Enzymes preparation

One cannot over-emphasize the importance of the enzyme preparation in the ability to develop an assay. First, purity is important because even the slightest contamination with another enzyme can lead one to screen with a measurement of the wrong activity. Specific reference inhibitors can be used to establish that the observed catalytic activity is the correct one. Of course, when working with novel targets, such reference inhibitors may not exist. With recombinant expression systems, one can generate catalytically inactive mutations to establish that the host cell is not the source of a contaminating phosphatase or kinase. In the end, however, the key requirement is to have an extremely pure and highly active enzyme preparation. 

If most of the enzyme molecules are inactive or if the enzyme molecules exhibit a very low catalytic efficiency (kcal/KJ, then one will need to have a high concentration of enzyme in the assay to obtain a good signal this can limit the ability to distinguish between weaker and stronger inhibitors. If one needs to have a 100 nM enzyme concentration in the reaction, then inhibitors with a Kt of 10 nM cannot be distinguished from those with a Kt of 100 nM in a steady-state IC50 experiment. 

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It should be noted that a number of different enzyme preparations can now be purchased directly from manufacturing chemists. It must be emphasised that the activity of an enzyme, whether purchased or prepared in the laboratory, may vary between rather wide limits. The activity is dependent on the source of the enzyme, the presence of poisons and also on the temperature. It appears, for example, that the quality of horseradish peroxidase depends upon the season of the year at which the root is obtained from the ground. It cannot be expected therefore that all the experiments described below will work always with the precision characteristic of an organic reaction proceeding under accurately known conditions. 

The rate of colour change will depend upon the activity of the enzyme preparation. The changes enumerated below are usually, however, easily observable. 

The sedimentation boundary of an enzyme preparation in an aqueous buffer at 20.6 C was measured after various times in an ultracentrifuge at 56,050 rpm. The following results were obtained ... 

Recovery. The principal purpose of recovery is to remove nonproteinaceous material from the enzyme preparation. Enzyme yields vary, sometimes exceeding 75%. Most industrial enzymes are secreted by a microorganism, and the first recovery step is often the removal of whole cells and other particulate matter (19) by centrifugation (20) or filtration (21). In the case of ceU-bound enzymes, the harvested cells can be used as is or dismpted by physical (eg, bead mills, high pressure homogenizer) and/or chemical (eg, solvent, detergent, lysozyme [9001 -63-2] or other lytic enzyme) techniques (22). Enzymes can be extracted from dismpted microbial cells, and ground animal (trypsin) or plant (papain) material by dilute salt solutions or aqueous two-phase systems (23). 

Purification. Enzyme purity, expressed in terms of the percent active enzyme protein of total protein, is primarily achieved by the strain selection and fermentation method. In some cases, however, removal of nonactive protein by purification is necessary. The key purification method is selective precipitation of the product or impurities by addition of salt, eg, sodium sulfate, or solvent, eg, ethanol or acetone by heat denaturation or by isoelectric precipitation, ie, pH adjustments. Methods have been introduced to produce crystalline enzyme preparations (24). 

There has been a steady growth ia the economic importance of therapeutic ensymes, with sales reaching hundreds of millions of dollars per year as of 1992. Table 2 Hsts the trade names and costs of some of the more commonly prescribed enzyme preparations. Despite the magnitude of use of these products, the manufacture and sale of therapeutic enzymes represents a comparatively small fraction of the production and profits of the pharmaceutical houses that market them. 

For enzymes intended for parenteral use, the manufacturer must assure that the enzyme preparation is essentially pure and free of endotoxins. Electrophoretic and immunologic tests provide the requisite evidence of purity and homogeneity. Most importandy, the manufacturer must remove toxic impurities, eg, bacterial hpopolysacchati.de (endotoxins) which might cause severe toxic reactions such as anaphylactic shock, fever, and vascular coUapse. 

L-lactate-cytochrome c-oxidoreductase (flavocytochrome was isolated for the first time from the thermo-tolerant yeast H. polymorpha. The mentioned above enzyme preparations were used for construction of the biorecognition elements of electrochemical sensors. 

Enzym-praparat, n. enzyme preparation, -wir-kung, /. enzyme action. 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

Enzyme preparations versus whole cell processes 13... 

In designing a process we have the choice of using the whole organism or specific enzymes isolated from it. As always both options have pro s and cons. Broadly speaking we could say that biosynthetic processes mostly rely on whole cells, whereas biotransformations can be catalysed by whole cells and by enzyme preparations. 

Benzene dioxygenase is a complex enzyme consisting of three protein components, that catalyse the conversion of benzene to benzene cis-dihydrodiol. Give two reasons why this biotransformation should be carried out using whole cells as opposed to using enzyme preparations. 

When compared to traditional chemical synthesis, processes based on biocatalysts are generally less reliable. This is due, in part, to the fact that biological systems are inherently complex. In bioprocesses involving whole cells, it is essential to use the same strain from the same culture collection to minimise problems of reproducibility. If cell free enzymes are used the reliability can depend on the purity of the enzyme preparation, for example iso-enzyme composition or the presence of other proteins. It is, therefore, important to consider the commercial source of the enzyme and the precise specifications of the biocatalyst employed. 

The multi-component enzyme system is likely to have much lower activity in enzyme preparations. 

For preparative purposes fermenting baker s yeast (Saccharomyces cerevisiae) is commonly used instead of a purified enzyme preparation. However, isolated pyruvate decarboxylates can also be used30. In this context, the most important substrate is benzaldehyde31 which is converted by n-glucosc fermenting yeast to (7 )-l-hydroxy-l-phenyl-2-propanone. This conversion has gained considerable industrial importance because ( )-l-hydroxy-1-phenyl-2-propanonc is an important precursor for the synthesis of (-)-cphedrin. 

Stimulation and inhibition of the enzyme by the GPCR-G-protein cycle occur by analogous mechanisms. Agonists induce hormone receptors to increase a Ga-GDP-GTP exchange and subsequent Ga 3y dissociation (GDP-a py + GTP GTP-ax + [3y + GDP) (Fig. 4). Consequently, agents that affect either the dissociation of either G or Gs, or the association of their respective as, a , or (3y subunits with adenylyl cyclase could affect rates of cAMP formation in enzyme preparations or in intact cells and tissues. There are several important examples. Gas is stably activated by poorly hydrolyzable analogs of GTP, e.g. GTPyS... 

The reaction was catalyzed by a partially purified enzyme preparation from a mutant of Bacillus pumillus (see the next subsection). From 1 g of D-glyceralde-hyde and 1 g of acetoin, 0.67 g of pure crystalline 1 -deoxy-D-r/treo-pentulose was obtained in one operation. The sugar was fully characterized. 

In these systems, solid enzyme preparations (e.g. lyophilized or immobilized on a support) are suspended in an organic solvent in the presence of enough aqueous buffers to ensure catalytic activity. Although the amount of water added to the solvent (as a rule of thumb <5% v/v) may exceed its solubility in that solvent, a visible discrete aqueous phase is not apparent because part of it is adsorbed by the enzyme. Therefore, the two phases involved in an organic solvent system are a liquid (bulk organic solvent and reagents dissolved in it) and a solid (hydrated enzyme particles). 

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

The mutagenic activity of the extracts was assayed using Salmonella typhimurium strain TA 1535, as described by McCann et al. (58). Ten ul of fish extract were spot-tested without addition of an S9-enzyme preparation (57). MNNG (20 ug/plate) was used as a positive control. 

The influence of enzyme maceration using pectinolytic enzyme preparations (Pectofruit and Pectofruit Press) on anthocyanin extraction at 43°C from two variants of black currant berries was studied. Enzymes accelerated the extraction yield the yield of anthocyanin extraction was similar for both enzymes and the duration did not influence the total content of released pigments. 

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