BIO 181
Enzyme Lab (pH)
GENERAL INTRODUCTION:



Alpha-amylase is a proteinic enzyme that hydrolyzes starch to produce maltose and
other small sugars. Our saliva contains alpha-amylase (it is produced by the
salivary glands), and so does our pancreatic juice (it is produced by the pancreatic
secretory cells).
Alpha-amylase is present in all organisms. We started this lab by extracting alphaamylase from three organisms: a thermophile bacteria (Bacillus licheniformis), a
microscopic fungus (Aspergillus oryzae), and from the pancreas of a pig.
Enzymes are affected by changes in pH and temperature. Each optimal has an
optimal T and pH values at which they are more active.
GENERAL OVERARCHING QUESTION:
Can optimal T and pH values for enzyme activity be used to distinguish organisms?
INTRODUCTION TO THE LAB:
(Have students whiteboard table of variables before they start the lab)
RESEARCH QUESTION:
Does pH affect α-amylase activity (production of maltose)?
HYPOTHESIS: pH has an effect on α-amylase activity. Optimal temperature
values can be used to identify the source of different α-amylase.
VARIABLES:
(Have students whiteboard table of variables before they start the lab; three variables
would be enough)
Table of Variables (Enzyme lab, pH)
Name
I/D/C
Symbol (Units)
Description
Temperature
C
T(°C)
Test tubes @ 23°C
Mass of maltose
D
m (g)
Change in the
amount of maltose
produced
Absorbance of maltose
D
Abs (Abs units)
Change in
absorbance values at
540nm
pH
I
pH (pH units)
Starch solution pH 5,
6, 7, 8, and 9
Substrate (starch) concentration
C
[c] (g/L)
Keep pH the same in
all tubes (1%)
Volume of substrate (starch)
C
V (L)
Keep pH the same in
all tubes (1mL)
α-amylase concentration
C
[c] (g/L)
Keep pH the same in
all tubes (XXX)
Time of enzymatic reaction
C
t (s)
Keep pH the same in
all tubes (12 min)
Wavelength
C
λ (nm)
Keep pH the same in
all tubes (540 nm)
Pre-incubation time
C
t (s)
Keep pH the same in
all tubes (10 min)
APPARATUS DRAWING:
This should be a neat drawing that clearly illustrates all equipment and how each
variable was measured. Draw and label!!
PROCEDURE:
The current protocol can be used as guideline.
 The procedure is the step-by-step process you go through to complete the
experiment
 This should be what you actually did as opposed to what the directions said.
 You may be brief, but you should be thorough – don’t skip important steps!!
DATA:
pH (pH units)
5
6
7
8
9
Abs 540nm
mg of maltose
Table 1. Production of maltose at different pH for α-amylase A (B, or C)
ANALYSIS:
(Provide examples in Lab 1 as to how to calculate mg of maltose using the standard
curve)

Calculate mg of maltose using the equation that relates
absorbance of maltose at 540 nm and mass of maltose (see maltose
standard curve, Lab 1):
Maltose Standard Curve
absorbance at 540 nm
0.4
0.35
y = 0.1743x
0.3
0.25
0.2
0.15
0.1
0.05
0
0
0.5
1
1.5
2
2.5
m g of m altose

Graph enzyme activity as measured by mg of maltose produced as a
function of pH. Graph by hand using graph paper provided by your
instructor (I have included Excel instructions just in case we decide they
graph with Excel):
Instructions for Graphing Using Excel 2007
 Type your X-values (T or pH in our case) in column A
 Type your Y-values (mg of maltose in our case) in columns B, C, and D as
shown above.
 Highlight values in all columns
 From the Insert menu, select “Chart”
 From “Chart Options” select “XY (Scatter).” Select first option “Scatter with
data points connected by smoothed lines.” Click “Next.” Once in the new
window, click “Series.” Name Series as alpha-amylase A, alpha-amylase B,
and alpha-amylase C. Click “Next”
 Go to “Gridlines” and unselect “Major Gridlines.” Go to “Titles” and give a
title to the two axis (X= Temperature (C) or pH; Y= mg of maltose). Title
your chart as “Effect of Temperature on alpha-amylase Activity.” “Click
Finish”
 Your graph is complete
CONCLUSION:
CLAIM:
Make a claim that answers your research question. pH affects α-amylase
activity (production of maltose). pH can be used to identify the source of our αamylase
EVIDENCE: Supported by results.
LAB SETTING:
(Four datasets for each enzyme. Each table conducts two experiments)
2x Enzyme A
2x Enzyme C
2x Enzyme B
2x Enzyme B
2x Enzyme C
2x Enzyme A