DE1227855B - Process for the production of enzyme bodies for the implementation of enzymatic reactions - Google Patents

Description

Process for the production of enzyme bodies for carrying out enzymatic Reactions The invention relates to a method for producing enzyme bodies for performing enzymatic reactions. It is characterized in that at least one enzyme or enzyme-containing microorganism in a dry, emulsified or dissolved form of a solution of a semipermeable material such as polyamide, polyvinyl acetate, Cellulose nitrate, cellulose acetate, ethyl cellulose, rubber chloride or lipoids or a solution of mixtures of these substances, incorporated and then the resulting solution, emulsion or suspension is formed while drying.

When using enzymes or enzyme-containing microorganisms is, even if the enzymes or microorganisms are present as colloids, the separation of enzyme or enzyme-containing microorganism and substrate after the reaction has ended difficult and uneconomical. It is known to add enzymes to carriers adsorb or take up in the aqueous stationary phase of a two-phase column, continuous procedures should be possible. The application is however limited to individual cases. There is a significant disadvantage to all of these procedures the partial inactivation of the enzymes during adsorption. Also missing in both Cases an effective protection against enzyme inactivating substances, such as proteases, the can very easily be introduced into the reaction mixture with parasites.

Finally, the enzymes loosely adhering to the wearer can easily be released the mobile substrate phase are entrained, so that the advantage of this method - the proper separation of the enzymes from the substrate - is not achieved.

So there is a need for enzymes or enzyme-containing microorganisms as solid bodies that they can be easily and cheaply removed from the substrate are detachable.

According to the invention, this is done in a novel way in that Enzyme bodies are produced for carrying out enzymatic reactions. These enzyme bodies consist of enzymes or enzyme-containing microorganisms, which are embedded in semipermeable material, so that the substrate through the semipermeable Penetrate membrane, with the enzyme or enzyme-containing microorganism in contact step and react and then exit again through the semipermeable membrane can. A separation of the corresponding enzyme bodies from the substrate liquid is necessary very easily possible, so that a simple retrieval of the corresponding Enzymes or enzyme-containing microorganisms as well as the multiple Application of the same or the successive use of different enzymes or enzyme-containing ones Microorganisms on the same substrate are possible. Above all, it is also a continuous Use easily achievable. It can be achieved without difficulty that the enzyme or the enzyme-containing microorganism is entrapped in the enzyme body remains by making the pore size of the embedding material smaller than the diameter of embedded materials. It can happen that the turnover rate is somewhat reduced by the embedding of the enzyme material, so that too the yield in individual reactions decreases. But the overall yield is still considerably higher, since the enzyme can now be separated and reused several times can do what was previously not possible.

Those embedded in semipermeable material and thus secured Enzymes or enzyme-containing microorganisms are the usual reaction approaches added. In addition to aqueous solutions, the application is also organic, with water Immiscible solvents possible. In this case the semipermeable material must be thoroughly watered beforehand. In addition, the solvent must be saturated with water be.

Any inert substance can be used as a semi-permeable material, which is able to embed the enzyme or the enzyme-containing microorganism in the reaction medium is insoluble, but the enzyme or the enzyme-containing microorganism free passage allowed. Have proven themselves z. B. modified natural substances, such as cellulose nitrate, acetate, ethyl cellulose and rubber hydrochloride, or plastics such as polyamide and polyvinyl acetate; lipoids are also suitable. To functionally perfect embedding substances To obtain, the mixture of semi-permeable substances is often necessary, such as. B.

Lipoid with cellulose nitrate. These mixtures allow a finer one Adaptation to the physical and chemical properties of the substrate and enzyme or enzyme-containing microorganism.

The enzymes or enzyme-containing microorganisms are dry, emulsified or incorporated dissolved into the dissolved embedding material. You get a Suspension, emulsion or solution of the material to be embedded in the solution of the Embedding material. The approach is based on taking into account the for that to be embedded Well required caution dried and possibly hardened. During the The solidification process gives the mixture the desired shape, for example by Spreading on, brushing on, spraying onto other types of material, by dipping of tools for the production of coatings, by pouring or rolling for the production of membranes, by spray drying for the production of hollow spheres or by cutting to produce lamellas or fillings for columns.

For embedding enzymes or enzyme-containing microorganisms in the semipermeable embedding material must be the lability of the enzymes or enzyme-containing Microorganisms are taken into account.

Enzymes or enzyme-containing microorganisms that are released during embedding can be inactivated in an open system and / or at room temperature processed at low temperatures, in a vacuum or in an inert gas phase.

The suspension of a dry micropowder has proven particularly useful in the semi-permeable material that is absorbed in an organic solvent. After the solvent has evaporated, the enzyme or enzyme-containing microorganism remains containing membrane back. Enzymes dissolved in water can also, depending on their properties of the semipermeable embedding material and its solvent included will. In this case, the dissolved enzymes in the dissolved semi-permeable embedding material finely emulsified.

The drying, in the case of plastics also the hardening, must be controlled in such a way that that the aqueous phase - mostly in the form of tiny droplets - remains with enzyme-containing Microorganisms are usually recommended to be freeze-dried beforehand from a gelatin solution.

The external shape of the enzyme body can meet practical requirements be adjusted. Lamellae, balls and covers are particularly suitable for tools, such as rollers, glass jars or tins. Balls prove their worth in reaction columns and porous material. Hollow spheres are often used for continuous operation in columns (Microballoons), so-called enzyme granules.

It is not necessary that the enzymes or enzyme-containing microorganisms can be embedded in pure form. Impure preparations can also be used. The impure enzymes or enzyme-containing microorganisms are often in the aqueous Environment caused by undesired accompanying enzymes (e.g.

Proteases) inactivated in a short time. Be in embedded form contaminated enzymes or enzyme-containing microorganisms, however, are much slower destroyed. The embedding also leads to an almost complete protection against inactivating Parasites in the reaction mixture. The enzyme bodies can be replaced by additives such as ion exchangers, Redox sub- punch or plastic hardeners or modifiers, are improved.

The enzyme bodies produced according to the invention have a good shelf life. They enable the implementation to be carried out continuously or in layers of substrates by biological means with long usability and quick separability the enzymes or enzyme-containing microorganisms. You can therefore get a substrate through successive contact with different embedded or enzyme-containing enzymes Also implement microorganisms in stages.

The enzyme bodies produced according to the invention can be, for example use for synthetic and analytical work or for waste water purification.

Appropriately sized ferment granules (microballoons) are for injections useful, as could be found in animal experiments.

Example 1 Recovery of l-alanine by transamination in the system 1-glutamic acid - pyruvic acid with enzyme lamellae l-glutamic acid + pyruvic acid = l-alanine + o; -ketoglutaric acid 10 g glutamic acid - pyruvic acid - transaminase (Dry powder from Schafherz according to K r e b s) are finely powdered and in 6000g suspended in an anhydrous cellulose nitrate solution. This solution consists of 2.1 0 / o cellulose nitrate, 52.8, 0 / o diethyl ether, 14.101, absolute ethyl alcohol, 20.9 0 / o acetone, 6.40 / o amyl alcohol and 3.7 01o ethylene glycol ethyl ether (all data in percent by weight). The mixture is placed on a horizontal, 4 m2 glass surface poured out and dried in a low-moisture atmosphere at 22 "C for 75 minutes. The membrane is then soaked for 12 hours and cut into 1 cm2 lamellae. The membrane thickness is 0.2 to 0.3 mm, the pore size 5 mp.

The enzyme lamellae are added to a reaction mixture, of 0.05 molar of sodium pyruvate and 0.05 molar of sodium glutamate (l-form) and on a pH of 7.4 is adjusted and buffered.

This approach is incubated for 5 to 6 hours at 25 ° C and then renewed when using the old slats. The same switch is made for 28 days. The aqueous, colloid-free reaction liquid becomes l-alanine by means of uniform multiplicative distribution separated according to 1 a n t z e n. Yield 1550 g of l-alanine.

For comparison, the results obtained with the previous one are given usual application of glutamic acid-pyruvic acid transaminase in colloidal Solution can be achieved. In this case, the enzyme can only be used once. From a In a 20 liter batch, an average of 30 g of l-alanine can be obtained.

If the enzyme lamellae are used for 28 days, the yield is about fifty times larger. In detail, the reusability shows the following Example: Example 2 Recovery of glutamic acid - pyruvic acid - transaminase from “enzyme lamellae” The enzyme lamellae were produced according to Example 1.

10 g enzyme lamellae with 5 g glutamic acid-pyruvic acid transaminase were in a mixture of 52.8 0 / o diethyl ether, 14.1 0 / o absolute alcohol, 20.9 % Acetone, 6.4% amyl alcohol and 3.74% ethylene glycol ethyl ether (all data in percent by weight) and then centrifuged at 10 to 150C0 g. That clear excess was decanted. The soil body was again in the ones listed above Solvent mixture taken up, shaken vigorously and then centrifuged.

After a third wash, the sediment was suspended in acetone, suctioned off, washed with diethyl ether and then dried over phosphorus pentoxide and weighed. The enzyme activity was determined according to Example 1. The loss the enzyme activity after this treatment is 0.82 g (mean from three experiments). This is 16.40 / »of the initial weight (5 g of ferment powder in enzyme lamellae).

Become 5 g of ferment powder without inclusion in enzyme lamellae in the same Treated wisely, the loss is 0.68 g (three attempts). This matches with 13.6% of the original weight.

To the applicability of the enzyme body in water-soluble and also To represent high molecular weight substrate, the following examples were tried: Example 3 Enzymatic breakdown of starch. Enzyme preparation Takamine Special Diastase HT Concentrate is included in the manner described above.

Enzyme formulation 50 mg diastase in particle form, 100 mg soluble starch Erg. B. 6 in 10 ml of 10 molar phosphate buffer (pH 7.0). Incubation time: 4 hours at 37 "C. The released glucose is used according to Fi s c h e r and Dö r f e l (Z. physiol. Chemie, 297 164 [1954]).

The enzyme particles are centrifuged off from the glucose determination and used in two other approaches. The detailed results are in the following Table has been compiled (mean values from five studies).

The following two approaches show almost unchanged hydrolytic properties Properties.

Starch conversion as a percentage of the amount used for 4 hours of incubation Control (enzyme particles) .................... <0.1% (starch) .................. .......... <1.0% enzyme particles with starch 1st batch .......................... 16.2% 2nd batch Approach .......................... 14.8% 3rd approach ................ .......... 12.3% Example 4 Enzymatic degradation of methyl butyrate by lipase from porcine pancreas Enzyme preparation 10 g lipase from pig pancreas (Mann Researoh Lab., New York) in The form of a micropowder is suspended in 500 ml of a cellulose nitrate solution and spray dried. The cellulose nitrate solution consists from 2.1% cellulose nitrate, 52.8% diethyl ether, 14.1% absolute ethyl alcohol, 20.9% acetone, 6.4% amyl alcohol and 3.70 / »ethylene glycol ethyl ether (all data in percent by weight). The middle Particle size is 1 to 5 p. Maximum temperature for spray drying 40 "C. After washing three times in distilled water (immediately after spray drying) and centrifugation, the particles are ready for use.

Enzyme formulation particles that contain an enzyme amount of 50 mg lipase, are in 7 mol of a freshly prepared solution containing 0.4 M glycocolla, 0.1 N NaOH and 10 / o methyl butyrate contains, suspended. In addition, approaches with a fifth, one-tenth and one-twentieth enzyme content checked. The vessels with the enzyme approach are closed with a cap and kept in a water bath at 40 ° C. for 5 hours. The enzyme particles are recovered by centrifugation after the incubation is complete and used again in two identical runs.

Determination of the enzyme activity by titration with 0.05 n-HCl and bromothymol blue as an indicator is carried out according to G 1 i c k (Z. f. physiol. chemistry, 223 252 [1934]).

From the following table it can be seen that the enzyme particles Have lipase activity and can be used multiple times.

Titration values (ml n / 20-HCl, mean values from three determinations) Enzyme dilution 1 5 10 20 Control .... 11.16 11.36 11.26 11.51 1st approach .... # 2.96 # 1.62 # 0.61 # 0.22 2nd approach .... # 2.18 # 1.51 # 0.62 # 0.29 3rd approach .... # 2.31 # 1.48 # 0.52 # 0.31 Example 5 Enzymatic degradation of keratin (wool) by trypsin 10 g of trypsin 1:80 (Mann Research Lab., New York) as micropowder are suspended in 500 ml of cellulose nitrate solution in the manner described above and spray-dried. The particles are washed immediately after spray drying.

Enzyme preparation The determination is carried out according to Go d d a T d and Micha-elis (J. biol. Chem., 106 605 {1934]) with native sheep wool, which by ether extraction is freed from fat and washed several times. 1 g of wool is in 100 ml of bicarbonate buffer (pH 8.5) incubated for 24 hours at 37oC with 1 g of trypsin (in enzyme particles). As a control, enzyme particles and wool are examined separately.

After the incubation, the protein is precipitated with sulfonic acid and the nitrogen content in the supernatant is determined according to K j e l d a h l.

Results The following table shows the cleavage rates in milligrams N have been compiled per approach.

It can be seen from this that the enzyme particles described above are keratin (Wool) able to hydrolyze.

Nitrogen content in milligrams per batch after incubation for 24 hours of trypsin in particle form in a keratin suspension (mean values from five studies) Keratin (wool) without enzyme particles <0.2 mg trypsin (enzyme particles) ......... <0.2 mg keratin and trypsin (enzyme particles) 6.4 mg Example 6 Separation of d, l-tryptophan by cleavage of acetyl-d, l-tryptophan with Aspergillus oryzae As an example for a reaction with an enzyme-containing microorganism becomes acetyl-d, l-tryptophan subjected to asymmetric hydrolysis by Aspergillus oryzae. l-tryptophan and acetyl-d-tryptophan are then separated. Aspergillus oryzae is in cellulose nitrate embedded.

Aspergillus oryzae (Ahlburg-Cohn) No. 4814 is made in the usual way on a homogenized nutrient medium made from glucose, peptone, glycerine and nutrient broth bred. The entire batch is spray-dried at 40 ° C and the dry powder, which has a moisture content of 2 to 50%, in cellulose nitrate according to the example 1 incorporated.

The cellulose lamellae, which contain 15 g of Aspergillus oryzae spray powder, are added to 400 ml of a reaction mixture containing 0.1 mol of acetyl-d, l-tryptophan and is adjusted to a pH of 7.2 and buffered. The approach is kept at 37 ° C for 48 hours and then using the old slats renewed. The same change can be made more than ten times. From the aqueous reaction liquid are l-tryptophan and acetyl-d-tryptophan in the usual manner (J. P. Greenstein and M. Winitz Chemistry of the Amino Acids, New York 1961, p. 2343). When using the Aspergillus oryzae lamellae ten times 0.38 mol of 1-tryptophan are obtained. Aspergillus is used in the known manner Separation is used, then only a single use is possible. The yield is then 0.046 moles of 1-tryptophan.

Example 7 Continuous separation of d, l-methionine by cleavage of acetyl-d, l-methionine on an enzyme column with amino acid acylase I acetyl-d, l-methionine is a continuous process of asymmetric hydrolysis by an enzyme made from pig kidney embedded in cellulose nitrate. Thereupon l-methionine and acetyl-d-methionine are separated.

1 g amino acid acylase I (dry acetone powder from pig kidney) is treated as in Example 1, taken up in a cellulose nitrate solution and poured into a 1 mm thick membrane. This is made with a cutting machine irregularly shaped particles of about 1 to 2 mm in diameter produced the it is filled into a glass column (diameter 2.5 cm, length 25 cm) as used for partition chromatography is needed. The column is washed with ice cold water for 6 hours (Pass about 50 ml / hour) and then with a 0.07 molar solution of acetyl-d, l-methionine charged, the pH of which is 7.4. The flow rate is 50 ml / hour. l-methionine and uncleaved acetyl-d-methionine are obtained by uniform multiplicative distribution after J a n t z e n separated in the solvent mixture n / 10-HCl-ethyl acetate. l-methionine remains in the aqueous phase. This solution is concentrated in vacuo and l-methionine is precipitated with absolute ethyl alcohol. The cleaning is done by Recrystallization from water-ethyl alcohol.

This enzyme column is used for 28 days for continuous racemate separation used. 140 g of l-methionine are obtained. Will the same amount of enzyme in the known way used for separation, then the yield is a single 24 hours Batch 15 g of l-methionine.

Example 8 Enzymatic Degradation of AD-Glucose in Fresh Egg White from hen's egg To improve the commercial properties of hen's egg white, the disruptive ß-d-glucose by an enzyme system that is embedded in cellulose nitrate is converted to d-d-gluconolactone.

After the reaction, the enzymes are completely recovered and can be reused multiple times. The egg white is almost glucose-free and can be further processed.

10 g of crude glucose oxidase and 1 g of partially purified horseradish peroxidase are taken up as in Example 1 in a cellulose nitrate solution and become a about 0.3 mm thick membrane poured out. The mean pore size is 7 mµ. These Membrane is cut into lamellae about 1 cm2 in size, which are kept for 5 hours at 25 ° C can be incubated with 2 kg of egg white with gentle stirring. After completion the implementation is the glucose concentration below 2 mg /%. Leave the enzyme lamellas easily separate from the egg white and in at least six to eight more batches continue to use.

Without this fixation in lamellae, the enzyme system can usually cannot be recovered.

Example 9 Enzymatic protection of mayonnaise from oxidative processes The enzyme system, which consumes O2 and glucose, prevents glucose oxidase-catalase in mayonnaise, as long as it is kept in airtight tin cans, odorless and loss of taste and the formation of peroxides. The tin cans are lined with a polyurethane film in which the enzyme system is embedded.

5 g of a penicillin-free, lyophilized strain of Penicillium notatum (after C. E. C o u t h a r d, R. Michaelis, W. F. Short, G. Sykes, G. E. H. Shrinshire, A.F.B. Standfast, J.H. Birkinshaw and H. Raistrick, Biochem. J., 39 24, 1945) mixed with 0.5 g of partially purified horseradish catalase H. Theorell, Arkiv. Kemi Mineral. Geol., 16A, No. 2, 1942, are in 200 ml of a Solution for the production of polyurethane films (100 parts Desmophen 1100, a branched Polyester made from adipic acid, triol and Butylene glycol, and 30 parts Desmodur L, a 75% strength solution of polyurethanes in ethyl acetate, Bayer plastics, 2nd edition, Leverkusen 1959). A 1000 ml can is made with this mixture lined with glass. The film takes about 24 hours to cure at room temperature.

800 to 1000 ml of weakly acidic mayonnaise can be sealed in this Can be stored for 5 months without the otherwise known signs of decomposition.

Example 10 Continuous cleavage of sucrose by invertase, which is embedded in polyvinyl alcohol, sucrose is produced in a continuous process the hydrolysis into glucose and fructose by the enzyme invertase, which is found in polyvinyl alcohol is embedded, subject.

2 g of partially purified invertase are in 200 ml of a 10 above aqueous solution of polyvinyl alcohol (Mowiol N 70-88) added and to an approximately 1 mm thick membrane processed according to known methods. This will result in a Cutting machine produces irregularly shaped particles from 1 to 2 mm in size and filled into a column as described in Example 2. This pillar is at room temperature with a 0.20 /, own solution of sucrose in aqueous n-butanol charged. The flow rate is 40 ml / hour.

This enzyme column was continuous with sucrose solution for 16 days loaded. Only traces of sucrose could be found in the run-through. the The yield of glucose was 670% of theory.

Example 11 In Example 1, the cellulose nitrate solution is through a Added 1.5% castor oil. This Improved lipoid addition to the membrane apparently the semipermeable properties. The alanine yield is thereby on average 70/0 increased. A disadvantage of this lipoid cellulose nitrate membrane is that it is somewhat greater susceptibility to bacterial decomposition with prolonged use.

Example 12 In Example 7, the cellulose nitrate solution was through a 2 0 / »ige solution of rubber hydrochloride in benzene replaced and a 1 mm thick membrane with filter paper as a carrier material made by immersion. - This membrane was processed as in Example 2 and filled into a column. The flow rate was 5 ml / hour. This enzyme column became continuous for 10 days Racemate used. 26 g of l-methionine were obtained. The advantage of this Column is a great resistance to bacterial destruction.

Claims (2)

Claims: 1. Process for the production of enzyme bodies for the implementation of enzymatic reactions, characterized in that at least an enzyme or enzyme-containing microorganism in dry, emulsified or dissolved form Form of a solution of a semi-permeable material such as polyamide, polyvinyl acetate, Cellulose nitrate, cellulose acetate, ethyl cellulose, rubber chloride or lipoids or a solution of mixtures of these substances, incorporated and then the resulting solution, emulsion or suspension is formed while drying. 2. The method according to claim 1, characterized in that the solution of the semipermeable material, ion exchangers, redox substances or plastic hardeners or modifiers are added.