PRACTICAL
BIOTECHNOLOGY
Glucoamylase production by
yeast
ORDINARY brewer’s yeast (Saccharomyces cerevisiae) is able to
utilise a variety of carbohydrates as an energy source. These include
glucose, sucrose and maltose. While sucrose is readily available (as
cane or beet sugar) glucose and the other sugars must be prepared by
enzymic or chemical hydrolysis of starch. This is the main puropse of
malting in traditional beer brewing. All these sugar feedstocks are
relatively expensive.
Unlike S. cerevisiae, the yeast S. diastaticus is able to grow on starch
and dextrins. This is because it makes an extra-cellular enzyme,
glucoamylase, which catalyses the hydrolysis of -1,4 glycosidic bonds
in starch. It does this by progressively chopping off single glucose
molecules from the ends of amylose chains. (Some glucoamylases can
also attack the branching -1,6 bonds of amylopectin, but at a much
slower rate than -1,4 bonds.)
Great interest has focussed in recent years on transferring the gene
for glucoamylase into S. cerevisiae by sexual hybridization with S.
diastaticus. Such hybrids can grow on cheaper substrates and better
utilize the carbon present in conventional feedstocks. This increases
the yield of ethanol and allows the yeast to out-compete any bacterial
contaminants which might lead to off-flavours in alcoholic drinks. (S.
diastaticus can not be used directly for brewing, as it produces a
compound which gives beer a spicy phenolic flavour.)
This investigation of glucoamylase production by S. diastaticus uses an
NCBE Bioreactor, although it may easily be adapted for use with
other fermenters. Alternatively, the yeast can be used for a simple
investigation of amylase production on a starch agar plate.
Materials
Culture of Saccharomyces diastaticus
(available from the NCBE)
Broth containing 1% starch, 1% yeast extract
and 1% peptone, 500 cm3 (put 20 cm3 of the
broth into a McCartney bottle, for use as an
inoculum; put 450 cm3 into the Bioreactor).
NCBE Bioreactor (+ air pump and water bath)
For the glucoamylase assay method
Centrifuge
Starch solution, 1%
Semi-quantitative glucose test strips e.g.
Boehringer Mannheim Diabur-Test® 5000
(available from high street chemists)
Waste container of disinfectant
diastaticus in a McCartney bottle containing 20
cm3 of starch yeast broth. Incubate at 28–30C.
Day 2
3.
Running the Bioreactor
4.
1.
2.
Set up and autoclave the NCBE Bioreactor as
described in the User Guide, using 450 cm3 of
starch yeast broth. After autoclaving, allow the
vessel to cool, and attach sterile syringes etc. as
instructed in ther Bioreactor manual.
Prepare an inoculum of Saccharomyces
Bubble air through the culture medium to
ensure that the conditions within remain
aerobic. Add antifoam as required to prevent
undue frothing. Take care to ensure that the air
filters do not become blocked with liquid.
Monitoring starch breakdown
5.
6.
7.
8.
9.
Take small samples of broth from the
Bioreactor at regular intervals, using the
method described in the Bioreactor manual.
Put the sample into a centrifuge tube, cover
with Cling film to prevent the formation and
escape of aerosols. Spin down the yeast cells in
a properly-balanced centrifuge. Take some of
the cell-free supernatant to test for
glucoamylase activity.
Add an equal volume of 1% starch solution to
the cell-free broth. Incubate at 30C for 6
hours or overnight if more convenient.
Use a semi-quantitative glucose test strip to
measure the concentration of glucose in the
reacted mixture.
Dispose of used test samples and glucose test
strips into disinfectant.
Safety
Standard microbiological safety procedures, including
aseptic techniques, must be observed by teachers,
technicians and students when carrying out this work.
Teachers are referred to: ‘Microbiology. An HMI guide
for schools and further education’ (1990) HMSO as
well as any safety guidelines produced by their LEA
and / or school governing body.
Particular care should also be taken when using the
centrifuge, and steps such as those described above
should be taken to prevent the formation of aerosols.
Further activities
1.
If a colorimeter or spectrometer is available,
measure the turbidity of the culture sample
before testing it for enzyme activity. This will
give an indication of the growth of the yeast.
2.
Before testing the culture samples for enzyme
activity, measure the concentration of glucose
in each, using a glucose test strip.
3.
Investigate the effect of growing S. diastaticus
on substrates other than starch. Does it still
produce glucoamylase in these conditions?
Practic al details
Advance preparation
Day 1
Place the Bioreactor in a water bath at 28C.
Allow the broth inside to warm to this
temperature, then inoculate the Bioreactor
using the culture prepared in (2) above.
Glucoamylase
production
Set up and inoculate the Bioreactor as described in
the User Guide and the accompanying notes.
Use starch broth in the flask and Saccharomyces
diastaticus as the inoculum. The yeast secretes an
enzyme, glucoamylase, into the culture medium.
This breaks down the starch to glucose.
Try to predict
— what will happen to the number of yeast cells
during the fermentation
— what will happen to the enzyme concentration
— what will happen to the glucose concentration
Enzyme assay
Record the
glucose
concentration,
then put the
used test
strips into
disinfectant
Mix equal volumes
of 1% starch
solution and cellfree broth
Spin down the yeast
cells, then take some
of the cell-free
supernatant
Incubate at
30C, then test
with a glucose
test strip
glucose units joined in an amylose chain
glucoamylase
hydrolyses -1,4 bonds,
splitting glucose units
from the end of the chain
CH 2 OH
CH 2OH
O
HO
O
OH
CH 2 OH
O
-1,4- glycosidic
bond
OH
CH 2 OH
O
O
OH
OH
glucose molecule
split from chain
Present your
findings as a
graph
O
O
OH
OH
O
OH
OH
How the amylose chains in
starch are broken down by
glucoamylase
© National Centre for Biotechnology Education, 1995