butyrate in cyclohexane as high as 68–78 fold.     

Resolution of 2-octanol via immobilized Pseudomonas sp. lipase in organic medium

Abstract

Pseudomonas sp. lipase (PSL) immobilization was performed using three different protocols. Lipase immobilized on Diaion HP20 (HP20-PSL) exhibited the highest catalytic activity and stability in the kinetic resolution of racemic 2-octanol. The reaction rate of HP20-PSL was approximately 20 times higher than that of free PSL and the residual activities of HP20-PSL and free PSL were respectively 84% and 19% after incubation in the reaction medium for 72 h. A study of the effect of different reaction parameters on HP20-PSL-catalyzed resolution of (R,S)-2octanol showed that the optimal water content of the immobilized matrix and the optimal molar ratio of vinyl acetate to 2-octanol were 60 ± 5% and 2.5:1, respectively. Under the optimized reaction conditions, (S)-2-octanol of high optically purity (enantiomeric excess > 99%) could be recovered at 53% conversion rate, and HP20-PSL could be reused for ten cycles without significant decrease in its activity and enantioselectivity.     

Biodiesel production from waste cooking oil by immobilized lipase on superparamagnetic Fe3O4 hollow sub-microspheres

Superparamagnetic Fe₃O₄ hollow sub-microspheres (FHSM) with strong response to an external magnet were prepared via a solvothermal method, followed by acid etching. Lipase from Candida sp. 99–125 was directly immobilized onto the amino-functional FHSM by simple adsorption, without glutaraldehyde linkage. The immobilized lipase was used to catalyze the esterification/transesterification of waste cooking oil with methanol to produce fatty acid methyl ester (FAME), a major source of biodiesel. FAME yield exceeded 93.4% over a wide range of temperatures from 10 to 40?°C. Notably, stability was clearly improved at the lower temperatures, in particular, giving a FAME yield of 89.6% after eight cycles of use at 10?°C.     

Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.     

Enhanced catalytic ability of Candida rugosa lipase immobilized on pore-enlarged hollow silica microspheres and cross-linked by modified dextran in both aqueous and non-aqueous phases

Candida rugosa lipase (CRL) is one of the most widely used lipases. To enhance the catalytic abilities of CRL in both aqueous and non-aqueous phases, hollow silica microspheres (HSMSs) with a pore size of 18.07 nm were used as an immobilization support, and aldehydecontaining dextrans were employed to further cross-link the adsorbed CRL. In the experimental ranges examined, the loading amount of lipase linearly increased to 171 ± 3.4 mg_protein/g_support with the CRL concentration and all the adsorption equilibriums were reached within 30 min. After simple cross-linking, the tolerance to pH 4.0 ～ 8.0 as well as the thermal stability of immobilized CRL at 40 ～ 80°C were both substantially increased, and 82 ± 2.1% activity remaining after the sixth reuse. The immobilized CRL was successfully applied to the resolution of racemic ibuprofen in non-aqueous phase. The initial reaction rate increased by 1.4- and 3.6-fold compared with the rates of adsorbed and native lipases, respectively. Furthermore, the R-ibuprofen was obtained at ee > 93%, and the enantiomeric ratio reached E > 140 at the conversion of 50 ± 1.5% within 48 h.     

Improved activity and stability of Rhizopus oryzae lipase via immobilization for citronellol ester synthesis in supercritical carbon dioxide

In present work, Rhizopus oryzae lipase immobilized on a film prepared using blend of hydroxylpropyl methyl cellulose (HPMC) and polyvinyl alcohol (PVA) was investigated for synthesis of citronellol esters with supercritical carbon dioxide (Sc-CO₂) as a reaction medium. The transesterification reaction was optimized for various reaction parameters like effect of molar ratio, acyl donor, time, temperature, enzyme concentration, effect of pressure and co-solvent to achieve the maximum yield of desired product. The results obtained signify remarkable increment (about eightfold) in the yield of citronellol acetate (91%) as compared to that of free lipase (11%) in Sc-CO₂. The developed biocatalytic methodology provides a substantial advantage of low biocatalyst loading (1.5%, w/v), lower reaction temperature (45 °C) and lower pressure (8 MPa) as compared to previous reports. The immobilization method has significantly enhanced the operational stability of lipase for ester synthesis under Sc-CO₂ conditions. The developed methodology was successfully applied for synthesis of three different industrially important citronellol esters namely citronellol acetate (91%), citronellol butyrate (98%), citronellol laurate (99%) with excellent yields using vinyl esters as acyl donor under Sc-CO₂ conditions. In addition, the immobilized biocatalyst was effectively recycled for three consecutive recycles.     

Silanized palygorskite for lipase immobilization

Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The K_m and V_max values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively.     

Study of lipase immobilization on zeolitic support and transesterification reaction in a solvent free-system

In order to understand the role of the acid–base, electrostatic and covalent interactions between enzyme and support, the catalytic behavior of the Rhizomucor miehei lipase (RML) immobilized on zeolite materials has been studied. The highest lipase activities were obtained when this enzyme, immobilized by adsorption, interacts through acid–base binding forces with the support surface, resulting in activation of the enzyme catalytic center. Due to the interest in biodiesel production by mild enzymatic transesterification, this heterogeneous biocatalyst has been used in transesterification of fatty acids contained in olive oil. The results show a high oleic acid conversion for several reaction cycles with a higher total biodiesel productivity compared to that using the free enzyme.     

Enzymatic synthesis of short-chain diacylated alkylglycerols: A kinetic study

Lipase-catalyzed transesterification of 1-O-octadecyl glycerol (batyl alcohol) with ethyl butyrate has been studied. The effect of vacuum on the rate of both transesterification and distillation of ethyl butyrate has also been investigated and at different reaction conditions, more than 85% of 1-O-octadecyl glycerol was consumed in only 5 min giving rise to the monoester. Then the monoester is again acylated to produce the diesterified product. In addition, the transesterification reactions were effected in solvent free reaction medium and it has been scaled-up to produce up to ca. 500 g of 2,3-dibutyroil-1-O-alkylglycerols in three consecutive cycles reutilizing the same batch of lipase. An efficient evaporation of ethanol was necessary to significantly reduce the reaction times of the transesterification reaction. Finally, a kinetics model describing both the rate of transesterification and the rate of inactivation of the immobilized lipase has been developed. The results indicate that the operational stability of the immobilized lipase confined into the mesh baskets (according to the value of k_d attained), was very high and that provides a half-life of the lipase higher than 1500 h.The present procedure is intended to be used for the synthesis of homogeneous alkylglycerols with biological activities and/or precursors of structured alkylglycerols.     

Synthesis of cinnamyl acetate catalysed by highly reusable cotton-immobilized Pseudomonas fluorescens lipase

Cinnamyl acetate as an important fragrance ingredient could be synthesized by lipase-catalysed transesterification in organic systems, but enzyme proteins tended to denature and inactivate for no water lubrication. To improve the non-aqueous stability of lipases, absorbent cotton was taken as an alternative “water” phase to stabilize enzyme proteins. In a mass ratio of 1:1, Pseudomonas fluorescens lipase was immobilized on cotton fibres by physical absorption in a column glass bottle, forming a facile cotton-lipase bioreactor in which the transesterification between cinnamyl alcohol and vinyl acetate processed efficiently. From the molar conversions after reaction for 2?h at 37?°C and 160?rpm, the ability of cotton-lipase to transform substrate was more than 5-folds of native lipase. And even in static state and at 4?°C, the conversion of reaction catalysed by cotton-PFL had 11-fold increase relative to native lipase after 8?h. Recycles showed that the cotton-lipase had an extra-long half-life of activity (t_1/2?=?693?h) and a negligible decay rate in the ability to transform substrate (D_r?=?0.08% h^?1). All these showed that this lipase had been effectively activated and stabilized by cotton fibres for the numerous hydroxyl groups and fluffy structure.     

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.     

High-yield synthesis of wax esters catalysed by modified <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase

Candida rugosa lipase (CRL) was applied in a non-solvent esterification reaction to yield twelve wax esters. All products were obtained in nearly 100% yield for 10 h at 50°C when immobilized PEG₂₀₀₀-activated C. rugosa lipase was added to the reaction mixture. The surfactant had also a beneficial effect on the stability of the biocatalytic preparation with 83% of its activity conserved after the seventh run of repeated batch reactions.