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.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m², however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m². The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K_m) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V_max) remained almost constant.     

Hen egg white as a feeder protein for lipase immobilization

Enzyme stabilization via immobilization is one of the preferred processes as it provides the advantages of recovery and reusability. In this study, Thermomyces lanuginosus lipase has been immobilized through crosslinking using 2% glutaraldehyde and hen egg white, as an approach towards CLEA preparation. The immobilization efficiency and the properties of the immobilized enzyme in terms of stability to pH, temperature, and denaturants was studied and compared with the free enzyme. Immobilization efficiency of 56% was achieved with hen egg white. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0 whereas the pH optimum for free enzyme was at pH 6.0. The immobilized enzyme was stable at higher temperature retaining about 83% of its maximum activity as compared to the free enzyme retaining only 41% activity at 70 °C. The denaturation of lipase in free form was rapid with a half-life of 2 h at 60 °C and 58 min at 70 °C as compared to 12 h at 60 °C and 2 h at 70 °C for the immobilized enzyme. The effect of denaturants, urea and guanidine hydrochloride on the free and immobilized enzyme was studied and the immobilized enzyme was found to be more stable towards denaturants retaining 74% activity in 8 M urea and 98% in 6 M GndHCl as compared to 42% and 33% respectively in the case of free enzyme. The apparent K_m (2.08 mM) and apparent V_max (0.95 μmol/min) of immobilized enzyme was lower as compared to free enzyme; K_m (8.0 mM) and V_max (2.857 μmol/min). The immobilized enzyme was reused several times for the hydrolysis of olive oil.     

A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

A novel method was developed for the immobilization of glucoamylase from Aspergillus niger. The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K_m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V_max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.     

Immobilization of acidic lipase derived from Pseudomonas gessardii onto mesoporous activated carbon for the hydrolysis of olive oil

Mesoporous activated carbon (MAC) derived from rice husk is used for the immobilization of acidic lipase (ALIP) produced from Pseudomonas gessardii. The purified acidic lipase had the specific activity and molecular weight of 1473 U/mg and 94 kDa respectively. To determine the optimum conditions for the immobilization of lipase onto MAC, the experiments were carried out by varying the time (10–180 min), pH (2–8), temperature (10–50 °C) and the initial lipase activity (49 × 10³, 98 × 10³, 147 × 10³ and 196 × 10³ U/l in acetate buffer). The optimum conditions for immobilization of acidic lipase were found to be: time—120 min; pH 3.5; temperature—30 °C, which resulted in achieving a maximum immobilization of 1834 U/g. The thermal stability of the immobilized lipase was comparatively higher than that in its free form. The free and immobilized enzyme kinetic parameters (K_m and V_max) were found using Michaelis–Menten enzyme kinetics. The K_m values for free enzyme and immobilized one were 0.655 and 0.243 mM respectively. The immobilization of acidic lipase onto MAC was confirmed using Fourier Transform-Infrared Spectroscopy, X-ray diffraction analysis and scanning electron microscopy.     

Forming nanoparticles of α-amylase and embedding them into solid surfaces

In the current work nanoparticles (NPs) of α-amylase were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on polyethylene (PE) films, or polycarbonate (PC) plates, or on microscope glass slides. The α-amylase NPs coated on the solid surfaces have been characterized by ESEM, TEM, FTIR, XPS and AFM. The substrates immobilized with α-amylase were used for hydrolyzing soluble potato starch to maltose. The amount of enzyme introduced in the substrates, leaching properties, and the catalytic activity of the immobilized enzyme were compared. The catalytic activity of the amylase deposited on the three solid surfaces was compared to that of the same amount of free enzyme at different pHs and temperatures. α-Amylase coated on PE showed the best catalytic activity in all the examined parameters when compared to native amylase, especially at high temperatures. When immobilized on glass, α-amylase showed better activity than the native enzyme over all pH and temperature values studied. However, the immobilization on PC did not improve the enzyme activity at any pH and any temperature compared to the free amylase. The kinetic parameters, K_m and V_max were also calculated. The amylase coated PE showed the most favorable kinetic parameters (K_m = 5 g L⁻¹ and V_max = 5E−07 mol mL⁻¹ min⁻¹). In contrast, the anchored enzyme-PC exhibited unfavorable kinetic parameters (K_m = 16 g L⁻¹, V_max = 4.2E−07 mol mL⁻¹ min⁻¹). The corresponding values for amylase-glass were K_m = 7 g L⁻¹, V_max = 1.8E−07 mol mL⁻¹ min⁻¹, relative to those obtained for the free enzyme (K_m = 6.6 g L⁻¹, V_max = 3.3E−07 mol mL⁻¹ min⁻¹).     

Immobilization of alkaline serine endopeptidase from Bacillus licheniformis on SBA-15 and MCF by surface covalent binding

An industrial enzyme, alkaline serine endopeptidase, was immobilized on surface modified SBA-15 and MCF materials by amide bond formation using carbodiimide as a coupling agent. The specific activities of free enzyme and enzyme immobilized on SBA-15 and MCF were studied using casein (soluble milk protein) as a substrate. The highest activity of free enzyme was obtained at pH 9.5 while this value shifted to pH 10 for SBA-15 and MCF immobilized enzyme. The highest activity of immobilized enzymes was obtained at higher temperature (60 °C) than that of the free enzyme (55 °C). Kinetic parameters, Michaelis–Menten constant (K_m) and maximum reaction velocity (V_max), were calculated as K_m = 13.375, 11.956, and 8.698 × 10^?4 mg/ml and V_max = 0.156, 0.163 and 0.17 × 10^?3 U/mg for the free enzyme and enzyme immobilized on SBA-15 and MCF, respectively. The reusability of immobilized enzyme showed 80% of the activity retained even after 15 cycles. Large pore sized MCF immobilized enzyme was found to be more promising than the SBA-15 immobilized enzyme due to the availability of larger pores of MCF, which offer facile diffusion of substrate and product molecules.     

Immobilization of acetylcholinesterase on new modified acrylonitrile copolymer membranes

The three new dual-layer matrices (polyacrylonitrile (PAN) membranes coated with physically bound chitosan (CHI)—PANCHI-A and chemically bound chitosan—PANCHI-B and PANCHI-C) for immobilization of acetylcholinesterase (AChE) were obtained. The chemical-modified PAN membrane (PAN-NaOH + ethylenediamine (EDA)) was used as a base for the prepared dual-layer membranes. For chemical chitosan bound membrane, chitosan was tethered onto the membrane surface to form a dual-layer biomimetic membrane in the presence of glutaraldehyde (GA). The basic characteristics (amount of amino groups, hydrophilicity and transport characteristics) of the chitosan-modified membranes were investigated. The SEM analyses were shown essential morphology change in the different chitosan membranes.The relative activities and V_max of the covalently immobilized enzyme on PANCHI-B and PANCHI-C membranes were higher than that on PANCHI-A membrane and chemical-modified membrane with NaOH + EDA. K_m values for the different modified membranes are lower for the chitosan-treated membranes. The pH and temperature optimum of immobilized enzyme were determined. The bound enzymes on PANCHI-B and PANCHI-C have higher thermal and storage stability in comparison with AChE on PANCHI-A membrane and free enzyme.     

Enhanced activity of immobilized pepsin nanoparticles coated on solid substrates compared to free pepsin

In the present work nanoparticles (NPs) of pepsin were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on low-density polyethylene (PE) films, or on polycarbonate (PC) plates, or on microscope glass slides. The pepsin NPs coated on the solid surfaces have been characterized by HRSEM, TEM, FTIR, XPS and DLS. The amount of enzyme introduced on the substrates, the leaching properties, and the catalytic activity of the immobilized enzyme on the three surfaces are compared. Catalytic activities of pepsin deposited onto the three solid surfaces as well as free pepsin, without sonication, and free pepsin NPs were compared at various pH levels and temperatures using a hemoglobin assay. Compared to native pepsin, pepsin coated onto PE showed the best catalytic activity in all the examined parameters. Pepsin immobilized on glass exhibited better activity than the native enzyme, especially at high temperatures. Enzyme activity of pepsin immobilized on PC was no better than native enzyme activity at all temperatures at pH 2, and only over a narrow pH range at 37 °C was the activity improved over the native enzyme. A remarkable observation is that immobilized pepsin on all the surfaces was still active to some extent even at pH 7, while free pepsin was completely inactive. The kinetic parameters, K_m and V_max were also calculated and compared for all the samples. Relative to the free enzyme, pepsin coated PE showed the greatest improvement in kinetic parameters (K_m = 15 g/L, V_max = 719 U/mg versus K_m = 12.6 g/L and V_max = 787 U/mg, respectively), whereas pepsin coated on PC exhibited the most unfavorable kinetic parameters (K_m = 18 g/L, V_max = 685 U/mg). The values for the anchored enzyme-glass were K_m = 19 g/L, V_max = 763 U/mg.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

A novel immobilization method for nuclease P1 on macroporous absorbent resin with glutaraldehyde cross-linking and determination of its properties

Microbial nuclease P₁ from Penicllium citrinum was immobilized on macroporous absorbent resins: strong polar poly (styrene-co-DVB) resin (SPPSD), polymethacrylic ester resin and poly (styrene-co-DVB)-Br resin. The results showed that SPPSD was the best carrier. Three methods of glutaraldehyde cross-linking were used and simultaneous immobilization and cross-linking (CIS) was demonstrated to be the best method. The functional properties of immobilized nuclease P₁ were studied and compared to those of the free enzyme. The highest enzyme activities of free and immobilized nuclease P₁ were obtained in 0.2 M acetate buffer at pH 4.5 and a temperature of 70 °C. An increase in K_m (from 3.165 to 18.125 mg mL^?1) and a decrease in V_max (from 1667.18 to 443.95 U min^?1 mL^?1) were recorded after immobilization. SPPSD-glutaraldehyde-nuclease P₁ exhibited better thermal stability than the free enzyme. The apparent activation energy (E_a) of the free and immobilized nuclease P₁ was 137.04 kJ mol^?1 and 98.43 kJ mol^?1, respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limit.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

Immobilization of α-galactosidase on galactose-containing polymeric beads

Industrial application of α-galactosidase requires efficient methods to immobilize the enzyme, yielding a biocatalyst with high activity and stability compared to free enzyme. An α-galactosidase from tomato fruit was immobilized on galactose-containing polymeric beads. The immobilized enzyme exhibited an activity of 0.62 U/g of support and activity yield of 46%. The optimum pH and temperature for the activity of both free and immobilized enzymes were found as pH 4.0 and 37 °C, respectively. Immobilized α-galactosidase was more stable than free enzyme in the range of pH 4.0–6.0 and more than 85% of the initial activity was recovered. The decrease in reaction rate of the immobilized enzyme at temperatures above 37 °C was much slower than that of the free counterpart. The immobilized enzyme shows 53% activity at 60 °C while free enzyme decreases 33% at the same temperature. The immobilized enzyme retained 50% of its initial activity after 17 cycles of reuse at 37 °C. Under same storage conditions, the free enzyme lost about 71% of its initial activity over a period of 7 months, whereas the immobilized enzyme lost about only 47% of its initial activity over the same period. Operational stability of the immobilized enzyme was also studied and the operational half-life (t_1/2 was determined as 6.72 h for p-nitrophenyl α-d-galactopyranoside (PNPG) as substrate. The kinetic parameters were determined by using PNPG as substrate. The K_m and V_max values were measured as 1.07 mM and 0.01 U/mg for free enzyme and 0.89 mM and 0.1 U/mg for immobilized enzyme, respectively. The synthesis of the galactose-containing polymeric beads and the enzyme immobilization procedure are very simple and also easy to carry out.     

Enhanced d-hydantoinase activity performance via immobilized cobalt ion affinity membrane and its kinetic study

Various immobilized metal ions affinity membranes (IMAMs) were prepared from the regenerated cellulose membrane (RC membrane) and chelated with various metal ions such as Co²⁺, Ni²⁺, Cu²⁺ and Zn²⁺. The D-hydantoin-hydrolyzing enzyme (DHTase) harboring a poly-His tagged residue was used as a model protein to be immobilized on the prepared IMAMs through the direct metal–protein interaction forces. The adsorption isotherm and the kinetic parameters V_max, K_m,app of DHTase on IMAMs were studied. The cobalt ions chelated IMAM (Co-IMAM) was found to yield the highest specific activity of DHTase. Under the immobilization condition, the cobalt ion chelated amount was 161.4 ± 4.7 μmol/disk with a DHTase activity of 4.1 ± 0.1 U/disk. As compared to the free DHTase, the immobilized DHTase membrane could achieve a broader pH tolerance and higher thermal stability. In addition, 98% of the residual activity could be retained for 7-times repeated use. Only little activity loss was observed within 36-day storage at 4 °C. This is the first report concerning about using cobalt ion as the effective chelated metal ion for simultaneous purification and immobilization operation.     

Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: Optimization and characterization

Laccase from Trametes versicolor was immobilized on Amberlite IR-120 H beads. Maximum immobilization obtained was 78.7% at pH = 4.5 and temperature T = 45 °C. Kinetic parameters, K_m and V_max values, were determined respectively as 0.051 mM and 2.77 × 10^?2 mM/s for free and 4.70 mM and 5.27 × 10^?3 mM/s for immobilized laccase. The Amberlite–laccase system showed a 30% residual activity after 7 cycles. On the other hand, the loss of activity for free laccase after 7 days of storage at 4 °C was 18.5% in comparison to Amberlite–laccase system with a loss of 1.4%, during the same period. Improved operational, thermal and storage stabilities of the immobilized laccase were obtained compared to the free counterpart. Therefore, the use of low-cost matrices, like Amberlite for enzyme immobilization represents a promising product for enzymatic industrial applications.     

Immobilizing cholesterol oxidase in chitosan–alginic acid network

Proton conducting biopolymer networks have potential use for bio-sensors. The cost-effective, non-hazardous and environmentally safe biopolymer, such as chitosan, is an attractive feature for bio-sensors. Cholesterol oxidase was immobilized in conducting network via complexation of chitosan with alginic acid. A method for the preparation of the complex along with characterization by elemental analysis, FTIR spectroscopy, TGA and DSC were reported. The proton conductivity chitosan–alginic acid network was studied via impedance spectroscopy under humidified condition. The complex polymer electrolyte with x = 1 exhibited maximum proton conductivity of 1.4 × 10^?3 S/cm at RT, RH ～ 50%. The potential use of this network in enzyme immobilization was studied by manufacturing cholesterol oxidase entrapped polymer networks. Additionally, the maximum reaction rate (V_max) and Michaelis–Menten constant (K_m) were investigated for the immobilized cholesterol oxidase. Also, temperature and pH optimization studies were performed, and operational stability and shelf life of the polymer network were examined.     

Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.     

Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures

Four immobilized forms of glucose oxidase (GOD) were used for biotransformation removal of glucose from its mixture with dextran oligosaccharides. GOD was biospecifically bound to Concanavalin A-bead cellulose (GOD-ConA-TBC) and covalently to triazine-bead cellulose (GOD-TBC). Eupergit C and Eupergit CM were used for preparation of other two forms of immobilized GOD: GOD-EupC and GOD-EupCM. GOD-ConA-TBC and GOD-EupC exhibited the best operational and storage stabilities. pH and temperature optima of these two immobilized enzyme forms were broadened and shifted to higher values (pH 7 and 35 °C) in comparison with those of free GOD. The decrease of V_max values after immobilization was observed, from 256.8 ± 7.0 μmol min⁻¹ mg_GOD⁻¹ for free enzyme to 63.8 ± 4.2 μmol min⁻¹ mg_GOD⁻¹ for GOD-ConA-TBC and 45 ± 2.7 μmol min⁻¹ mg_GOD⁻¹ for GOD-EupC, respectively. Depending on the immobilization mode, the immobilized GODs were able to decrease the glucose content in solution to 3.8–15.6% of its initial amount The best glucose conversion, was achieved by an action of GOD-EupCM on a mixture of 100 g dextran with 9 g of glucose (i.e. 98.7% removal of glucose).     

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.