Enzyme purity

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). 

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

Different enzymes exhibit different specific activities and turnover numbers. The specific activity is a measure of enzyme purity and is defined as the number of enzyme units per milligram of protein. During the purification of an enzyme, the specific activity increases, and it reaches its maximum when the enzyme is in the pure state. The turnover number of an enzyme is the maximal number of moles of substrate hydrolyzed per mole of enzyme per unit time [63], For example, carbonic anhydrase, found in red blood cells, is a very active enzyme with a turnover number of 36 X 106/min per enzyme molecule. It catalyzes a very important reaction of reversible hydration of dissolved carbon dioxide in blood to form carbonic acid [57, p. 220],... 

Perhaps of first concern in determining the overall design of a particular assay is the actual method used for product identification (or for substrate depletion) per unit time. Many different methods have been utilized (e.g., radiometric, spectrophotometric, fluorometric, pH-stat, polarimetric, etc.) No matter which method is used, the product has to be clearly identified (or substrate, if substrate depletion is being measured). With stopped-time assays, it may be necessary to separate product(s) from substrate(s) prior to determination of the amounts of the metabolite(s) present (as well as demonstration that product(s) and substrate(s) are truly separated). If so, the investigator should be able to demonstrate that the assay procedure clearly measures true initial rates (see below). Closely related to these issues are concerns about purity (See Substrate Purity Enzyme Purity Water Purity, etc.) and stability (See Substrate Stability Enzyme Stability, etc.. If the components of the assay mixture are not stable over the time course of the experiment (or, if certain side reactions occur), then corrections have to be made in analyzing the rate behavior. 

PROGRESS CURVE INITIAL RATE CONDITION SUBSTRATE PURITY ENZYME PURITY WATER PURITY SUBSTRATE STABILITY ENZYME STABILITY MIXING TIME INITIAL RATE CONDITION QUENCHING EXPONENTIAL EXPONENTIAL BREAKDOWN Extended Debye-Huckel equation, DEBYE-HUCKEL TREATMENT EXTENSIVE PROPERTY EXTENT OF REACTION RATE OF CONVERSION... 

REAGENT PURITY SUBSTRATE PURITY WATER PURITY ENZYME PURITY Receptor complex,... 

It is usually difficult to express the enzyme concentration in molar unit because of difficulties in determining enzyme purity. Thus, the concentration is sometimes expressed as a unit, which is proportional to the catalytic activity of an enzyme. The definition of an enzyme unit is arbitrary, but one unit is generally defined as the amount of enzyme that produces 1 pmol of the product in 1 min at the optimal temperature, pH and substrate concentration. 

By international agreement, 1.0 unit of enzyme activity is defined as the amount of enzyme causing transformation of 1.0 gmol of substrate per minute at 25 °C under optimal conditions of measurement. The term activity refers to the total units of enzyme in a solution. The specific activity is the number of enzyme units per milligram of total protein (Fig. 3-23). The specific activity is a measure of enzyme purity it increases during purification of an enzyme and becomes maximal and constant when the enzyme is pure (Table 3-5). 

Several factors are critical for the accuracy and reproducibility of (3-glucan determination. As with any other analysis, some of these factors relate to the skills of the analyst. Parameters critical for (3-glucan analysis include the following discussed below sample homogeneity, particle size, enzyme purity, glucose standard, pipetting technique, and absorbance measurement. 

Subtilisin BPN was prepared through a series of protein purification steps applied to the fermentation broth. These steps included ultrafiltration ethanol precipitation DEAE (diethyl-aminoethyl) Tris Acryl batch anionic exchange SP (sulfopropyl) Tris Acryl column cationic exchange and, concentration with an Amicon stirred cell. The enzyme purity was determined to be -951 via spectroscopic assays that measure the ratio of active enzyme to total protein. In addition, purity was verified via HPLC and SDS-page (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 

The selection of the most suitable enzyme for a certain purpose mainly depends on its biocatalytic characteristics. Once a correct choice has been made, it is important to minimize the expenses associated with the enzyme use, as the economic feasibility of enzymatic processes is likely to depend on the cost of the enzyme production. In this context, several authors showed that the performance of various peroxidase processes was independent of enzyme purity [1,2], even suggesting that the crude enzyme was protected from inactivation [3, 4]. Microfiltration and subsequent ultrafiltration stages are sufficient to separate biomass and concentrate the enzyme for an economically viable operation [2, 5]. 

In 1971 and 1972 Halliwell and co-workers (21, 57) separated the T. viride cellulase into four fractions—Ci, C2, CM-cellulase, and cello-biase. It was suggested that the Ci enzyme was a cellobiohydrolase since the principal product of its action was cellobiose. Addition of cellobiase with the Cl enzyme permitted 70% solubilization of cotton after 21 days. No evidence of the enzyme purity was presented. 

Early steady state kinetic studies established techniques for monitoring the overall reaction and for determining substrate specificity. The most generally applicable method for determining steady state rates of the oxidases is O2 consumption. Oxygen electrode techniques (28) have now superseded earlier manometric methods. The enzyme preparations must either be completely free of catalase activity, as a result of high enzyme purity or addition of cyanide, or catalase must be added in amounts sufficient to prevent transient H2O2 accumulation. 

Fig. 1.—Solubility Test for Enzyme Purity. [Amount of protein dissolved in supernatant liquor, against amoimt of solid protein added for (a) a pure protein, and (b) an impure protein.]...

Functional tests, for enzyme purity, 286 Fungi, polysaccharides of, 367—417 2-Furaldehyde, 5-(hydroxymethyl)-, from cellulose on pyrolysis, 432 Furan, tetrahydro-, as solvent in lithium... 

Solubility test, for enzyme purity, 285 Solvation, effect on ring structure and conformation, 33 Solvents... 

Electronic absorption spectra of flavocytochrome 62 were first reported in 1942 (72). Since then, visible absorption has been an important tool for measuring enzyme concentration. The electronic absorption spectra for S. cerevisiae flavocytochrome 62 in both oxidized and reduced states are shown in Fig. 7. The sharp peaks at 557, 528, and 423 nm (reduced) are characteristic of a 6-type cytochrome. The ratio of the absorbances at 269 and 423 nm (reduced) provides a useful indication of enzyme purity (20). The absorption coefficients generally accepted as most accurate are listed in Table II. There are no significant... 

Fractionation of beef liver shows an aggregation of zinc as glutamic dehydrogenase activity to protein ratio reaches its maximum value. Simultaneously, the metal protein ratio of all other elements studied decreases with increasing enzyme purity. The zinc/protein and activity/protein ratios become maximal in the third crystallization. 

Regulatory assessments for enzymes used in biotransformation are not clearly stipulated. At present, food assessments of microbial enzymes are provided by AMFEP, which has suggested microbial enzyme purity and immobilization as given below. 

The purification of heparinase has been followed by SDS gel electrophoresis. The crude sonicate gave more than 20 major bands the HA purified enzyme, 3 major bands and the IEF purified enzyme, 2 major bands. A summary of the specific activities, protein recoveries, and enzyme purity obtained using our purification procedures is listed in Table I. 

The importance of enzyme purity has already been emphasized. When enzyme preparations are used that contain more than one enzyme acting on the substrate, or its degradation products, the results obtained will, at the best, be ambiguous and, more likely, completely meaningless. The same holds true when inadequately characterized enzymes are used. 

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