1. A Review of Published α-Amylase Assays Used in Bacterial & Fungal
Production Systems
Philip Tucker
[C11484968]
BSc Degree in Biosciences
School of Biological Sciences
Dublin Institute of Technology
Kevin Street
Dublin 8
May 2014
Supervisor: Gwilym Williams
2. ii
ACKNOWLEDGEMENTS
I would like to thank the following people for making this dissertation possible. I would like
to thank my supervisor, Gwilym Williams, for giving me all the necessary information and
guidelines to produce the best paper that I could. I would like to thank our course co-
ordinator, Orla Howe, for providing the clear and concise instructions as to how to best lay
out my dissertation. I would like to thank Robert Lynch for providing me with relevant
information related to my topic as well as important references to help further my research. I
would finally like to thank Eugene Kelly for providing useful data that was essential for
optimising my results.
ABSTRACT
This dissertation investigates the methods used to measure alpha-amylase activity and how
they differ from one another with regard to principle. The aim is to provide a comprehensive
review of all the available information on amylase assays, differentiate them and compare
them according to cost, efficiency and ease of execution. Extensive research was carried out
using all available resources, including relevant books, journals and websites, to provide a
clear picture of the scope of variation between methodologies. The significant findings were
recorded, described and compared. Some focus was put on highlighting the advantages and
disadvantages of each assay, as well as the potential direction for future development.
4. 1
CHAPTER 1
INTRODUCTION
1.1 α-amylase (E.C.3.2.1.1)
Amylase is an enzyme that acts on polysaccharides such as starch and glycogen.
Starch is the form that plants use to store glucose for energy and glycogen is the storage form
for humans. Any living organism that utilises glucose must be able to break down these
energy stores and, for this reason, amylase is one of the most common enzymes to be found
in nature. It comes in three different forms; α-amylase, β-amylase and γ-amylase, which is
more commonly named glucoamylase [Pandey & Ramachandran, 2006]. They are each given
their own enzyme classification as each has its own specific function. When breaking down
starch or glycogen α-amylase (AA) produces dextrins, β-amylase produces maltose and
glucoamylase produces glucose units. The focus of this dissertation is on AA specifically.
AA was first discovered in 1833 by two scientists named Anselme Payen and Jean-
Francois Persoz [Needham, 1970]. Since then, vast study has been carried out on the activity
of the enzyme, for example, in catabolite repression and in enzyme induction. There has been
major development in its utilisation, especially in the industry sector, and as a result the
market for AA has grown exponentially. It is currently being used in the textile, bakery, sugar
syrup and detergent industries and has an estimated market value of $1.6 billion [Mohbini-
Dehkordini et al, 2012]. Amylases alone make up approximately 30% of enzyme production
throughout the world. Any of the above mentioned areas that are utilising AA need to be able
to test its activity in order to ensure it is performing to the desired standard. This test is
known as an enzyme assay and because AA is such a popular enzyme, various different
assays have been produced in order to test it. The aim of this dissertation is to carry out a
comprehensive review of the assays available and to provide a good comparison of each,
focussing on areas such as cost and efficiency.
5. 2
1.1.1 Activity
An enzyme is classified by the reaction that it catalyses. It is given a specific number,
called an Enzyme Commission or EC number for short, which describes its activity. AA is
given the classification number E.C.3.2.1.1 and its official name is 1,4-α-D-Glucan
glucanohydrolase. According to enzyme nomenclature there are six different categories of
enzymes, which are as follows: 1. Oxidoreductases 2. Transferases 3. Hydrolases 4. Lyases
5. Isomerases 6. Ligases [Palmer, 1995]. AA is classified as a hydrolase enzyme and
therefore its first EC number is three. It carries out hydrolysis, which is the process whereby a
molecular bond is cleaved by the addition of a water molecule [Palmer, 1995]. The second
number in the EC code denotes the type of bond that undergoes hydrolysis. If an ester bond is
hydrolysed, for example, the number one is given. In the case of AA, it hydrolyses glycosidic
bonds which connect carbohydrate units and thus, is given the number two. The third digit
further describes the bond being hydrolysed. An enzyme with the code E.C.3.2.1 is referred
to as a glycosidase enzyme which hydrolyses O- and S-glycosyl compounds specifically
[Palmer, 1995]. AA is a glycosidase that breaks down large polysaccharides in to smaller
constituents. When starch is broken down by AA, hydrolysis occurs on the α-amylose and
amylopectin molecules that make it up, resulting in a lower molecular weight product known
as dextrin [Ophardt, 2003].
1.1.2 Structure
Although the sequencing of AA has been shown to vary greatly between different
domains and species, there are certain characteristics displayed that are common to all
[Janecek, 1994]. Research shows that the catalytic domain is in the form of a (beta/alpha)(8)-
barrel, which consists of a beta barrel with 8 parallel strands, surrounded by 8 alpha helices
[Wierenga, 2001]. It forms a doughnut shape and is more commonly known as a
triosephosphate isomerase (TIM) barrel, named after the first enzyme discovered to utilise the
structure [Macgregor et al, 2001; Wierenga, 2001]. It is the most recurring enzyme fold on
the Protein Data Bank and is used by some of the most effective enzymes [Wierenga, 2001].
This suggests that it plays an important part in the catalytic ability of AA. The active site of
AA is located at the C-terminal end of the beta barrel and contains two aspartic acid residues,
as well as a glutamic amino acid residue [Macgregor et al, 2001; Goodsell, 2006]. These take
part in the cleavage of the sugars in the polysaccharide. Another common characteristic of the
AA enzyme, is two histidine residues that act to stabilise it during transition state [Macgregor
et al, 2001].
6. 3
1.1.3 Function
AA is an endo-enzyme, meaning that it does not act on the non-reducing end of a
molecule but rather cleaves the internal bonds [Sivaramakrishnan et al, 2006]. The bonds that
AA hydrolyses specifically, are α1→4 glycosidic bonds that covalently link glucose
monomers in polysaccharides such as glycogen and starch [Walsh, 2002]. Starch consists
completely of glucose units, which are arranged in to the two previously mentioned forms, α-
amylose and amylopectin. α-amylose is a linear polymer that is made up of successive D-
glucose monomers, covalently linked by a glycosidic bond. The bonds are in the α1→4
conformation. Amylopectin is similar to amylose, in that it too has a linear portion made of
α1→4 linked monomers, however it also has branch points every 25-30 residues
approximately, as a result of an α1→6 linkage [Walsh, 2002]. Glycogen contains higher
levels of glucose and has more frequent branch points than amylopectin, occurring every 8-10
glucose units [Ophardt, 2003]. The complexity of the molecule impacts how efficiently the
enzyme can carry out hydrolysis, for example a glycogen molecule has significantly more
glucose units than a starch molecule, so therefore requires more time and energy to be broken
down.
Figure 1: Structure of alpha-amylase highlighting three acidic groups (green), five sugars (yellow), the site of
cleavage (pink) and a calcium ion (grey) [Goodsell, 2006].
7. 4
1.1.4 Sources
AA is manufactured by plants, animals and microorganisms alike due to the
abundance of polysaccharides in nature, particularly starch and glycogen, which need to be
broken down for energy. For humans, starch is a main dietary constituent and they, therefore,
produce their own type of AA. It is produced in two forms, salivary and pancreatic, which aid
in the breakdown of starch to maltose in the digestive system [Goodsell, 2006]. The
production of AA by fungi, yeasts and bacteria has proven to be of more use in the industry
sector than plant and animal amylases. The reason for this is that the stability of microbial
enzymes is greater than that of plants and animals, and they have a broader spectrum of use
[Mohbini-Dehkordhi et al, 2012]. Enzyme production companies are able to utilise
microorganisms’ ability to produce enzymes in bulk. This is a great advantage and is very
economically beneficial. They are able to produce enzymes with specific characteristics by
manipulating the microorganisms. This proves to be a cost effective, consistent and fast way
for the companies to achieve their desired results.
The range of AA producing organisms for use in industry is large, with many
different species available to choose from. The Bacillus spp. is one of the most common
species used for the industrial production of AA, due to its thermostability in reactions
[Sivaramakrishnan et al, 2006]. The most popular strains for use are B. amyloliquefaciens, B.
subtilis, B. licheniformis and B. stearothermophilus. A thermostable enzyme provides certain
advantages for industrial processes, such as minimising D-glucose polymerisation to iso-
Glucose
Molecule
Branch point
Glucose
Molecule
Branch Point
Figure 2: Picture showing the structure of amylose and amylopectin, highlighting the α1→4 bonds hydrolysed by alpha-
amylase [El-Fallal et al, 2012].
8. 5
maltose, and decreasing contamination risks by using increased temperature reactions
[Kavanagh, 2005]. Fungi are not as thermostable as bacteria and are therefore not as
commonly used, however certain filamentous fungi, such as Aspergillus spp. are good
amylase producers. Aspergillus oryzae is an example of a popular fungus that has high
amylase activity and strong starch degrading capability [Silambarasan et al, 2013].
1.1.5 Commercial Use
Development of biotechnology over recent decades has caused a huge advance in
enzyme technology. Microbial AA has found use in a wide range of industrial applications. It
is currently being used for starch conversion and fuel alcohol production, as well as in the
detergent, food, textile and paper industries. Starch conversion involves gelatinisation,
liquefaction and saccharification and is the process used to convert starch in to glucose and
fructose syrup [Gupta et al, 2003]. Fructose is a very sweet molecule that is widely used in
carbonated soft drinks. Gelatinisation uses water and heat to form a viscous solution, which
can then be acted on by AA in the process of liquefaction [de Souza et al, 2010]. The partial
hydrolysis of the starch by AA causes the loss in viscosity. The amylases used in this process
are required to perform at high temperatures and therefore, those produced by B.
stearothermophilus and B. licheniformis are popular. Saccharification is utilised for the
production of ethanol. A glucoamylase enzyme produces glucose and maltose sugars after
liquefaction, and from there, fermentation of those sugars to ethanol can take place [Mojsov,
2012]. An ethanol fermenting microorganism such as yeast Saccharomyces cerevisiae can
carry out the fermentation procedure [Moraes et al, 1999].
Detergent industries are the primary consumers of enzymes with 90% of all liquid
detergents containing amylases [Mitidieri et al, 2006]. Amylases are very useful in removing
starchy food stains such as chocolate, as well as keeping particulate soils that are attracted to
starch from dulling white clothes. Their popularity comes from their ability to perform at
lower temperatures and to withstand the alkaline pH of the oxidising environment in the
washing machine [de Souza et al, 2010]. The food industry uses AA in areas such as baking.
It can increase fermentation of yeast which in turn reduces the viscosity of dough, improving
its texture. It also increases the amount of sugar in the dough, improving the taste, and has an
anti-staling effect which increases its shelf life [Mojsov, 2012]. The paper and textile
industries use a technique known as sizing that often uses starch. For textiles this technique
provides a fast and secure weaving process and for the paper industry it provides a protective
coating. Desizing involves the breaking down of the starch by AA. It removes the starch from
9. 6
woven fabric without affecting the fabric itself. The starch used to strengthen paper must be
partially broken down so as not be too rigid and to allow some elasticity, thus AA is used.
1.2 Measurement of enzyme activity
An enzyme is a biological catalyst that speeds up a chemical reaction without being
used up in it. The reaction would still take place regardless of the enzyme’s presence, only it
would occur at a much slower rate. Most enzymes are proteins. They bind to a molecule
known as the substrate, causing a conformational change and, in turn, a chemical reaction to
take place, forming a product. The rate at which the enzyme carries out this function can be
measured in a process known as an assay. Two standard units of measurement are widely
used in the measurement of enzyme activity; the International Unit and the Katal. The
International Unit was devised first in 1961 by the International Union of Biochemistry. They
described it as the amount of enzyme that would transform one micromole of substrate into
product in one minute at 25°C [International Union of Biochemistry, 1979]. The Commission
on Biochemical Nomenclature later described their own unit of enzymatic activity to adhere
to that of the Système International unit. It measures the increase of reaction rate in an
enzyme assay, given in micromoles per minute and is called the Katal. Although these two
units are considered the standard units of measurement, they are not the only ways in which
enzymatic activity is described. With the development of new assays containing different
enzymes, reagents and components, new units specific to these assays have been devised.
1.2.1 Assay Procedure
Determining an enzyme’s catalytic activity is the simplest approach to carrying out an
enzyme assay. The catalytic activity is the amount of substrate an enzyme can convert to
product in a given time under specified conditions [Palmer, 1995]. When it comes to kinetic
assays, problems with results may arise if the linear dependence of measured catalytic
activity on enzyme concentration cannot be ensured. Thus an appropriate time interval must
be chosen in order to provide an ‘initial’ rate [Gul et al, 1998]. To perform an adequate assay,
it is necessary to keep the reaction conditions constant. These conditions include temperature,
pH, buffer composition, organic co-solvent and ionic strength [Walsh, 2002]. When
designing an assay procedure, it is preferable to try get as close as possible to the in vivo
conditions of the enzyme being tested, however this may not always be practical. The
conditions at which an enzyme is most active are known as the optimum conditions [Gul et
al, 1998]. They can vary greatly from enzyme to enzyme but generally, in regards to
temperature, assays are carried out at 25°C, 30°C or 37°C. In order to calibrate instruments
10. 7
used to measure enzyme activity, a ‘blank’ must be used. The ‘blank’ is identical to the test
sample only it does not contain the analyte being measured [Needham, 1970]. For example in
spectrophotometry, where the absorbance of light is measured, the ‘blank’ ensures that the
solvent does not interfere with results obtained and that only light absorbed by the analyte is
taken into account. It also prevents the scattering of any light, which would also affect the
result.
For an enzyme assay to yield adequate and accurate results, certain practical
considerations need to be made. The purpose of the assay, the nature of the reaction involved,
the apparatus and instruments needed and the purity of the enzyme are all elements of an
assay that must be reviewed [Walsh, 2002]. An enzyme’s stability in various reaction
mixtures can implicate the performance of an assay, which as a result may need to be
adjusted. The stability of the substrate, as well as its purity, should also be considered, as it
too can impact on the quality of results [Gul et al, 1998]. Depending then, on the relative
stability between the two, a decision can be made on whether to add the enzyme or the
substrate to initiate the reaction. If it is a membrane-bound enzyme being used, there is a
possibility of activity loss or change in kinetic activity once removed from the membrane
[Palmer, 1995]. Also isoenzymes and analogous enzymes from different species can have
varying levels of specific activity, which may require the adjustment of the assay conditions.
1.2.2 Types of Assay
There is a vast array of assay methods and techniques available for the
measurement of all kinds of enzymes. Some are more popular than others and used on a more
regular basis. A continuous assay is one that provides the results, in the form of a curve, as
the reaction occurs [Scopes, 2002]. This proves advantageous in that any deviation from
linearity of the initial rate can be observed immediately. There are three variations of the
continuous assay. They are direct, indirect and coupled [Needham, 1970]. A direct assay is
used when the product produced can be measured itself, without the need for a reagent in the
reaction mixture. An indirect assay does not produce a suitable signal to be read by the
instrument, and therefore does need a reagent [Gul et al, 1998]. This reagent must not
interfere with the activity of the enzyme, while still reacting with the product to produce a
detectable signal. The continuous assay uses additional enzymes to transform the initial
product of the reaction in to a final product that can be detected. The advantage of this kind
of assay is that product inhibition does not take place (as the initial product is transformed),
and therefore the measured rate can be kept constant over long periods [Scopes, 2002]. In
11. 8
contrast, the end-point assay uses a fixed time and is focussed on the substrate. It is designed
so that most of the substrate gets converted to product. This allows the estimation of substrate
concentration by measurement of the resulting product [Palmer, 1995]. A third common
enzyme assay is the kinetic assay. The principle of this assay is based on the fact that initial
velocity is directly proportional to enzyme concentration, once other factors are kept
constant. The measured reaction rate can be used to determine the concentration of the
substance of interest.
12. 9
CHAPTER 2
METHODOLOGY
2.1 Study Design
Over the past few decades there has been major development in technological as well
as analytical aspects in the biotechnology sector. In regard to assay techniques, the
engineering of new enzymes and substrates, as well as the discovery of better chemical
reagents has allowed scientists to produce faster, more accurate and more reliable procedures.
This has resulted in a large increase in assay number and variation. For this reason, it was
necessary to review the project outline briefing, supplied by the supervisor, in order to
identify key words that would aid in refining the search for literature on AA assays. After
some deliberation, it was decided that the following phrases would provide a broad spectrum
from which adequate data on the specific topic could be obtained. The key words were as
follows: ‘alpha-amylase’, ‘α-amylase’, ‘assay’, ‘activity’, ‘review’, ‘industry’. Iterative
search routines were then carried out using these words, both singularly and in combination,
to establish an idea of the scope of literature available. From there, a literature review could
be carried out to evaluate the quality of information. This was done through a range of
sources including books, journals, patent databases and the web. The end goal of the
literature review was to gain comprehensive background knowledge of AA and of assay
systems, in order to provide a strong basis for the intended study. It allowed for the
development of an appropriate study design with suitable parameters.
The designing of an adequate study required the acknowledgement of certain aspects,
such as the purpose of the research and its expected results. What was to be achieved by
carrying out the study? It led to the observation that there are many different ways in which
AA can be measured for enzymatic activity. Numerous assays currently exist that can
perform this task, with variations in their components and procedures. It was observed that
these variations can range from the very slight to the very obvious; obvious being the entire
principle of the assay was different from one to the next. Slight differences included pH,
temperature, incubation times or reagent buffers, among others. The conclusion was drawn
that the different forms of assay needed to be critically analysed and compared. Features such
as the ease and efficiency of the assay, the cost of reagents, the equipment needed, the
necessary time and labour requirements, as well as the potential demand for automation were
13. 10
considered. A plan then had to be devised on how best to retrieve the desired information. It
involved the consideration of all available resources and a review of all databases that could
knowingly be accessed. Once achieved, this helped to give direction and structure to the
study.
2.2 Key Steps
As mentioned previously, initial research began with the retrieval of as much
background information as possible. In order to understand the assays for measuring AA
activity it was necessary to first get a complete understanding of the enzyme itself, along with
its mechanisms. Beginning with a basic textbook, Understanding Enzymes, the activity of AA
was uncovered. It was used to supply the enzyme class in which AA is included as well as the
type of bonds on which it acts. Further research continued on the Protein Data Bank, as well
as in the European Journal of Biochemistry, which gave information about the structure of
the enzyme. This related back to the function of AA, clarifying its catalytic ability. Another
textbook was consulted, Proteins: Biochemistry and Biotechnology, which highlighted the
substrate specificity of AA. It described the type of substrates that AA can and cannot break
down. Once a clear picture had been created of how the enzyme works, it was important to
understand its sources and how it is utilised. Sivaramakrishnan et al were able to describe in
the Journal of Food Technology and Biotechnology, the main types of microorganism that are
used to produce AA in bulk, underlining those most popularly used and the reasons for their
popularity. A journal called the Journal of Biology and today’s world, found while mining
the internet, contained information on the market value of AA, which contextualised the
importance of the above mentioned production processes.
The next task was to understand exactly how enzyme activity could be measured. The
European Journal of Biochemistry provided the two standard units of measurement for
enzyme activity, the International Unit and the Katal, as well as providing some history of
their origins. The book Enzyme Assays was studied and it explained the common variables
and the necessary requirements of a valid assay. This tied in with the writings of Walsh and
of Palmer, which described the considerations that needed to be made in order to carry out a
reliable and accurate assay. Finally, the workings of Scopes as well as that of Gul et al
showed the types of assay that are mainly used. They elucidated the main steps in these
assays and gave some good explanation of their use. This completed the background research
and allowed an informed study of AA assays to proceed.
14. 11
When beginning the study of AA assays specifically, it was decided that the best area
to begin was distinguishing the significantly different assays, based on their fundamental
principles. The internet was scoured for papers and studies either based on AA assay
procedures or that used different procedures within them. Certain aspects of the procedure
were focussed on, such as the substrate that was used or the pH and temperature at which it
was performed. The online databases used to carry out this task included Science Direct,
PubMed, Web of Knowledge, Springer and Knovel. The research led to papers on the
development of new assay techniques, the refinement of pre-existing procedures and
reagents, as well as evaluations and comparisons on each. The protocols were recorded and
compared, and the notable observations were tabulated in the results section of the study.
Whilst exploring the protocols, attention was paid to the different reagents used and the
different units of measurement for enzyme activity. Assay components were analysed and
compared, with regard to their relative efficiencies as well as their convenience of use. Sigma
Aldrich proved to be a valuable resource when inspecting factors such as cost of reagents and
their range of uses. Other aspects of methodology that were considered were the necessary
instruments, length and ease of execution along with skill and labour requirements. Next, the
various areas in which the AA assay has found use were investigated. This was to help
establish an idea of the importance of the assay in the scientific and medical industries. It also
linked in with previous research done on the amylase industry.
Finally, it was decided that an adequate study would not be complete without
referring to relevant patent literature. The Google Patents search engine was chosen as the
main source of information for this task. The reason for this is that it was easily navigated. By
using key words in combination as well as singularly, it scanned both the titles and the
literature to yield adequate and relevant search results. It also had a Prior Art Finder tool
which allowed access to related material while also providing additional key words to refine
the search. It contained links to other patent databases such as Espacenet and USPTO for
further verification. A thorough exploration was carried out of literature related to AA assay
methods and technologies. It gave an idea of the new ideas and inventions that have been
produced over recent decades. The patents went in to great detail of the invention itself, the
workings and abilities of the invention, as well as other related claims. Particular attention
was paid to the filing dates of each patent explored. This enabled a good analysis of the
development of different techniques as well as the analysis of interest growth over a given
time period. Relevant patents were noted and referred to in the results section.
15. 12
CHAPTER 3
RESULTS
A thorough investigation was conducted of all of the available literature on AA
assays. The first objective of the study was to breakdown the assays to their basic principles,
so that they could be easily separated, categorised and tabulated. Once this was done, the
assays could be individually inspected and scrutinised accordingly. The significant assays
have been recorded in Table 1 below.
Method Assay
Type
Enzyme
Activity
Substrate Conditions
(Temp. &
pH)
Description Reference
Wohlgemuth
(1908)
Indirect,
end-
point/kinetic
Amount of
starch
(1.0%)
hydrolysed
by 1ml of
enzyme
Starch 40-60°C
6.0-6.9
Measures digestion
time required for
enzyme to produce
definite iodine
colouration
[Yoo et al,
1986]
Somogyi-
Nelson
(1944)
Indirect,
end-point
Amount of
enzyme that
releases
1mole of
reducing
sugar per
minute
Starch 50°C
6.5
A reducing sugar
(maltose) reacts
with an alkaline
copper solution to
produce a product
that forms a
measurable blue
colour when reacted
with
arsenomolybdate
[King &
Garner, 1947]
Fuwa (1954) Indirect,
end-point
1 unit is
equal to a
10% decline
in OD when
measured
against a
substrate
control
Starch 37°C
5.5
Measures
dextrinising activity
by using starch-
iodine complex and
comparing against a
control tube
[Alves-Prado
et al, 2008;
Satyanarayana
et al, 2006]
Bernfeld
(1955)
Indirect,
end-point
1 unit counts
as 1mg of
maltose
released
from 1.0%
starch
solution in 3
minutes
Starch,
Amylose,
Amylopectin
100°C
6.9
Measures reducing
sugars produced
using dintrosalicylic
acid
[Yoo et al,
1986; Sigma
Aldrich]
Jansen and
Wydeveld
(1958)
Direct,
kinetic
Amount of
enzyme that
releases
1mole of
measurable
product from
a blocked
substrate per
min
Blocked
chromogen-
bound
oligosaccharide
40°C
5.2-5.4
Release of a
chromogen-bound
oligosaccharide is
measured, which
relates to enzyme
activity
[Chavez et al,
1990;
Megazyme]
16. 13
Method Assay
Type
Enzyme Activity Substrate Conditions
(Temp. &
pH)
Description References
Rinderknecht,
Wilding and
Haverback
(1967)
Direct,
end-point
1 unit is the
amount of
enzyme that
releases 1mole
of measurable
product from
dyed substrate
Starch,
Amylose,
Amylopectin
40°C
6.0
Release of blue
pigment
proportional to
amount of
enzyme.
[Park &
Wang, 1991;
Morrison et
al, 1993]
Tietz, Miranda
and Weinstock
(1972)
Coupled,
kinetic
1 unit measures
the amount of
substrate in
moles to be
transformed
through coupled
enzyme reactions
Starch,
Maltotetraose,
Maltopentaose
30-37°C
6.3-6.9
The amount of
NADH produced
through coupled
enzyme reactions
is proportional to
enzyme activity
[Lorentz et
al, 1999;
Kaufman et
al, 1980]
Table 1: Table highlighting the main types of assay used to measure alpha-amylase activity based on different
procedures, with some emphasis on the originators.
3.1 Description
As can be seen in Table 1, there are seven distinct types of assay for measuring AA
activity according to different procedures. Most of these are no longer carried out as they
were originally formulated, and have been modified and optimised over the past few decades.
The assay developed by Wohlgemuth in 1908 was the first serum assay to be
introduced to clinical chemistry [Karmen et al, 1955]. It involved measuring the digestion
time necessary for AA to convert limit dextrin to products that form a red-brown colour with
iodine, while in the presence of excess β-amylase [Perten, 1965]. The method would involve
using a Hellige Comparator to compare a coloured disk that corresponded with the colour
produced by a known amount of enzyme [Hagberg, 1960]. The problem with the procedure
was that it was very time consuming and not particularly sensitive. In 1964, Harald Perten
devised a colorimetric method for determining AA activity under the direction of the
International Association for Cereal Chemistry [Perten, 1965]. It was based on Hagberg’s H-
unit method that was, itself, a modification of the Wohlgemuth design. It involved removing
enzyme-substrate samples from incubation at timed intervals, then adding them to iodine and
measuring their extinction in a colorimeter.
The next distinct assay is that of Nelson and Somogyi, which originated in 1944. It is
known as the colorimetric copper method as it uses the cuprous ions present in a copper
reagent to oxidise maltose [Sushma et al, 2013]. This forms cupric ions, which are then
transformed back to cuprous ions by the addition of arsenomolybdate. The arsenomlybdate is
reduced in the process and forms a measurable blue colour. This assay is still widely used
17. 14
today and there have been some efforts to improve on it. Green et al have attempted to
modify the procedure to a microassay by using microtiter plates. According to their work, this
can have many advantages such as reduced sample and reagent volumes, automated
measurement, and increased range and reproducibility, among others [Green et al, 1989].
It is also starch’s reaction with iodine that provides the basis for the next procedure,
only in this case, Fuwa used a different method of measuring dextrinising activity. The
reaction takes place using soluble starch as substrate and is stopped with HCl [Yaldagard et
al, 2008]. Iodine is added and the absorbance is measured at 620nm. Fuwa considered one
unit of enzyme as a ten percent drop in optical density when compared against a control
[Satyanarayana et al, 2006]. The disadvantage of this assay is that certain media components,
for example corn steep liquor, can interfere with the result. The addition of copper sulphate
and hydrogen peroxide can counteract this problem. Some variations have been made to the
assay in more recent years such as the work of Gupta et al which modified the units, so that
one unit of enzyme is equal to a one percent drop in optical density rather than a ten percent
drop [Yaldagard et al, 2008]. Also, Carlsen et al succeeded in incorporating it with flow
injection analysis which has proven to increase sample rates and response time, as well as
improve its flexibility, while using a simpler apparatus [Satyanarayana et al, 2006].
The dinitroslalicylic acid (DNS) assay was first described by Bernfeld in 1955 and it
is still very much in use today. It works on the principle that the reducing sugar being
produced by AA has its free carbonyl group acted on by DNS [Negrulescu et al, 2012]. This
forms the aromatic compound 3-amino-5-nitrosalicylic acid that can be measured at 540nm.
The limitation of Bernfeld’s method was that colour production would slowly decrease and
also some of the glucose would be destroyed by certain components of the acid
[Satyanarayana et al, 2006]. Miller resolved these issues by removing the Rochelle salts from
the acid and adding 0.05% sodium sulphite. Further advancement has been made, similar to
that of the Nelson-Somogyi method, in that it has been modified for microtiter plates
[Negrulescu et al, 2012]. It uses a microwave in order to heat the reagent instead of the
general boiling procedure. According to the developers, it can benefit the environment
through reduced reagent volume and can allow relatively high numbers of samples.
Jansen and Wydeveld first described using p-nitrophenyl α-maltoside as a substrate
for AA. They replaced the anomeric hydroxyl group of maltose with p-nitrophenol which,
once cleaved, could be monitored at 410nm [Chavez et al, 1990]. Wallenfels et al developed
18. 15
this further by removing interference caused by endogenous glucose and pyruvate. Although,
according to Menson et al, as well as Chavez et al, these developments still do not produce a
satisfactory AA assay [Menson et al, 1978; Chavez et al, 1990]. They and their colleagues
have separately worked on methods of improving the assay by inventing more efficient and
reliable substrates, an example of which is 2-chloro-4-nitrophenyl-alpha-D-maltotrioside
(CNPG3).
The sixth substantial assay found in the literature was that of Rinderknecht et al in
1967. The original process involved staining starch with a dye called Remazol Brilliant Blue
R. This resulted in a covalently dyed substrate that could determine α- and -amylase activity
[Wahlefeld, 1974]. The enzyme would cleave the starch, producing a coloured pigment in the
reaction mixture which, once separated, could be measured photometrically. There have been
some slight variations in the method over the years, depending on the circumstance.
However, due to its simplicity, the method has mostly stayed the same. It is currently called
the starch azure or amylose azure assay [Doehlert & Duke, 1982]
Finally, the most recently popular technique for measuring AA activity is using
coupled assays. The technique was first described by Tietz et al in 1972, whereby AA was
used to break down starch to maltose, maltotriose and dextrins [Kaufman & Tietz, 1980].
From there, α-glucosidase would break down these products to glucose. Then, the glucose
oxidase would oxidise glucose to hydrogen peroxide and δ-gluconolactone. In this way, the
oxygen that was removed from the solution, relating to amylase activity, could be measured
using a Clark electrode. There have been some adaptations to this method, which enable the
production of NADH as the measurable signal, rather than oxygen removal. Some examples
are described in the variation section below.
3.2 Variation
Throughout the study, the literature revealed that there can be significant variation
between methodologies carried out under the same principle. The following is a summary of
some of the more prominent variations that were observed.
Beginning with the procedures for measuring reducing sugars, a similar method to
that of Nelson and Somogyi, based on copper ion reduction, was found. It was developed by
Folin and Wu in 1920 and, similarly, uses a hetero-polymolybdate complex to produce a blue
colour that can be measured [King & Garner, 1947; Sushma et al, 2013]. The complex uses
sodium tungstate in order to react with the reducing sugar and the reagent is known as
19. 16
phosphmolybdic acid. A less frequently used reagent is 2-2’-bicinchoninate, although it
performs to the same effect as the Nelson-Somogyi assay [Doner & Irwin, 1992].
After the improvements made of Bernfeld’s assay by Miller, no further modifications
have been made to the DNS assay procedure, however some experimentation has been
carried out using substrates other than starch. An amylose substrate was developed by
Kurimoto et al which, according to them, improves the uniformity and reproducibility of the
amylase assay [Kurimoto & Yoshida, 1971]. Amylopectin is another substrate that has been
used in previous methods [Walker & Harmon, 1996]. A variant of reducing sugar
measurement, first described by Cole, uses potassium ferricyanide. Ferricyanide is yellow in
colour and is reduced by hemiacetals or hemiketals, forming ferrocyanide which is colourless
[Cole, 1933]. Lastly, a technique involving p-hydroxybensoic acid hydrazide can be used
because in alkaline solution, it displays a yellow colour when in the presence of reducing
carboydrates. This is as a result of anions that are produced in the reaction [Lever, 1972]
The protocol using blocked substrates with chromogenic labels is the most
straightforward, in that the enzyme acting upon them produces a directly measurable signal.
In this way there is not much variation in procedure, except in few cases, where an additional
enzyme may be required, such as with ethylidene-blocked 4-nitrophenylmaltoheptaoside
(EPS-G7) and α-glucosidase [Lorentz, 2000]. There are, however, a large number of
synthetic substrates available to be used for this procedure, some examples of which can be
seen in the patent filed by Chavez et al in 1986. Substrates such as, 1-beta-Chloro-2-
tricloroacetylmaltotriose, 1-beta-Chloro-2-hydroxymaltotriose nonaacetate and 4-Chloro-2-
nitro-1-naphthyl-alpha-maltotrioside are just three of the many examples given [Chavez et al,
1990]. One notable experiment involved the use of supramolecular chemistry to create four
different fluorophore-modified cyclodextrins [Murayama et al, 2006]. They each had a 4-
amino-7-nitrobenz-2-oxa-1,3-diazole (NBD-amine) moiety that caused fluorescence in
acetate buffer. The cyclodextrin ring was hydrolysed by AA, exposing the hydrophobic
NBD-amine to the aqueous solution, decreasing fluorescence. This allowed for direct
measurement of AA activity.
Over the past few decades there has been much development in substrate staining for
use in amylase assays. Not only is stained starch used, but stained amylose and amylopectin
have also been reported. An example is the Cibachron Blue F3GA dye, which is a sulfonated
polyaromatic compound that can be used to stain amylose [Kopperschlager et al, 1982; Klein
20. 17
et al, 1969]. It can also stain soluble starch, causing it to become insoluble by crosslinking
1,4-butanediol glycide ether [Wahlefeld, 1974]. This technique has been used with black
drawing ink as well and has been reported to work well in the presence of both dextran and
polyethylene glycol, which are used in aqueous two-phase systems [Safarik & Safarikova,
1992; Park & Wang, 1991]. Dyes that are used commonly for the staining of amylopectin
include Reaktone Red 2 B or Procion Brilliant Red M-2 BS.
As mentioned previously, there are varied versions of the coupled assay used to
produce a detectable signal in order to measure AA activity. The DuPont aca amylase method
uses maltopentaose as the substrate, which is broken down to maltotriose and maltose by AA.
Further hydrolysis by α-glucosidase produces glucose and then this can be measured by the
hexokinase or glucose-6-phosphate dehydrogenase method [Balkcom et al, 1979]. The
Beckman Enzymatic Amylase-DS Method uses maltotetraose as a substrate that, when
broken down, forms 2 maltose molecules [Quast Hanson & Yasmineh, 1979]. Coupled with
maltose phosphorylase, then -phosphoglucomutase and finally glucose-6-phosphate
dehydrogenase, the assay produces NADH to be measured at 340nm. The Eskalab Method is
similar to the original protocol of Tietz et al only the hexokinase technique is used instead of
glucose oxidase [Kaufman & Tietz, 1980]. The α-Amyl Harleco Method too uses the
hexokinase technique, only short chain oligosaccharides approximately 5 to 15 glucose
residues in length, provide the substrate.
3.3 Comparison
3.3.1 Cost
A table was constructed in order to highlight the cost of the main constituents that
make up the different assays. The purpose of Table 2 is to provide a general comparison of
the assay costs relative to one another. The creators’ names are used rather than the principle,
in order to represent the difference in amounts of components used between similar assays.
Table 2 is located on the following page
.
21. 18
Assay Substrate Price
(per g)
Reagent Price
(per
mL or
per g)
Additional
Enzymes
Price
(per
250
units)
Wohlgemuth Starch €0.26 Iodine €0.17 - -
Nelson-
Somogyi
Starch €0.26 Arsenomolybdate
(Ammonium
molybdate &
sodium arsenate)
(€19.42
&
€1.04)
- -
Fuwa Starch €0.26 Iodine €0.17 - -
Bernfeld Starch €0.26 DNS acid €4.54 - -
Amylose €83.10
Amylopectin €0.20
Jansen &
Wydeveld
CNPG3 €1026.00 None - - -
Rinderknecht,
Wilding &
Haverback
Starch Azure €52.20 None - - -
Tietz, Miranda
& Weinstock
Starch €0.26 None - MPase
-PGMase
G-6-PDH
€127.00
Maltotetraose €979.00 €99.50
Maltopentaose €1030.00 €92.88
Table 2: Theoretical pricing of main components of the various assay procedures.
Note: All prices were obtained from the Sigma Aldrich website, so cost may vary depending
on the product source. Although particular assays use some of the same components,
depending on the procedure, the amounts of these components differ. The table does not
account for any of the other reagents and buffers that are used, as some of the assays are very
diverse. CNPG3 was taken as a single example of a blocked p-nitrophenyl substrate. Many
more exist, that may vary in price.
MPase = maltose phosphorylase from Enterococcus spp.
-PGMase = -phosphoglucomutase from Lactococcus spp.
G-6-PDH = glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides
CNPG3 = 2-chloro-4-nitrophenyl-alpha-D-maltotrioside
Calculations were carried out based on published experimental methods to provide
theoretical costs for the different substrates. In the experiment referred to, regarding starch,
10g were used for the substrate control, which would amount to €2.60 [Perten, 1965]. No
such experiment could be found for amylose. However, research showed that it costs
considerably more than starch and amylopectin on a weight by weight basis. The price of the
synthetic oligosaccharides, per assay, was notably higher. CNPG3 was calculated at €6.90
22. 19
using the 4.4mmol/L concentration described by Winn-Deen et al [Winn-Deen et al, 1988].
The starch azure assay, using Remazol Brilliant Blue R, proved to be cheaper at €5.22 per
assay in accordance with Doehlert’s method [Doehlert & Duke, 1982]. Referring to work by
Whitlow et al, 7mmol/L concentration of maltotetraose would cost €10.28, which is the most
expensive substrate overall [Whitlow et al, 1979]. Based on the previous concentration, a
hypothetical cost was calculated for maltopentaose, which was priced at €8.70, the second
highest costing substrate. Atop the synthetic substrate costs must be added the price of any
additional enzymes used in the assay. MPase, -PGMase and G-6-PDH prices were
calculated, by their units/mL, to be €1.50, €0.64 and €2.22 respectively [Kaufman et al,
1980].
Similar calculations were carried out for the reagents. Although it would appear as
though DNS acid is one of the more expensive reagents, the price is quite low in relation to
the amount required for the procedure. Cost was calculated at approximately €0.05 per assay
[Worthington Biochem. Corp., No Date]. The price for iodine is negligible when calculated,
due to how little is required. It was calculated to cost €0.005 per assay using the referred
protocol [Yaldagard et al, 2008]. Finally, the arsenomolybdate assay was assessed by
breaking it down to its two constituents, ammonium molybdate and sodium arsenate, and
pricing these [Educational Portal, No Date]. It was proven to be the most expensive reagent,
costing €0.67 per assay. Overall the starch-iodine assay was shown to be the cheapest method
for measuring AA activity, according to the data. This would be followed by the DNS assay,
using either amylopectin or starch as the substrate. Next would be the starch azure assay,
followed by the arsenomolybdate method, and finally the synthetic oligosaccharides.
3.3.2 Performance and Efficiency
When comparing assays, there are certain aspects that need to be considered in order
to establish their quality. Aspects such as time taken to perform the assay, sample and reagent
volumes, and reproducibility are all considered, among others. With respect to AA assays,
there is great variation seen in these aspects, even between assays using the same principle.
Scientists are constantly experimenting with different methods in an attempt to fully optimise
them, which explains the high number of variations.
According to the literature regarding starch-iodine assays, it was observed that sample
size ranged from 1mL to 4mL and reagent volumes ranged from 1mL of iodine up to as high
as 10mL. Instruments such as a spectrophotometer, a Hellige comparator and a water bath
23. 20
were shown to be necessary in order to carry out the assay. Assay times varied from as little
as 20 minutes to as long as 960 minutes in older procedures. In most methods it was noted
that a standard curve was required, which is a disadvantage of the assay. The starch-iodine
assay has been reported to lack sensitivity and reproducibility when compared to the likes of
chromogenic assays such as the DyAmyl method [Chung et al, 1971].
The assays involving dyed substrates were also reported to require a standard curve in
their procedure. Some substrate volumes were recorded to be 200mg, 2ml and 5ml in various
protocols. The amount of enzyme used did not exceed 1ml in any of the papers researched,
with some using as little as 0.1ml of enzyme. Time taken to perform the assay ranged from
10 minutes to 50 minutes at most. A spectrophotometer was necessary for the procedure, as
well as a water bath, or a Dubnoff shaker in certain cases [Klein et al, 1970]. Sample removal
at timed intervals was a necessary step in some experiments, which is an increased labour
requirement. Precision of the technique appeared to be good, with a coefficient of variation
(CV) of 3.5% reported in one test [Klein et al, 1970]. -amylase has, however, been noted to
interfere with the assay if plant extracts are used, which can cause an over estimation of AA
activity [Doehlert & Duke, 1982].
Blocked substrate assays would generally be one of the quickest forms and would be
the most straightforward in terms of steps involved. This is due to the fact that few additional
components are needed other than the enzyme and the substrate. Time taken, according to
literature sourced, can be between 5 and 40 minutes depending on number of samples. The
amount of enzyme ranged from 10L to 100L, and substrate volumes ranged between
100L and 1mL. The assay seems to be very specific, with one case reporting a greater than
95% recovery of AA activity [Winn-Deen et al, 1988]. Also, reported CVs of the assay were
as low 1.4% in certain experiments. Spectrophotometers, pH meters and centrifugal analysers
were all mentioned in different studies as required apparatus.
The coupled assay techniques are also quick, running between 7 and 35 minutes in
length. Few steps are necessary in this procedure once the substrate, the amylase and any
additional enzymes have been prepared or acquired. Sample sizes required were between 10
and 100L, and reagent volumes between 1.0 and 4.9mL. Depending on the type of coupled
assay used, certain interference could take place. For example the aca amylase assay can be
affected by glucose interference, and so gel filtration must be carried out in order to
counteract this [Kaufman & Tietz, 1980]. The Beckman assay on the other hand does not
24. 21
suffer from endogenous glucose interference. A spectrophotometer or a fluorescence detector,
along with a water bath is essential for the procedure.
The DNS assay is a timed assay and, therefore, requires more skill and labour from
the operator. A standard curve also needs to be produced in order to obtain readings. The
assay lasts between 20 and 60 minutes depending on the number of samples. Sample size can
range from 0.6mL to 3mL, according to the literature, and reagent volumes tend to be
between 0.9mL and 3mL. The apparatus required for the test include a spectrophotometer and
a water bath. Dinitrosalicylic acid can be harmful if swallowed and is a respiratory and skin
irritant as stated by Sigma Aldrich, therefore care must be taken when handling this
substance.
Most literature sourced followed the Somogyi method when using the
arsenomolybdate technique, which used 2ml measurements for both reagent and sample
volumes. In some cases 1ml volumes of sample were used instead. Somogyi reported that, in
the case of maltose, the reaction time was less than 20 minutes but included a 20 minute
heating procedure prior to incubation [Somogyi, 1959]. According to his research the
technique shows good proportionality, and in reference to work done by King and Garner, it
also shows good conformability with Beer’s Law [King & Garner, 1947]. Neslon reports
good sensitivity and reproducibility of the technique in his works [Nelson, 1944]. A
drawback of this assay is that a standard curve needs to be produced in order to obtain results.
Great care must be taken when handling sodium arsenate and ammonium molybdate as they
are both very toxic if inhaled or ingested, according to Sigma Aldrich.
3.3.3 Units
While investigating the different assay techniques, particular attention was paid to the
types of units that were used. Many were dependent on the procedure being carried out as can
be seen in Table 1. Some units, however, occurred more frequently in the literature, with
certain protocols applying conversion rates to their results, in order to express them in these
units. The following are examples of the main units observed.
Modified Wohlgemuth Unit. This unit is used in the determination of liquefying
amylase in a sample [Pomeranz, 1991]. It is described as the amount of enzyme that
dextrinises 1mg of soluble starch to a definite size dextrin in 30 minutes under the specified
assay conditions. The specific dextrins released produce a definite blue colour with iodine
25. 22
that can be compared with a standard [Verenium, 2011]. This unit has found use in industry
and textile sectors.
One of the most common units referred to in the literature was the SKB unit. This
stands for Sandstedt Kneen Blish unit, after the three people that derived it. The effect of α-
and - amylases on starch mixtures has been related to the diastatic power of grain [Briggs,
1961]. Their individual effects were unable to be quantified through the measurement of
reducing power. Sandstedt et al discovered that once a certain amount of -amylase was
present in a substrate mixture, the addition of any more would not cause further reduction in
the blue colour produced by the starch-iodine complex. In this way, reduction in colour
would be proportional to the amount of AA present. The SKB unit is defined as 1g of starch
converted by 1g of malt in 1 hour at 30°C [Lallemand, 1996].
Other units observed in the literature include, the Somgyi unit, Ceralpha unit and the
H-unit. The Somogyi unit is still in use today, mostly in clinical laboratories for the use of
measuring blood serum amylases. It is defined as the amount of enzyme required to
completely hydrolyse 5mg of starch in 15 minutes at 37 or 40°C [Chary et al, 2004]. It can be
related to the International Unit by multiplying by a factor of 0.185 [Chattopadhyay, 1999].
The Ceralpha method was designed by Megazyme as a microplate assay for measuring AA
activity. They describe the Ceralpha unit in this procedure as the amount of enzyme, in the
presence of α-glucosidase, required to release one micromole of p-nitrophenol from a blocked
p-nitrophenyl maltoheptaoside [Megazyme, 2004]. Finally, the H-unit was described, in a
study by Hagberg, as a measurement of the period of half-life of AA, which indicated
enzyme activity. This could be related to the SKB unit by multiplying by a factor of 0.42
[Hagberg, 1960].
26. 23
CHAPTER 4
DISCUSSION
The extensive research that has been conducted, on the various methods for amylase
measurement, has allowed a picture to be established of the advantages and disadvantages of
each. It has also helped in determining the popularity of the various techniques. The methods
are discussed below in order of their prevalence, based on citation frequency.
According to number of citation, the DNS assay procedure is that which is most
commonly used [Sakac et al, 2010]. When the words ‘alpha-amylase assay’ were searched
using the Google search engine, the top two results were from the Sigma Aldrich and the
Worthington Biochemical Corporation websites. Both sites referred to the DNS procedure,
emphasising its popularity. Its advantages include that it is a relatively cheap assay and the
procedure is not overly time consuming. There are not an overwhelming number of steps
involved in the protocol and highly technical equipment is not required. The fact that it is a
timed assay is a disadvantage, however, as it means careful attention needs to be paid to each
step and there is a greater margin for error, when compared with other methods. The Nelson-
Somogyi method is included in the same bracket of popularity as the DNS assay, with many
references to it in the literature. There are a number of examples of performance comparisons
between the two assays available. It has been noted, in some, that the DNS assay is 10 times
less sensitive than the alkaline copper method, yet is still used just as much [Gusakov et al,
2011]. Possible reasons for the latter not being used more, may include the higher price,
greater preparation requirements or the potentially harmful substances used. A disadvantage
of using the copper reagent is that it can be re-oxidised by atmospheric oxygen, which would
impact on the accuracy of results [Somogyi, 1959]. In order to combat this, the reagent needs
to be saturated with sodium sulphate, increasing labour and material requirements of the
assay. Iron reagents using ferricyanide are a considered alternative, although, according to
Somogyi they do not oxidise sugars as selectively.
Citation frequency suggests that assays using iodine to stain residual starch after a
reaction has taken place are the second most widely used, even though it has been reported in
several studies that this method can lack reliability, linearity and reproducibility [Sakac et al,
2010; Pointe Scientific Inc, No date]. One explanation for the aforementioned limitations is
the fact that starch is not a well-defined substrate. The molecular structure and composition
27. 24
of starch can vary greatly depending on the source [Kurimoto & Yoshida, 1971]. It is made
up of components of different molecular weight, amylose and amylopectin, with varying
susceptibility to AA, which causes starch to be a less consistent substrate. The suggestion has
been made by Kurimoto et al to use amylose as a substrate instead, however, as has been
shown (Table 2), it is much more costly to do so. Possible reasons for the method’s
popularity, despite its drawbacks, may include that the components are cheap, widely
available, and easily obtained. Also, the protocol is generally not comprised of a number of
complicated steps, therefore there is not much operator skill required. The widespread use of
starch-iodine methods has led to attempts of standardisation. The Indian Pharmacopoeia
method, for example, involves incubating AA in a range of dilutions of starch, then treating
them with iodine. The tube that lacks a blue colour is then used to calculate starch in grams
digested by a given volume of enzyme [Indian Standard, 1982]. It has been attempted to have
the starch-iodine assay modified to microplate form. Xiao et al describe a procedure that has
allowed significant reduction in sample and reagent volumes [Xiao et al, 2006]. They have
succeeded in determining the wavelength for maximum absorbance of the starch-iodine
complex, which is 580nm, as well as the optimum iodine concentration required when using a
given amount of starch. They have developed the assay to be highly reproducible and highly
efficient, with over 95% of enzyme activity being detected.
The next most commonly used technique is that which utilises dye covalently bonded
to the substrate [Sakac et al, 2010]. This form of assay is simple and straightforward to
perform in relation to the steps involved, and the techniques required while performing them.
However, it has been stated that some methods can be technically cumbersome [Klein et al,
1970]. The fact that an insoluble substrate is used means that centrifugation or filtration is a
necessary step, a disadvantage in regards to length of time of the assay [Pointe Scientific Inc,
No date]. It has also been reported to lack linearity, which may likely be caused, in part, by
the use of starch as the substrate. As mentioned in the results section, -amylase can interfere
in certain assays, causing inaccuracies in results. Doehlert et al attempted to remove these
interfernces by using a similar procedure to that carried out by Sandstedt et al when
developing the SKB unit. They used the addition of -amylase until saturation to try to
counteract interference, and also tried diluting the enzyme as another test. In both cases the
interference was not removed completely, however it was successfully reduced, which
improved the accuracy of the results [Doehlert & Duke, 1982].
28. 25
The fourth most common methods, based on citation frequency, are ones that are
generally used in the bakery sector [Sakac et al, 2010]. They have not been included in the
results section, as they are used to test the quality of flour and not normally to estimate AA
activity, although some procedures have been developed in order to do so [Gupta et al, 2003].
They are known as viscometric methods, and include those such as the
Amylograph/Farinograph test and the Falling Number method. The Amylograph and the
Farinograph are both instruments designed to measure the viscosity of starch slurry. Enzyme
activity correlates with the measured viscosity, which is displayed graphically. The units of
measurement are given in Brabender units, defined as the peak viscosity on a scale of up to
1000 for flour heated 1.5°C per minute up to 93°C [Lallemand, 1996]. For example, the
typical viscosity value of untreated flour would be between 800 and 900 Brabender units. The
Falling Number method was developed by Hagberg as a way to determine sprout damage in
grain [Hagberg, 1958]. It involved a stirrer-viscometer to gelatinise a heated starch
suspension, forming a viscous paste, and a measurement of the time taken to form this paste.
Next, the time taken to form a thin paste while being heated was also measured, called the
liquefaction time. The two thicknesses were differentiated by the time taken for the stirrer to
pass through the paste. Hagberg showed that the relationship between gelatinsation time and
liquefaction time was linked to enzyme activity. The units are given in seconds taken for the
stirrer to fall through the suspension after 1 minute at 100°C [Lallemand, 1996]. It is now an
approved International Association of Cereal Science and Technology (ICC) method
[Kweon, 2010]. Advances in technology have allowed for the reduction in size of starch
samples required for viscometric measurement, however the disadvantage is that the
necessary equipment, for example a U–tube viscometer, is quite specialised [Tipples, 1969].
Also, the differences in starch from different sources need to be considered as they can
influence the quality of results.
The blocked substrate assays are advantageous in many ways. They provide a well-
defined substrate, unlike those that use starch. Also, the speed at which the reaction takes
place is quicker than most of the other techniques available. Two common substrates used for
this method are 2-chloro-4-nitrophenyl-alpha-D-maltotrioside (CNPG3) and 4,6-ethylidene-
p-nitrophenol-alpha-D-maltoheptaoside (EPS-G7). Both have been reported to be very
sensitive, reproducible and accurate. Based on the research carried out it would appear that
CNPG3 is the preferred substrate, with some claiming it has greater sensitivity and greater
reagent stability when compared with EPS-G7 [Foo & Bais, 1998]. According to Foo et al it
29. 26
has a low background absorbance, is minimally affected by pH variation and has little to no
lag phase. It has also been reported to not be inhibited by endogenous factors [Pointe
Scientific Inc., No date]. Despite these many advantages, this form of assay is not especially
popular. There are several potential reasons for this. Firstly, there are a vast number of
possible substrates available, making the assay difficult to standardise. The preparation
procedures for such substrates, should that be a step included in the assay, can be extensive
and very complicated. If not, the cost to buy the substrates is significantly higher than other
methods, meaning it is not suited to large sample sizes.
Similarly, the coupled assays are not used as often as other methods, despite their
advantages. There are numerous commercial kits available for purchase, which eliminate the
need to devise a novel procedure. The manuals in these kits provide a clear explanation of the
principle as well as a step by step guide of the correct method, with no interpretation
required. The protocol also does not require a standard curve to be produced, adding to its
simplicity. These kits have been shown to be reliable and reproducible in the examples
studied [Kaufman & Tietz, 1980]. A main factor of the lack of popularity is the use of the
additional enzymes. The enzymes typically used for these assays are expensive to purchase
and can also have residual effects on the reagents being used. Due to the cost, the assay is not
suitable for large procedures involving high sample volume or sample number.
Overall, the research of the different papers and patents suggests that there is much
need for standardisation in the measurement of AA activity. The sheer volume of procedures
available using different techniques, substrates, reagents or instrumentation, makes it
exceptionally difficult to compare the results obtained. This also applies to the various units
in which AA activity is expressed. Many of these units are specifically tailored to the
particular assay being described and even those that are well-defined and more commonly
used, are not all relatable to the International Unit of measurement. There is a lot that can still
be done to optimise and improve on the methods that are already out there. A relative
example, is that involving the Beckman Enzymatic Amylase-DS method, which was changed
to use maltotetraose as the substrate instead of soluble starch [Quast Hanson et al, 1979]. It
succeeded in doubling the sensitivity of the assay, by doing so. In more recent decades, the
need for automation has been realised. As was already mentioned this process has begun to
take place with the more popular assay techniques. The DNS, alkaline copper and starch-
iodine assays have all been modified to perform as microplate assays. This is a promising
sign for the following reasons: reduced sample size will greatly decrease the cost
30. 27
requirements of the assays, reduced reagent volumes will benefit the environment, and
reduced labour requirements will cut down on time taken to carry out the procedures as well
as allow for more samples to be tested. Potential future development may involve mass
production of CNPG3 or related substrates with an eventual price decline or possibly the use
of potentiometric methods that use electrochemical sensors to measure amylase activity
[Lorentz, 1979; Sakac et al, 2010].
31. 28
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![A Review of Published α-Amylase Assays Used in Bacterial & Fungal
Production Systems
Philip Tucker
[C11484968]
BSc Degree in Biosciences
School of Biological Sciences
Dublin Institute of Technology
Kevin Street
Dublin 8
May 2014
Supervisor: Gwilym Williams](https://image.slidesharecdn.com/6ce1ee79-298a-4cd0-b60c-c68bcdbbfd95-150622165615-lva1-app6892/85/The-Dissertation-1-320.jpg)
