Heavy metal removal by novel CBD-EC20 sorbents immobilized on cellulose

Heavy metals are major contributors to pollution of the biosphere, and their efficient removal from contaminated water is required. Biosorption is an emerging technology that has been shown to be effective in removing very low levels of heavy metal from wastewater. Although peptides such as metallothioneins or phytotchelatins are known to immobilize heavy metals, peptide-based biosorbents have not been extensively investigated. In this paper, we describe the construction and expression of bifunctional fusion proteins consisting of synthetic phytochelatin (EC20) linked to a Clostridium-derived cellulose-binding domain (CBD(clos)), enabling purification and immobilization of the fusions onto different cellulose materials in essentially a single step. The immobilized sorbents were shown to be highly effective in removing cadmium at parts per million levels. Repeated removal of cadmium was demonstrated in an immobilized column. The ability to genetically engineer biosorbents with precisely defined properties could provide an attractive strategy for developing high-affinity bioadsorbents suitable for heavy metal removal.     

Solid phase peptide synthesis on epoxy-bearing methacrylate monoliths.

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.     

Harvesting of Microalgae Biomass with Polyethylenimine-Based Sorbents

The purpose of this work was to investigate the sorbents on the basis of polyethylenimine (PEI) intended for collecting biomass of microalgae (MA). For this purpose, a series of porous and insoluble polymeric materials were synthesized by cross-linking of PEI with epichlorohydrine. The analysis of kinetics and efficiency of immobilization assessed for the model culture Chlorella vulgaris, revealed that already within 3 h of incubation, 39–75% of MA cells attached to the surface of tested sorbents. It was shown that on the initial stage of immobilization the sorption activity of polymeric materials depended on the “PEI:crosslinker” ratio. One of the tested sorbents was additionally quartenized by alkylation with dimethyl sulphate resulting in sharp increase of its sorption activity. The estimation of the MA desorption from polymeric surface showed that most Ch. vulgaris cells were practically irreversibly immobilized on all tested sorbents based on the PEI cross-linked with epichlorohydrine.     

Immobilization of bacteria on polymer matrices for degradation of crude oil and oil products

Organic polymer materials (OPM) differing in sets of functional groups, fiber surface thickness and character, and density of fiber packing in fabric were synthesized. OPM were studied for assessing the possibility of their application as sorbents for oil spills in water bodies. The synthesized OPM were used for the creation of bio-hybrid materials as matrices for immobilization of bacteria of the genus Rhodococcus sp. capable of petroleum degradation. Actively dividing bacterial cells forming clusters were shown to be present at the surface of fibers. Active attachment of the cells to polymeric surface due to intrusion and/or excretion of extracellular biopolymeric matrix were detected. The modification of polymer sorbents was shown to influence bacterial immobilization. The peculiarity of growth and the specificity of cell morphology of bacterial culture were noted.     

Immobilization of EcoR1, Pae1 and Lpl1 restriction enzymes]

In search for sorbents (silica gels, styrene-divinylbenzene copolymers), for immobilization of some restriction endonucleases, derivatives of trityl-containing silochroms are shown to bind EcoRI, PaeI and LplI endonucleases with the retention of 10-20, 60-70 and 40-60% activity, respectively. The immobilized restriction endonucleases have the unchanged substrate specificity, can be used several times and are stable at storage. Tritylaminopropylsilochrom is suggested to be the sorbent of choice.     

The development of methods for obtaining monospecific ingredients for immunoenzyme analysis

The methods of the modification of Salmonella O- and H-antigens and the preparation of biologically active sorbents on their basis have been developed. The use of these sorbents has permitted the isolation of affinity antibodies with strictly defined specific activity. The work shows the possibility of the successful use of carriers obtained on the basis of porous glass, chemically modified by acrylic copolymers containing activated carboxylic groups, and intended for the immobilization of antigens of both protein and carbohydrate nature.     

Methods and prospects of development of the affinitive sorbents for plasminogen isolation from human blood plasma

The review paper was dedicated to development of the promising photochemical synthesis of affine sorbents for plasminogen isolation from the human blood plasma. Some most interesting, from the viewpoint of practice, types of sorbents and carriers based on high-molecular compounds of natural (organic) or synthetic origin have been considered. The advantages of the use of photochemical synthesis of biospheric sorbent as compared with thermochemical method have been shown. The most promising methods of creation of affine sorbents on the basis of oligomer-polymeric photoinitiators and oligomerpolymeric photosensibilizing (donor-acceptor) systems are presented.     

Immobilization of proteolytic complexes on pectin-containing carriers

A number of sorbents were synthesized on the basis of pectin and then used for immobilization of proteolytic complexes--pancreatin and protosubtilin. The best properties were shown by the enzyme preparations based on pectin, formaldehyde and melamin (PFM). Thus immobilization of pancreatin on PFM through Fe(III) ions gave a preparation with the activity of 79 000 mumole/g X h with respect to methyl ester of L-tryptophane (the activity yield is 91%). The pH optimum for all immobilized preparations was shifted towards the alkaline region. The thermostable fraction of the immobilized preparations retains the activity at 60 degrees for a long time.     

DNA immobilization on modified inorganic sorbents

Fragments of denaturated DNA having the length of about 500 nucleotides were immobilized, using silica carriers modified by organic polymers. It was shown that the organic coating allows to obtain sorbents, which practically exclude non-specific sorption with respect to DNA. The immobilization was carried out on hydroxyl-containing sorbents and on their phrosphorylated derivatives by means of water-soluble carbodiimide. The resulting preparations contained up to 60 units at A260 of DNA per 1 g of carrier. The effects of endonuclease A236, exonuclease A5 and pancreatic DNAse on the immobilized RNA were studied.     

Selective adsorption of endotoxin inside a polycationic network of flat-sheet microfiltration membranes

For the removal of remaining amounts of endotoxin, sorbents with high selectivity for endotoxin are required. Typically, particulate sorbents with positively charged ligands, such as histidine, polymyxin B poly-L-lysine and poly(ethyleneimine) (PEI), display moderate to high removal efficiencies in an environment of low ionic strength. It was found that polycationic ligands are most suitable to meet an endotoxin concentration which is below the threshold level required for parenteralia. Furthermore, protein recoveries close to 100% are obtained if the decontamination is performed at a pH close to the pI of acidic proteins. The high selectivity is probably caused by complexation of the polycationic ligand with the polyanionic endotoxin, leading to interactions with KD < 10(-9) M using PEI and assuming M(r) = 10 kDa for monomeric endotoxin; with BSA the same ligand reveals only KD = 4 x 10(-6) M. Using polymer-coated microfiltration membranes, immobilization of positively charged ligands leads to membrane adsorbers which are generally superior to chromatographic adsorbers and allow faster processing. Since immobilization takes place at polymer chains, low-molecular-weight ligands mainly add positive charges to the hydrophilic polymer. Consequently, membrane adsorbers with low-molecular-weight ligands, even DEAE, demonstrate similar selectivity to PEI or poly-L-lysine.     

Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA)

Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.     

Dye-ligand affinity systems.

Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.     

Preparation and use of magnetic sorbents for studying microorganism antigens

In this work simple techniques for obtaining polyacrylamide sorbents with magnetic properties are described. These techniques have permitted obtaining block and microgranulated sorbents with the immobilization of antibodies from plague antiserum in the cellular gel structure for the specific sorption of killed and live Yersinia pestis cells and their first fraction; pig brain gangliosides have also been incorporated into the gel structure with a view to the sorption of cholera toxin from the filtrate of Vibrio cholerae culture. The magnetic properties of sorbents, obtained by the copolymerization of powdered magnetic ferric oxides in gel, have made it possible to increase the effectiveness of specific sorption due to mixing and rapid separation in different magnetic fields, as well as to facilitate and accelerate manipulations with the sorbent at all stages. The capacity of different types of sorbents and the time of sorption have been determined.     

Immobilization of Lipase from Rhizopus Niveus: A Way to Enhance its Synthetic Activity in Organic Solvent

Lipase (EC 3.1.1.3) from Rhizopus niveus was immobilized by physical adsorption on various carriers, including different types of Celite, Spherosil and Duolite. After the enzyme immobilization, the recovered hydrolytic and synthetic activities on the different carriers were then determined. The results showed that the highest synthetic activity was obtained when Duolite XAD 761 was used as the carrier. However the recovered hydrolytic activity after the immobilization on this resin was relatively low although this carrier showed the best protein loading capacity. The highest recovered hydrolytic activity was observed when the lipase was immobilized on Celite Hyflo-Supercel using an immobilization buffer adjusted to pH 4. The comparison of the free and immobilized lipase specific activities suggest that the immobilization on Celite Hyflo-Supercel, Spherosil XOA 200 and silica has enhanced the lipase hydrolytic activity. On the other hand, the use of the lipase immobilized on Duolite XAD 761 as biocatalyst of synthetic reaction, compared to that of the free enzyme, allows the reaction initial velocity to be increased 12.2-fold. In addition, the synthetic activity of the lipase immobilized on Duolite XAD 761 was shown to be maximum at a water activity in the range of 0.32-0.52.     

Affinity chromatography of nucleases

The review concerns isolation and purification of nucleases by affinity chromatography. Different stationary ligands and the methods for their immobilization on supports are described, along with diverse eluents and various procedures for a nuclease detachment from the affinity sorbents. The data on the affinity chromatography application for measuring the dissociation constants of the enzyme complexes with either immobilized or soluble ligands are compiled.     

Lead biosorption by waste biomass of Corynebacterium glutamicum generated from lysine fermentation process

Biomass waste, mainly Corynebacterium glutamicum, is generated from large-scale lysine fermentation process. In this study, protonated C. glutamicum biomass was evaluated as a biosorbent for the removal of lead from synthetic wastewater. As Pb2+ were bound to the biomass, the solution pH deceased, indicating that protons in the biomass were exchanged with lead ions. The Corynebacterium biomass bound Pb2+ at up to 2.74 mmol g(-1) at pH 5, where lead does not precipitate. Compared with other biosorbents and conventional sorbents, such as natural zeolite, activated carbon and synthetic ion exchange resin, the protonated C. glutamicum biomass was considered to be a useful biomaterial for lead biosorption.     

Preparation of affinity sorbents with immobilized synthetic ligands for therapeutic apheresis

The practical aspects of preparation and stability of medical sorbents are considered. A simple and convenient technique has been developed for synthesis of highly effective biospecific autoclavable sorbents based on the polysassharide matrix; synthetic ligands (amino acid, oligopeptide, or oligosaccharide) containing primary amino group were immobilized to the matrix via a spacer. The developed approach may be used for preparation of various affinity sorbents suitable for application in medicine and biotechnology.     

Immobilization of cyanobacteria and microalgae on polyethylenimine-based sorbents

Immobilization of phototrophic microogranisms: microalgae (MA) and cyanobacteria (CB) on polyethylenimine (PEI)-based sorbents was studied. For this purpose, 3 insoluble porous polymeric materials were synthesized by cross-linking of PEI with epichlorohydrine and immobilization of PEI on the surface of styrene–divinylbenzene copolymer. The sorbent on the basis of cross-linked PEI was also alkylated with hexadecyl bromide to achieve hydrophobicity of its surface. The analysis of kinetics and efficiency of immobilization assessed for the model MA and CB cultures revealed the significant difference in the sorption activity of different types of sorbents depending on their synthesis procedure, chemical composition and hydrophilic-hydrophobic properties of polymeric surface. The hydrophobic sorbent obtained by immobilization of PEI on the surface of styrene–divinylbenzene copolymer characterized by very low sorption activity towards CB and MA cells. The highest immobilization efficiency of phototrophic cells was achieved for the hydrophilic sorbent on the basis of PEI cross-linked with epichlorohydrine, which provided the attachment of 50–70% of cells during 3 h of incubation. The hydrophobic sorbent based on alkylated cross-linked PEI effectively immobilized CB cells, while the colonization of the polymer surface by MA cells was very scarce. The noticed effect is explained by difference in prokaryotic (CB) and eukaryotic (MA) types of surface structures organization. Assessment of photosynthetic activity of immobilized MA cells by pulse-modulated fluorometry showed that hydrophobic sorbents had no toxic effect on the cells, while toxicity of hydrophilic cross-linked PEI-based sorbent was observed only after long-term cultivation ofphototrophic cells with this sorbent.     

Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.     

The use of Fab-masking antigens to enhance the activity of immobilized antibodies

This study demonstrates that masking the Feb regions of a monoclonal antibody (Mab) with synthetic antigens prior to covalent immobilization efficiency. Water-soluble adducts of poly(2-methyloxazoline) polymers and a syntheticpeptide epitope for the Mab were constructed. These synthetic antigens are referred to as Fab-masking antigents (FMAs). The antibody used in this study is a Ca(2+)-dependent murine monoclonal lgG directed against the plasma protein, human protein C (hPC). The FMAs were pre-equilibrated with Mab in the presence of calcium prior to immobilization and were then removed by EDTA, which destabilized the FMA-Mab complexes. The antigen binding efficiency and accessibility of the Fab domain of the immobilized antibody was significantly increased for Mab immobilized in the presence of FMA relative to those Mab immobilized without FMA. The increase in binding efficiency was most pronounced for the largest FMA employed. No appreciable differences were detected in the avidity of hPC-Mab complexes formed by immunosorbents produced by either masked or unmaked antibody. These results provide evidence that orientgation may play an important role in the binding activity of immobilized antibodies.