Investigating The Quality Of A New Batch Of ?í

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Introduction:

Most biological reactions require catalysts such as enzymes to accelerate the rate of the product formation. Enzymes are proteins that have highly specific and complex structures that enable them to catalyze reactions in a highly effective and regulated manner. One of the most important biological catalysts is β-galactosidase. β-galactosidase is essential in the breakdown of the substrate lactose. Lactose is a disaccharide found in milk. β-galactosidase catalyzes this catabolic reaction, breaking lactose into two monosaccharides; glucose and galactose. Hydrolysis is used to cleave the oxygen bridge connecting the two sides of the lactose molecule.

β-galactosidase is widely used in the food industry, especially in the dairy
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Therefore, to determine those various constants, kinetic enzymatic activity must be measured. The absorbance will be measured using a a Genesys 10S UV-VIS spectrophotometer which uses VISIONlite software.

Fortunately the substrate specificity of the enzyme is such that a variety of different substrates, each with a β-Dgalactopyranoside moiety, can be acted upon. Investigators have taken advantage of this by synthesizing compounds which when hydrolyzed by β-galactosidase result in a colored product. One such compound is o-nitrophenol-β-Dgalactoside
(ONPG), which when hydrolyzed forms galactose and o-nitrophenol (Figure 1).
Figure 1. Enzymatic hydrolysis of o-nitrophenol-β-Dgalactoside
(ONPG) to of o-nitrophenol (ONP) and galactose by β-galactosidase. β-galactosidase catalyses the hydrolysis of the terminal β-D-galacto-pyranoside moiety resulting in the formation of the yellow (λmax 420 nm) compound ONP.
The compound ONP absorbs light at 420 nm whereas the precursor molecule ONPG does not. Therefore, the increase in light absorbance at 420 nm can be used to monitor β- galactosidase when ONPG is used as a substrate.

Materials and Methods: Safety considerations:
• Eating and drinking are not permitted in the laboratory at any time.
• Lab coats, safety glasses and gloves must be worn.
• DO NOT wear contact lenses in the lab.
• All students must have gone through WHMIS training and
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