Effect of Temperature, Study and Measure of Enzymes Activity

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Basic Biochemistry CP2085

Effect of Temperature, Study and Measure of Enzymes Activity

Abstract
This experiment investigates the effect that temperature has on the rate of activity of enzyme β-galactosidase and also the rate of β-galactosidase activity in different concentration of substrate over time. Ο-nitrophenylgalactoside (ONPG) is used as a substrate for β-galactosidase. A spectrophotometer is used to detect the change in colour of the substrate. Results show that increase in temperature up to 50oC speeds up the rate of enzyme activity and any increase in temperature after 50oC reduces rate of enzyme activity drastically. Results also show that an increase in concentration of substrate also increases the rate of enzyme activity.

Objective
The objective of experiment 5 is to study the effect of temperature on the rate of activity on enzyme β-galactosidase using a spectrophotometer.
The objective of experiment 6 is to study and measure the rate of enzyme β-galactosidase activity over time using a spectrophotometer.

Introduction
This report investigates the effect of temperature and different substrate concentrations have on the rate of enzyme activity in 2 separate experiments. The enzyme used in both experiments is β-galactosidase. ONPG replaces lactose as a substrate for the experiments.
ONPG is an abbreviation for (ortho) 2-Nitrophenyl-β-D-galactopyranoside. ONPG is used in colourimetric and spectrophotometric assays for the detection of β-galactosidase activity which are of interest in clinical, environmental, food and molecular biology arenas.1 ONPG is hydrolysed by β-galactosidase to yield galactose and ortho-Nitrophenol, which is yellow in colour and absorbs light at 420 nm. The intensity of the colour can be used to determine the rate at which β-galactosidase can hydrolyse lactose.
(Carbosynth.com n.d.)

(University of California Santa Cruz 2005)

(University of Massachusetts Amherst n.d.)
A spectrophotometer is used to detect and



References: Carbosynth.com. n.d.. Untitled. [online] Available at: http://www.carbosynth.com/carbosynth/website.nsf/(w-productdisplay)/FFFCC074F681C8D980256DF0006BE44A [Accessed: 20 Jun 2013] University of California Santa Cruz. 2005. ONPG. [image online] Available at: http://bio.classes.ucsc.edu/bio105l/EXERCISES/ASSAY/handout.pdf [Accessed: 20 Jun 2013] University of Massachusetts Amherst. n.d.. A.Absorption Spectrum of o-nitrophenolate. [image online] Available at: http://bcrc.bio.umass.edu/intro/manual/index.php/Enzyme_Activity [Accessed: 20 Jun 2013] Biology.clc.uc.edu. 2003. Spectrophotometer Use. [online] Available at: http://biology.clc.uc.edu/fankhauser/labs/microbiology/Growth_Curve/Spectrophotometer.htm [Accessed: 20 Jun 2013] New Mexico Environment Department. n.d.. Chapter 11 Spectrophotometry. [online] Available at: http://ftp://ftp.nmenv.state.nm.us/www/swqb/UOCP/StudyManuals/WWLabStudyGuide/11.pdf [Accessed: 20 Jun 2013] Academic.brooklyn.cuny.edu. n.d.. Effect of temperature on enzyme activity. [online] Available at: http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm [Accessed: 20 Jun 2013] BBC UK. n.d. Graph of enzyme activity against temperature. [image online] Available at: http://www.bbc.co.uk/schools/gcsebitesize/science/add_aqa/proteins/proteinsrev3.shtml [Accessed: 20 Jun 2013] Biotek. 2007. Kinetic reaction of various ß-galactosidase enzyme concentrations. [image online] Available at: http://www.biotek.com/resources/articles/kinetic-analysis-beta-galactosidase.html [Accessed: 20 Jun 2013]

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