Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads

Silica gel bead coated with macroporous chitosan layer (CTS-SiO₂) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu²⁺, Zn²⁺, Ni²⁺ ions, respectively on CTS-SiO₂, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu²⁺-CTS-SiO₂ with the value of 4980 ± 125 IU g⁻¹ of the adsorbent, while the maximal adsorption capacity for trypsin on Zn²⁺ and Ni²⁺ loaded adsorbent was 3762 ± 68 IU g⁻¹ and 2636 ± 53 IU g⁻¹, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO₂ beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO₂ could effectively digest BSA revealed by HPLC peptide mapping.     

Bifunctional adsorbents for hydrophobic displacement chromatography of proteins

The separation of pancreatic trypsinogen and alpha-chymochypsinogen A by displacement chromatography was tested on a bifunctional adsorbent containing both butyl and carboxymethyl groups. Methacrylic triblock copolymer was synthesized and used as the displacer. Compared to the displacement results on commercial Butyl-Sepharose, it was found that both the separation and recovery of trypsinogen and alpha-chymochypsinogen A were improved. Adsorption isotherms of proteins were measured on both the commercial Butyl-Sepharose and the synthesized bifunctional adsorbents. It was found that the improvement of protein separation on bifunctional adsorbents was attributed to the alteration of the adsorption of trypsinogen. Charge repulsion between trypsinogen and the negatively charged carboxymethyl groups may account for the alteration. In addition, taking advantage of the effect of charge repulsion, the column regeneration became much easier on the column packed with bifunctional adsorbents.     

One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports.

Epoxy supports covalently immobilize proteins following a two-step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, in a simple manner, the purification, immobilization, and stabilization of a poly-His-tagged protein. To fulfill this objective we developed a new kind of multifunctional epoxy support (chelate epoxy support [CES]), which was tested using a poly-His-tagged glutaryl acylase as a model protein (an alphabeta-heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly-His-tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low-density chelate groups (e.g., 5 micromol/g) and metals with a low affinity (e.g., Co). However, the rate of covalent immobilization of the protein by its reaction with the epoxy groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fold more stable than the soluble enzyme. By taking advantage of the selectivity properties of the novel support, we were able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

Removal of heavy metals from water effluents using supermacroporous metal chelating cryogels

Applications of IDA in, for example, immobilized metal ion affinity chromatography for purification of His-tagged proteins are well recognized. The use of IDA as an efficient chelating adsorbent for environmental separations, that is, for the capture of heavy metals, is not studied. Adsorbents based on supermacroporous gels (cryogels) bearing metal chelating functionalities (IDA residues and ligand derived from derivatization of epoxy-cryogel with tris(2-aminoethyl)amine followed by the treatment with bromoacetic acid (defined as TBA ligand)) have been prepared and evaluated on capture of heavy metal ions. The cryogels were prepared in plastic carriers, resulting in desired mechanical stability and named as macroporous gel particles (MGPs). Sorption and desorption experiments for different metals (Cu2+, Zn2+, Cd2+, and Ni2+ with IDA adsorbent and Cu2+ and Zn2+ with TBA adsorbent) were carried out in batch and monolithic modes, respectively. Obtained capacities with Cu2+ were 74 μmol/mL (TBA) and 19 μmol/mL gel (IDA). The metal removal was higher for pH values between pH 3 and 5. Both adsorbents showed improved sorption at lower temperatures (10°C) than at higher (40°C) and the adsorption significantly dropped for the TBA adsorbent and Zn2+ at 40°C. Desorption of Cu2+ by using 1 M HCl and 0.1 M EDTA was successful for the IDA adsorbent whereas the desorption with the TBA adsorbent needs further attention. The result of this work has demonstrated that MGPs are potential treatment alternatives within the field of environmental separations and the removal of heavy metals from water effluents.     

Physico-chemical boundaries in the continuous and one-step discontinuous affinity-chromatographic isolation of proteins.

From a physico-chemical point of view, affinity chromatography has no unambiguous definition. It is generally understood as the one-step chromatographic isolation of a protein from a biological sample. For such processes the protein recovery and the adsorption capacity for a given adsorption time is limited by static and dynamic physico-chemical properties of the system. The protein recovery is limited by the ratio of the static capacity, n(s), and the dissociation constant, K, for the interaction with the immobilized binding site. The limits of these quantities for 90% and 99% protein recovery were estimated. The residence time required to reach 90% of the adsorptive capacity of an adsorbent is a function of the above static properties, the pore-diffusion coefficient, D(p), and the diffusion distance in the adsorbent. It was estimated and was found to correlate well with experimental data. The one-step discontinuous or continuous chromatographic isolation of one protein from a biological sample by means of adsorbents that separate with respect to different properties is reviewed. This is only possible with selective specific adsorbents and, in special cases, also with bifunctional adsorbents that use hydrophobic interactions for the adsorption, and electrostatic repulsion for the desorption.     

Integrated enzyme purification and immobilization processes with immobilized metal affinity adsorbents

Silica-based immobilized metal affinity chromatography adsorbents with various ligand densities were prepared for the purification and immobilization of poly(His)-tagged d-hydantoinase (DHTase). An adsorbent with a ligand density of 13.0 μmol Cu²⁺/g gel exhibiting the optimal selectivity and a capacity of 1.4 mg/g gel toward the poly(His)-tagged enzyme was identified. The adsorbent was used for the one-step purification of His-tagged enzymes from crude cell lysate with a purity above 90%. The silica-based affinity adsorbents are particularly well suited for industrial scale operations due to their robustness. A packed-bed bioreactor with the DHTase-loaded adsorbents was used for the continuous conversion of d,l-p-hydroxyphenylhydantoin (d,l-HPH) to N-carbamoyl-d-hydroxyphenylglycine, an intermediate for the production of d-hydroxylphenylglycine. Under optimal conditions, 60 °C and pH 8.0, a conversion of 60% was obtained at a residence time of 30 min. Upon extended operation, the catalytic activity of the biocatalysts declined significantly due to enzyme leakage and enzyme denaturation. The extent of enzyme leakage can be attenuated by crosslinking with glutaraldehyde. In this study, we successfully demonstrate that a packed-bed bioreactor containing silica-based IMAC adsorbents can be used for the direct purification and immobilization of poly(His)-tagged enzymes for biotransformation.     

Immobilization of horseradish peroxidase onto Purolite® A109 and its anthraquinone dye biodegradation and detoxification potential

Horseradish peroxidase (HRP) is a highly specific enzyme with great potential for use in the decolorization of synthetic dyes. A comprehensive study of HRP immobilization using various techniques such as adsorption and covalent immobilization on the novel carrier Purolite® A109 with a special focus on enzymatic decolorization and toxicity of artificially colored wastewater. The immobilized preparations with an activity of 156.21 ± 1.41 U g⁻¹ and 85.71 ± 1.62 U g⁻¹ after the HRP adsorption and covalent immobilization, respectively, were obtained. Stability and reusability of the immobilized preparations were also evaluated. A noteworthy decolorization level (~90%) with immobilized HRP was achieved. Phytotoxicity testing using Mung bean seeds and acute toxicity assay with Artemia salina has confirmed the applicability of the obtained immobilized preparation in industrial wastewater plants for the treatment of colored wastewater.     

Heterofunctional Magnetic Metal‐Chelate‐Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity

In this study, the combined use of the selectivity of metal chelate affinity chromatography with the capacity of epoxy supports to immobilize poly‐His‐tagged recombinant benzoylformate decarboxylase from Pseudomonas putida (BFD, E.C. 4.1.1.7) via covalent attachment is shown. This was achieved by designing tailor‐made magnetic chelate–epoxy supports. In order to selectively adsorb and then covalently immobilize the poly‐His‐tagged BFD, the epoxy groups (300 µmol epoxy groups/g support) and a very small density of Co²⁺‐chelate groups (38 µmol Co²⁺/g support) was introduced onto magnetic supports. That is, it was possible to accomplish, in a simple manner, the purification and covalent immobilization of a histidine‐tagged recombinant BFD. The magnetically responsive biocatalyst was tested to catalyze the carboligation reactions. The benzoin condensation reactions were performed with this simple and convenient heterogeneous biocatalyst and were comparable to that of a free‐enzyme‐catalyzed reaction. The enantiomeric excess (ee) of (R)‐benzoin was obtained at 99 ± 2% for the free enzyme and 96 ± 3% for the immobilized enzyme. To test the stability of the covalently immobilized enzyme, the immobilized enzyme was reused in five reaction cycles for the formation of chiral 2‐hydroxypropiophenone (2‐HPP) from benzaldehyde and acetaldehyde, and it retained 96% of its original activity after five reaction cycles. Chirality 27:635–642, 2015. © 2015 Wiley Periodicals, Inc.     

Evaluation of cellulose-based biospecific adsorbents as a stationary phase for lectin affinity chromatography

Macroporous cellulose Granocel was evaluated as a matrix for the immobilization of two lectins Concanavalin A (ConA) (108 kDa) and Wheat Germ Agglutinin (WGA) (36 kDa). Two different methods were employed for the immobilization of the lectins via their protein moieties by a Schiff's bases reaction. One of them results in covalent coupling of the lectin directly to the support and the other gives the attachment through a long spacer arm which benefits the immobilization of voluminous ConA molecules. The adsorbents were characterized by the glycoproteins sorption recording adsorption kinetic data and isotherms. The adsorbents demonstrated high affinity to glycoproteins with a sorption capacity in the column up to 7.4 mg/ml support and a high recovery (up to 93%). The adsorption isotherms of glucose oxidase (GOD) onto ConA adsorbents reveals an adsorption behavior with high and low affinity binding sites. The dissociation constant K(d) of the ligand-sorbate complex is approximately 1 x 10(-6) and 0.4 x 10(-5)M, respectively. It was supposed that the second step is related to the sorption of solvated GOD onto already adsorbed GOD forming sorbate dimers.     

Improvement of enzymatic performance of Asclepias curassavica L. proteases by immobilization. Application to the synthesis of an antihypertensive peptide

The aim of this work was to study different immobilization strategies on silica supports in order to obtain robust biocatalysts from latex proteases of Asclepias curassavica L., a South American native plant. Immobilized enzyme performance was evaluated under harsh reaction conditions such as the synthesis of the antihypertensive peptide N-α-CBZ-Val-Gly-OH.Proteases from A. curassavica, named asclepain, were immobilized (0.51–5.56 mg of protein/ g of support) in non-functionalized silica (S), in glyoxyl-silica (GS) and in octyl-glyoxyl-silica (OGS), by adsorption, and multi-point covalent attachment on mono and hetero-functional supports, respectively, under previously determined optimal immobilization conditions. Immobilization yields were expressed as activity yield (Y_a) and protein yield (Y_p).Asclepain-OGS showed the highest Y_a (178 ± 1.62 %) meaning an expressed activity 1.8 times higher than the offered activity, while Y_p was 75 ± 0.4 %. Y_a for asclepain-S and -GS were 64 ± 1.45 % and 16 ± 0.37 %, respectively. Best results were attributed to the ability of OGS support to guide the enzyme before covalent attachment, increasing its reactivity. Asclepain-OGS led to product yield of 95.5 ± 0.14 %, five times higher than soluble asclepain in the synthesis of N-α-CBZ-Val-Gly-OH, after 3 h in 30 % methanol in 0.1 M Tris-HCl buffer pH 8.     

Effects of poly(ethylene glycol) and salt on the binding of α-amylase from the fermentation broth of Bacillus amyloliquefaciens by Cu2+-β-CD affinity adsorbent

α-Amylase from Bacillus amyloliquefaciens was purified by the immobilized metal ion affinity adsorbent, β-CD_cl-IDA-Cu²⁺. The adsorbent was prepared by reacting the cross-linked β-cyclodextrin (β-CD) with the ligand, iminodiacetic acid (IDA). The copper ion was further linked to the adsorbent. Poly(ethylene glycol) (PEG) was added to the fermentation broth to improve the adsorption efficiency of the adsorbent toward α-amylase. The effort was to provide hydrophobic interactions with the impurities which might interfere with the adsorption of α-amylase. It also provided a polymer shielding effect to prevent non-specific interactions. With the addition of PEG, the adsorption efficiency could be increased to 98%. Imidazole containing a phosphate buffer and NaCl was used to elute the bound α-amylase. By consecutive adsorption/desorption steps, up to 81% of the α-amylase activity could be recovered. Regarding the reutilization of the affinity adsorbents, α-amylase could be adsorbed and desorbed six times consecutively without a significant loss of α-amylase activity.     

Synergistic Removal of Pb(II), Cd(II) and Humic Acid by Fe3O4@Mesoporous Silica-Graphene Oxide Composites

The synergistic adsorption of heavy metal ions and humic acid can be very challenging. This is largely because of their competitive adsorption onto most adsorbent materials. Hierarchically structured composites containing polyethylenimine-modified magnetic mesoporous silica and graphene oxide (MMSP-GO) were here prepared to address this. Magnetic mesoporous silica microspheres were synthesized and functionalized with PEI molecules, providing many amine groups for chemical conjugation with the carboxyl groups on GO sheets and enhanced the affinity between the pollutants and the mesoporous silica. The features of the composites were characterized using TEM, SEM, TGA, DLS, and VSM measurements. Series adsorption results proved that this system was suitable for simultaneous and efficient removal of heavy metal ions and humic acid using MMSP-GO composites as adsorbents. The maximum adsorption capacities of MMSP-GO for Pb(II) and Cd (II) were 333 and 167 mg g⁻¹ caculated by Langmuir model, respectively. HA enhances adsorption of heavy metals by MMSP-GO composites due to their interactions in aqueous solutions. The underlying mechanism of synergistic adsorption of heavy metal ions and humic acid were discussed. MMSP-GO composites have shown promise for use as adsorbents in the simultaneous removal of heavy metals and humic acid in wastewater treatment processes.     

Immobilization of yeast cells on polyphenyleneoxide

Summary The potential of a polymeric product of 2, 6-dimethylphenol as a support for immobilized intact yeast cells was investigated. The procedure used is based on modification of the polymeric adsorbent by adsorption of glutaraldehyde, and the immobilization of cells is probably accomplished by their adsorption and covalent linkage.     

One-step purification,covalent immobilization,and additional stabilization of a thermophilic poly-His-tagged beta-galactosidase from Thermus sp. strain T2 by using novel heterofunctional chelate-epoxy Sepabeads

Using the poly-His-tagged-beta-galactosidase from Thermus sp. strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification. The aforementioned enzyme could not be immobilized onto standard epoxy supports with good yields, and after purification and storage, it exhibited a strong trend to yield very large aggregates as shown by ultracentrifugation experiments. That preparation could not be immobilized in any support, very likely because the pores of the solid became clogged by the large aggregates. These novel epoxy-metal chelate heterofunctional supports contain a low concentration of Co(2+) chelated in IDA groups and a high density of epoxy groups. This enabled the selective adsorption of poly-His-tagged enzymes, and as this adsorption step is necessary for the covalent immobilization procedure, the selective covalent immobilization of the target enzyme could take place. This strategy allowed similar maximum loadings of the target enzyme using either pure or crude preparations of the enzyme. The enzyme derivative presented a very high activity at 70 degrees C (over 1000 IU in the hydrolysis of lactose) and very high stability and stabilization when compared to its soluble counterpart (activity remained unaltered after several days of incubation at 50 degrees C). In fact, this preparation was much more stable than when the same enzyme was immobilized onto standard epoxy Sepabeads.     

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.     

A novel heterofunctional epoxy-amino sepabeads for a new enzyme immobilization protocol: immobilization-stabilization of beta-galactosidase from Aspergillus oryzae

The properties of a new and commercially available amino-epoxy support (amino-epoxy-Sepabeads) have been compared to conventional epoxy supports to immobilize enzymes, using the beta-galactosidase from Aspergillus oryzae as a model enzyme. The new support has a layer of epoxy groups over a layer of ethylenediamine that is covalently bound to the support. This support has both a great anionic exchanger strength and a high density of epoxy groups. Epoxy supports require the physical adsorption of the proteins onto the support before the covalent binding of the enzyme to the epoxy groups. Using conventional supports the immobilization rate is slow, because the adsorption is of hydrophobic nature, and immobilization must be performed using high ionic strength (over 0.5 M sodium phosphate) and a support with a fairly hydrophobic nature. Using the new support, immobilization may be performed at moderately low ionic strength, it occurs very rapidly, and it is not necessary to use a hydrophobic support. Therefore, this support should be specially recommended for immobilization of enzymes that cannot be submitted to high ionic strength. Also, both supports may be expected to yield different orientations of the proteins on the support, and that may result in some advantages in specific cases. For example, the model enzyme became almost fully inactivated when using the conventional support, while it exhibited an almost intact activity after immobilization on the new support. Furthermore, enzyme stability was significantly improved by the immobilization on this support (by more than a 12-fold factor), suggesting the promotion of some multipoint covalent attachment between the enzyme and the support (in fact the enzyme adsorbed on an equivalent cationic support without epoxy groups was even slightly less stable than the soluble enzyme).     

Modelling regeneration effects on protein A affinity chromatography

The effect of in-place regeneration of protein A adsorbents on protein adsorption characteristics is investigated. Regeneration with sodium hydroxide and time of exposure determined the protein capacity of the adsorbent, but no effect was observed on the adsorbent protein affinity. Fixed-bed adsorption of human immunoglobulin G was studied. Breakthrough curves were measured for protein adsorption on fixed-bed columns. These data were analyzed by a simple kinetic model to determine the rate constants for the adsorption process. It was found that forward adsorption rate constant remained constant along the chemical treatment exposure time. Protein A adsorbent selectivity was determined using mouse serum immunoglobulins G ₁ and G ₃ . Column linear gradient elution showed that adsorbent selectivity decreased with the exposure time chemical treatment. The implications of these results on the design and optimization of protein A chromatographic process are discussed.     

A new metal chelate affinity adsorbent for cytochrome C

We have prepared a novel metal-chelate adsorbent utilizing N-methacryloyl-L-histidine methyl ester (MAH) as a metal-chelating ligand. MAH was synthesized by using methacryloyl chloride and l-histidine methyl ester dihydrochloride. Spherical beads with an average diameter of 75-125 microm were produced by suspension polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAH carried out in an aqueous dispersion medium. Then, Cu(2+) ions were chelated directly on the chelating beads. Cu(2+)-chelated beads were used in the adsorption of cytochrome c (cyt c) from aqueous solutions. The maximum cyt c adsorption capacity of the Cu(2+)-chelated beads (658.2 micromol/g Cu(2+) loading) was found to be 31.7 mg/g at pH 10 in phosphate buffer. The nonspecific cyt c adsorption on the naked PHEMA beads was 0.2 mg/g. Cyt c adsorption increased with increasing Cu(2+) loading. Cyt c adsorption capacity was demonstrated for the buffer types with the effects in the order phosphate > HEPES > MOPS > MES > Tris-HCl. Cyt c molecules could be adsorbed and desorbed five times with these adsorbents without noticeable loss in their cyt c adsorption capacity.