Enzyme Assays Lactase Activity

Application and Principle This procedure is used to determine the neutral lactase activity of enzyme preparations derived from Kluyveromyces marxianus var. lactis and Saccharomyces sp. The assay is based on a 10-min hydrolysis of an o-nitrophenyl-fl-D-galactopyranosidc (ONPG) substrate at 30.0° 0.1° and at pH 6.50. [Pg.911]

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.

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

Calculation for NLU Activity One Neutral Lactase Unit (NLU) is defined as that quantity of enzyme that will liberate 1.30 (imol/min of o-nitrophenol under the conditions of the assay. Calculate the activity of the enzyme preparation taken for the analysis as follows ... [Pg.913]

The method is proposed to be applicable to solutions of lactase between 2000 and 5000 units/g, where one enzymatic unit is defined as the quantity of enzyme that liberates 1.30 pmol o-nitrophenol/min under assay conditions. Lactase decomposes the artificial substrate o-nitrophenyl-p-D-galactopyranoside into o-nitrophenol and galactose. Absorbance of o-nitrophenol (yellow in alkaline medium) is measured to estimate the enzymatic activity, at 420 nm. [Pg.340]

This is actually a very convenient method for determining activity of such class of enzymes, since organic coenzymes (i.e. FAD or NADH) are usually very easy to determine analytically. An example of a coupled system considering coenzyme determination is the assay for lactase ((3-galactosidase EC 3.2.1.23). The enzyme catalyzes the hydrolysis of lactose according to ... [Pg.11]

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