Enzyme capable of hydorlyzing plaque, microorganism...

Details Enzyme capable of hydorlyzing plaque, microorganism... Enzyme capable of hydorlyzing plaque, microorganism...

2000-07-18

2002-11-26

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S203000, C435S211000, C424S050000

Reexamination Certificate

active

06485953

ABSTRACT:

This application is a 371 of PCT/KR00/00191 filed Mar. 9, 2000.
TECHNICAL FIELD
The present invention relates to an enzyme capable of hydrolyzing dental plaque, a microorganism producing the same and a composition comprising the same.
BACKGROUND OF THE INVENTION
Plaque formed on the surface of a tooth is composed of compactly packed bacteria and non-cellular materials. The main polysaccharide component of plaque is water-insoluble glucan or mutan, which constitutes approximately 20% of the dried mass of plaque and is a main cause of dental caries. Structural studies of glucans produced by Streptococos mutans revealed that the insoluble glucans are mainly composed of &agr;-1,3-, &agr;-1,4-, &agr;-1,6-D-glucoside. Therefore, to eliminate plaque effectively, mutanolytic, amylolytic. and dextranolytic activities are required.
Conventionally, methods of reducing the growth of
Streptococos mutans
(
S. mutans
) in mouth have been suggested to prevent the formation of plaque or dental caries. To achieve this, antiseptics or fluoride which inhibit the growth of
S. mutans
have been added in oral compositions such as toothpaste or mouthwash. Fluoride is one of the most widely used chemicals because it reduces the growth of
S. mutans
. Although fluorine can inhibit the growth of the caries inducing bacteria, it can cause caries-like lesions (formation of orthodontic white spots on tooth enamel) as well as severe side effects such as strong toxicity and air pollution. Enzymes such as dextranase have been used to prevent dental caries, however, its effect has yet to be proven.
U.S. Pat. No. 5,741,773 discloses a toothpaste composition comprising glycomacropeptide having antiplaque and anticaries activities.
These conventional techniques relate to the reduction of the bacteria growth that causes dental caries. This invention can prevent the formation of plaque and hydrolyze pre-formed plaque.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide an enzyme which can hydrolyze or inhibit dental plaque.
Another object of the present invention is to provide a microorganism which produces a dental plaque hydrolyzing or inhibiting enzyme.
The present invention relates to a dental plaque hydrolyzing or inhibiting enzyme, a preparation method thereof, a composition containing the same and microorganism producing the same.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
It has been reported that
Lipomyces starkeyi
(
L. starkeyl
) produces endo-dextranase (EC 3.2.1.11) which degrades dextran and &agr;-amylase which degrades starch. This microorganism has been used in food related applications and is not known to produce antibiotics or toxic metabolites.
Except for a few bacterial dextranases, dextranases which are produced by microorganisms are known to be inducible enzymes. The present inventors have reported that
L. starkeyi
ATCC 74054 produces both dextranase and amylase (U.S. Pat. No. 5,229,277) while disclosing the characteristics of the enzyme produced by the same microorganism. The present inventors have also reported that these microorganisms can produce small molecular weight dextrans by using sucrose and/or starch.
The present invention relates to an enzyme that can inhibit the formation of or degrade dental plaque.
The enzyme of the present invention degrades dextran and starch as well as insoluble glucans and will be referred to as DXAMase hereinafter.
DXAMase according to the present invention mainly produces glucose, isomaltose and branched tetrose and small amounts of branched pentose and hexose when dextran is used as a substrate. When starch is used as a substrate, DXAMase can mainly produce glucose, maltose, maltotriose and maltotetraose as well as a variety of malto-oligosaccharides.
Since DXAMase can degrade levan which is a polymer of &bgr;-fructan, DXAMase according to the present invention can effectively degrade fructan forming plaque.
DXAMase according to the present invention, therefore, can effectively degrade soluble as well as insoluble glucans and fructan. DXAMase can effectively prevent dental caries since plaque formation can be inhibited by preventing the aggregation of glucan and microorganisms or pre-formed plaque can be eliminated.
Experimental results using hydroxyapatite, which is similar in composition with dental materials, indicate that DXAMase has a stronger binding with hydroxyapatite than
P. funiculosum
dextranase. Therefore, it is expected that DXAMase will have a higher chance to retain on the tooth surface.
DXAMase according to the present invention is stable in a variety of mouthwash. Moreover, DXAMase does not lose its enzymatic activity in the presence of by chlorhexidine which is currently used as a remedy of periodontal disease.
DXAMase can be isolated from
L. starkeyi
ATCC 74054 or
L. starkeyi
KSM 22. In other words, DXAMase is isolated from a culture medium of
L. starkeyi
ATCC 74054 or
L. starkeyi
KSM 22, and is identified by double bands of 94K and 60K on a SDS-PAGE (10%) eletrophoresis whose pl is 6.0 for both bands.
The present invention also relates to a new microorganism that produces DXAMase.
L. starkeyi
KSM 22 was obtained by mutating
L. starkeyi
ATCC 74054 and has a higher productivity of DXAMase than
L. starkeyi
ATCC 74054. The present microorganism,
L. starkeyi
KSM 22 (Taxonomy: Eukaryota; Fungi; Ascomycota; Saccharomycotina; Saccharomycetes; Saccharomycetales, Lipomycetaceae; Lipomyces;
Lypomyces starkeyi
) has been deposited with Korean Federation of Culture Collections (KFCC) located at Shinchon-dong 134, Seodaemun-ku, Seoul, Korea on Jan. 19, 1999 and was given a deposit number KFCC-11077. The same strain was also deposited according to the Budapest Treaty at the international depository Korean Culture Center of Microorganisms (KCCM) located at 361-221, Yurim B/D, Hongje-1-dong, Seodaemon-gu, Seoul 120-091, Republic of Korea, and was given a deposit number KCCM-10181 on Mar. 7, 2000.
The present invention also relates to a method of producing DXAMase. The method of the present invention comprises culturing
L. starkeyi
ATCC 74054 or
L. starkeyi
KSM 22 and recovering DXAMase from the culture medium. Since
L. starkeyi
ATCC 74054 and
L. starkeyi
KSM 22 can produce DXAMase from not only the expensive dextran but also relatively cheap glucose, fructose, sucrose or starch, it is economical for practical use. DXAMases obtained from
L. starkeyi
ATCC 74054 and isolated from
L. starkeyi
KSM 22 are practically identical.
Also the present invention relates to an anticaries composition comprising DXAMase. The composition according to the present invention can be used as an additive of a composition for oral hygiene such as toothpaste and mouthwash and food such as candy, chewing gum and beverage. The enzyme according to the present invention maintains its enzymatic activity for a long period of time in commercially available mouthwash solutions and has a strong resistance against enzyme inhibitors. It will be understood that the components of the oral composition and of food ingredients can be verified without difficulties within known conventional limits as will be apparent to those skilled in this art.
Definitions
Dextranase activity is determined by measuring the amount of isomaltose produced by the reaction of an enzyme solution in a buffer solution containing 2% dextran for 15 min at 37° C. Dextranase 1 IU is defined as the amount of the enzyme that produces 1 &mgr;mol of isomaltose when reaction is carried out by using dextran as a substrate for 1 min at 37° C.
Amylase activity is measured by reacting the enzyme solution in a buffer solution containing 2% starch for 15 min at 37° C.
Minimum salt medium: (NH
4
)
2
SO
4
0.5%(w/v); KH
2
PO
4
0.1 5%(w/v); MgSO
4
.7H
2
O 0.01%(w/v); NaCl 0.01%(w/v); CaCl
2
2H
2
O 0.01%(w/v).
LW medium: yeast extract 0.3%(w/v); KH
2
PO
4
0.3%(w/v).
Glucan hydrolysis rate
=
(
reducing sugar produced
with enzyme treatment,
μmol/mL
)
-
(
reducing sugar produced
without enzyme treatment,
μmol
/
mL
)
[
total glucose
in polysaccharide
sample, μmol

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