DE2553649A1 - METHOD FOR CARRYING OUT ENZYMATIC REACTIONS - Google Patents

Description

Process for carrying out enzymatic reactions

The invention relates to a method for carrying out enzymatic Reactions and in particular for carrying out enzymatic reactions with the aid of insoluble enzymes which are present in the Pores of microporous membranes are networked.

The advantage of using insoluble enzymes for enzymatic Reactions lies in the possibility of acting catalytically with an enzyme on a substrate flow in a continuous manner can be given without the need to separate the enzyme from the product obtained by the catalytic reaction is.

The use of insoluble enzymes in performing enzymatic reactions is known. So, for example, are the following known methods of such an application called:

1. The insoluble enzyme particles are suspended in a container with stirring, with the exiting flow passes through a filter;

2. The insoluble enzyme particles are packed into a column or column through which the substrate is continuous flows through.

Both systems have been studied by different groups, and diffusion restrictions have been observed in numerous cases, those of non-agitated layers within the particles and appeared all around the particles. These limitations become more important when the rate of diffusion increases of the substrate are slowest and the speeds of the enzymatic reaction are the highest. So it was found that this Limitations on diffusion lower the effectiveness of the unsolubilized enzyme particles, and sometimes only a small percentage of their potential activity is used. Therefore it would be advantageous to have a method for Carrying out enzymatic reactions with the help of insoluble enzymes to have available, in which these disadvantages the known working methods are essentially avoided.

The object of the invention is to create such a method without the disadvantages mentioned above.

Surprisingly, there has now been a method of carrying it out found from enzymatic reactions, this process being the application of pressure to an aqueous solution of a substrate comprises and wherein the substrate can be chemically changed by means of an enzymatic reaction, including the solution below a differential pressure flows through a cross-linked enzyme membrane and the products obtained from the solution are removed by methods known per se, the crosslinked enzyme membrane being a is a microporous membrane in the pores of which an enzyme has been crosslinked with the aid of a bifunctional coupling agent.

An aqueous solution as used in the method according to the invention means a solution in which the amount of water is sufficient to make the enzymatic reaction possible. In some cases the substrate is not sufficiently soluble in water and an additional solvent such as ethanol must then be added. In most cases, however, the solvent will contain at least 50 % water.

Enzyme membranes similar to those used in the method of the invention are already made and shown in FIG in the literature, e.g. by Goldman et al., Science Vol. 150 (1965) pp. 758-760 and DT-OS 1 915 970 (Avrameas et al.), but these publications relate to the production and investigation of biological membranes under diffusion conditions and the microenvironmental conditions of such membranes, and this does not anticipate the process according to the invention and the surprising results achieved thereby.

Furthermore, the preparation and use of insolubilized enzymes is described in British patent specification 1,183,260. In particular, a method for carrying out enzymatic reactions is included in this patent Described use of enzymes which are chemically bound to an insoluble carrier. Of particular interest is the The fact that in this patent the aforementioned article by Goldman et al. is mentioned and that is set out with reference thereto that an attempt to use crosslinked membrane-entrapped enzymes to carry out enzymatic reactions would not be practical and that enzymes directly covalently bound to membranes would be selected instead became.

Contrary to the teachings and assumptions in this patent has now surprisingly been used in the method according to the invention for carrying out enzymatic reactions found this to have numerous advantages compared to

CU9826M030

■ - 2553549

the methods previously described, which rely on the use of insoluble enzyme particles or covalently bound Enzyme membranes are based, owns.

Most of the prior art designs for reaction vessels with immobilized enzyme touching enzyme particles, in which the diffusion resistances play a major role.

In contrast to soluble enzymes, enzymes carried by a matrix act and can in a heterogeneous environment A distinction can be made between five individual stages in the overall enzymatic process:

1) Diffusion of the substrate from the phase present as a mass to the carrier surface (external diffusion),

2) transport of the substrate from the support surface to the area of the enzyme (internal diffusion);

3) enzyme catalyzed conversion of the substrate;

4) Transport of the product from the inner pores filled with enzyme to the particle surface,

5) Diffusion of the product from the particle surface in the phase present as a mass.

Levels 1 and 5 are external diffusion effects or room diffusion effects, resulting from the presence of a near-standing layer of liquid around the surface of the enzyme carried by the matrix. The transport of substrate through this area occurs mainly through Molecular diffusion and gives diffusion resistances. Room diffusion restrictions were observed in the case of small, packed columns and in the case of enzyme membranes. High molecular weight substrates are most affected by diffusion resistances because of their low diffusivity. So there was decreased activity for

• 0 U y 8 2 4/10 3 0

bound papain, ficin and chymotrypsin on exposure observed on casein compared to its activity on low molecular weight substrates.

Levels 2 and 4 are internal diffusion effects or pore diffusion effects, which result from the fact that the greater part of the immobile enzyme is inside the porous support matrix is bound and that the substrate or product diffuse into and out of narrow, internal enzyme-containing pores must in order to reach or leave the reaction site. In numerous cases such internal diffusion resistances set the stated catalytic activity of the enzyme substantially down. Enolase covalently bound to a porous support shows only a quarter of its potential activity, since only the part of the enzyme is near the surface of the support and shows activity.

Limitations on internal diffusion can also result in increased product inhibition due to slow diffusion of the reaction products from the microenvironment. This was done for the hydrolysis of esters of amino acids proteolytic enzymes observed. The hydrogen ions formed by the enzymatic reaction cause a decrease the local pH compared to the pH of the mass, resulting in a shift in the pH activity curve. Such shifts in the profiles of pH activity were observed for papain, trypsin, chymotrypsin, and glucose oxidase observed.

As previously described, however, it has surprisingly been found that most, if not all, of these Diffusion restrictions can be overcome in an enzymatic reaction vessel based on pressure filtration based on microporous membranes in which the pore walls are covered with a layer of cross-linked enzyme molecules. The substrate flow into the pores is controlled by a pressure

o υ a 8 2 u 4 ro 3 0

served controlled. The enzyme-catalyzed conversion takes place inside the pores, and the products are depending on their Education removed. The diffusion-based stages of the procedure described above are completely switched off, when microporous membranes with small pore sizes are used. Outer, unmixed layers are missing because the substrate is forced to flow into the pores by a pressure gradient, and the internal pore diffusion is negligible because of the small radius of the pores.

The elimination of restrictions on external and internal mass transport in the above-mentioned pressurized ones Reaction vessels with an enzymatic membrane result in a significant increase in biochemical performance:

a) because the reaction process is kinetically controlled and not controlled by diffusion; and

b) because the enzymes bound to the pore walls of the membrane for the catalytic reaction are much more effective Ways are available as enzymes that are bound to porous particles, which because of the restrictions imposed by Pore diffusion the reaction is often limited to the particle surface.

In addition, the large ratio of pore wall surface to pore volume (2 χ Λθτ for a pore with 0.1 η diameter) gives a very high local concentration of enzyme in a pore. If it is assumed that the pores exist as equal, straight cylinders, and if it is assumed that the enzyme molecules cover the pore walls in the form of a dense, monomolecular layer, the enzyme concentration can be calculated as follows:

60 ^ 824/1030

Enzyme concentration (M / _ccm ) = ψβ- =

A = pore wall surface

a = surface of the enzyme molecule = X ² (X = 100 Å)

V = pore volume

N = Avogadro's number

r = pore radius.

The enzyme concentration in a 0.1 µm pore size membrane would be 0.6 µm / cc. Converted to mg, this would be 15 mgu of an enzyme with a molecular weight of 25,000 (i.e. chymotrypsin). This high local concentration results in a high conversion speed of substrates.

The use of membranes with crosslinked enzyme according to the The invention has, inter alia, the following advantages compared to the membranes containing covalent enzyme bound, such as them are described in British patent specification 1,183,260 on:

1. The loading capacity of the membrane, i.e. the amount of cross-linked, insolubilized enzyme per membrane unit greater. The enzyme molecules form a tightly packed layer on the pore surface, so that a very high local concentration of enzyme is reached in the pore;

2. The three-dimensional network of the enzyme in the membrane strengthens the matrix and increases the resistance of the membrane compared to compaction, as occurs in the method according to the invention;

3 · The crosslinked enzyme is against heat denaturation and more stable than covalently bound enzymes towards longer storage times. In addition, by networking the Enzyme molecules with an inactive protein have the protective effect of inactive surrounding proteins with the stabilizing one Combine the effect of networking on a matrix;

G Uy 824/1 030 '

- 8 - λ r r- λ λ ί

/ h; ■> .1 Ώ α

2 5 5

4. The cross-linking of enzymes within a previously trained Membrane is a general and non-specific method that applies to any matrix or any Enzyme is suitable. It avoids the need for the formation of active places on the membrane and requires just a suitable, bifunctional, reactive agent.

It can be seen from this that the method according to the invention represents a great advance and numerous advantages in carrying out enzymatic reactions.

The invention also relates to an optimal method of manufacture of membranes with crosslinked enzyme for use in the method of the invention. The invention includes hence a process for the production of cross-linked enzyme membranes, which are particularly suitable for use in the method according to the invention, wherein an enzyme solution by the pores of a membrane is pressed and then the membrane loaded with enzyme into a solution of a bifunctional crosslinking agent is immersed.

In contrast to the information from Goldman and in DT-OS 1 915 97O _{5, in} which both times these enzyme membranes were produced by means of a diffusion process, this method not being completely satisfactory, it was considered advantageous to use a new method for producing the To develop an enzyme membrane that eliminates the disadvantages of the diffusion process. After numerous investigations it was found that the use of pressure to introduce the enzyme molecules into the membrane pores made it possible to control the loading capacity of the membrane and to increase it in comparison to the known diffusion process. In addition, it has been found that the process according to the invention is simpler and faster

GU a 8 2 U / 1 Π 3 0

25 5 3

3549

Way to manufacture is. In addition, it has particular advantages in the production of enzymatic membranes in
large scale.

The process for making the crosslinked enzyme membrane comprises the following steps: taking a membrane and the
Forcing an enzyme solution through the pores of this membrane under a pressure differential, for example in a pressure cell, the pores of the membrane being filled with the enzyme solution, and then immersing the membrane loaded with enzyme in a solution of a bifunctional crosslinking agent.

Preferably the pressure applied is such that the flow rate the enzyme solution through the membrane is about 0.05 to about 0.5 cnr / min-cm.

The enzyme solution preferably has a concentration of 1 to 10 mg / cc and a pH of 5 ~ 9 · the enzyme solution may also contain an inactive protein, e.g. albumin. This protein protects labile enzymes from inactivation. In addition, the solution preferably also contains a reversible inhibitor or a substrate that does not contain any Contains groups that can react with the crosslinking agent.

Immersing the membrane in the solution of the crosslinking agent requires about 1 to 10 hours at about 1 to 3O ⁰ C.

The membrane is then advantageously placed in a pressure filtration cell washed to remove unbound enzyme and crosslinking agent. Free aldehyde or other active groups can with glycine or another suitable blocking agent blocked.

The membrane used can be any prefabricated membrane with pore sizes in the range of ultrafiltration or micro-

euys 2 u / 10 3 0

filtration, ie. with pore sizes of 0.1 to 10 microns and preferably between about 0.025 and 0.5 microns, e.g. made of cellulose acetate, cellulose triacetate, cellulose nitrate,
mixed ester celluloses, nylon, polyelectrolyte complexes, etc.

Suitable "crosslinking agents are, for example: dialdehydes, e.g. glutaraldehyde, dioxobenzidine, hexamethylene diisocyanate, 1,5-difluoro-2,4-dinitrobenzene, N, N'-hexamethylene bisiodacetamide, etc..

The enzyme can be of animal or vegetable origin. Examples are: hydrolases, e.g. chymotrypsin, Trypsin, amilases, carboxypeptidase, aminoacylase and carboxypeptidase; Oxidases such as glucose oxidase, catalase, alcohol dehydrogenase and peroxidase; Amidases, e.g., urease, asparaginase; alkaline phosphatase etc ..

If an inhibitor is used, such an inhibitor is specific for each enzyme. For example, glucose is used to To protect glucose oxidase and ßr-phenylpropionate to chymotrypsin to protect, etc.

As previously described, the method includes performing of enzymatic reactions according to the invention that an aqueous solution of a substrate chemically by enzymatic Response can be changed under a pressure differential passed through a cross-linked enzyme membrane, and any enzyme that will cause enzymatic reactions Can be used as part of the enzyme membrane. So there can be an unlimited number of possibilities for the implementation of enzymatic reactions should be envisaged. For example, the method according to the invention can be used be to racemic mixtures of N- or O-acylamino acids to split. For this purpose, such a mixture is used by the

6 U y 8 2 4/1 0 3 0

suitable enzyme membrane pressed through, in which an isomer is hydrolyzed, as a mixture of an L- or D-optically pure Amino acid and the D- or L-acyl amino acid of the non-hydrolyzed Isomers is obtained, which can be split or separated by methods known per se.

Furthermore, it is possible to use glucose to glucuronic acid of glucose oxidase as an enzyme to oxidize.

Urease can also be used to decompose urea.

Another option is to use catalase for Decomposition of peroxides.

The one required to carry out the enzymatic reaction Pressure depends on the enzyme membrane, the substrate concentration, the degree of conversion desired and / or the desired Reaction speed. However, there is no difficulty in determining the optimal pressure differential for any enzymatic reaction as directed by the reactant used and the desired results are given. Thus, with a specific, cross-linked enzyme membrane, increased Increased reaction rates along with decreasing degrees of conversion, and optimal results can be achieved in pressure Agreement can be achieved herewith. In practice it has been found that the pressure differences from about 0.05 to about 10 atm and that they preferably range from about 0.05 to about 5 atm. These pressure differences of the Pressure differentials can be created either by applying direct pressure to the substrate solution or by maintaining a vacuum can be achieved on the other side of the crosslinked enzyme membrane.

The invention is hereinafter described in connection with certain preferred embodiments explained in more detail using examples, however, it should be noted that the invention

G ü BS 24/1030

2553549

is not limited to these specific embodiments. The following examples show preferred embodiments, the practical implementation of the process according to the invention explain in more detail and the principles of the invention Show procedure.

As will be seen from the examples below, several crosslinked enzyme membranes were prepared and each membrane was made under different flow conditions, which the continuous Flow of the substrate under pressure through the membrane was analyzed. In addition, each membrane was under conditions a batch-wise process management investigated, which the turbulent stirring of the membrane in a batch of the substrate included. It should be noted that the crosslinked enzyme membrane in the latter pail in a similar way to a Particle bound enzyme behaves. The reaction rates for identical degrees of conversion under flow conditions and under batch process conditions were compared in each case, where a higher value was found under flow conditions than under conditions of a batch process. This difference in speeds results from the lack of diffusion restrictions on substrate movement in one case and from the existence of these restrictions in the other. Under flow conditions will the substrate is forcibly inserted into the membrane and easily enters with the enzyme molecules immobilized in the pores Contact. On the other hand, under batch conditions, the substrate must diffuse into the membrane, first through a stationary (not stirred) layer that forms along the membrane surface, and then into the membrane pores. Therefore, the result is the increase in the reaction rate under flow conditions compared to that under the conditions of a rudimentary process management from overcoming the diffusion restrictions, and that

CUaö2-.4 / 10 3 0

2 5 5 3 6 A 9

Ratio of the two speeds - V / V ^ - that as "Speed ratio" reported in the examples is a measure and determination of increased performance for the utilization of the enzyme under flow conditions.

example 1

This example describes the production and testing of a chymotrypsin membrane.

A chymotrypsin solution containing β-phenylproprionate as an inhibitor (6 mg chymotrypsin and 7 mg inhibitor / ccm) in 0.5 M phosphate buffer, pH = 7.0, was passed through a cellulose triacetate membrane under a pressure of 0.1 atm with pore sizes of approximately 0.1 η (manufacturer's information). The membrane was briefly washed with water and immersed in a 2.5 # glutaraldehyde solution for 1 hour. It was then placed in a pressure cell and washed with water, 10 ^ N HCl (to break up the enzyme-inhibitor complex) and buffer until no UV absorption and no enzymatic activity were found in the wash water. The membrane was then tested for enzymatic activity using N-benzoyl-L-tyrosine ethyl ester (BTEE) in buffer of 30 % AtOH 70 % Tris, pH = 7.8 (0.05 M), of N-acetyl-L- tryptophan methyl ester in Tris buffer, pH = 7.8 (0.05 M) containing CaCl ₂ (0.05 M), and of N-acetyl-DL-phenylalanine ethyl ester in Tris buffer, pH = 7.3 (0 0.05 M) containing CaCl ₂ (0.05 M) as substrates. These substrates were pressed through the membrane under various pressures at a temperature of 26 ^° C., and the product formed was measured in the exiting liquids.

The membrane was then examined under the conditions of a batch procedure, i.e. with a batch of the sub-

GU y 8 2 U f 1 0 3 0

2553549

strates with turbulent stirring. The results and the ratio the reaction rates under flow conditions and conditions of a batch process, where the same conversion percentages were obtained, are given in the following tables, using the following abbreviations:

P = pressure differential across the membrane Q = flow rate V = reaction rate % = % of substrate converted into product

Approach = carrying out the enzymatic reaction under conditions a rudimentary procedure.

Reaction speed

under the conditions of a batch procedure

LO LTj O!

Table I.

Substrate: P.
atm N-benzoyl-lyrosine ethyl ester M / iain.cm Conversion
lung 5- P.
atm ccm / min-cm V 2
um / min.cm Conversion
lung Concentration: o, 5 5 ^# 1O ~ M 0.07 100 0.5 0.21 0.73 100 1.0 c demmin, cm yes 0.11 100 1 0.42 1.29 90 1.5 0.21 0.16 100 1.5 0.62 1.7 72 2 0.42 0.36 100 3 0.62 0.52 88 3 1.25 2.3 50 3.5 0.95 0.58 61 - 1.58 0.036 75 - - 0.375 50 2.23 75 6th 50 approach - Fast
employment relationship
nis 15th

Table II

Substrate

N-acetyl-L-tryptophan methyl ester

concentration

2 · 1Ο ^{5 sts}

P.
atm ccm / min.cm 13th V ρ
nM / min.cm Conversion
lung
% 0.5
1
2
5.5 0.125
0.21
0.83
1.46 0.23
0.375
1.45
2.3 100
100
100
90 approach - - 0.18 90 Fast
behavior- 90

Table III

Substrate

N-acetyl-DL-phenylalanine ethyl ester

Concentration
tion • 1-10 ⁵ sts 6th W.cm ² Conversion
lung P.
atm , ^Q 2
c cm / min, cm iiM / 0.198
0.36
0.56
0.895 50
50
50
46 0.5
1
1.5
2 0.125
0.26
0.36
0.625 0.146 46 approach - - 46 G-speedy-
keÜB ratio-
nis

⁺ only the L-ester is hydrolyzed by the enzyme

M Π 30

From the values in the tables given above, it can be seen that conversion percentages of 75%,> 50 #, 90 % and 4-6 % were achieved at a reaction rate that was 15 times, 6 times, 13 times and 6 times faster, respectively Application of the method of the invention compared to a batch process in which the same respective substrates were used in the same concentration and with the same membranes.

Example 2

This example shows the production and testing of a trypsin membrane.

A solution of trypsin (5 mg / cc) in 0.05 M CaCIp was pressed through at a pressure of 0.1 atm by a cellulose triacetate membrane with a pore size of about 0.1 η. The membrane was then washed briefly with water and then immersed for 1 h at room temperature in a 2.5% glutaraldehyde solution which was brought to pH = 7 with 0.1 M NaOH.

The membrane was then inserted into an ultrafiltration cell and washed with water and buffer (phosphate, pH = 7.0) until UV absorption and enzyme activity were no longer found in the washing water. The membrane was then tested for activity using N-Benzoylargininäthylester in Tris buffer, pH = 8.0 (0.05 M), CaCl ₂ (O, O1 M) containing and N-benzoyl-DL-lysinäthylester in Tris buffer, pH 8.0 (0.05 M), containing CaCl _p (0.01 M), examined as substrates. The substrates were sent through the membrane at 26 ^° C. under pressure, and the amount of product formed was measured in the exiting liquid. The membrane was also examined under the conditions of a batch procedure, ie with a batch of the substrate with turbulent stirring. The ratio of the velocities under flow conditions and batch processing conditions for the same degree of conversion was calculated. The results are summarized in Tables IV and V below.

• 6US82W1030

Table IV

⁺ by the enzymatic reaction
only the L-ester is hydrolyzed

cn cz cc

Substrate P.
atm N-benzoyl arginine ethyl ester 4th Conversion
lung
O/
/ 0 P.
atm 1x10 " ² M. 2.5 V ρ
nM / min.cm Conversion
lung
i concentration 0.5
1.5
2.0 3x10 "" ⁵ M. 100
92
76 0.5
1.0
1.5
2.0 ccm / min.cm 0.35
1.25
1.48
1.71 100
97
63
41 - Qo ^V P
ccm / min / cm yuM / rnin.cm 74 - 0.125
0.187
0.34
o, 62 0.63 97 0.18 0.41
0.31 0.63
0.62 1.09 75 - 97 approach 0.25 Fast
employment relationship
nis

Substrate

Table V N-Benzoyl-DL-lysine ethyl ester

concentration P.
atm 2x10 " ⁵ M. Conversion
lung atm 2x10 " ² M. 2 πΜ / min.cm Conversion
lung 0.5
1.6
2.1 QPV ρ
ccm / min / cm yuM / min.cm 50
48
43 0.4
1
1.5
2.0 c cm / min, cm 0.33
1.46
1.77
2.19 50 ι ro
46 J ^
37 <£ - 0.16 0.43
0.33 0.75
0.63 1.15 43 - 0.08
1.16
0.31
0.63 0.78 45 approach - '0.16 43 - 46 Fast
speed relationship 7th

5 5 3 6

Example 5

This example shows the production and testing of a carboxypeptidase B membrane.

A solution of carboxypeptidase-B (3.3 mg / ccm) in LiCl (10 wt .- / vol .- #) was under pressure (0.1 atm) through a microfiltration membrane (product Millipore MF-GS) with a pore size of 0.22 Hm sent through. The membrane was washed briefly and immersed in a crosslinking solution of glutaraldehyde (2.5 #, pH = 7.0) for 1 hour at room temperature. The membrane was then placed in an ultrafiltration cell and washed with V / ater and veronal buffer (0.05 M, pH = 7.0) until no UV absorption and no enzyme activity were found in the Y / ash water. The membrane was then tested for activity under flow and batch conditions as in the previous examples, and the rate ratios were calculated. The substrate used was
N-Hippurilarginine in Tris buffer, pH = 7.6 (0.03 M), the
NaCl (0.1 M). The results are shown in Table VI below.

Table VI

Substrate P.
atm N-Hippuril-L-arginine 1X1O "% 8th Conversion
lung concentration 0.3
1.5 2 ^V 2
yuM / min.cm 100
56 ccn / min.cm 0.31
1.04- 56 0.1
1.88 0.13 56 approach Speed
Relationship

GU 9 8 2 L / 1 Π 3 0

2 5 5 3 6 Λ 9

Example 4

This example shows the manufacture and investigation of a Catalase membrane.

A solution of catalase (3 mg / ccm) in phosphate buffer, pH = 7.0 (0.05 M), which contained 1% NaCl, was passed through a microfiltration membrane with a pore size of 0.22 µm (product Millipore MP-GS) sent through. The membrane was washed briefly with water and ^{immersed in a glutaraldehyde solution (2.5 0} P) in phosphate buffer 9 (pH = 7.0), (0.05 M) for two hours. The membrane was then washed under pressure in an ultrafiltration cell with v / ater and 10 % NaCl until no UV absorption and no enzymatic activity were found in the wash water. The membrane was then examined for its enzymatic activity with a solution of HpO ₂ (0.035 M) in phosphate buffer (0.05 M), pH = 7.0, under flow conditions and under conditions of a batch procedure as described in Example 1. The ratio of the velocities under flow conditions and batch processing conditions for the same conversion was calculated.

The results are shown in Table VII below.

Table VII

Substrate P.
atm ccm / mxn.cm H ₂ O ₂ 10 Conversion
lung
% concentration 0.5
2 0.37
2.7 0.035 M. 98
55 - V ρ
yuM / min.cm 55 - - - 10.5
55.4- 55 approach 5.5 Speed
Acceptance ratio

25 5 3 6

Example 5

This example shows the production and testing of a glucose oxidase membrane.

A solution of glucose oxidase (5 mg / cc) in 0.05 M phosphate buffer with pH = 6, which contained 1 ρ glucose, was in the Pores of a Millipore membrane with a pore size of 0.45 ^ m (Product HA-MP) pressed. The membrane was then immersed in a glutaraldehyde solution (2.5 #) for 2 hours at room temperature. It was then washed with v / ater and a phosphate buffer of pH 6.8 until no UV absorption and none more enzymatic activity was found in the wash water.

The membrane was examined by using a 5 # solution of glucose flow through them in a phosphate buffer of pH = 6.0 (0.05 M) and the amount of HpOp formed in the effluent liquid was measured. The results were compared with those for the same reaction under normal operating conditions. the Results are summarized in Table VIII below.

Table VIII

Substrate

Glucose

concentration P.
atm ccm / min. 4th
1
3 2, cm 6x10 ¹ st Conversion
lung 0.5
2
4th 4,
22
59, V ρ
liM / min.cm 0.01 25th 0.016
0.03
0.05 0.01 approach 0.002 0.01 Speed
keits ^ rarhälinis

6 U-y 824/1030

Example 6

This example shows the production and testing of a urease membrane.

A solution of urease (5 mg / ccm) in 0.02 M phosphate buffer of pH = 7, polyglot 10 ~ * M EDTA contained, was in a membrane (Gelman-Metrcel TGM 200 product) pressed with a pore size of 0.2 µm. The membrane was then filled with water and Phosphate buffer washed until no UV absorption and none enzymatic activity was found more in the wash water.

The membrane was then tested for its activity with a 0.3 solution of urea in a phosphate buffer, pH = 7.0 (0.1 M), which contained EDTA (10 ~ ^ M), under flow conditions and conditions of a batch procedure, as described in Example 1, investigated.
The results are shown in Table IX below.

Table IX

Substrate P.
atm urea 4th Conversion
lung concentration 0.05
0.5 5x10 " ² st 5
0.5 Q ₂ V ρ
c cm / min.cm uM / min.cm vji 0.04 0.02
0.42 0.02 5 approach 0.005 Fast
ability
relationship

6U982 4/1030

Example 7

This example shows the production and investigation of chymotrypsin membranes with different pore sizes.

A chymotrypsin solution containing ß-phenylproprionate as an inhibitor contained (3> 3 mg chymotrypsin and 7 mg inhibitor / ccm) in 0.05 M phosphate buffer of pH = 7.0, was carried out under pressure Millipore membrane with different pore sizes sent through:

1) O, O25yum (VS-MF-Millipore)

2) 0.1 ma (VC-MF-Millipore)

3) 0.22 µm (GS-MF-Millipore)

The pressure differentials used to pass the enzyme solution and load the membrane were about 0.4 atm, 0.1 atm and 0.02 atm, respectively. The membranes were washed briefly and immersed in a glutaraldehyde solution (2.5 #) in 0.05 M phosphate buffer of pH 7 _{5 O.} After 2 hours, the membranes were placed in a pressure cell and washed with 10 ^ M HCl to break up the enzyme-inhibitor complex and with buffer until no UV absorption and no enzymatic activity were found in the wash water. The membranes were then tested for enzymatic activity ^ by a solution of N-benzoyl-tyrosinäthylester (2.5 x 1O ^ M) under pressure in the buffer of 30 f & EtOH - 70% Tris-buffer of pH = 7.8 ( 0.05 M) and the amount of product formed in the outflowing liquid was measured. The membranes were also examined under "batch processing conditions, ie with a batch of the substrate with turbulent stirring, the ratio of the velocities under flow conditions and batch processing conditions then being calculated for the same conversion. The results are shown in the following" ^ abelle X compiled.

1030

Table X

Substrate

2.5x10 M benzoyl tyrosine ethyl ester

Pores
size 0 approach P. O, 025 / in 16 ι Umw.
% 0 P. 0.1 around Umw.
% 0 P. 0.22 / in V etc.
% 1 Speed
dignity
relationship , 5 Q V 87 0 , 4 Q V 37 0 , 2 Q 2.33 30th 2 , 5 0.13 0.29 92 1 , 75 0.29 0.63 89 0 , 3 1,4-3 3.46 66 3 , 5 0.33 0.78 80 2 , 5 0.53 1.19 88 1 , 5 2.8 4.47 56 8th ,8th 0.55 1.14 80 3 , 5 1.06 2.36 85 , 0 3.17 9.39 60 -16 0.83 1.72 80 , 6 1.97 3.62 80 6.06 1.00 2.2 70 2.7 5.55 2.0 4.0 80 30th 0.17 80 0.14 80 0.18 80 30th 31 17th

Example 8

This example shows the production and testing of chymotrypsin membranes made of non-cellulosic materials.

A chymotrypsin solution containing ß-phenylproprionate (3 ^ g chymotrypsin and 7 W> inhibitor / ccm) in 0.05 ^M phosphate buffer of pH 7.0 was passed through membranes made from the following non-cellulosic materials:

a) inert, synthetic, non-cellulose-like polymer - Diaflo ultrafiltration membrane XM 300 (MW = limit value 300,000, product of Amicon) and

b) Polycarbonate nucleopore membrane with a pore size of 0.4yum and a thickness of 11 / µm.

The membranes were briefly washed and placed in a glutaraldehyde solution (2.5 #) immersed for 2 h. They were then washed and with BTEE (2.5 x 10 "~ * M) as described in Example 1, examined .

2 5 5 3 R 4 9

The results are shown in Table XI below

Table XI

Substrate P. approach N-benzoyl-tyros 4th Uraw. in ethyl ester (2.5 x 10 " ³ M) Membrane-
Type 0.1
0.2 Speed
dignity
relationship Polycarbonate
Nucleopore
membrane 36
24 Membrane off
synthetic,
loose-like inert,
non-cellu-
Polymer Q V 36 PQV 0.3 0.29
0.93 P \ 45 36 2.0 0.22 0.55
3.0 0.36 0.87 0.07 0.07 12.4 Umv /. 100
98 98 98

Of course, the method according to the invention is not limited to the details of the embodiments described above by way of example limited.

- claims

Claims (16)

Claims 1 «Procedure for carrying out enzymatic reactions, characterized in that pressure is applied to an aqueous solution of a substrate, this being chemically can be changed by an enzymatic ^ reaction, whereby the flow of the solution under a Pressure differential caused by a cross-linked enzyme membrane is, and that the solution products obtained are removed by methods known per se, the cross-linked enzyme membrane is a microporous membrane, in whose pores an enzyme by means of a bifunctional coupling agent has been networked. 2. The method according to claim 1, characterized in that that a pressure differential in the range of about 0.05 until about 10 atm is applied. 3 · Method according to claim 2, characterized in that it is marked e t that a pressure differential between about 0.05 and 5 is created. 4-. Method according to claim 1, characterized in that that the pore size of the membrane is between about 0.1 and 10 microns. 5. The method according to claim 1, characterized in that that the pore size of the membrane is between about 0.025 and 0.5 microns. 6. The method according to claim 1, characterized in calibration n et that the enzymatic ^ reaction is a hydrolytic reac tion is. 6U9824M030 7. The method according to claim 1, characterized in that that the hydrolytic reaction is used for the resolution of racemic mixtures of IT or 0-acylamino acids for which the mixture is pressed through the appropriate enzyme membrane, where an isomer is hydrolyzed and a mixture an optically pure amino acid and the acylamino acid des unhydrolyzed isomers is obtained, which then differ from each other be separated according to methods known per se. 8. The method according to claim 1, characterized n et that the enzymatic reaction is an oxidation reaction is. 9. The method according to claim 1, characterized in that that chymotrypsin or trypsin is used as the enzyme. 10. The method according to claim 1, characterized in that that the crosslinked enzyme membrane is produced by forcing an enzyme solution through the pores of a membrane and then the membrane loaded with enzyme is immersed in a solution of a bifunctional crosslinking agent will. 11. The method according to claim 1, characterized in that a membrane made of cellulose acetate, Cellulose triacetate, cellulose nitrate, mixed ester cellulose, nylon or polyelectrolyte complexes is used. 12. The method according to claim 1, characterized in that that as a crosslinking agent a dialdehyde, diazobenzidine, hexamethylene diisocyanate, 1,5-difluoro-2,4-dinitrobenzene or ^ N'-hexamethylene bisoodacetamide is used. 13. The method according to claim 12, characterized in that that glutaraldehyde is used as a crosslinking agent. 14-. Method according to claim 10, characterized in that net that the enzyme solution is a reversible inhibitor or a substrate which does not react with the crosslinking agent contains. 15. The method according to claim 10, characterized in that e t that the enzyme solution still contains an inactive protein. 16. The method according to claim 10, characterized in that the enzyme solution has a concentration of 1 to 10 mg / ecm owns. «247 10 30