Enzyme Kinetics of Beta-Galactosidase

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Abstract
This experiment is to study and measure the enzyme activity of β-galactosidase in the different concentrations of o-Nitrophenylgalactoside (ONPG) using a spectrophotometer. The spectrophotometer was also set at 420nm, a wavelength which is best for recording the absorbance values for the experiment. From the results, 0.9mM ONPG solution has the highest absorbance and 0.1mM ONPG solution has the least. Also, 0.5mM ONPG solution has the highest rate of enzyme activity and it is the most efficient as the enzyme activity of the ONPG solution continues even though the other concentrations of ONPG solution has already stopped the enzymatic reactions as the substrate is already used up.

Introduction
This experiment is to study and…show more content…
However, the rate of reaction only increases for a certain period of time until there is lesser substrate molecules than the enzyme molecules. The increase of enzyme concentration does not have effect if there are lesser substrate molecules than enzyme molecules initially.

A spectrophotometer is an instrument which measures the amount of light of a specified wavelength which passes through a medium. This instrument is usually used for the measurement of reflectance of solutions. Light is separate into different wavelengths and is being passed through the sample solution. The sample solution will have its own wavelength and will absorb a certain amount of light. The higher the molecular concentration, the higher the absorbance value.

Materials and methods
Materials needed for Experiment 6:
Samples: 0.1mM, 0.5mM, 0.7mM, 0.9mM and 1.0mM o-Nitrophenylgalactoside (ONPG) solutions
Reagents: Grade 6 β-galactosidase (SIGMA), sodium phosphate buffer pH 7.3
Apparatus: Spectrophotometer (UV-1201), cuvettes, water bath (set at 37°C), 200µl and 1000µl micropipettes and test tube
Methods for Experiment 6: 1. 5 different concentrations of ONPG solutions was provided. 2. The different concentrations of ONPG solutions and buffer solutions were incubated in the water bath at 37°C for 5 minutes. 3. The spectrophotometer was set at 420nm. Distilled water was also used as the ‘blank’. 4. 200µl of ONPG solutions, which is the
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