B-Galactosidase Enzyme Activities - Essay Example

B-Galactosidase Enzyme Activities Abstract This review delivers a summary of the structure, function, and catalytic mechanism of lacZ β-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it easy to appreciate why the removal of certain sums to detach into dimers but also eradicated activity. Substrate formerly binds near the top of the active site but then moves deeper for the reaction. The acceptor reaction with glucose is essential for the formation of allolactose, the natural inducer of the lac operon. Introduction The ideas on the regulation of protein synthesis in bacteria have mainly arose from the studies of Perse Monod on the induced synthesis of B-galactosidase in E.coli. It resulted in the discovery of regulatory events to the transcription of DNA and the assemblage of functionally related genes into clusters called operons. The specific mechanisms exist to regulate the levels of expression in the cell. The regulation may be as a result of transcription, translation or messenger RNA stability. In this experiment, there is the regulation of transcription of bacterial genes that are either inducible or repressible enzyme systems. In induced enzymes, there is the breakdown of complex molecules for example lactose induces synthesis of the proteins B-galactosidase, galactose permease and thiogalactoside transacetylase in E.coli. B-galactosidase enzyme found in E.coli is an example of an inducible enzyme. It catalyzes the hydrolysis of a wide variety of B-galactosides such as lactose into its constituent substances Methods/ procedure There was of sharing of the reagents labeled control, and lactose (L) flask. It was vital that the lactose does not enter the control bottle. Separate, a labeled pipette was reserved to make an addition to, or to sample from the flask. Time was a important factor and lactose was timed at zero time, ninety minutes, hundred and twenty and one hundred and eighty minutes up to two hundred and forty minute. From there, sampling procedure included. At the appropriate time, 3ml aliquots were removed from both control(C), and lactose (L) culture and further 0.5mL aliquots from lactose (L) culture placed in a plastic centrifuge tube. Dilution of 0.5mL aliquot one in five by adding 2ml of culture medium was done and labeled the cell L/5. (Michael & Nelson. 2008) The absorbance was then read at550 & 620nm as a measure of cell growth using culture medium as once blank. The average values were tabulated in the table. The samples were chilled for about 2-3minutes and centrifuged at 3,500 rpm for ten minutes. They poured off and discarded the supernatant into the water bottle provided. A 1.0mL of Tris HCL/KCL (0.05M Tris HCL, 0.05M KCL, pH 7.0) buffer was added. Using Pasteur pipette, was resuspended by pipetting up and down until you have a uniform suspension. 0.5ml aliquot of this bacterial suspension was taken into a fresh labeled glass test tube and 2drops of toluene was added and vortex vigorously for 30-50seconds.it was then placed in a shaking water bath for at least 3o minutes to liberate the enzymes. (Michael & Nelson. 2008) With all the tubes being ready, one should note the time, and then add 0.0049M 0-nitrophenyl-ß-D-galactoside (ONPG) and incubate at 37oC for exactly 20 minutes. Incubation was stopped by an addition of 4.0ml of triethanolamine acetate 2.0M, pH 8.8), and the contented was poured into a plastic centrifuge tube. Centrifugation was then repeated at 3,500r rpm for the same time which was 10-25 minutes. The absorbance of the supernatant at 420nm & 450 nm was read against distilled water. (Henry 2014)Results Table1. Cell growth     A620   cell density   Time (min) Control(C) Induced(L) induced(1/5) control induced 120 0.24 0.23 0.05 0.26 0.26 150 0.235 0.245 0.055 0.2209 0.2303 180 0.2 0.19 0.04 0.188 0.1786 210 0.22 0.19 0.05 0.2068 0.1786 Table2. B-Galactosidase Activity   A420     activity (unit g/ml)     specific activity   Time Control Induced Induced(1/5) Control Induced Induced(1/5( Control Induced 120 0.07 1.5 0.07 0.31 6.67 15.56 1.38 68.83 150 0.07 1.5 0.85 0.31 6.67 18.89 1.41 82.02 180 0.08 1.3 0.61 0.36 5.78 13.56 1.89 75.90 210 0.07 0.95 0.311 0.311 4.221 6.91 1.50 78.72 Graph of B-galactosidase enzyme activity (g/ml) against time (mins) From table1, the values of the control & density are not the same at 60min, 120min and 150min respectively. Hence, there is a significant change in the values of the induced of the cell mass during that time it’s evident from the table 1. Show a positive reaction between B-galactosidase enzyme and the substrate. (Michael & Nelson. 2008) From table2 (B-galactosidase activity), the activity of the induced is a slight difference between the values of 60mins, 120 mins, and 150 min. The activities of the induced 1/5 are also not the same in 60mins and 120 min. Hence, there is a sharp increase in the induced rate of the specific activity compared to the particular activity of the control. The absorbance for the control of the B-galactosidase activity at a wavelength of 420-450nm is near same in 60min and 150min. From the graph above, the values of B-galactose activity remained steady during the 0 min; it started to increase as time increase. It was due to the rise of lactose concentration and also enough time to allow the breakdown of the lactose into its constituent components. It indicates the action of enzyme B-galactosidase found in E.coli to the conversion of lactose into its component. Can be possible only if optimum conditions favoring the conversation of galactose into pure sugar. Others such as inhibitor can result in the small level of concentration or at time decrease in the concentration over a period as shown on the graph above. Discussion It was expected that the activity of the control from the results of the table2 which is B-galactosidase activity to remain the same over time, as lactose to be absent in the in the control flask, but from the experiment it was not the case. Therefore, the mechanism of transcription will not be induced as expected. From the class result, some mistakes such as inaccuracy in measuring of reagents and also error that may have occurred during dilution of the reagents and samples, wrong timing also contributed to some errors in the results collected not forgetting inhibitors which competes with the substrate to occupy the active site of the enzyme B-galactosidase. From the graph of B-galactosidase activity of control and induced against time, I expected the value should be increasing gradually from 60 minutes. It was as expected but when it reached 150min it begin to reduce. It could have resulted from the error committed during either dilution or timing or sample collection. The lac genes were successfully transcribed, after adding the lactose hence B-galactosidase activity increased. The transcription of these genes required lactose to be present in the medium. The activity of B-galactosidase is diminutive, when E.coli is grown in a media lacking lactose. Lactose is a good example of an inducer. Expression of the lac genes is induced by the twelve carbon sugar lactose The activity of the control from the results of the table2 which is B-galactosidase activity to remain the same over time, as lactose to be absent in the in the control flask, but from the experiment it was not the case. Therefore, the mechanism of transcription will not be induced as expected. Conclusion From the data obtained during the experiment of B-galactosidase activity, I can conclude that the primary objective of the experiment was successful done. It was due to the quantification of the result and also the graph drawn showing the rate of reaction. There were also some errors that arise from the experiment. It is explained by variance in the tabulated values in the two table tabulated in the results. To reduce this error, it is required to avoid some small error for example dilution error led to the dilution of the reagent, correct timing of the effect should precise since the changes occur at narrow time change. Also the main aim of the experiment was achieved since quantification of B-galactosidase enzyme found in the E.coli was successful. In other words the experiment can to accomplishment successfully. References Clark, D. P. (2013). Molecular biology. Waltham: Academic press. Henry, N. (2014). Introductory molecular biology and biochemistry. Practical manual: university of New England. Michael, D. & Nelson, L. (2008). Lehninger principle of Biochemistry. USA: oxford . Read More