FR2617867A1 - GLUCANASE, IMMOBILIZED ON APATITIS AND METHOD FOR ITS PREPARATION - Google Patents

Abstract

Apatite on which glucanase is immobilized and process for its preparation, which process consists in adding drop by drop glutaraldehyde to an aqueous solution in which glucanase, protein and apatite are present in a mixed state.

Description

The present invention relates to an apatite on which is immobilized

glucanase, and a method

  immobilization of glucanase in apatite.

  "Glucanase", which is a general term for enzymes breaking down glucan, covers various enzymes. It should be noted however that the term "glucanase" here designates the levanase which breaks down the levan, the dextranase which

  breaks down dextran and mutanase which breaks down muta-

  born. In addition, the term "apatite" denotes both hydro-

xyapatite and fluoro-apatite.

  It is well known that the appearance of dental caries

  is silent due to dental plaque formed by the action of

  polysaccharides such as levan, dextran and mu

  tane, which are produced by various bacteria in the mouth.

  As a result, it is believed that polysaccharides

  produced by these bacteria should be eliminated for supp-

  take precedence over the appearance of dental plaque in order to prevent

  dental caries. The present invention relates to a statelessness

  te on which enzymes are immobilized for the decomposition

  of polysaccharides associated with tooth decay, and a

  process to prepare this apatite.

  In the prior art, various methods have been developed for removing dental plaque in order to pre

  come tooth decay. These processes include the elimination

  tion by scraping the dental plaque using a polishing agent such as zeolite, calcium carbonate, alumina and silica, using dextranase in

  same time as a stabilizer, etc ... So far, however

  However, there was no method of immobilizing a child.

  zyme that is capable of breaking down a responsible polysaccharide

  sand from the appearance of tooth decay such as levana-

  se, mutanase and dextranase, with apatite, and it

  there was no apatite in which these enzymes were im-

  mobilized, as envisaged in the present invention.

  Glucanase is an enzyme and it is relative-

  unstable. Consequently, when glucanase is

  mixed with a toothpaste without being modified, its activity

  over time and finally disappears. Dextranase

  is used today with toothpaste, and various

  bilisants are prepared to prevent its deactivation. By

  example, combinations of dextranase with aluminum oxide

  minium, carvone or e-menthol, and gelatin or

  peptone, are proposed in the specifications of the

  Japanese patent applications N 56-63915 and 56-110609 and in

  Japanese patent N 52-49005. As the toothpaste is desti-

  born to be used in the mouth, various limitations are

  imposed, including considerations regarding the

  fluence on the human body and the demand for a refreshing sensation after use. As it is obvious that

  these limitations are imposed on the stabilizers used, the choice.

  stabilizers pose difficult problems. The use of enzymes other than dextranase poses the same problems

than the use of dextranase.

  Following extensive and extensive studies on glucose stabilization, it has been found that immobilization of glucanase with apatite, which is used as a polishing agent, produces a glucanase which does not require stabilizer and which has activity on

  an extended period. The present invention provides an apa-

  titan on which is immobilized glucanase, and a pro-

  yielded to prepare this apatite.

  It is well known that there is a method of immobi-

  lization of enzymes based on the fact that hydroxyapatite, active carbon, kaolinite, kaolin etc ... physically absorb enzymes for immobilization, and it is generally known that immobilized enzymes are so stable that they have a activity change over time lower than the untreated enzyme and are convenient to handle. It has therefore been known for a long time that apatite

  securely absorbs and binds to a certain type of protein.

  In view of the fact that immobilization of glucanase can lead to a reduction in the variation in activity over time, it is assumed that if glucanase was immobilized with

  apatite used as a polishing agent for dentin

  fried by the physical adsorption process, an im-

  mobilized could be easily obtained, and it might then be possible to obtain an interesting toothpaste

  which combines a polishing capacity with a decom-

  position of the polysaccharides, and dispenses with any choice of ca

  talyseur. Following studies carried out on this presumption,

  it has been found that the adsorption and immobilization of glucana-

  getting on apatite are not achievable due to the fact that

  the adsorption of glucanase on apatite is extremely limited

  tee. For this reason, other studies have been carried out on the immobilization of glucanase by means of a protein which is strongly adsorbed on and bound to apatite, and to the

  following these, we found an apatite on which is immo-

  of glucanase and a process for preparing it.

  The present invention provides apatite in lacquer

  the immobilized glucanase and a method of immobilizing

  tion of glucanase in apatite at the same time as a cer-

  a type of protein which is strongly adsorbed on apatite.

  The apatite is suspended in an aqueous solution or in a phosphate buffer solution having a concentration of 0.01 to 0.05 mole, in which the glucanase and the protein having a strong adsorption on apatite and

  having no harmful effect on the human organism are dis-

  bunkers, then a bifunctional aldehyde is added dropwise

  drop, shaking vigorously, to the suspension obtained,

  below room temperature. The protein used can be chosen from albumin, casein, lysosyme, cytochrome C, etc. Depending on the type of protein, there is a difference in the titer of the immobilized enzyme obtained.

  and in the bonding strength for apatite. As we cons-

  will be based on the results of the elution test which will be

  given below, however, the lysosyme, cyto-

  chromium C etc ... The glucanase used can be chosen arbi-

  trairement among levanase, dextranase and mutanase,

  as already indicated. If desired, we can use

  be mixtures of these enzymes. The reaction involved is

  operates at a pH for which there is no decomposition

  tion of apatite, that is to say at a pH of 5.6 or more.

  However, as a higher pH exerts a negative influence

  vorable on protein adsorption, pH above 9.0

  is not preferred. The preferred pH is around 7.0.

  It is desirable that the particle size of

  the apatite used is as uniform as possible, however

  in the apatite particles generally used as

  polishing agent for toothpaste suitable for this-

  te particular application. An apatite having a particle size of 2 to 200 microns is preferred because it is easy to handle. The amount of apatite used is 10 to 100 times

  greater than the amount of protein. For an agitation e-

  effective, it is desirable to use a larger amount

  of water compared to the amount of apatite used. The quanti-

  The amount of water is adjusted so that the solids content in the reaction phase is 4 to 20%. It is best to use almost equivalent amounts of protein and glucanase. A large difference in the amount of the two constituents, especially the use of glucanase in a smaller amount compared to the protein, should be avoided, as there will be a decrease

  the titer of the immobilized enzyme obtained. In general, we prefer

  re use glutaraldehyde as a bifunctional aldehyde. The amount of bifunctional aldehyde used is the factor that

  has the greatest influence on the title of the enzyme immo-

  stabilized. Too little glutaraldehyde in front

  being added generally results in the enzyme immobi-

  sée has a weak bonding force for apatite and experiences considerable deactivation over time. A high amount of glutaraldehyde leads to a reduction in

  titer of the immobilized enzyme obtained, and a further increase

  additional quantity of it leads to deactivation

  tion of this enzyme. Although the amount of glutaraldehyde

  used is slightly different depending on the type of gluca-

  nase and protein, in general, the amount of glutaraldehy-

  used is 3 to 60 mg, preferably 6 to 20 mg per

  gram of protein used. The reaction involved is carried out

  killed at a temperature not higher than ambient temperature

  te, preferably around 5 C. While stirring vigorously

  only a suspension in which the protein coexists,

  glucanase and apatite, we add it slowly, drop by

  drop, an aqueous solution of glutaraldehyde. After the addi-

  drip, the stirring is carried out at the same temperature

  cross off for several hours to complete the reaction. Lofs-

  that the reaction is finished, we filter. The apatite obtained is washed thoroughly with water or with the buffer solution used for the elimination of the protein and the enzyme entrained, then it is maintained at a lower temperature or lyophilized for solidification, then stored at the annual temperature. This apatite immobilized in accordance with the present invention can also be prepared in the following manner.

  Apatite is added, with sufficient stirring, to a solution.

  aqueous solution or to a buffer solution in which the protein

  is not dissolved, so that the protein is adsorbed on the titite for saturation. Next, the apatite which adsorbs the protein is collected and added to water or a buffer solution in which glutaraldehyde is dissolved. All in

  vigorously stirring the solution obtained, drop therein

  slowly, an aqueous solution of glutaraldehyde. The temperature and other conditions used for this purpose itself

the same as before.

  The apatite on which glucani is immobilized is obtained in this way is stable, it experiences less

  variations over time and it's easy to handle.

  Apatite has been shown to absorb some

  a certain type of protein. It is also known that gluta-

  aldehyde is used as a crosslinking agent for immo

  stabilization of enzymes. Although the mechanism by which im.

  mobilization of glucanase with apatite is carried out remains unclear, it is assumed that the adsorption of a protein which is ai: mentally adsorbed by apatite on apatite, takes place in

  at the same time as the crosslinking of protein and gluca-

  nase, thus giving immobilized apatite.

  For a better understanding of the invention, and to show how it can be put into practice, we

  refer to the following working example as an example.

  EXAMPLE 1 Immobilization of levanase on hydroxyapatite.

  100 mg of lysozyme and 100 mg of levanase are dissolved in 50 ml of pure water and 2 g are added to the solution obtained.

  hydroxyapatite as a polishing agent, after which

  cools to 4 C. While maintaining the temperature at 4 C and stirring vigorously, 2 ml of a solution containing 28 mg of glutaraldehyde in

  ml of water. When the drip addition is complete

  born, the stirring is carried out for 2 hours while holding

  keeping the temperature at 4 C. A subsequent centrifugation gives a solid product, which is washed in turn, with stirring,

  three times with 50 ml of pure water to obtain a hydroxyapa-

  tit immobilized in the undried state. (If desired, this product can be used as is). Lyophilization gives 2.05 g of a powder. To 1 g of this powder is added 10 ml of a potassium phosphate buffer solution having a pH of 6.8 and a concentration of one mole. We perform the agitation

  for 1 hour, then centrifuged to collect the wire-

  trat. The same operation is repeated twice with the residue.

  The filtrates obtained are combined to determine the protein content by the Lowry method. After washing with water, we

  dries the residue to weigh it. We find that 11.7 mg of pro-

  Teins are linked per gram of hydroxyapatite. The measurement of the levanase activity of the immobilized hydroxyapatite in the undried state, by the following method, indicates that 0.47 g of levan are broken down per gram of protein bound to the hydroxyapatite. EXAMPLE 2 Immobilization of levanase on fluoro-apatite The procedure is carried out under the conditions as in example 1, except that fluoro-apatite is used in place of

  hydroxyapatite, and 2.05 g of a lyophilic product are obtained

  read. The measurement of the bound protein, carried out in the same way

  denier that in example 1, indicates that 13.4 mg of protein are bound per gram of fluoro-apatite. The results of the measurement of the levanase activity of non-fluoro-apatitis

  dried also indicate that 0.54 g of yeast are broken down

  per gram of the bound protein.

  EXAMPLE 3 Immobilization of the Mutanase on Hydroxyapatite

  We operate under the same conditions as in example-

  ple 1, except that mutanase is used in place of levanase, to obtain an immobilized hydroxyapatite. The decomposition is carried out in the same way as in the example

  ple 1. It is confirmed that 15.0 mg of protein are bound per gram-

  hydroxyapatite. The mutanase activity of the product is

  ensured by the method for measuring the activity of mutanase which will be described below. We find that 0.48 g of mutan are

  broken down per gram of protein bound to hydroxyapatite.

  EXAMPLE 4 - immobilization of the mutanase on fluoro-apatite.

  We operate under the same conditions as in example-

  ple 2, except that mutanase is used in place of levanase, to obtain an immobilized fluoro-apatite. The results of the analysis and the measurement of the titer thereof, carried out in the same manner as in Example 3, indicate that 17 mg of protein are bound per gram of fluoro-apatite,

  and that 0.45 g of mutan is broken down per gram of pro-

linked tein.

  EXAMPLE 5 Immobilization of the dextranase on hydroxyapatite 5 g of hydroxyapatite used as a polishing agent and 50 ml of a potassium phosphate buffer solution having a concentration of 0.05 mol and a pH of 6.8 to 10 are added.

  mixing 50 mg of lysozyme with 50 mg of dextranase. We re-

  the product obtained cools down to 4 ° C. and is stirred vigorously.

  While maintaining this temperature, we add drop by drop

  te 125 μl of a 0.2% aqueous glutaraldehyde solution,

  stirring, after which additional stirring is carried out

  shut up for 5 hours. The pro-

  of the reaction and washed three times with 100 ml of the above buffer solution to obtain an immobilized hydroxyapatite in the undried state. Lyophilization gives 5.07

  g of dextranase immobilized on powdered hydroxyapatite.

  1 ml of the immobilized hydroxyapatite is centrifuged

to get a precipitate.

  2 ml of a potassium phosphate buffer solution having a concentration of 1 mol are added to the precipitate and

  a pH of 6.8, and the solution obtained is stirred for 3 hours

  res for desorption of the bound protein, then centrifuged.

  The same operation is repeated with the precipitate obtained. We managed

  nit the filtrates to measure the quantity of the protein which is contained by the Lowry method, the precipitates are washed with water, then dried to weigh them. We observe that 13.8 mg

  of protein are bound per gram of hydroxyapatite. The results

  states of measurement of dextranase activity by a mete

  method of measuring the activity of dextranase which will be described

  te below indicates that 0.415 g of dextran is broken down

per gram of bound protein.

  EXAMPLE 6 Immobilization of dextranase on fluoro-apatite The same preparation process is used, the same

  analysis and the same titer measurement as in Example 5, except

  we use fluoro-apatite instead of hydroxy-

  apatite, which gives 5.08 g of immobilized fluoro-apatite in which 15.2 mg of protein are bound per gram of

  fluoro-apatite, and 0.43 g of dextran are broken down per gram-

me from the protein bound.

  The results obtained under various conditions are given in Table 1. The treatment conditions are

  the same as in the previous examples.

Measurement of enzyme titers.

  1 ml of the non-dried immobilized apatite obtained in each experiment is added to 10 ml of a 1% solution

  of the corresponding substrate. After having stirred the solution

  held at 37 ° C. for 2 hours, the amount of the mono-

  mother who is trained therein, and the protein linked to 1 ml

  of the undried immobilized apatite is determined by the

  above method. The titer of each immobilized enzyme is then expressed by the amount of the monomer per gram of the

protein linked to each apatite.

  The substrates used were dextran for dextranase, mutane for mutanase and levan for levanase. The amounts of dextranase and mutanase were determined from the glucose obtained by decomposition by the glucose oxidase method, and the amount of levanase was determined from the fluctose obtained by decomposition using high performance liquid chromatography

  (column: sugar pack I) in the usual way.

  Elution test for bound prcteine. The immobilized apatite obtained is introduced into

  a column and washed with various concentrations of a solution

  buffer to determine the concentration for elution of the bound protein. The binding strength of the protein on

  apatite is then expressed by the result obtained. The Your-

  bleau 2 gives the results obtained with hydroxyapatite on which dextranase is immobilized. The eluate used was a potassium phosphate buffer solution having a concentration of 1 millimole to 1 mole and a pH of 6.8, and the control used was a hydroxyapatite to which only dextranase was linked. The results indicate that the

preferred carrier is lysozyme.

  Activity of enzymes immobilized over time.

  The variations in activity over time were examined of several samples of immobilized apatite obtained.

  in accordance with the present invention. The samples used

  These were all non-dried immobilized products. The acti-

  The vity of each sample was measured by the above method. The results are given in Table 3. It was found that the immobilized apatites obtained all saw

  their activity increase over time and exhibited a maximum

  mum of activity after a while, after which it turns

  produced a gradual decline in activity.

  According to the present invention, the glucanase can be immobilized in apatite by a very simple operation. Immobilized apatite is easy to handle, stable, and experiences less variation over time. In

  in addition, the apatite of the present invention brings together the properties

  polishing tees of apatite with an ability to decompose

  the polysaccharides responsible for tooth decay. In con.

-11

  sequence, the use of the product as toothpaste is preferred

  given the desire to prevent tooth decay. For

  For these reasons, the invention is extremely advantageous for

maintain good oral hygiene.

TABLE 1

  Type and quantity of Type and quantity of Type and quantity Glutar- ABN protein glucanase of apatite aldehyde 7 lysozyme 0.1g levanase 0 lg hydroxyapatite 2g 1.12 mg 10.3 mg 0.47g 8 lysozyme Olg mutanase lig fluoroapatite 2g l12mg 14.7 mg 0 , 47g 9 lysozyme Olg levanase Oflg hydroxyapatite 2g 0.56mg 9.7mg 0.42g casein Oelg levanase Ojlg hydroxyapatite 2g 0.56mg 103mg OJ44g 11 cytochrome C OQlg l1vanase Ojlg hydroxyapatite 2g 0.56mg 13.2mg OJ49g 12 lysozyme 2g 2 24mg 13.7mg O045g 13 lysozyme O / lg l1vanase Ojlg hydroxyapatite 2g 4.48mg 15.3mg OJ32g 14 lysozyme 0O1 mutanase OJlg hydroxyapatite 2g 8.96mg 16.4mg OJ13g lysozyme 0, lg levanase Olg fluoromgatite 2 Omg OO4g 16 lysozyme O, lg mutanase Olg hydroxyapatite 2g 18> 0mg 17 4mg Oj02g 17 lysozyme 0.2g dextranase 0O2g hydroxyapatite 5g. 0 31.4mg 0O40g 18 albumin 0.05g dextranase 0O05g hydroxyapatite 5g 1, Omg 10.9mg Oj51g 19 lysozyme 0.05g dextranase 0.2g hydroxyapatite 5g 0.5mg 9.94mg OJ62g lysozyme 0.05g dextranase 0.2g hydroxyapatite 5g 10g 15.2mg 0.46g 21 lysozyme 0.025g dextranase 0O025g hydroxyapatite 2j5g 50mg 8.75mg 0.06g 22 lysozyme 0.0125g dextranase 0, 0125g hydroxyapatite 2_5g 50mg 12.37mg 0 23 lysozyme 0.05g dextranase 0, 05g fluoroapmg 5.5g fluoroapatite mg 0.50g 24 cytochrome C 0O5g dextranase 0.05g hydroxyapatite 5g 0.135mg 12.16mg 0.46g lysozyme 0.05g dextranase 0.05g fluoroapatite 5g 1, Omg 12.3mg 0 / 53g A: Total bound protein / gram of apatite; B: Amount of decomposed substrate / gram of co protein

TABLE 2

  Immobilization force of hydroxyapatite for dextranase Support Elution concentration Control 0.08 mole Lysozyme 0.18 mole Albumin 0.12 mole Casein 0.15 mole

TABLE 3

  Variation in the enzymatic activity of apatite immobilized over time (specific activity) Immediately after N the preparation 4 C - 20 days 4 C - 60 days 36 "C - 20 days 360C - 60 days

0; 0 154 0 705

1 100 213d 6 98.0 154, 0 7 0D 5

2,100,303.3,110.4 136.7 57/2

3 100 278 8 121, 9 150, 6 68J4

4 100 241, 6 95 8 15 530 70 165

100 293., o 02, 9 3271o 126.8

6 100 287J4 108/7 320; 4 120/8

A 100 - - 68.4 27.3

B 100 - - 65, 3 25, '

C 100 - - 58 7 20, 3

  A: Levanase only; B: Mutanase only; C: Dextranase

  only; all stored as an aqueous solution.

  The present invention is not limited to the exemplary embodiments which have just been described, it

  is on the contrary susceptible to modifications and varian-

  those who will appear to those skilled in the art.

Claims (7)

  1. Apatite on which gluca- is immobilized   nase. 2. Process for the preparation of an apatite on which gluzanase is immobilized, which consists in adding dropwise glutaraldehyde to an aqueous solution in which the glucanase, the protein and the apatite are present in the mixed state . 3. The method of claim 2, wherein   the protein used is lysozyme.   4. The method of claim 2, wherein the aqueous solution used is a buffer solution having a pH 5.6 to 9.0.   5. The method of claim 2, wherein the amount of glutaraldehyde is 0.3 to 6% relative to protein.   6. The method of claim 2, wherein   apatite has a particle size of 2 to 200 microns.   7. The method of claim 2, wherein   a suspension is obtained in a buffer solution, which   the buffer solution has a pH of 5.6 to 9.0 and contains quan-   equivalent amounts of lysozyme and glutanase, and apati-   te having a particle size of 2 to 200 microns in an amount 10 to 100 times greater than the amount of lysozyme, and   glutaraldehyde is added dropwise in a quantity   0.3% to 6% relative to lysozyme, cooling and waving.