Universiti Tunku Abdul Rahman

UDBM 2114 Microbial Biochemistry and Physiology

MAY2017

Experiment 1

Title: The Effect of Environment on Growth

Objectives/Goals

1. To demonstrate how osmolarity, pH, organic acids, and sulfa drugs and their antagonists
can affect the growth of bacteria

Introduction

Although the construction of media for the cultivation of microbes is based on well-
established nutritional and physiological considerations, unexpected effects of concentration and
interactions among medium components can produce unpleasant surprises. Compounds that are
good, readily used sources of carbon, and energy at one concentration or at a given pH can
become inhibitory at another concentration or pH. Compounds can be inert themselves yet can
reverse the growth inhibition by a second compound or act with it to halt growth. In this
experiment, some of these effects are demonstrated using Escherichia coli B.
All the effects seen in this experiment have known chemical and physiological bases. In
this deceptively simple experiment, you will (1) use your knowledge of experimental design to
describe the effects of medium variables and (2) do some reading to learn the mechanisms of the
effects and to explain them.

Materials

Supplies
Bunsen burner
Spectrophotometer set at 660 nm and
cuvettes Shaking incubator set at 30C
Sterile 16 x 150 mm test tubes

Solutions
30 ml of Medium A: standard mineral base (SMB) medium containing 0.05 M potassium
phosphate buffer and 0.1% NH4Cl, final pH 7.4
6 ml of Medium B: Medium A + 40% (w/v) glucose
10 ml of Medium C: same as Medium A except that the final pH is
6.1 6 ml of sterile water
1 ml of 1.2% sodium benzoate, pH
6.5 1 ml of 50 mM sulfanilamide
0.5 ml of 50 M p-aminobenzoic acid (PABA), sodium
salt 0.9 ml of E. coli culture

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM 2114 Microbial Biochemistry and Physiology
MAY2017

Method/Procedures

Part 1: Preparing culture media

1. Make up a series of 9 tubes according to the table below.

Medium Medium Medium Benzoate Sulfanilamide PABA Sterile

Tube
A (ml) B (ml) C (ml) (ml) (ml) (ml) H2O (ml)

1 5.00 - - - - - 1.00
2 5.00 0.005 - - - - 1.00
3 4.95 0.05 - - - - 1.00
4 - 5.00 - - - - 1.00
5 - 0.05 4.95 1.00 - - -
6 - 0.05 4.95 - - - 1.00
7 4.95 0.05 - 1.00 - - -
8 4.95 0.05 - - 0.50 - 0.50
9 4.95 0.05 - - 0.50 0.50 -

Part 2: Inoculation and incubation

1. Inoculate each tube with 50 l of culture and incubate at 30C with shaking at 200 rpm for
two or more days. Make certain that the tubes are placed on a slant in the test tube rack.

Part 3: Measuring turbidity

1. After 48 h, measure the turbidity at 660 nm. Measure the turbidity again at 72 h to ensure
that growth has ceased.
2. Describe and explain the effects of glucose concentration, pH, benzoate at different pH
values, sulfanilamide, and PABA on the growth of E. coli.

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM 2114 Microbial Biochemistry and Physiology
MAY2017

Experiment 2
Title: Assay of Amylase and Protease Secreted by Bacillus subtilis

Objectives/Goals

1. To learn how to assay for amylase and protease and determine if they are exoenzymes

Introduction

Bacillus subtilis can utilize starch as a source of carbon and energy. In order to do this,
the cells must secrete amylase to degrade the starch to smaller molecules that can enter the cells.
In fact, certain strains of B. subtilis secrete so much amylase and protease that the strains are
used as a commercial source of the enzymes.
The degradation of starch is monitored by the disappearance of material that stains with
iodine. The assay detects -amylase, which hydrolyzes -1,4-glucoside linkages at random in
starch, glycogen, and other polyglucosans. Initially, there is a rapid decrease in the molecular
weight of the starch, resulting in a decrease in its iodine-staining properties. The final products
are primarily small-molecular weight oligosaccharides. (In contrast, -amylases catalyze an
exolytic attack and degrade starch by cleaving off maltose (a disaccharide) units from the ends of
the starch chains). The enzyme reaction is measured spectroscopically. The amylase is stable at
room temperature and can be kept in the refrigerator for at least 48 hours.
The protease assay you will use is a qualitative method which it is based upon the fact
that ninhydrin reacts with the amino groups of -amino acids and peptides to give a product with
a blue color. The blue color develops as a result of chemical reactions during which an amino
group is transferred to the ninhydrin which then reacts with a second ninhydrin molecule to give
the blue product.

Materials

Supplies
9 sterile 16 x 150 mm test tubes
4 sterile 13 x 100 mm test tubes
Bunsen burner
Centrifuge and tubes
Water baths at 30C and 37C
Spectrophotometer set at 550 nm and cuvettes

Solutions
5 ml of B. subtilis culture
40 ml of 0.1 M potassium phosphate buffer, pH 6.5
8 ml of iodine reagent (0.2% I2 + 0.3% KI)
1 ml of 2% soluble starch in 0.1 M potassium phosphate buffer, pH 6.5
50 ml of distilled water

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM 2114 Microbial Biochemistry and Physiology
MAY2017

2 ml of bovine serum albumin (BSA), 1 mg/ml in water

2 ml of ninhydrin reagent (3.5 g in 100 ml of 1:1 mixture of acetone and butanol)

Method/Procedures

Part 1: Preparing test samples

1. Pipette 1 ml of B. subtilis culture into each of two 1.5-ml microtubes. Label the first tube
CT (culture).
2. Centrifuge the second tube at 8000 g for 20 min. Carefully transfer the resulting
supernatant to a fresh microtube. Label this tube SN (supernatant).
3. Resuspend the cell pellet (second tube) in 1 ml of 0.1 M potassium phosphate buffer. Label
this tube CL (cells).
4. CT, SN, and CL are your test samples.

Part 2: Amylase assay

1. Pipette 1 ml of the iodine reagent into each of six 16 x 150 mm test tubes. Label the tubes
0CT, 20CT, 0SN, 20SN, and 0CL, 20CL.
2. Place 10 ml of phosphate buffer and 150 l of starch solution into each of three 16 x 150
mm test tubes. Mix by gently invert the tubes a few times. Label one tube CT, the second
SN, and the third CL.
3. Remove 2 ml from each of the three test tubes (step 2) and pipette into 1 ml of iodine
reagent in the appropriately labeled 0 tube (step 1). Add 7 ml of water and mix. Read
absorbance at 550 nm. Use water as blank.
4. Add 0.2 ml of test sample (CT, SN, or CL) to the remaining mixture of phosphate buffer
and starch solution in the test tube, as labelled. Mix and place in the 30C water bath.
5. At 20 min, transfer 2 ml to 1 ml of iodine reagent in the 20CT, 20SN, or 20CL test tube,
as labelled). Add 7 ml of water and mix. Read absorbance at 550 nm.

Part 3: Protease assay

1. Add 0.4 ml of BSA to each of four 13 x 100 mm test tubes. Label the tubes 0 (no enzyme),
CT, SN, and CL.
2. Add 0.2 ml of test sample (CT, SN, or CL) to each tube except the tube labeled 0. Incubate
at 30C for 30 min.
3. Add 0.2 ml of ninhydrin reagent and mix. Incubate at 37C for 10 min and observe the
color change.

Part 4: Preparation and analysis of data

1. Subtract the reading at 20 min from the reading at 0 min and define an enzyme unit as a
change of 0.1 A550 units per 20 min. Record your data as units (U)/ml of enzyme for the
amylase or +, for the protease.
2. According to your data, where are the amylase and protease located?
3. Can you think of an explanation of why the protease does not destroy the amylase?

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM 2114 Microbial Biochemistry and Physiology
MAY 2017

Experiment 3

Title: Concentration of Amylase from Bacillus subtilis by Ammonium Sulfate

Precipitation and Separation from Protease by Affinity Purification

Objectives/Goals

1. To concentrate amylase by ammonium sulfate precipitation and also separate it from

protease by adsorbing it to insoluble starch and eluting it with maltose

Introduction

As shown in Experiment 3, Bacillus subtilis secretes protease and amylase into the growth
medium. The enzymes can be separated from each other by adsorbing the amylase to starch and
subsequently eluting it with maltose. You will also concentrate the proteins by ammonium sulfate
precipitation. The amylase will be assayed using the iodine assay described in Experiment 3.
Protein can be concentrated or purified by ammonium sulfate purification. The process is
called salting out of proteins. This is because salts at very high concentrations neutralize surface
charges on the proteins and reduce the effective concentration of water. As a consequence the
proteins interact with each other rather than with water and come out of solution. This can be
used to purify proteins because the concentration of salt required to precipitate a particular
protein reflects the number of charges and their distribution on the protein as well as other
characteristics such as the number and distribution of hydrophobic amino acids that become
exposed as the surface charges are neutralized, as well as the size and shape of the protein. At a
sufficiently high concentration of ammonium sulfate, for example, 85% saturation at 0C, most
proteins precipitate and therefore it is possible to concentrate bulk proteins from a crude extract
using ammonium sulfate.
It is possible to separate the amylase from the protease by adding insoluble starch to the
culture supernatant to adsorb the amylase. The protease is not adsorbed. The amylase can be
subsequently eluted from the starch with maltose (or soluble starch). Schwimmer and Balls were able
to purify the amylase from barley using a similar procedure. The technique of purifying a protein by
adsorbing it to an insoluble ligand is called affinity purification and has been used to purify other
proteins. The principle is that the protein is applied to an immobilized ligand to which the protein has
an affinity. For example, the ligand may be an antibody to the enzyme or a molecule resembling the
substrate, or a cofactor for an enzyme. If column chromatography is used, then the ligand is attached
to a solid support (a resin or gel). The advantage to affinity chromatography is that the protein can be
greatly purified is a single step from a crude extract. Since the amylase will attach to insoluble potato
starch, which can be sedimented by centrifugation, the enzyme can be separated from the protease
and purified without the use of a column.

Materials

Supplies
8 sterile 50-ml centrifuge tubes
16 sterile 15-ml centrifuge tubes

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM 2114 Microbial Biochemistry and Physiology
MAY 2017
2 100-ml beakers
2 glass rods
2 ice buckets containing
ice Bunsen burner
Refrigerated centrifuge
Water baths at 30C and at 37C
Spectrophotometer set at 550 nm and cuvettes

Solutions
50 ml of B. subtilis subsp. spizizenii culture
40 ml of 0.1 M potassium phosphate buffer, pH 6.5
5 mg of solid bovine serum albumin (BSA)
5 g of insoluble potato starch
10 ml of 10% maltose in 0.1 M potassium phosphate buffer, pH
6.5 15 ml of iodine reagent (0.2% I2 + 0.3% KI)
1 ml of 2% soluble starch in 0.1 M potassium phosphate buffer, pH 6.5
50 ml of distilled water
2 ml of BSA, 1 mg/ml in water
2 ml of ninhydrin reagent (3.5 g in 100 ml of 1:1 mixture of acetone and butanol)

Method/Procedures

Part 1: Preparing culture supernatants

1. Pipette 25 ml of B. subtilis culture into each of two 50-ml centrifuge tubes.
2. Centrifuge at 8,000 g for 20 min in a refrigerated centrifuge (set at 4C). Place the resulting
culture supernatants in an ice bath.

Part 2: Precipitating the protein

1. Pipette 25 ml of one of the culture supernatants into a 100-ml beaker sitting in an ice bath
and slowly stir the solution for about 5 min.
2. Add 5 mg of solid BSA and dissolve by stirring. This is added as a carrier protein to ensure
a significant precipitate. What is its final concentration?
3. Add 5 g of solid ammonium sulfate a little at a time and dissolve by stirring. Wait until the
amount you have added dissolves before adding the next increment. It should take around
10 min to add all the ammonium sulfate. This makes an 85% saturated solution at 0C.

4. Continue stirring in the ice bath for 30 min or longer. Transfer the preparation to a 50-ml
centrifuge tube and centrifuge at 10,000 g for 10 min. Carefully decant the supernatant.
Resuspend the pellet in 5 ml of phosphate buffer. Label it N (ammonium sulfate
precipitation). Keep on ice until ready to assay.

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM 2114 Microbial Biochemistry and Physiology
MAY 2017

Part 3: Purification of amylase using insoluble starch

1. Pipette 20 ml of the other culture supernatant into a 100-ml beaker sitting in another ice
bath and slowly stir the solution. The enzyme must be cold during the adsorption to starch
so that the starch is not degraded, which might result in the release of enzyme. Pipette 1 ml
of the remaining supernatant into a 50-ml centrifuge tube. Label this tube C (crude). Keep
on ice until ready to assay.
2. Add 4 g of insoluble potato starch to the supernatant in the beaker. Stir for 30 min so that
starch does not settle to the bottom.
3. Transfer the preparation to a 50-ml centrifuge tube and centrifuge at 10,000 g for 10 min to
sediment the starch.
4. Remove the clear supernatant carefully to a 50-ml centrifuge tube with a pipette. The starch
pellet is not firm. It is all right if you leave just a little bit of supernatant on the starch.
Label the tube U (unadsorbed). You will assay it for enzyme that is not adsorbed. Keep
on ice until ready to assay.
5. Resuspend the starch pellet in 10 ml of maltose solution and transfer the resulting
suspension into a beaker. Stir at room temperature for 30 min. The enzyme binds to
maltose and is released from the insoluble starch.
6. Transfer the preparation to a 50-ml centrifuge tube and centrifuge at 10,000 g for 10 min.
Remove the clear supernatant carefully to a 50-ml centrifuge tube with a pipette. Label this
tube P (purified). This should contain the eluted purified enzyme. Keep on ice until ready
to assay.

Part 4: Amylase and protease assays

1. Perform the amylase assay on N, C, U, and P samples as in Experiment 3. Assay 50 l of
sample each.
2. Perform the protease assay on C, U, and P samples as in Experiment 3. Assay 0.2 ml of
sample each. Remember to include the 0 (no enzyme) tube.

Part 5: Preparation and analysis of data

1. Calculate the % of amylase in the crude that was adsorbed to the starch, the % of amylase
that was eluted from the starch, and the % of amylase precipitated by ammonium sulfate.
For example, suppose 50 l of the crude enzyme catalyzed an absorbance change of 0.76
units in 20 min, then the number of units of enzyme per ml is 0.76/0.05 or 15.2 units per
ml. If you started with 20 ml of enzyme, then you started with 304 units.

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM2114 Microbial Biochemistry And Physiology
MAY 2017

Experiment 4

Title: Protein Separation by Ion Exchange Chromatography

Introduction:
Ion exchange chromatography is used to separate charged molecules, including proteins,
from complex biological samples. Charged substances are separated by column chromatography
with resins that carry charged ionic groups. Biomolecules, such as proteins, with an opposite
charge will bind to the resins. The ionic groups of the columns are covalently bound to a gel
matrix and are protected by small concentrations of counter ions that are present in the buffer.
When a sample is added to the column, an exchange with the weakly bound counter ions takes
place and the charged molecules bind to the solid support.
Proteins contain regions of charged groups on their surface that are formed by the side
groups of charged amino acids, the amino and carboxyl termini of the polypeptide
chains, and other interacting groups. These charged groups are available for interaction and
exchange with ionic groups of the ion exchange columns.
Proteins are multivalent anions or cations and the protonation (addition of a proton) of
protein molecules changes with changes in pH. Under strongly acidic pH conditions, all proteins
are present as cations as a result of the suppression of the dissociation of the carboxy groups and
protonation of the amino groups. At pH values above 12, proteins are present as anions due to
the amino group being a free base and the carboxy group is dissociated. Depending on the total
(net) charge of a protein and the wide range of ion exchange columns available it is simple to
bind proteins of interest to a corresponding charged stationary phase for purification.
During the practical application of ion exchange chromatography it is important to use
pH values that ensure the ionic exchange resins are in an ionized state and the proteins contain
an excess of positive or negative charges, i.e. they are not near their pI (isoelectric point) value,
the net charge is zero.
Increasing the salt concentration results in the shielding of the charges on the proteins
surface and effective binding to an exchanger is inhibited. Also changing the pH of the binding
buffer changes the ionization of the protein charged groups and results in the breaking of the
interaction with the ion exchange columns. As a result, proteins immobilized on an ion
exchange column can be eluted either by increasing the salt concentration or by altering the pH
of the binding buffer, or a combination of the two.
This lab activity involves using a protein mix, consisting of hemoglobin and
Cytochrome C, and running ionexchange chromatography to separate the proteins.

Materials:
1 Cationic Chromatography Column
1 vial Protein Mix
8 ml Equilibration Buffer
1 ml Elution Buffer

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM2114 Microbial Biochemistry And Physiology
MAY 2017

1 bottle CB Reagent (Shares with whole class)

20 centrifuge tubes (1.5 ml)
Stand and clamp

Methods:
Part 1: Ion Exchange Chromatography

1) Add 0.2ml Equilibration Buffer to the Protein Extract vial. Periodically vortex the
tube until the protein completely dissolves.
2) Centrifuge the tube for 2 minutes at maximum speed in a micro centrifuge to remove the
froth.
3) Clamp the Cationic Chromatography Column in an upright position to the stand.
4) Open the top cap first and then the bottom cap of the column to prevent air entering the
resin. Allow the buffer to drain out of the column, under gravity, to a waste container.
Make sure that the column resin evenly settles down in the column.
5) Equilibrate the column: Apply 2 volumes (0.5ml each) of Equilibration Buffer.
Add 0.5ml Equilibration Buffer, allow the buffer to drain out and then apply the
second volume. Let the buffer drain out into a waste container.
* Add the Equilibration buffer slowly to avoid disturbing the resin in the column.
6) Carefully load all the Protein Extract prepared in step 2 to the column.
7) Once the protein sample has entered the column, wash the column 3 times (0.5ml each)
with Equilibration Buffer to remove unbound protein from the column: Collect 0.5ml
wash fractions as the buffer freely drains into labeled 1.5ml collection tubes. Change to a
fresh tube before applying the next wash volume.
8) Elute the sample using a salt gradient: Prepare a salt gradient by mixing
Equilibration Buffer and Elution Buffer, a high salt buffer, as detailed in the table 1.

Salt
Fraction Equilibration Elution
Concentration
# Buffer (l) Buffer (l)
(mM)
1 60 485 15
2 100 475 25
3 140 465 35
4 200 450 50
5 300 425 75
6 400 400 100
7 500 375 125

Table 1: Gradient elution buffer

Bachelor of Science (Hons) Microbiology

Revised May 2017
Universiti Tunku Abdul Rahman
UDBM2114 Microbial Biochemistry And Physiology
MAY 2017

9) Apply 0.5ml of each prepared gradient elution buffer to the column, starting with the
lowest salt concentration (Fraction #1) first.
10) Collect 0.5ml fractions as the buffer freely drains into labeled 1.5ml collection tubes.
Change to a fresh tube before applying the next elution buffer. Collect all 7 elutions in 7
separate 1.5ml tubes.
11) Determine the protein concentration of the fractions by following the protocol in Part II.

Part II. CB Protein Assay

1) Label 10 tubes and transfer 50l elute from each fraction (three washes and
seven elutions).
2) Mix the CB Reagent gently by inverting the bottle several times.
*To avoid foaming, do not shake the bottle
3) Add 1ml CB Reagent to each tube and vortex briefly to mix the content. Incubate the
tubes at room temperature for 5 minutes.
4) Add 1ml distilled water or CB Reagent to a cuvette to zero the absorbance of the
spectrophotometer. Measure the absorbance (595nm) of each tube and record the
values in the results section.

Analysis and result:

1) Prepare a graph showing protein elution profile of ion-exchange chromatography by
plotting the absorbance against the fraction number.

Bachelor of Science (Hons) Microbiology

Revised May 2017