Enzyme assays are performed to serve two different purposes: (i) To identify a special enzyme by proving its presence or absence in a distinct specimen. (ii) To determine the amount of the enzyme in the sample by monitoring the disappearance of substrate or appearance of product. Enzymes speed up reaction rate by decreasing the activation energy required to start the reaction. Activation energy is the energy required to break certain bonds in the substrate so that other bonds can form. The formation of these new bonds results in the formation of the product by measuring the changes in absorbance due to the substrate (starch) being changed into product by the amylase enzyme.
After isolation of DNA from the source it is digested enzymatically with the help of restriction endonucleases. Enzyme treated DNA is then separated by size in an agarose gel and shifted to a membrane. A radioactive or fluorescently labeled probe is bonded with the DNA on the membrane. They target specific sequences that are marked by the restriction enzyme sites. The size of these fragments varies hence generate a biological bar code of restriction enzyme- digested DNA fragments.
They are used both within and outside the cells as biological catalysts to increase the rate of biological reactions. They increase the rate of reactions by reducing the amount of activation energy; hence the barrier to a reaction is lower when an enzyme is being used. Enzymes have an active site to which specific substrate binds. The combination of enzyme and substrate creates a new energy outline with a lower amount of activation energy (Lesson 6.5 A Catalyst and the Rate of Reaction). Once a product has been formed, they leave the active site of the enzyme hence more reactions can occur.
1.1 Abstract The purpose of quantitative analysis of protein using a spectrophotometer is to measure the concentration of proteins in a given sample. The experiment is conducted by laboratory method (Biuret Test) and using spectrophotometer to analyze the absorbance of reactants at 540 nm, hence determining the concentration of the proteins in a given sample. The purpose of stopped enzyme assay to study B-galactosidase is to determine the effect of temperature and concentrations of substrate on enzyme activity. B-galactosidase breaks down the disaccharide lactose into simple sugars glucose and galactose. However, glucose is a colorless compound hence it has to be substituted with a compound that is detectable by a visible color change.
This organism was cultured under solid-state fermentation for 72 h using wheat bran as the substrate. After 72 h, crude enzyme was extracted from the culture medium. The fibrinolytic enzyme was purified from the crude sample by various steps: ammonium sulfate precipitation, dialysis, ion exchange chromatography, and casein-agarose affinity chromatography. All purification steps were performed at 4°C until otherwise stated. The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min).
Turel and Patil (1996)  have established a rapid and selective method for the extraction of molybdenum with malachite green into nitrobenzene. The influence of solvent extraction variables on molybdenum extraction such as effect of pH, time of equilibration, solvents, effect of various anions and cations have been studied. On the basis of substoichiometric extraction method the constituent ratio of the metal-organic complexes was found as 1:1. The slope ratio method was also in agreement with the
The Cu1+ then react to bicinchoninic acid assay forming a purple water soluble complex. Moreover, the total volume of protein concentration can be measured by the colorimetric technique which will change the colour of sample solution from green to purple in proportion to protein concentration. SDS-PAGE electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis) is probably the most common analytical technique widely used to separate biological molecules, usually a nucleic acid or protein based on their electrophoretic mobility. The motility is a function of conformation, the length of their peptide chain and charge of the molecule. Depending on their size, small biomolecules move faster and more easily fit through the pores in the gel than larger ones.
Without a catalyst a reaction will take a long period of time to be broken down. Only a small amount of catalyst is needed to increase the rate of reaction. All Enzymes are proteins; they are a substance produced by a living organism which act as a catalyst. They are found in all living cells where they help speed up the chemical reactions. Enzymes are also proteins that are folded into complex shapes
The third method is stab culture. Escherichia coli and Bacillus subtilis are grown by stabbing vertically into nutrient gelatine in a test tube. Gelatine which contains Escherichia coli remains solid while the gelatine that contains Bacillus subtilis turns into liquid. This shows that Bacillus subtilis produces an enzyme called gelatinase which will turn the solid gelatine into liquid form while Escherichia coli do not produce gelatinase. The last method is plate culture whereby Escherichia coli are grown by streaking on agar plates in Petri dishes.
Enzymes are homogeneous biological catalyst that work by lowering the activation of a reaction pathway or providing a new pathway with a low activation energy. Enzymes are special biological polymers that contain an active site, which is responsible for binding the substrates, the reactants, and processing them into products. As is true of any catalyst, the active site returns to its original state after the products are released. Many enzymes consist primarily of proteins, some featuring organic or inorganic cofactors in their active sites. However, certain ribonucleic acid (RNA) molecules can also be biological catalysts, forming ribozymes.