FR2653444A1 - Process for inhibiting the activity of the enzyme ss-glucanase - Google Patents

Abstract

The subject of the invention is a process for inhibiting the activity of the enzyme ss-glucanase content in a medium. The process of the invention is characterised by the addition, to the medium, of a compound capable of liberating a borohydride ion of formula [ZBH3] in which Z is either hydrogen or a CN group.

Description

 The present invention relates to a method for inhibiting the activity of β-glucanase enzymes.

 13-glucans, glucose polymers linked by ss linkage, are polysaccharides which have interesting rheological properties and, in particular, a high viscosity, which ensure them applications in various fields and in particular in the petroleum field.

 However, certain enzymes are capable of making cuts in the polysaccharide chains and thus very quickly lower their molecular weight and, consequently, their rheological properties, including the viscosifying power.

 It is therefore important to be able to inhibit these enzymes and in particular the β-glucanases which are specific for β-glucans, considered previously.

 It is well known that enzymes present in the media are capable of attacking certain sites or functions of macromolecular compounds and thus impairing the properties of these compounds. In order to overcome these drawbacks, means have been sought to inhibit the activity of these contaminating enzymes. Thus, in the case of proteases, particularly in the food and pharmaceutical industries, much has been done with the inhibition processes, of which many agents are now known.

 On the other hand, in the field of osidases (and that of lipases), the work has been less thorough and few inhibitors are known.

The subject of the present invention is a method for inhibiting β-glucanase enzymes present in an aqueous medium, containing a glucose polymer, characterized in that a compound capable of releasing a borohydride ion of formula is added to the medium.
[Z - BH3 in which Z represents either hydrogen or the C = N group and containing an X + cation making it possible to solubilize the compound in the medium.

 The cation used will preferably come from an alkali metal Li, K, Na for example or an alkaline earth metal or will be the ammonium ion NH4 +.

 The amount of compound to be added to the medium will naturally depend on the concentration of β-glucanase; it will be such that the concentration of borohydride ion medium will be between 0.01 and 20% by weight.

The preferred compound of the process of the invention will, for economic reasons, sodium borohydride N2BH4
The process of the invention is usable for the protection of all the polysaccharides of the B-linked glucan family: celluloses, scleroglucans, xanthan, etc.

 It will be particularly well suited to the protection of the polysaccharide during the processes of obtaining and purifying from source microorganisms, for example, Sclerotium rolfsii for scleroglucan.

 The use of the borohydride ion appears as a particularly effective means for reducing the activity of 13-glucanases.

 It also seems that this action is endowed with specificity, since under the same conditions, the glucanase activity of a complex osidase preparation is completely inhibited, whereas the xylanase activity, for example, of the same preparation is much less sensitive to the action of the borohydride ion.

The method of the invention is illustrated by the following examples
EXAMPLES 1 TO 3
Preparation of the enzyme solution
A glucanase of Botrytis cinerea is treated in solution by
6% NaBH4 for 1 hour. The reaction mixture is then neutralized with CH3COOH and then centrifuged. The supernatant is adjusted in volume with distilled water to give an enzyme solution at 10 mg / ml.

Preparation of the polymer solution: 100 mg of scleroglucan obtained by fermentation of Sclerotium rolfsii are dissolved in 50 ml of distilled water to give a solution at 2 g / l (with stirring at 4 ° C for 24 hours).

Then 3 ml of stock solution of polysaccharide at 2 g / l and 3 ml of 0.2 M phosphate buffer solution at pH 7 are mixed for 2 hours at ambient temperature to give a solution of 0.1 M scleroglucan pH 7 at 1 g / l of polysaccharide. .

Three test portions of 2 ml of the said solution are supplemented with 0.5 ml of concentrated enzyme solution at 10 g / l (NaBH4 treated enzyme solution, native enzyme solution without treatment and control without enzyme).

These three outlets are placed in the measuring cell of a Low Shear viscometer. The substrate concentration (scleroglucan) is 0.8 g / l and the temperature maintained at 30 ° C.

The results obtained are described in Table 1
Table 1

<tb><SEP> Solution <SEP> Viscosity <SEP> (mPa.s)
<tb><SEP> after <SEP> 5mn <SEP> after <SEP> 1h <SEP> ≈ <SEP> after <SEP> 4h
<tb> Solution <SEP> enzymati- <SEP> I <SEP>
<tb> than <SEP> processed <SEP> NaBH4 <SEP> | <SEP> 155 <SEP> | <SEP> 95 <SEP> 55
<tb> Solution <SEP> enzymati <SEP> I <SEP> 0 <SEP> I <SEP> 0 <SEP> 1 <SEP><SEP> 0
<tb> than <SEP> native
<tb> Witness <SEP> 160 <SEP> 160 <SEP> 160
<Tb>
We can note that
In the absence of 13-glucanase, the viscosity remains virtually unchanged (control).

In the presence of the native enzyme, there is immediate degradation and abrupt drop in viscosity.

In the presence of the enzyme treated with sodium borohydride, the activity decreases very slowly with time and remains a residual activity. The inhibition of enzymatic activity is considerable. This activity was evaluated at 1 AU / min for the solution treated with NaBH4, whereas it is 20 for the native enzyme solution.

EXAMPLES 4 TO 6
Comparison of the inhibition of the activities xvlanase / slucanase
For these examples, the following 3 substrates were used: glucuronoxylan extracted from birch wood (Betula papyrifera Marsh.) - carboxymethyl cellulose (CMC): cellulose blanose gum 7H3
SXF HERCULES Ltd.

- ss-glucan (scleroglucan sclerotium rolfsii) that is subjected to the action of an enzymatic solution (raw industrial complex comprising several enzymes).

Preparation of substrates
The substrates were prepared as follows - xylan and CMC at 2.5 g / l in 0.1 M acetate buffer pH 5 - 1 g / l scleroglucan in 0.01 M phosphate buffer pH 7 Enzymatic preparation with inhibitor
The enzyme is treated for 3 hours (50 g / l) in an aqueous solution containing 20 g / l NaBH4 and an antifoaming agent.

The reaction mixture is then neutralized with acetic acid, and then centrifuged, the supernatant containing the enzymes is lyophilized for preservation.

The activity tests are carried out at 40 ° C., with stirring, on 1.9 ml of solution or suspension of substrate and 0.1 ml of appropriately diluted, native enzyme solution then treated with NaBH.sub.4.After the time given, the reaction is stopped. by addition of Somogyi reagent, and the reductive ends appeared (characteristics of the number of cleavages made by the enzyme) are determined spectrophotometrically at 500 nm (method of Somogyi and Nelson
Somogyi S., J. Biol. Chem. 195, p 19.23, 1952).

 The incubation time is 30 minutes with the native enzyme and 5 hours with the NaBH4 treated enzyme.

The results obtained are shown in Table 2 below.
Table 2

3 glucanase (sur sclérogucane) est totale et l'inhibition de l'activité 131

4 glucanase (sur CMC) est presque totale. En revanche, l'activité xylanase n'est affectée qu'à 50% environ par le traitement d'inhibition proposée. <tb><SEP> I <SEP> I
<tb> SUBSTRATE <SEP> I <SEP> I <SEP> SCLERO
<tb><SEP> ACTIVITY <SEP> XYLANE <SEP> | <SEP> C <SEP> M <SEP> C <SEP> GLUCAN
<tb> Absolute <SEP> activity <SEP>: <SEP> 1 <SEP><SEP> 9.3 <SEP> 1 <SEP> 29.3 <SEP> 1
<tb><SEP> enzyme <SEP> native <SEP> I <SEP> I
<tb><SEP> (nKat / mg) <SEP> I <SEP>
<tb> Activity <SEP> Relative <SEP> 100 <SEP> 1 <SEP> 100 <SEP> 100
<tb><SEP> I <SEP> I
<tb> I <SEP><SEP> Relative <SEP> Activity: <SEP> 1 <SEP> 52 <SEP> 0.02 <SEP> 1 <SEP><SEP> 0 <SEP>
<tb><SEP> enzyme <SEP> treated <SEP> with <SEP> NaBH4
<Tb>
It can be seen that under the effect of
NaBH4, inhibition of ss activity 1

3 glucanase (on sclerogucan) is complete and the inhibition of 131

4 glucanase (on CMC) is almost total. On the other hand, the xylanase activity is only affected by about 50% by the proposed inhibition treatment.

Claims (3)

1 - Inhibition method of the enzyme ss-glucanase present  in an aqueous medium containing a glucose polymer  characterized in that a compound is added to the medium  capable of releasing a borohydride ion of formula  [Z - BH3 i wherein Z represents either hydrogen  is the radical CN and having an X + cation allowing  to solubilize the compound in the medium. 2 - Process according to claim 1 characterized in that the  cation X + will be selected from NH4 + and those from a  alkali metal or alkaline earth metal. 3 - Process according to claim 2 characterized in that the  compound is NaBH4 4 - Process according to one of claims 1 to 3 characterized  in that the concentration of the medium in borohydride ion  will be between 0.01 and 20% by weight.