Co-immobilized Enzymes Research Articles

Co-Immobilization of Alcalase/Dispase for Production of Selenium-Enriched Peptide from Cardamine violifolia.

Enzymatically derived selenium-enriched peptides from Cardamine violifolia (CV) can serve as valuable selenium supplements. However, the industrial application of free enzyme is impeded by its limited stability and reusability. Herein, this study explores the application of co-immobilized enzymes (Alcalase and Dispase) on amino resin for hydrolyzing CV proteins to produce selenium-enriched peptides. The successful enzyme immobilization was confirmed through scanning electron microscopy (SEM), energy dispersive X-ray (EDX), and Fourier-transform infrared spectroscopy (FTIR). Co-immobilized enzyme at a mass ratio of 5:1 (Alcalase/Dispase) exhibited the smallest pore size (7.065 nm) and highest activity (41 U/mg), resulting in a high degree of hydrolysis of CV protein (27.2%), which was obviously higher than the case of using free enzymes (20.7%) or immobilized Alcalase (25.8%). In addition, after a month of storage, the co-immobilized enzyme still retained a viability level of 41.93%, showing fairly good stability. Encouragingly, the selenium-enriched peptides from co-immobilized enzyme hydrolysis exhibited uniform distribution of selenium forms, complete amino acid fractions and homogeneous distribution of molecular weight, confirming the practicality of using co-immobilized enzymes for CV protein hydrolysis.

Development of a Multi-Enzymatic Biocatalytic System through Immobilization on High Quality Few-Layer bio-Graphene

In this work, we report the green production of few-layer bio-Graphene (bG) through liquid exfoliation of graphite in the presence of bovine serum albumin. Microscopic characterization evaluated the quality of the produced nanomaterial, showing the presence of 3-4-layer graphene. Moreover, spectroscopic techniques also confirmed the quality of the resulted bG, as well as the presence of bovine serum albumin on the graphene sheets. Next, for the first time, bG was used as support for the simultaneous covalent co-immobilization of three enzymes, namely β-glucosidase, glucose oxidase, and horseradish peroxidase. The three enzymes were efficiently co-immobilized on bG, demonstrating high immobilization yields and activity recoveries (up to 98.5 and 90%, respectively). Co-immobilization on bG led to an increase of apparent KM values and a decrease of apparent Vmax values, while the stability of the nanobiocatalysts prevailed compared to the free forms of the enzymes. Co-immobilized enzymes exhibited high reusability, preserving a significant part of their activity (up to 72%) after four successive catalytic cycles at 30 °C. Finally, the tri-enzymatic nanobiocatalytic system was applied in three-step cascade reactions, involving, as the first step, the hydrolysis of p-Nitrophenyl-β-D-Glucopyranoside and cellobiose.

Multienzymatic conversion of monosaccharides from birch biomass after pretreatment

This study presents proof-of-concept validation of a multi enzymatic biocatalytic system, capable of producing monosaccharides from a birch biomass pretreated liquor with simultaneous cofactor regeneration. To improve the stability of the proposed biocatalyst, co-immobilization of NAD + -dependent xylose dehydrogenases, glucose dehydrogenase, and NADH-dependent 3-hydroxybutyrate dehydrogenase was performed on mesoporous silica SBA-15. The results of the physicochemical analysis confirmed the effectiveness of enzymes deposition on the support material and changes in the surface area and porous structure of silica before and after the process. Process conditions in which the three-enzymes system allowed to achieve the highest efficiency of the conducted reactions were optimized at 20 °C, pH 7, 90 min of process duration, 400 mM of levulinic acid addition and ratio of NAD + :NADH 1:5. Results from this study demonstrate excellent xylonic acid, gluconic acid and 4-hydroxyvaleric acid production at conversion rate of 98.7%, 95.6% and 99.2%, respectively. In addition, the high stability and reusability of the developed biocatalytic system was confirmed based on tests in ten successive catalytic cycles and the storage time of 10 days at 4 °C. At the end of the tests, co-immobilized enzymes were capable of catalyzing the process with >70% efficiency. The presented data confirmed the effectiveness of co-immobilization, improvement of enzyme properties including their high reusability, as well as indicated the application potential of the proposed multienzymatic system in the conversion of components of pretreated biomass solutions. • Three types of dehydrogenases have been successfully co-immobilized using silica SBA-15. • Monosaccharides from the birch biomass pretreated liquor were effectively converted. • Addition of levulinic acid ensured the creation of a cofactor regeneration system. • The production of three organic acids was carried out with over 95% of efficiency. • Co-immobilized enzymes retained 70% of acids productivity after 10 catalytic cycles.

Co-immobilization of lipase and laccase on agarose-based supports via layer-by-layer strategy: Effect of diffusional limitations

Co-immobilized enzymes present desirable advantages to performing cascade reactions when employed in one-pot processes, mainly because of the shorter lag times owing to better reagent diffusion across the active sites of the different enzymes. But high product yields require high enzyme loads, usually achieved with high surface area and porous supports, where diffusional limitations may be introduced or increased. Heterogeneous biocatalysts were prepared by co-immobilizing Pseudomonas fluorescens lipase (PFL) and Aspergillus sp. Laccase (Novozymes 51003) on two agarose-based supports, DEAE-Sepharose and Octyl-agarose. A layer-by-layer strategy was used by adding polyethyleneimine (PEI) to allow the attachment of the upper enzyme layer onto the lower adjacent layer. A final step, crosslinking with glutaraldehyde, was performed to prevent enzyme leaching under harsh environmental conditions. The heterogeneous biocatalysts showed lower temperature dependence and a significant increase in thermal stability at 50 ºC. No internal mass transfer effects were observed when a low protein load was used during co-immobilization (2 mgprotein/gsupport of each enzyme). However, increasing the enzyme load (more than 10 mgprotein/gsupport) promoted internal diffusion limitations. The layer-by-layer strategy defined in this work enabled the preparation of a multi-enzymatic heterogeneous biocatalyst with high activity, thermal stability, and no diffusional limitations, depending on enzyme concentration.

Multi-Combilipases: Co-Immobilizing Lipases with Very Different Stabilities Combining Immobilization via Interfacial Activation and Ion Exchange. The Reuse of the Most Stable Co-Immobilized Enzymes after Inactivation of the Least Stable Ones

The lipases A and B from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) or Rhizomucor miehei (RML), and the commercial and artificial phospholipase Lecitase ultra (LEU) may be co-immobilized on octyl agarose beads. However, LEU and RML became almost fully inactivated under conditions where CALA, CALB and TLL retained full activity. This means that, to have a five components co-immobilized combi-lipase, we should discard 3 fully active and immobilized enzymes when the other two enzymes are inactivated. To solve this situation, CALA, CALB and TLL have been co-immobilized on octyl-vinyl sulfone agarose beads, coated with polyethylenimine (PEI) and the least stable enzymes, RML and LEU have been co-immobilized over these immobilized enzymes. The coating with PEI is even favorable for the activity of the immobilized enzymes. It was checked that RML and LEU could be released from the enzyme-PEI coated biocatalyst, although this also produced some release of the PEI. That way, a protocol was developed to co-immobilize the five enzymes, in a way that the most stable could be reused after the inactivation of the least stable ones. After RML and LEU inactivation, the combi-biocatalysts were incubated in 0.5 M of ammonium sulfate to release the inactivated enzymes, incubated again with PEI and a new RML and LEU batch could be immobilized, maintaining the activity of the three most stable enzymes for at least five cycles of incubation at pH 7.0 and 60 °C for 3 h, incubation on ammonium sulfate, incubation in PEI and co-immobilization of new enzymes. The effect of the order of co-immobilization of the different enzymes on the co-immobilized biocatalyst activity was also investigated using different substrates, finding that when the most active enzyme versus one substrate was immobilized first (nearer to the surface of the particle), the activity was higher than when this enzyme was co-immobilized last (nearer to the particle core).

Co-immobilized enzymes for the pre-treatment of wastewater

Co-immobilized enzymes for the pre-treatment of wastewater

Aroma Release in Wine Using Co-Immobilized Enzyme Aggregates.

Aroma is a remarkable factor of quality and consumer preference in wine, representing a distinctive feature of the product. Most aromatic compounds in varietals are in the form of glycosidic precursors, which are constituted by a volatile aglycone moiety linked to a glucose residue by an O-glycosidic bond; glucose is often linked to another sugar (arabinose, rhamnose or apiose). The use of soluble β-glycosidases for aroma liberation implies the addition of a precipitating agent to remove it from the product and precludes its reuse after one batch. An attractive option from a technological perspective that will aid in removing such constraints is the use of immobilized glycosidases. Immobilization by aggregation and crosslinking is a simple strategy producing enzyme catalysts of very high specific activity, being an attractive option to conventional immobilization to solid inert supports. The purpose of this work was the evaluation of co-immobilized β-glycosidases crosslinked aggregates produced from the commercial preparation AR2000, which contains the enzymes involved in the release of aromatic terpenes in Muscat wine (α-l-arabinofuranosidase and β-d-glucopyranosidase). To do so, experiments were conducted with co-immobilized crosslinked enzyme aggregates (combi-CLEAs), and with the soluble enzymes, using an experiment without enzyme addition as control. Stability of the enzymes at the conditions of winemaking was assessed and the volatiles composition of wine was determined by SPE-GC-MS. Stability of enzymes in combi-CLEAs was much higher than in soluble form, 80% of the initial activity remaining after 60 days in contact with the wine; at the same conditions, the soluble enzymes had lost 80% of their initial activities after 20 days. Such higher stabilities will allow prolonged use of the enzyme catalyst reducing its impact in the cost of winemaking. Wine treated with combi-CLEAs was the one exhibiting the highest concentration of total terpenes (18% higher than the control) and the highest concentrations of linalool (20% higher), nerol (20% higher) and geraniol (100% higher), which are the most important terpenes in determining Muscat typicity. Co-immobilized enzymes were highly stable at winemaking conditions, so their reutilization is possible and technologically attractive by reducing the impact of enzyme cost on winemaking cost.

Rational Design of Nanoparticle Platforms for "Cutting-the-Fat": Covalent Immobilization of Lipase, Glycerol Kinase, and Glycerol-3-Phosphate Oxidase on Metal Nanoparticles.

The aggregates of nanoparticles (NPs) are considered better supports for the immobilization of enzymes, as these promote enzyme kinetics, due to their unusual but favorable properties such as larger surface area to volume ratio, high catalytic efficiency of certain immobilized enzymes, non-toxicity of some of the nanoparticle matrices, high stability, strong adsorption of the enzyme of interest by a number of different approaches, and faster electron transportability. Co-immobilization of multiple enzymes required for a multistep reaction cascade on a single support is more efficient than separately immobilizing the corresponding enzymes and mixing them physically, since products of one enzyme could serve as reactants for another. These products can diffuse much more easily between enzymes on the same particle than diffusion from one particle to the next, in the reaction medium. Thus, co-immobilization of enzymes onto NP aggregates is expected to produce faster kinetics than their individual immobilizations on separate matrices. Lipase, glycerol kinase, and glycerol-3-phosphate oxidase are required for lipid analysis in a cascade reaction, and we describe the co-immobilization of these three enzymes on nanocomposites of zinc oxide nanoparticles (ZnONPs)-chitosan (CHIT) and gold nanoparticles-polypyrrole-polyindole carboxylic acid (AuPPy-Pin5COOH) which are electrodeposited on Pt and Au electrodes, respectively. The kinetic properties and analytes used for amperometric determination of TG are fully described for others to practice in a trained laboratory. Cyclic voltammetry, scanning electron microscopy, Fourier transform infra-red spectra, and electrochemical impedance spectra confirmed their covalent co-immobilization onto electrode surfaces through glutaraldehyde coupling on CHIT-ZnONPs and amide bonding on AuPPy/Pin5COOH. The combined activities of co-immobilized enzymes was tested amperometrically, and these composite nanobiocatalysts showed optimum activity within 4-5s, at pH 6.5-7.5 and 35°C, when polarized at a potential between 0.1 and 0.4V. Co-immobilized enzymes showed excellent linearity within 50-700mg/dl of the lipid with detection limit of 20mg/dl for triolein. The half life of co-immobilized enzymes was 7 months, when stored dry at 4°C which is very convenient for practical applications. Co-immobilized biocatalysts measured triglycerides in the sera of apparently healthy persons and persons suffering from hypertriglyceridemia, which is recognized as a leading cause for heart disease. The measurement of serum TG by co-immobilized enzymes was unaffected by the presence of a number of serum substances, tested as potential interferences. Thus, co-immobilization of enzymes onto aggregates of NPs resulted in improved performance for TG analysis.

Preparation of Genipin by Hydrolysis of Geniposide with Co-Immobilized Enzyme

Co-immobilize enzyme by cross-linking and embedding, optimize conditions for immobilizing, determinate the enzymatic properties of co-immobilized enzyme and study the methods for preparation of genipin using co-immobilized enzyme to hydrolyze geniposide. Optimized immobilizing conditions include glutaraldehyde concentration being 0.15%, cross-linking temperature being 20°C, cross-linking time being 2 hours, the activity of co-immobilized β-glucosidase and cell reaches to 65.33U/mg and the enzyme activity recovery being 52.63%. Enzymatic properties of co-immobilized enzyme are following: optimum temperature is 55°C and optimum pH is 5.0. The transformation experiments are carried out with co-immobilized enzyme. The results show that half-life of co-immobilized enzyme reaches around 40 days, higher than the normal immobilized enzyme. The conversion rate of geniposide is above 95% after 8 hours. The genipin is isolated, purified and recrystallized to reach more than 98% of purity by High Performance Liquid Chromatography. Advantages to prepare genipin using co-immobilized enzyme include low cost, high yield, environmental friendly and easy to manufacturing.

Preparation of Gardenia Red Pigment by Hydrolysis of Geniposidic Acid with Co-Immobilized Enzyme

Geniposidic acid is hydrolyzed to produce genipinic acid by the co-immobilized enzyme,and the type of amino acids which react with genipinic acid to produce gardenia red pigment and some reaction conditions are optimized,we also studied on the methods of purifying gardenia red pigment by macroporous resin. Optimized conditions are: pH of reaction solution of genipinic acid and amino acid is 4.0, the temperature is 40 °C, reaction time is 42 hr, glutamic acid is better for producing gardenia red pigment than other amino acids, molar ratio of glutamic acid and genipinic acid is 2:1,molar ratio of citric acid and genipinic acid is 6:1.Gardenia red pigment is adsorbed by HPD100 macroporous resin,then elute by ammonia water of pH 11, the eluent is dried under 50°C,the violet black powder is obtained,it’s color value can reach 92.32 (E1%1cm,528nm). To prepare gardenia red pigment from geniposidic acid by the co-immobilized enzyme has advantages of low cost, environmental friendly and easy to mass production.

Co-immobilized enzymes in magnetic chitosan beads for improved hydrolysis of macromolecular substrates under a time-varying magnetic field

Glucoamylase and α-amylase co-immobilized with gamma ferric oxide powders in chitosan beads for consecutive starch liquefaction and saccharification under different magnetic fields was investigated. The chitosan concentration in the beads was found to greatly affect the immobilized enzyme performance. Superior immobilization efficiency and enzyme stability were noted when 2% instead of 4% chitosan was utilized. Using confocal microscopy and scanning electron microscopy, the beads with 2% chitosan were seen to exhibit a more rugged surface topology with more macropores and accommodate more protein near the external surface than with the 4% chitosan beads. An optimum loading ratio between α-amylase and glucoamylase exists that gives the highest glucose production, and this ratio varies with the size of the beads. The inclusion of the gamma ferric oxide powders renders the beads magnetically anisotropic and causes them to tumble under a single-phase alternating magnetic field, resulted in increased overall reaction rates. When exposed to a three-phase alternating magnetic field, these beads were stirred vigorously, also leading to enhanced reaction rates. The use of multi-enzyme co-immobilization in magnetic anisotropic chitosan beads may be extended to other practical applications that involve coordinated enzymatic reactions of macromolecular substrates.

Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: Activity, stability and co-immobilization

In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.

Colorimetric Assay for Lecithin Using Two Co-immobilized Enzymes and an Indicator Dye Conjugate

According to the new assay for lecithin, phospholipase D (PL), choline oxidase (ChOX), and the indicator conjugate bromothymol blue−glutathione (BTB−GSH) are co-immobilized on glutaraldehyde-activated polyacrylamide transparent gel. When a sample containing lecithin is added to the biocatalytic gel, lecithin is hydrolyzed by PL to choline, which is oxidized to betaine by ChOX. The organic acid betaine changes the microenvironmental pH of the enzymes, thus leading the optical absorption of the biocatalytic gel to a hypsochromic shift because of the co-immobilized indicator conjugate, BTB−GSH. The change of the optical absorption at λmax (622 nm) is related linearly to lecithin concentration in the range 20−120 μM. When the optical effect is quantified in a spectrophotometer, the analyte concentration can be determined. The biocatalytic gel is used successfully for lecithin determinations in bovine serum and soybean granules. The principle of the method is applicable to the construction of optical biosensor...

Flow-Injection Method for the Simultaneous Determination of d-Glucose and 3-Hydroxybutyrate with Co-Immobilized Enzyme Reactors and a Chemiluminometer

The simultaneous determination of D-glucose and 3-hydroxybutyrate in a plasma and serum by a split-stream flow-injection method using two immobilized enzyme reactors is described. One reactor for the determination of D-glucose was packed with glucose dehydrogenase and NADH oxidase co-immobilized on poly(vinyl alcohol) beads; the other reactor for 3-hydroxybutyrate was packed with 3-hydroxybutyrate dehydrogenase and NADH oxidase co-immobilized on the beads. The hydrogen peroxide produced by the enzymatic reactions was detected by measuring the chemiluminescence emitted upon admixing with luminol and potassium hexacyanoferrate(III). For 3-hydroxybutyrate the calibration graph was linear over the range 1×10-6-5×10-4M; for D-glucose it was 1×10-5-1×10-3M. One assay could be completed within 6min with satisfactory precision (<2.9% relative standard deviation).