Covalent Immobilization Of Lipase Research Articles

Covalent immobilization of lipase on magnetic biochar for one-pot production of biodiesel from high acid value oil

Magnetic biochar was synthesized via chelation of Fe3+ with carboxymethyl cellulose and pyrolysis for covalently immobilizing Eversa® Transform lipase. The magnetic biochar had 75.8 mg/g lipase loading that was 54.1 % higher than that without magnetism. The immobilized lipase achieved 91.3 mg/g lipase loading with 19.2 U/mg lipase activity after optimization. It showed good thermal and acid stability with 82.5 % and 98.2 % relative activity at 45 °C and pH 4, respectively. Its relative activity was 90.8 % after stored for 30 d at 4 °C. After magnetically separated for 10 cycles, it still kept 70.1 % activity due to the strong covalent bonding. The lipase further catalyzed one-pot esterification and transesterification of high acid value oil (38 mg KOH/g) with 95.7 % biodiesel yield and cycled for 10 times at 85.7 % yield. Kinetic study gave the activation energy of 28.7 kJ/mol. The covalently immobilized lipase could find practical applications.

Covalent immobilization of lipase on an ionic liquid-functionalized magnetic Cu-based metal-organic framework with boosted catalytic performance in flavor ester synthesis.

Enzymatic esterification plays an important role in the fields of chemistry and biotechnology. In this study, lipase was immobilized on an ionic liquid (IL)-modified magnetic metal-organic framework (MOF) and used to synthesize isoamyl acetate. The immobilized lipase (PPL-ILs/Fe3O4@MOF) showed 2.1-fold and 1.8-fold higher activity compared to the free and immobilized lipase without ILs (PPL-Fe3O4@MOF), respectively. In addition, the anti-denaturant ability and reusability of the PPL-ILs/Fe3O4@MOF were also higher than those of other samples. The ester yield reached 75.1% when the biocatalyst was used to synthesize isoamyl acetate in hexane. The synthesized supports supplied a good microenvironment for the immobilized lipase through multiple interactions. Results of the structural analysis showed that the conformation state of lipase molecules changed after immobilization. The magnetism of the prepared biocatalyst makes it easy to recycle so that PPL-ILs/Fe3O4@MOF maintained 70.2% of the initial activity after eight cycles. The prepared composite materials exhibited good potential in lipase immobilization with enhanced catalytic ability and stability.

Covalent Immobilization of Lipase on Bacterial Cellulose Membrane and Nanocellulose

The cellulose from Komagataeibacter hansenii is synthesized as a thin film at the surface of glucose based media. Strong acid hydrolysis release sections of crystalline cellulose chains in nanometric scale that leads to persistent suspensions in water. The cellulose anhydro-glucose hydroxyls are suitable to receive functional groups as enzymes, and lipases have great economic value being a valuable model for protein immobilization. In this work both, the membrane of bacterial cellulose as well the nanocellulose produced trough acid hydrolysis, was functionalized with a lipase. The bacterial cellulose membranes were produced by Hestrin-Schramm medium, and nanocelluloses produced from the pristine material was characterized using techniques as 13C solid state NMR and transmission electron microscopy (TEM). The pristine membranes and nanocellulose were functionalized with succinic acid as linker, then lipase was conjugated using EDC (N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride), and NHS (N-Hydroxysuccinimide). The effectiveness of the chemical process was characterized, and the lipase activity were measured. The presence of the succinic acid and amide linkage, as well physical-chemical changes on the functionalized polysaccharide. Hence, we inferred that after immobilization the enzyme maintained its activity in both cellulose and the cellulose membrane.

Lipase on carbon nanotubes – an active, selective, stable and easy-to-optimize nanobiocatalyst for kinetic resolutions

An efficient biocatalyst was obtained by covalent immobilization of lipase B from C. antarctica on functionalized SwCNTs for the kinetic resolution of some aryl-ethanols in batch and continuous-flow modes.

Metal-Organic Frameworks Conjugated Lipase with Enhanced Bio-catalytic Activity and Stability.

Covalent immobilization of lipase onto a solid carrier is an effective way to enhance stability. Immobilization inhibits the activity of lipase due to decreased flexibility of enzyme structure via the covalent bond. In this study, monomer of the metal-organic frameworks (MOFs) material ZIF-8 (2-methyl imidazole-4-carboxylic acid) was innovatively used as a chemical modifier of Candida nrugosa lipase (CRL). The circular dichroism spectra results show that the CRL molecule was altered by chemical modification and thus its catalytic activity was 1.3 times higher than that of the free CRL. The modified CRL molecule was further immobilized in the "skeleton" of ZIF-8 through the monomer while in situ forming the cell skeleton of the MOFs, which prevent the active center from being destroyed. The results show that conjugation of chemical modification and immobilized enzymes ensure that there was no obvious reduction in the activity of CRL after immobilization and the stability of CRL was improved. Especially, the organic solvent stability of the modified immobilization CRL in isopropanol was significantly improved and retained more than 148% of its activity.

Enantioselective Resolution of (R, S)-2-Phenoxy-Propionic Acid Methyl Ester by Covalent Immobilized Lipase from Aspergillus oryzae.

(R)-2-Phenoxy-propionic acid methyl ester (PPAM) is an important chiral precursor of aryloxy phenoxy propionate herbicides. The covalent immobilization of lipase from Aspergillus oryzae WZ007 and the catalysis of enantioselective (R, S)-PPAM resolution by the immobilized A. oryzae lipase (AOL) were investigated in this study. The primary amino resin LX-1000HA was selected as the support for the covalent immobilization of AOL. The Km and Vmax of the immobilized lipase were 1.97mM and 4.84 × 103μmol/mgmin, respectively. The key reaction parameters (pH, temperature, rotation speed, and substrate concentration) for the lipase-catalyzed resolution of (R, S)-PPAM were optimized. An e.e.s of 99.5% and conversion rate of 50.8% were achieved under the optimal conditions of pH7.5, 30°C, and substrate concentration 500mM. The immobilized lipase retained 87.3% of its initial activity after 15cycles of the repeated experiments. The results demonstrated that the covalent immobilized AOL has potential industrial applications.

Covalent Immobilization of Lipase on Novel Nanofibrous Membrane for Catalysis of the Organic Synthesis

Covalent Immobilization of Lipase on Novel Nanofibrous Membrane for Catalysis of the Organic Synthesis

Bisepoxide-activated Hollow Silica Microspheres for Covalent Immobilization of Lipase from Burkholderia cepacia

An efficient and easy-to-perform method was developed for covalent immobilization of lipase from Burkholderia cepacia (Lipase PS) on hollow silica microspheres (M540) by bisepoxide activation. For immobilization, various bisepoxides of different length, rigidity and hydrophobicity in their linkers were applied to activate the amino groups on the M540 support. Effect of the individual bisepoxides on the catalytic performance of the immobilized Lipase PS was studied by using lipase-catalyzed kinetic resolution (KR) of racemic 1-phenylethanol (rac-1) with vinyl acetate in batch mode. Catalytic activity, enantiomer selectivity, recyclability and thermal stability of the new immobilized Lipase PS biocatalysts were investigated. The optimal enzyme / support ratio with the support activated by the most efficient bisepoxide, i.e. poly(ethylene glycol) diglycidyl ether (PDE), was 1:5. The most efficient Lipase PS on PDE activated M540 showed an almost five fold higher biocatalytic activity value (rbatch = 42.8 U/g) with enhanced selectivity (ee(R)-2 = 99.1 %) to the free form of Lipase PS (rbatch = 9.0 U/g; ee(R)-2 = 98.9 %). The Lipase PS on PDE-M540 was compared to a commercially available immobilized Lipase PS biocatalyst (Lipobond Lipase PS) and also applied in a packed-bed enzyme reactor operated in continuous-flow mode, where the optimal temperature of M540-PDE-PS reached the 70 °C, while the optimum for Lipobond Lipase PS was 50 °C.

Covalent immobilization of lipase onto chitosan-mesoporous silica hybrid nanomaterials by carboxyl functionalized ionic liquids as the coupling agent.

Chitosan-mesoporous silica SBA-15 hybrid nanomaterials (CTS-SBA-15) were synthesized by means of carboxyl functionalized ionic liquids as the coupling agent. The as-prepared CTS-SBA-15 support was characterized by TEM, FTIR, TG and nitrogen adsorption-desorption techniques. Porcine pancreas lipase (PPL) was then bound to the hybrid nanomaterials by using the cross-linking reagent glutaraldehyde (GA). Further, the parameters like cross-linking concentration, time and ratio of supports to enzyme were optimized. The property of immobilized lipase were tested in detail by enzyme activity assays. The results indicated that the hybrid nanomaterials could form three-dimensional (3D) structure with homogeneous mesoporous structures and immobilized PPL revealed excellent enzymatic performance.

Covalent immobilization of lipase onto aminopropyl-functionalized hydroxyapatite-encapsulated-γ-Fe2O3 nanoparticles: A magnetic biocatalyst for interesterification of soybean oil.

Hydroxyapatite-encapsulated γ-Fe2O3 nanoparticles were prepared, and lipase from Candida rugosa was then covalently bound onto the magnetic materials via covalent linkages. The magnetic carrier and immobilized lipase were characterized by enzyme activity assays, XRD, FT-IR, TEM, VSM and N2 adsorption-desorption techniques. Results demonstrated that γ-Fe2O3 nanoparticles were coated with the hydroxyapatite, and the lipase was indeed tethered to the magnetic carriers without damage to their structure. The immobilized lipase showed a strong magnetic responsiveness and displayed high catalytic activities towards the interesterification of soybean oil. The interesterified products were evaluated for their total fatty acid (FA) composition, slip melting point (SMP), iodine value, triacylglycerols (TAGs) profile and FA composition at sn-2 position in TAGs. The FA positional distributions and TAG species significantly changed after the enzymatic interesterification. Besides this, the interesterified products showed an obvious reduction in their SMP in comparison with the physical blends.

Physical and Covalent Immobilization of Lipase onto Amine Groups Bearing Thiol-Ene Photocured Coatings.

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37°C (free lipase) to 50-55°C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70% of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56% of its hydrolytic activity and 67% of its synthetic activity in the same cycle period. In the case of hydrolysis V max values slightly decreased after immobilization. For synthetic assay, the V max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.

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

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.

Covalent Immobilization of Lipase onto Citric Acid-Esterified Loofah Sponge

Loofah sponge was activated by thermochemical esterification between the carboxyl groups of citric acid and hydroxyl groups of cellulose to introduce free carboxyl groups, which were further reacted with lipase amino groups with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as the condensing reagent. This resulted in loofah sponge–immobilized lipase. Under the optimized immobilization conditions, the highest activity of immobilized lipase per gram of dry carrier was found to be 45.8 U/g. The immobilized lipase exhibited maximum activity at 40 °C, pH 8.0, while the optimal temperature and pH for the free lipase were 37 oC and 7.5, respectively. The immobilized lipase showed better thermal stability, storage stability, and reusability than free lipase.

Use of lipase immobilized on celluse support for cleaning aged oil layers

The present study reports (i) the covalent immobilization of lipase on different cellulose supports (cotton buds, make-up remover pads, cellulose powder, cotton and tissues) using sodium periodate as activating agent and (ii) its application on the aged linseed oil removal from canvas. The optimization of experimental conditions such as pH, temperature and reaction time was performed for both, immobilization procedure and the biocatalyst application. Thus optimal conditions of immobilization were pH 7.0, 20 ºC, 0.3 mg lipase loading per mg support and 200 min reaction time, while those for treating canvas surface, having stratified aged linseed oil were pH 6, 40 ºC and 45 min reaction time. The ability of the immobilized lipase to remove aged oil films was confirmed by UV-Vis spectroscopy, high performance liquid chromatography (HPLC) and scanning electron microscopic (SEM) analysis.

Conformational Changes of Lipases in Aqueous Media: A Comparative Computational Study and Experimental Implications

AbstractConformational changes occurring to the open form of five different lipases were studied by means of molecular dynamic simulations in explicit water. The conformational changes indicate remarkable differences among the lipases considered, not only in terms of accessibility of the active site but also of modification of the geometry of the catalytic machinery. Lipase B from Candida antarctica undergoes minor conformational change at either level, so that it appears to be the most suitable lipase for being applied and formulated in aqueous environments or other hydrophilic media. On the other side, lipase from Pseudomonas cepacia undergoes the most relevant conformational variations both at the level of the catalytic triad and the residues involved in the oxyanion stabilization, suggesting that its “interfacial activation” is not simply related to a variation of the accessibility of the active site. Indeed, preliminary experimental data here reported indicate that covalent immobilization of lipase from Pseudomonas cepacia performed in the presence of hydrophobic solvent allows one to achieve a more than 10‐fold increase in immobilization yield as compared to similar protocols performed in simple aqueous buffer. On the contrary, the benefit coming from immobilizing lipase B from Candida antarctica in hydrophobic solvent appears more limited (two‐fold higher immobilization yield).

Covalent immobilization of lipase onto amine functionalized polypropylene membrane and its application in green apple flavor (ethyl valerate) synthesis

In this study, a functionalized hydrophobic polypropylene chloride membrane (PPC) was prepared by the amination of chlorinated polypropylene with hexamethylene diamine (APP). The PPC and APP membranes were characterized using SEM, FTIR and contact angle studies. The aminated polypropylene (APP) membrane was used for covalent immobilization of Candida rugosa lipase via glutaraldehyde coupling. The retained activity of the immobilized lipase was 76%. Kinetic analysis shows that the dependence of lipolytic activity of both free and immobilized lipase on tributyrin substrate concentration can be described by Michaelis–Menten model. The estimated apparent K m values for the free and immobilized lipase were 2.9 and 8.4 mM, respectively. The V max values of free and immobilized enzymes were calculated as 926 and 741 U/mg enzyme, respectively. Optimal temperature was 5 °C higher for immobilized enzyme than that of the free enzyme. Thermal and storage stabilities were found to be increased upon immobilization. Finally, the immobilized lipase was used for the production of green apple flavor (i.e., ethyl valerate) in hexane medium.

Covalent immobilization of lipase from Candida rugosa onto poly(acrylonitrile- co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application

A simple way of fabricating enzymatic membrane reactor with high enzyme loading and activity retention from the conjugation between nanofibrous membrane and lipase was devised. Poly(acrylonitrile- co-2-hydroxyethyl methacrylate) (PANCHEMA) was electrospun into fibrous membrane and used as support for enzyme immobilization. The hydroxyl groups on the fibrous membrane surface were activated with epichlorohydrin, cyanuric chloride or p-benzoquinone, respectively. Lipase from Candida rugosa was covalently immobilized on these fibrous membranes. The resulted bioactive fibrous membranes were examined in catalytic efficiency and activity for hydrolysis. The observed enzyme loading on the fibrous membrane with fiber diameter of 80–150 nm was up to 1.6% (wt/wt), which was as thrice as that on the fibrous membrane with fiber diameter of 800–1000 nm. Activity retention for the immobilized lipase varied between 32.5% and 40.6% with the activation methods of hydroxyl groups. Stabilities of the immobilized lipase were obviously improved. In addition, continuous hydrolysis was carried out with an enzyme-immobilized fibrous membrane bioreactor and a steady hydrolysis conversion (3.6%) was obtained at a 0.23 mL/min flow rate under optimum condition.

Immobilization of lipase and penicillin acylase on hydrophobic acrylic carriers

This paper describes the immobilization of lipase and penicillin acylase (PA) on hydrophobic acrylic carriers crosslinked with ethylene glycol dimethacrylate (EGDMA) and diethylene glycol dimethacrylate (DEGDA). Two methods of immobilization were used for lipase—physical adsorption on unmodified copolymers and covalent attachment on carriers with amino groups, whereas penicillin acylase was only covalently linked to carriers with amino groups. Physical adsorption gave preparations with high activity and specific activity but poor storage stability. In the best case the immobilized lipase had specific activity 36,352 U/L/mg, that is two times higher than the native enzyme used in an immobilization procedure. However, physically immobilized lipase retained only 20–35% of the initial activity after 3-month storage. Covalent attachment gave less active but stable preparations. Highly hydrophobic carrier composed of 2-ethylhexyl acrylate (2EHA)/EGDMA and modified with 1,4 diaminobutane gave the best results in covalent immobilization of lipase since enzyme retained relatively high activity (10,587 U/L/mg) and storage stability (62.5% of initial value). The best carriers for penicillin acylase immobilization are crosslinked with DEGDA as they display preferential sorption and then immobilization of penicillin acylase, what leads to highly active preparations. The best carrier – BA/DEGDA modified with 1,4-diaminobutane – immobilized 5.46 mg of protein/mL with specific activity 1.59 U/mg. More than 70% of this initial activity was retained after 1-month storage of this enzymatic preparation.

Covalent immobilization of lipase from Candida rugosa on Eupergit®

An approach is presented for the stable covalent immobilization of Upase from Candida rugosa on Eupergit? with a high retention of hydrolytic activity. It comprises covalent bonding via lipase carbohydrate moiety previously modified by periodate oxidation, allowing a reduction in the involvement of the enzyme functional groups that are probably important in the catalytic mechanism. The hydrolytic activities of the lipase immobilized on Eupergif1 by two conventional methods (via oxirane group and via glutaralde-hyde) and with periodate method were compared. Results of lipase assays suggest that periodate method is superior for lipase immobilization on Eupergit? among methods applied in this study with respect to both, yield of immobilization and hydrolytic activity of the immobilized enzyme.

Covalent immobilization of lipase onto hydrophobic group incorporated poly(2-hydroxyethyl methacrylate) based hydrophilic membrane matrix

In this study, a hydrophobic group containing monomer, 2-methacrylamidophenyalanine (MAPA) was prepared by using methacrylochloride and phenylalanine. Then, poly(2-hydroxyethyl methacrylate-co-methacrylamido-phenlyalanine) (pHEMA-MAPA) membranes were prepared by UV-initiated photopolymerization of HEMA and MAPA in the presence of an initiator α-α ′-azobisisobutyronitrile (AIBN). The lipase was immobilized onto these membranes by covalent bonding through carbodiimide activation. The amount of enzyme loading on the membranes was increased as the MAPA ratio increased in the membrane structure. Immobilization improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with immobilization and at 60 ° C the thermal stability constants were 1.1×10 −1min for free enzyme and 1.2×10 −2min for the immobilized enzyme. The immobilized enzyme activity was found to be quite stable in repeated experiments.