WO1979000875A1

WO1979000875A1 - Method and means for carrying out an enzymatic process

Info

Publication number: WO1979000875A1
Application number: PCT/SE1979/000081
Authority: WO
Inventors: K Martensson; R Axen
Original assignee: Svenska Sockerfabriks Ab; K Martensson; R Axen
Priority date: 1978-04-03
Filing date: 1979-04-02
Publication date: 1979-11-01
Also published as: GB2043076A; FR2421909B1; FR2421909A1; SE416314B; SE7803732L; JPS55500188A; GB2043076B; DK511779A

Abstract

A method for carrying out an anzymatic process and a soluble enzyme complex for use in this method. The method resides in a new technique for providing modified characteristics of enzymes and is characterized by being performed with an enzyme which is in the form of a soluble complex, wherein the enzyme is bound to a substance including at least one (1) hydrophobic part for giving the binding and at least one (1) functional part for modifying the kinetic productivity characteristics of the enzymes. The soluble enzyme complex is in a corresponding manner characterized by including an enzyme which is bound to a substance of the type indicated above.

Description

TITLE OF INVENTION

METHOD AND MEANS FOR CARRYING OUT AN ENZYMATIC PROCESS.

The present invention relates to a method for carrying out an enzymatic process and the invention also includes a soluble enzyme complex for use in this method. Enzymes are to-day prepared and utilized industrially in several different areas. Within the foodstuff area many processes, in inter alia the starch, dairy, bakery and brewery industries, are based on enzymes, but also outside these areas the enzyme technology finds use, for example in the washing agent, tannery and pharmaceutical industries. The enzymes are also utilized to a large extent for analytical purposes in biochemical or clinical laboratories and to some extent also for medical applications. During the last fifteen years a nearly explosive development has taken place within the area of immobilized enzymes, i.e. enzymes which chemically or physically have been attached to insoluble carriers, and this technique has resulted in new interesting applications within the above-mentioned areas. All enzymes heretofore known are, as to their structure, proteins having a relatively high molecular weight of up to about 1000 000. The protein structure is arranged in such a manner spacewise that the amino acids involved in the catalytic activity come into close contact with each other and can form the so-called active center. The remainder, usually the major part of the enzyme protein, is stabilized in its space structure inter alia by electrostatic bonds, hydrogen bonds, van der Waal's forces and hydrophobic interaction. The latter phenomenon means that certain hydrophobous (water-repellant) parts of the protein structure cooperate and are localized adjacent to each other, so that the enzyme molecule will consist of regions of more or less pronounced hydrophobic or hydrophilic (water-compatible) characteristics.

The characterizing features of an enzyme, such as solubility, stability, catalytic activity, pH- and temperature dependency, antigenic characteristics, cofactor dependence etc., are dependent not only from the amino acids present in the active center but also from the amino acid sequence as a whole and the space configuration of the protein structure, which in turn has to do with the hydrophobic/hydrophilic balance, the charge distribution etc. This has for a result that all parts of the enzyme molecule to a larger or smaller extent effect the characteristics of the enzyme. In a corresponding manner a change in structure, for example by introducing a substance, results in a more or less pronounced change on the characteristics of the enzyme.

Limitations in the characteristics of an enzyme often impart limitation to its use. In the practical utilization of enzymes, both such that are used to-day and those having potential usefulness but from different reasons are not used at present, it is therefore often desirable to change the characteristics of the enzyme in some desired direction. As an example of such changes there may be mentioned improvement of the stability per- forms of an enzyme, the displacement of the pH-optimum of an enzyme towards higher or lower pH-values, changing the solubility characteristics, introducing functional groups that may be used in immobilization of an enzyme, increasing the catalytic activity, changing its antigenic characteristics. It is therefore no doubt that a sufficiently simple technique for effecting the function of the enzymes in a simple manner would constitute important advantages.

The most decisive characteristics of the enzyme with regard to its practical utilization may be summarized under the term kinetic productivity characteristics. This means such characteristics which directly express the catalytic ability of the enzyme in its use as a catalyst, which in reality is a function of basic characteristics of the total enzyme system, for example substrate concentration, inhibitory concentration, enzyme quantity, temperature, pH etc., and are expressed by such traditional parameters as pH-optimum, heat stability, operational stability, Michaeli's constant (K_m), inhibitory constant (K_i), maximum reaction rate (V_max) etc., as distinguished from other characteristics of the enzyme which do not affect its catalytic function in its practical use. To the latter characteristics belong such which either may be considered as general molecular, such as molecular weight solubility, available reactive or adsorptive groups at the enzyme surface, or such biochemical characteristics which have nothing to do with catalysis, such as for example antigeneicity, or characteristics which are expressed by kinetic terms but not have anything to do with the process use, such as for instance storage stability. By the concept kinetic productivity characteristics are not either meant such physical parameters in the reaction vessel as stirring rate, reactor vessel dimensions etc.

By displacing the pH-optimum of an enzyme to a value most suited to the process, the productivity of the enzyme may be increased, i.e. the quantity of product formed per unit of time is increased afe a constant enzyme consumption, which means possibilities of significant saving of time and thereby also economic advantages. A lowering of the K_m of the enzyme vis-à-vis the substrate in question, an increase of K_i vis-à-vis inhibitors present in the process and an increase of Vmax does, of course, effect in a corresponding manner the productivity in a favourable direction accompanied by essential advantages thereof. The improvement of the heat stability of an enzyme means that a higher process temperature may be used, V_max for the enzyme being increased, at the same time as other advantages may be obtained, for exampled reduced risk for microbiological contamination, precipitations etc. The operational stability of an enzyme signifies the catalytic activity of the enzyme as a function of time when used in a process, from which follows that increased operational stability results in production of a larger amount of product from a certain quantity of enzyme, which clearly is of utmost importance.

Some attempts to change the characteristics of an enzyme are already described in literature. It is, for example, well known that by covalently binding a soluble dextrane, polyethylene glycol or the like activated with BrCN or HIO₄, in certain cases one may for example obtain improved stability, activity or solubility characteristics. In the same way microenvironmental effects are known which arise in immobilization of enzymes to different carrier materials. In the latter case one has, for example, been able to observe displaced pH-optima in immobilization to electrostatically charged polymer materials, as well as changed stability, activity, solubility characteristics, kinetic parameters etc.

However, the present invention is based on a totally different technique to provide for change in characteristics of enzymes. The technique of this invention is based on binding the enzyme to a chemical substance containing at least one hydrophσbous part and at least one functional part, by using the hydrophobic interaction between the enzyme and the substance in question, whereby the desired change in the kinetic productivity characteristics of the enzyme is obtained. The actual substance is hereby brought into contact with the enzyme in a polar solvent under suitable conditions, the hydrophobic parts of the substance and the hydrophobic regions of the enzy approaching each other and cooperating to the formation of a highly stable enzyme complex. The functional part of the substance is not primarily intended for providing bonds to the enzyme and may, therefore, be fully or partly utilized for the intended purpose, i.e. to effect the characteristics of the enzyme in direction to improved productivity kinetics.

The suggested technique offers several advantages. It is generally useful and may be used for the introduction of most functional groups on enzymes possessing different types of catalytic activity. The technique is, moreover, mild to the enzyme, since it specifically is directed towards utilization of the hydrophobic structure of the protein which often are not directly involved in the catalytic activity. The technique is very simple and thereby also cheap but still sufficiently effective to provide for a sufficiently strong binding of the substance to the enzyme. The technique is also easily applied in practice in view of the fact that several useful substances with different types of functional groups are commercially available.

Thus, the present invention relates to providing a soluble enzyme complex for use in enzymetically catalyzed processes. In this enzyme complex the enzyme is bound to a substance containing at least a hydrophobous part for providing the binding and at least a functional part for modifying the kinetic productivity characteristics of the enzyme. In the present context the term "hydrophobic part" of the substance means a structure which has the ability of forming a practically useful stable complex together with the enzyme in question. The hydrophobic part should, in order that the desired effectivity in the hydrophobic interaction with the enzyme protein shall be obtained but still a reasonably mild binding with regard to the enzyme, contained at least one structural element of a straight or branched, saturated or unsaturated alkyl or cycloalkyl chain having between 6 and 12 carbon atoms. Preferably a straight saturated alkyl chain having 8 carbon atoms is used. The functional part of the substance may be any structure which has the ability of imparting to the enzyme complex such characteristics which result in modification or changing the original productivity kinetic of the free enzyme. The functional part of the substance has, in this invention, an operational character and must therefore in this manner be given an operational definition in contrast with the above structural description of the hydrophobic part of the substance. Examples of different basic structures of the functional part resulting in a certain type of change of the kinetic productivity characteristics are further described under the separate sections below, which deal with displacement of pH-optimum, improvement of the stability characteristics, changes in catalytic activity etc.

It is known that hydrophobic interaction between surface-active substances and proteins may take place under certain conditions while forming a complex, wherein the ratios of surface-active substance to protein, the structure of and fehe concentration of the former, the ion strength in the solution, the temperature etc. determine the extent of binding of the surface-active substance, the conformation of the protein in the complex etc. At a relatively small supply of surface-active agent to certain proteins with hydrophobic surface structures formation of specific complexes may take place, wherein often also ion binding effects contribute. This complex formation is rather an exception than a rule for proteins in general. When increasing the amount of surface-active substance there is obtained, with regard to most substances and also in principle with all proteins, at a certain concentration a cooperative binding, where the final complex will consist of a relatively large amount of substance bound to the protein in question. This type of bond is general and applicable to all proteins in principle. Mainly with regard to non-ionic surface-active substances in certain cases a relatively large amount of substance may be part of the complex without the binding being cooperative. If the concentration of surface-active substance is further increased the critical micell formation concentration (CMC) will be obtained, where micell formation of the surface- -active substance competes with the binding of the substance to the protein in question.

The invention also provides for a process for the preparation of the enzyme complex described above, and in this process the enzyme in question is brought together with a chemical substance including hydrophobic and functional parts in a polar solvent to the formation of the desired enzyme complex. In the preparation the concentration range should be selected in such a way that a sufficiently great amount of substance is bound to the enzyme so that its kinetic productivity characteristics will be changed to the desired degree but at the same time micell formation of the substance will be avoided. Micell formation is often no hindrance to the formation of the complex but results in an unnecessary waste of substance which cannot be utilized for the intended purpose. Preferably, the concentration of substance should therefore lie above the limit for cooperative binding if such can take place but below CMC. Initiation of complex formation may in certain cases take place by smooth heating of the reaction mixture, alternatively by adding a minor quantity of denaturing substance, for example urea. The enzyme complex formed may either be used directly in the solution obtained or may if desired be isolated and recovered.

Enzymes may depending on the type of reaction to be catalyzed be systematically divided-up into the following six main groups: Oxidoreductasae, hydrolasae, isomerasae, transferasae, lyasae and ligasae. Since the hydrophobic complex formation described in connection with this invention is general and thus is applicable to in principle all proteins it is also generally useful to modify the kinetic productivity characteristics of all enzymes. The types of enzymes utilized in practical use may, however, almost exclusively be systematically found in the groups oxidoreductasae, hydrolasae or isomerasae, in view of which the invention is considered to have the highest value for enzymes belonging to any of said groups. Below there are briefly given some different applications of the technique of this invention which, however, must not be considered as limiting. These applications have only for a purpose to somewhat more specifically illustrate the usefulness of the invention. Displacement of the pH-optimum of an enzyme. The changing of pH-optimums of enzymes is of a great technical interest in view of the fact that this enables for example simultaneous carrying through of several enzyme reactions with the advantages this means in the form of saving of time, advantageous enzyme kinetic with a lower energy consumption etc., or in order that one can carry out an enzymatic process at a pH-value where the risk of microbial infection is lower than where competing reactions are less pronounced. By introducing in complex form into the immediate environment of the enzyme anionic groups there occurs attraction of hydrogen ions from the surrounding medium at a low ionic strength, in view of which a local hydrogen ion excess is obtained in the micro environment where the enzyme operates. An apparent increase of the pH-optimum of the enzyme may then be measured in the outer solution where substrate and reaction products are present. In a corresponding manner a lowering of the pH-optimum may be obtained by introduction of cationic groups.

By using this technique the modification may easily be carried out by treating, in the former case, the enzyme with for example sodium octyl sulphate, caproic acid sodium salt, potassium hexyl sulphonate or the like, whereas in the latter case for example decyltrimethyl ammonium bromide, diethylhexylamine, octylammonium chloride or the like may be used. The hydrophobic part of the substance used is selected in view of inter alia necessary binding strength, enzyme stability etc., whereas the functional part, i.e. the anionic or cationic group, together with concentration conditions and the like in the treatment are decisive for the change of the net charge of the enzyme obtained and thereby the degree of pH-displace ment resulting from the change in micro environment.

The principle described above may be used also to displace the isoelectric point of an enzyme and to change kinetic parameters, such as Michaelis constant, inhibitor constant etc. in view of a corresponding change of affinity towards electrostatically charged substrates, inhibitors etc.

Improvement of the stability characteristics of an enzyme In the practical use of enzymes, particularly immobilized enzymes, the operational stability is nearly always the decisive factor which determines whether a economically certain enzyme may be technically and/useful in a certain process. Enzymes may be destroyed when used as a result of different mechanisms, for example thermodenaturing, inhibition from certain substrate components, as heavy metals etc., decomposition as a result of microbiological attacks etc. It is known that certain substances, frequently of a carbohydrate nature, sometimes have a stabilizing effect on the enzyme with regard to thermodenaturing, such as dextrane, starch, lactose, saccharose, polyethylene glycol etc. In the same way it is known that protein-protein interaction when adding an external protein to an enzyme in solution often has a stabilizing effect on the enzyme structure. By means of different complexing agents, such as ethylenediamine tetraacetic acid (EDTA) or compounds containing sulphhydryl groups, such as cystein, glutation etc., one may stabilize an enzyme towards inhibition in view of different heavy metals. Different bactericides, such as antibiotics, sodium azide etc., may, in the corresponding manner, decrease the risk of bacterial attack on the enzyme thereby providing a stabilizing effect on the catalytic ability of the enzyme.

While using the technique of this invention stabilizing agents of for example the above mentioned types may be introduced in the enzyme structure by means of hydrophobic interaction. The functional part of the substance used is thereby for example any of the above-mentioned structures, whereas the hydrophobic part in accordance with this invention consists of a structure in accordance with what has been previously described which is chemically bound to the functional part in question. Examples of substances that may be used to improve the stability characteristics of enzymes in accordance with this invention are octyl dextrane, hexyl saccharose, octyl polyethylene glycol, decyl glutation, hexylthiol, octyl-EDTA, phenyl cystein etc. Change in the catalytic activity of an enzyme.

The catalytic activity of an enzyme is, of course, the most central factor among the characteristics possessed by the enzyme, and the practical value of an increase of the catalytic ability is easily understood. In special cases a change of the affinity of an enzyme towards different substrates may also be desirable. General methods of providing a directed change in the form of for example an increase in activity cannot be formulated, but suitable measures must be judged from case to case and adapted to each single enzyme in dependence on the substrate in question, possible inhibitors etc. However, different measures of the type described under the applications mentioned above may in different ways change the activity and the specificity of an enzyme in view of change in charge distribution, solubility, surface structure etc. In this manner an enzyme may, for example, be protected against macromolecular inhibitor to advantage for a low molecular substrate by the fact that the functional part as stated above consists of a polymer which sterically blocks the surface of the enzyme from the attack of the inhibitor. The introduction of a compound, the functional part of which is electrostatically charged, may in a corresponding manner attract the substrate of opposite charge, so that the activity against this substrate will be selectively increased. If the functional part introduced is strongly hydrophilic the water-solubility of the enzyme may be increased and thereby also the affinity towards particularly hydrophilic substrates. By using the technique of this invention there is obtained a practically useful means for providing this change and the invention thereby constitutes an essential enrichment of the technique in question.

The invention will in the following be described further by specific examples, example 1 illustrating the preparation of chemical substances useful in the invention. EXAMPLE 1.

Preparation of hexyl and decyl dextrane. Dextrane (T 10 and T 500, respectively, from Pharmacia) (200 mg) is dissolved in 2 M NaOH (20 ml), 1-bromohexane (1.0 ml) and 1-bromodecane (1.0 ml), respectively, being then added. The mixture is vigorously shaken at 70°C for 12 hours and then neutralized with 2 M HC1. After short centrifugation the organic phase is separated-. The aqueous phase is dialyzed against distilled water for two days at room temperature, and the solution is then freeze-dried and hexyl dextrane and decyl dextrane, respectively, are recovered. EXAMPLE 2.

Improvement of the operational stability of xylose isomerase immobilized to DEAE-cellulose.

To a solution of xylose isomerase in 0.02 M tris-HCl-buffer (pH 8.5) (10 ml; 20 mg/ml) is added hexyl dextrane (T500) (1.0 g), the solution being than shaken a room temperature for three hours. The enzyme-dextrane complex formed is then adsorbed onto DEAE-cellulose (What- man DE-52) (1.0 g) by adding the cellulose to the enzyme solution obtained according to the above under careful mixing which is maintained for 2 hours at room temperature. The immobilized enzyme dextrane complex hereby obtained is then packed in a column (∅ 9 mm) and then a substrate solution of glucose in 0.02 M tris-HCl-buffer (pH 8.5) (40 % weight/weight), which is 0.4 mM with regard to M_gSO₄. 7H₂0, is continuously pumped through the column at 60°C. The flow through the column is continuously controlled in such a manner that the conversion degree with regard to a fructose formed is kept constant at 44 %. The immobilized enzyme dextrane complex hereby shows a half time of 30 days with a total productivity of 18.7 g of fructose per mg of enzyme calculated at the ratio between quantity of fructose formed and quantity of utilized enzyme until the enzyme shows 25 % of its initial activity. In a reference experiment it is found that the corresponding figures for non-

-hexyldextrane treated enzyme is 8 days and 4.3 g fructose per mg enzyme, respectively. EXAMPLE 3.

Improvement of the heat stability of a pullulanase. To a solution of pullulanase in 0.05 M phosphate buffer (pH 7.0) (2.0 ml; 5.0 mg/ml) there is added decyl dextrane (TI0) (100 mg), and the solution is then shaken at room temperature for 17 hours. The enzyme dextrane complex formed is isolated by gel filtration (Sephadex G 200) in the above-mentioned buffer solution. The eluate is diluted to exactly 10 ml, and the enzyme-dextrane complex solution is then heated in a boiling water bath for 5 minutes. The solution is allowed to stand at room temperature for 30 minutes and the remaining enzyme activity is then determined. A reference solution of untreated enzyme is treated in the same manner. Before the heat treatment the enzyme-dextrane complex shows an activity corresponding to 84 % of the originally added enzyme quantity, whereas 95 % of this remaining quantity remains after the heat treatment. The remaining activity of the reference sample after corresponding treatment is only 17 % . EXAMPLE 4.

Displacement of the pH-optimum of amyloglucosidase and xyloisomerase. To a solution of amyloglucosidase in 0.1 M phosphate-citrate buffer (pH 4.5) (5.0 ml; 8 mg/ml) which is 0.5 M with regard to NaCl there is added a solution of sodium octyl sulphonate i distilled water (1.0 ml; 40 mg/ml), and the mixture is then allowed to stand under stirring at room temperature for 17 hours. In a corresponding manner a solution of xylose isomerase in 0.1 M tris-HCl-buffer (pH 8.5) (5 ml; 8 mg/ml) which is 0.5 M with regard to NaCl is treated with a solution of octyl trimethyl ammonium bromide in distilled water (1.0 ml; 40 mg/ml). The enzyme preparations treated in this manner are freed from excess of surface-active agents and are desalted by gel filtration (Sephadex G 50) in a 0.05 M phosphate buffer, pH 6.5. The amyloglucosidase preparation treated in this manner shows a pH-optimum at 6.1 as compared to 4.5 for the non-treated preparation. The xylose isomerase, the optimum of which towards the native enzyme lies at pH 8.5_* shows in a corresponding manner after the treatment optimum activity at pH 7.1.

A 4 % starch solution is prepared by suspending native potato starch (20 g) in distilled water (500 ml), the suspension being then boiled under vigorous stirring for 30 minutes. The starch solution is rapidly cooled to 60°C, is made 0.4 mM with regard to M_gSO . 7 H₂0 and to the solution is immediately added enzyme solutions treated as described above in 0.05 M phosphate buffer (pH 6.5) (10 ml) of each, and a solution of α-amylase in the same buffer (10 ml; 2 mg/ml). The mixture is incubated at 60°c and pH 6.5 under stirring and fructose formed as a function of time is measured by regular analyses. A reference sample prepared in the same manner but with untreated enzyme preparations is incubated and analyzed in parallel. The incubation is maintained until the yield of fructose based on starting starch is 30 %, which in the former case (by treated enzyme) takes 6.5 hours, whereas the corresponding time in the latter case (untreated enzyme) is 18 hours. EXAMPLE 5.

Increase of K_i for trypsine towards the trypsine inhibitor in soja bean.

To a solution of trypsine in 0.05 M phosphate buffer (pH 7.6) (2.0 ml; 5 mg/ml) there is added decyl polyethylene glycol (50 mg), the mixture being then shaken for 20 minutes at 4°C. The inhibitor constant K_i towards the trypsine inhibitor in soja bean is then determined for the decylpolyethylene glycoltreated enzyme by determining the enzyme activity towards N-bensoyl-arginine-ethylester (BAEE) in the presence of different inhibitor concentrations and is compared with a reference sample which under the corresponding conditions has been treated with non-modified polyethylene glycol. K. is found in the former case to be >50 mg/ml, whereas the corresponding value in the latter case is 2.2 mg/ml. EXAMPLE 6.

Lowering of K_m for trypsine towards BAEE.

To a solution of trypsine in 0.05 M phosphate buffer (pH 7.6) (2.0 ml; 50 mg/ml) 0.5 M with regard to NaCl, there is added a solution of sodium decyl sulphonate in distilled water (4.0 ml; 25 mg/ml), the mixture being then shaken for 5 minutes at 4°C. The enzyme is then freed from non-bound sodium decyl sulphonate and salts by gel filtration (Sephadex G 50). The activity of the enzyme treated in this manner at different concentrations of

N-bensoyl-arginine-ethyl ester (BAEE) is then determined, from which K_m BAEE may be calculated. K_m BAEE is found to be 0.52 mM whereas the corresponding value for the non-treated enzyme is 4.3 mM.

Claims

CLAIMS :

1. A method for carrying out an enzymatic process, characterized thereby, that it is performed using an enzyme which is in the form of a soluble complex, wherein the enzyme is bound to a substance including at least one (1) hydrophobic part and at least one (1) functional part for modifying the kinetic productivity characteristics of the enzyme.

2. A method according to claim 1, characterized in that it is performed while using the soluble enzyme complex immobilized to an insoluble carrier.

3. A method according to claim 1 or 2, characterized in that the modification resides in displacement of the pH-optimum of the enzyme.

4. A method according to claim 1 or 2, characterized in that the modification resides in changed values of K_m, K_χ or V_maχ,

5. A method according to claim 1 or 2, characterized in that the modification resides in changed stability characteristics of the enzyme.

6. A soluble enzyme complex for use in the method according to claim 1, characterized by including an enzyme which is bound to a substance containing at least one (1) hydrophobic part and at least one (1) functional part for modifying the kinetic productivity characteristics of the enzyme.