In this study, a three step purification of alkaline phosphatase from non-pasteurized milk was reported. It included cream extraction, n-butanol treatment and acetone precipitation. Different parameters like buffer concentration, temperature, pH, substrate concentration, acetone and n-butanol treatment were optimized to maximize the enzyme activity. The enzyme was fruitfully purified upto homogeneity from the milk, with percentage recovery and fold purification of 56.17 and 17.67 respectively. The kinetic parameter were determined to be 0.927 (Km) and 55.86 (Vmax), with specific activity of 11.31 U/mg. Other optimized parameters were estimated as buffer concentration of 0.5 M with pH 9.0, temperature optima at 37ºC, with n-butanol and acetone concentration of 20% (v/v) and 50% (v/v) respectively. This approach provides a simple and effective method for the purification of alkaline phosphatase from non-pasteurized milk.
INTRODUCTION
Alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) is a non-specific monoesterases that catalyze the hydrolysis of various phosphate esters and anhydrides of phosphoric acid, under alkaline conditions (Junior et al. 2008). They are the non-specific phosphatases which are apparently ubiquitous in nature. They are widely used in recombinant DNA technology (Kopetzki et al. 1994) and DNA sequencing. It is also an important component of enzyme-linked immunosorbent assay (ELISA) based kits. So it’s purification on large scale is required for different commercial and research purposes. The enzymes have been isolated from different sources, which include bacteria, fungi, organs of mammals and invertebrates, but a little have been reported in plants. Commercially available alkaline ...
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Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
The affects of pH, temperature, and salt concentration on the enzyme lactase were all expected to have an effect on enzymatic activity, compared to an untreated 25oC control. The reactions incubated at 37oC were hypothesized to increase the enzymatic activity, because it is normal human body temperature. This hypothesis was supported by the results. The reaction incubated to 60oC was expected to decrease the enzymatic activity, because it is much higher than normal body temperature, however this hypothesis was not supported. When incubated to 0oC, the reaction rate was hypothesized to decrease, and according to the results the hypothesis was supported. Both in low and high pH, the reaction rate was hypothesized to decrease, which was also supported by the results. Lastly, the reaction rate was hypothesized to decrease in a higher salt concentration, which was also supported by the results.
The Effect of Temperature on the Activity of Rennin in Milk Aim: To find out what effect different temperatures have on the enzyme, rennin, in milk. Introduction An enzyme is a biological catalyst. It speeds up a reaction by lowering the activation energy required to start the reaction. It speeds up a reaction, but remains unchanged unless certain limiting factors are introduced.
While the tube for specimen Cb turned a tannish white in the lower half of the tube while the top stayed the lavender inoculated tube color. Do to this evidence I determined that both specimens Ca and Cb cannot use the process Casein hydrolysis or Casein coagulation due to lack of soft or firm curds in both tubes. Since there was no casein curds formed, I concluded that specimens Ca and Cb also cannot perform the process of proteolysis. My conclusion is supported by the fact that there was no clearing of the medium. I have also determine that neither of my organisms can make the enzymes rennin, proteolytic or even proteases. I know my specimens cannot produce proteases due to the fact that there was no blue coloring in the tubes which means that the byproduct Ammonia was not produced to increase the pH. Since neither of my specimens can make these enzymes, I concluded that my specimens cannot break down lactose or casein. Although I did learn that specimen Cb can reduce litmus due to the evidence that the lower part of the tube turned a tannish white color with a purple ring at the top. This color change from a purple to a white means that the litmus was reduced turning it clear and leaving the white of the milk to show. Finally I know that specimen Ca cannot reduce litmus due to the fact that the tube had no change in
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
Alkaline Phosphatase (APase) is an important enzyme in pre-diagnostic treatments making it an intensely studied enzyme. In order to fully understand the biochemical properties of enzymes, a kinetic explanation is essential. The kinetic assessment allows for a mechanism on how the enzyme functions. The experiment performed outlines the kinetic assessment for the purification of APase, which was purified in latter experiments through the lysis of E.coli’s bacterial cell wall. This kinetic experiment exploits the catalytic process of APase; APase catalyzes a hydrolysis reaction to produce an inorganic phosphate and alcohol via an intermediate complex.1 Using the Michaelis-Menton model for kinetic characteristics, the kinetic values of APase were found by evaluating the enzymatic rate using a paranitrophenyl phosphate (PNPP) substrate. This model uses an equation to describe enzymatic rates, by relating the
Since the beginning of time, people have been drinking milk. Even today you will find a gallon of milk in almost every refrigerator in America. Milk is, and has always been, a staple of our diet. Because it contains essential proteins, carbohydrates, fats, minerals and vitamins, milk is considered one of nature's perfect foods. Unfortunately, throughout the last century milk has been subjected to many forms of modern processing practices, which deprive milk from many of its natural qualities and benefits. Therefore many essential vitamins and enzymes are lost. Processing milk has altered one of nature’s perfect foods and changed it into something nature did not intend. Because of the abundant health benefits in raw milk, this report will explain why it should be made legal for consumers to buy throughout the United States.
The idea of pasteurizing milk bagan in the 1920s, and later became an aspect of everyday life in the 1950s. Milk that has undergone this process is normally prefered since it is sterilized, therefore lowering the chance of human illness. However, it’s not the 1950s anymore, and the idea of pasteurizing milk has lost its luster for the people that now prefer raw milk. Unlike the milk that most Americans consume, raw milk has not been pasteurized, or quickly heated to a high temperature to kill harmful bacteria. In raw milk, these bacterias haven’t been removed, leaving people at risk. E. Coli, salmonella, and listeria are only some of the bacteria that raw milk carries, all of which can cause sickness, or even death. Common affects of consuming raw milk are diarrhea, stomach cramping, and vomiting, but it's the rare ones: kidney failure,paralysis, and death that causes raw milk to be illegal in half of the states and illegal to carry over state lines in its final form. Nevertheless, people still actively seek out and consume raw milk because they believe its nutritional values to be greater. Controversies surround this topic on whether organic food
By comparing the data from both the β-galactosidase activity assay experiment and the complementation experiment, we can determine the likely gene encoded in the lac operon, which had been mutated by UV exposure, and thus prevent lactose usage, in each of the two mutants.
In order to identify the presence of lipids in these samples, we use the Sudan IV solution. If the unknown A, B, C milk samples turn from clear to dark pink color
From looking at the results I can conclude that when the pH was 3 and 5. No oxygen was produced, therefore no reactions were taking place. This was because the pH had a high hydrogen ion content, which caused the breaking of the ionic bonds that hold the tertiary structure of the enzyme in place of the syringe. The enzyme lost its functional shape.
Milk Milk is an almost complete food. It consists of proteins (mainly casein), salts, fat and milk sugar, or lactose. It also contains vitamins A, C, D, certain B vitamins, and small amounts of others. Factors That May Affect The Reaction Ø The concentration of Rennin Ø
What Makes Human Milk Special? (Mar-Apr 2006). New Beginnings Vol. 23 No.2 , pp 82-3.
Purpose: The purpose of this lab is to explore the different factors which effect enzyme activity and the rates of reaction, such as particle size and temperature.
Since when does sexy conduct healthier and better milk? The two print ads that I am introducing to you is a milk from the Coca-Cola Company. The milk is called Fairlife and comes in different flavors: 2% reduced fat, 2% chocolate, fat free, and whole. Their tag line is “Believe in a better milk”.