E. G. Vlakh

Flow-through immobilized enzyme reactors based on monoliths: II. Kinetics study and application.

In the last decade, the application of monolithic materials has rapidly expanded to the realization of flow-through bioconversion processes. Up to these days, different classes of enzymes such as hydrolases, lyases, and oxidoreductases have been immobilized on organic, inorganic, or hybrid monolithic materials to prepare the effective flow-through enzymes reactors for application in proteomics, biotechnology, pharmaceutics, organic synthesis, and biosensoring. Current review describes the results of kinetic study and specialties of flow-through immobilized enzyme reactors based on the existing monolithic materials.

Flow‐through immobilized enzyme reactors based on monoliths: I. Preparation of heterogeneous biocatalysts

The application of monoliths for realization of solid-phase biocatalytic processes was dramatically extended since the beginning of new century. Different enzyme immobilization techniques regarding these modern stationary phases have been developed, adapted, and optimized within last decade. The choice of enzyme immobilization method depends on material nature and monolith manufacturing. The present review collected, analyzed, and discussed the accessible published data on existing approaches and specialties of preparation of flow-through enzyme reactors based on monoliths.

HPLC analysis of synthetic polymers on short monolithic columns

Ultrashort monolithic columns (disks) were thoroughly studied as efficient stationary phases for precipitation-dissolution chromatography of synthetic polymers. Gradient elution mode was applied in all chromatographic runs. The mixtures of different flexible chain homopolymers, such as polystyrenes, poly(methyl methacrylates), and poly(tert-butylmethacrylates) were separated according to their molecular weights on both commercial poly(styrene-co-divinylbenzene) disks (12 id × 3 mm and 5 × 5 mm) and lab-made monolithic columns (4.6 id × 50 mm) filled with supports of different hydrophobicity. The experimental conditions were optimized to reach fast and highly efficient separation. It was observed that, similar to the separation of monoliths of other classes of (macro)molecules (proteins, DNA, oligonucleotides), the length of column did not affect the peak resolution. A comparison of the retention properties of the poly(styrene-co-divinylbenzene) disk-shaped monoliths with those based on poly(lauryl methacrylate-co-ethylene dimethacrylate), poly(butyl methacrylate-co-ethylene dimethacrylate), and poly(glycidyl methacrylate-co-ethylene dimethacrylate) supports demonstrated the obvious effect of surface chemistry on the resolution factor. Additionally, the results of the discussed chromatographic mode on the fast determination of the molecular weights of homopolymers used in this study were compared to those established by SEC on columns packed with sorbent beads of a similar nature to the monoliths.

Biocatalytic reactors based on ribonuclease A immobilized on macroporous monolithic supports

AbstractImmobilized enzyme reactors (IMERs) produced by the covalent attachment of ribonuclease A to macroporous methacrylate-based monolithic supports using different experimental approaches are discussed and compared. Enzyme immobilization was carried out by direct covalent binding, as well as through attachment via a polymer spacer. The kinetic properties of an IMER operating in either recirculation mode or zonal elution mode were studied. Additionally, the effect of flow rate on the bioconversion efficiency of each IMER sample was examined. FigureEnzyme immobilization via aldehyde-bearing macromolecular spacer on the surface of epoxy-containing monoliths

Polymer monoliths as efficient solid phases for enzymatic polynucleotide degradation followed by fast HPLC analysis

Two ribonuclease A bioreactors based on lab-made macroporous monolithic columns and intended for polynucleotide degradation were prepared using in situ free-radical polymerization. Different methods of enzyme immobilization were applied. In the first case, the biocatalyst molecule was attached to the solid surface via direct covalent binding, while in the second bioreactor the flexible-chain synthetic polymer was used as an intermediate spacer. The effect of temperature, substrate flow rate, and loaded sample volume on the biocatalytic efficiency of the immobilized enzyme was examined. The kinetic parameters of the enzymatic degradation of synthetic polycytidylic acid were calculated and compared to those found for hydrolysis with soluble ribonuclease A. The monitoring of substrate splitting was carried out by means of fast anion-exchange HPLC on an ultra-short monolithic column (disk) using off- and on-line analytical approaches.

Molecular imprinting: a tool of modern chemistry for the preparation of highly selective monolithic sorbents

Characteristic features of the synthesis of molecularly imprinted polymer systems used as sorbents for separation of complex mixtures into components are discussed. The data about the molecules imprinted in monoliths of various natures and shapes are integrated. Examples of application of new-generation separating media are discussed. Data on the utilization of the molecular imprinting principle for the fabrication of supermacroporous monolithic cryogels specific to particular molecules, which is important for the design of smart biomaterials, are analyzed. The bibliography includes 293 references.

Xylan degradation improved by a combination of monolithic columns bearing immobilized recombinant β-xylosidase from Aspergillus awamori X-100 and Grindamyl H121 β-xylanase.

Synergistic action of exo‐ and endohydrolazes is preferred for effective destruction of biopolymers. The main purpose of the present work was to develop an efficient tool for degradation of xylan. Macroporous lab‐made monolithic columns and commercial CIM‐Epoxy disk were used to immobilize the recombinant β‐xylosidase from Aspergillus awamori and Grindamyl β‐xylanase. The efficiency of xylan degradation using the low‐loaded β‐xylosidase column appeared to be four times higher than for the in‐solution process and about six times higher than for the high‐loaded bioreactor. Disk bioreactor with the Grindamil β‐xylanase operated in a recirculation mode has shown noticeable advantages over the column design. Additionally, a system comprised of two immobilized enzyme reactors (IMERs) was tested to accelerate the biopolymer hydrolysis, yielding total xylan conversion into xylose within 20 min. Fast online monitoring HPLC procedure was developed where an analytical DEAE CIM disk was added to the two‐enzyme system in a conjoint mode. A loss of activity of immobilized enzymes did not exceed 7% after 5 months of the bioreactor usage. We can therefore conclude that the bioreactors developed exhibit high efficiency and remarkable long‐term stability.

Affinity Chromatography of Proteins on Monolithic Columns

At present, monolithic stationary phases, because of their morphology, are widely used for development and realization of fast dynamic and static processes based on mass transition between liquid and solid phases. These are liquid chromatography, solid phase synthesis, microarrays, flow-through enzyme reactors, etc. High-performance liquid chromatography on monoliths, including bioaffinity mode, represents a unique technique appropriate for fast and efficient separation of biological (macro)molecules of different sizes and shapes (proteins, nucleic acids, peptides), as well as such supramolecular systems as viruses.In this work, the examples of application of commercially available macroporous monoliths for modern affinity processing are presented. In particular, the original methods developed for efficient isolation and fractionation of monospecific antibodies from rabbit blood sera, the possibility of simultaneous affinity separation of protein G and serum albumin from human serum, the isolation of recombinant products, such as protein G and tissue plasminogen activator from E. coli cell lysate and Chinese Hamster Ovary cell culture supernatant, respectively, are described in detail. The suggested and realized multifunctional fractionation of polyclonal pools of antibodies by combination of several short monolithic columns (disks) with different affinity functionalities stacked in the same cartridge represents an original and practically valuable method that can be used in biotechnology.

A multienzyme bioreactor based on a chitinase complex

A heterogeneous biocatalyst containing a complex of chitinolytic enzymes isolated from the culture medium of bacteria Clostridium paraputrificum on the surface of macroporous monolithic minidisc was obtained. The complex of chitinolytic enzymes was immobilized on the polymer matrix using a multistep method involving the introduction of an intermediate macromolecular spacer. The endochitinase and N-acetylglucosaminidase activity of the heterogeneous biocatalyst was studied.

The preparation and study of the properties of macroporous monolith-based continuous flow bioreactors

The surfaces of macroporous monolithic disks were modified with hydrolytic enzymes, viz., α-chymotrypsin and ribonuclease A. The effect of the enzyme-immobilization approach and the amount of the aldehyde groups in the polymer spacer, as well as the flow rate of the substrate solution on the efficiency of the heterogeneous biocatalysis was explored using the reactions of hydrolysis of both low and macromolecular substrates as examples.