Tannase 's Protein Purification : The Cloning, Expression, And Purification Of Tannase Enzyme Obtained From Bacterium L

40 mL of a concentrated solution of sucrose was prepared at 200mg/mL. Using appropriate dilutions of the stock, 11 solutions including a control solution were made in plastic tubes. The enzyme reaction with sucrose was run in 2 mL volume at room temperature in water. The enzyme constituted half the volume of the stock solution. The substrate was added to the enzyme in order to start the reaction. Each reaction ran for 5 min after which 2 mLs of DNS reagent was added. The solution was boiled for 10 min and the results were read using a spectrophotometer.

The extent at which environmental factors affect the rate of catalase activity was discovered in this lab. The assay system, in which a filter paper disc was dipped into the enzyme and submerged using a stirring rod in a test tube filled with 20mL of hydrogen peroxide, was used to test several enzyme factors. As the saturation of hydrogen peroxide increased the rate of reaction increased as well. When the enzyme concentration increased the rate of catalase activity increased too. When catalase was subjected to an increase of temperature changes, the rate of reaction increased. Once the protein denatured around 100ºC the catalase activity decreased. Catalase operated with a high efficiency

The purpose of this experiment was to record catalase enzyme activity with different temperatures and substrate concentrations. It was hypothesized that, until all active sites were bound, as the substrate concentration increased, the reaction rate would increase. The first experiment consisted of five different substrate concentrations, 0.8%, 0.4%, 0.2%, 0.1%, and 0% H2O2. The second experiment was completed using 0.8% substrate concentration and four different temperatures of enzymes ranging from cold to boiled. It was hypothesized that as the temperature increased, the reaction rate would increase. This would occur until the enzyme was denatured. The results from the two experiments show that the more substrate concentration,

The results recorded in (table 30) and (figure 32) Indicated that Cu+2 activated the enzyme at 0.01M concentration by 1.2 fold and the activity gradually decrease by increasing the metal concentration to 0.1M with activity 51.29U/mg protein. Na+ ion activate the enzyme when added with 0.1M by

Restriction digest involves the use of restriction enzymes (also known as restriction endonucleases) to locate specific base pair sequences in DNA. These enzymes cut, or cleave, DNA only at their designated sequence, which is referred to as a recognition sequence. While there are four different types of restriction enzymes (1), the only type that was worked with in the following experiment were type II restriction enzymes (2). These enzymes have recognition sites that are mostly palindromic and usually consist of around four to eight base pairs. They also require only magnesium (Mg2+) as a cofactor to operate. Cofactors are molecules that bind to enzymes in order to activate them (3). Additionally, they cut DNA only at, or very near to, their specified restriction site, unlike other types, which cleave at various distances from their recognition site (1). The restriction enzymes that will be used in following experiment are Hind III, PVU II, and Bgl I (2). Hind III recognizes and cuts DNA at the sequence AAGCTT. It is isolated from Haemophilus influenzae (4), which is a bacteria that is the cause of several diseases, including pneumonia, and meningitis. (5) When Hind III is used to cleave DNA, the end result will have “sticky ends,” which means that there will be a few unpaired nucleotide bases on each end of

The results of the three-part experiment provide a deeper knowledge about the factors that influence the rate of the reaction of the enzyme activity and how the factors influence the structure or function of the enzyme.

Significance: One of the most popular techniques known to man when determining proteins and biological macromolecules is x-Ray crystallography. This procedure has advanced mankind in that it helps to structure drug designs, and to expand knowledge on the subject of the elucidation

Phosphorus-32 labeling of tRNA. In order to visualize the sample, -32P radiolabeled ATP was added to the yeast tRNAPhe by means of enzymatic catalysis by NT CCA-adding enzyme. The 15 µL reaction mix consisted of 30 µM yeast tRNAPhe, 1.1 µM -32P ATP, 100 µM CTP, 10 mM glycine, 2 mM MgCl2, 200 µM dithiothreitol, and enough reagent-grade water to initially make up a volume of 14 µL. After a 30 second incubation at 37 °C in a water bath, NT in glycerol was added to the reaction and mixed by finger-flicking. The reaction was allowed to carry on for 5-10 minutes, at which time 5 µL chilled formamide containing bromphenol blue and xylene cyanole was added to the reaction and mixed in order to inhibit the NT from catalyzing further addition of nucleotide

Atomic charges were assigned to the receptor using AMBER7 FF99 force field. The protein complex was minimized using AMBER7 FF99 force field. Finally the 3D structure of the prepared protein was saved as PDB file.

The purpose of this lab report is to investigate the effect of substrate concentration on enzyme activity as tested with the enzyme catalase and the substrate hydrogen peroxide at several concentrations to produce oxygen. It was assumed that an increase in hydrogen peroxide concentration would decrease the amount of time the paper circle with the enzyme catalase present on it, sowing an increase in enzyme activity. Therefore it can be hypothesised that there would be an effect on catalase activity from the increase in hydrogen peroxide concentration measured in time for the paper circle to ride to the top of the solution.

In this specific experiment, the pH of the environment in which the reaction took place was altered in order to observe how the rate of the enzyme catalase’s activity would be affected. Like most other enzymes, catalase performs optimally at a pH of 7. For the purposes of this experiment, the varying pH levels were assigned as the independent variable and the rate of enzymatic activity was the dependent variable. From a molecular standpoint, pH levels can change the shape of an enzyme, causing the active site’s shape to change and not allowing the substrate to fit in correctly. The pH levels may also change properties of the substrate, in which case it may not bind properly and result in an ineffective catalyzation (Nishiura). In this way, varying the pHs allows for the rate of enzymatic activity to be observed since there will be an optimal pH at which the enzyme and substrate’s shape and integral properties are retained. Any deviation from this pH will

The structure of carboxypeptidase A was determined by high resolution X-ray crystal structural study (Figure 2). The zinc ion is located well inside the surface of the protein. The zinc is coordinated to His-69 and His-196 by two imidazole side chains and also to the bidentate carboxylate group of Glu-72. And the coordination geometry is accomplished by a water molecule which leads to the completion of five coordination of zinc (II).

The extracellular thermostable phytase gene was isolated from the soil bacteria Bacillus subtilis ARRMK33 (BsPHYARRMK33). The gene had an open reading frame of 1152 bp and it encoded 383 amino acid residues of a protein with a putative leader peptide of 27 amino acids. According to the insilico analysis of the phytase gene, the phytase from BsPHYARRMK33 is closely related to Bacillus subtilis sp. phytase proteins and has no similarities to other phytases. In silico analysis of this phytase disclosed β propellar structure of phytase. The Phytase encoding cDNA was subcloned into the pET-28a (+) expression vector. The recombinant plasmid pET-28 a (+) BsPHYARRMK33 was expressed in Escherichia coli BL21 (DE3). The expressed protein was analysed by SDS-PAGE and a specific band with a molecular mass of approximately 43 kDa was found. Recombinant phytase enzyme activity assay was verified, with sodium phytate substrate. The maximum phytase activity was 2.25 U ml-1 obtained from the cellular extract of E. coli BL21 (DE3) harbouring pET-28 (a) BsPHYARRMK33. The optimal pH and temperature of the crude recombinant phytase were 7.0 and 55 ᴼC, respectively.

The correlation method is based upon the fact that a particular mode can be infrared active only if its symmetry species of vibration is same as that of at least one of the dipole moment or equivalent of a vector like translation. Similarly Raman activity for a particular mode is possible if its symmetry species of vibration is same as that of at least one component or combination of polarizability tensor α.

A similar approach has been used for obtaining cellulases from anaerobic beer lees converting consortium, wherein the metagenome was sequenced and then screened for cellulase sequences. Thereafter, three cellulase genes, when cloned and expressed in E. coli, were found exhibiting considerable cellulase activities (Yang et al., 2016).