DE2528617A1 - PROCESS FOR IMMOBILIZING ENZYMES, THE PRODUCTS RECEIVED THEREOF AND THEIR USE - Google Patents

Description

PATENT ADVOCATES A. GRÜNECKER

-DIPL.-ING.

H. KINKELDEY

DR.-INO.

W. STOCKMAlR

DR.-ING. ■ AaE (CALTECH)

K. SCHUMANN

DR. RER. NAT. · DIPL.-PHYS.

P. H. JAKOB

DIPL.-INQ.

G. BEZOLD

DR. RER. NAT. · DIPU-CHEM.

MUNICH

E. K. WEIL

DR. RER. OEC. INQ.

LINDAU

MUNICH 22

MAXIMILIANSTRASSE 43

June 25, 1975
P 9330

Research Corp.

Lexington Avenue

Hew York, Ii.Y. 10017, USA

Process for immobilizing enzymes obtained thereby Products and their uses

The invention relates to a method for physically immobilizing (immobilizing) or holding Enzymes within the pores of microporous carriers to isolate the enzymes or use the immobilized ones Enzymes in continuous catalytic processes to control substrate conversion and the like.

The enzyme technology is very promising for the future, the isolation of the enzymes was according to the previously known methods however difficult and costly. The use of enzymes as heterogeneous specific catalysts in the

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TELEPHONE (089) 355962 TELEX O5-2933O TELEGRAMS MONAP

Research and industry is increasing and becoming increasingly important. It is known that enzymes for their effective use can be artificially immobilized (immobile) by various methods: (1) by adsorption on inert carriers or synthetic ion exchange resins,

(2) by inclusion in Gelgitteui whose pores are too small, to allow the trapped protein to escape,

(3) by covalently attaching the enzyme to a suitable water-insoluble carrier via functional groups that are responsible for the biological activity of the enzyme is not essential, (4) by covalently attaching the enzyme to an appropriate substrate using a suitable bifunctional reagent, (5) by adsorbing the enzyme on a suitable carrier material and then intermolecular crosslinking of the enzyme. These methods generally result in a large decrease in the activity of the enzyme, in many cases by more than half the activity that the enzyme has in solution.

US Pat. No. 3,576,760 describes the inclusion of enzymes in water-soluble hydroxyalkyl acrylate or methacrylate polymers. In US Pat. No. 3,766,013, a polytetrafluoroethylene matrix is described, within which a carrier insoluble in serum is dispersed with a catalytically active enzyme chemically bound to the carrier. British Patent 953,414 states that natural or synthetic polymeric materials or lipids may contain embedded enzymes, the pores of the polymer or lipoid being impermeable to the embedded enzyme.

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Sine overview of the manufacture of different types of immobilized enzyme systems and their uses is in "Biochemical Aspects of Reactions an Solid Supports", edited by George R. Stark, Academic Press, 1971 (Chapter 1, "Water-Insoluble Enzyme Derivatives and Artificial Enzyme Membranes ").

It is known that polyelectrolytes have their hydrodynamic properties in solution Change volume when, for example, a change in the environmental conditions in which the polyelectrolyte occurs, and that this change of the hydrodynamic The volume can be used to remove the polyelectrolyte molecule to physically retain (entrap) in the micropores of microporous solids, such as for example in the article by Allan et al, "Physical Entrapment of Polyelectrolytes within Microporous Solids: The Jack-in-the-Box-Effect ", Nature, Volume 225, No. 5228, Pp. 175-176 (Jan. 10, 1970).

The main object of the present invention is to provide an improved method for physically immobilizing (immobilizing) enzymes within the pores of a preformed microporous carrier in which the chemical structure of the enzyme is not altered and the enzyme does not suffer any loss of activity. Other objectives are to provide a immobilis5-ertes enzyme system in which the enzyme undergoes little or no enzymatic activity loss, and a method for isolating and concentrating of known and unknown enzymes from natural sources rnzugeben _o

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The invention relates to a method for the physical immobilization (immobilization) of a catalytically active Enzyme within the pores of a water-insoluble, pre-formed, microporous carrier without affecting the chemical structure of the enzyme is changed and this has little or no loss of enzymatic Activity, which is characterized by the fact that one

1) decreases the hydrodynamic volume of the enzyme by adjusting its environment,

2) a microporous support with micropores, their volume is equal to or greater than the reduced hydrodynamic volume, of the enzyme, so that it brings the enzyme into contact can freely enter the pores of the carrier, and

3) the environment of the enzyme, while in contact with the microporous support, readjusts to its hydrodynamic volume increased and the enzyme enlarged within the pores of the microporous The carrier is physically held (trapped).

The method of the invention is useful for isolating known ones and unknown enzymes and for the use of the enzymes in continuous catalytic processes, for Controlling the substrate conversion, for the detection and investigation of chemical compounds, as specific Adsorbents and the like.

The invention also relates to artificially immobilized (immobilized) enzyme systems in which the enzyme within the micropores of a microporous solid

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is enclosed (held) without the chemical Structure of the enzyme is changed ^ and being the enzyme only suffers little or no loss of enzymatic activity.

The terms used here are defined below: Under "enzyme" is a catalyst produced by living cells to understand, these are generally complexes, relatively large protein molecules with both positive as well as negative reaction centers. Examples of enzymes are Arayl3.se, catalysis, erepsin, ß-glucosidase, Lipase, Melibiase, Maltese, Papain. Pectolase, pepsin, Phosphatase, protease, 2enin, invertase, trypsin, tyrosinase and urease. The enzymes can be divided into the following general classes: (1) hydrolytic enzymes or Hydrolases, which hydrolyze complex compounds into simpler compounds, (2) fermenting enzymes or 2 / ymasen, which are generally monosaccharides in lactic acid, butyric acid or converting alcohol and the like, (3) oxidizing ferments or oxidases, (4) reducing enzymes or reductases, (5) catalyzing enzymes or Kataüase ^ and (6) Antikatalasen, which counteract the catalytic effect.

Under the "isoelectric point" the point is the electrical one! Understanding neutrality or ITull potential. In the event of of an enzyme, the isoelectric point is the pH value at which the enzyme is neutral. Below or above this pH value, the enzyme acts either as a base or as an acid.

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The expression “microporous carrier” is to be understood as meaning a material which is insoluble in water and has pores whose size is in the order of magnitude of the enzyme molecule, for example from 20 to 1000 Å. lies. The size of the micropores in the carrier that can entrap the enzyme depends on the hydrodynamic volume of the particular enzyme used. Microporous supports that can be used include natural and synthetic polymeric materials, metals with proous surfaces, and inorganic materials with microporous surfaces such as ceramic materials. Specific materials that can be used _Λ include: B. cellulose materials, cellulose esters, polyolefins, polyamides, polyesters, polyfluoro compounds, polycarbonates and the like. The microporous carrier is preferably electrically neutral and chemically inert to the enzyme (s) to be immobilized therein.

As used herein, the term "hydrodynamic volume" means the volume occupied by the enzyme molecule in solution to understand. Enzymes change their hydrodynamic volume when the environmental conditions surrounding the enzyme are changed, for example by changing the pH the solution in which the enzyme is dissolved. On the isoelectric At this point, the enzyme generally occupies its smallest volume.

Enzymes can be trapped (held in place) within the pores of preformed microporous supports without becoming changes their chemical structure with little or no loss of enzymatic activity by

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get the hydrodynamic volume dsr enzymes up to that Downsized points where they can enter the micropores of the microporous support, and then the hydrodynamic Enlarged volume of the enzyme in order to physically immobilize (immobile close). The hydrodynamic volume of the Enzjnns is due to Adjustment of the environment of the enzyme, generally the pH of the solution containing the enzyme, varies. The volume occupied by the enzyme is minimal at its isoelectric point and increases to a maximum hydrodynamic value at pH values on both sides of the isoelectric point. The isoelectric point of many enzymes is known and has already been determined.

When the hydrodynamic volume of the enzyme is more minimal t has or is close to, the microporous support will be in with the enzyme for a sufficient period of time brought physical contact to allow the enzyme molecules can penetrate into the micropores of the carrier. In general, to achieve maximum enzyme content a contact time of only a few minutes is required within the micropores of the microporous support. the Movement of the enzyme or enzymes in the enzyme solution which also contains the preformed microporous carrier, is arbitrary (statistical), so that a normal statistical distribution of the enzyme molecules within and occurs outside the micropores of the carrier. After a suitable contact time, the pH becomes the Enzyme or the solution containing the enzymes adjusted to increase the hydrodynamic volume of the enzyme.

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When the hydrodynamic volume increases, it increases the molecule of the enzyme, making the molecule physically in its enlarged state within those micropores is included (held), the volume of which is smaller than the volume of the enlarged enzyme molecule.

It has now surprisingly been found that entrapped in microporous carriers in the manner described above (Trapped) enzymes lose little or no enzymatic activity. This is a major benefit compared to systems for immobilizing enzymes, as they have been used hitherto, which generally lead to a severe loss of enzymatic activity.

Another aspect of the present invention is the use of the method described above for Isolating known or unknown enzymes from natural substances, e.g. B. a cellular material containing the enzymes contain. The conventional methods of isolating enzymes from a cellular material make the treatment of large volumes of the material and the subsequent filtration, sieving, extraction, filtration and the like. necessary. The end result is a very small amount of active enzyme. The procedures are time consuming, ineffective and 'difficult to do. The "method according to the invention can be used for the simple and cheap separation and isolation of enzymes from sources in which they are used occur naturally. The material containing the enzyme is crushed and slurried in an aqueous solution, the enzyme is produced using conventional extraction methods

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extracted from the crushed hainrial and separated. The pH of the extraction solution is then set to the isoelectric Point of the enzyme contained in the solution or to a value near the isoelectric point set. A microporous carrier, the pore size of which is sufficient to allow the enzyme to enter the pores, while its hydrodynamic volume is reduced, and within the pores while increasing the hydrodynamic Volume of the enzine this physically includes (hold on) is then brought into the solution. After a contact time of a few minutes, the pH will be the solution adjusted to increase the hydrodynamic volume of the enzyme and this within the pores to physically enclose (hold) the microporous support. The one containing the entrapped enzyme Preformed microporous supports can then be removed from the remaining solution by filtration, decanting, or any other by other suitable methods.

The method described above can also be used to isolate enzymes, their chemical structure and isoelectric point are not known. To achieve this, a number of fractions (e.g. 10 or 20) an aqueous solution containing the dissolved unknown enzyme is each adjusted to a different pH value

included
according to the theory that one of the solutions is adjusted to a pH of or near the isoelectric point of the unknown enzyme. All solutions are brought into contact with a microporous carrier for a few minutes and the pH values of each of the solutions are then adjusted to a different value so that the solution,

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-lO-whose pH-value corresponds to the iso-electrical point of the unknown Closest to the enzyme, enough ste.rk is changed, around the unknown enzyme within the micropores of the microporous To physically enclose the carrier (to be held in place). Each carrier is then aqueous from its remaining Solution separated and recovered and the ensynthetic activity of each carrier is then determined by known methods determined and calculated. The microporous carrier fraction with the greatest enzymatic activity is that which immobilizes (immobilizes) most of the unknown enzyme.

The microporous support that holds the immobilized enzyme or which contains immobilized enzymes can be in the form of particles, in the form of a suspension, in the form of membranes or in the form of filled columns in a continuous catalytic process to control the substrate conversion, Used for detection and investigation of chemical compounds, as a specific adsorbent and the like will.

The invention will be further elucidated by the following examples is explained without, however, "being limited thereto.

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- ii -

example 1

Isolation of the enzyme urease by customary methods iia Comparison to the isolation of the enzyme urease according to the invention Procedure '

1 kg of chopped soybean meal in a 30% aqueous solution Acetone solution mixed with a citrate buffer (2000 ml) "at a pH was maintained at 6 was "stirred at 5 G" for 2 hours and then centrifuged and filtered to give an extract containing the enzyme urease.

The enzyme urease was isolated in the usual way by a 500 ml portion of the extract containing the enzyme It cooled down to -10 C at night to precipitate the urease enzyme, and collected the enzyme by filtering and freeze drying. The obtained crude enzyme (38 mg) showed urease activity of 40 units / µg protein as determined using Urea. This corresponded to a total enzyme activity of 304 units / 100 ml of the original extract. The raw one Enzyme (30 mg) was purified by dissolving the crude enzyme in double distilled water (10 ml) and precipitating the dissolved one Enzyme with 10 ml of acetone and cool. After repeating this procedure three times, a more highly purified one became Urease (18 mg) obtained, which has an activity of 95 units / -mg Protein, corresponding to total enzyme activity of 433 units / 100 ml of the original extract.

A part of the same extract containing the enzyme (I5OO ml) was isolated by the method according to the invention by bringing I5OO ml of the extract containing the enzyme into contact with 100 g of an oc-cellulose pulp which had previously been successively mixed with 100 ml of 50% aqueous Acetone and 2000 ml of distilled water. The enzyme-containing extract / a-cellulose pulps mixture was left ü ^ oer ITacht at -10 G

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calmly. The fibers were then collected and sequentially with 3000 ml of a tris buffer solution (pH 8), 1000 ml of 30 tiger aqueous acetone and 2000 ml of double-distilled water (pH 7). After! Filtration, the pulp that 0.5 mg protein per gram laser contained, "stored at -10 ° C. The pulp was then left to thaw at tree temperature to determine its activity. The immobilized Fibers containing enzyme urease (immobilized) were then prepared from a solution of 20 g of urea and 1000 ml of 0.02 M Sodium potassium phosphate (pH 7) was added and occasionally Stirring held at 22 ° C for exactly 5 minutes. After removal of the fibers by filtration, the ammonia content of the filtrate was determined by titration using a standardized hydrochloric acid solution. The urease activity the fiber was 5,700 units / 100 g fiber, accordingly a total enzyme activity of 380 units / 100 ml of the original extract.

Example 2

A 100 ml portion of the extract of Example 1 containing the enzyme urease, which was kept at pH 6 with a citrate buffer, was poured onto a 15 cm χ 15 cm sheet made of a porous polytetrafluoroethylene polymer (Geigard), which lasted 2 hours was kept at 5 ° C and stored overnight at -10 ⁰ C. · The polymeric film was drawn off and 1000 ml of a tris-buffer solution (pH 8), 100 ml of 30% aqueous acetone and 100 ml of "double-distilled water . The urease activity of the membrane was 102 units / 100 g fibers.

Example 3
Convention ^ nell-e ^ IsojLation of

1 kg of fresh veal liver was chopped up and with 2000 ml one

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0.01 M manganese sulfate solution adjusted to pH 4 using maleic acid, extracted. A 500 ml portion of the centrifuge extract \ vurde overnight at -10 ⁰ C and kept then brought ml by freeze-drying to a volume of 100th The concentrate was treated with 30 'ml of pyridine, 3 minutes, heated at 55 to 59 ° C, cooled, decolored by filtration and! Laughing at 0 ⁰ C stored.

The precipitate was then dissolved in 100 ml v / ater, dialyzed against distilled water for 15 hours at 5 ° C. in a seamless cellophane tube, diluted with 200 ml of methanol and left to stand at -10 ° C. for 2 hours. The material that deposited was collected by decantation and centrifugation. Each freeze-drying 9 gave mg arginase ^ ¹⁰ ^ calculated \ etunäen ^{10 '4 / |} "*>" ^{Which had an} activity of 260 arginine units / mg protein, corresponding to 468 units / 100 nl of the original extract.

Example 4

100 g of α-cellulose pulp, which had previously been washed successively with 100 ml of 50% aqueous acetone and 200 ml of distilled water, were added to a 1500 ml portion of the centrifuged arginase extract of Example 3 described above, which was kept at 5 ° C , admitted. Each 1 hour at the same temperature, the mixture was! Laughing at -10 ⁰ C stored, thawed at room temperature and filtered. The moist fibers were then washed successively with 300 ml of distilled water (pH 7), 2000 ml of water containing 10% pyridine and again with 2000 ml of distilled water (pH 7) Suspended for hours in 1000 ml of a 0.05 M manganese maleate buffer (pH 7) "at room temperature, filtered and mixed with 2000 ml of double-distilled water (pH 7)

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washed. The fibers were then stored at -10 C and thawed at room temperature to determine the arginase activity. This was carried out by adding 100 g of the fibers to 1000 ml of a solution of 10 g of arginine in water, those with a mixture of tris (hydro ~ ethyl) aminomethane and sulfuric acid 'was buffered to pH 8. After exactly 30 For minutes at 22 ° C. with occasional stirring, the mixture was filtered and 2 mg of urease was added to the filtrate. After standing at hem temperature overnight, this became released ammonia is distilled off, in 100 ml of a 1 ^ igen Boric acid solution absorbed and titrated against a standardized hydrochloric acid. The released ammonia corresponded to an arginase enzyme activity of 92 units / g Fibers, 4-60 units / mg protein in the fibers or 680 units / 100 al of the original veal liver extract. This corresponded to an increase in the activity of the collected enzyme by 24%. A repetition of the determination described above the effectiveness of the enzyme after 3 days showed that no loss of activity had occurred.

Example 5

Wirksamkeit- the lt_er after erfindungsgemäüen method immobilized _sierten i.ii2beweg _i li £ h_gjemacht_en) _ £ use Enzyme__b_ei_wiederh

10 g of an α-cellulose pulp were distilled in 250 ml Water containing 10 mg horseradish peroxidase, suspended, diluted with 50 ml water, adjusted to pH 7 »2, 4 hours left to stand for a long time at room temperature and filtered and with 1000 ml of acidic water (pH 5.8), the 1 ml of 0.1% aqueous Containing hydrogen peroxide, washed. Those containing the enzyme Fibers were treated with a solution of 20 mg isoeugenol in 250 ml n-butanol / ethanol / water (l / l / l) for 5 minutes mixed and then treated with 1 ml of 0.3% aqueous hydrogen peroxide. After 4 hours and after 16 hours the

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Repeated addition of hydrogen peroxide and the mixture was left to stand at room temperature for 3 days.

The oxidation effects obtained were followed by quantitative thin layer chromatography on silica gel with evolution with benzene. The surface of the plate containing isoeugenol was scraped, suspended in 25 ml of methanol and filtered. The isoeugenol content of the filtrate was determined spectrophotometrically. The constituent of the second main spot, possibly dehydroisoeugenol, was determined in the same way Order followed with a rate constant of 1.67 x 10 -4 minutes The reusability of the enzyme peroxidase immobilized (immobilized) within the pasers was demonstrated by the retention of enzyme activity over a series of reactions repeated 3 days apart of isoeugenol under these conditions were 100%, 98%, 96% and 96%. in addition, as the following table in comparison with a non-bound enzyme was, shows that / effectiveness of the present invention immobilized (immobilized) enzyme system is not reduced The ones in the following table The results summarized show that although the unbound enzyme initially showed a higher rate of oxidation, the conversion eventually achieved in the enzyme system was practically the same.

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- 16 table

Comparison of the effectiveness of the unbound enzyme with

Conversion of isoeugenol after. 2-.SM. 18 hours

Immobilized in cellulose

Peroxidase 21 % . 90 &

Peroxidase 29 % 88%

The invention has been described above with reference to preferred embodiments explained in more detail, but it is obvious to a person skilled in the art that they are by no means thereon is limited, but that these can be changed and modified in many ways without affecting the Is left within the scope of the present invention.

Patent claims:

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Claims (12)

Pat ent a η s υ rü ch e r \ J A method for the physical immobilization (immobilization) of a catalytically active enzyme within the pores of a water-insoluble, preformed, microporous support without changing the chemical structure of the enzyme and with a relatively small loss of enzyme activity, characterized in that one reduces the hydrodynamic volume of the enzyme by adjusting its environment, a microporous support with Kil: roporen whose volume is equal to or greater so as brings than the reduced hydrodynamic volume of the enzyme into contact, that the enzyme en freely into the Po ^r> is the carrier occur and the environment of the enzyme, while is in contact with the microporous support, so that its hydrodynamic volume increases and the enlarged enzyme is physically retained within the pores of the microporous support . 2. The method according to claim 1, characterized. That the enzyme in a relative to the enzyme inert solvent dissolves. 3. The method according to claim 2, characterized in that the solvent used is water. 4-, The method according to at least one of claims 1 to 3, characterized in that the microporous carrier used is an electrically neutral, natural or synthetic polymeric material in paser or film form with micropores having a size within the range of 20 "to 1000 £ or more. 509884/1132 5. The method according to at least one of claims 1 to 4, characterized in that the hydrodynamic volume of the enzyme by adjusting the pH-T / ertes of the solution to isoelectric point of the enzyme decreased. 6.Method of physical immobilization (immobilization) a catalytically active enzyme within the pores of a preformed, electrically neutral, insoluble in water, microporous carrier without the chemical structure of the enzyme to change and with little or no loss of enzyme activity, characterized in that one aqueous adjusts the pH of a dissolved enzyme containing / solution to the isoelectric point of the enzyme in order to reduce the hydrodynamic volume of the enzyme to a minimum, ie, e solution with a preformed, electrically neutral, water-insoluble, microporous carrier with Micropores, the volume of which is equal to or _{greater than the reduced hydrodynamic volume of the enzyme s brought} into contact so that the enzyme can freely enter the pores of the carrier and the pH of the solution containing the enzyme to a value outside the isoelectric point adjusts the same to increase the hydrodynamic volume of the enzyme and to hold the enzyme physically within the pores of the microporous support »-. 7. Heterogeneous enzyme-catalyst composition, characterized in that it consists of an immobilized (immobilized), catalytically active enzyme which is present within the pores of a microporous support having micropores with a pore volume equal to or greater than the hydrodynamic volume of the enzyme its isoelectric point or near it and which is less than the hydrodynamic volume of the Enz7 / ms outside its isoelectric point, 509884/1132 is held physically without thereby his Enzyme activity suffers a substantial loss and without changing its chemical structure. 8. Catalyst composition according to claim 7 »characterized characterized in that the microporous support is an electrically neutral, water-insoluble substance from the group of natural and synthetic polymers acts in fiber and / or film form. 9 · Catalyst composition according to claim 7 and / or 8, characterized in that the pore volume of the microporous support is within the range from about 20 to about 1000 n_ . 10. A method for isolating a water-soluble, catalytically active enzyme from a material in which it occurs in the nature, characterized in that one extracts the enzyme from the material to form an extractant solution containing the enzyme, adjusts the environment of the extractant solution containing the enzyme so that the hydrodynamic volume of the enzyme reduced v / ird, - · brings the extractant solution into contact with a microporous carrier, so that the enzyme with the reduced hydrodynamic Volume can penetrate into the pores of the microporous carrier, readjusts the environment of the enzyme while in contact with the microporous support to be hydrodynamic Increase volume so that the enzyme is expanded physically within the micropores of the microporous support recorded v / ird, and separating the microporous carrier containing the entrapped enzyme from the remainder of the solution. 509884/1132 11. The method according to claim 10, characterized in that it is carried out in an aqueous solution. 12. The method according to claim 11, characterized in that that the pH of the solution containing the enzyme is adjusted to the isoelectric point of the enzyme to decrease hydrodynamic volume, and that then the pH of the solution to one of the isoelectric point of the Enzyme different value is set again to be un increase hydrodynamic volume. 509884/1132