1
Introduction:
The
efficiency
and
complexity
of
biochemistry
in
the
human
body
is
due
in
large
part
to
enzymatic
activity.
Enzymes
are
catalysts,
usually
composed
of
a
protein,
which
promote
specific
reactions
or
groups
of
reactions.1
In
other
words,
enzymes
speed
up
processes
(such
as
the
digestion
of
food)
so
that
our
bodies
can
function
at
a
normal
and
healthy
rate.2
Enzymes
are
extremely
selective
in
the
reactions
that
they
catalyze,
and
some
are
absolutely
specific,
operating
for
only
one
substance
in
one
reaction.1
The
rate
at
which
an
enzyme
catalyzes
a
reaction
can
depend
on
various
environmental
factors
such
as
concentration
of
enzyme
or
substrate,
pH,
and
temperature.11
The
substance
that
undergoes
the
reaction
is
called
a
substrate
(in
the
Beano®
experiment,
the
substrate
is
the
raffinose
sugar
from
the
split
green
pea
extract).1
As
efficient
as
the
human
body
is,
some
enzymes
do
not
contain
the
necessary
materials
needed
to
break
down
certain
complexes.
Beano®
contains
the
sugar
digesting
enzyme
that
our
body
lacks
to
digest
the
sugar
in
beans
and
many
vegetables,
such
as
peas.3
This
enzyme
is
called
alpha
galactosidase,
which
comes
from
the
fungus
Aspergillus
niger.4
In
further
detail,
the
enzyme
in
Beano®
,
Alpha
galactosidase
,
breaks
down
olgiosacchrides
(complex
sugars
including
raffinose,
stachyose,
and
verbascose),
that
are
found
in
peas,
brown
beans,
cauliflower,
brussels
sprouts,
bran,
broccoli,
and
cabbage.5
The
enzyme
yields
β‐glucose
as
a
product
of
the
chemical
reaction.
A
glucometer
(aka.
Glucose‐meter),
is
a
medical
device
that
is
used
to
calculate
β‐glucose
in
the
blood
by
determining
concentration
levels
in
mg/dL.6
Therefore,
with
the
help
of
a
glucometer,
the
level
of
β‐glucose
in
the
sample
can
be
calculated.
That
glucose
level,
in
turn,
helps
to
determine
the
efficiency
of
the
alpha
galactosidase
enzyme
in
Beano®
under
various
2
conditions.
Taking
into
account
that
blood
is
not
being
used
in
this
lab,
a
calibration
curve
for
the
glucometer
is
necessary
to
improve
the
accuracy
of
the
readings.5
Beano®
‘s
ultimate
purpose
from
these
enzymatic
reactions
is
to
prevent
flatulence
by
reducing
gas
in
the
digestive
tract.4
The
product
contains
an
enzyme
to
break
down
the
polysaccharides
in
certain
foods
that
otherwise
would
only
be
broken
down
by
specific
bacteria
through
fermentation
in
our
large
intestine.4
In
the
case
that
these
complex
sugars
are
left
solely
to
be
fermented
by
the
bacteria
in
our
large
intestine,
various
gases
are
given
off
as
products
,
causing
flatulence.7
Flatulence
is
the
passage
of
air
or
gas
from
the
intestines
through
the
rectum.8
A
proportion
of
the
gas
is
nitrogen
(20%
to
90%),
with
the
remainder
portion
consisting
of
H2
(hydrogen
gas),
CO2
(carbon
dioxide),
and
CH4
(methane).8
The
average
person
passes
gas
through
the
rectum
8
to
20
times
a
day.6
Flatulence
is
influenced
by
the
following
conditions:
the
amount
of
air
that
is
swallowed,
the
efficiency
with
which
the
gastrointestinal
tract
moves
or
expels
gas,
and
the
amount
of
gas
that
is
produced
by
the
bacteria
in
living
in
the
large
intestine
that
act
on
incompletely
digested
food.5
Beano®
works
to
prevent
or
at
least
lessen
the
gas
products
of
large
Intestine
bacteria.
The
production
of
gas
in
the
lower
intestine
occurs
when
some
foods
are
not
completely
digested
in
the
small
intestine
(this
often
occurs
because
the
specific
enzyme
needed
for
digestion
is
not
located
in
the
small
intestine).8
When
the
undigested
bits
of
food
reach
the
lower
intestine,
the
mixture
of
nutrients
and
stomach
acid
is
fermented
by
the
bacteria
that
live
in
the
large
intestine,
eventually
causing
gas.8
At
this
point,
the
alpha
galactosidase
enzyme
in
Beano®
steps
in
and
digests
the
“problem
food”
before
it
reaches
3
the
large
Intestine,
thus
preventing
the
gaseous
products.
In
this
lab,
the
study
of
the
reaction
between
Beano®
and
split
green
peas,
which
contain
a
high
concentration
of
raffinose
sugars
,
has
shown
a
lot
of
interesting
data
regarding
the
effectiveness
of
the
enzyme.5
The
main
goal
of
the
Beano®
lab
is
to
study
the
way
the
reaction
rate
of
the
enzyme
in
Beano®
is
affected
when
environmental
changes
in
concentration,
pH,
and
temperature
occur.
This
lab
includes
a
series
of
tests,
using
the
ReliOn®
Ultima
(Wal‐Mart
Corp.)
glucometer,
an
over‐the‐counter
enzyme‐based
dietary
supplement
Beano®
and
its
reaction
with
split
green
peas.5
I
hypothesize
that
the
galactosidase’s
rate
will
be
greatly
affected
by
all
of
these
changes,
either
by
hindering
or
speeding
up
the
production
on
β‐glucose.
I
think
that
an
increase
in
concentration
of
enzyme
or
substrate
will
also
correlate
with
increase
in
temperature.
And
lastly,
I
believe
that
a
rise
in
pH
will
speed
up
the
reaction
rate
(provided
it
stays
around
pH=8)and
increase
β‐glucose
production.
Procedure:
The procedure for the Beano® lab is explicitly taken from: Chemistry 113B- Biological
Laboratory Manual, Spring 2010; Heckman, Kimberly A. and Keiser, Joseph T.; Prentice Hall
Inc., Englewood Cliffs, NJ; pp. 6-1 to 6-16.5 A complete record of my observations can be found
in my notebook, pp.10-14.9 My partner in this lab was Morgan Brown.10
In the Beano® lab, there were six main objectives. The first goal was to familiarize use of
a 100 µL “air displacement” pipette (micropipette). This was accomplished by practicing tip
ejection and accurate solution delivery, followed by determining the standard deviation for the
4
measurements. The second goal was to calibrate the ReliOn® Ultima (Wal-Mart Corp.)
glucometer by using five standard glucose solutions. Using the recorded readings from each of
the five solutions, a calibration graph was produced comparing the five known concentrations to
the five values given by the glucometer. The third goal of the lab was to determine the
relationship between substrate concentration (pea extract) and the rate of reaction. Using the
gathered data, a graph was created comparing glucose concentration vs. time for each of the
three different pea extracts. For the fourth goal, the main concepts of the experiment were used
to explore the relationship between enzyme (Beano®) concentration and the rate of reaction. For
the fifth goal, the relationship between pH and rate of reaction was determined using critical
thinking. The final goal of the Beano® lab was to determine the relationship between
temperature and the rate of reaction. This was explored by producing a graph of of glucose
concentration vs. time with each of the temperatures (10°C and 40°C), including the room temp
(25°C) from the previous section.
All of these findings helped shed light on how enzymes work and what specific
conditions and variables change their effectiveness.
Results:
Practice with Micropipetting:
Before beginning experimentation with enzymes and substrates, practice with micropipettes was
deemed necessary in order to establish good lab technique.
5
Table 1.1: Practice with 100µl Pipetting:
Delivery # Weight in grams
1 0.098
2 0.100
3 0.0995
4 0.1005
Figure 1.1 shows four trials of 1 drop from the pipet and its respective weight in grams.
Example of calculating standard deviation:
Standard deviation is calculated by adding all four values up and dividing by 4 to find the mean,
which is 0.0995g: (
0.098+0.100+0.0995+0.1005)/4=0.0995g.
Next, each value is subtracted from the mean, squared, and then added to the other values.
(0.098-0.0995) 2 +(0.100-0.0995) 2+(0.0995-0.0995)2+(0.1005-0.0995) 2=3.50x10-6
Standard Deviation= ±3.50x10-6
Calibration of the Glucometer Data:
The first table and graph demonstrate the calibration step first taken to make sure that the
glucometer was reading measurements correctly.
Table 1.2: Calibration graph of Glucose Concentrations
Glucose Solution (mg/dL) Concentration of Glucose (mg/dL)
50 67
100 143
150 254
200 331
250 438
Table 1.2 shows the relationship between glucose concentration and the glucometer’s reading of the concentration.
6
Figure 1.1:Calibration Graph of Glucose Concentrations
Calibration
Graph
of
Glucose
Concentration
500
Glucose
Concentration
(mg/dL)
from
450
400
350
300
Glucometer
250
200
Glucose
concentration
data
150
points
(mg/dL)
100
y
=
1.86x
‐
32.4
50
0
0
50
100
150
200
250
300
Glucose
Concentration
(mg/dL)
Figure 1.1 shows the correlation between glucose concentration and the concentration measured on the glucometer
in mg/dl, with a slope of 1.86x.
Varying Substrate Concentration Data:
The following chart and graph show the glucose concentrations found for the different pea
extract solutions over a ten-minute span of time.
Table 1.3: Time vs. Concentration of Glucose
Time (min) Glucose Conc. Glucose Conc. Glucose Conc.
(mg/dL) for 25% (mg/dL) for 50% (mg/dL) for 100%
Pea Extract Pea Extract Pea Extract
0 Too Low Too Low 28
2 31 22 129
4 65 69 137
6 97 127 256
8 132 193 278
10 166 241 356
Table 1.3 shows both the relationship of time vs. glucose concentration in mg/dL, as well as measuring the
concentrations found using varying potencies of the Pea Extract.
7
Figure 1.2:Time vs. Varying Glucose Concentration graph
Time
vs.
Glucose
Concentration
@
25º
C
400
Glucose
Concentration
)mg/dL)
350
300
25%
Pea
Extract
Solution
y
=
16.85x
‐
2.9
250
50%
Pea
Extract
Solution
200
y
=
28.1x
‐
38.2
150
100
%
Pea
Extract
Solution
100
y
=
29.75x
+
52.7
50
0
0
2
4
6
8
10
12
Time
(minutes)
Figure 1.2 shows the relationship of glucose concentration vs. time.
Varying Temperature on Enzyme Activity Data:
The following table and figure show the affect of varying temperatures on enzyme activity and
glucose concentrations produced.
Table 1.4.Time vs. Glucose Concentration Data
Time (minutes) Glucose Concentration Glucose Concentration
(mg/dL) of 50% Pea Extract (mg/dL) of 50% Pea Extract
Solution at 40 ° C Solution at 10 ° C
0 Too Low Too Low
2 31 Too Low
4 118 21
6 174 49
8 239 73
10 284 88
Table 1.4 again shows that time and glucose concentration are directly related, as well as showing that temperature
and glucose concentration are also directly related.
8
Figure 1.3: Time vs. Glucose Concentration with Varying Temperature graph
Time
vs.
Glucose
Concentration
with
varying
400
Temperature
Glucose
Concentration
(mg/dL)
350
50%
Pea
Extract
Solution
300
at
40°
C
y
=
31.35x
‐
18.9
250
50%
Pea
Extract
Solution
200
at
25°
C
y
=
28.1x
‐
38.2
150
50%
Pea
Extract
Solution
at
10°
C
100
y
=
11.25x
‐
21
50
0
0
2
4
6
8
10
12
Time
(minutes)
Figure 1.3 shows that glucose concentration increases as temperature increases.
Table 1.5: Slope Values of the Varying Substrate Concentrations and Temperatures
Substrate Concentrations Slope Equation from slope
25% Pea Extract at 25ºC 16.9 16.9x-2.9
50% Pea Extract at 25ºC 28.1 28.1x-38.2
100% Pea Extract at 25ºC 29.75 29.75x+52.7
50% Pea Extract at 40ºC 31.35 31.35x-18.9
50% Pea Extract at 10ºC 11.25 11.25x-21
Figure 1.5 shows the relationship between varying substrate concentration and slope equations.
Example Calculation of Finding the Slope:
Slope is used to determine the rate at which these reactions occur. The equation used for slope
equation is Y=mx+b.
To find slope, two points selected from the 25% Pea Extract equation:
2 min 31 mg/dL
4min 65 mg/dL
9
Y2-Y1 / X2-X1 = 65-31 / 4-2 =17
(note: Excel produced a more exact result of 16.9)
Example Calculation of Ratio of Initial Rates of Reaction:
Theoretically, the ratio for the 100% and 50% Pea Extracts would be 100:50, or 2:1. The results
found in this experiment, however are different.
Slope of 100% Extract= 29.75
Slope of 50% Extract=28.1
29.75: 28.1=1.06, which is significantly lower than the theoretical ratio of 2.
Theoretically, the ratio for the 100% and 50% Pea Extracts would be 100:25, or 4:1. The results
found in this experiment are as follows:
Slope of 100% Extract= 29.75
Slope of 25% Extract= 16.9
29.75:16.9=1.76, which is significantly lower than the theoretical ratio of 4.
Discussion:
The results presented in Table 1.1 of the four different sample weights of of 100µL
of
distilled
water
from
a
micropipette.
These
results
are
used
to
calculate
the
average
weight,
and
standard
deviation
of
one
100µL
drop.
The
average
weight
of
one
drop
is
0.0995
grams
with
a
standard
deviation
of
±3.50×10−6grams.
Given
these
results,
a
drop
of
100µL
Beano®
from
the
micropipette
will
be
very
accurate
and
precise
each
time,
because
the
standard
deviation
amount
is
extremely
insignificant.
The
table
and
graph
showing
calibration
of
the
glucometer
present
the
calibration
curve
for
the
Wal‐Mart
brand
instrument,
ReliOn®
Ultima.
This
calibration
curve
is
essential
to
the
accuracy
of
the
lab,
because
glucometers
are
built
to
measure
β‐glucose
concentration
in
human
blood,
not
aqueous
solutions
or
pea
extract.
Therefore,
the
glucometer
allows
the
experiment
to
provide
realistic
numbers
in
terms
of
how
Beano®
10
would
work
in
the
body.
The
points
on
the
graph
are
linear
for
the
most
part,
indicating
that
the
glucometer
is
fairly
accurate
in
its
ability
to
measure
β‐glucose
concentration.
By
using
the
equation
of
the
line
of
best
fit
from
this
graph,
even
more
accurate
results
can
be
obtained.
Figure
1.2
and
Table
1.3
demonstrate
the
effects
of
varying
substrate
concentration
(pea
extract)
on
the
rate
of
reaction.
Due
to
substrate
concentration
having
a
direct
affect
on
enzyme
activity,
the
amount
of
glucose
measured
increased
as
the
pea
extract
concentration
increased.
Furthermore,
the
rate
of
reaction
is
fastest
for
100%
pea
extract,
followed
by
50%
extract
and
lastly
25%
extract.
The
slopes
for
these
values
are
16.85,
28.1,
and
29.75
respectively.
This
increase
in
rate
of
reaction,
proven
by
the
increase
in
glucose
production,
illustrates
that
the
more
raffinose
sugar
from
the
extract
present,
the
more
will
be
broken
down
by
the
enzyme
in
Beano®,
called
galactosidase.
Theoretically,
the
relationship
between
the
increase
in
substrate
concentration
and
the
increase
in
rate
of
reaction
should
be
proportional,
resulting
in
final
glucose
concentrations
of
4:2:1
for
the
100%,
50%,
and
25%
concentrations
respectively.
However,
due
to
human
error
and
possible
contamination
of
equipment,
the
results
from
the
lab
experiment
are
not
quite
proportional.
For
example,
the
final
β‐glucose
ratio
for
100%
and
25%
extracts
was
356:166,
or
2.14.
That
ratio
is
significantly
lower
than
the
theoretical
value
of
4.
This
is
due
in
part
to
the
fact
that
the
glucometer,
although
inexpensive
and
easy
to
use,
is
not
the
most
precise
way
to
measure
glucose
concentration.
Using
the
theory
presented
with
this
data,
if
the
substrate
was
held
constant
and
the
concentration
Beano®
was
decreased,
the
reaction
rate
would
also
decrease
because
there
would
be
less
enzyme
present
to
break
11
down
the
raffinose
sugars.
However,
if
the
experiment
ran
to
completion,
the
concentration
of
β‐glucose
would
be
the
same.
Figure
1.3
and
Table
1.5
demonstrate
the
relationship
between
temperature
and
the
rate
of
reaction.
There
is
a
positive
correlation
between
increased
temperature
reaction
rate.
The
three
samples,
all
50%
pea
extract
concentration,
were
measured
for
glucose
concentration
at
40,
25,
and
10
degrees
Celsius.
From
the
results,
one
can
conclude
that
the
galactosidase
enzyme
works
optimally
at
40°
C,
producing
more
glucose
than
the
lower
temperatures.
This
result
makes
sense
from
a
physiological
standpoint,
because
Beano®
is
ingested
with
food
and
is
processed
in
the
small
intestine,
which
is
normally
around
37°
C.
A
decrease
in
temperature
has
a
direct
effect
on
enzymatic
activity,
slowing
the
rate
at
which
raffinose
is
broken
down,
which
ultimately
lowers
the
overall
concentration
of
of
β‐
glucose.
Although
the
experiment
did
not
include
any
direct
testing
on
the
relationship
between
pH
and
reaction
rate,
knowing
where
Beano®
is
most
effective
in
the
body
helps
to
determine
its
optimal
pH
range.
Since
galactosidase
activity
works
to
break
down
sugars
in
the
small
intestine
where
pH
levels
are
generally
around
8,
one
can
assume
that
Beano®
works
the
best
in
a
similar
basic
pH.
A
pH
level
more
similar
to
the
stomach
at
the
acidic
level
of
2
would
be
less
conducive
to
the
enzymatic
efficiency
of
galactosidase,
slowing
the
rate
of
reaction.
Overall,
this
experiment
seemed
fairly
accurate
and
precise
in
its
results.
Considering
the
fact
that
most
of
our
data
was
dependant
on
the
inexpensive
and
somewhat
precarious
glucometer,
all
of
the
expected
results
proved
true
to
the
hypothesis.
12
However,
the
ratio
between
the
rates
of
reactions
(slopes)
and
the
ratio
between
final
glucose
concentrations
were
significantly
different
than
the
theoretical
ratios
discussed
above.
This
is
due
to
human
error
in
the
lab,
such
as
differing
pipette
technique
and
imprecise
timekeeping.
It
is
also
important
to
remember
that
staging
a
reaction
between
the
enzymes
in
Beano®
and
the
substrate
(pea
extract)
in
a
graduated
cylinder
will
never
be
as
accurate
or
efficient
as
those
similar
reactions
that
occur
in
the
small
intestine,
due
to
the
body’s
complex
nature
of
systematic
processing
and
desire
for
homeostasis.
Conclusion:
In
Beano®.
the
galactosidase
enzyme’s
ability
to
break
down
raffinose
sugars
is
indeed
affected
by
environmental
changes
in
substrate/enzyme
concentration,
temperature
change,
and
change
in
pH.
Both
an
increase
in
substrate/enzyme
concentration
and
an
increase
in
temperature
result
in
an
increased
reaction
rate.
The
optimal
temperature
for
galactosidase
is
around
40°C
and
the
optimal
pH
is
basic,
around
8.
Hence,
my
hypothesis
is
proven
correct.
References:
1Folchetti,
Nicole,
Ed;
Chemistry
The
Central
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