WO2006091118A2

WO2006091118A2 - A NEW STRAIN OF PAENIBACILLUS CURDLANOLYTICUS, A METHOD OF MUTANASE PRODUCTION, THE APPLICATION OF α-L ,3-GLUCAN FOR THE PRODUCTION OF MUTANASE, AND AN ENZYMATIC PREPARATION

Info

Publication number: WO2006091118A2
Application number: PCT/PL2006/000012
Authority: WO
Inventors: Janusz Szczodrak; Adrian Wiater; Malgorzata Pleszczynska
Original assignee: Uniwersytet Marii Curie-Sklodowskiej
Priority date: 2005-02-28
Filing date: 2006-02-20
Publication date: 2006-08-31
Also published as: WO2006091118A3

Abstract

The invention is a new strain of bacteria Paenibacillus curdlanolyticus MP-I, deposited at the Instytut Biotechnologii Przemyslu Rolno-Spozywczego in Warsaw, Poland under the no. KKP 2017p. This strain is particularly well- suited for the production of mutanase. The invention also concerns the application of a glucan of fungal origin as inductor of mutanase for human- targeted preparations, in particular as a component of dental hygiene products. The glucan comes from fungi. A method was also invented to produce mutanase based on the new strain and the glucan of fungal origin. The invention also covers an enzymatic preparation containing mutanase.

Description

A new strain of Paenibacillus curdlanolyticus, a method of mutanase production, the application of α-l ,3-glucan for the production of mutanase, and an enzymatic preparation

The invention covers a new strain of Paenibacillus curdlanolyticus, a method of mutanase production, the application of α-l,3-glucan for the production of mutanase, and an enzymatic preparation intended primarily for application in dental agents designed for people, in particular in oral hygiene products.

PRIO R ART

Dental caries is a destructive disease consisting in gradual demineralisation of tooth hard tissue. The cause of the demineralisation is lactic acid, the main product of anaerobic decomposition of food carbohydrates under the influence of bacteria accumulated in dental plaque. Dental plaque, similarly to denture plaque, develops due to inappropriate hygiene. Henceforth, plaque signifies dental or denture plaque. In dental plaque, there are various bacteria which are harmful to humans, including Streptococcus mutans and Streptococcus sorbinus. These are doubly responsible for the development of caries as they also produce glucans, i.e., dextrans and water-insoluble mutans, which form the skeleton of dental plaque. For many years, numerous methods have been used to remove dental plaque, ranging from daily hygiene of the oral cavity or the use of chemical agents inhibiting the development of bacterial flora, to the use of biochemical agents containing enzymes. The best-known group of the enzymes are mutanases.

Mutanases (α-l,3-glucanases) are the only group of enzymes which have the ability to effectively decompose mutans - the biopolymers forming the plaque skeleton. Their practical application in dentistry as an additional component of dental hygiene products leads to disintegration of the structural element of plaque, and thus facilitates effective treatment of dental caries. However, a more widespread use of mutanases encounters numerous difficulties, the most prominent being the fact that most of the microorganisms which produce these enzymes produce them only when there is an appropriate inducer in the cultivation medium.

The inducers of mutanases are α-l,3-glucans of bacterial and fungal origin. A bacterial glucan insoluble in water, called mutan, is produced by microorganisms which occur in dental plaque and are responsible for the development of dental caries. Unfortunately, the mi croorganisms occurring in the oral cavity are incapable of producing mutanase which would destroy the skeleton of dental plaque.

Mutan is a polymer of glucose built from the main chain, in which glucose residues are linked by α-l,3-glycosidic bonds, and from a varying number of side chains. As known from the patent description no. US 4,353,891, mutan is a strong stimulant of mutanase production, and the obtained enzyme effectively hydrolyses streptococcal mutans in vitro and in vivo. However, the production of mutanases on a large scale based on this inducer is practically impossible due to the lack of a commercial mutan preparation on the market. For mutan synthesis, pathogenic bacteria (mutans streptococci) need to be used, grown on complex, expensive media which contain hazardous materials of animal origin (e.g., cow Brain Heart Infusion). Thus, this mutan is ill-fitted for producing agents to be used in humans as it may be dangerous to their health or even lives. Additionally, the production is a multi-stage process which proceeds with low efficiency, and the obtained product is characterised by great structural variability. α-l,3-glucans also occur in the cell wall of fungi including yeast, e.g., Cryptococcus albidus, Schi∑osaccharomyces pombe, filamentous fungi, e.g., Aspergillus niger, Paracoccidioides hrasiliensis, and fungi which form fruiting bodies (basidiomycetes), e. g., Piptoporus betulinus, Polyporus tumulosus, Boletus edulis, Schizophyllum commune.

Henceforth, α-l,3-glucan will be referred to in short as glucan. To date, there have been few papers in the world literature concerning the application of fungal glucans as mutanase inducers. The use of a glucan from Aspergillus niger has been discussed in articles published in Agric. Biol. Chem., 41, 1977 and J. Gen. Microbiol., 118, 1980. This glucan, also known as pseudonigeran, consists in 98% of glucose residues linked with α-l,3-glycosidic bonds. Obtaining its pure form is very arduous and is characterised by low efficiency. Inducing mutanase of Streptomyces KI-8 with whole yeast cells of Schizosaccharomyces pombe has been described by Meyer and Phaff (J. Gen. Microbiol., 118, 1980). Equally scarce are studies on the stimulation of mutanase production with glucans from the cell wall of basidiomycetes. Fruiting bodies of Lentinus edodes, Piptoporus betulinus, and Schizophyllum commune were used (Proc. IV IFS: Ferment. Technol. Today, 735-742, 1972; Agric. Biol. Chem., 41, 1977; Biosci. Biotechnol. Biochem., 67, 2003). It should be emphasised though that most of the authors of the above-mentioned papers were interested in the problem of the use of the enzyme induced with fungal glucans to obtain protoplasts. They have not determined the activity of the obtained mutanase with regard to the streptococcal mutan, nor have any of the enzymes found application in human-targeted products, including those applied in dentistry.

The publication J. Oral Rehabil., 1978, no. 5, pp. 35-39 discusses a substance which inhibits and removes plaque, and is composed of mutanase and dextranase with concentrations of 1,000 and 10,000 units per gram of preparation, respectively. Patent description no. US 4,438,093, in turn, presents a preparation consisting of dextranase from Corynebacterium and mutanase from Pseudomonas, containing from 0.5 to 100 units per gram of each of these enzymes, while the ratio of one enzyme to the other ranges from 1:2 to 2:1.

In order to achieve substantial effectiveness of these preparations, it is necessary to use high concentrations of enzymes in the preparation.

DI S CLO U S URE OF INVEN TI ON

The invention covers a new strain of the bacterium Paenibacillus curdlanolyticus MP-I deposited in the Instytut Biotechnologii Przemyshi Rolno- Spozywczego in Warsaw, Poland under the no. KKP 2017p. This strain is particularly well-suited for production of mutanase. The invention also concerns the application of a glucan of fungal origin as an inducer of mutanase for human-targeted preparations. Glucan of specially quality comes from Laetiporus sulphweus or Piptoporus betulinus. 100 The glucan can be used in any form, i.e., both as a freely prepared mycelium, a fruiting body, or their extract.

The essence of the method of producing mutanase by means of microorganism cultivation on glucan- containing medium, consists in the fact that the microorganism is Paenibacillus curdlanotyticus while the glucan comes 105 from a fungus, from the class of basidiomycetes.

The best microorganism is Paenibacillus curdlanolyticiis no. KKP 2017p. The best fungus, in turn, is Laetiporus sulphureus or Piptoporus betulinus.

The essence of the method of producing mutanase through cultivation of 110 Trichoderma harzianum on a glucan-containing medium consists in the fact that the glucan comes from a fungus, the especially class of basidiomycetes. The best basidiomycete is Laetiporus sulphureus or Piptoporus betulinus.

The dental plaque-removing preparation containing mutanase and dextranase consists of 0.1 to 100 U/ml dextranase and 0.05 to 100 U/ml 115 exomutanase from Trichoderma harzianum CCM F-340 and/or 0.05 to 100 U/ml endomutanase. Endomutanase is obtained from Paenibacillus sp.

The invention has made it possible to obtain mutanase produced from non-hazardous materials, and, it has also utilized a cheap and easily-available

120 source of glucan in the form of mycelium. Taken together, the invention permits to obtain more efficient and safer hygiene agents for humans. With regard to toothpaste, this would result in a reduction of the content of abrasive substances, which rub off not only the dental plaque but also the enamel, which is their disadvantage. The activity of fungal mutanase obtained according to the

125 invented method is comparable with the activity of the enzyme induced with the bacterial mutan.

Example I. Induction of bacterial mutanase with α-1, 3 -glucan from Laetiporus sulphureus 130 The composition of the cultivation medium (g/1): KH ₂PO₄ - 1; (NHU)₂PO₄

- 1.5; MgSO₄ • 7H₂O - 0.5; CaCl₂»2H₂O - 0.5; NaCl - 0.01; FeCl₃^H₂O - 0.01; MnCl₂ ^«4H₂O - 0.01; pH 6.8 was supplemented with an addition of 0.25% (w/v) of dried and ground fruiting bodies of Laetiporus sulphureus. The medium, inoculated with bacteria Paenibacillus curdlanolyticus MP-I isolated

135 from soil, was incubated for 48 hours in optimum conditions (temperature - 30⁰C, shaking - 170 revolutions per minute). Next, after centrifugation of the bacteria cells, mutanase activity was measured in the supernatant against streptococcal mutan which had lost its α-l,6-glycosidic bonds as a result of the activity of commercial dextranase from SIGMA-ALDRICH. The obtained

140 activity equalled 0.23 U/ml .

A unit of mutanase activity [U] was defined as the amount of the enzyme that in one minute releases from mutan reducing sugars equivalent to 1 micromole of glucose. Example II.

145 In the conditions as in Example I, streptococcal mutan was used as inducer and the obtained activity equalled 0.47 U/ml.

Example III. Induction of fungal mutanase with α-l,3-glucan from Piptoporus betulinus.

Cultivation medium C (J. Bacteriol, 1962, 83, 400-408) with the addition

150 of 0.5% (w/v) peptone, was supplemented with 0.3% (w/v) of dried and ground fruiting bodies of Piptoporus betulinus. The medium, inoculated with conidia Trichoderma harzianum CCM F-340, was incubated for 72 hours in optimum conditions (temperature 30°C, shaking 220 revolutions per minute). Next, after centrifugal removal of the mycelium, mutanase activity was measured in the

155 supernatant against streptococcal mutan mostly without α-l,6-glycosidic bonds due to the activity of commercial dextranase. The obtained activity equalled 0.32 U/ml.

Example IV. Induction of fungal mutanase with α-l,3-glucan from Laetiporus sulphurous.

160 Cultivation medium C (J. Bacteriol., 1962, 83, 400-408) with the addition of 0.5% (w/v) peptone, was supplemented with 0.3% (w/v) of dried, ground and three times boiled fruiting bodies of Laetiporus sulphureus. The medium, inoculated with conidia Trichoderma harzianum CCM F-340, was incubated for 72 hours in optimum conditions (temperature 30⁰C, shaking 220

165 revolutions per minute). Next, after centrifugal removal of the mycelium, mutanase activity was measured in the supernatant against streptococcal mutan with α-l,6-glycosidic bonds mostly removed to the activity of commercial dextranase. The obtained activity of 0.4 U/ml is higher than the one obtained in the mutan of streptococcal origin.

170 Example V.

In the conditions as in Example III, streptococcal mutan was used as mutanase inducer and the obtained activity equalled 0.33 U/ml. Example VI.

Obtaining mutanase from Streptomyces sp. KI-8 according to Imai et al.,

175 Agric. Biol. Chem., 1977, 41, 1339-1346.

Table. A comparison of results obtained in Examples I - VI

Example VII. Hydrolysis of streptococcal mutan with the use of mutanases 180 obtained in Example I.

Streptococcal mutan (1 mg/ml), suspended in 0.1 M Clark-Lubs buffer at pH 6,2 and homogenised, was subjected to the activity of mutanase from P. curdlanolyticns MP-I used at the concentration of 0.1 U/ml (enzyme units per millilitre). The reaction was conducted at 40°C for 120 minutes. The degree of 185 glucan saccharification was measured based on the amount of reducing sugars released from the glucan. Substrate solubilisation was measured with the turbidimetric method, by measuring the decrease in turbidity caused by the presence of the water-insoluble polysaccharide in the assay at 560 nm wavelength.

190 It was determined that after 2-h incubation with the enzyme from P. curdlanolyticns MP-I the mutan almost completely degraded into soluble products (92% solubilisation), and, simultaneously, approximately 0.17 mg of reducing sugars were released from 1 mg of mutan.

Example VIII. Hydrolysis of streptococcal mutan (lmg/ml) with the use of 195 mutanase obtained in Example IV.

The mutan, suspended in 0.2 M acetate buffer at pH 5.5 and homogenised, was submitted to the activity of mutanase from T. harziamtm

CCM F-340 used at the concentration of 0.1 U/ml (enzyme units per millilitre).

The reaction was conducted at 40°C for 120 minutes. The degree of mutan 200 saccharification was measured based on the amount of reducing sugars released from the glucan. The solubilisation of glucan was measured with the turbidimetric method, by measuring the decrease in turbidity caused by the presence of water-insoluble polysaccharide in the assay at 560 nm wavelength.

24-hour hydrolysis of the mutan showed that this polymer solubilised in 68%, 205 and 0.79 mg of reducing sugars were released from 1 mg of mutan.

Example IX. Hydrolysis of streptococcal mutan with the use of mutanase obtained in Example III.

The mutan (lmg/ml), suspended in 0.2 M acetate buffer at pH 5.5 and homogenised, was subjected to the activity of mutanase from T. harzianum 210 CCM F-340 used at the concentration of 0.1 U/ml (enzyme units per millilitre).

The reaction was conducted at 40⁰C for 120 minutes. The degree of glucan saccharification was measured based on the amount of reducing sugars released from the glucan. The solubilisation of glucan was measured with the turbidimetric method, by measuring the decrease in turbidity caused by the

215 presence of water-insoluble polysaccharide in the assay at 560 nm wavelength. It was determined that after 24-hour incubation the mutan solubilised in 83%, and 0.84 nig of reducing sugars were released from 1 mg of mutan. Example X.

The new strain of Paenibacillus curdlanofyticus MP-I was isolated from

220 soil. This strain was described with the use of Bergey's manual and a set of biochemical tests: API 50CH for measuring the , metabolism of carbohydrates, and API 2OE test. The obtained results permitted inclusion of the strain in the genus Bacillus. The strain Paenibacillus curdlanofyticus MP-I was deposited at the Instytut Biotechnologii Przemyslu Rolno-Spozywczego in Warsaw, Poland

225 under the no. KKP 2017p. Detailed test results are included in the deposit documentation.

Isolated bacteria are rod-shaped and stain Gram-negatively even in young cultures. They occur individually or in pairs. Cells are motile. Mesophilic aerobes. Produce catalase. Form oval endospores placed in the terminal or

230 subterminal region, which distinctly swell the mother cell. Example XI.

10 units of exomutanase from Trichoderma harzianum CCM F-340, 10 units of endomutanase from Paenibacillus sp. , and 100 units of dextranase were introduced into 100 millilitres of 0.1 M acetate buffer pH 6. A unit of

235 dextranase activity was defined as the amount of enzyme which in one minute released from dextran reducing sugars equivalent to 1 micromole of isomaltose measured as maltose. Subsequently, plaque fixed in vitro on a plate of acrylic material used in prosthodontics was placed in the prepared enzymatic mixture. Plaque was generated through 24-hour incubation of the plate in a medium

240 containing Brain Heart Infusion (BHI) and 3% saccharose, inoculated with the following bacteria strains: Streptococcous sorbinus, Streptococcosis mutans, Fusobacterium nucleatum, Peptostreptococcus anaerobius, Peptostreptococcus magnus, Propionibacterium acnes, Prevotella intermedia as well as the yeast Candidia albicans. 12 hours after the placement of plaque in the preparation, the 245 mutan contained in it was completely decomposed and plaque residues, in the form of microorganism cells, were removed in a stream of water, with no additional actions.

Example XII.

A sample prepared as in Example XI was immersed in a preparation 250 containing a mixture of exomutanase and dextranase. After 12-hour hydrolysis and rinsing of the plate, plaque residues were found on it.

Example XIII.

A sample prepared as in Example XI was immersed in a preparation containing a mixture of endomutanase and dextranase. After 12-hour hydrolysis 255 and rinsing of the plate, plaque residues were found on it.

Claims

1. The new strain of bacteria Paenibacilliis curdlanolyticus MP-I deposited at 260 the Instytut Biotechnologii Przemysiu Rolno-Spozywczego in Warsaw,

Poland under the no. KKP 2017ρ.

2. The application of α-l,3-glucan of fungal origin as mutanase inducer intended for application in human-targeted preparations.

3. α-l,3-glucan of claim 2, characterised in that it comes from Laetiporus 265 sulphureus.

4. α-l,3-glucan of claim 2, characterised in that it comes from Piptoporus betulinus.

5. The method of producing mutanase by means of microorganism cultivation on an α-l,3-glucan-containing medium, characterised in that the

270 microorganism is Paenibacillus curdlanolyticus and the α-l,3-glucan comes from a fungus.

6. The method of claim 5, characterised in that the microorganism is Paenibacillus curdlanolyticus no, KKP 2017p.

7. The method of claim 5, characterised in that the fungus belongs to the class 275 ofbasidiomycetes.

8. The method of claim 7, characterised in that the basidiomycete is Laetiporus sulphureus.

9. The method of claim 7, characterised in that the basidiomycete is Piptoporus betulinus.

280 10. The method of producing mutanase by means of cultivation of Trichoderma harzianum on an α-l,3-glucan-containing medium, characterised in that the α-l,3-glucan comes from a fungus.

11. The method of claim 10, characterised in that the fungus belongs to the class ofbasidiomycetes. 285

12. The method of claim 11, characterised in that the basidiomycete is

Laetiporus sulphureus.

13. The method of claim 11, characterised in that the basidiomycete is Piptoporus betulinus. \ - I i -

14. The dental plaque-removing preparation containing mutanase and dextranase, 290 characterised in that it consists of 0.1 to 100 U/ml dextranase and 0.05 to

100 U/ml exomutanase from Trichoderma harzianum CCM F-340 and/or 0.05 to 100 U/ml endomutanase.

15. The preparation of claim 14, characterised in that the endomutanase is obtained from Paenibacillus sp.

295