How Proteins May Be Refined By Controlled Enzymatic Hydrolysis

Most chemical reactions speed up as temperature is raised. As the temperature is raised, kinetic energy within the molecules increases as well to endure the reaction. Because enzymes are catalysts, meaning they make a chemical reaction react faster, enzyme reactions also tend to go faster with increase temperature. However, if the temperature of an enzyme-catalyze is raised to a temperature optimum, the kinetic energy in the water molecules and the enzyme becomes too high and the enzyme starts to denature, meaning the molecules become disputed. Most enzymes are denatured by around 104 degrees Fahrenheit to about 122 degrees Fahrenheit. Denaturing is disabling an enzyme by changing the normal qualities or the nature of it, for instance when

Background and Introduction: Enzymes are proteins that process substrates, which is the chemical molecule that enzymes work on to make products. Enzyme purpose is to increase the rate of activity and speed up chemical reaction in a form of biological catalysts. The enzymes specialize in lowering the activation energy to start the process. Enzymes are very specific in their process, each substrate is designed to fit with a specific substrate and the enzyme and substrate link at the active site. The binding of a substrate to the active site of an enzyme is a very specific interaction. Active sites are clefts or grooves on the surface of an enzyme, usually composed of amino acids from different parts of the polypeptide chain that are brought together in the tertiary structure of the folded protein. Substrates initially bind to the active site by noncovalent interactions, including hydrogen bonds, ionic bonds, and hydrophobic interactions. Once a substrate is bound to the active site of an enzyme, multiple mechanisms can accelerate its conversion to the product of the reaction. But sometimes, these enzymes fail or succeed to increase the rate of action because of various factors that limit the action. These factors can be known as temperature, acidity levels (pH), enzyme and/or substrate concentration, etc. In this experiment, it will be tested how much of an effect

As stated in the introduction, three conditions that may affect enzyme activity are salinity, temperature, and pH. In experiment two, we explored how temperature can affect enzymatic activity. Since most enzymes function best at their optimum temperature or room temperature, it was expected that the best reaction is in this environment. The higher the temperature that faster the reaction unless the enzyme is denatured because it is too hot. Similarly, pH and salinity can affect enzyme activity.

Proteins are the metabolic workhorses of the cell; they engage in a variety of essential activities ranging from enzymatically catabolizing macromolecular food sources to serving as structural components that maintain cell stability. Maximizing protein function relies on intricate non-covalent interactions occurring on the secondary, tertiary, and quaternary levels that help determine the overall shape of the protein. In their native states, proteins will assume the most energetically favorable configuration. Occasionally however, cells are exposed to exogenous disruptions such as heat stress. Heat Stress can compromise protein three-dimensional structure. Hydrophobic residues tend to be buried in the interior of the protein but when

Hydrolysis is a type of chemical reaction in which water is used to break bonds within a molecule. Since hydrolyzed proteins have been broken into smaller molecules they are able to absorbed by the hair strand filling in gaps and smoothing the cuticle layer. Smoother cuticles reduce the risk of knots and tangles being formed as the cuticle sheets are closed and so not able to snag unto other sheets from neighboring strands. Types of hydrolyzed proteins to look out for are hydrolyzed silk protein, hydrolyzed wheat protein and hydrolyzed soy

The use of the Protease Inhibitor, 1.0mM PMSF, resulted in the highest amount of Tyrosinase activity, which was 13.9 units per milliliter of latent enzyme. The Protease Inhibitor, 0.1mM PMSF was therefore, most effective at extracting Tyrosinase. The Protease Inhibitor, 1.0mM EDTA, resulted in the second highest amount of Tyrosinase activity, which was 12.9 units per milliliter. The Protease Inhibitor, 1.0mM EDTA was therefore the second most effective at extracting Tyrosinase. The use of the Protease Inhibitor, E-64, resulted in the third highest amount of Tyrosinase activity, which was 12.3 units per milliliter. The Protease Inhibitor, E-64, was therefore the third most effective at extracting Tyrosinase. The use of the Protease Inhibitor, Pepstatin, resulted in 9.9 units per milliliter of Tyrosinase activity. The Protease Inhibitor, Pepstatin, was therefore the least effective at extracting the enzyme Tyrosinase. The concentration of hydrogen ions, or as it is more commonly known, the pH, had a huge effect on the extraction of Tyrosinase. At a pH concentration of 5.5 and lower (pH = 3), the Tyrosinase enzyme activity was very low to nothing at all. At pH concentration of 6 to 8, the enzymatic activity of Tyrosinase greatly increased. It is known that every enzyme has its own optimum pH at which it performs at its best. According to the graph in this journal, it is observed that this optimum pH for the

Introduction: Enzymes are protein catalysts facilitating the conversion of substrates into products (Alexander and Peters, 2011). They go through a whole chemical reaction which starts off with the substrate and then ends up with a product. The only way this reaction can be adjusted or not even work is if they end up going through some sort of affect which only temperature and pH levels can do determining the environment. When enzymes are in an environment that is too acidic or alkaline, their chemical properties, sizes and shapes can become altered (Magher, 2015) Chemical modification of proteins is widely used as a too; to maintain a native conformation, improving stability (Rodriguez-Cabrera, Regalado, and Garcia-Almendarez, 2011) In this experiment, four trials were conducted and recorded every 15 seconds for 5 minutes in order to calculate the optimum levels and IRV.

Organisms cannot depend solely on spontaneous reactions for the production of materials because they occur slowly and are not responsive to the organism's needs (Martineau, Dean, et al, Laboratory Manual, 43). In order to speed up the reaction process, cells use enzymes as biological catalysts. Enzymes are able to speed up the reaction through lowering activation energy. Additionally, enzymes facilitate reactions without being consumed (manual,43). Each enzyme acts on a specific molecule or set of molecules referred to as the enzyme's substrate and the results of this reaction are called products (manual 43). As a result, enzymes promote a reaction so that substrates are converted into products on a faster pace (manual 43). Most enzymes are proteins whose structure is determined by its sequence of its amino acids. Enzymes are designed to function the best under physiological conditions of PH and temperature. Any change of these variables that change the conformation of the enzyme will destroy or enhance enzyme activity(manual, 43).

Trypsin. Trypsin (Cat: T4549, Sigma-Aldrich) from porcine pancreas was another protease enzyme used in this research. It was obtained in liquid (solution) form with an activity of 1485.9 U/ml. For our research purpose, we used trypsin in two different concentrations, the original (1485.9 U/ml) and diluted to 426.4

Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the

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

Enzymes, proteins that act as catalysts, are the most important type of protein[1]. Catalysts speed up chemical reactions and can go without being used up or changed [3] Without enzymes, the biochemical reactions that take place will react too slowly to keep up with the metabolic needs and the life functions of organisms. Catecholase is a reaction between oxygen and catechol [2]. In the presence of oxygen, the removal of two hydrogen atoms oxidizes the compound catechol, as a result of the formation of water [2]. Oxygen is reduced by the addition of two hydrogen atoms, which also forms water, after catechol is

The hydrophobic nature of sesame proteins limits its use in certain food formulation, particularly in fluids and beverages. Sesame is rich in sulfur containing amino acids and limited in lysine and contains significant amounts of oxalic (2.5%) and phytic (5%) acids (Kapadia et al., 2002), which are responsible for making certain minerals like calcium unavailable for absorption.

Over the years, it has become a known fact that health and nutrition are intricately linked. Not only do food nutrients supply the necessary biomolecules for various metabolic activities, but, in some cases, food nutrients are able to trigger certain desirable physiological responses in the body. Food proteins and hydrolysates thereof are amongst the most well studied bioactive molecules (Danquah and Agyei, 2012). Bioactive peptides have been defined as protein hydrolysates which, upon entry and absorption into the body, have the ability to induce certain desirable and physiologically measurable ‘hormone-like’ activities(Korhonen and Pihlanto, 2006). Some biological functions induced by these peptides include immunomodulatory, cytomodulatory, opiate, antihypertensive, antimicrobial, antithrombotic and metal-chelation activities (Möller et al. , 2008). As natural products of food origin, bioactive peptides have a huge potential in health-promoting functional foods and therapeutic products(Korhonen, 2009).However, this potential is not being realised as a result of certain bioprocess challenges. The lack of commercially-viable processes for large-scale

Denaturing RTA at 45 ◦ C did not make it a better substrate for degradation (Figure 3A). The use of Gdn-HCl-denatured methylated RTA and Gdn-HCl-denatured non-methylated RTA ensured that methylation was not the cause of the resistance to proteasomal degradation as it showed that there was no loss of protein (Figure 3C). Similarly, when RTA was denatured using an acid treatment it also showed little to no effect on proteasomal degradation. Taken together this suggests that denaturation of RTA, either by heat or acid treatment, does not make it a better substrate for proteasomal degradation.