Is The Relationship Between Β-Galactosidase And Hydrochloric Acid

Main aim of experiment 5 is to use the biuret test to quantify the concentration of the Bovine Serum Albumin (BSA) then constructing a standard curve graph and to use the spectrophotometer to perform an enzyme assay using different concentration of the BSA. Experiment 5 also verifies the Beer-Lambert Law, which is the linear relationship between absorbance and concentration of an absorbing species. Absorbance formula is shown in fig. 1.1. However, the Beer-Lambert Law is not obeyed at high concentration as solution with high concentration may alter the refractive index of the solution which in turn may affect the absorbance reading. The limitation of the Beer-Lambert Law is shown by the non-linear part of the graph in fig 1.2.

The absorbance
readings of the BSA are taken from the spectrophotometer, which is an equipment that measures the amount of light of a specified wavelength which passes through a medium. Spectrophotometer is used to determine the concentration of a coloured solute in a solution in a laboratory by measuring the absorbency of light at a given wavelength. Example of industrial usage for spectrophotometer is the quality control for paper sheets.

Experiment 6 is the stopped enzyme assay where the study of β-galactosidase, which is a hydrolase enzyme, was conducted.
The main aim of the experiment is to study the effect of temperature and substrate concentration on enzyme activity, using the enzyme β-galactosidase to convert the substrate into its individual products, specifically its coloured product for the measurement of its absorbance reading as enzyme assay relies on measuring the increase in product. The substrate used is o-Nitrophenylgalactoside (ONPG) which produces galactose and a yellow coloured monomer, o-Nitrophenol (ONP), when it is being hydrolyzed. This experiment measures the absorbance reading of the coloured product, ONP, with the spectrophotometer set at 420nm to study the effect of temperature and substrate concentration on enzyme activity by measuring the absorbance reading of the coloured product monomer at the end of the
reaction.

O-Nitrophenylgalactoside (ONPG) is a replacement artificial substrate for lactose, it is colourless initially and ONPG will produce galactose and a yellow coloured monomer, ONP, after being hydrolyzed. This is an important property as the yellow coloured monomer can then be used as an indication of the reaction and also enable the study of the correlation of the absorbance reading of the enzymatic activity with temperature change.

β-galactosidase, which is more commonly known as lactase, is a hydrolases enzyme that hydrolyses its natural substrate, lactose, into its monomers which are glucose and galactose.
Because of this property, the food processing industry uses it to its advantage to break down lactose in milk for consumers that are lactose intolerant. Hydrolysis of whey also converts lactose into a very useful product like sweet syrup that can be used in various processes of dairy. The enzyme β-galactosidase can also be used in transglycosylation of lactose to synthesize galacto-oligosaccharides (GOSs) from whey lactose. GOSs is a nondigestible oligosaccharides which are not hydrolyzed in the upper intestinal tract, are however fermented selectively by beneficial intestinal bacteria which is a prebiotic
effect.

Experiment 7 is a continuous enzyme assay to study β-galactosidase. Its main aim is to conduct a continuous enzyme assay for the hydrolysis by β-galactosidase and also to study the rate of velocity of reaction with different substrate concentration by measuring the absorbance reading of the product. A spectrophotometer is used to measure the absorbance readings of the solution. Multiple absorbance readings are then taken down during the process of the hydrolysis, this enables the comparison of different substrate concentration and the rate of the reaction.

In the process of the assay, buffer solution is added to the solution. A buffered solution is preferred in any enzyme assay as most enzymes have an optimal pH where the reaction they catalyze is the fastest, thus buffer solution can ensure the constancy of the pH through the reaction and prevent any pH changes in the solution. In experiment 6, phosphate buffer solution of pH 7.2 is used, while in experiment 7 sodium phosphate buffer solution of pH 7.2 is used. This is then used to facilitate the reaction of the hydrolysis.