Enzyme Lab Report

An unknown bacterium was handed out by Dr. Honer. The appropriate tests were prepared and applied. The first procedure that was done was the gram stain. Under a microscope, if the gram stain is purple, the bacterium is gram positive, if the stain is red, it is gram negative. The next test was the fermentation tests for glucose, sucrose and

This lab was performed in order to discover the activity of the enzyme catecholase in different pH levels as well as its absorbance in differently concentrated solutions. A spetrophotometer was used to measure the absorbance of the enzyme catecholase in different pH solutions as well as to measure the absorbance of catecholase in solutions with different concentrations of potato juice and phosphate buffers. Absorbance of the enzyme catecholase was at an optimum level when pH was close to neutral. When pH was acidic or basic, the catecholase was less effective. Also, when there was a higher concentration of potato juice and a lower concentration of phosphate buffer, absorbance of the enzyme increased.

From the stock substrate solution of 2.5 mM, each group serially diluted at least one different substrate concentration for a total of four different substrate concentrations to be investigated: 1.25 mM, 1.0 mM, 0.75 mM, 0.25 mM. The enzyme concentration was kept constant at 2.0 mM while experimenting on the affect of varying enzyme concentration on the rate and product formation of ONP. Enough 2.0 mM enzyme solution was prepared in the previous part of the project to supply this assay. Using similar procedure to collect absorbance data as the first part, 0.5 mL of 2.0 mM enzyme concentration was placed into the cuvette and used to calibrate the spectrometer at 420 nm. Data was then started, with the immediate addition of 0.5 mL of varying substrate concentrations. Each varying substrate concentration was split between the team and run for a total of 10 minutes, with the exception of the 1.25 mM run. Upon completion, data from each varying substrate concentration was copied to a single Excel sheet and used to produce an absorbance vs. time graph, product formation vs. time graph, Michaelis Menten plot, and Lineweaver-Birk plot. This analysis was used to calculate the V0,Vmax, and Km for β-Galactosidase

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.

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

Enzymes react differently under different conditions and concentrations, being the most productive at the enzymes specific optimum condition and concentration. The enzyme sucrase, extracted from yeast, breaks down the complex sugar sucrose into the simple sugar glucose. Testing for sucrase’s optimum environment, multiple reactions were ran using varying amounts and concentrations of sucrose and sucrase at different pHs and temperatures. The product was then treated with Benedicts solution to visually observe what amount of glucose was present after the reaction was ran; negative results being little to no glucose present and positive results being

The products produced by the first reaction were used as a substrate for the second. In this case the enzyme used NADH, which resulted in the decreased absorbance due to the NADH oxidation to NAD+. In addition, the spectrophotometer was used as a measuring device to follow the change in absorbance of the NADH molecules at 340nm.

In the exercise # 2 we observed the effect of substrate concentration, enzyme concentration, pH and temperature on enzyme activity. All the data showed that once potato extract was added to catechol and water the reaction varied dependent on the level of catechol. As in

40 mL of a concentrated solution of sucrose was prepared at 200mg/mL. Using appropriate dilutions of the stock, 11 solutions including a control solution were made in plastic tubes. The enzyme reaction with sucrose was run in 2 mL volume at room temperature in water. The enzyme constituted half the volume of the stock solution. The substrate was added to the enzyme in order to start the reaction. Each reaction ran for 5 min after which 2 mLs of DNS reagent was added. The solution was boiled for 10 min and the results were read using a spectrophotometer.

The experiments involved PH buffers of different pH were added to potato juice, water, and the enzyme catecholase. The mixture was then subjected to spectrophotometer at a wavelength of 420nm taking the absorbance readings. In the second experiment, a phosphate buffer of PH 7.0 was used in different measures together with different measurement of potato juice and the enzyme catecholase then subjected to the spectrophotometer at a wavelength of 420nm. The data collected inform of table and analyzed using descriptive statistics such as line graph and later interpreted, showing that PH and enzyme concentration do affect the rate of enzyme reaction

The use of multiple test tubes and Parafilm was used for each experiment. Catechol, potato juice, pH 7 phosphate buffer, and stock potato extract 1:1 will be used to conduct the following experiments: temperature effect on enzyme activity, the effect of pH on enzyme action, the effect of enzyme concentration, and the effect of substrate concentration on enzyme activity. For the temperature effect on enzyme activity, three test tube were filled with three ml of pH 7 phosphate buffer and each test tube was labels 1.5 degrees Celsius, 20 °C, and 60 °C. The first test tube was placed in an ice-water bath, the second test tube was left at room temperature, and the third test tube was placed in approximately 60°C of warm water. After filling the test tubes with three ml of the

The purpose of this experiment was to record catalase enzyme activity with different temperatures and substrate concentrations. It was hypothesized that, until all active sites were bound, as the substrate concentration increased, the reaction rate would increase. The first experiment consisted of five different substrate concentrations, 0.8%, 0.4%, 0.2%, 0.1%, and 0% H2O2. The second experiment was completed using 0.8% substrate concentration and four different temperatures of enzymes ranging from cold to boiled. It was hypothesized that as the temperature increased, the reaction rate would increase. This would occur until the enzyme was denatured. The results from the two experiments show that the more substrate concentration,

The purpose of this lab report is to investigate the effect of substrate concentration on enzyme activity as tested with the enzyme catalase and the substrate hydrogen peroxide at several concentrations to produce oxygen. It was assumed that an increase in hydrogen peroxide concentration would decrease the amount of time the paper circle with the enzyme catalase present on it, sowing an increase in enzyme activity. Therefore it can be hypothesised that there would be an effect on catalase activity from the increase in hydrogen peroxide concentration measured in time for the paper circle to ride to the top of the solution.

In this lab or experiment, the aim was to determine the following factors of enzymes: (1) the effects of enzymes concentration the catalytic rate or the rate of the reaction, (2) the effects of pH on a particular enzyme, an enzyme known and referred throughout this experiment as ALP (alkaline phosphate enzyme) and lastly (3) the effects of various temperatures on the reaction or catalytic rate. Throughout the experiment 8 separate cuvettes and tubes are mixed with various solutions (labeled as tables 1,3 & 4 in the apparatus/materials sections of the lab) and tested for the effects of the factors mentioned above (concentration, pH and temperature). The tubes labeled 1-4 are tested for pH with pH paper and by spectrophotometer, cuvettes 1a-4a was tested for concentration and cuvettes labeled 1b-4b was tested for temperature in four different atmospheric conditions (4ºC, 23ºC, 32ºC and 60ºC) to see how the enzyme solution was affected by the various conditions. After carrying out the procedures the results showed that the experiment followed the theory for the most part, which is that all the factors work best at its optimum level. So, the optimum pH that the enzymes reacted at was a pH of 7 (neutral), the optimum temperature that the reactions occurs with the enzymes is a temperature of 4ºC or

To prevent fluctuation in the pH, a solution known as a “buffer solution” was used in the experiment. Buffer solutions are mixtures of at least two chemicals which counteract the effect of acids and alkalis. Therefore, when a small quantity of alkali or acid solution is added the pH of the enzyme doesn’t change.