Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system

This paper reports a simple method for producing macroporous silica-monoliths with controllable porosity that can be used for the immobilization of lipases to generate an active and stable micro-reactor for biocatalysis. A range of commercially available lipases has been examined using the hydrolysis reactions of 4-nitrophenyl butyrate in water–decane media. The kinetic studies performed have identified that a similar value for k_cat is obtained for the immobilized Candida antarctica lipase A (0.13 min⁻¹) and the free lipase in solution (0.12 min⁻¹) whilst the immobilized apparent Michaelis constant K_m (3.1 mM) is 12 times lower than the free lipase in solution (38 mM). A 96% conversion was obtained for the immobilized C. antarctica lipase A compared to only 23% conversion for the free lipase. The significant higher conversions obtained with the immobilized lipases were mainly attributed to the formation of a favourable biphasic system in the continuous flowing micro-reactor system, where a significant increase in the interfacial activation occurred. The immobilized C. antarctica lipase A on the monolith also exhibited improved stability, showing 64% conversion at 80 °C and 70% conversion after continuous running for 480 h, compared to 40 and 20% conversions under the same temperature and reaction time for the free lipase.     

Enhancing the catalytic properties of porcine pancreatic lipase by immobilization on SBA-15 modified by functionalized ionic liquid

The mesoporous silica SBA-15 was modified by carboxyl-functionalized ionic liquid (COOH-IL-SBA). The prepared support was used to immobilize porcine pancreatic lipase (PPL) by physical adsorption (PPL-COOH-IL-SBA) and covalent attachment (PPL-CON-IL-SBA). Enzymatic properties of the immobilized PPL were investigated in the triacetin hydrolysis reaction. It was found that carboxyl functionalized ionic liquid modification of the support surface was an effective method to improve the properties of immobilized PPL. Incorporating into the functionalized SBA-15 made PPL more resistant to temperature and pH changes, compared with PPL immobilized on parent SBA-15 (PPL-SBA). Especially, after the covalent attachment to a functionalized support, the stability of PPL was improved obviously, which retained 81.25% and 52.50% of the original activity after incubation for 20 days and four times recycling, respectively, whereas PPL-SBA exhibited only 58.80% and 27.78% of the original activity under the same conditions. In addition, physical and chemical properties of the supports and immobilized PPL were characterized by small-angle X-ray powder diffraction (SAXRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), nitrogen adsorption, nuclear magnetic resonance (NMR) and thermogravimetry (TG). The images and data confirmed chemical modification in SBA-15 and PPL immobilization on the tested support.     

Immobilization and characterization of human carbonic anhydrase I on amine functionalized magnetic nanoparticles

In this study, we synthesized magnetic nanoparticles (MNPs) by co-precipitation method. After that, silica coating with tetraethyl orthosilicate (TEOS) (SMNPs), amine functionalization of silica coated MNPs (ASMNPs) by using 3-aminopropyltriethoxysilane (APTES) were performed, respectively. After activation with glutaraldehyde (GA) of ASMNPs, human carbonic anhydrase (hCA I) was immobilized on ASMNPs. The characterization of nanoparticles was performed by transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and vibrating sample magnetometer (VSM). The immobilization conditions such as GA concentration, activation time of support with GA, enzyme amount, enzyme immobilization time were optimized. In addition of that, optimum conditions for activity, kinetic parameters (K_m, V_max, k_cat, kcat/K_m), thermal stability, storage stability and reusability of immobilized enzyme were determined.The immobilized enzyme activity was optimum at pH 8.0 and 25 °C. The K_m value of the immobilized enzyme (1.02 mM) was higher than the free hCA I (0.48 mM). After 40 days incubation at 4 °C and 25 °C, the immobilized hCA I sustained 89% and 85% of its activity, respectively. Also, it sustained 61% of its initial activity after 13 cycles. Such results revealed good potential of immobilized enzyme for various applications.     

Enzyme, β-galactosidase immobilized on membrane surface for galacto-oligosaccharides formation from lactose: Kinetic study with feed flow under recirculation loop

The work focuses on producing galacto-oligosaccharides (GOS) through an enzymatic reaction with lactose under a partial recirculation loop by utilizing membrane-immobilized β-galactosidase. Cross-linking through covalent bonding, using gluteraldehyde, was employed to immobilize enzyme on a microporous polyvinylidene fluoride membrane. GOS synthesis was carried out in a laboratory fabricated reaction cell, whereby three immobilized membranes were housed in series. The reaction was conducted at varying initial lactose concentrations (ILCs) and feed flow rates at pH 6 and 40 °C. A maximum GOS of 30% (dry basis) was obtained after 60 h of reaction time, 50 g/L ILC, 241 U of enzyme (specific loading of 600 U/g-membrane), and 0.5 mL/min of feed flow rate at 56% lactose conversion. The GOS yield increased with increased ILC and decreased feed flow rate. The selectivity of GOS formation increased by increasing both the ILC and the feed flow rate, whereas the reverse was true for mono-saccharides. The immobilized enzyme retained ∼50% of its initial activity after 30 days of storage at 20 °C, while the native enzyme lost 100% of its activity within 21 days. Furthermore, a five-step, nine-parameter model was developed, and simulated results showed excellent agreement with the experimental data.     

Glucose oxidase enzyme immobilized porous silica for improved performance of a glucose biosensor

High activity of glucose oxidase (GOD) enzyme (immobilized in porous silica particles) is desirable for a better glucose biosensor. In this work, effect of pore diameter of two porous hosts on enzyme immobilization, activity and glucose sensing was compared. The hosts were amine functionalized: (i) microporous silica (NH₂-MS) and (ii) mesoporous silica (NH₂-SBA-15). Based on whether the dimension of GOD is either larger or smaller than the pore diameter, GOD was immobilized on either external or internal surface of NH₂-MS and NH₂-SBA-15, with loadings of 512.5 and 634 mg/g, respectively. However, GOD in NH₂-SBA-15 gave a higher normalized absolute activity (NAA), which led to an amperometric sensor with a larger linear range of 0.4–13.0 mM glucose. In comparison, GOD in NH₂-MS had a lower NAA and a smaller linear range of 0.4–3.1 mM. In fact, the present GOD-NH₂-SBA-15 electrode based sensor was better than other MS and SBA-15 based electrodes reported in literature. Thus, achieving only a high GOD loading (as in NH₂-MS) does not necessarily give a good sensor performance. Instead, a host with a relatively larger pore than enzyme, together with optimized electrode composition ensures the sensor to be functional in both hyper- and hypoglycemic range.     

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.     

Immobilized β-glucosidase on magnetic chitosan microspheres for hydrolysis of straw cellulose

β-Glucosidase immobilized on magnetic chitosan microspheres for potential recycling usage in hydrolysis of cellulosic biomass was investigated. The immobilized enzyme had an activity of 6.4 U/g support under optimized condition when using cellobiose as substrate. Immobilization resulted in less increase of the apparent K_m, low drift of the optimal pH, as well as improved stability relative to the free enzyme. The immobilized β-glucosidase was applied to enzymatic hydrolysis of corn straw to produce 60.2 g/l reducing sugar with a conversion rate of 78.2% over the course of a 32-h reaction. This conversion rate was maintained above 76.5% after recycling the enzyme for use in eight batches (total 256 h), showing favorable operational stability of the immobilized enzyme.     

Lipase immobilized microstructured fiber based flow-through microreactor for facile lipid transformations

A facile continuous flow-through Candida antarctica lipase B immobilized silica microstructured optical fiber (SMOF) microreactor for application in lipid transformations has been demonstrated herewith. The lipase was immobilized on the amino activated silica fiber using glutaraldehyde as a bifunctional reagent. The immobilized lipase activity in the SMOF was tested calorimetrically by determination of p-nitrophenyl butyrate hydrolysis products. The specific activity of the immobilized lipase was calculated to be 0.91 U/mg. The SMOF microreactor performance was evaluated by using it as a platform for synthesis of butyl laurate from lauric acid and n-butanol in n-hexane and n-heptane at 50 °C, with products identified by gas chromatography–mass spectrometry (GC–MS). Different substrate mole ratios were evaluated, with 1:3, lauric acid:n-butanol showing best performance. Remarkably, percentage yields of up to 99% were realized with less than ∼38 s microreactor residence time. In addition, the SMOF microreactor could be reused many times (at least 7 runs) with minimal reduction in the activity of the enzyme. The enzyme stability did not change even with storage of the microreactor in ambient conditions over one month.     

Ethyl isovalerate synthesis using Candida rugosa lipase immobilized on silica nanoparticles prepared in nonionic reverse micelles

Uniform and monodispersed silica nanoparticles were synthesized with a mean diameter of 100 ± 20 nm as analyzed by Transmission Electron Microscopy (TEM). Glutaraldehyde was used as a coupling agent for efficient binding of the lipase onto the silica nanoparticles. For the hydrolysis of pNPP at pH 7.2, the activation energy within 25–40 °C for free and immobilized lipase was 7.8 and 1.25 KJ/mol, respectively. The V_max and K_m of immobilized lipase at 25 °C for pNPP hydrolysis were found to be 212 μmol/min/mg and 0.3 mM, whereas those for free lipase were 26.17 μmol/min and 1.427 mM, respectively. The lower activation energy of immobilized lipase in comparison to free lipase suggests a change in conformation of the enzyme leading to a requirement for lower energy on the surface of the nanoparticles. A better yield (7 fold higher) of ethyl isovalerate was observed using lipase immobilized onto silica nanoparticles in comparison to free lipase.     

Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium

Racemic _DL-tert-leucine (_DL-Tle) was resolved to obtain enantiopure _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative with immobilized penicillin G acylase (PGA). The effects of pH, reaction temperature, substrate concentration and reaction time on the reaction were investigated. The reaction was conveniently carried out at 0.4 M substrate concentration in water at pH 8.0 and 30 °C. Under the optimized reaction conditions, _L-Tle was obtained in an enantiopure form (>99% ee) with 45.8% substrate conversion after 4 h. The thermal stability and operational stability of immobilized PGA were examined. Furthermore, the preparation of _L-Tle was successfully performed in a recirculating packed bed reactor (RPBR) system and immobilized PGA exhibited a long-term stability for 51 days with a slight decrease of activity. The isolated _D-enantiomer was racemized at 160 °C for 15 min and reused as substrate. The results obtained clearly demonstrated a potential for industrial application of immobilized PGA in the preparation of _L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative.     

Baeyer–Villiger oxidation with peracid generated in situ by CaLB-CLEA catalyzed perhydrolysis

Candida antarctica lipase B, immobilized as cross linked enzyme aggregates (CLEAs) was used to mediate the Baeyer–Villiger oxidation of cyclohexanone to ɛ-caprolactone, and the reaction was compared with the one using Novozym^® 435 as catalyst. The conversion was dependent on the initial concentration of cyclohexanone, and was about 90% after 48 h at concentrations of up to 0.25 M but was decreased at higher concentrations. Caprolactone concentrations up to 0.6 M had no effect on the reaction efficiency. Among the cyclic ketones tested, the highest degree of conversion was achieved for cyclopentanone (88%) and the lowest for cyclooctanone (about 2%). The effect of methyl substitution and position of substitution on the cycloketone was studied using methylcyclohexanone and it has shown to influence the conversion efficiency. Both hydrogen peroxide and the reaction by-product acetic acid had a deleterious effect on the stability of the biocatalyst.     

Ordered mesoporous materials containing Mucor Miehei Lipase as biocatalyst for transesterification reaction

Herein, we report the design of a biocatalyst by the immobilization of Mucor Miehei Lipase (Mm-L) onto mesoporous silica materials. Supports with different pore diameters have been considered. Infrared spectroscopy was used to determine the adsorption isotherms in different pH conditions.Then, the biocatalyst was tested for the methanolysis of colza oil. The production of methyl esters was monitored over time by gas chromatography coupled to a mass spectrometer. The results show that to reach maximum performance of the biocatalyst, a certain amount of water is required (5 wt.%). By using ratio lower than the stoichiometry (1:1), the methanol conversion is completed and high transesterification yields could be obtained even in the absence of non polar solvents (i.e. hexane). Herein, the lipase uses the fatty substrate as lipophilic interface required for the opening of the active site of the enzyme.     

Lipase immobilization on O-propargyl and O-pentynyl dextrans and its application for the synthesis of click beetle pheromones

Lipase of Rhizopus arrhizus was immobilized on O-propargyl dextran (PgD) and O-pentynyl dextran (PyD). Compared with Lewatit VP OC 1600 cation ion exchange resin, wood shaves, fuller earth, silica and alumina, PgD with degree of substitution (DS) of 0.68 and a surface of 10 m²/g was found to be the most effective immobilization support and an excellent biocatalyst for esterification reactions in organic solvents as the synthesis of click beetle pheromone geranyl octanoate. PyD (DS 0.44) with a surface of 3.3 m²/g was of similar high efficiency. For the enzymatic esterification the optimum concentration of geraniol and octanoic acid was 0.4 mol L^?1 each. The biocatalyst worked the best in hexane at a moisture level of 0.02%. The enzyme could be repeatedly used and conversion dropped from 80% to 70% after four cycles, while reaction rate even increased when repeatedly employed.     

A chip-based method for studying the effects of exogenous proteins on neuronal axons

It has been reported that the activity of protein improved when it was adsorbed inside the pores of mesoporous silica (MPS). The current study investigated the activity of immobilized avidin to the biotin on MPS with various pore sizes (diameter=2.4-45.0 nm). The binding amount of immobilized avidin to biotin is 123 to 160 ng biotin/10 μg avidin on 2.7- to 5.4-nm pore MPS, but that on 12- to 45-nm pore MPS was markedly decreased (33-42 ng biotin/10 μg). Moreover, the binding amount was approximately 2- and 3-fold higher on the glycidoxypropyl (Gly)-functionalized 5.4- and 45-nm pore MPS in comparison with methyl (Me)-functionalized 5.4- and 45-nm pore MPS, respectively. Furthermore, avidin immobilized in native and Gly-grafted 45-nm pore MPS retained more than 70% and 50% binding activity to biotin, respectively, after incubating at 90°C for 3 h. In contrast, the activity was greatly reduced in the native and Gly-grafted 5.4-nm pore MPS under the same conditions (<36.9%). The immobilization also protected against effects of 0.01 M HCl and 50% MeOH; all of immobilized avidin proteins showed high activity (>50%) with biotin compared with that observed with free avidin (MeOH [<18.2%] and HCl [<32.7%]).     

Combination use of ultrasound irradiation and ionic liquid in enzymatic isomerization of glucose to fructose

The synthetic effect of the combination use of ultrasound irradiation (UI) and ionic liquid (IL) on improving enzyme activity was studied when they were employed in isomerization of glucose to fructose by immobilized glucose isomerase (IGI, produced from streptomyces murinus and immobilized on silica). Both of UI and IL [EMIM][Cl], which was screened as the best medium for this reaction, were found to increase the enzyme activity in isomerization reaction. And a further increase of enzyme activity was observed by combination use of UI and IL. A systematic screening and optimization of the reaction parameters in ILs under UI on the IGI activity were performed. Under the optimum reaction conditions, 45.3% yield of fructose was achieved in 10 h under UI in [EMIM][Cl], compared to only 41.5% yield under stirring in [EMIM][Cl], 44.2% under UI without [EMIM][Cl] and 38.9% under stirring without [EMIM][Cl] in 12 h, respectively. High thermal stability and reusability of IGI was also observed under UI in [EMIM][Cl]. These results indicated that the combination use of UI and IL might be a fast and efficient method for enzymatic isomerization of glucose to fructose.     

Evaluation of adsorbents for separation and purification of paclitaxel from plant cell cultures

In this study, we evaluated the efficiency of different adsorbents for the removal of plant-derived impurities during the pre-purification of paclitaxel from plant cell cultures. Using the synthetic adsorbents sylopute and active clay and their major components SiO₂ and MgO, we performed adsorbent treatment and analyzed the paclitaxel precipitates recovered from hexane precipitation. When SiO₂ was used, the highest purity (～58.1%) and yield (～91.5%) of paclitaxel were obtained. We also determined differences in the effectiveness of the adsorbent treatment according to changes in the surface area, pore volume and pore diameter of SiO₂. Adsorbent treatment was more effective when pore diameter was larger (silica I [2.19 nm] < silica II [4.92 nm] < silica III [9.07 nm]). The highest purity (～74.3%) and yield (～92.9%) of paclitaxel were obtained when silica III was used in the adsorbent treatment. Pore diameter had a greater effect on the removal of plant-derived impurities during the pre-purification of paclitaxel compared with surface area and pore volume. This result could be confirmed by HPLC analysis of the absorbent after treatment and TGA of the organic substances that were bonded to the adsorbent.     

Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads

The para-nitrobenzyl esterase (PNBE), which was encoded by pnbA gene from Bacillus subtilis, was immobilized on amino-functionalized magnetic supports as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE-CLEA immobilized on the magnetic beads using glutaraldehyde as a coupling agent was 31.4 mg/g of beads with a 78% activity recovery after the immobilization. The performance of immobilized PNBE-CLEA was evaluated under various conditions. As compared to its free form, the optimal pH and temperature of PNBE-CLEA were 1 unit (pH 8.0) and 5 °C higher (45 °C), respectively. Under different temperature settings, the residual enzyme activity was highest for the PNBE-CLEA, followed by covalently fixed PNBE without further cross-linking and the free PNBE. During 40 days of storage pried, the PNBE-CLEA maintained more than 90% of its initial activity while the free PNBE maintained about 60% under the same condition. PNBE-CLEA also retained more than 80% activity after 30 reuses with 30 min of each reaction time, indicating stable reusability under aqueous medium.     

Xylitol production in a bubble column bioreactor: Influence of the aeration rate and immobilized system concentration

This work evaluated the xylitol production from sugarcane bagasse hemicellulosic hydrolysate in a bubble column bioreactor using cells of the yeast Candida guilliermondii immobilized in calcium-alginate. The fermentation runs were performed according to a 2² full factorial design with three replicates at the center point in order to determine the effect of the variables: aeration rate (0.66–1.33 vvm) and immobilized system concentration (20–40% v/v), on the efficiency of xylose-to-xylitol conversion and on the xylitol volumetric productivity. The results indicated a significant influence of both variables on xylitol production. The highest conversion efficiency (41%) was attained using 1.33 vvm aeration rate and 40% immobilized system. Under these conditions, the volumetric productivity was 0.21 g l⁻¹ h⁻¹.     

Class-specific immunoaffinity monolith for efficient on-line clean-up of pyrethroids followed by high-performance liquid chromatography analysis

A class-specific monolithic immunoaffinity column was developed for on-line clean-up of pyrethroid insecticides in complex samples. Deltamethrin was oxidized with ozone to generate the hapten of (RS)-α-cyano-3-phenoxybenzyl (RS)-cis,trans-2,2-dimethyl-3-formyl-cyclopropane carboxylate. Class-specific antibodies against pyrethroids were produced using the conjugate of above hapten with bovine serum albumin as the immunogen. Poly(ethylene dimethacrylate-glycidyl methacrylate) monolith was synthesized in a 50 mm × 4.6 mm i.d. stainless steel cartridge with two auxiliary pipette tips. The polymerization method was proved to be economic and reproducible. Antibodies against pyrethroids were covalently immobilized onto the monolithic support via Schiff base reaction. With a column-switching valve system, the immunoaffinity monolith (IAM) could be readily adapted to the reversed-phase high-performance liquid chromatography (HPLC) system. Under the optimum loading, washing and eluting conditions, the IAM specifically retained deltamethrin, flumethrin, flucythrinate and cis/trans permethrin, which were further baseline separated by C18 column using acetonitrile–water (83:17, V/V) as the mobile phase at a flow rate of 1.0 mL/min. The established system provides a highly efficient approach for high-throughput on-line clean-up of pyrethroid in various samples.     

Synthesis of geranyl acetate in non-aqueous media using immobilized Pseudomonas cepacia lipase on biodegradable polymer film: Kinetic modelling and chain length effect study

Pseudomonas cepacia lipase (PCL) was immobilized on ternary blend biodegradable polymer made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alcohol (PVA). Immobilized biocatalyst was characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), % water content, protein and lipase activity assay. The lipase activity assay showed enhanced activity of immobilized lipase than crude lipase. Higher half life time (t_1/2) and lower deactivation rate constant (K_d) was found for the n-hexane among various tested solvent. Influence of various reaction parameters on enzyme activity were studied in detail. When geraniol (1 mmol) and vinyl acetate (4 mmol) in toluene (3 mL) were reacted with 50 mg immobilized lipase at 55 °C; then 99% geraniol was converted to geranyl acetate after 3 h. Various kinetic parameters such as r_max, K_i(A), K_m(A), K_m(B) were determined using non-linear regression analysis for ternary-complex and Bi–Bi ping-pong mechanism. The kinetic study showed that reaction followed ternary-complex mechanism with inhibition by geraniol. Activation energy (Ea) was found to be lower for immobilized lipase (13.76 kCal/mol) than crude lipase (19.9 kCal/mol) indicating better catalytic efficiency of immobilized lipase. Immobilized biocatalyst demonstrated 4 fold increased catalytic activity than crude lipase and recycled five times.