FR2754269A1 - Drying of heat-sensitive substances, especially enzymes - Google Patents

Abstract

Process for preparing a composition containing at least one heat-sensitive substance comprises drying the composition at a temperature above the degradation temperature of the substance for a time such that the substance is not degraded.

Description

PROCESS FOR THE PREPARATION OF A COMPOSITION COMPRISING A
THERMOSENSITIVE SUBSTANCE
The present invention relates to a process for the preparation of a composition comprising at least one heat-sensitive substance.

 A more particular subject of the invention is the preparation of a composition comprising an enzyme, optionally immobilized on an appropriate support.

 Usually, the preparation of compositions comprising one or more thermosensitive substances, and optionally a support, comprises in most of the cases a drying step. The purpose of this step may be the drying of the active substance alone, at the end of its process for example, or else the elimination of the solvent or of the dispersant generally used when the active material is brought into contact. with its possible support.

 It is this drying step that generally poses difficulties. Indeed, we then find ourselves faced with the problem of not degrading the heat-sensitive substance, and that of speeding up the drying process as much as possible in order to maintain good productivity in the process for preparing the composition. We therefore note that we are in the presence of two contradictory interests because the first leads to reducing the temperature used so as to avoid any risk of degradation of the substance, while the second is to increase this same drying temperature. to speed up the process.

 If we take the particular case of the preparation of compositions based on enzymes immobilized on a support, the conventional drying methods consist in placing the composition in an oven at an ambient temperature lower than that of denaturation of the enzyme, or else to evaporate the solvent or the dispersant under vacuum.

 Proceeding in this manner is the cause of various disadvantages.

 The first is linked to the low temperature at which drying is carried out, since the temperatures above which the tolerance threshold of the enzyme is exceeded are relatively low, which results in long drying times, which can range up to several days.

 In addition, the surface used for drying is very large, all the more important since these methods hardly lend themselves to a mode of continuous operation.

 An additional problem may arise in the case where a third compound is necessary to obtain good immobilization of the heat-sensitive substance on the support. Indeed, in certain cases, these third compounds, which can also be called binders, are chosen from prepolymers which crosslink with heat, so as to create a bridging between the substance and the support. However, to achieve good crosslinking, sufficient times and temperatures of contacting with heat are necessary. It is therefore not possible to modify the operating conditions without taking this fact into account, which further complicates the drying operation.

 The present invention makes it possible in particular to solve the aforementioned problems without observing degradation of the heat-sensitive substance.

 Thus, the process according to the invention, for preparing a composition comprising at least one thermosensitive substance, consists in carrying out at least one step of drying said composition at a temperature higher than that of degradation of said substance and for a period such that there is substantially no degradation of this substance.

 It has indeed been found, against all expectations, that despite the fact that the drying temperature is higher than that of degradation of the heat-sensitive substance, said substance did not undergo any detrimental change in its structure or its activity.

 This method therefore makes it possible to considerably reduce the duration of drying (a few days to less than a minute), which represents an obvious saving of time but also of energy. In addition, the area devoted to this drying step is also considerably reduced because the equipment used no longer needs to be as large as that used before. It should also be noted that if the drying in an oven lends itself very little to continuous use, the drying means proposed by the present invention can be fully exploited in this way. It is even recommended to do so because this further reduces the size of the necessary equipment in which drying is carried out.

 However, other characteristics and advantages of the present invention will appear more clearly on reading the description and the examples which follow.

 As indicated above, the drying according to the invention is carried out at temperatures higher than those conventionally reached. In fact, the drying temperatures of the process according to the invention are higher than those of degradation of the substance present.

 In the particular case of enzymes, the drying is carried out at a temperature higher than that of denaturation of the enzyme for a sufficiently short time to avoid this denaturation. Consequently, the resulting composition retains its own enzymatic catalytic properties, which is completely unexpected.

 In addition, in the case where the enzyme is immobilized on a support, in particular by means of a binder, it has been found that, despite this very short drying time, the bonding of the enzyme to the resin was correct. This indicates that the polymerization of the binder used in the composition does indeed take place sufficiently.

 Thus, the method according to the invention more particularly comprises a drying step carried out at a temperature between 120 and 2600C, knowing that the drying temperature remains higher than that of degradation of the heat-sensitive substance.

 According to a particular embodiment of the invention, the drying is carried out at a temperature greater than or equal to 1500C. In addition, the drying temperature is preferably less than 200 "C.

 The drying time is more precisely less than 30 seconds. According to a particular embodiment of the invention, the duration of the drying is less than 10 seconds.

 The drying process as just specified is particularly suitable for obtaining compositions comprising at least one enzyme as a heat-sensitive substance.

 Preferably, the enzyme is associated with a support. In general, and although the invention is not limited thereto, the support used is a resin, preferably macroporous.

 Furthermore, the enzyme support bond can advantageously be carried out by means of a binder.

 It should be noted that the drying conditions are adaptable by a person skilled in the art, taking into account the fact that the higher the drying temperature, the shorter the drying time must be in order to avoid any degradation of the substance. thermosensitive, or any denaturation of the enzyme.

 It is also necessary to take into account the capacity of the binder to crosslink, if such an agent is present in the composition. Consequently, the duration of the drying must be sufficient to allow crosslinking, and in fact to allow satisfactory contact of the substance and its support.

 In fact, the conditions of temperature and duration of drying are such that everything happens as if the composition, and more particularly the heat-sensitive substance, remained subjected to a temperature below that of degradation of the latter.

 This type of drying, which can be described as flash drying, is used in any type of apparatus making it possible to reach temperatures as high as those mentioned above for very short contact times.

 For example, one can use induction ovens on conveyor belts, or optionally fluidized beds. It is possible to use devices in which are arranged scraped thermostatically controlled plates. Generally, these devices are operated under vacuum, which makes it possible to reduce the drying temperatures. Turbospheres can also be used, which will also be used under vacuum.

 Also suitable for the implementation of the invention, the atomization towers.

According to this route, the composition to be dried is generally sprayed into a vortex created by the drying flow, the injections usually being carried out against the current. The particles are then collected in a collector which is usually a cyclone, although any other type of conventional apparatus is suitable.

 In a completely advantageous manner, this flash drying is implemented in an apparatus called "spin flash", developed by the company APV-Baker (article published in Chemistry Information No. 342 - October 1992 - p. 141 and following).

 According to a preferred embodiment, this device is constructed so that the composition to be dried and the drying flux are injected into a co-current oven.

 The supply of the drying flux is carried out below the supply of the composition. In this way, an ascending vortex is created in the oven enclosure, which drives the dried particles to a collector.

 In a conventional manner, the collector is a cyclone, but any other type of apparatus intended to recover solid particles entrained in a gas flow is suitable.

 This type of dryer is very interesting because it reduces the attrition phenomena to which the grains of the composition are subjected. This point is particularly advantageous when the composition comprises a resin coated with an enzyme.

 The drying flow, in each of the cases mentioned above, is generally hot air, but any other gas would be suitable, such as nitrogen.

 The method according to the invention allows solutions, suspensions or solids comprising a heat-sensitive substance to be dried.

 The solvents or dispersants are very variable depending on the heat-sensitive substances used.

 According to a very advantageous embodiment of the present invention, and as has been mentioned previously, the drying process is carried out for heat-sensitive substances chosen from enzymes.

The present invention is particularly suitable for the preparation of a composition comprising at least one enzyme immobilized on a support by means of a binder, this composition being subsequently used for the preparation of acid.
L-aspartic.

 The preparation of this particular catalyst will now be described in more detail.

Thus, the present invention is particularly suitable for drying compositions comprising, as a heat-sensitive substance, at least one enzyme produced by a microorganism chosen from the following: Pseudomonas fluorescens, Pseudomonas dacunha e, Bacillus megaterium, Arthrobaster species,
Arthrobacter simplex, Streptomyces pha echromogen es, Rhodospondium torulcides,
Escherichia coli, Aerobacter aerogenes, Bacterium succinum, Micrococcus sp, Bacillus subtilis, Serratia marcescens. Preferably, the enzyme produced by the microorganism E. Coli is used. We can in particular use the strain whose ATCC number is 11303.

 Preferably, the enzymes obtained by fermentation of the microorganisms in suitable nutritive media and then separated from the nutritional medium, are immobilized on a support.

 According to a variant1 the support is chosen from macroporous resins which exchange cations or anions.

 Among the cation exchange resins, resins of the strong acid or weak acid type can be used. These resins can be in an acid form (against hydrogen ion), or salified (against ammonium ion or alkali or alkaline earth metal).

Preferably, they are in the hydrogen form.

 By way of example of resins of the weak acid type, mention may be made of resins which are copolymers of acrylic, methacrylic acid, esters or even corresponding nitriles. Mention may likewise be made of phenolic resins.

Among the resins of the strong acid type, mention may very particularly be made of the resins being copolymers of divinylbenzene styrene having sulfonated graft functional groups. Such resins are sold in particular under the trade name Dowex of the company Dow, or else Purolite of the company
Purolite, Amberlyst from Rohm & Haas. According to a preferred embodiment of the invention, macroporous resins IRA 938 from the company Rhom & Haas are used,
Purolite A 501 P, Duolite ES 181 from the company Duolite.

 According to a preferred embodiment, resins of the strongly acid type are used.

 Among the anion exchange resins, mention may be made, for example, and without intending to be limited, of resins based on polystyrene, polyacrylate, or copolymers based on styrene or acrylate having hydroxyl, carbonate groups or alternatively chlorine atoms.

 Usually, the diameter of the resin beads is between 0.1 and 1 mm.

According to this variant, it is necessary to introduce into the composition comprising the enzyme a binding agent capable of reacting with the resin and the enzyme. Especially suitable for polyazetidines. You can find this type of product in the range
Polycup marketed by Hercules.

 The respective amounts of these various compounds are governed by the ratios R1 corresponding to the weight ratio of the biomass used (expressed in dry matter) to the dry matter of resin and R2 corresponding to the weight ratio of the biomass used (expressed in dry matter) to the dry binder material.

 In general, these two ratios R1 and R2 are each between 0.1 and 1 and are not necessarily equal.

 Such compositions are particularly suitable for the preparation of L-aspartic acid.

 According to a first method for preparing these compositions, the contacting of the enzyme, the resin and the binder is carried out by spraying an enzyme-binder mixture on the resin.

 According to a second variant, the various constituent elements of the above-mentioned catalyst are brought into contact in an indifferent order.

 Whatever the variant chosen, the three abovementioned elements of the catalyst are brought into contact in any type of mixer such as, in particular, concrete mixers, drageers, turbospheres, rotating balloons, mixers, bicones or stirred reactors.

 The contacting takes place at a temperature lower than that of denaturation of the synthesized enzyme.

 The duration of the contacting is very variable and lasts long enough to obtain a homogeneous flowing mixture.

 With regard to this type of enzymatic catalyst immobilized on resins, or any other type of support, as well as their method of contacting, reference may also be made to European patent EP 89 165.

 Once this contacting operation has been carried out, drying is carried out according to the invention. Everything that has just been described is therefore valid and will not be repeated here.

 It should also be noted that the drying is preferably carried out so as to obtain a catalytic composition having a residual moisture which is not less than the hydrophobic threshold of the enzyme. Conventionally, the humidity is between 10 and 30%.

The resulting catalyst can then be used in the synthesis of the acid.
L-aspartic. It can also be stored dry or advantageously in rehydrated form in ammonium aspartate, at a temperature always lower than that of the denaturation of the enzyme. This is usually close to the ambient or slightly lower.

 The process for preparing aspartic acid consists in bringing ammonium fumarate into contact with the catalyst to give ammonium aspartate. The latter is separated from the reaction mixture and acidified to obtain L-aspartic acid.

 According to a conventional method, the catalyst can be used in the form of a fixed bed. However, according to a particularly advantageous mode, the contacting is carried out in a fluidized bed of catalyst.

 Usually, in a first step, the ammonium fumarate is prepared in one or more cycles consisting of adding water and then ammonia.

 The respective amounts of the compounds are such that an ammonium fumarate solution is obtained having a concentration lower than that causing poisoning of the enzyme used by an excess of substrate (ammonium fumarate). Those skilled in the art are quite capable of accessing such information, the enzymes being known.

 However, by way of illustration only, and in the case where the enzyme used is obtained from E. Coli, this concentration is about 0.5 to 2 mol / l.

 In addition, it may be advantageous to control the pH of the ammonium fumarate solution obtained. Indeed, the pH thereof should preferably be in the optimum pH range for proper functioning of the enzyme. Here again, this pH is entirely accessible to those skilled in the art. By way of example and still in the case of the abovementioned enzyme, this pH is between 6 and 11, and preferably between 7 and 10.

 To adapt the pH of the solution, ammonia, soda, potash, or hydrochloric, sulfuric, phosphoric acid will be added in the conventional manner.

 The resulting fumarate solution is then brought into contact with the catalyst which is in a fixed bed, or preferably in a fluidized bed.

 The contacting takes place in an upward flow of reagent if the bed is fluidized, or in an upward or downward flow of reagent if the catalytic bed is fixed.

 In the case of the use of a fluidized bed, the speed of the reactant flow is preferably greater than the minimum fluidization speed. By minimum fluidization speed is meant the minimum speed from which the catalytic bed is lifted ". In addition, the maximum speed of the flow is preferably less than the speed at which the catalytic bed is driven. More particularly, the speed maximum of the gas flow is at most 90% of the entrainment speed of the catalytic bed. In fact, one operates in such a way as to preferably have an ultra-laminar flow within the reactor (piston-type flow). correspond for example to gas flow velocities of less than 1 mm traveled per second.

 Obviously such conditions are they that there is a homogeneous distribution of ammonium fumarate in the reactor, for obvious reasons of efficiency of the process.

 The quantity of catalyst used can be determined by a person skilled in the art according to the productivity which he wishes to achieve.

 The temperature at which the catalyst is brought into contact with the reagent is lower than the denaturation temperature of the enzyme.

 In the case of the abovementioned enzyme (E. Coli), this is usually less than 40 "C. It is preferably around 30 - 35" C. These values are once again purely indicative and must be determined according to the enzyme used.

 According to an advantageous embodiment, the contacting is carried out in the presence of an additional catalyst. In the particular case of the abovementioned enzyme, this catalyst is magnesium sulfate. This catalyst can be introduced directly into the reactor or even be added to the ammonium fumarate solution. The amount of this additional catalyst is generally less than 0.01 mol / l.

 Quite advantageously, the reaction is carried out continuously.

 This process, and more especially when it is carried out in a fluidized bed, makes it possible to achieve degrees of conversion as high as 99.5 or even 99.99%.

 An ammonium aspartate solution having a concentration of the same order of magnitude as that of the incoming fumarate is therefore obtained at the outlet of the reactor.

 The L-aspartic acid is recovered from the reaction medium by any known means.

 For example, according to a first variant, the solution can be mixed with sulfuric acid. This is done so that you are at the isoelectric point of L-aspartic acid (about 2.9). The addition takes place at a temperature between 90 and 95 "C. A primer for crystallization of aspartic acid is then obtained. The crystallization is conventionally continued in a crystallizer, batchwise or continuously. The crystals obtained are then separated. .

 According to another variant, the ammonium aspartate solution can be treated with an alcoholic solution of fumaric acid. On this subject, reference may be made to patent application EP-A-678 499.

 According to another possible route, the separation of the L-aspartic acid is carried out by adding fumaric acid under conditions as described in patent application EP-A-588 674.

 The aspartic acid obtained is an optically active acid. It finds inter alia an application in the synthesis of polyaspartic anhydride and polyaspartates, compounds which can be used in particular in detergent compositions.

Claims (8)

1. A method of preparing a composition comprising at least one thermosensitive substance, characterized in that at least one step of drying said composition is carried out at a temperature higher than that of degradation of said substance and for a period such that 'there is substantially no degradation of this substance. 2. Method according to the preceding claim, characterized in that the drying is carried out at a temperature between 120 and 260 "C. 3. Method according to any one of the preceding claims, characterized in that the drying step is carried out for a duration of less than 30 seconds, preferably less than 10 seconds. 4. Method according to any one of the preceding claims, characterized in that the drying is carried out of a composition comprising, as heat-sensitive substance, at least one enzyme. 5. Method according to the preceding claim, characterized in that the drying is carried out of a composition comprising, as heat-sensitive substance, at least one enzyme produced by a microorganism chosen from the following Pseudomonas fluorescens, Pseudomonas dacunhae, Bacillus megaterium, Aflhrobaster species, Arthrobacter simplex, Streptomyces phaechromogenes, Rhodospoddium toruloides, Escherichia coli, Aerobacter aerogenes, Bacterium succinum, Micrococcus spia, Bacillus subtilis. 6. Method according to claim 5, characterized in that the drying is carried out of a composition comprising, as a heat-sensitive substance, an enzyme produced by the microorganism E. Coli, and in particular the strain whose ATCC number is 11303. 7. Method according to any one of claims 4 to 6, characterized in that one carries out the drying of a composition further comprising a support chosen from resins. 8. Use of the composition obtained by the process according to any one of claims 1 to 7, for the preparation of L-aspartic acid.