Classification And Discussion Of Esterification

The protein molecules in many foods provide the amino acid building blocks required by our own cells to produce new proteins. To determine whether a sample contains protein, a reagent called Biuret solution is used. Biuret solution contains copper ions. However, the chemical state of the copper ions in Biuret solution causes them to form a chemical complex with the peptide bonds between amino acids (when present), changing the color of the solution. Biuret solution is normally blue, but changes to pink when short peptides are present and to violet when long polypeptides are present.

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

Additionally, three separate Bradford samples were prepared under the same conditions with the exception of using mushroom protein extract (15-25 μL) in lieu of the BSA standard. The solutions were placed in a Thermo Scientific Genysys 20 spectrophotometer to collect their respective absorbance data at 595 nm. The data was compiled into Microsoft Excel 2017® to generate a standard curve using the absorbance data of the BSA solutions with their corresponding masses (μg). The standard curve was then used to generate a line best fit equation where Y was equal to the amount of protein present and X was equal to absorbance. This then made it possible to calculate the amount of protein (μg) present within the mushroom extract samples by simply plugging in their respective absorbance values as X. after determining the total amount of protein it was then possible to determine the concentration (μg/μL) of protein by dividing the total amount by its respective volume

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

Gelatin is observed to have a lower absorbance reading than lysozyme in only the Bradford assay, while in the BCA assay gelatin was observed to have about the same absorbance as lysozyme. In Bradford assay, the color yield for proteins with higher content of tryptophan, tyrosine, or cysteine residue, will be higher. The observed protein sensitivities did not correlates with the amino acid content in the Bradford assay. As presented in Table I, the mole fractions of lysine and arginine in BSA were 10 mole percent and 4 mole percent, respectively. The sensitivity should have been higher in BSA than in the Bradford assy. However, the observed sensitivities did correlates with the amino acid contents in the BCA assay.

The role of an enzyme is to catalyse reactions within a cell. The enzyme present in a potato (Solanum Tuberosum) is catechol oxidase. In this experiment, the enzyme activity was tested under different temperature and pH conditions. The objective of this experiment was to determine the ideal conditions under which catechol oxidase catalyses reactions. In order to do this, catechol was catalyzed by catechol oxidase into benzoquinone at diverse temperatures and pH values. The enzyme was exposed to its new environment for 5 minutes before the absorbance of the catechol oxidase was measured at 420 nm using a spectrophotometer. The use of a spectrophotometer was crucial for the collection of data in this experiment. When exposed to hot and cold temperatures, some enzymes were found to denature causing the activity to decrease. Similarly, when the pH was too high or low, then the catechol oxidase enzyme experienced a significant decrease in activity. It can be concluded after completing this experiment that the optimal pH for catechol oxidase is 7 and that the prime temperature is 20º C. Due to the fact that the catechol oxidase was only tested under several different temperatures and pH values, it is always possible to get a more precise result by decreasing the increments between the test values. However, our experiment was able to produce accurate results as to the

vulgaris plants, via the formation of a standard curve prepared using varying concentrations of bovine serum albumin (BSA) solution. Following absorbance readings of the various BSA solutions, they were plotted against their concentrations providing an indirect measure for determining protein concentrations of the plant samples within the assay tubes, and through further calculations the sample protein concentration. The mean protein concentration for the control group was calculated to be 3.34 ± 1.30 mg/mL, while the mean treated group concentration was 2.01 ± 1.26 mg/mL. These results similarly like the chlorophyll results correlate with the literature articles, as a reduced protein content within the Paraquat treated plants can be expected to some extent (Chia et al., 1981). This reduction in protein concentration is the result of those superoxide anions produced by Paraquat, disrupting the chloroplast membranes and allowing for intracellular components including some proteins to leak out, hence the decrease in protein concentration in comparison to the non-treated plants (Qian et al., 2009). A slight outlier may exist within the treated groups protein concentrations as one of the groups provided a negative value for protein concentration which is not valid, but even after exclusion of that data value, results are still supportive of the expected outcome. Though these results support the claim of Paraquat toxicity causing membrane deterioration and leakiness, protein concentration values are rather more purposeful when used to analyze malondialdehyde (MDA) values on per mg of protein

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

Catechol, in the presence of oxygen is oxidized by catechol oxidase to form benzoquinone (Harel et al., 1964). Bananas and potatoes contain catechol oxidase that acts on catechol which is initially colorless and converts it to brown (Harel et al., 1964). In this experiment, the effect of pH on the activity of catechol oxidase was conducted using buffers ranging from pH2 to pH10. Two trials were conducted due to the first trial results being altered by an external factor. The results were acquired by taking readings every 2 minutes for 20 minutes from a spectrophotometer and then recorded on to the table. The data collected in the table were then made into graphs to illustrate the influence of pH on the catechol oxidase catalyzed reaction. After analysis, the data revealed that pH did have a significant influence on the enzyme as recorded by absorbance per minute. However, the data was collected was not accurate due to external factors, thus the results are debatable and should be experimented again for validation.

The Effect of pH on the Digestion of Casein by Trypsin When planning the experiment, the equipment and method had to be well thought-out in order for the experiment to be accurate and efficient. Firstly, I have chose to use a 1% trypsin concentration then altered it to 0.8%, because a higher concentration means more trypsin molecules in the solution and therefore more enzyme substrate complexes are likely to occur with the casein in the milk, causing digestion of the casein to be faster. However, I don’t wantowever digestion to occur too quickly as I will not be able to analyse the effect of PH. Therefore, I chose a lower concentration which would allow me to test the percentage transmission at

The objective of this lab was to learn the principle of affinity chromatography by isolating a carbohydrate-binding protein from an extract of jack bean meal.

Based on the class data of the pH of phosphorylase reaction, the potato phosphorylase is reached the endpoint which the phosphorylase active at pH 6, it started active within 6 minutes. The optimal pH of phosphorylase is at pH 7 which active just within 4 minutes. At pH 6, it started to breaking down the starch primer +glucose-1-phosphase into starch + phosphate which reacted with the iodine test to formed the blue precipitate. At the optimal pH 7, it shows that it maximized its activity. Comparing the data of the pH of the potato phosphorylase reaction with the study published by Russell Pressey from Plant

GST- and MBP- tagged proteins were overexpressed at the similar levels, however, protein recovery in a 100.000g supernatant (soluble fraction) was very poor (data not shown). Additionally GST-tagged proteins were irreversibly sticking to the column which made us focus exclusively on the MBP-tag proteins. Temperature variations (160C – 370C), IPTG concentration (0.5mM – 1mM), duration of induction (3h – 18h), as well as the addition of various molecules (arginine, DTT, glycine) to the supernatant, were tested in a number of experiments to optimize the expression and recovery of recombinant proteins (data not shown). In short, protein expression at 160C with an induction with 0.5mMIPTG for 18h was chosen as optimal. Addition of 1M arginine to the homogenate was also shown to be beneficial for protein recovery, especially after freeze/though cycles. In Figure 1A a representative example of one such an experiment is shown.

In general, aminopeptidases are efficient in reducing the bitterness and play a vital role in enzymatic hydrolysis of protein. Bitter flavor of tryptic hydrolysate of casein was minimized after it was incubated with aminopeptidase from Erwinia ananas. The crude enzyme extract of L.casei (mixer of aminopeptidase, x-prolydipeptidyl peptidase, and proline-iminopeptidase) mixed with neutrase produced cheddar cheese without any bitter taste or bad flavor. Aminopeptidase T from Thermus aaquaticus YT-1 decreased the bitterness of casein hydrolysates by producing more free amino acids(Saha and Hayashi

Part A of this experiment examines the extraction and absorption of casein within milk. The foremost objective is to effectively remove the casein-rich curds from the milk by altering the pH using acetic acid. Then, the objective is to dry the curds so that only the proteins and various other materials remain. Afterwards, this part aims to evaluate the spectrum of copper (II) sulfate pentahydrate, then use this spectrum in comparison to a solution of Casein sodium potassium tartrate and NaOH in a copper (II) sulfate solution, also known as the biuret test. This denotes the presence of protein.1 The ultimate objective of this part is to characterize the protein Casein.