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Immobilized Enzymes
Published in Debabrata Das, Debayan Das, Biochemical Engineering, 2019
Different types of supporting matrices are available for immobilization processes. Examples of some carriers are as follows: Carriers used for the adsorption of enzymes: alumina, bentonite, calcium carbonate gel, carbon, cellulose, clay, collagen, sepharose, glass (porous), hydroxyapatite, ion-exchange resins, kaolin, phenolic polymers, silica gel, PVC.Carriers used for the covalent attachment of enzymes: agarose (sepharose), cellulose, dextran (sepharose), glass, polyacrylamide co-polymer, polyaminostyrene.Carriers used for the entrapment of enzymes/cells: polyacrylamide gels, calcium alginate gels, kappa-carrageenan, agar gel.
Chromatography
Published in Pau Loke Show, Chien Wei Ooi, Tau Chuan Ling, Bioprocess Engineering, 2019
Kirupa Sankar Muthuvelu, Senthil Kumar Arumugasamy
Emil Starkenstein published an article in 1910 about resolving macromolecule complexes based on their specific interactions with an immobilized substrate. This article describes the effect of chloride on the enzymatic activity of liver α-amylase, and it encouraged several researchers to work on it (Arsenis and Mccormick, 1964; Bautz and Hall, 1962; Campbell et al., 1951). In 1968, the name, affinity chromatography, was introduced by Pedro Cuatecasas, Chris Anfinsen, and Meir Wilchek in an article that dealt with the purification of enzymes using immobilized substrates and inhibitors (Wang et al., 2017). Other articles illustrated the use of sepharose matrix by activating it using a cyanogen bromide (CNBr) reaction (Axén et al., 1967) and the applicability of spacer arm to lessen steric hindrance (Clonis et al., 2000).
Protocols for Key Steps in the Development of an Immunoassay
Published in Richard O’Kennedy, Caroline Murphy, Immunoassays, 2017
Caroline Murphy, Richard O’Kennedy
High-resolution purification using chromatographic methods can be used as a final polishing step following initial purification procedures. Size exclusion chromatography (SEC) or gel filtration uses a cross-linked beaded form of agarose (or other material) as a separation media for molecules based on their size. There are many types of chromatographic media and GE Healthcare provides a range of these to facilitate antibody purification. SEC can be used for ‘group separation’ and ‘fractionation’. Group separation is used to remove high or low molecular weight contaminants (e.g. desalting), while fractionation is used to purify biomolecules according to their size. To perform SEC, a column is packed with porous spherical particles (e.g. agarose or sepharose). A suitable equilibration buffer is added to the column, which fills the spheres and the space between. A heterogeneous sample (containing antibody) is added to the column, the molecules move through the column at varying speeds according to their size. The smaller molecules move into the porous spheres, slowing their progress through the column, while the larger molecules move into and out of the beads to a lesser degree, therefore passing through the column at greater speed. A protocol using SEC for the purification of antibody follows (Protocol 12.2).
Recent developments in purification techniques and industrial applications for whey valorization: A review
Published in Chemical Engineering Communications, 2020
Navpreet Kaur, Poorva Sharma, Seema Jaimni, Bababode Adesegun Kehinde, Shubhneet Kaur
Ounis et al. (2008) conducted a study involving the purification of minor proteins from WPIs using the heparin affinity chromatography technique. In the course of their study, it was observed that heparin affinity chromatography can also be used for the concentration of the minor cationic proteins and growth factors of milk. Using the affinity chromatography technique, Atasever et al. (2013) studied the one-step separation of the LPO enzyme from bovine milk. It was discovered that sulfanilamide is a competitive inhibitor of LPO with IC50 inhibition value of 0.848 × 10−5 M. This mechanism was used to design a suitable affinity matrix by binding a ligand (sulfanilamide) and spacer arm (l-tyrosine) to an activated-Sepharose 4B matrix consisting of cyanogen bromide (CNBr). Results showed that the newly synthesized sulfanilamide affinity matrix, purified 409-fold of LPO with a yield of 62.3% in a one step. Baieli et al. (2014) explained a low-cost purification method by using integrated chromatographic technique for separation of LF from sweet whey. Chitosan was used as mini spheres and low cost immobilized yellow dye (HE-4R) as ligand. Highest adsorption obtained ranged from 51.14 to 58.28 mg of LF/g affinity matrix. Chitosan mini spheres adsorbed 95% and eluted >80% of absorbed LF. A high yield with over 90% was achieved in purification involving only a single step. The purification operation was found to be adequate for three subsequent cycles without any step involving regeneration. Casein glycomacropeptide (CMP), a peptide with an amino acid chain length of 46 was productively purified from bovine whey by Baieli et al. (2017).
Production of polyclonal antibody against human Neuritin and its application of immunodetection
Published in Preparative Biochemistry and Biotechnology, 2019
Na Wang, Yu Wei, Wen Zhang, Xingyi Li, Jingling Zhu, Liya Shan, Chunyan Liu, Wumei Yuan, Jin Huang
Antiserum was purified with immune affinity (Sigma, Cyanogen bromide-activated-Sepharose® 4B) according to the manufacturer’s instructions. Briefly, sepharose was activated by 1 mM HCl and coupled with rhNeuritin and then incubated with antiserum overnight at 4 °C. Effluent was collected and columns were washed with ten volumes of elution buffer (0.1 M glycine, pH 3.2). Sepharose beads coupled with expressed rhNeuritin were washed with buffers according to the manufacturer’s protocol and stored at 4 °C.
Increase in cysteine-mediated multimerization under attractive protein–protein interactions
Published in Preparative Biochemistry & Biotechnology, 2022
Leo A. Jakob, Tomás Mesurado, Alois Jungbauer, Nico Lingg
The impact of protein-protein interactions on multimerization in the adsorbed phase was investigated by batch adsorption of CASPON enzyme on Ni Sepharose FF resin. The protein was adsorbed for 6 h under either highly attractive (1 M ammonium sulfate, pH 8) or slightly repulsive (1 M NaCl, pH 8) conditions and subsequently eluted and evaluated. This contact time is typical for a purification process using IMAC.[40–42]