∗

Malgorzata Pleszczyńska1
Enzymes in therapy of biofilm-related oral Adrian Wiater1
diseases Teresa Bachanek2
Janusz Szczodrak1

1 Department of Industrial Microbiology, Maria Curie-Sklodowska

University, Lublin, Poland
2 Department of Conservative Dentistry and Endodontics, Medical
University of Lublin, Lublin, Poland

Abstract
Biofilm-related infections of the oral cavity, including dental of the enzymes. Oxidative salivary enzymes inhibit or limit the
caries and periodontitis, represent the most prevalent health growth of oral pathogens, thereby supporting the natural host
problems. For years, the treatment thereof was largely based defense system; polysaccharide hydrolases (mutanases and
on antibacterial chemical agents. Recently, however, there has dextranases) degrade important carbohydrate components of
been growing interest in the application of more preventive the biofilm matrix, whereas proteases disrupt bacterial
and minimally invasive biotechnological methods. This review adhesion to oral surfaces or affect cell–cell interactions. The
focuses on the potential applications of enzymes in the efficiency of the enzymes in in vitro and in vivo studies,
treatment and prevention of oral diseases. Dental plaque is a advantages and limitations, as well as future perspectives for
microbial community that develops on the tooth surface, improving the enzymatic strategy are discussed.  C 2016

embedded in a matrix of extracellular polymeric substances of International Union of Biochemistry and Molecular Biology, Inc. Volume 64,
bacterial and host origin. Both cariogenic microorganisms and Number 3, Pages 337–346, 2017
the key components of oral biofilm matrix may be the targets

Keywords: antimicrobial enzymes, caries control, dental plaque, glucan

hydrolases, proteases

1. Introduction [3]. These diseases have a significant effect on the quality of

life and may deteriorate systemic health [4].
Three quarters of all microbial infections found in humans are
Various conventional strategies have been developed
associated with microbial biofilm [1], for instance oral diseases,
for the treatment of oral biofilm related diseases (Table 1).
dental caries, and periodontal diseases caused by oral biofilm
Currently, there are three prevailing approaches as follows:
known as dental plaque. Dental caries is a health problem in a
(i) mechanical elimination of dental plaque as a basic method to
majority of developed countries (mainly due to cariogenic diet)
combat the diseases, (ii) maintenance of a constant low level of
and, as shown by a report of the World Health Organization, it
fluoride in the oral cavity by regular wide-scale use of fluoride-
affects people of all ages, that is, 60−90% of school age children
containing oral care products and water fluoridation as the
and most adults [2]. In most countries, the periodontal disease
most effective methods for prevention of dental caries, and
burden also remains high, with approximately 15−20% of
(iii) a widespread use of biocides with chlorhexidine being
adults and 2% of young people affected by severe periodontitis
the most frequently used antiseptic [5, 6]. In recent years,
increasing emphasis has been placed on more preventive and
minimally invasive concepts of caries treatment based on an-
Abbreviations: EPS, extracellular polymeric substances; Ftf, ticariogenic diet, proper oral hygiene, and development of
fructosyltransferase; Gbp, glucan-binding protein; Gtf, glucosyltransferase..
∗ Address for correspondence: Malgorzata Pleszczyńska, PhD, Department
various kinds of new, human-friendly approaches to prevention
of cariogenic biofilm formation such as replacement thera-
of Industrial Microbiology, Maria Curie-Sklodowska University, 19
Akademicka Street, 20–033 Lublin, Poland. Tel.: +48 81 537 5929; pies and the use of probiotics and prebiotics, antimicrobial
Fax: +48 81 5375959; e-mail: [email protected]. peptides, or natural substances such as plant-based extracts,
Received 30 January 2016; accepted 3 March 2016 and essential oils [7–9]. For some time now, the application
DOI: 10.1002/bab.1490 of enzymes has been considered as an alternative strategy to
Published online 21 September 2016 in Wiley Online Library fight with various kinds of biofilm, for example, that associated
(wileyonlinelibrary.com) with medical devices or industrial biofilm [10–12]. This review

337
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Biotechnology and
Applied Biochemistry

Current strategies of dental plaque prevention and control

TABLE 1

Antidental plaque Examples of antiplaque agents

strategy [key reference] Mechanism

Protection of dental hard Inorganic and organic fluoride [13] Inhibition of demineralization and promotion of
tissues remineralization of enamel
Established plaque Personal and professional Removal of immature and mature plaque and
removal mechanical cleaning [14] calculus
Interference with the Polyphenolics from Camellia Inhibition of synthesis of mutans streptococci glucans
attachment of oral sinensis, flavonols (e.g., by Gtfs activity inhibition or enzyme inactivation
microorganisms to the kaempferol and myricetin),
pellicle or to each other flavones (e.g., apigenin) [8]

Zinc compounds [15], delmopinol Inhibition of bacterial adhesion

hydrochloride [16], chlorhexidine
[17]

Inhibition of growth or Chlorhexidine [6], essential oils (e.g., Damage to the integrity of the cell membrane
killing oral thymol, eugenol); propolis
microorganisms components (tt-farnesol) [8]

Stannous and amine fluoride [16], Antibacterial activity of nonfluoride components;

triclosan [15] interference with membrane function

Alteration of metabolic Propolis components (apigenin and Disruption the accumulation of an intracellular
activity of the dental tt-farnesol) [8] storage polymer of Streptococcus mutans
plaque

Chlorhexidine [18], zinc, triclosan Inhibition of sugar transport, acid production, and
[19], delmopinol hydrochloride various membrane functions of mutans
[16] streptococci; inhibition of Porphyromonas
gingivalis protease activity
Stannous and amine fluoride [20] Inhibition of activity of essential enzymes of oral
bacteria

focuses on the use of enzymes as an unconventional approach Dental plaque is a complex biofilm formed on the tooth
to control plaque-associated oral diseases. surface, tongue, oral mucosa, hard palate, gingival pockets, or
on the artificial surfaces of orthodontic appliances or dentures
(denture plaque). The biofilm consists of a multispecies mi-
crobial community (over 700 species), which is, however, only
2. Dental Plaque as an Etiological Factor less than 10% of the biofilm dry mass. Microorganisms dis-
of Oral Diseases tributed nonrandomly in a bacterial and host-origin matrix of
Dental caries involves localized progressive demineralization highly hydrated extracellular polymeric substances (EPS) form
of the tooth hard tissues caused by acids produced by cario- microcolonies. The plaque is a natural form of colonization
genic bacteria in supragingival plaque. It has a multifactorial of teeth and is considered as part of the host defense system
etiology and thus the presence of the dental plaque itself is preventing colonization of exogenous (frequently pathogenic)
not sufficient to cause the disease [21]. Periodontal diseases microorganisms [9].
affect the supporting structures of teeth and are a result of As illustrated in Fig. 1, dental plaque formation is a mul-
inflammatory reaction in response to formation of subgingival tistep process involving acquired pellicle formation, initial and
plaque consisting of various bacteria (gingivitis) or specific polysaccharide-mediated attachment of cells to the surface,
Gram-negative bacteria (periodontitis) at or under the gingival biofilm maturation, and dispersion of biofilm cells [23–26].
margin [22]. Gingivitis is manifested as bleeding of the gingival Early dental plaque contains a limited commensal microflora,
or gum tissues. Periodontitis results in progressive loss of the but factors such as prolonged sucrose intake may induce a
alveolar bone around the teeth [21]. shift in the composition of the predominant species toward

338 Enzymes in Therapy of Oral Diseases

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Diagrammatic representation of dental plaque
FIG. 1 development on the tooth surface. A cleaned tooth
capability of rapid synthesis of adherent exopolysaccharides
surface is very quickly covered with an acquired
pellicle (A) that, on the one hand, protects tooth from dietary sucrose due to production of glucosyltransferases.
enamel against harmful agents and, on the other Streptococcus mutans produces three glucosyltransferases
hand, allows bacterial adhesion. Commensal (Gtfs): GtfD synthesizes water-soluble (1→6)-α-d-glucans (dex-
streptococci and other early colonizers attach trans), GtfB synthesizes water-insoluble (1→3)-α-d-glucans
reversibly to the pellicle (B). Bacteria (mainly
(mutans), and GtfC produces a mixture thereof [28–30]. Ini-
mutans streptococci, in the presence of sucrose
and starch hydrolysates) begin to produce tially, bacteria adhere to the acquired pellicle by a sucrose-
extracellular polymeric substances helping them independent mechanism mediated by lectin-like interactions
to bond closely to pellicle-coated surfaces and to and then produce Gtfs. Enzymes adsorbed to the pellicle imme-
each other (C) and modify the local environment diately utilize sucrose for in situ synthesis of water-soluble and
allowing survival of late colonizers, for example,
-insoluble glucans, which provide binding sites for adhesion
periodontal bridging bacteria Fusobacterium
nucleatum, Aggregatibacter of subsequent bacteria by a sucrose-dependent mechanism
actinomycetemcomitans, and Prevotella [31]. In this manner, highly coherent and adherent plaque is
intermedia, and highly pathogenic bacteria quickly formed and accumulated, even if S. mutans are not the
Tannerella forsythia, Treponema denticola, and dominant microflora in the oral cavity. Moreover, S. mutans
Porphyromonas gingivalis [26, 35]. As a result of
can grow at low pH values and ferment sugars to produce
coaggregation, coadhesion, communication, and
growth of attached cells, organized bacterial lactic acid [27]. By taking advantage of these abilities, the
complexes surrounded by EPS form microcolonies bacteria are very successful colonizers. They can effectively
and can also leave the biofilm by single cell or cell compete with other microorganisms in the dental plaque and
cluster detachment (D). are probably the only microorganisms to initiate the carious
process [32], although many other bacteria in the dental plaque
increased numbers of cariogenic bacteria. Streptococcus mu- produce different matrix exopolysaccharides or are equally po-
tans is generally considered to be a prime causative agent of tent acid-producing and acid-tolerant organisms, for example
dental caries [27]. However, its role in dental plaque formation Lactobacillus spp., Bifidobacterium dentinum, and Scardovia
is related not to the large number, but rather to the bacterial spp. [33, 34].

Biotechnology and Applied Biochemistry 339

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Biotechnology and
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3. Extracellular Polymeric Substances: 4. Enzymes as Alternative Antibiofilm

A Home for Microbial Community Agents
The biofilm matrix is composed of polysaccharides, microbial Enzymes, mainly microbial, added to oral hygiene products
and mammalian proteins, lipids, nucleic acids, and inorganic such as mouth rinses, toothpastes, and chewing gums effec-
ions such as Ca, Mg, P, and F [36]. The polysaccharides mainly tively support mechanical cleaning of teeth through different
include glucose and fructose homopolymers, that is, (1→3)-α- mechanisms (Table 2) [16]. Enzymes act gently and specifically
d-glucan, (1→4)-α-d-glucan, and (1→6)-α-d-glucan (10−40% and, therefore, they hardly affect the biological environment of
dry weight of dental plaque) and (2→6)-β-d-fructans (levans the oral cavity. A great advantage of this type of strategy is that
1−10%) [36]. They are synthesized from ingested sucrose resistance of bacteria to enzymes, in contrast to chemicals, is
by glucosyltransferases (Gtfs) and fructosyltransferases (Ftfs) rare.
secreted by oral bacteria (Streptococcus spp., Lactobacillus
spp., and Actinomyces spp.). Mutans and dextrans are par- 4.1. Antimicrobial enzyme system
ticularly important glucans in the formation of dental plaque. One of the important functions of saliva is to protect oral tissues
Mutans have a highly branched structure with main chains from the harmful effect of microorganisms. Therefore, saliva
composed of glucose molecules linked with (1→3)-α bonds and contains a spectrum of proteins with antibacterial properties.
(1→6)-α-glucosidic linkages in their side chains [37–40]. Dex- Among the nonimmunologic salivary protein components,
trans are also high molecular weight polymers of glucose there are peroxidases and lysozyme, that is, part of the innate
containing numerous consecutive (1→6)-α-linkages in their defense system [47]. Using a number of different mechanisms,
backbone and side chains, which begin from the (1→3)-α- these enzymes exhibit bactericidal or bacteriostatic activity
linkage [41]. The synthesis of glucans is an important factor of against a broad spectrum of microorganisms. They are found
virulence of mutans streptococci, as these compounds deter- in almost all secreted body fluids and tissues of human and
mine the properties of EPS and enable the matrix to support animal organisms, such as milk, tears, and saliva [48].
biofilm functioning [36, 42]. First, glucosyltransferases and Human whole saliva contains a salivary peroxidase enzyme
glucans produced by mutans streptococci are responsible for secreted by the salivary glands. It is a porphyrin-containing gly-
sucrose-dependent adherence of the bacteria to the tooth sur- coprotein forming an antimicrobial peroxidase system with its
face and to each other [43–45]. Second, glucans can interact cofactors. The active component of this system is, depending on
with many other biofilm components, for example, proteins, the pH, a hypothiocyanite ion (OSCN− ) or hypothiocyanous acid
lipids, lectins, and other polysaccharides forming a gelatinous (HOSCN), which is formed by peroxidase-catalyzed oxidation of
network in which microbial cells are embedded [46]. This salivary thiocyanate (SCN− ) using hydrogen peroxide derived
network keeps the bacteria bonded together, allowing their from bacteria and host cells [49, 50]. The oxidation products
interactions, and maintains them on the surface. Furthermore, of SCN− inhibit growth of Gram-negative and Gram-positive
EPS can protect microbial cells within the biofilm by hindering bacteria by oxidation of the sulphydryl groups of enzymes and
the diffusion of antimicrobials inside the biofilm or by binding other proteins [48]. The peroxidase system interacts, often
harmful agents [36]. Binding of metal ions ensures stronger at- synergistically, with other immunologic and nonimmunologic
tachment of the developing biofilm and its mechanical stability salivary constituents, for instance IgA, lysozyme, and lacto-
and creates a local nutrition-rich environment favoring specific ferrin [51]. Lysozyme splits the β-(1→4)-glucosidic linkages in
microorganisms [43, 46]. Some polysaccharides also serve as peptidoglycan and thus can degrade the bacterial cell wall. The
an additional source of energy for biofilm microorganisms. antibacterial activity of the enzyme also includes aggregation
Other important components of the dental plaque ma- of bacteria, activation of bacterial autolysins, and inhibition of
trix are proteins, including enzymes and structural proteins. bacterial adherence and glucose metabolism [52]. Lysozyme
Many EPS enzymes are involved not only in the synthesis of has higher antibacterial activity toward Gram-positive bacte-
matrix polymers (Gtfs and Ftfs) but also in modification of ria; however, the peptidoglycans of some of these bacteria are
the matrix composition as well as degradation of a preformed resistant to being hydrolyzed by this enzyme. In the case of
matrix to disperse bacteria from biofilms (dextranases, levan Gram-negative bacteria, the inner membrane composed of pro-
hydrolases, proteases, nucleases, or lipases) [36]. Examples teins, phospholipids, and lipopolysaccharides is a barrier for
of structural proteins are cell surface associated and extra- the enzyme [53]. However, lysozyme activity can be enhanced
cellular carbohydrate-binding proteins involved in bacterial by certain substances such as lactoferrin, which is a glycopro-
adhesion, coadhesion, and coaggregation. For instance, at least tein belonging to the transferrin family and is thus capable
four glucan-binding proteins (Gbps) have been identified in of binding and transferring Fe3+ ions. Lactoferrin has a wide
S. mutans. Particularly, two cell wall bound proteins, Gbp C variety of biological functions, many of which do not appear to
and B, which function as cell surface glucan receptors, have a be connected with its iron-binding ability. It affects the growth
role in sucrose-dependent adhesion and biofilm formation by and proliferation of both Gram-positive and Gram-negative
the bacteria [43]. bacteria, viruses, protozoa, or fungi. The bacteriostatic effects

340 Enzymes in Therapy of Oral Diseases

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Enzymes in dental plaque control
TABLE 2

Enzyme targets Potential effects Example of enzyme

Viability of Killing or bacterial growth limiting Oxidative enzymes: salivary

microorganisms peroxidases, lysozyme,
lactoferrin
Components of biofilm Removal of the existing plaque by degradation of Glucan hydrolases:
matrix biofilm-bonding extracellular matrix components or dextranase, mutanase
disruption of biofilm formation by interference with
sucrose-dependent adhesion of bacteria

Colonization of oral Prevention of biofilm formation by disruption of Proteases: Krillase, papain

surfaces bacterial adhesion to oral surfaces and/or
coaggregation and coadhesion of microorganisms

of lactoferrin are associated with sequestration of iron because free glucose from dextrins produced via hydrolysis of starch
the absence of iron inhibits the growth of iron-dependent bacte- residues by salivary and bacterial amylases [48, 66].
ria [54]. Moreover, the N-terminal cationic region of lactoferrin The effectiveness of the products has been studied in
binds to specific receptors on the surface of some microor- clinical trials. However, their results were controversial as
ganisms inducing cell death in Gram-negative bacteria due to indicated by the example of the history of Biotene products
disruption of the cell wall [55]. In addition, lactoferrin exhibits (Laclede Professional Products, Rancho Dominguez, CA, USA)
proteolytic activity against a variety of bacterial proteins [56]. (for a review, see Tenovuo [48]). Biotene products contained
The above-mentioned proteins, or rather their active coun- all the components of the peroxidase system, that is lactoper-
terparts such as milk lactoperoxidase, which is structurally oxidase, KSCN, hydrogen peroxide generating glucose oxidase
somewhat different but catalytically similar to salivary peroxi- (from added glucose), and additionally lysozyme and lactofer-
dase, can be purified in a cost-effective way from, for instance, rin. They were available in the form of toothpaste, mouth rinse,
bovine milk or colostrum or produced from microorganisms moisturizing gels (Oral Balance, without lysozyme and lactofer-
[57]. In vitro studies have confirmed the anticaries activity of rin), and a denture adhesive (Denture Grip). The products were
these enzymes. For example, lactoperoxidase and lysozyme especially intended for patients who suffer from dry mouth
worked synergistically to restrict S. mutans metabolism and but also for normal oral hygiene. Numerous clinical studies
hence reduced acid production in the plaque environment revealed that the Biotene mouth rinse and toothpaste used by
[58–60]. Lactoferrin inhibited adherence of S. mutans to saliva- healthy volunteers effectively generated HOSCN/OSCN− but no
coated hydroxyapatite beads [61]. Hence, an idea emerged to bactericidal effect was observed in most cases [65, 67, 68].
produce oral care products formulated with saliva-borne pro- Better results, although only with respect to alleviating dry
teins to potentiate or restore host defense against oral biofilm mouth symptoms, were achieved in patients suffering from
related diseases [48]. Their active counterparts including hyposalivation. Xerostomia (dry mouth) is related to a number
lactoferrin, lysozyme, as well as lactoperoxidase and its sub- of chronic conditions and diseases. It affects patients taking
strate (KSCN) have been introduced in various combinations systemic medications, undergoing radical radiotherapy for the
to oral hygiene products (toothpastes, mouth rinses, chewing treatment of head and neck neoplasms, and suffering from
gums, moisturizing gels, and sprays) such as Biotene, BioXtra, other diseases, such as Sjögren’s syndrome. Such patients have
and Zendium. It was found in in vitro studies that a denture difficulties in chewing, swallowing, tasting, or speaking and are
adhesive Biotene Denture Grip exhibited antimicrobial activity at increased risk of developing dental caries [69]. Three-week
against oral malodor related microorganisms [62], and BioXtra trials with xerostomic pediatric patients using Biotene mouth
and Biotene toothpastes were effective against S. mutans [63]. rinse combined with Biotene gel or chewing gum showed that
However, hydrogen peroxide proved to be a limiting factor the lactoperoxidase-system significantly relieved subjective
for the formation of the oxidation products of SCN− in whole oral symptoms [70]. The palliative effects of Oral Balance gel
saliva [64]. Enhancement of the peroxidase system in vivo was and Biotene toothpaste, superior to the effects of a placebo,
achieved by combining it with the glucose oxidase system in have also been reported in patients with xerostomia following
which fungal (generally Aspergillus niger) glucose oxidase uti- radiation therapy and some diseases [71–77]. However, since
lized glucose to synthesize H2 O2 needed by lactoperoxidase to some of these studies lacked proper controls [75, 76], it is
oxidize halides [65]. Some products like Zendium additionally difficult to say what factors contributed to the improvement
comprised another enzyme, that is, amyloglucosidase, to in- of the patients’ condition: the addition of the enzymes or the
crease the concentration of glucose. This exo-enzyme liberates generally gentler action of the tested products due to the

Biotechnology and Applied Biochemistry 341

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Biotechnology and
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nonfoaming and alcohol-free or sodium lauryl sulfate-free were comprehensively described by Khalikova et al. [41].
formula. At present, Biotene products are marketed by Glax- Among others, these enzymes are produced by a variety of oral
oSmithKline and they do not contain the above-mentioned bacteria, mainly from the genus Streptococcus [85–88]. The
enzymes (http://www.biotene.com). Human Oral Microbiome Database reveals 19 genes encoding
dextranolytic enzymes. However, the role of the enzymes in oral
4.2. Glucan hydrolases bacteria is not entirely clear; it is believed that they are one of
Mutans and dextrans are considered the most important dental the streptococci virulence factors, that is, they facilitate the use
plaque matrix polysaccharides. Water-insoluble mutans pro- of dextran as an additional source of carbon for bacteria and
duced by surface-adsorbed glucosyltransferases are essential modify the structure of dextran polymers synthesized by Gtfs
to the assembly of the three-dimensional structure of the EPS [87, 89].
matrix [44]; they easily adsorb to pellicle-coated enamel, pro- In the 1970s and 1980s, high expectations were associated
mote bacterial coaggregation, and substantially enhance the with anticaries application of dextranases. In in vitro studies,
cohesive and adhesive properties of plaque [78]. Because of these exogenous enzymes partially hydrolyzed water-insoluble
the presence of (1→3)-α-glucoside links, which are resistant and -soluble dextrans, and even inhibited mutan synthesis and
to the action of enzymes present in the oral cavity, mutans degraded artificial biofilms of mutans streptococci [90–92].
provide dental plaque with stability and durability [78, 79]. Marotta et al. [93] showed that small amounts of a commercial
Water-soluble dextrans together with other matrix polysac- fungal enzyme Dextranase 50 L inhibited, to a limited ex-
charides function as a reserve of carbohydrates and retain tent, production of streptococcal water-insoluble glucans and
water to prevent desiccation of the biofilm [42]. Therefore, deprived the synthesized polymer of its adhesive properties.
both these glucans may be promising targets for antibiofilm The enzyme also degraded presynthesized glucans up to 20%.
agents including exogenous glucan hydrolyzing enzymes such However, the results of animal and human studies point to a
as mutanases and dextranases. dubious effectiveness of the enzyme in prevention/reduction of
(1→3)-α-Glucanases are a poorly understood class of caries. Guggenheim et al. [94] found that the fungal dextranase
glycoside hydrolases, which catalyze the hydrolysis of glu- inhibited only in a low degree the development of dental caries
cosidic bonds in (1→3)-α-d-glucans of different origins, that in rats and, in addition, only in conditions of relative gnoto-
is, fungal cell wall and/or oral bacterial glucans. Enzymes biosis. In clinical trials carried out by Caldwell et al. [95], the
degrading bacterial mutans are referred to as mutanases. enzyme used in the form of a mouth rinse had no significant ef-
Generally, (1→3)-α-glucanases are inducible enzymes pro- fect on plaque scores and plaque dry weight, although Lobene
duced in the presence of mutans or other glucans containing [96] showed that similar application of dextranase reduced
(1→3)-α-type bonds by fungi, mainly Trichoderma spp., and soil plaque dry weight. However, at the same time, it turned out
bacteria of the genera Paenibacillus and Bacillus [80–82]. All that mutans rather than dextrans played a crucial role in the
(1→3)-α-glucanases (E.C. 3.2.1.59) characterized experimen- formation of dental plaque [97].
tally so far belong to glycoside hydrolase family 71 (fungal Mutanases have been shown to modify the dental plaque
enzymes) or 87 (bacterial enzymes). Fungal glucanases release matrix by affecting the amount, structure, aqueous solubil-
glucose moieties as main products of glucan degradation. An ity, and adhesive properties of extracellular water-insoluble
example is the well-characterized T. harzianum mutanase, (1→3)-α-glucans and, consequently, they lead to destruction of
an endo-type processive enzyme, which after the initial in- biofilm architecture [44]. In in vitro studies, mutanases effec-
trachain attack hydrolyzes glucan in a repetitive manner tively degraded and solubilized water-insoluble streptococcal
toward the nonreducing end releasing β-glucose residues as mutans [98–103]. For instance, a Pseudomonas enzyme, at a
the major digestion product [83]. Bacterial glucanases degrade very low concentration, removed nearly all adherent mutan
(1→3)-α-d-glucans in an endo-pattern releasing short-chain preformed on a glass surface and markedly inhibited (up to
nigerooligosaccharides, for instance nigerose, trinigerosac- 50%) de novo formation of insoluble glucan. The enzyme frag-
charide, and tetranigerosaccharide as in the case of the mented large aggregates of (1→3)-α-linked double-stranded
Paenibacillus curdlanolyticus enzyme [84]. fibrils of mutan, by which glucan became less adherent [104].
Dextran-degrading enzymes commonly called dextranases Some mutanases have the ability to bind to insoluble substrates
form a diverse group of different carbohydrases and trans- because of the presence of a glucan-binding domain [81, 105],
ferases. They specifically hydrolyze (1→6)-α-glucosidic link- which ensures the possibility of prolonged action even after
ages in dextrans, their derivatives, and isomaltodextrins. removal of the vehicle (mouth rinse, toothpaste) via which they
Dextranases have often been classified as endo- and exodex- are supplied from the oral cavity. Additionally, it was found
tranases. Exodextranases remove successively one or more that the enzymes degraded heterogeneous material of dental
glucose units from the nonreducing ends of the dextran chains. plaque EPS more effectively when they were used together with
Endodextranases hydrolyze (1→6)-α-d-glycoside linkages in dextranases or other hydrolases [106].
random sites of dextran, removing isomaltooligosaccharides The first attempts at using mutanase in the form of a
usually containing two to five units of glucose [41]. The diversity mouth rinse for humans did not bring spectacular results
of the sources, properties, and modes of action of dextranases [107]. Reduction of dental plaque was only achieved by

342 Enzymes in Therapy of Oral Diseases

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application of a T. harzianum OMZ 779 mutanase formula- disruption of proteinaceous connections or weakening of the
tion [108, 109]. Bacterial mutanase from Pseudomonas sp. structure of the biofilm matrix by proteolytic enzymes.
reduced the growth of dental plaque on the labial and lin- For this purpose, attempts were made to use microbial,
gual surfaces of the front teeth as well [110]. A similar effect plant, and animal proteases singly and in mixtures. Unfortu-
was achieved using a T. harzianum CCM F-340 enzyme [111], nately, the results of clinical studies conducted mainly in the
which, in contrast to other mutanases, can be produced on a 1950s and 1960s did not fully confirm the above-mentioned
large scale, since an easily accessible (1→3)-α-d-glucan from a suppositions [119, 120]. Conflicting results were achieved by
cultivable fungus Laetiporus sulphureus is used as an inducer assessing the effectiveness of Bacillus subtilis preparations
of the enzyme synthesis [80, 112]. The necessity of using mutan containing proteases and amylase. In contrast to the reports
produced by mutans streptococci as an inducer has so far of Mollé [121] and Shaver and Schiff [122], two other clinical
been a serious limitation in the wide application of these en- studies showed no effect of these preparations on reduction
zymes. However, constitutively produced glucan hydrolase from and accumulation of dental plaque and on gingivitis [123, 124].
Lipomyces starkeyi with broad substrate specificity toward the Harrison et al. [125] found that highly active proteolytic and
(1→3)-α-glycoside, (1→6)-α-glycoside, and (1→4)-α-glycoside amylolytic enzymes from fungi formulated in toothpastes re-
linkages has also been tested. The enzyme-containing mouth tarded calculus deposition. Similarly, anticalculus activities of
rinse resulted in significantly lower plaque formation com- mucinase (proteolytic enzymes and amylase) were shown in
pared to the control and chlorhexidine mouth rinses [113, 114]. clinical studies in a small number of participants [126, 127].
More recently, enzyme-based Biotene products, that is, tooth- In 1959, Viokase, a preparation of enzymes isolated from
paste, chewing gum, and mouth rinse, are available on the dehydrated pancreas containing trypsin, chymotrypsin, car-
market. They contain mutanase and dextranase together with boxypeptidase, amylase, lipase, and nucleases was tested in a
the above-mentioned oxidative antimicrobial enzymes. Unfor- long-term study with 134 subjects using the preparation once
tunately, there are no reports proving the antiplaque matrix a day for 10–15 min. Jensen [128] reported a 60% decrease in
efficiency of these products. calculus formation. Viokase was then introduced to chewing
There have been attempts at application of glucan hydro- gums and sticks to ensure long-term exposure necessary for
lases for removal of denture plaque accumulating on removable enzyme effectiveness. The formulations produced a 22–26% re-
prosthetic devices. The structure of this plaque is very similar duction in calculus formation compared with the control groups
to that of dental plaque [115]. Denture plaque occurs in a ma- [129]. On the other hand, Packman et al. [130] compared the
jority of patients with dentures and is a cause of troublesome efficacy of Viokase-containing chewing gums or fungal en-
oral inflammations caused primarily by biofilm-colonizing Can- zymes and concluded that combined enzymes of fungal origin
dida albicans yeasts isolated from 50 to 100% of patients with worked more efficiently in retarding calculus deposition. More
inflammatory lesions. A key to maintenance of the oral mucosa recently, satisfactory results have been obtained using pro-
in a proper condition is the effective denture cleansing method. teases isolated from the digestive tract of the Antarctic krill
A Danish research team headed by Budtz-Jörgensen conducted (Euphausia superba). Krillase is a multienzyme preparation
comprehensive investigations on enzymatic cleansing of both obtained as a purified extract of krill hydrolytic enzymes, which
new and old, unkempt dentures using single or combined contains a mixture of serine proteinases, acidic endopeptidases
enzymes. It was found that a mixture of mutanase and protease (trypsin and chymotrypsin-like enzymes), and exopeptidases
was more efficient than the enzymes applied separately, which (carboxypeptidase A and B) [131]. This preparation had no
was reflected in improvement of the condition of the palatal antimicrobial activity; therefore, it did not alter oral microbiota
mucosa, reduction of the yeast cell count in smears, and a lower but inhibited the binding of oral bacteria to saliva-covered
concentration of leukocytes [116, 117]. Simultaneously, long- surfaces and detached bacterial plaque [132]. Krillase was
term application of the enzymatic method for everyday denture used in a chewing gum formulation (for 10 min, four times
cleansing used for a long time by elderly people with visible a day for 10 days) in healthy volunteers [133]. It was shown
inflammatory palatal lesions caused by infected dentures and that already the minimal dose (0.06 U) of proteases in the gum
poor hygiene reduced the size of plaque and improved the significantly disrupted bacterial adherence to the dental pellicle
condition of oral mucosa already within 6 weeks [116, 118]. and reduced gingivitis in 54%, compared with the control. The
effectiveness of the enzyme mixture was probably a result of its
unique synergistic action and the wide specificity of different
4.3. Antiplaque, anticalculus, and whitening proteases proteases [134].
Generally, dental plaque is formed by (i) colonization of the Proteases of plant origin have also been tested, mainly
acquired protein pellicle (composed of mucins, other glycopro- for removal of tooth stains and calculus. The color of teeth is
teins, and proteins) that covers the surface of the teeth with influenced by a combination of their intrinsic color and the
early colonizers and (ii) extensive coaggregations and coadhe- presence of any extrinsic stains that may form on the tooth
sions mediated by different adhesion proteins synthesized by surface. Enzymatic removal/prevention of stains proceeds
secondary colonizers [23]. Therefore, a promising antibiofilm likewise calculus removal through hydrolytic degradation or
option seems to be to interfere with plaque accumulation by alteration of the protein salivary pellicle, which is essential

Biotechnology and Applied Biochemistry 343

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Biotechnology and
Applied Biochemistry

in the attachment of stains and calculus to the tooth surface important components of oral biofilm matrix, such as glucans
or/and in disturbance of plaque formation [135]. On the market, or proteins.
there are several products with plant proteolytic enzymes, for However, like other treatments, the application of enzymes
example, Rembrandt and Janina toothpastes. They contain has some limitations. Enzymes are characterized by sensitivity
papain and/or bromelain derived from papaya (Carica papaya) to proteolysis and a limited possibility of penetration. The low
and pineapple (Ananas comosus), respectively. The Rembrandt activity of wild-type enzymes and their insufficient stability in
toothpaste containing citroxain (a combination of papain and oral hygiene products limit their efficiency. Due to their spe-
citrate chelate) was shown to reduce plaque (19%) and calculus cific mode of action, formulations containing several different
(17%), and whitened teeth significantly (55%) in relation to enzymes may be necessary to be successful, for instance, in
the control [136, 137]. The toothpaste also effectively removed degradation or inhibition of formation of the complex and het-
chlorhexidine-induced stains [138]. In vitro and in vivo studies erogeneous dental plaque matrix. Another problem is the low
have demonstrated that toothpaste with both fruit enzymes was availability and high cost of production of the enzymes, partic-
effective in removing established stains [139, 140]. Recently, a ularly in contrast to chemical antimicrobial agents. Therefore,
papain-based product developed in Brazil has been reported before the full potential of the enzymes may be realized, it is
as an excellent noninvasive chemomechanical method for necessary to improve their efficiency and cost effectiveness.
removal of caries, which eliminates infected dentin and simul- Among others, it is advisable to search for new enzyme for-
taneously preserves a healthy dental structure. The papacarie mulations, as conventional toothpastes or mouthwashes are
gel is composed of papain, chloramines, toluidine blue, salts, characterized by an insufficient time of retention in the oral
and thickening vehicle, which together are responsible for its cavity. A method for overcoming this problem could be the
bactericidal, bacteriostatic, and anti-inflammatory character- application of liposomes as enzyme carriers [149]. Additionally,
istics. Papain digests only infected tissues by breaking down biotechnological advance facilitates creation of enzymes with
collagen molecules partially degraded by bacteria and mak- improved or new catalytic properties by strategies based on
ing the infected dentine softer, and thereby, easier to remove rational, semirational, and randomly directed evolution; the
[141–143]. discovery of new microbial enzymes from nature using modern
As in the case of mutanases, there have been attempts techniques, that is, metagenomics and genomics, and produc-
at employing proteases for the fight against oral biofilms, for tion of recombinant proteins in microbial hosts [150]. Recently,
example, plaque accumulated on dentures. Enzyme denture for example, Otsuka et al. [151] have constructed and produced
cleansers available on the market (such as Corega Tabs with a chimeric glucanase associating the Paenibacillus humicus
papain) contain proteolytic and/or yeast lytic enzymes [144]. NA1123 mutanase and the S. mutans ATCC 25175 dextranase.
The effectiveness of the enzymes in removing food debris, The chimeric enzyme reduced the formation of the total amount
denture plaque, and Candida present in the plaque was studied. of water-insoluble glucan and was able to decompose biofilm,
Connor et al. [145] showed that incorporation of enzymes in being four times more effective than a mixture of dextranase
a denture cleanser enhanced its effectiveness. Tamamoto and mutanase. There should be also a new way to look at the
et al. [146] reported two lytic and several proteolytic enzymes possibility of the use of the enzymes in dentistry. The enzymes
removing Candida in in vitro tests. Similarly, papain was found could destroy the physical integrity of the biofilm matrix, and
to be effective in elimination of fungi from denture plaque consequently deprive it of the function of a protective barrier
[147]. Clinical findings also confirmed that Krillase efficiently for the microorganisms contained therein. Matrix-targeting
detached bacteria from dentures [132, 133] and a mixture strategies for mature biofilm control may facilitate penetration
of proteases and mutanases reduced denture plaque and of biofilm by antimicrobial agents, enhancing their therapeutic
improved the condition of the palatal mucosa [118]. Ödman effects. There are single examples of complementary appli-
[148] showed that soaking dentures in alcalase was ineffective cations of enzymes and antimicrobials, that is, the use of in
in contrast to using the enzyme in combination with brushing. situ produced dextranase to enhance the susceptibility of oral
On the other hand, Nakamoto et al. [144] did not confirm the biofilm to bacteriocins with activity against mutans streptococci
efficiency of several enzyme denture cleansers tested under the [152].
same conditions against a control alkaline peroxide cleanser.
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