Introduction
An eEnzyme is kind of a protein and, like all proteins, proton is composed of a chain (or chains) made up of the 20 different amino acids found in most living organisms. The sequence of the amino acid chain(s) determines the 3-D shape of the protein, and its shape to a great extent determines its properties. and it has a 3-D shape. Enzymes act in aqueous solutions to facilitate used in biochemical reactions by lowering the energy barrier, but they are not themselves changed or consumed by the reaction. This is referred to as catalysis, and enzymes are biological catalysts. to make the reaction accrue but it don't consume by the reaction. this kind of reaction is called Catalysts. Enzyme activity is affected by physical-chemical parameters such as temperature, enzyme concentration and pH. Raising the temperature increases molecular motion and therefore makes it more likely an enzyme molecule will encounter other molecules (known as the enzyme’s substrates) with which it can carry out catalysis. Increasing the enzyme’s concentration has a similar effect. Enzymes work efficiently only within a limited pH range; outside that range, their activity rapidly falls off and they may undergo irreversible chemical changes, destroying their activity.There is some condition that effect on the enzyme activity like the temperature and the concentration. if the temperature increase the enzyme activity will increase too. That happens because the increase f molecules motion increase the chance of substrata and enzyme molecules encounters. Also, when the concentration increase the enzyme will increase and it happen because enzyme molecules will hydrolyze with the substrata molecules.
For these experiments, in this particular experience the enzyme used is bβ-galactosidase (bβ-galGz). will be used. It is found in cells as diverse as bacteria and humans where it functions by catalyzing the hydrolysis of β-galactosides into monosaccharides like, for example, splitting lactose (milk sugar) into its component monosaccharides, glucose and galactose.This kind of enzyme can be found on some bacteria and in the intestinal mucousal cells of in infant mammals. This enzyme used to hydrolyze some substances like lactouse ( the sugar milk) and the enzyme hydrolyze it to become gluctose. β-gal will also hydrolyse ortho-Nitrophenyl-β-galactoside (ONPG), a colorless compound that is converted into a yellow pigment by the catalytic activity of the enzyme and thus can serve as a specific indicator of β-gal activity. Also (b-gz) can be found in hydrolyzing the Ortho-nitrophenyl-glacto-pyranoside (ONPG) this is a yellow pigment substrata. .In the experiments described below, the enzyme reaction is carried out at physiological pH (around pH 7) and then base is added to raise pH and stop the reaction. in this experience allows the reaction to happen in physiological condition than base should be added to stop the reaction. That happen because the enzyme will not function well in physiological condition (pH 7).
Mmaterials:
in the effects of concentration experience this materials should be used. tThe enzyme was β-gal (b-Gz) that found in from Escherichia coli, the substrate was. a substrata ONPG, and the , reaction assay bufferwas carried out in phosphate buffer, pH 7. Sodium carbonate solution (pH around 11-12) was used to stop the reaction. All solutions were prepared in deionized water. ( Sadiume phosphate, pH7, Na2Co3 , solution and this solution must be prepared in deionized distill water. Other equipment consisted of18 plastic tubes, water proof marker, pip, pipetteit, electronic balance and, tissues to clean the tubes before measuring their absorbancem in a spectrophotometer.
Methods:
Reactions were carried out at room temperature. Before beginning the experiments, we measured the room temperature which . The room temperature was 23Cc the day of the experience. We used the enzyme at Use three concentrations: to 100%, 75%, and 50%. For each enzyme concentration, we used 6 tubes and set the reaction times of time 4, 8, and 12 minutes, measuring two tubes at each time point.for every two tubes. We usedIn this time at the 100% concentration use 1.00ml 100 µL (100%), 75 µL (75%) and 50 µL of the enzyme, and the substrata and for added Na2COo3 solution to stop for stopping the reaction at the appropriate time. In the 75% use 75ml for the same solution and in the 25% use 25 ml as well. The amount of ONPG converted into the yellow pigment by the enzyme was found by measuring the absorbance of the tubes at 420 nm (A420). For each concentration, we also measured and subtracted the A420 of a tube in which no reaction had taken place (blank). We measured the A420 of two tubes for each time point and calculated the average value. Also, measure the tubes at A420 and subtracted with the blank reagent that been found for every concentration. Finally find the average for every time you used in every concentration.
Discussion:
The A420 was proximity close for each time in every temperature and that was what should be expected as result for the experience. Also, the data shows and approved that when the consecrations increase the enzyme activity will increase too.The results show that the higher the enzyme concentration, the faster the reaction rate. This is the result expected (see Introduction)
