Enzyme Assays Invertase Activity

Aqueous invertase solutions (soluble and immobilized enzyme) were incubated in acetate buffer (0.010 M, pH5.5) for 20,40,60,80, and 120 min at 30,37,40,45,50, and 55°C. After incubation at each specified time, both forms were assayed for residual activity according to the standard procedure (see Standard Assay for Measuring Invertase Activity). 

Application and Principle This procedure is used to determine the invertase activity of enzyme preparations from yeast Saccharomyces sp (Kluyveromyces). The assay is based on a 30-min hydrolysis of sucrose at 30° 0.1° and at pH 4.62. The degree of hydrolysis is determined by measuring the optical rotation of the solution with a polarimeter. 

Table-TV shows the effect ot fhe LMW fraction on the activity of some of these enzymes in vitro. Maltase, lactase and invertase were competitively inhibited at a concentration of 10 mg/ ml. When the effectsof a range of concentrations (2.5-20 mg/ml) of the LMW fraction were studied, it was revealed that the inhibition was not of the pure competitive type. Table V shows the effect of the HMW fraction. Low concentrations had to be used in the assays, as the intense brown color of this fraction interfered with the spectrophotometric measurements. In spite of this a strong competitive inhibition of lactase and of invertase was found. Maltase was also inhibited, and, to a lesser extent, even trehalase. a-Amylase from saliva was not affected at the concentration tested.

The activities of free and immobilized invertase for various substrate concentrations are plotted in a Lineweaver-Burk graph, from which Vmax and Km values were calculated (Fig. 9). The Km of free enzyme was 40.3 mM, while the apparent Km was 38.2 mM for the immobilized one. These similar Km values for both forms indicate that enzyme-substract interaction is not substantially altered after immobilization. However, the Vmax for immobilized invertase (0.0489 U/mL) was 35% higher than the Vmax for soluble invertase (0.0320 U/mL). Such a difference could be explained by the predominance of supramolecular aggregates (hexamer, octamer forms) guaranteed under the conditions of the immobilization assay. 

Enzyme solution. Powdered invertase obtained from yeast is available from mai r biochemical supply houses, such as Sigma Chemical Co., P.O. Box 14508, St. Louis, MO 63178. A sterile solution of invertase must be prepared with great care. All apparatus used to make up this solution must be sterilized prior to use. More than 1 L of distilled water is boiled for 10 min, covered with aluminum foil, and chilled in an ice bath. Then add a precisely weighed amount of invertase (approx. 5-10 mg) to water in a 1-L volumetric flask and make up to the mark. The enzyme solution should be kept tightly stoppered and chilled at all times. The correct invertase concentration is very sensitive to the specific activity of the enzyme as purchased it is necessary to carry out the standard assay and adjust the solution to an appropriate final concentration. 

Using the value of a determined above, the results of the standard assay made initially to check the enzyme activity, the assay in part C, and the given concentration of the enzyme stock solution in g L , calculate the specific activity of the enzyme— that is, the number of micromoles of sucrose hydrolyzed per minute per gram of enzyme present. (The specific activity of an enzyme preparation is of course a function of the purity of the enzyme. As inactive protein is removed from the preparation, the specific activity will rise. When the specific activity can no longer be increased by any purification method, a homogeneous enzyme preparation may have been achieved but proof of this depends on other criteria.) The exact chemical composition of invertase is still unknown, but its molar mass has been estimated at 100,000 g mol Combining this datum with your calculated specific activity, estimate the turnover number for the enzyme. 

Polarimetry is a simple and accurate method for determining optically active compounds. A polarimeter is a low cost instrument readily available in many research laboratories. The detector can be integrated into an HPLC system if separation of substrates and products of reaction is required. Invertase ((3-D-fructofurano-side fructohydrolase EC 3.2.1.26), a commodity enzyme widely used in the food industry, can be conveniently assayed by polarimetry (Chen et al. 2000), since the specific optical rotation of the substrate (sucrose) differs from that of the products (fructose plus glucose). 

Rubisco activity was measured by radiochemical method and enzyme quantity by immunorocket as in (3). Extracts for Sucrose-P-synthase were desalted on G-25 Sephadex column and the reaction was run as in (3), except that UDP glucose and Fructose 6 P were adjusted to 25 and 10 mM respectively.The spectrophotometric assay was used to measure ADPgIucose pyrophosphorylase (3). Sucrose synthase and neutral invertase were assayed as in (4). 

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