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.     

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.     

Optimization and immobilization of amylase obtained from halotolerant bacteria isolated from solar salterns

The objective of the present study was to isolate halotolerant bacteria from the sediment sample collected from Marakanam Solar Salterns, Tamil Nadu, India using NaCl supplemented media and screened for amylase production. Among the 22 isolates recovered, two strains that had immense potential were selected for amylase production and designated as P1 and P2. The phylogenetic analysis revealed that P1 and P2 have highest homology with Pontibacillus chungwhensis (99%) and Bacillus barbaricus (100%). Their amylase activity was optimized to obtain high yield under various temperature, pH and NaCl concentration. P1 and P2 strain showed respective, amylase activity maximum at 35 °C and 40 °C; pH 7.0 and 8.0; 1.5 M and 1.0 M NaCl concentration. Further under optimized conditions, the amylase activity of P1 strain (49.6 U mL^?1) was higher than P2 strain. Therefore, the amylase enzyme isolated from P. chungwhensis P1 was immobilized in sodium alginate beads. Compared to the free enzyme form (49.6 U mL^?1), the immobilized enzyme showed higher amylase activity as 90.3 U mL^?1. The enzyme was further purified partially and the molecular mass was determined as 40 kDa by SDS–PAGE. Thus, high activity of amylase even under increased NaCl concentration would render immense benefits in food processing industries.     

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.     

Covalent immobilization of ω-transaminase from Vibrio fluvialis JS17 on chitosan beads

Chitosan beads were prepared by emulsion method and used for the immobilization of ω-transaminase of Vibrio fluvialis. The yield of enzyme immobilization (54.3%) and its residual activity (17.8%) were higher than those obtained with other commercial beads. ω-Transaminase was effectively immobilized on the chitosan beads at pH 6.0. The optimal pH of the immobilized enzyme was pH 9.0, which is the same as that of the free enzyme. The immobilized enzyme on chitosan beads retained ca. 77% of its conversion after five consecutive reactions with the 25 mM substrate, while the immobilized enzyme on Eupergit^® C retained 12%. Also, the immobilized ω-transaminase on chitosan bead retained 70% of initial activity when it's stored at 4 °C for 3.5 weeks. Addition of the co-factor, pyridoxal 5-phosphate (PLP), was needed to maintain the stability of the immobilized ω-transaminase.     

Immobilization of urease on vapour phase stain etched porous silicon

Porous silicon layers fabricated by the reaction-induced vapor phase stain etch method were coated with 5% polyethylenimine. Urease from Canavalia brasiliensis beans was immobilized on this support through covalent linking with 2.5% glutaraldehyde. The pH and temperature profile of the immobilized and free urease exhibited higher activity at pH 6.5 and 37 °C. After being stored for 30 days at 4 °C, the immobilized enzyme had 75% of the initial activity. The maximum apparent Michaelis constant for free urease (K_m) was 94.33 mM whereas for immobilized urease was 53.04 mM. The maximum reaction velocity (V_max) for free urease was 3.51 mmol/min and for immobilized urease was 1.57 mmol/min.     

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 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 apricot pectinesterase (Prunus armeniaca L.) on porous glass beads and its characterization

Pectinesterase isolated from Malatya apricot pulp was covalently immobilized onto glutaraldehyde-containing amino group functionalized porous glass beads surface by chemical immobilization at pH 8.0. The amount of covalently bound apricot PE was found 1.721 mg/g glass support. The properties of immobilized enzyme were investigated and compared to those of free enzyme. The effect of various parameters such as pH, temperature, activation energy, heat and storage stability on immobilized enzyme were investigated. Optimum pH and temperature were determined to be 8.0 and 50 °C, respectively. The immobilized PE exhibited better thermostability than the free one. Kinetic parameters of the immobilized enzyme (K_m and V_max values) were also evaluated. The K_m was 0.71 mM and the V_max was 0.64 μmol min^?1 mg^?1. No drastic change was observed in the K_m and V_max values. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Thermal and storage stability experiments were also carried out. It was observed that the immobilized enzyme had longer storage stability and retained 50% of its initial activity during 30 days.     

Stabilization of ficin extract by immobilization on glyoxyl agarose. Preliminary characterization of the biocatalyst performance in hydrolysis of proteins

A protein extract containing ficin was immobilized on glyoxyl agarose at pH 10 and 25 °C. The free enzyme remained fully active after 24 h at pH 10. However the enzyme immobilized on the support retained only 30% of the activity after this time using a small substrate. After checking the stability of ficin preparations obtained after different enzyme-support multi-interaction times, it was found that it reached a maximum at 3 h (40-folds more stable than the free enzyme at pH 5). The immobilized enzyme was active in a wide range of pH (e.g., retained double activity at pH 10 than the free enzyme) and temperatures (e.g., at 80 °C retained three-folds more activity than the free enzyme). The activity versus casein almost matched the results using the small substrate (60%) at 55 °C. However, in the presence of 2 M of urea, it became three times more active than the free enzyme. The immobilized enzyme could be reused five cycles at 55 °C without losing activity.     

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.     

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.     

A comparison of the kinetic properties of free and immobilized Aspergillus oryzae β-galactosidase

The objective of this work was to compare the properties of free and immobilized β-galactosidase (Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90 g/L and 140 g/L, respectively, a temperature of 35 °C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The K_m and V_m values of the free enzyme were 52.13 ± 2.8 mM and 2.56 ± 0.3 g_glucose/L min mg_enzyme, respectively, and were 60.30 ± 3.3 mM and 1032.07 ± 51.6 g_lactose/min m³_catalyst, respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH 4.5 and 55 °C. Alternatively, the immobilized form was most active at pH 5.0 at 60 °C. The free and immobilized enzymes presented activation energies of 6.90 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer.     

High activity and selectivity immobilized lipase on Fe3O4 nanoparticles for banana flavour synthesis

Lipase (E.C.3.1.1.3) from Thermomyces lanuginosus (TL) was directly bonded, through multiple physical interactions, on citric acid functionalized monodispersed Fe₃O₄ nanoparticles (NPs) in presence of a small amount of hydrophobic functionalities. A very promising scalable synthetic approach ensuring high control and reproducibility of the results, and an easy and green immobilization procedure was chosen for NPs synthesis and lipase anchoring. The size and structure of magnetic nanoparticles were characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The samples at different degree of functionalization were analysed through thermogravimetric measurements. Lipase immobilization was further confirmed by enzymatic assay and Fourier transform infrared (FT-IR) spectra. Immobilized lipase showed a very high activity recovery up to 144% at pH = 7 and 323% at pH = 7.5 (activity of the immobilized enzyme compared to that of its free form). The enzyme, anchored to the Fe₃O₄ nanoparticles, to be easy recovered and reused, resulted more stable than the native counterpart and useful to produce banana flavour. The immobilized lipase results less sensitive to the temperature and pH, with the optimum temperature higher of 5 °C and optimum pH up shifted to 7.5 (free lipase optimum pH = 7.0). After 120 days, free and immobilized lipases retained 64% and 51% of their initial activity, respectively. Ester yield at 40 °C for immobilized lipase reached 88% and 100% selectivity.     

One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca²⁺ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.     

Preparation and characterization of mixed-mode magnetic adsorbent with p-amino-benzamidine ligand: Operated in a magnetically stabilized fluidized bed reactor for purification of trypsin from bovine pancreas

The magnetic beads were synthesized using glycidylmethacrylate (GMA) and methylmethacrylate (MMA) monomers. A multimodal ligand (i.e., p-amino-benzamidine) was covalently immobilized onto magnetic beads after glutaraldehyde activation, and consequently used for purification of the trypsin from bovine pancreas. The p-amino-benzamidine ligand immobilized magnetic beads were characterized by FTIR, VSM, SEM, and analytical methods. Trypsin adsorption experiments were investigated under different experimental conditions (i.e., medium pH, initial trypsin concentration, temperature, and ionic strength) in a batch system. Maximum trypsin adsorption capacity was found to be 75.9 ± 2.6 mg/g beads. Adsorbed trypsin was eluted by using (0.1 M acetate buffer, pH 3.0) with a 97% recovery. The purification factor of trypsin from crude pancreas extract was 8.7 folds. The purity of the eluted trypsin from p-amino-benzamidine functionalized magnetic beads was determined as 86% by HPLC. The method developed in this report was successfully applied for purification of the trypsin from crude pancreas extract in a magnetically stabilized fluidized bed reactor.     

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.     

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.     

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and K_m of the enzyme were ∼55.0 kDa and 151 μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe²⁺ and Pb²⁺ at 0.1 mM of each acted as inhibitors, while Ag⁺, Ca²⁺, Ni²⁺ and Zn²⁺ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.