Polaina J., MacCabe A.P. (ed.). Industrial Enzymes. Structure, Function and Applications
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As regards sequence, these two types of amylase do not contain any of the
conserved regions characteristic of the -amylase family (Fig. 2). Although they are
both exo-amylases their amino acid sequences and three-dimensional structures are
different (Aleshin et al., 1992; Mikami et al., 1993). Structurally, -amylase (Fig. 5a)
ranks along with -amylase among the large family of parallel (/
8
-barrel
proteins (Pujadas and Palau, 1999), while glucoamylase (Fig. 5b) belongs
to a smaller family of proteins adopting the (/
6
-barrel fold (Aleshin et al., 1992).
Family GH14 includes -amylases (EC 3.2.1.2) and hypothetical proteins with
sequence similarity to -amylases. Half of the family members are experimentally
verified enzymes having -amylase activity. -Amylases are especially produced
by plants: Arabidopsis thaliana, Oryza sativa, Triticum aestivum and Solanum
tuberosum. Family GH15 includes glucoamylases (EC 3.2.1.3), two glucodex-
tranases (EC 3.2.1.70) and hypothetical proteins with sequence similarity to GH15.
Again, about 50% of the family members are experimentally verified enzymes
having glucoamylase or glucodextranase activities.
The first determined three-dimensional structure of a -amylase was that of
soybean (Mikami et al., 1993). At present, the structures of -amylases from
sweet potato (Cheong et al., 1995), barley (Mikami et al., 1999b) and Bacillus
cereus (Mikami et al., 1999a; Oyama et al., 1999) are also known. The core of
the -amylase structure is formed by the catalytic (/
8
-barrel domain (Fig. 5a)
followed by the C-terminal loop region. Although this loop surrounds the N-terminal
side of the (/
8
-barrel and may stabilise the whole -amylase molecule, it is
not involved in catalysis (Mikami, 2000). As has been pointed out above, the
-amylase (/
8
-barrel differs from that of -amylase and all other enzymes
of clan GH-H, resembling more the single-domain structure of triosephosphate
isomerase (Mikami, 2000). The two amino acid residues responsible for catalysis are
the two glutamates, Glu186 and Glu380 (soybean -amylase numbering), positioned
Figure 5. Three-dimensional structures of (a) GH14 -amylase from soybean (1BYA; Mikami
et al., 1993) and (b) GH15 glucoamylase from Aspergillus awamori (1AGM; Aleshin et al., 1992)
AMYLOLYTIC ENZYMES 11
near the C-terminus of strands 4 and 7 of the (/
8
-barrel domain, respec-
tively (Mikami et al., 1994). Totsuka and Fukazawa (1996) described further the
indispensable roles for Asp101 and Leu383 in addition to the two catalytic gluta-
mates. Analyses of the (/
8
-barrel fold of -amylases from both the evolutionary
and structural points of view are available (Pujadas et al., 1996; Pujadas and
Palau, 1997).
Glucoamylase structures have been solved for two fungal enzymes: Aspergillus
awamori (Aleshin et al., 1992) and the yeast Saccharomycopsis fibuligera (Sevcik
et al., 1998), and one bacterial enzyme from Thermoanaerobacterium thermosac-
charolyticum (Aleshin et al., 2003). The glucoamylase catalytic domain is composed
of 12 -helices that form the so-called (/
6
-barrel fold (Fig. 5b). It consists of
an inner core of six mutually parallel -helices that are connected to each other
through a peripheral set of six -helices which are parallel to each other but approx-
imately antiparallel to the inner core of the -helices (Aleshin et al., 1992). This
fold is not as frequent as the TIM-barrel fold (Farber and Petsko, 1990; Jane
ˇ
cek
and Bateman, 1996; Pujadas and Palau, 1999), however, the (/
6
-barrel has also
been found in different proteins and enzymes, for example in the enzymes from
families GH8 and GH9 (Juy et al., 1992; Alzari et al., 1996). Some glucoamylases,
like some -amylases (and related enzymes from the clan GH-H) and -amylases,
contain starch-binding domains (Svensson et al., 1989; Jane
ˇ
cek and Sevcik, 1999)
which can be of various types (for a review, see Rodriguez-Sanoja et al., 2005).
The starch-binding domain may be evolutionarily independent from the catalytic
domain (Jane
ˇ
cek et al., 2003). It should also be possible to add a starch-binding
domain artificially to an amylase (or eventually to any other protein) to improve its
amylolytic and raw starch-binding and degradation abilities (Ohdan et al., 2000; Ji
et al., 2003; Hua et al., 2004; Levy et al., 2004; Kramhøft et al., 2005; Latorre-
Garcia et al., 2005). Recently, it seems evident that some amylases may contain
starch-binding activity without a specific structural module (Hostinova et al., 2003;
Tranier et al., 2005).
Based on the analysis of glucoamylase amino acid sequences, Coutinho and
Reilly (1997) described seven subfamilies taxonomically corresponding to bacterial
(1), archaeal (1), yeast (3) and fungal (2) origins. As evidenced by the crystal
structures of the glucoamylases from Aspergillus awamori (Harris et al., 1993;
Aleshin et al., 1994, 1996; Stoffer et al., 1995) and Saccharomycopsis fibuligera
(Sevcik et al., 1998), the two glutamates, Glu179 and Glu400 (Aspergillus
enzyme numbering), act as the key catalytic residues. The next most well-studied
glucoamylase is that from Aspergillus niger (Christensen et al., 1996; Frandsen
et al., 1996) which is highly similar to the Aspergillus awamori counterpart.
5. FAMILY GH31
There are some glucoamylases that have been classified into family GH31 together
with -glucosidases, -xylosidases and glucan lyases (Yu et al., 1999; Lee
et al., 2003; 2005b). These enzymes act through a retaining mechanism like the
12 MACHOVI
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C AND JANE
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CEK
Figure 6. Three-dimensional structure of GH31 -xylosidase from Escherichia coli (1XSI;
Lovering et al., 2005)
members of clan GH-H (Chiba , 1997; Nakai et al., 2005). GH31 was considered to
be a member of clan GH-H because of remote sequence homologies between GH31
and GH13 enzymes (Rigden, 2002). This assumption has recently been supported
by the resolution of the three-dimensional structure of a GH31 -xylosidase from
Escherichia coli (Lovering et al., 2005) and -glucosidase from Sulfolobus solfa-
taricus (Ernst et al., 2006) showing the expected (/
8
-barrel catalytic domain
(Fig. 6). Interestingly, the domain arrangement of the GH31 members strongly
resembles that of GH13 enzymes (Fig. 3), especially regarding domain B protruding
out of the (/
8
-barrel in the place of loop 3 (Lovering et al., 2005).
6. FAMILY GH57
For a long time GH57 has been one of the most popular GH families, attracting
much scientific interest. More than 15 years ago the sequence of a heat-stable
-amylase from the thermophilic bacterium Dictyoglomus thermophilum was
published (Fukusumi et al., 1988). Despite the fact that this sequence encoded an -
amylase, its analysis did not reveal any detectable similarity with GH13 -amylases.
Later, a similar sequence encoding the -amylase from the hyperthermophilic
archaeon, Pyrococcus furiosus, was determined (Laderman et al., 1993). These two
sequences became the basis for the new amylolytic family, GH57, established in
1996 (Henrissat and Bairoch 1996). In the last few years, when entire genomes
AMYLOLYTIC ENZYMES 13
of many micro-organisms have been sequenced, family GH57 has expanded. Its
members are all prokaryotic enzymes, most of them from hyperthermophilic archaea
(Zona et al., 2004). At present the GH57 family consists of about 100 members
(Coutinho and Henrissat, 1999) and five enzyme (Jane
ˇ
cek, 2005; Murakami et al.,
2006): -amylase (EC 3.2.1.1), -galactosidase (EC 3.2.1.22), amylopullulanase
(EC 3.2.1.1/41), branching enzyme (EC 2.4.1.18) and 4--glucanotransferase (EC
2.4.1.25). Only about 10% of the family sequence entries are enzymes; all others are
hypothetical proteins without known activity (Zona et al., 2004). GH57 sequences
are highly heterogeneous: some of them have less than 400 residues whereas others
have more than 1,500 residues (Zona et al., 2004).
Structural information for GH57 members is scarce. To date, only the structures
of the 4--glucanotransferase from Thermococcus litoralis (Imamura et al., 2003)
and AmyC enzyme from Thermotoga maritima (Dickmanns et al., 2006) have been
determined. They both revealed a (/
7
-barrel fold (Fig. 7), i.e. an incomplete
TIM-barrel. Glu123 and Asp214 (T. litoralis enzyme numbering) which define the
Figure 7. Three-dimensional structure of GH57 4--glucanotransferance from Thermococcus litoralis
(1K1W; Imamura et al., 2003)
14 MACHOVI
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C AND JANE
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catalytic centre of the enzyme, are arranged at a distance of less than 7 Å (Imamura
et al., 2003), thus confirming that GH57 also employs a retaining mechanism for
-glycosidic bond cleavage.
New information about GH57 has arisen from a bioinformatic study focused on
the conserved sequences containing the pair of catalytic residues (Zona et al., 2004).
In addition to T. litoralis 4--glucanotransferase, both catalytic residues were
experimentally identified in two amylopullulanases from Thermococcus hydrother-
malis (Zona et al., 2004) and Pyrococcus furiosus (Kang et al., 2005). The catalytic
nucleophile was found also in the -galactosidase from Pyrococcus furiosus (Van
Lieshout et al., 2003). Biochemical analysis indicates that family GH57 enzymes
may lack a genuine -amylase specificity (Jane
ˇ
cek, 2005).
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CHAPTER 2
THE USE OF STARCH PROCESSING ENZYMES
IN THE FOOD INDUSTRY
JÓZEF SYNOWIECKI
∗
Department of Food Chemistry, Technology and Biotechnology, Chemical Faculty, Gdansk University
of Technology, Gdansk, Poland
∗
synowiec@chem.pg.gda.pl
1. INTRODUCTION
Starch, the main component of many agricultural products, e.g. corn (maize),
potatoes, rice and wheat, is deposited in plant cells as reserve material for the
organism in the form of granules which are insoluble in cold water. This carbo-
hydrate is the main constituent of food products such as bread and other bakery
goods or is added to many foods for its functionality as a thickener, water binder,
emulsion stabilizer, gelling agent and fat substitute. Starch granules consist of two
types of molecules composed of -D-glucose units called amylose and amylopectin.
In amylose almost all the glucose residues are linked by -1,4-glycosidic bonds,
whereas in amylopectin about 5 % of the carbohydrate units are also joined by -1,6-
linkages forming branch points. The relative contents of amylose and amylopectin
depend on the plant species. For example, wheat starch contains about 25% amylose
while waxy corn starch is more than 97–99% amylopectin. Starch origin also makes
differences to the size, shape and structure of the polysaccharide granules, their
swelling power, gelatinisation temperature, extent of esterification with phosphoric
acid, and the amounts of lipids and other compounds which are retained inside the
hydrophobic inner surface of the amylose helices.
Expanding starch functionality can be achieved through chemical or enzymatic
modifications. The most important methods of enzymatic starch processing (Fig. 1)
are the production of cyclodextrins and the hydrolysis of starch into a mixture of
simpler carbohydrates for the production of syrups having different compositions
and properties. These products are used in a wide variety of foodstuffs: soft drinks,
confectionery, meats, packed products, ice cream, sauces, baby food, canned fruit,
19
J. Polaina and A.P. MacCabe (eds.), Industrial Enzymes, 19–34.
© 2007 Springer.