Enzyme solution

Mix 3 g. of starch well with loml. of water in a test-tube and pour the mixture into 90 ml. of boiling water contained in a 300 ml. conical flask, stirring at the same time. Cool to about 70 and then place in a water-bath maintained at 65-70 , but not higher. Now add 2-3 ml. of the malt extract prepared as above, mix well and allow the hydrolysis to proceed. Take a series of test -tubes and in each put 10 ml. of water and 2 drops of a 1 % iodine solution. At intervals of about 4 minutes (depending upon the activity of the enzyme solution), remove 1 ml. of the reaction mixture, cool and add it to one of the test-tubes and note the colour obtained. At the beginning of the experiment the colour will be blue due to the starch alone. As the reaction proceeds, the colour gradually becomes violet, reddish, yellowish and finally colourless. 

To this yellow solution add the enzyme solution, mix well and allow to stand at room temperature. The mixture becomes red as the pH rises to 8. 

With the aid of a small pipette or a fine-bore dropping-tube (Fig. 30, p. 60), add about 4 drops of the filtered enzyme solution to the amine acetate solution. Using another dropping-tube add i drop of 20 volume hydrogen peroxide solution and shake well. Note the colour change which takes place. 

Repeat one or two of the above experiments using however, in place of the active enzyme, a sample of the enzyme solution which has been thoroughly boiled for 2 minutes. Note that the above colour changes do not now occur. 

The enzyme can be immobilized on the electrode by several techniques (53). The simplest method, first used in 1962, is to trap an enzyme solution between the electrode surface and a semipermeable membrane. Another technique is to immobilize the enzyme in a polymer gel such as polyacrylamide which is coated on the electrode surface. Very thin-membrane films can be obtained by electropolymerization techniques (49,54,55) using polypyrrole, polyindole, or polyphenylenediamine films, among others. These thin films (qv) offer the advantage of improved diffusion of substrate and product that... 

Enzymatic determinations of the detection limit where the chromatograms are first sprayed with an enzyme solution Then after appropriate incubation the enzymatically altered components are detected by reaction with a suitable reagent... 

The susceptibility of biological systems, including procaryotic and eucaryo-tic cultures and enzyme solutions, to the forces prevailing under normal processing conditions has been extensively studied and is the subject of comprehensive reviews [12-16], including other chapters in this volume. Downstream processing operations, as well as routine pumping, will expose cell suspensions... 

Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HCl buffer (pH 8.0) with ImM CaCl2, were incubated at 37 C for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms Ipmol of 4,5-unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M cm [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37°C. Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HCl buffer (pH 8.0) with 0.5 mM CaCl2. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min under the conditions of reaction. Specific viscosity (n p) is (t/to)-l, where t is the flow time (sec) of the reaction mixture and t is the flow time of the buffer. The inverse pecific viscosity (n p ) is proportional to the incubation time and the amount of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. 

Fig. 12. On-line enzyme reactor system designs, merging stream system (Top) and immobilized-enzyme reactor system (Bottom). A = mobile phase, B = enzyme solution...

Enzyme solutions can be stabilized using sugars, polyhydric alcohols, polymers, or salts [65]. These compounds affecting the enzyme stability are ligands (substate, product, inhibitor, coenzymes) or nonspecific additives. 

Add 1 part enzyme solution to 5 parts aspirated material. 

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The final step is a hydrolyzing step with sulfatase enzymes (E.C. number 3.1.6.1), such as limpet sulfatase, Aerobacter aerogenes sulfatase, Abalone entrail sulfatase, or Helixpomatia sulfatase. This step was suggested to be carried out in a CSTR or fluidized bed reactors, with counter-current flow between the aqueous and the oil phase. A more efficient removal of the sulfate into the aqueous stream is expected to occur in this cross-flow manner. A final separation of the reacting mixture was suggested to obtain sulfur-free product and aqueous enzyme solution for recycle. 

Dissolve 18 mg of HRP in 0.1M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 20-30mg/ml. The more highly concentrated the enzyme solution, the more efficient will be the modification reaction. For conjugating smaller quantities of enzyme and antibody, proportionally decrease the amount of the reagents used, while attempting to maintain the same relative concentrations in solution. 

In a fume hood, add 10 pi of 25 percent glutaraldehyde (Sigma) per ml of antibody/enzyme solution. Mix well. 

Immediately add 100 pi of the sodium periodate solution to each ml of the enzyme solution. This ratio of addition results in an 8mM periodate concentration in the reaction mixture. Mix to dissolve. Protect from light. 

Mix the antibody solution with the enzyme solution at a ratio of 1 1 (v/v). Since an equal mass of antibody and enzyme is present in the final solution, this will result in a 3.75 molar excess of HRP over the amount of IgG. For conjugates consisting of greater enzyme-to-antibody ratios, proportionally increase the amount of enzyme solution as required. Typically, molar ratios of 4 1 to 15 1 (enzyme antibody) give acceptable conjugates useful in a variety of ETISA techniques. 

Add 25 pi of the SATA stock solution to each ml of lOmg/ml enzyme solution. For different concentrations of enzyme in the reaction medium, proportionally adjust the amount of SATA addition however do not exceed 10 percent DMSO in the aqueous reaction medium. 

Dissolve alkaline phosphatase at a concentration of lOmg/ml in 0.1M sodium bicarbonate, 3M NaCl, pH 8.5. Dialyze against this solution if the enzyme is already dissolved in another buffer. This protocol requires at least 0.4 ml of the enzyme solution. 

Negatively charged species such as carboxylic acid group in acid-treated CNTs can attract positively charged enzymes from solution as long as the pH value of the enzyme solution is controlled to be lower than the iso-electric point of the enzyme thus, multilayer films of the enzyme can be formed by the layer-by-layer technique. For example, five layers of GOx can be immobilized on the electrode surface by alternatively dipping a poly(diallyldimethylammonium chloride (PDDA))-functionalized GC into a CNT solution and a GOx solution (pH 3.8). Figure 15.15 illustrates the preparation process for the formation of a multilayer film of GOx on the electrode. 

In addition to the aforementioned quantitative methods, the activity of pectic depolymerases is often identified by the cup-plate method.113 Cups are cut out from solidified agar containing the substrate, and are filled with the enzyme solution. After elapse of a cer-... 

Other authors (e.g., G10, W18) define the unit as micromoles X 10-3 NADPH2 (or NADH2) formed per 1.00ml enzyme solution diluted by the indicated test volume per hour, or as the quantity of enzyme in the test system reducing 0.01 pinole NADP (or NAD) per minute at a given temperature. Furthermore, it has been suggested that the unit be defined as the number of micromoles of substrate converted per milliliter of serum per one hour at room temperature. 

Since the enzyme solution used for the assay consists of different media (e.g., blood, serum, urine, cerebrospinal fluid, tissue homogenate, saliva), the activity must be related to different volumetric or gravimetric units. 

The variability of these correlation systems necessitates the use of an exact definition in expression of units. In the present paper all activity units will be defined as recommended by IUPAC, i.e., one unit equals the substrate conversion in micromoles per minute at 25° C per milliliter enzyme solution unless indicated otherwise. The referred data given in the literature have been recalculated for these units wherever possible. 

Their specimen of cholinesterase was prepared from horse serum by the method of Stedman and Stedman,1 and the method of estimation was that of Ammon.2 The enzyme solution was placed in the right-hand flask of a Barcrofb manometer, in a total volume of 3 ml. of 0-2 per cent NaHC03 solution the gas phase was 5 per cent C02 in Na. The reaction, carried out at 20°, was started by adding a solution containing 2 mg. of acetylcholine chloride. The C02 output was usually linear until about 100 fi. had been produced. 

Reversibility. It is known that the effect of eserine on cholinesterase can be completely reversed by prolonged dialysis against water. On the other hand, it proved impossible to obtain any reversal of the poisoning by the phosphorofluoridate esters (see table below). The enzyme solution (5 ml.) was treated with the inhibitor for 15 min. at 38° 1 ml. was used at once for activity estimation, and the remainder dialysed against running water for 24 hr. in the case of the eserine experiment, 36 hr. in the others. It was clear that the combination between the phosphorofluoridate esters and the enzyme is much firmer than that between eserine and the enzyme. 

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