Amylases

a Amylases are starch hydrolases with several

amino acid sequences that are highly conserved
amongst family members. Amylases of A.
hydrophila strains are about 48–49 kDa in size,
although a larger amylase (70 kDa) is found in A.
hydrophila JMP636. The a amylase of A. hydrophila
MCC-1 shows conservation in catalytic- and
substrate-binding residues. In addition, three of
four calcium-binding residues (Asn100, Asp167,
His201) present in other a amylases are retained in
MCC-1, consistent with the fact that this enzyme
requires calcium for activity.

a-Amylases split the a-1,4 glycosidic linkages in

amylose to yield maltose and glucose, but they do
not act on maltose, a disaccharide composed of two
glucose subunits linked by an a-1,4 linkage. In
theory a-amylase will ultimately degrade a solution
of amylose to maltose, and glucose which can be
released from the ends of the chains (Fig. 8.5).
Intermediate oligosaccharides (dextrins) are formed
in the process. a-Amylases also attack amylopectin
and glycogen at their a-1,4 linkages. Intermediate
unbranched oligosaccharides and branched
oligosaccharides (a-limit dextrins) are formed. Thus
a mixture of products is produced (Fig. 8.5).
Salivary amylase starts the digestion of starch. It
continues to act for up to half an hour in the
interior of the food bolus after it has arrived in the
stomach. It is eventually inactivated at the low pH
produced by the gastric acid when it penetrates the
food bolus. It can digest up to 50% of the starch
present in food. Pancreatic juice that contains a
second a-amylase is released into the duodenum
when a meal is present in the digestive tract.
Pancreatic amylase continues the digestion of
starch and glycogen in the small intestine. It is
produced in larger amounts than salivary amylase.
The a-amylases from the two sources have similar
catalytic properties, despite having different amino
acid sequences. They both require Cl- for optimum
activity and both act at neutral or slightly alkaline
pH values.
4.5.2.2. Effect of co-ingredients
a-Amylases are all calcium metallo enzymes with
each protein chain requiring the presence of at least
one Ca2+ ion to impart stability to the tertiary
structure against pH and temperature variation and
against proteolysis. It is thus possible to inhibit
amylase action with strong builders/sequestrants
such as EDTA or EGTA. Nevertheless, amylases seem
less sensitive than proteases and are tolerant of the
strong builders used in machine dishwashing. As
discussed above with regard to bleaches, the initial
a-amylases for detergent applications were
sensitive to the oxygen bleaches present in these
formulations. Recently, the major detergent
suppliers, Novozymes and Genencor International,
have utilized protein engineering to improve the
bleach stability of their products [85]. Replacing the
oxidation sensitive amino acid methionine at
position 197 by leucine in Bacillus licheniformis
resulted in an amylase with improved storage and
wash resistance to oxygen bleaches [87]. Genencor
markets this mutant enzyme under the trade name
Purafect OxAm, while Novozyme sells their product
as Duramyl [74,85].

Advances in Applied Microbiology

A HYDROLYTIC ENZYMES
a-Amylase is also known as Taka-amylase A, named
after its discoverer J. Takamine, and its structure
has been analyzed in great detail, which contributed
to advances in protein chemistry. Genes encoding
the a-amylase were independently isolated from
four laboratories in 1989 (Gines et al., 1989; Tada et
al., 1989; Tsukagoshi et al., 1989; Wirsel et al.,
1989). Unexpectedly, A. oryzae has three genes
encoding a-amylase with the same amino acid
sequence. Since a-amylase is one of the most
important enzymes for sake brewing, A. oryzae has
evolved to have three copies of genes due to the
selection of strains with high amyloytic activity at
the tane koji preparation companies for more than
a several hundred years. These three copies (amyA,
amyB, amyC) are located on different chromosomes
and separated from each other by physical mapping
using pulse field gel electrophoresis (Kitamoto et al.,
1994). Interestingly, other koji molds (A. shirousamii
and A. awamori) have the same a-amylase gene
with high identity of more than 98% at the
nucleotide level. Since most genes cloned and
analyzed from shochu koji mold (A. awamori and A.
shirousamii) have 60–80% of identity with sake koji
mold (A. oryzae), it has been suggested that
horizontal transmission from A. oryzae to A.
awamori and A. shirousamii occurred during the
past few hundred years or less. A transposase
encoding gene has been found near an amylase
gene in A. oryzae (Gomi, 2001).

As shown in Table III, four amylase genes including

a-amylase, two types of glucoamylase, and a-
glucosidase have been isolated from A. oryzae.
Glucoamylase activity is a very important factor for
the production of a high-quality sake named ginjou-
shu. Therefore, after Hata et al. (1991b) cloned the
glucoamylase gene (glaA), transformants with high
copies of glaA gene were constructed and examined
for their glucoamylase activity. The transformants
showed about 20–30-fold higher activity of
glucoamylase in submerged culture, but almost no
increase in the solid-state culture associated with
koji making. Next, Hata et al. (1998) isolated
another glucoamylase gene (glaB) from A. oryzae,
and showed that glaB is expressed specifically under
solid-state culture conditions (Ishida et al., 1998,
2000). Since GlaA contains the glucoamylase
catalytic domain along with a starch-binding
domain, which is comparable to GlaA from A.
awamori, it can hydrolyze raw starch. On the
contrary, GlaB, without a starch-binding domain,
hydrolyzes only steamed starch-like materials
during sake brewing. An A. oryzae transformant
with multiple copies of glaB revealed higher
glucoamylase activity in solid-state culture like koji
making. Thus, A. oryzae has two types of
glucoamylases, glaA is prominently expressed in
submerged culture, while glaB is expressed in solid-
state conditions. It is expected that A. oryzae most
likely contains similar genes specifically expressed
under solid-state culture conditions. In fact, several
genes encoding tyrosinase, acid protease,
hydrophobin, etc., were identified as solid-state
culture specific genes by the A. oryzae EST project
(Obata et al., 2000; Akao et al., 2001; Hata, 2001).

In comparison to the two amylases, a-amylase and

glucoamylase, it was long thought that a-
glucosidase did not contribute as much to sake
brewing. However, after the a-glucosidase encoding
gene (agdA) was isolated and analyzed (Minetoki et
al., 1995), it was found that it plays an important
role during the formation of ethyl-a-D-glucoside and
a-D-glucosylglycerol compounds, which contribute
to the taste and flavor of sake (Hayakawa et al.,
2000, Takenaka et al., 2000). Kaneko et al. (1996)
isolated another gene (asaA) encoding a-amylase
from the koji mold, A. kawachii, utilized for shochu
making, which contained the catalytic domain and
the starch-binding domain akin to that of GlaA. This
acid stable a-amylase has raw starch-digesting
activity. Aspergillus oryzae and A. shirousamii may
also have homologs for asaA and glaB genes.

Proteinases are important for shoyu and miso

making. Proteinase genes cloned from A. oryzae are
summarized in Table IV. Several studies on these
neutral and alkaline proteinases have been
reported (Ikegaya et al., 1992; Tatsumi et al., 1994;
Fushimi et al., 1999; Ichishima, 2000). Plant cell wall
degrading enzymes such as cellulase, xylanase etc.
play vital role during the filtration stage of shoyu
making. Genes encoding these enzymes from A.
oryzae are shown in Table V. During miso making,
lipases of koji contribute to flavoring of the final
products and such genes encoding the lipases from
A. oryzae are shown in Table VI.
THE MOUTH, SALIVARY GLANDS AND OESOPHAGUS
Digestion
a-Amylase is the major digestive enzyme in saliva. It
hydrolyses a-1,4 glycosidic linkages in starch (see
Ch. 8). The efficiency of mastication is important for
salivary amylase to penetrate the food bolus.
Despite the short exposure of saliva in the mouth,
the salivary digestion of starch is important because
it continues after the food has reached the
stomach. Gastric acid in the stomach inactivates a-
amylase but as the bolus of food takes time to
disintegrate in the stomach, salivary digestion can
continue within it for as long as half an hour. When
the acid has completely penetrated the food the
enzyme is inactivated. a-Amylase works best at a
slightly alkaline pH. The starch in potatoes or bread
may be digested to the extent of up to 75% by
salivary a-amylase before the enzyme is inactivated
by acid in the stomach.
Small amounts of other enzymes are also present in
saliva, including lysozyme, sialoperoxidase, lingual
lipase, ribonuclease, deoxyribonuclease and
kallikreins. These salivary components are not
important for the digestive process, although
lysozyme and sialoperoxidase provide important
protective functions (see below).

Aspergillus in Biomedical ResearcH

Amylase
a-Amylases (E.C.3.2.1.1) are enzymes that catalyze
the hydrolysis of the internal a-1,4-glycosidic
linkages in starch, converting starch into low-
molecular-weight products such as glucose,
maltose, and maltotriose units (Rajagopalan and
Krishnan, 2008; Gupta et al., 2003; Kandra, 2003).
These enzymes are among the most important
industrial enzymes occupying approximately 25% of
the world enzyme market (Rajagopalan and
Krishnan, 2008; Reddy et al., 2003). They can be
derived from several fungi, yeasts, and bacteria.
However, mostly enzymes from fungi and bacteria
are used in industrial sectors (Reddy et al., 2003).
The ease in cultivation as well as desirable
physicochemical properties have made Aspergillus a
very important organism for commercial production
of a-amylases. Various species of Aspergillus
produce large varieties of extracellular enzymes,
among which amylases are the ones with the most
significant industrial importance (Hernández et al.,
2006). Fungal a-amylases are usually preferred over
other microbial sources because of their higher
“Generally Recognized as Safe” status (Gupta et al.,
2003). Thus, species of Aspergillus such as
Aspergillus oryzae and Aspergillus niger are studied
extensively for industrial production of a-amylases.

The initial step in the digestion of starch occurs in

the mouth with the secretion of a-amylase that
catalyses the digestion of carbohydrates into
smaller oligosaccharides (Robyt, 2008). Efficient
secretion of amylase by the salivary glands reduces
the workload on the small intestine (Leibowand and
Rothman, 1975). Studies reported that secretion of
amylase and lipase decreased in women over 45
years of age, but increased in men of the same age.
On the other hand, a study in rats showed a
decrease in pancreatic amylase secretion by 41% in
aging rats (Tiscornia et al., 1986). Individuals with
higher salivary amylase activity were recorded to
have lower postprandial blood glucose
concentrations as compared to those individuals
with lower salivary amylase activity, and better
adapted to ingested starches and have lower risk
for insulin resistance and diabetes (Mandel and
Breslin, 2012). Low amylase secretion can be one of
the plausible causes of diabetes and blood sugar
imbalances (Kei et al., 2011). Thus, one way of
preventing and treating unwanted physiological
disorders could be by having sufficient intake of
enzymatic supplements containing a-amylases.
Purified mixtures of pancreatic
proenzymes/enzymes, trypsinogen/trypsin,
chymotrypsinogen/chymotrypsin, and amylase have
been reported to have potent antimetastatic and
antitumor effects in animal and human systems.
Such proenzyme/enzyme mixtures have been
implicated to have inhibitory effects on tumor cell
migration at the cellular level (Beard, 1906, 1911;
Trnka et al., 1999; Novak and Trnka, 2005). These
findings suggested that with more research and
standard trials, supplements containing a-amylases
and other enzymes will be of valuable use in
ameliorating diverse health problems.
Phytohormonal quantification based on biological
principles

13.3.1.9 Amylase activity bioassay for gibberellins

The a-amylase activity test for GAs has been
developed based on the release of a-amylase by the
aleurone layers of barley seeds induced by GA
treatment (Yomo, 1960; Varner and Chandra, 1964;
Jones and Varner, 1966). The highly specific
bioassay has proven to be reproducible and
insensitive to other soluble residues or substances
present in crude plant extracts and has been
successfully applied to the quantification of
gibberellins. The amount of a-amylase released
from embryoless half-seeds of barley in response to
GA3 application is proportional in the range from
0.0005 µg/mL to 0.05 µg/mL. GA1, GA4, and GA7
were found to be comparable to GA3 with respect
to their activity in a-amylase release (Jones and
Varner, 1966).

MALT | Chemistry of Malting

a-Amylases
The a-amylases are able to hydrolyze intact starch
granules with the formation of soluble products.
They are responsible for the initial degradation of
starch granules during malting. a-Amylases (EC
3.2.1.1) are endoenzymes that cleave internal a-(1 ?
4) glucosyl linkages of amylose and amylopectin in a
random fashion. They are unable to hydrolyze the
a-(1 ? 6) glucosyl linkages at the branch points in
amylopectin. Their hydrolytic action is slower on
short chains, and they vary in their ability to
hydrolyze the a-(1 ? 4) linkages in close proximity to
the branch points. a-Amylases, acting on their own,
are able to degrade amylose to a mixture of shorter
linear a-glucan chains (linear a-dextrins),
oligosaccharides, maltose, and glucose.
Amylopectin, however, is degraded to a lesser
extent, yielding a mixture of still large branched
fragments (branched a-dextrins) as well as some
linear oligosaccharides, maltose and glucose (Figure
6). Two isoenzymes of a-amylases with the same
apparent substrate specificities and action patterns,
but different properties, are synthesized in
germinating barley (Table 2).

Production, Purification, and Application of

Microbial Enzymes

2.7.1.2 Baking Industry

Alpha-amylases have been most widely studied in
connection with improved bread quality and
increased shelf life. Both fungal and bacterial
amylases are used. The added amount needs to be
carefully controlled as overdosage may lead to
sticky dough. One of the motivations to study the
effects of enzymes on dough and bread qualities
comes from the pressure to reduce other additives.
In addition to starch, flour typically contains minor
amounts of cellulose, glucans, and hemicelluloses,
such as arabinoxylan and arabinogalactan. There is
evidence that the use of xylanases decreases the
water absorption and thus reduces the amount of
added water needed in baking. This leads to more
stable dough. In particular, xylanases are used in
wholemeal rye baking and dry crisps common in
Scandinavia.

Proteinases can be added to improve dough-

handling properties; glucose oxidase has been used
to replace chemical oxidants and lipases to
strengthen gluten, which leads to more stable
dough and better bread quality.