CH615933A5 - Process for carrying out enzymatic reactions - Google Patents

Abstract

A microporous membrane is used, the pores of which contain an enzyme, fixed by crosslinking using a bifunctional bonding agent, and an aqueous solution of a product capable of undergoing an enzymatic reaction is passed through the membrane by applying a pressure, and the products formed are removed from the solution. The process can be applied, especially, for carrying out the hydrolysis reaction of a racemic mixture of N- or O-acylated amino acids.

Description

The present invention relates to a method for carrying out enzymatic reactions and, more particularly, to carrying out enzymatic reactions using insoluble enzymes fixed by crosslinking in the pores of microporous membranes.

The advantage of using insoluble enzymes for enzymatic reactions lies in the possibility of making an enzyme play a catalytic role on a stream of substrate continuously, without it being necessary to separate the enzyme from the product. resulting from the catalytic reaction.

The use of insoluble enzymes to carry out enzymatic reactions is known. Thus, for example, the following methods of use should be mentioned:

1) The insoluble particles of the enzyme are suspended in a tank with agitation, and the current which leaves the tank passes through a filter.

2) The insoluble particles of the enzyme line a column which is continuously traversed by the substrate.

Both systems have been studied by various groups and, in many cases, scattering limitations have been observed from non-agitated layers in and around the particles. These limitations have become more important when the diffusion rate of the substrate is the slowest and the enzymatic reaction rate is the highest. Thus, it has been found that these limitations due to diffusion decrease the efficiency or yield of the insolubilized enzyme particles, and sometimes only a small percentage of their potential activity is used. Therefore, it has been considered desirable to find a method for carrying out enzymatic reactions using insoluble enzymes, substantially avoiding the disadvantages of known methods.

According to the present invention, a method has been found for carrying out enzymatic reactions, according to which a pressure is applied to an aqueous solution of a product which can be chemically modified by an enzymatic reaction, causing the flow of the solution. under the influence of a pressure difference across a membrane comprising the enzyme fixed by crosslinking and by removing, by methods known in themselves, the products in solution thus obtained. According to this method, the membrane in which the enzyme is fixed by crosslinking is a microporous membrane in the pores of which an enzyme has been fixed by crosslinking thanks to a difunctional binding agent.

For the present invention, an aqueous solution designates a solution in which the quantity of water is sufficient to allow the enzymatic reaction. In certain cases, the substrate is not soluble in water and it is then necessary to add another solvent, for example ethanol. In most cases, however, the solvent will include at least 50% water.

Membranes containing enzymes, similar to those used in the process of the present invention, have been prepared and described in the literature, for example in Goldman et al., "Science", vol. 150, pp. 758-760 (1965), and in the patent application of the Federal Republic of Germany DOS No. 1915970. However, these publications aim at the preparation of biological membranes, their study under diffusion conditions and the study of micro- environment of these membranes, and they do not or do not suggest the process of the present invention or the superior results thereby obtained.

Another interesting publication is British Patent No. 1183260, which relates to insolubilized enzymes, their preparation and their use. More particularly, this British patent describes a process for carrying out enzymatic reactions using enzymes which are chemically linked to an insoluble support. It is particularly interesting to note that this British patent specifically mentions the article by Goldman and his collaborators and, after having considered the teachings, it supposes that the method consisting in using crosslinked enzymes enclosed in a membrane to perform enzymatic reactions is not practical and, on the contrary, he chooses to directly covalently attach the enzymes to the membranes.

Contrary to the teachings and assumptions of the aforementioned British patent, it has just been found that the method for carrying out enzymatic reactions according to the present invention has many advantages when compared with the methods mentioned above, which are based on the use of insoluble particles of an enzyme or on membranes to which an enzyme is covalently attached.

Most reactors of the prior art comprising an immobilized enzyme are based on particles of the enzyme and resistance to diffusion then plays an important role.

Unlike soluble enzymes, the enzymes supported in a gangue or matrix act in a heterogeneous environment, and we can identify 5 distinct stages in the overall enzymatic process:

1) diffusion of the product from the bulky phase towards the support surface (external diffusion);

2) transport of the product from the surface of the support to the domain of the enzyme (internal diffusion);

3) transformation of the product, catalyzed by the enzyme;

4) transport of the product from the internal pores filled with the enzyme towards the surface of the particles;

5) diffusion of the product from the surface of the particles towards the bulky phase.

Stages 1 and 5 are effects of external diffusion or in the bulky mass, which result from the presence of a layer of almost stagnant fluid around the surface of the enzyme supported by the gangue or matrix. The transport of the product in this region is mainly by molecular diffusion and it leads to resistance to diffusion. Mass diffusion limitations have been observed in the case of small packed columns and in the case of membranes containing an enzyme. High molecular weight products experience greater difficulties due to the

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diffusion, due to their low diffusivity. Thus, there is a decrease in activity for papain, ficin and chymotripsin linked and which act on casein, in comparison with their activity on products with low molecular mass.

Stages 2 and 6 are effects of internal diffusion or in the pores, which result from the fact that the major part of the immobilized enzyme is fixed inside the gangue or porous matrix serving as support and that the product must enter by diffusion into and from narrow pores containing the internal enzyme by diffusion, to reach or leave the reaction site. In many cases, these resistances to internal diffusion significantly decrease the catalytic activity indicated for the enzyme. Thus, the enolase covalently linked to a porous support only exhibits% of its potential activity, due to the fact that only a portion of the enzyme is located in the vicinity of the surface of the support and exhibits activity.

Restrictions on internal diffusion can also lead to increased inhibition of product diffusion, due to the slow diffusion of reaction products from the microenvironment. This has been observed for the hydrolysis of esters and amino acids by proteolytic enzymes. The hydrogen ions formed by the enzymatic reaction cause a decrease in the local pH, compared to the global pH, which results in a displacement in the activity curve as a function of the pH. Such shifts in the activity curve as a function of pH have been observed for papain, trypsin, chymotrypsin and glucose oxidase.

As indicated above, it has just been discovered according to the present invention that most, if not all, of these diffusion limitations can be overcome in an enzyme reactor based on filtration under pressure through microporous membranes including the pores have walls coated with a layer of the molecules of enzymes fixed by crosslinking. The input flow rate of the substrate into the pores is regulated by a pressure gradient. The enzyme catalyzed transformation occurs inside the pores and the products are removed as they are formed. The diffusion-based stages of the above processes are completely eliminated when microporous membranes with small pore sizes are used. Unstirred outer layers are absent because a pressure gradient pushes the substrate into the pores, and internal diffusion to the pores is negligible due to the small pore radii.

The elimination of external and internal restrictions on mass transport, in the reactors described above and in which an enzymatic membrane acts under the action of pressure, results in a significant increase in biochemical efficiency:

a) because the reaction process will be dependent on kinetics and not dependent on diffusion, and b) because enzymes bound to the walls of the membrane pores are available for a catalytic reaction much more efficiently than bound enzymes to porous articles in which, due to the limitations of diffusion in the pores, the reaction is often limited to the surface of the particles alone.

In addition, the large ratio between the surface area of the pores and the volume of the pores (2 × 10 5 for a pore of 0.1 p. In diameter) results in a very high local concentration of the enzyme in the pore. If we assume that the pores are identical straight cylinders and if we assume that the enzyme molecules cover the walls of the pores in a dense monomolecular layer, we can calculate the concentration of the enzyme as follows :

Enzyme concentration (M / cm3) =

or

A: surface of the pore walls a: surface of the enzyme molecule = X2 (X = 100 Å)

V: pore volume N: Avogadro number r: pore radius.

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The concentration of the enzyme in a membrane with pores of 0.1 µx would be 0.6 pmol / cm3. Translated into milligrams, this would indicate 15 mg of an enzyme with a molecular weight of 25,000 (i.e. chymotripsine). This high local concentration results in a high rate of transformation of the substrates.

The use of the enzyme membrane fixed by crosslinking according to the present invention in particular also has the following advantages in comparison with the enzyme membrane covalently linked which has been described in the aforementioned British patent No. 1183260:

1) The carrying capacity of the membrane, that is to say the quantity of enzyme fixed by crosslinking and insolubilized per unit of membrane is greater. The enzyme molecules form a tightly packed layer on the surface of the pores, which achieves a very high local concentration of the enzyme in the pore.

2) The three-dimensional network of the enzyme in the membrane strengthens the gangue or matrix and increases the resistance of the membrane to compaction as encountered in the process according to the present invention.

3) The enzyme fixed by crosslinking is more stable with respect to heat denaturation and with regard to long periods of storage than enzymes covalently fixed. In addition, by fixing the enzyme molecules by crosslinking using an inactive protein, the protective effect of the inactive proteins in the environment can be added to the stabilizing effect when fixed by crosslinking on a matrix. .

4) The fixing of enzymes by crosslinking inside a preformed membrane constitutes a general and non-specific process which is suitable for any matrix and any enzyme. It avoids the need for the formation of active sites on the membrane and it only requires an appropriate bifunctional reactive agent.

Thus, it will be understood that the process of the present invention constitutes a major advance and provides numerous advantages in carrying out enzymatic reactions.

To prepare the membranes comprising the enzyme fixed by crosslinking and to be used in the process according to the invention, preferably an enzyme solution is discharged into the pores of a membrane, then the membrane charged with the liquid is immersed. enzyme in a solution of a bifunctional crosslinking agent.

With the distinction of the works described in the aforementioned article of "Science" and in the aforementioned patent application of the Federal Republic of Germany N ° DOS 1915970, according to which the membranes containing the enzyme are prepared by a diffusion process, which is not entirely satisfactory, it appeared desirable to find, for the preparation of membranes containing the enzyme, a new process which can overcome the drawbacks of the diffusion process. After much research, it has been found that the use of pressure to introduce the molecules of the enzyme into the pores of the membrane makes it possible to regulate the loading capacity of the membrane and to increase it in comparison with the known process. by broadcast. In addition, it has also been found that the new process provides an easier and faster route for preparation. In addition, this process has particular advantages in the case of large-scale preparation of the enzyme membranes.

More particularly, the process for preparing the membrane comprising of the enzyme fixed by crosslinking consists in taking a membrane and in discharging an enzyme solution through the pores of this membrane under a certain pressure difference, for example in an element or cell operating under pressure, when the pores of the membrane are filled with the enzyme solution and then the membrane loaded with the enzyme is immersed in a solution of a bifunctional crosslinking agent.

Preferably, the pressure used will be such that the flow rate of the enzyme solution through the membrane will be from about 0.05 to about 0.5 cm3 / min / cm2.

The enzyme solution preferably has a concentration of 1 to 10 mg / cm3 and a pH of 5 to 9. The enzyme solution can equal3

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understand an inactive protein, such as albumin. This protein protects labile enzymes against inactivation. In addition, this solution also preferably contains a reversible inhibitor or a substrate which does not contain groups capable of reacting with the crosslinking agent.

Immersion of the membrane in the solution of the crosslinking agent requires approximately 1 to 10 hours at approximately 1 to 30 ° C.

The membrane is then advantageously washed in a pressure filtration cell or unit in order to remove the enzyme and the non-fixed crosslinking agent. Free aldehyde groups or other free active groups can be blocked by glycine or another suitable blocking agent.

The membrane used can be any membrane whose pores have dimensions lying in the range of ultrafiltration or microfiltration, that is to say that the membrane has pore dimensions of 0.01 at 10 | i and preferably between 0.025 and 0.5 ji. These are, for example, membranes made of cellulose acetate, cellulose triacetate, cellulose nitrate, mixed cellulose esters, nylon, polyelectrolyte complexes, etc.

As agents suitable for crosslinking, there may be mentioned, for example, dialdehydes such as glutaraldehyde; dioxobenzidine; hexamethylene diisocyanates; 1,5-difluoro-2,4-dinitrobenzene; N, N'-hexamethylenebisiodoacetamide, etc.

The enzyme can be of animal or vegetable origin. Thus, mention may be made, for example, of hydrolases such as chymotripsine, tripsine, amylases, carboxypeptidase, amino acylase and carboxypeptidase; oxidases for example glucose oxidase, catalase, alcooldéhydrogenase and per-oxidases; amidases such as urease, asparaginase; alkaline phosphatase, etc.

If an inhibitor is used, this inhibitor is specific for each enzyme. So, for example, glucose is used to protect glucose oxidase; P-phenyl propionate to protect chymotrypsin, etc.

As indicated above, the method for carrying out enzymatic reactions according to the present invention consists in causing a solution of a product (which can be chemically modified by enzymatic reaction) to flow under a pressure difference through a membrane containing enzyme attached by cross-linking, and any enzyme that is suitable for causing enzyme reactions can be used to be part of the membrane containing the enzyme. Thus, one can consider an unlimited number of possibilities for carrying out enzymatic reactions. For example, the method according to the present invention can be used for the resolution of racemic mixtures of N- or O-acylamino acids. Such a mixture is then discharged through the membrane containing the appropriate enzyme; an isomer is hydrolyzed therein, which gives a mixture of an optically pure L- or D-amino acid and of the D- or L-acylamino acid of the non-hydrolyzed isomer, which can be separated by known methods. themselves.

It is also possible to oxidize glucose to glucuronic acid by using glucose oxidase as an enzyme.

In addition, urease can be used to decompose urea.

Another possibility is to use catalase for the decomposition of peroxides.

The pressure required to carry out the enzymatic reaction depends on the membrane containing the enzyme, the concentration of the substrate, the desired degree of transformation and / or the desired reaction rate. There is however no difficulty in determining the optimal pressure difference for each enzymatic reaction, according to the bodies which one wishes to react and the results which one wishes to obtain. Thus, for example, with a membrane comprising a particular enzyme fixed by crosslinking, the increase in pressure will determine an increase in the reaction rates with a decrease in the degrees of transformation, and one can thus make adjustments in order to get the best results. In practice, it has in fact been found that the pressure differences can be between approximately 0.05 and approximately 10 bars and preferably between approximately 0.05 and approximately 5 bars. It is known that such pressure differences can be obtained by applying direct pressure to the solution of the substrate or else by maintaining a vacuum on the other face of the membrane comprising the enzyme fixed by crosslinking.

As will be seen in the examples below, several membranes have been prepared with enzymes attached by crosslinking and each membrane has been analyzed under various flow conditions involving the continuous flow of the substrate under pressure through the membrane. In addition, each membrane was analyzed under discontinuous conditions involving turbulent agitation of the membrane in a batch or batch of substrate. It should be noted that, in the latter case, the membrane comprising the enzyme fixed by crosslinking behaves similarly to an enzyme linked to a particle. The reaction rates were compared for identical degrees of transformation, under discontinuous conditions and those of a continuous flow, and in each case, we found in the conditions of (continuous) flow a greater value than under discontinuous conditions. This difference between the speeds results from the absence of the diffusion limitations imposed on the movement of the substrate in one case and from the existence of these limitations in the other case. Under the conditions of a (continuous) flow, the substrate is pushed back into the membrane and it easily comes into contact with the molecules of the enzyme immobilized in the pores. In the conditions of discontinuous batch operation, on the contrary, the substrate must penetrate by diffusion into the membrane, firstly through an unstirred layer which forms along the surface of the membrane and then into the pores of the membrane. Thus, the increase in the reaction rate under the conditions of a continuous flow compared to the discontinuous conditions comes from the fact that the limitation of diffusion has been overcome. The ratio between the two velocities (Ve / Vd) described in the examples as being the ratio between the velocities, measures and determines the increase in the efficiency of the use of the enzyme under conditions of continuous flow.

Example 1:

Preparation and analysis of a membrane containing chymotrypsin

A solution of chymotrypsin containing P-phenyl propionate as an inhibitor (6 mg of chymotrypsin and 3 g) is passed through a membrane of cellulose triacetate, the pores of which are about 0.1 μm. 7 mg inhibitor per cubic centimeter) in 0.5M phosphate buffer at pH 7.0. The membrane is briefly washed with water and immersed in a 2.5% solution of glutarodialdehyde for 1 h. It is then placed in a cell operating under pressure and washed with water, with 10 "3N HCl (to break the enzyme / inhibitor complex) and with buffer until it is no longer found in the washing water neither absorption of ultraviolet nor enzymatic activity. The membrane is subjected to tests of its enzymatic activity using the ethyl ester of N-benzoyl-L-tyrosine (EEBT) in a mixture at 30% of ethanol EtOH and with 70% Tris buffer at pH 7.8 (0.05 M); N-acetyl-L-tryptophan methyl ester in Tris buffer (0.05 M; pH 7.8) containing 0.05 M CaCl2 and ethyl ester of N-acetyl-DL-phenylalanine in Tris buffer (0.05 M; pH 7.8) containing 0.05 M CaCl2 as substrates. substrates through the membrane under various pressures at a temperature of 26 ° C, and the product thus formed is measured in the effluent.

The membrane is then analyzed under discontinuous conditions, that is to say with a substrate charge under turbulent stirring. The following tables give the results and the ratio between the reaction rates in the case of a continuous flow and a discontinuous operation when the same values are obtained4

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transformation percentages. Here are the meanings of the column heads and abbreviations of these tables:

P: pressure difference between the two faces of the membrane Q: flow rate V: reaction speed

%: percentage of the substrate transformed into a loaded product: carrying out the enzymatic reaction under discontinuous conditions (in a batch or a load).

reaction speed under conditions

5, continuous flow

Ratio between the speeds =: ——

reaction rate under discontinuous conditions

Table I

Product

N-benzoyl-L-tyrosine ethyl ester

Concentration

5.10-4M

AT

5.10-3M

P

Q

V

% of

P

Q

V

% of

atm cm3 / min / cm2

liM / mn / cm2

transform atm cm3 / mn / cm2

pM / min / cm2

transform

mation

mation

0.5

0.21

0.07

100

0.5

0.21

0.73

100

1.0

0.42

0.11

100

1

0.42

1.29

90

1.5

0.62

0.16

100

1.5

0.62

1.7

72

2

0.95

0.36

100

3

1.58

0.52

88

3

1.25

2.3

50

3.5

2.23

0.58

61

Charge

discontinuous

-

-

0.036

75

-

-

0.375

50

Ratio

the speeds

15

75

6

50

Table II

Table III

Product Concentration

N-acetyl-L-tryptophan methyl ester

2-103M

, ",

P Q V% of atm cm3 / min / cm2 (iM / min / cm1 transformation

0.5

0.125

0.23

100

1

0.21

0.375

100

2

0.83

1.45

100

3.5

1.46

2.3

90

Charge

discontinuous -

-

0.18

90

Report

between the

gears

13

90

It appears from the tables that transformation percentages of 75.50.90 and 46% are obtained at a reaction rate which is 15,6,13 and 6 times as fast, respectively, when using the process of the present invention in comparison with a batch process involving the same respective substrates at the same concentration and with the same membranes.

Example 2:

Preparation and analysis of a membrane comprising trypsin A solution comprising 5 mg of trypsin / cm3 of 0.05 M CaCl 2 is passed under a pressure of 0.1 bar through a membrane of cellulose triacetate having a pore size about 0.1 (x. The membrane is then briefly washed with water and then immersed for 1 h at room temperature in a 2.5% glutaraldehyde solution brought to pH 7 using 0.1 M NaOH

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Product Concentration

N-acetyl-DL-phenylalanine ethyl ester * M0 ~ 3M

P

ATM

Q

cm3 / min / cm2

V

(iM / min / cm2

% transformation

0.5

0.125

0.198

50

1

0.26

0.36

50

1.5

0.36

0.56

50

2

0.625

0.895

46

Charge

discontinuous

-

-

0.146

46

Ratio

the speeds

6

46

* Only ester L is hydrolyzed by the enzyme.

The membrane is then placed in an ultrafiltration cell, 55 and this membrane is washed with water and buffer (phosphate, pH 7.0) until no more absorption of ultraviolet or activity is found. of enzyme in the wash water. The activity of the membrane is then analyzed using ethyl ester of N-benzoyl-arginine in Tris buffer at pH 8.0 (0.05M) containing 60 CaCl1 (0.01M) and using N-benzoyl-DL-lysine ethyl ester in Tris buffer at pH 8.0 (0.05M) containing CaCU (0.01 M) as substrates. The substrates are passed under pressure through the membrane at 26 ° C., and the quantity of product formed is measured in the effluent. The membrane 65 is also analyzed under conditions of use of a discontinuous charge, that is to say with a substrate charge under turbulent stirring. One calculates the ratio between the velocities in the case of a continuous flow and under conditions of a discontinuous load, for the

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same degree of transformation. Results obtained are shown in Tables IV and V.

Table IV

Product

N-benzoylarginine ethyl ester

Concentration

3-

10-3M

1 '

• 10_2M

/ '"

P

ATM

Q

cm3 / min / cm2

V% of juM / mn / cm2 transformation

P

ATM

Q

cm3 / min / cm2

V

pM / min / cm2

% transformation

0.5 1.5 2.0

0.18 0.31 0.52

0.41 0.63 1.09

100 92 76

0.5 1.0 1.5 2.0

0.125 0.187 0.34 0.62

0.85 1.25 1.48 1.71

100 97 63 41

Discontinuous charge

_

_

0.25

74

_

0.68

97

Ratio between speeds

4

75

2.5

97

Table V

Product N-benzoyl-DL-lysine ethyl ester *

Concentration

2-10 "3M

2-10_2M

P

ATM

Q

cm3 / min / cm2

V

| iM / min / cm2

% transformation

P

ATM

Q

cm3 / min / cm2

V

pM / min / cm2

% transformation

0.5 1.6 2.1

0.16 0.33 0.63

0.43 0.75 1.15

50 48 43

0.4 1

1.5 2.0

0.08 1.16 0.31 0.63

0.83 1.46 1.77 2.19

50 46 37 30

Discontinuous charge

_

0.16

43

_

0.78

46

Ratio between speeds

7

43

2

46

* Only ester L is hydrolyzed by the enzyme.

Example 3: Table VI

Preparation and analysis of a membrane comprising carboxy- ™

peptidase B

A solution comprising 3.3 mg of carboxypeptidase B per cubic centimeter of 10% (w / v) solution of LiCl is passed under pressure (0.1 bar) through a membrane for micro-filtration (Millipore MF-GS ; pore size: 0.22 | i).

The membrane is briefly washed and immersed for 1 h at room temperature in a 2.5% solution of glutarodialdehyde (pH 7.0; crosslinking agent). The membrane is then placed in an ultrafiltration cell and washed with water and with veronal buffer (0.05M; pH 7.0) until it is no longer found in the washing water. neither absorption of ultraviolet nor activity of the enzyme. The membrane is then analyzed to determine its activity under the conditions of a continuous flow and in the presence of a discontinuous charge, as described in the preceding examples, and the ratio between the speeds is calculated. The substrate used is N-hippurylarginine in Tris buffer (0.03 M pH 7.6) containing 0.1 M NaCl. The results obtained are shown in Table VI.

Product

N-hippuryl-L-arginine

Concentration

1-10 "3M a

P cm3 / min / cm2 | 4.M / min / cm2% of atm transformation

Discontinuous charge

65 Ratio between speeds

0.3 0.1 1.5 1.88

0.31 1.04

0.13

100 56

56 56

7

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Example 4:

Preparation and analysis of a membrane containing catalase

A solution comprising 3 mg of catalase per cubic centimeter of a phosphate buffer (0.05M; pH 7.0) containing 1% NaCl is passed through a membrane for microfiltration (Millipore MF-GS; pore size: 0.22 n). The membrane is briefly washed with water and immersed in a 2.5% solution of glutarodialdehyde in a phosphate buffer (0.05 M; pH 7.0) for 2 h. The membrane is then washed under pressure in an ultrafiltration cell with water and with a 10% NaCl solution until no more absorption of ultraviolet or enzymatic activity in the washing water can be detected. . The enzymatic activity of the membrane is then analyzed using a solution of 0.035M of H2O2 in phosphate buffer (0.05M; pH 7.0) under the conditions of a continuous flow and a discontinuous charge, as described in example 1. One calculates, for the same degree of transformation, the ratio between the velocities in the case of conditions corresponding to a continuous flow and conditions corresponding to a discontinuous charge.

The results obtained are shown in Table VII.

Table VII

Product H202

Concen- 0.035M

tration *

P

ATM

Q

cm3 / min / cm2

V

pM / min / cm2

% transformation

0.5

0.37

10.5

98

2

2.7

55.4

55

Charge dis

keep on going

5.5

55

Report

between the

gears

10

55

Example 5:

Preparation and analysis of a membrane comprising glucose oxidase

A solution comprising 5 mg of glucose oxidase per cubic centimeter of a 0.05M phosphate buffer (pH 6), containing 1% of glucose, is discharged into the pores of a Millipore HA-MF membrane (pore size : 0.45 (d.) The membrane is then immersed for 2 h at room temperature in a 2.5% solution of glutaraldehyde. It is then washed with water and with a phosphate buffer (pH 6, 8) until no more absorption of ultraviolet or enzymatic activity is found in the washing water.

The membrane is tested by flowing a 5% solution of glucose in a phosphate buffer (0.05M; pH 6.0) through this membrane and measuring the amount of H2O2 formed in the eluent. The results are compared with those of the same reaction carried out in the case of a batch charge. The results obtained are presented in Table VIII.

(Table at the top of the next column)

Example 6:

Preparation and analysis of a membrane containing urease

A solution comprising 3 mg of urease per cubic centimeter of a 0.02M phosphate buffer (pH 7), containing 10 3M of EDTA, is discharged into a Gelman Metrocel TCM 200 membrane having a pore size of 0.2 | i. The membrane is then washed with water and with phosphate buffer until no longer found

Table VIII

Glucose Product

Concen- 2.6-10 "'M

tration, * s

P Q V% of atm cm3 / min / cm2 pM / min / cm2 transformation

0.5

4.4

0.016

2

22.1

0.03

4

59.3

0.05

0.01

Charge dis

keep on going

0.002

0.01

Report

-

between the

gears

25

0.01

absorption of ultraviolet 'nor enzymatic activity in the wash water.

The activity of the membrane is then analyzed using a 0.3% urea solution in a phosphate buffer (0.1M; pH 7.0) containing 10 "3M of EDTA, under the conditions of a continuous flow and under the conditions of a discontinuous load, as described in Example 1.

The results obtained are shown in Table IX.

Table IX

Product

Concentration

Urea 5-10 "2M

AT

P

ATM

Q

cm3 / min / cm2

V

(xM / min / cm2

% transformation

0.05

0.04

0.02

5

0.5

0.42

0.02

0.5

Charge dis

keep on going

0.005

5

Report

between the

gears

4

5

Example 7:

Preparation and analysis of membranes containing chymotrypsin and having various pore sizes

A solution of chymotrypsin, containing P-phenyl propionate as an inhibitor (3.3 mg of chymotrypsin and 7 mg of inhibitor per cubic centimeter) is placed under pressure in phosphate buffer (0.05M; pH 7.0 ) through Millipore membranes with various pore sizes:

1) 0.025 p (VS-MF-Millipore)

2) 0.1 n (VC-MF-Millipore)

3) 0.22 n (GS-MF-Millipore).

The pressure differences that are used to pass the enzyme solution and to charge the membranes are approximately 0.4 bar, 0.1 bar and 0.02 bar, respectively. The membranes are quickly washed and immersed in a 2.5% solution of glutarodialdehyde in 0.05 M phosphate buffer (pH 7.0). At the end

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for 2 h, the membranes are placed in a cell under pressure and washed with 10 "3M HCl (to break the enzyme / inhibitor complex) and with buffer until no longer found in the washing water or absorption of ultraviolet or enzymatic activity. The enzymatic activity of the membranes is then analyzed by passing under pressure a solution of ethyl ester of N-benzoyl-tyrosine (2.5 x 10 "3M) in 30% of ethyl alcohol— 70% of

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Tris buffer (0.05M; pH 7.8) and by measuring the quantity of product formed in the effluent. The membranes are also tested under discontinuous conditions, that is to say with a discontinuous loading of substrate under turbulent stirring. The ratio between the speed under turbulent flow conditions and under the conditions of discontinuous loading is calculated for the same degree of transformation. The results obtained are presented in Table X.

Paintings

Product

Pore size

0.025 p

Benzoyltyrosine ethyl ester (2.5 x 10 3) 0.1 d

0.22 n

% transformation

% transformation

P Q

% transformation

0.5

0.13

0.29

87

0.4

0.29

0.63

87

0.2

1.43

2.88

80

1.5

0.33

0.78

92

0.75

0.53

1.19

89

0.3

2.8

3.46

66

2.5

0.55

1.14

80

1.5

1.06

2.36

88

0.5

3.17

4.47

56

3.8

0.83

1.72

80

2.5

1.97

3.62

85

1.0

6.06

9.39

60

8

1.00

2.2

80

3.6

2.7

5.55

80

16

2.0

4.0

70

0.14

80

0.18

80

0.17

80

Discontinuous charge Ratio between speeds

16

80

31

80

17

80

Example 8:

Preparation and analysis of membranes made of non-cellulosic materials containing chymotrypsin A solution of chymotrypsin, containing P-phenyl propionate (3 mg of chymotrypsin and 7 mg of inhibitor per cubic centimeter) is passed through phosphate buffer ( 0.05M; pH 7.0) through membranes made up of the following non-cellulosic materials:

a) inert non-cellulosic synthetic polymer: membrane

35 for ultrafiltration (Diaflo XM 300 molecular mass: 300,000), produced by Amicon, and b) Nucleopore polycarbonate membrane; pore size: 0.4 | x; thickness: 11 p.

The membranes are briefly washed and immersed in a 2.5% glutarodialdehyde solution for 2 h. They are then washed and tested with N-benzoyl-tyrosine ethyl ester (EEBT; 2.5 x 10 "3M) as described in Example 1. The results obtained are shown in Table XI .

Table XI

Product N-Benzoyltyrosine Ethyl Ester (2.5 x 10 3M)

Type of membrane

Nucleopore

AT

Amicon

AT

P

Q

V

% transformation

P

Q

V

% transformation

0.1

0.3

0.29

36

2.0

0.122

0.55

100

0.2

0.93

0.45

24

3.0

0.36

0.87

98

Discontinuous charge

0.07

36

-

0.07

98

Ratio between speeds

4

36

12.4

98

R

Claims (6)

615,933 1. Process for carrying out enzymatic reactions, according to which a pressure is applied to an aqueous solution of a product which can undergo a chemical modification by an enzymatic reaction, which causes the flow of the solution under a pressure difference at through a membrane comprising an enzyme fixed by crosslinking, and the products obtained are removed from the solution, this process being characterized in that the membrane comprising an enzyme fixed by crosslinking is a microporous membrane in the pores of which an enzyme has been fixed by crosslinking using a bifunctional binding agent. 2. Method according to claim 1, characterized in that a pressure difference between 0.05 and 10 bars and in particular between 0.05 and 5 bars is applied. 2 3. Method according to claim 1, characterized in that the dimension of the pores of the membranes is between 0.01 and 10 n and in particular between 0.025 and 0.5 p .. 4. Application of the method according to claim 1, for carrying out an enzymatic hydrolysis reaction, characterized in that a racemic mixture of N- or O-acyl-amino acids is forced through the membrane containing the appropriate enzyme and in which an isomer is hydrolyzed, which gives a mixture of an optically pure amino acid and the acylamino acid of the non-hydrolyzed isomer and which is separated from one another. 5. Application according to claim 4, characterized in that the enzyme is chosen from chymotrypsin and trypsin. 6. Membrane for implementing the method according to claim 1, characterized in that it consists of a microporous membrane in the pores of which an enzyme has been fixed by crosslinking using a bifunctional binding agent.