Enzyme concentration and purification

Enzyme Concentration and Purification. The number of existing enzymes is estimated to be more than 10,000 of which more than 100 have been purified in crystalline form and over 600 in fairly purified form. The molecular weight of enzymes varies from 12,700 (ribonuclease) to over 1,000,000 (L-glutamate dehydrogenase, d-carboxylase). All enzymes are proteins, conjugated proteins or metalloproteins containing one or more active sites per molecule. 

Enzymes are highly specific biological catalysts which have an optimum pH and temperature (usually fairly mild conditions) for maximum activity. From a chemical engineering point of view they are the ideal catalyst. Inorganic catalysts are seldom as specific (enzyme catalysts yield almost no by-products) and require high temperatures and/or extremes in pH to be effective. 

Because of their protein structure, enzymes may be inactivated temporarily or permanently by heat, chemicals, or shear forces. UF systems can be designed to recover enzymes athermally with minimum shear inactivation (using diaphragm pumps). 

The majority of microbial enzymes are produced in relatively small batch fermenters. After separation from the biomass with open membranes, filter presses, or centrifuges, UF is used to concentrate the enzyme and remove small peptides, oligosaccharides and salts. 

Crude extracts of enzymes are also obtained from macerated tissues, germination seeds, fermented bran, or the beer from the submerged fermentation of selected strains of microorganisms. After extraction and prefiltration to remove suspended solids and particulates, UF is applied as above. 

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Enzyme extraction and purification steps The crude enzyme was extracted from the solid state culture with 100 ml of 0.33% toluene at 4°C. The enzyme was concentrated with 60-90% (NH4)2S04, then the dialysed enzyme... 

Frequently however, enzymes found in the fermentation broth at the time of harvesting are quite different from the state in which they can be used as industrial biocatalysts, reagents in clinical chemistry or as therapeutic agents, The recovery of an enzyme from microbial culture, its concentration and purification will require careful and effective sequential operations, which ate called downstream processing (Wheelwright, 1989). 

Ultrafil- tration Asymmetric micro-porous polymer membrane. Pore size 1-50 nm Hydrostatic pressure 2-10 bar Sieving mechanism, pore size, and particle diameter determine separation characteristics Separation, concentration and purification of macro-molecular solutions such as proteins, enzymes, polypeptides, etc. 

After the removal of the nucleic acid, cell debris, unwanted contaminants, and other proteins, water then needs to be removed. Different methods are available for enzyme concentration and initial purification. Among them are precipitation using ammonium sulfate, organic solvents or polymers, manbrane separation, or an aqueous biphasic system (Janson, 2011 Schmauder and Schweizer, 1997). 

Abstract Natural and synthetic polyelectrolytes have acquired notable importance in recent years due to their increasing application in different areas. One of these is downstream process methods which include the recovery, separation, concentration and purification of target enzymes from their natural sources. Polyelectrolytes interact with proteins to form soluble or non-soluble complexes. The interaction is driven by experimental variables of media such as pH, protein isoelectrical value, polyelectrolyte pKa, ionic strength and the presence of salts. The concentration of polyelectrolytes necessary to precipitate a protein completely is of the order of 10 " - 10 % p/v. Precipitation of protein by PE is a novel technique integrating clarification, concentration and initial purification in a single step. This chapter presents some properties of aqueous solutions of natural and synthetic PE as a tool to use them in the protein downstream process. 

Retention and concentration of valuables (concentration and purification of enzymes, etc.)... 

Advantages Better reaction control No side reactions Higher substrate and product concentration tolerance Simple and cheap Cofactor regeneration within cells No enzyme isolation and purification Higher stability of many redox enz3Tnes... 

If the activity is controlled by the existence of a slowest step, then two different approaches can be considered for optimization. The first approach consists of extraction and purification of the enzymes or proteins, followed by formulation of a new biocatalyst containing the enzyme or protein involved in the slowest step at a higher concentration. A second approach is to over express that enzyme or protein by GE tools in the WC. 

The mixture was filtered and the enzyme washed with MTBE until the filtrate turned colorless. The filtrate was then successively washed with 1 M aqueous HCl, saturated NaHCOa and brine and then concentrated and deprotected directly without further purification (typical yield 80 %). 

Figure 5.2 Flow diagram of purification of hydrogenase from D. fructosovorans. At each stage of fractionation, the enzyme activity and protein concentration are measured for each fraction.The fractions with the greatest specific activity (S.A., units/milligram protein) are taken onto the next stage.

Removing Interfering Polyphenols. The most troublesome problem encountered was high polyphenol content. If not removed, this dark-colored wood extractive decreased resolution during chromatographic separations, quickly render expensive chromatographic media (e.g., HPLC columns) nearly useless, and often precipitated enzymes during subsequent purification steps (14,15), We found it best to remove the bulk of this material prior to the first concentration step. 

Unfractionated Preparatioii. Each harvest of the enzyme was concentrated to 401 and diafiltered with 0.01 M sodium acetate, pH 6.0. Enzyme concentrate was bound to Q-Sepharose and eluted with 0.4 M sodium acetate, pH 6.0. Fractions with activity were pooled, sterile filtered and stored in the cold in 10 mM veratryl alcohol. Before further purification, the solution was dialyzed. 

Isozymes are also a common presence in enzyme preparations and they can often be detected via polyacrylamide gel electrophoresis. The detected presence of isozymes may result in the need for further purification steps and the kinetic characterization of each isozyme. It may be necessary to use nondenaturing electrophoretic procedures to separate the different isozymes. See Isozymes Enzyme Concentration... 

In order to be exploitable for extraction and purification of proteins/enzymes, RMs should exhibit two characteristic features. First, they should be capable of solubilizing proteins selectively. This protein uptake is referred to as forward extraction. Second, they should be able to release these proteins into aqueous phase so that a quantitative recovery of the purified protein can be obtained, which is referred to as back extraction. A schematic representation of protein solubilization in RMs from aqueous phase is shown in Fig. 2. In a number of recent publications, extraction and purification of proteins (both forward and back extraction) has been demonstrated using various reverse micellar systems [44,46-48]. In Table 2, exclusively various enzymes/proteins that are extracted using RMs as well as the stability and conformational studies of various enzymes in RMs are summarized. The studies revealed that the extraction process is generally controlled by various factors such as concentration and type of surfactant, pH and ionic strength of the aqueous phase, concentration and type of CO-surfactants, salts, charge of the protein, temperature, water content, size and shape of reverse micelles, etc. By manipulating these parameters selective sepa-... 

The binding of milk lipases to casein micelles apparently imparts some stability to the enzyme, for as purification progresses, the milk lipase becomes less stable, and more so as the concentration of casein decreases (Downey and Andrews 1966 Egelrud and Olivecrona 1972). 

The enzyme was originally found to be membrane bound and resisted solubilization and purification (26). Lundblad and Moore (27), however, have reported solubilizing it using dilute (5 mM) sodium borate buffer at pH 9 after 16 hr at 37°. Studies on regional and subcellular distribution using density gradient techniques have revealed that the 2, 3 -cyclic phosphate diesterase concentrates in those fractions containing myelin (28, 29), and the conclusion has been reached that the enzyme is localized in the myelin sheath or intimately associated structures. Kurihara and... 

The enzyme was purified from Candida utilis in 1965 by Rosen et al. (8Q). Dried yeast was allowed to autolyze in phosphate buffer at pH 7.5 for 48 hr, and the enzyme was isolated in crystalline form from these autolysates by a procedure which included heating to 55° at pH 5.0, fractionation with ammonium sulfate, and purification on phospho-cellulose columns from which the enzyme was specifically eluted with malonate buffer containing 2.0 mM FDP. Crystallization was carried out by addition of ammonium sulfate in the presence of mM magnesium chloride. The Candida enzyme was more active than the mammalian FDPases at room temperature and pH 9.5 the crystalline protein catalyzed the hydrolysis of 83 /nnoles of FDP per minute per milligram of protein. The enzyme was completely inactive with other phosphate esters, including sedoheptulose diphosphate, ribulose diphosphate, and fructose 1- or fructose 6-phosphates. Nor was the activity of the enzyme inhibited by any of these compounds. Optimum activity was observed at concentrations of FDP between 0.05 and 0.5 mM higher concentrations of FDP (5 mM) were inhibitory. 

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