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    JJ

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    Eureca Parra
    The document describes an experiment measuring the enzyme activity of beta-glucosidase. Samples were taken from the reaction mixture at various time points and glucose concentration was measured. This data was plotted as concentration vs time, yielding a linear trendline with a slope of 0.033 μmol/mL-min, indicating the initial reaction rate. Using this slope and the volumes and masses involved, the enzyme activity was calculated as 1.65 units/mL of enzyme solution and 165 units/mg protein.

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    JJ

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    Eureca Parra
    369 views4 pages
    The document describes an experiment measuring the enzyme activity of beta-glucosidase. Samples were taken from the reaction mixture at various time points and glucose concentration was measured. This data was plotted as concentration vs time, yielding a linear trendline with a slope of 0.033 μmol/mL-min, indicating the initial reaction rate. Using this slope and the volumes and masses involved, the enzyme activity was calculated as 1.65 units/mL of enzyme solution and 165 units/mg protein.

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    The document describes an experiment measuring the enzyme activity of beta-glucosidase. Samples were taken from the reaction mixture at various time points and glucose concentration was measured. This data was plotted as concentration vs time, yielding a linear trendline with a slope of 0.033 μmol/mL-min, indicating the initial reaction rate. Using this slope and the volumes and masses involved, the enzyme activity was calculated as 1.65 units/mL of enzyme solution and 165 units/mg protein.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    369 views4 pages

    JJ

    Uploaded by

    Eureca Parra
    The document describes an experiment measuring the enzyme activity of beta-glucosidase. Samples were taken from the reaction mixture at various time points and glucose concentration was measured. This data was plotted as concentration vs time, yielding a linear trendline with a slope of 0.033 μmol/mL-min, indicating the initial reaction rate. Using this slope and the volumes and masses involved, the enzyme activity was calculated as 1.65 units/mL of enzyme solution and 165 units/mg protein.

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    © All Rights Reserved
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    The activity and initial rate of a beta-glucosidase enzyme reaction were measured.

    The glucose concentration was measured at various time points after initiating the reaction of cellobiose with beta-glucosidase.

    Michaelis-Menten kinetic parameters were evaluated using Langmuir, Lineweaver-Burk, Eadie-Hofstee plots and nonlinear regression.

    2.

    1 In order to measure the enzyme activity and the initial rate of reaction, 5 mL of cellobiose
    (100mumol/mL) and 44 mL of buffer solution were placed in a stirred vessel. The reaction was
    initiated by adding 1 mL of enzyme (beta-glucosidase) solution which contained 0.1 mg of protein
    per mL. At 1, 5, 10, 15, and 30 minutes, 0.1 mL of sample was removed from the reaction mixture
    and its glucose content was measured. The results were as follows:

    Time (min) Glucose Concentration (µmol/mL)


    1 0.05
    5 0.23
    10 0.38
    15 0.52
    30 1.03

    a. What is the activity of the β-glucosidase in units/mL of enzyme solution and in units/mg
    protein? A unit is defined as the enzyme activity which can produce Imumol of product
    per minute.
    b. What is the initial rate of reaction?

    Concentration vs Time
    1.5

    1
    [S]

    0.5 y = 0.033x + 0.0391


    R² = 0.9973
    0
    0 5 10 15 20 25 30 35
    t

    a. Vt = Vcellobiose + Vbuffer solution + Venzyme 1.65 mumol/min


    Vt * m  =
    = 5 mL + 44 mL + 1 mL 1 mL of enzyme
    = 50 mL 1.65 units/mL of enzyme = 165
    Plotting [S] vs time yields an units/mg protein
    equation with a slope of m = 0.033
    (mumol/mL-min). therefore, the b. Initial reaction rate is the slope of the
    activity is equation. Thus, reaction rate is
    Vt * m = (50 mL)(.033) m=0.033 mumol/mL-min
    = 1.65 mumol/min
    2.5 Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholine (substrate) by
    dog serum (source of enzyme) and obtained the following data:

    Substrate Concentration Initial Reaction Rate (mol/L min)


    (mol/L)
    0.0032 0.111
    0.0049 0.148
    0.0062 0.143
    0.0080 0.166
    0.0095 0.200

    Evaluate the Michaelis-Menten kinetic parameters by employing (a) the Langmuir plot, (b) the
    Lineweaver-Burk plot, (c) the Eadie-Hofstee plot, and (d) non-linear regression procedure.

    (a) Langmuir Plot

    Langmuir
    LANGMUIR PLOT
    0.06 Cs/r
    Cs
    0.05
    0.028829
    0.04 0.0032
    CS/R

    0.03 y = 3.3133x + 0.0191 0.033108


    R² = 0.8837 0.0049
    0.02
    0.043357
    0.0062
    0.01
    0.048193
    0 0.008
    0 0.002 0.004 0.006 0.008 0.01
    CS 0.0475
    0.0095
    (b) Lineweaver-Burk Plot

    Lineweaver-Burk
    LINEWEAVER-BURK PLOT
    10 1/Cs 1/r
    9
    8
    7
    312.5 9.009009
    6
    5 204.0816 6.756757
    1/R

    y = 0.0172x + 3.6342
    4 R² = 0.9146
    3 161.2903 6.993007
    2
    1
    125 6.024096
    0
    0 50 100 150 200 250 300 350
    1/CS 105.2632 5

    (c) Eadie-Hofstee Plot


    Eadie-Hofstee

    EADIE-HOFSTEE PLOT r/Cs r


    0.25
    34.6875 0.111
    0.2
    30.20408 0.148
    0.15
    R

    23.06452 0.143
    0.1
    y = -0.0043x + 0.2645
    R² = 0.6584
    0.05 20.75 0.166

    0 21.05263 0.2
    0 5 10 15 20 25 30 35 40
    R/CS
    (d) Nonlinear Regression

    Nonlinear Regression
    0.25

    0.2

    0.15

    0.1

    0.05

    0
    0 0.002 0.004 0.006 0.008 0.01

    Vmax = 0.2

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