© 2005 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION

Printed in U.S.A. Vol. 33, No. 6, pp. 420 –425, 2005

Laboratory Exercises

A Series of Enzymology-based Experiments Designed to

Mimic an Applied Research Project
Received for publication, April 19, 2005, and in revised form, July 19, 2005

Angela Boyce and Gary Walsh‡

From the Industrial Biochemistry Program, Chemical and Environmental Sciences Department,
University of Limerick, Limerick, Ireland

Four mini-practicals are described in which the effects of temperature and pH on phytase activity are
assessed, as well as the enzyme’s thermostability and the effect upon stability of simulated digestive tract
conditions. Phytase is routinely incorporated into monogastric animal feed to ameliorate the negative
nutritional and environmental consequences of its substrate, dietary phytic acid. In addition to illustrating
selected basic concepts in enzymology, the combined experiments allow the students to determine the
suitability of the test phytase for inclusion in animal feed. As such the practical mimics an applied research
project and is particularly suited to biotechnology students undertaking courses in basic biochemistry.
Students may be segregated into groups of 4, with each team member charged with undertaking one of the
mini-experiments. In this way students are given individual responsibility and learn to work as part of an
integrated research grouping.
Keywords: Enzyme, phytase, environmental biotechnology, biotechnology education.

INTRODUCTION AND EXPERIMENTAL OVERVIEW
Previously we published a paper in this journal outlining
a biochemistry laboratory practical that entailed assay of
the enzyme phytase [1]. Phytase is now routinely included
in monogastric animal feed to ameliorate the negative
nutritional and environmental consequences of its natural
substrate, phytic acid (Fig. 1). As part of that paper the
rationale for addition of selected enzymes to animal feed
was outlined, and a synopsis of the feed enzyme industry
was presented. A number of relevant literature and com-
pany references, which provide comprehensive back-
ground detail of the area, was also provided. Refs. 2– 4
herein provide additional background detail. The practical
described involved the generation of a phosphate (phytase
reaction product) standard curve followed by phytase as-
say and hence determination of its activity.
Herein we describe 4 follow-on phytase-related experi-
ments designed to mimic an applied research project in
which selected physicochemical characteristics of a phy-
tase are determined to assess the enzyme’s suitability for
inclusion in animal feed. The effect of temperature and pH
upon phytase activity is determined and discussed in the
context of physiological relevance. The thermostability of
the enzyme at 90 °C is determined and discussed in the
context of feed heat treatment. Finally the effect of simu-
lated digestive tract conditions upon phytase activity is
investigated. The 4 experiments may be undertaken over
the course of two 2.5-hour practical slots. Alternatively
and preferentially, the students may be segregated into
‡ To whom correspondence should be addressed. E-mail:
gary.walsh@ul.ie.
420
groups of 4, with each member of the “research team”
charged with undertaking 1 experiment. The experiments
are technically straightforward, require only basic labo-
ratory equipment, and provide the scope for a multifac-
eted post-laboratory discussion in the context of applied
enzymology.
EXPECTED LEARNING OUTCOMES
1. Students will gain an enhanced overall understand-
ing of enzymology, enzyme assays, and in particular
the influence of various physicochemical character-
istics upon enzyme activity.
2. Students will gain direct laboratory experience in
spectrophotometry.
3. Students will gain an understanding of buffers and
their importance in the context of pH control.
4. Students will gain an understanding of selected ba-
sic statistical concepts (mean, S.D., statistical sig-
nificance), how to calculate them, and how to pres-
ent the resultant data in tabular and graphical
format.
5. Students will gain an appreciation of working as part
of an integrated research team.
6. Given the number of assay samples involved, stu-
dents will gain an appreciation of the importance of
clear labeling of all reagent and reaction vessels.
EXPERIMENT 1: THE EFFECT OF TEMPERATURE ON
PHYTASE ACTIVITY
Experimental Protocol—The phytase assay is under-
taken exactly as outlined in the previous paper [1] with the
exception of the assay temperature employed. Assays are
This paper is available on line at http://www.bambed.org
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421

FIG. 1. The reaction catalyzed by the enzyme phytase.

carried out at 20, 30, 40, 50, 60, and 70 °C. For each assay
time point the assay is carried out in duplicate and with an
associated assay blank.
Calculation, Presentation, and Discussion of Results—
Students may calculate enzyme activity levels at each
temperature using the inorganic phosphate standard curve
generated in the initial practical [1]. As described therein,
activity may be calculated either in international units or in
nkat. Once activity levels at all temperatures are calculated FIG. 2. The effect of temperature upon the activity of the test
the highest activity level is assigned a relative activity of phytase.
100%. Activity levels at other temperatures are calculated
relative to this. The results are presented graphically as
shown in Fig. 2. Students are then asked to discuss the
results in the context of the enzyme’s intended use. The
discussion would be expected to include points such as
the enzyme’s optimum temperature (50 °C) is not a phys-
iologically relevant temperature. However, the enzyme dis-
plays significant activity (60% of optimal activity) at phys-
iological temperatures. Moreover, some may make the
point that the retention of catalytic activity at temperatures FIG. 3. The effect of pH upon the activity of the test phytase.
below physiological temperature is also important as the
animal feed is normally ingested cold and as such the intended use. The discussion should center on the likely
initial stages of digestion will occur at temperatures below activity levels within the pH range generally encountered
37 °C. within the upper gastrointestinal tract (salivary gland, pH
5.0 –7.0; stomach, initial fed state circa pH 6.5, dropping to
EXPERIMENT 2: EFFECT OF pH UPON PHYTASE ACTIVITY pH 3.5– 4.5 upon stimulation of gastric acid secretion; the
Experimental Protocol—The phytase assay is carried duodenum, circa pH 6.0 – 6.8).
out at pH 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0.
Substrate (0.1% w/v phytic acid) is made up using the EXPERIMENT 3: ENZYME THERMOSTABILITY AT 90 °C
following range of 0.2 M buffers: KCl-HCl (pH 2.0), glycine- Experimental Protocol—200-!l aliquots of suitable en-
HCl (pH 3.0), sodium acetate (pH 4.0 and 5.0), Tris-male- zyme dilutions (see technical/preparatory notes) are pipet-
ate-NaOH (pH 6.0, 7.0, and 8.0), and the initial enzyme ted into 200-!l PCR tubes. A standard laboratory PCR
dilution is prepared in water (see technical/preparatory notes block is used to heat individual samples at 90 °C for 5, 10,
section). The actual assay is conducted as described in Ref. 15, and 20 min. Samples are placed on ice immediately
1. For each assay pH value, the assay is carried out in upon removal from the PCR block. An unheated sample,
duplicate and with an associated assay blank. kept on ice for the duration of the experiment, serves as a
Calculation, Presentation, and Discussion of Results— control. All samples are diluted 20-fold in sodium acetate
Students may calculate enzyme activity determined at buffer, pH 5.5, and are subsequently assayed [1].
each pH value using the inorganic phosphate standard Calculation, Presentation, and Discussion of Results—
curve generated in the initial practical [1]. Once all activity Once activity levels of the control (unheated) and all heated
values are calculated, the highest activity level is assigned samples are calculated, the activity level of the unheated
a relative activity of 100%. Activity levels at other pH sample is assigned a relative activity of 100%. Activity
values are calculated relative to this. The results are pre- levels of all other samples are calculated relative to this.
sented graphically as shown in Fig. 3. Students are then Typical results obtained are presented in Fig. 4. Students
asked to discuss the results in the context of the enzyme’s then are asked to discuss the results in the context of the
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422 BAMBED, Vol. 33, No. 6, pp. 420 –425, 2005

FIG. 5. Stability of test phytase upon its incubation under

simulated stomach and small intestine conditions.
FIG. 4. Typical thermostability profile (90 °C over 20 min) of
the test phytase. machine (although a water bath or standard heating block
will suffice if necessary).

enzyme’s intended use. In this context students need to be Chemicals/Reagents Required

made aware that all animal feed is now routinely heat
The chemicals and reagents required to undertake the
treated as a pathogen control measure. Treatment condi-
phytase assay are described in our previous paper [1].
tions vary, but generally temperatures of 75–95 °C with
Enzyme—Phytase derived from Aspergillus ficuum,
holding temperatures ranging from several seconds to
(Sigma product number P-9792). Different dilutions of the
several minutes are employed [5]. Therefore the more ther-
commercial enzyme are prepared for the 4 experiments as
mostable the enzyme is, the better.
follows:
EXPERIMENT 4: EFFECT OF SIMULATED DIGESTIVE TRACT
A suitable dilution for experiments 1 and 2 falls within the
CONDITIONS UPON PHYTASE ACTIVITY 1/10,000 –1/25,000 dilution range. For experiment 1 the
commercial enzyme is diluted using 200 mM sodium ace-
Experimental Protocol—Determination of simulated gas-
tate buffer, pH 5.5 (dilution to be prepared within 24 h of
tric conditions entails the co-incubation of 5.0 ml of suit-
practical commencement and stored at 4 °C until imme-
ably diluted enzyme and 5.0 ml of pepsin solution (see
diately prior to practical commencement). For experi-
technical/preparatory notes) at 37 °C for 15 min followed
ment 2 the enzyme is diluted using distilled water only
by dilution to 25 ml with 0.2 M sodium acetate buffer, pH
and immediately prior to commencement of the practi-
5.5. The resultant solution is immediately subjected to the
cal. For experiment 3 a 1/500 –1/1,250 dilution is pre-
standard phytase assay [1].
pared in 200 mM sodium acetate buffer, pH 5.5. For
Determination of simulated small intestinal tract condi-
experiment 4 a 1/2,500 –1/5,000 dilution of the enzyme is
tions entails co-incubation of 5.0 ml of suitably diluted
prepared in distilled water immediately prior to practical
enzyme with 5.0 ml of pancreatin solution (see technical/
commencement.
preparatory notes) at 37 °C for 15 min followed by dilution
Additional Chemicals/Reagents—For experiment 1 and
to 25 ml with 0.2 M sodium acetate buffer pH 5.5. The
3: none. For experiment 2: substrate (0.1% w/v phytic
resultant solution is immediately subjected to the stand-
acid) made up in the following 0.2 M buffers: KCl-HCl (pH
ard phytase assay [1]. A control for both experiments is
2.0), glycine-HCl (pH 3.0), sodium acetate (pH 4.0 and 5.0),
undertaken by substitution of the 5.0 ml of pepsin (or
Tris-maleate-NaOH (pH 6.0, 7.0, and 8.0). Buffers are pre-
pancreatin) with 5.0 ml of 0.2 M sodium acetate buffer,
pared as described in Ref. 6.
pH 5.5.
For experiment 4: Pepsin solution (3.2 g of porcine pep-
Calculation, Presentation, and Discussion of Results—
sin/liter, prepared in 0.1 M glycine-HCl buffer pH 2.5).
For each experiment the control activity value is assigned
Porcine pepsin purchased from Sigma (product number
a relative activity of 100%, and activity levels of the other
P-7000). Pancreatin (porcine) solution: 10.0 g of porcine
samples are calculated relative to this. Typical results ob-
pancreatin (Sigma, P-1500)/liter, prepared in 0.1 M Tris-
tained are presented in Fig. 5. Students then should dis-
malate-NaOH buffer, pH 6.8.
cuss the findings in terms of the enzyme’s intended use.
Their discussion should center on the likely stability of the SAFETY AND REAGENT DISPOSAL ISSUES
enzyme in the context of physiological parameters asso-
Material and Safety Data Sheets for all the chemical
ciated with the stomach and small intestine, in particular
reagents are available from the manufacturer/distributor,
pH values and the presence of proteolytic enzymes.
and are generally supplied automatically along with the
TECHNICAL/PREPARATORY NOTES product. Most reagents/equipment items pose relatively
minor safety hazards. Students should however be re-
Equipment Required
minded of the corrosive nature of the trichloroacetic acid
A spectrophotometer capable of measuring sample ab- solution and that phytase assay reagent A contains H2SO4.
sorbency values at a wavelength of 660 nm. Several water Contact of these reagents or buffers of low pH with skin
baths (water baths set at 6 different temperatures are should be followed at minimum by thorough rinsing of the
required to undertake experiment 1, although the number effected area. Students should also take basic precautions
of data points can be reduced and/or the assays can be to avoid burns when using the water baths or exposed
undertaken sequentially using the same water bath se- heating blocks set at elevated temperatures. As under-
quentially set at increasing temperatures). A standard PCR taken in our laboratories, contents of assay tubes are
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423
disposed of by pouring down the sink concurrently with information in the practical manual should be sufficient. On
running water. Contact of pepsin or pancreatin-containing the other hand, if the students have little or no prior expo-
solutions with skin should be followed by thorough rinsing sure to statistical analysis, a more comprehensive back-
of the effected area (in case of potential dermatological ground treatment will be required. This may require at least
sensitivities). Students with known sensitivities should one dedicated lecture prior to the practical coupled with a
wear gloves. detailed treatment in the practical manual. Basic statistical
principles and calculation methodologies may be obtained
ASSOCIATED PRACTICAL TASKS
from a wide range of statistics textbooks but are often
Statistical Analysis presented in a more appropriate context in books specif-
As described, each assay requires 3 reaction tubes (du- ically aimed at students of the biological sciences (e.g.
plicate assay tubes and an assay blank). From a pragmatic Refs. 7 and 8). Students may calculate the appropriate
standpoint large student numbers, limited time, and often mean and S.D. values manually but (assuming availability)
limited resources render impractical the inclusion of a may more conveniently use basic statistical software
greater number of assay replicates. Students, however, packages such as the statistical functions associated with
should be made aware of the desirability of using greater the widely used Microsoft Excel package. Again, depend-
numbers of replicates when generating datasets. Further- ing upon prior exposure levels, students may require a short
more, at some stage of their program of study, students computer-based demonstration and may find it helpful to
should gain an understanding of basic, relevant statistical consult the “help” function of the program in use and/or the
analysis and how to carry out such analyses in practice. As explanatory booklets often provided along with the software
part of this practical, we provide some application-relevant or other relevant publications (e.g. Refs. 9 and 10).
data that the students can manipulate statistically. The
manipulation required entails the calculation of mean val- Questions for Students
ues, standard deviations and determination of statistical
significance. Table I provides a (theoretical) phytase ther- Listed in our previous paper [1] is a series of 4 questions
mostability dataset on which the calculations may be that are directly relevant to this practical. Additional ques-
based. The students should be asked to calculate mean tions could include:
values and standard deviations (S.D.) and should present 1. The enzyme’s pH profile displays twin activity
these in tabular form (Table II) and in graphical form (Fig. peaks, one at 2.0 and one at 5.0. Generally pure
6). Students can also be asked to investigate: (a) whether enzymes display single pH activity peaks with ac-
the observed differences in thermostability after 1 versus 5 tivity levels falling off on either side. The phytase you
min are statistically significant (answer is no, p ! 0.05) and are working with is an unpurified, extracellular prep-
(b) whether the observed differences in thermostability aration. It is produced by growing the producing
after 1 versus 10 min are statistically different (answer is fungus in a fermentation vessel followed by removal
yes, p " 0.05). of the fungus and drying of the enzyme-containing
Prior to undertaking these calculations the students will fluid left behind. Can you think of any potential expla-
require appropriate background tuition in statistical anal- nation(s) as to why two activity peaks are evident?
ysis. If students have already undertaken an appropriate
Answer: In this particular instance, the reason for the
maths/stats module, a 3– 4 min “refresher” incorporated
twin activity peaks relates to pH-dependant differences in
into the pre-practical talk and/or the inclusion of summary
charge distribution at the substrate binding site, which
TABLE I influences substrate interaction. This detailed biochemical
Thermostability of a phytase upon heating to 90 °C over the reason will almost certainly be beyond undergraduate bio-
indicated time course chemistry students. However, obtaining the correct result
For each time point, the assay was undertaken in quadruplicate. is secondary to developing some type of plausible theory.
The data are presented as percent residual activity remaining, as Students (particularly those also taking modules in basic
compared to an unheated sample
microbiology) for example may conjecture that there may
Residual activity be two different phytases present in the crude preparation
1 min 5 min 10 min 15 min 20 min and that the result obtained is a composite profile of both.
% % % % % Other students may conjecture that a proportion of the
90 86 73 66 50 phytase molecules present have been modified (oxidized,
85 85 75 62 54 subjected to partial proteolysis, etc.) and that the modified
91 84 70 66 55
88 88 71 63 57
enzyme molecules, while retaining activity displays an al-
tered pH versus activity profile.
TABLE II
Thermostability of a phytase upon heating to 90 °C over the indicated time course
The residual activity data are presented as mean values # S.D. (n $ 4).
Residual activity
1 min 5 min 10 min 15 min 20 min
% % % % %
88.50 # 2.65 85.75 # 1.71 72.25 # 2.22 64.25 # 2.06 54.00 # 2.94

424
FIG. 6. Thermostability profile of a theoretical phytase (con-
structed using thermostability dataset presented in Table I).
Each point represents mean value # S.D. (n $ 4).
2. Incubation of the enzyme under simulated small
intestine conditions results in a greater activity loss
than incubation under simulated gastric conditions.
Can you think of any theoretical reasons as to why
this is so?
Possible answer: The enzyme is likely far more stable at
low pH values than at neutral pH values. Although this was
not tested directly, the results from the pH versus activity
experiment showed the enzyme to be maximally active at
low pH while retaining little/no activity at pH values ap-
proaching 7. In general you would expect an enzyme to be
most stable at pH values similar to those at which it is
maximally active. Also, the enzyme may be far more sus-
ceptible to proteolytic degradation by pancreatin as com-
pared with stomach pepsin.
3. For experiments 1 and 3 you have been provided
with an enzyme solution prepared in buffer, but for
experiment 2 the enzyme has been prepared in
water. Why do you think that is so?
Answer: Experiment 2 entails assay of the enzyme at
various specific pH values. The substrate is prepared in
buffers set at the desired pH values. By preparing the
enzyme in water, the buffering effect of the resultant solu-
tion will be minimal, and you can be sure that the final
assay pH (upon mixing substrate and enzyme) will be at
the correct, intended value.
4. Explain how in practice you would make up 500 ml
of the following buffer solutions:
(a) 0.1 M sodium phosphate buffer, pH 7.4
(b) 250 mM citric acid-sodium citrate buffer, pH 5.6
Answer: To answer this question the students should be
provided with the molecular weight of the various con-
stituent buffer components, i.e. Na2HPO4. 2 H2O $
178.05, NaH2PO4. H2O $ 138.01, C6H8O7. H2O $
210.14, C6H5O7Na3. 2 H20 $ 294.12. They also should
be provided with a table (for both buffers) showing the
ratios in which both buffer components need to be
mixed to achieve a specific pH. Such tables are avail-
able in Ref. 6. Students would then have to calculate
how much of each buffer component to make up and
demonstrate how to make up that volume of solution at
the appropriate concentration (0.1 M for the phosphate
buffer and 0.25 M for the citrate buffer). If students have
not previously studied the theory of buffers and how
they are prepared in practice, such information must be
detailed prior to commencement of the practical.
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BAMBED, Vol. 33, No. 6, pp. 420 –425, 2005
5. Commercial phytase preparations are sold in liquid,
powder, and granulated forms. In addition, some
granulated forms are coated with lipid to enhance
their in-feed stability. Can you think of any potential
advantages of powdered over liquid formulations
and how do you think a lipid coating might enhance
enzyme stability?
Answer: By removing the water chemical reactivity can
be reduced, therefore the enzyme may not be as suscep-
tible to chemical inactivation or inactivation by e.g. con-
taminating proteases or due to microbial spoilage. Also by
removing the water the product’s weight is reduced which
can reduce shipping costs. Lipid coating could enhance
stability by for example shielding the enzyme from (hydro-
philic) denaturants/inactivation agents.
DISCUSSION
This practical provides students with an opportunity to
directly determine the effects of temperature, pH, and
proteolysis upon enzyme activity. In addition to reinforcing
selected principles of enzymology, it demonstrates the
practical significance of such properties in the context of
applied/industrial enzymology. All students can undertake
all 4 experiments over 2 laboratory sessions. It is arguably
preferable to group the students into teams of 4, with each
member of the “research team” charged with undertaking
1 experiment. In this way students are given individual
responsibility and learn to work as part of an integrated
research grouping. Individual students are generally com-
fortable with this as the phytase assay, which they have
undertaken as part of the previous practical [1], forms the
core of each current experiment.
The students must then share their results and prepare
a laboratory report. Again, in keeping with the research
team theme, preparation of the report as well as answering
the associated questions should be a team effort.
As designed and presented here, each team is given the
same enzyme to work with. The “research feel” of the
practical could be strengthened if each team were pro-
vided with a different phytase to evaluate. This would
depend upon local availability of additional phytases. Po-
tential sources could include enzyme samples from com-
mercial phytase manufacturers (listed in Ref. 1). Sigma
also stocks a plant (wheat)-derived phytase (product num-
ber P 1259). Alternatively various phytase producing fungal
strains (e.g. listed in Refs. 11–13) could be purchased from
the American Type Culture Collection, cultured in phytase-
inducing media (Ref. 13), and the resultant crude extra-
cellular phytase used as the test enzyme. For most de-
partments, however, limited preparatory time and
resources render the use of the single commercially
available phytase the most pragmatic. The research feel
could also be enhanced by (rather than providing the
pre-designed practical) asking the students to design
their own set of experiments to assess the suitability of a
candidate phytase for in-feed suitability. Should time,
flexibility, and experimental design allow, students could
then pursue their own experiments.
The statistical analysis section of the proposed practical
can be omitted, if desired. However in our experience, a
significant proportion of students undertaking degrees in

biological sciences graduate with a less than comprehen-
sive grasp of statistical methodologies. Our experience
would also indicate that teaching statistical principles in a
discipline-related context is more effective than the use of
an off the shelf, generic statistical module offered to mul-
ticohorts of students by maths/stats departments. The
relevance of the statistical analysis undertaken as part of
this practical would be immediately obvious to the stu-
dents, and an understanding of and the ability to under-
take such statistical calculations would be particularly im-
portant in the case of those students who undertake a
career in research.
The fourth student question included relates to buffer
preparation. Buffers play a particularly significant role in
experiments 2 and 4. We find it helpful to engage students
in practicing buffer calculations well before they get to the
stage of undertaking independent laboratory research.
Overall the practical is technically straightforward and
requires relatively basic laboratory equipment and inex-
pensive chemicals. The reagents by and large present little
safety hazard, and prior reagent preparation by technical
personnel is relatively straightforward. The results gener-
ated, along with the student questions, provide scope for
an in depth analysis and discussion of the topic under
consideration.
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425
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[12] E. J. Mullaney, C. B. Daly, A. H. J. Ullah (2000) Advances in phytase
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