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Effect Of Increasing Concentration Of Yeast Invertase On Sucrose

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Student’s Name
Instructor’s Name
CHEM 202
Date of Submission
Biochemistry Laboratory
Experiment: Enzyme Kinetics
Abstract
The aim of the study is two-fold: to study the rate of absorbance with increasing concentration of glucose, and to measure the activity of enzyme yeast invertase on sucrose. In task 1, the product formation was measured using 3, 5-dinitrosalicyclic acid that reacts with glucose leading to a change in colour from yellow to reddish brown. In task 2, the enzyme kinetics of yeast invertase on sucrose was studied. The absorbance values of the corresponding volumes of the solutions were measured using a spectrophotometer. Michaelis-Menten curve and Lineweaver-Burk Plot were made in order to estimate the values of Vmax and Km
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concentration was plotted in order to estimate the concentration of the unknown solution (Figure 3). Figure 3: A graph of absorbance vs. Concentration
From the graph, R2 can be extrapolated from the y-axis and the corresponding value on the x-axis (the point of intersection on the curve) gives the concentration of the unknown solution.
R2 = 0.695.
By extrapolating from the curve, the concentration of the unknown solution is ≈ 1.9mg.mL. The rate of reaction increases linearly with an increase in the substrate concentration. Task 2: Enzymatic activity of yeast invertase
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. Results and Analysis
Table 1: The reaction scheme/ enzymatic activity of yeast
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Discussion
Using Michaelis-formula Vmax can be obtained as follows: VO=VMax ((substrate concentration)/(subtrate concentration +km))
Vo=absorbence/min for example, 0.018/5min =0.0036/min.
From Figure 4, the maximum velocity is the highest point on the curve and the constant (km) is ½ the maximum velocity. However, it is difficult to estimate Vmax using Michaelis-Menten curve with precision since the limits of the hyperbolic curve deviate at varying concentrations of the substrate. The data can be linearized using Lineweaver-Burk Plot to obtain Vmax and Km as follows: Slope=Km/V_max
From Figure 5, Vmax = 0.0172 and Km = 0.3144. At ½ Vmax (Figure 4), the rate of reaction increases steadily with an increase in substrate concentration. At this point, the rate of product formation is partially limited by substrate availability. As the curve approaches Vmax, the enzyme catalyzed saturation increases. In effect, the enzyme activity increases the rate of product formation up to a point whereby the reaction rate remains constant as more substrate is added (saturation).
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