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in Oral Biology & Medicine
Salivary -Amylase: Role in Dental Plaque and Caries Formation
Frank A. Scannapieco, Guillermo Torres and Michael J. Levine
CROBM 1993 4: 301
DOI: 10.1177/10454411930040030701
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Critical Reviews in Oral Biology and Medicine, 4(3/4):301-307 (1993)

Salivary a-Amylase: Role in Dental Plaque and Caries

Formation
Frank A. Scannapieco, * Guillermo Torres, and Michael J. Levine
Department of Oral Biology and Dental Research Institute, Foster Hall, State University of New York at Buffalo,
Buffalo, NY 14214
ABSTRACT: Salivary a-amylase, one of the most plentiful components in human saliva, has at least three distinct biological
functions. The enzymatic activity of a-amylase undoubtedly plays a role in carbohydrate digestion. Amylase in solution binds
with high affinity to a selected group of oral streptococci, a function that may contribute to bacterial clearance and nutrition. The
fact that a-amylase is also found in acquired enamel pellicle suggests a role in the adhesion of a-amylase-binding bacteria. All
of these biological activities seem to depend on an intact enzyme conformation. Binding of a-amylase to bacteria and teeth may
have important implications for dental plaque and caries formation. a-Amylase bound to bacteria in plaque may facilitate dietary
starch hydrolysis to provide additional glucose for metabolism by plaque microorganisms in close proximity to the tooth surface.
The resulting lactic acid produced may be added to the pool of acid in plaque to contribute to tooth demineralization.
KEY WORDS: a-amylase, oral streptococci, Streptococcus gordonii, S. mitis, saliva, dental caries, dental plaque.

To whom all correspondence should be addressed.

1. INTRODUCTION
Components of saliva play an important role in
the colonization and metabolism of bacteria in the
oral cavity. At least 50 macromolecular components
have been reported to be secreted from salivary glands
(for reviews, see Bennick, 1982; Cohen and Levine,
1989; Hay and Moreno, 1987; Levine et al., 1987;
Mandel, 1987; Oppenheim, 1989; Scannapieco and
Levine, 1990). a-Amylase, one of the most plentiful
components in saliva (Aguirre et al., 1987), is a calcium-containing metalloenzyme that hydrolyzes the
al,4 linkages of starch to glucose and maltose.
a-Amylase is actually a family of proteins, consisting of several isoforms that differ in charge and
glycosylation (see Table 1). Traditionally, this salivary enzyme was thought only to initiate the digestion of starch in the oral cavity. However, evidence
obtained over the last several years has shown that
a-amylase has a heretofore overlooked bacterial interactive function. For example, a-amylase can directly inhibit the growth of Neisseria gonorrhoeae
(Mellersh et al., 1979), whereas Legionella
pneumophila appears to be inhibited by substances in
starch-containing culture media that are enzymatically unmasked by the enzyme (Bortner et al., 1983).
a-Amylase also binds specifically with high affinity

to several numerically prominent species of oral streptococci. Once bound to these bacteria, a-amylase
retains its enzymatic activity. Furthermore, because
a-amylase is found in acquired enamel pellicle and
dental plaque, it may serve to promote adhesion of
streptococci to the tooth surface. Bacterial adhesion
to a-amylase on the tooth may be considered undesirable if it contributes to dental plaque formation. For
example, starch digestion could provide dietary substrates for colonized flora, leading to acid production
and dental caries progression. Conversely, the binding of a-amylase to bacteria in solution may be considered protective if it leads to bacterial clearance
from the oral cavity. To date, it is unclear which of
these alternate hypotheses are correct.
The goal of this article is to relate recent work on
the interaction of salivary a-amylase with oral bacteria to dental plaque formation and caries development. Preliminary studies of the binding of streptococci to a-amylase-coated hydroxyapatite will be
presented. Previous studies of the cariogenicity of
starch will also be reviewed as they relate to
a-amylase-microbial interactions. The study of
a-amylase provides one example of the potential of
salivary molecules to possess heretofore unrecognized functions of potential significance to oral
microbial ecology.

1045-441 1/93/$.50
1993 by CRC Press, Inc.

301
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TABLE 1
Biochemical Properties of Human Salivary a-Amylase
Molecular weight (kDa)
Glycosylateda
Nonglycosylated
Concentration in saliva (Jig/ml)b
lsoformsa,c
Glycosylated
Nonglycosylated
Type of glycosylationd
Oligosaccharide unitsd
Size of oligosaccharides (kDa)d
Substrate binding sitese
Bacterial binding sitesf
a

c
d

61 (electrophoretic)
56 (electrophoretic), 55.9 (deduced)
100-2600
3
2-3
N-linked
1
2-2.5
2?
2?

Electrophoretic size from Keller et al. Biochemistry 10:4867-4874

(1971). Deduced amino acid sequence from Nishide et al. Gene
41:299-304 (1986).
Aguirre et al. Arch. Oral Biol. 32:297-301 (1987).
Rosenmund and Kaczmarek Clin. Chim. Acta 71:185-189 (1976).
Yamashita et al. J. Biol. Chem. 255:5635-5642 (1980).
Buisson et al. EMBO J. 6:3909-3916 (1987).
Scannapieco et al. Biochem. Biophys. Res. Commun. 173:11091115 (1990).

II. CHARACTERISTICS OF a-AMYLASE

BINDING TO ORAL STREPTOCOCCI
Oral streptococci are a phenotypically, biochemically, and genetically heterogeneous group of organisms found in high numbers in early dental plaque
(Coykendall, 1989; Guggenheim, 1968; Kilian et al.,
1989). In agreement with recent changes in taxonomy
(Coykendall, 1989; Kilian et al., 1989), a-amylase
binds only to Streptococcus gordonii (formerly
S. sanguis genotype 1), S. mitis (formerly S. mitis genotype 2), S. crista, and S. anginosus but not to S. sanguis,
S. oralis, S. mutans, Actinomyces viscosus, and several
other Gram-negative oral bacteria (Douglas etal., 1990;
Kilian and Nyvad, 1990; Scannapieco et al., 1989).
Thus, a-amylase binding may prove useful to differentiate these streptococci.
To begin to assess the proportion and identity of
cz-amylase-binding bacteria in the oral cavity, a replica plate assay system was used to enumerate
a-amylase-binding bacteria in dental plaque (Tseng
et al., 1992). This assay enabled the screening of a
large number of primary dental plaque strains for
interaction with 1251-a-amylase with minimal in vitro
transfer of bacterial cultures. On average, results
showed that 10 to 14% of all colonies derived from
supragingival plaque-bound a-amylase. Only strains
identified as S. gordonii and one strain of A. viscosus

were found to bind a-amylase. The fact that a significant proportion of the dental plaque flora can
bind a-amylase suggests a potentially important
role for a-amylase in plaque development.
The binding of radiolabeled a-amylase to
S. gordonii G9B is saturable, calcium independent,
and inhibitable with excess unlabeled a-amylases from
genetically diverse sources (Scannapieco et al., 1989).
Approximately 15,000 to 30,000 molecules of
a-amylase bind to the bacterial surface with a dissociation constant of approximately 10-" M. Once bound,
radiolabeled a-amylase cannot be eluted from the bacterial surface with excess unlabeled a-amylase, further
supporting the high affinity of this interaction. Binding
of a-amylase to S. gordonii is inhibitable with substrates such as maltotriose, maltoheptaose, malto-oligosaccharides, limit dextrins, and starch (Douglas,
1990; Scannapieco et al., 1989). Binding is dependent
on an intact a-amylase secondary and/or tertiary structure. a-Amylase bound to the bacterial surface retained approximately 50 to 60% of the added enzymatic activity (Scannapieco et al., 1990). The bacterial-bound a-amylase can hydrolyze starch to glucose,
which is then metabolized to lactic acid by the bacteria
(Douglas, 1990; Scannapieco et al., 1990). Collectively, these results suggest that a-amylase contains
multiple sites for bacterial binding and enzymatic activity that share structural similarities.

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Reduction and alkylation of a-amylase disulfide

bonds was found to abolish both enzymatic and bacterial-binding activity (Scannapieco et al., 1989). Circular dichroism studies found the reduced and alkylated material had a significantly altered secondary
structure. Similar results were obtained when a-amylase was treated with 8 M guanidine-HCl. These data
suggest that both biological activities of a-amylase
are conformation dependent. Because previous studies have implicated histidine residues in the enzymatic activity of pancreatic a-amylase (Kita et al.,
1982; Nakatani, 1988). Therefore, the histidine residues of salivary a-amylase were selectively modified
with diethyl pyrocarbonate (DEP) (Scannapieco et al.,
1990). Protective modification by DEP was also performed in the presence of excess maltotriose. It was
reasoned that maltotriose would block the modification of histidines in the enzymatic site. Also, if histidines were involved with bacterial binding, these
would be spared by maltotriose protection. Results
showed that DEP modification of a-amylase abolished both enzymatic and bacterial-binding functions,
whereas both activities were retained when the substrate-binding site(s) was protected with maltotriose.
Collectively, these studies suggested that both enzymatic and bacterial-binding activities of a-amylase
are conformation dependent and involve histidine
residue(s).
A full understanding of the structural basis of aamylase's function(s) can only be accomplished following precise determination of its three-dimensional
structure. In this regard, the three-dimensional structure of porcine pancreatic a-amylase has been solved
by X-ray crystallography to 2.9 A (Buisson et al.,
1987). Recently, crystals of nonglycosylated salivary
a-amylase suitable for X-ray crystallographic analysis
have been obtained (Ramasubbu etal., 1991) and structural analysis is underway. Because both salivary and
porcine pancreatic a-amylase have 496 amino acid
residues, bind 1 mole of calcium per mole of enzyme,
and share over 97% sequence homology (Horii et al.,
1987), the porcine enzyme can serve as a reasonable
model of salivary a-amylase's structure. In porcine
pancreatic a-amylase, the enzymatic site is proposed
to be located in a cleft within the N-terminal central
domain. On the basis of studies in which crystals were
soaked with substrate analogs, Asp 197 and Asp 300
were proposed as the catalytic residues. In addition to
the enzymatic cleft, a secondary substrate-binding site
was also located on the surface of the molecule about
20 A away from the active site cleft (Buisson et al.,
1987). This site may function as an anchoring site for
starch, a regulatory site that binds bile salts (O'Donnell
etal., 1975), or as a bacterial binding site (Scannapieco
et al., 1990).

Construction of mutant Bacillus stearothermophilus a-amylases (Holm et al., 1990) showed that
diminished enzymatic activity was associated with
amino acid changes that clustered around the active
site cleft. A model of B. stearothermophilus a-amylase, built on the crystal structure ofAspergillus oryzae
a-amylase (Matsuura et al., 1984), suggested the active site catalytic residues to be Glu 264, Asp 234, and
Asp 331. It is interesting to note that two of these
residues (Asp 234 and Asp 331) are likely homologues
of the proposed catalytic residues in porcine pancreatic
a-amylase (Asp 197 and Asp 300, Buisson et al.,
1987). Similar results were obtained by site-directed
mutagenesis in B. stearothermophilus a-amylase
(Vihinen et al., 1990), where Asp 331 was also found
to be a catalytically active residue. A consistent finding of all of these studies was that mutations in amino
acids buried within the molecule were typically more
prone to affect function than mutations on the surface.
Furthermore, it seems that several noncatalytic residues located within the enzymatic cleft and a secondary site can bind substrate. It is therefore possible that
these noncatalytic residues may also participate in
bacterial binding as well as in substrate binding. Construction of similar mutations in recombinant salivary
a-amylase will allow assignment of bacterial binding
and enzymatic functions to specific residues and allow
identification of each functional domain.

III. ROLE OF a-AMYLASE IN MICROBIAL

ADHESION AND DENTAL PLAQUE
FORMATION
Several lines of evidence indicate that a-amylase
may play a role in plaque formation. First, a-amylase
has been identified as a constituent of the acquired
enamel pellicle (Orstavik and Kraus, 1973; 1974; AlHashimi and Levine, 1989) and may act as a receptor
for bacterial adhesion to the tooth surface. Second,
a-amylase has been detected in dental plaque by immunochemical (DiPaola et al., 1984), enzymatic, and
electrophoretic methods (Birkhed and Skude, 1978).
Third, as previously stated, the enzyme has also been
found to interact with several species of oral streptococci, which are among the first to colonize dental

plaque.
Based on the available data, an interesting concept
of a-amylase function in the oral cavity has emerged.
Rather than having a single function, it seems a-amylase possesses at least three distinct biological functions. The enzymatic function is self-evident, and as
such, the enzyme undoubtedly plays a role in carbohydrate digestion. In addition, a-amylase binds with high
affinity to a selected group of oral streptococci, a
function that may contribute to bacterial clearance and

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nutrition (Douglas, 1983; Douglas, 1990; Scannapieco

et al., 1989; Scannapieco et al., 1990). Finally, the fact
that a-amylase binds to teeth as a constituent of enamel
pellicle (Al-Hashimi and Levine, 1989) and promotes
the adhesion of amylase-binding bacteria to hydroxyapatite (HAP) in vitro (see the following) argues for a
potential role in bacterial adhesion. These properties
of a-amylase provide yet another example of
multifunctionality that may prove to be the rule for
most, if not all, salivary molecules (Bennick, 1982;
Cohen and Levine, 1989; Gibbons and Hay, 1988;
Levine et al., 1987; Tabak et al., 1982).
In this context, experiments were performed to
determine if salivary a-amylase promotes adhesion of
amylase-binding bacteria to HAP (Torres et al., 1992).
Amylase-enriched fractions obtained by Bio-Gel P60
chromatography of human parotid saliva promoted the
adhesion to HAP of S. gordonii G9B and Challis, but
not the a-amylase nonbinding strain S. sanguis 10556.
Additional experiments demonstrated that purified
a-amylase promoted adhesion of S. gordonii G9B as
did proline-rich protein 1 (PRP1) (Table 2). The later
data are in agreement with the recent findings of Gibbons etal., (1991). Because amylase binds to S. gordonii
in solution and these bacteria also bind to HAP-immobilized a-amylase, it is plausible that the streptococcal
and HAP binding sites are distinct.

IV. ROLE OF a-AMYLASE IN DENTAL

CARIES
Although the interaction of a-amylase with oral
bacteria is becoming better defined, little is known
concerning the significance of this interaction to plaque

formation or dental caries. Because starch is widespread in the human diet, a knowledge of its relationship with salivary a-amylase and the subsequent
cariogenic potential of this interaction is important to
understanding the cariogenicity of foods. Numerous
studies of the cariogenicity of starch-containing foods
have been performed using a variety of in vitro and in
vivo measurements of pH or tooth demineralization,
animal model systems, as well as cross-sectional and
longitudinal clinical studies. Most of the in vitro and
animal model systems, however, involved the use of
mutans streptococci as the cariogenic organism. It is
known that mutans streptococci do not produce endogenous a-amylase (Edwardsson, 1968) nor do they bind
salivary a-amylase (Douglas et al., 1990; Kilian and
Nyvad, 1990; Scannapieco et al., 1989). Thus, the
evaluation of the cariogenic potential of starch-containing foods in these model systems may not accurately reflect in vivo conditions.
Although there is evidence for the presence of aamylase-producing bacteria in dental plaque (Ruby
and Gerencser, 1974), it is generally accepted that
most plaque a-amylase activity is of salivary origin. In
an earlier study, approximately 25% of the total plaque
a-amylase activity was found to be bound to plaque
bacteria (Fiehn and Moe, 1983). More recent studies
have shown that only about 20 to 60% of a-amylase
bound to S. gordonii remain in active form (Douglas,
1990; Scannapieco et al., 1990). Starch metabolism by
oral bacteria appears to require salivary ca-amylase.
Stephan and Hemmens (1947) found that starch, in
comparison with mono- and disaccharides, was the
poorest substrate for acid formation by pure cultures of
plaque bacteria. When the same bacteria were mixed
with saliva and then exposed to starch, however, acid

TABLE 2
Adhesion of S. gordonll G9B to HAP Coated with Salivary

Componentsa
Component Amount of protein
Buffer
HPS
Amylasec
PRPJ d
a

c
d

0
1500
45
158

(gg)

Bacteria bound x 106

34.5 5.2b
17.8 4.3
50.7 0.3
56.0 0.3

Assays were run following the procedure of Clark and Gibbons

Infect. Immun. 18:514-523 (1977).
Values represent bacteria bound x 106 + SD per 30 mg of HAP. All
assays were run in duplicate.
Prepared according to Scannapieco et al. Infect. Immun. 57:28532863 (1989).
Prepared according to Ramasubbu et al. Proteins 11:230-232
(1991).

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was produced at a rate comparable with glucose, presumably due to processing of the starch by salivary
a-amylase (Volker and Pinkerton, 1947). Other studies found that S. mutans alone was unable to ferment
potato starch, whereas fermentation by a dental plaquesaliva mixture was extensive (Toors and Herczog,
1978). Interestingly, in vitro studies have demonstrated
that bacteria coated with a-amylase produce acid following starch exposure (Douglas, 1990; Scannapieco
et al., 1990). Thus, it is likely that a-amylase specifically bound to plaque bacteria may foster dietary starch
hydrolysis with subsequent acid formation in close
proximity to the tooth.
In situ measurements have demonstrated acid production from potato starch by dental plaque (Toors and
Herczog, 1978) with the fermentation of cooked starch
being intermediate between that of raw starch and
sucrose (Neff, 1967). Other studies showed that severe
processing of starch (e.g., extrusion cooking or autoclaving) increased its fermentation in human dental
plaque (Lingstrom et al., 1989). Interestingly, starch
fermentation may be enhanced by prior exposure of
plaque to sucrose (Dodds and Edgar, 1986). Also,
sweetened starches (e.g., cookies and sweetened cereals) are more cariogenic in rats than sucrose alone
(Bowen et al., 1981). These observations may be explained by a synergistic effect between starch and
sucrose or that sweetened starch is more retentive than
sucrose alone. In light of these observations, it is interesting to note that a-amylase activities were reported
to be higher in dental plaques from subjects on a high
sucrose diet than from subjects on low sucrose (Dodds
and Edgar, 1986). Thus, a synergistic effect between
sucrose and starch may be due to the enhanced fermentation of starch by plaque-bound a-amylase with a
subsequent increase in caries activity.
The ability of cooked starch and raw starch to
cause enamel demineralization was also investigated
using an intraoral demineralization test (Brudevold et
al., 1985). This model involves the use of a palatal
prosthesis holding bovine enamel slabs coated with a
layer of S. mutans. Changes in demineralization were
measured by iodine permeability, following rinsing by
the test subjects with various starch solutions, as was
maltose clearance from the oral cavity. The fact that
mild demineralization was caused by cooked starch
and resulted in higher initial maltose concentrations
while little effect was noted for raw starch suggested
that cooked starch can be hydrolyzed in saliva by
a-amylase and the released simple sugars metabolized by the bacteria to cause enamel demineralization.
It would be interesting to perform a similar experiment
using this model system with enamel slabs coated with
a-amylase-binding bacteria such as S. gordonii in place
of S. mutans.

The potential cariogenicity of various starches has

also been tested in animal models. Rats fed raw wheat
starch had low caries experience in contrast to rats fed
glucose or sucrose (Grenby, 1963), further supporting
the feeble fermentability of this form of starch. However, rats fed processed (pregelatinized) starches had
several-fold more carious lesions than those fed unprocessed corn starch (Frostell and Baer, 1971). In
addition, the highly branched starch amylopectin was
more cariogenic than the linear starch glucose polymer
amylose. Further support for an effect of starch on
caries development is provided by the findings that a
glucosidic ax-amylase inhibitor (BAY e 4609) significantly decreased caries experience in rats fed a processed starch diet (Mormann et al., 1983).
In a study of the relationship between a-amylase
activity and caries experience, dental plaque, and saliva from caries inactive, low-caries active and highcaries active young adults were assessed for levels of
total cultivable bacteria, Streptococcus salivarius,
S. mutans, sucrase activity, and a-amylase activity
(Fiehn et al., 1986). The only significant difference
noted between the caries inactive and high-caries active subjects was the level of ca-amylase activity. In
some individuals, a-amylase activity was higher in
dental plaque than in saliva. It is possible that this
finding was due to an increased proportion of a-amylase-binding bacteria in these plaques when compared
with plaques from patients having a higher a-amylase
activity in saliva than in the plaque.
In summary, the available evidence from in vitro,
animal, and human studies implicates dietary starch
as a cariogenic substrate. a-Amylase bound on the
surface of plaque bacteria may promote the processing of dietary starch. To obtain a more meaningful
picture of the role of starch and a-amylase in the
caries process, the relationships between a-amylase,
a-amylase-binding bacteria in plaque, and starch intake should be assessed in caries active and inactive
subjects. Bacterial plaques having high levels of aamylase-binding bacteria may concentrate salivary
a-amylase within the plaque matrix to provide more
glucose from dietary starch in close proximity to the
tooth surface. Such plaques would likely be more
cariogenic in the presence of starch-containing foods
than plaques having low numbers of a-amylase-binding bacteria.

ACKNOWLEDGMENTS
We thank Dr. Narayanan Ramasubbu for helpful
discussions of the structure of a-amylase. This work
was supported by grants DE09838, DE07585, and
DE08240 from the National Institutes of Health.

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