CH624714A5 - Process for improving the activity of oxidoreductase enzymes encapsulated in filamentary structures - Google Patents

Abstract

The technical problem of improving the activity of oxidoreductase enzymes is solved by encapsulating the enzyme and a coenzyme in a filamentary structure, the coenzyme having previously been functionalised and attached to a water-soluble polymer of high molecular weight.

Description

624714

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CLAIM Procedure for improving the activity of oxide-reductase enzymes consisting in incorporating the enzyme and a coenzyme in filamentous structures which has been previously functionalized and therefore attached to water-soluble polymers with a high molecular weight.

The present invention relates to a process for improving the activity of oxidation-reducing enzymes consisting of incorporating the enzyme and a coenzyme in filamentous structures which has been previously functionalized and therefore attached to water-soluble polymers of high molecular weight.

It is known that enzymes can be immobilized with various techniques on water insoluble supports and therefore be used as heterogeneous catalysts which can be easily separated from the reaction mixture and therefore reusable.

In the case of enzymes or enzyme complexes which require a coenzyme of the NAD + type, it was necessary to add this coenzyme to the reaction mixture. In fact, given the low molecular weight of the coenzyme, it is difficult to immobilize it together with the enzyme on the same support so as to avoid a continuous addition of the coenzyme itself in the reaction mixture. Furthermore, the covalent attachment of the coenzyme to the same support to which the enzyme is attached is not convenient for steric hindrances which make it impossible or severely limit the interactions between coenzyme and enzyme necessary for the activity. Since these coenzymes are quite expensive, the fact of having to continually add them to the reaction mixture severely limited the use of fiber oxidoreductase systems.

It is also known that it is possible to functionalize the NAD + in the 6-amino purine group and attach the functionalized NAD + to water-soluble high molecular weight polymers, according to processes described in the Swiss patents numbers 618 169 and 605 951 in the name of the same applicant.

It has now been found that such polymers having coenzymatic activity can be incorporated in water insoluble polymeric matrices, in particular in filamentous structures, together with the enzyme.

The simultaneous incorporation of enzymes and coenzymes involves a stabilization of the oxide-reductases which in the absence of coenzymes are subject to association and dissociation equilibria of the subunits with loss of activity.

The practical application of the process as defined in the single claim allows the realization of a polyenzymatic bioreactor where the water-soluble polymeric derivative of NAD + is continuously and cyclically converted from the oxidized form to the reduced form, and vice versa, with the use of two oxidoreductases. The derivative of NAD + can be converted cyclically, as well as by enzymatic means, that is, by using two oxidoreductases, also by enzymatic and chemical way by using an oxidoreductase and a chemical compound. The recycling system is chosen based on the product to be obtained.

The way of incorporating enzyme and coenzyme is that described in the Swiss patent 520 715 in the name of the same applicant, in which further technical details are exposed.

The applications of macromolecular coenzymes that can be incorporated together with enzymes in polymeric matrices can be the most varied. In the preparatory field, synthesis of steroids or transformations of steroid nuclei can be performed in specific positions; moreover, stereospecific syntheses of amino acids can be carried out starting from hydroxyacids or ketoacids. In the analytical field, enzymatic-coenzyme systems can be incorporated in which the last oxidation reaction of the reduced coenzyme or reduction of the oxidized coenzyme

it is carried out by a chemical whose absorption spectrum depends on the reduced or oxidized state in which it is found; the color of this substance can be easily measured and related to the quantity of substance to be determined.

The following examples demonstrate some embodiments of the process as defined in the single claim.

Example 1

A solution in glycerine buffer (75: 25) of Polyethylenimine-NAD + (PEI-NAD1), Lactic Dehydrogenase (LDH) (from rabbit: Boeh-ringer, Mannheim, GmbH) and Alanine Dehydrogenase are available for incorporation Ala DH) (from B. Subtilis, Boehringer, Mannheim, GmbH), with the following characteristics:

PEI-NAD + 37.5 mg / ml (= 0.63 NADVml thymols) LDH 17.5 mg / ml (70 I.U., NAD * as substrate)

Wing DH 3.75 mg / ml.

10 g of cellulose tri-acetate (Fluka AG. Buchs) are dissolved under stirring in 133 g of methylene chloride. 20 grams of enzymatic solution are added to the polymer solution and the emulsion of the two phases is favored with vigorous stirring at 0 ° C for 30 '.

The emulsion is poured into a small fuser kept at 0 ° C and spun under nitrogen pressure.

The wire coagulates in toluene at 0 ° C and collects it in a winding machine.

It dries in the air to remove organic solvents. 2 grams of fiber thus obtained, corresponding to about 1 g of dry polymer, are washed with bicarbonate buffer pH 8.0 to remove the enzymes and PEI-NAD + adsorbed on the surface and placed in 10 ml of aqueous solution of the following composition:

ammonium L-lactate 3% (w / v) pH 7.4.

The mixture is stirred at room temperature (-22 ° C). After 10 hours the quantity of L-alanine formed, equal to 0.163 g (96% with respect to the theoretical), is determined by means of an auto-analyzer of amino acids.

Once the mixture has been discharged, a fresh solution of ammonium lactate is put in contact with the same fiber for a new reaction cycle. After 7 days of operations the percentage of L-alanine is 94% at 10 hours, after 30 days always at 10 hours there is 90%. L-alanine is separated by precipitation with ethanol at pH 6.2. [a] D25 = + 14.6 ° (c = 2.0; 5 N HCl).

Example 2

A fiber is prepared as in example 1 using Formil-PEI-NAD + instead of PEI-NAD +.

2 grams of fiber thus obtained, corresponding to -1 of dry polymer, are washed with 0.05 M phosphate buffer pH 8.0 and placed in 10 ml of aqueous solution of the following composition: 0.05 M phosphate buffer pH 8.0 containing 5% (w / v) ammonium DL-lactate. After 10 hours the alkaline formed is 98% of the theoretical. The same fiber is put into another 10 ml of solution and every 10 hours the solution is changed. After 30 days of continuous operations, the percentage of alanine formed at 10 hours is 95%.

Example 3

A fiber is prepared as in Examples 1 and 2 using Polysine-NAD +. Operating exactly as in 1 and 2 at 10 hours, 92% of theoretical alanine is obtained. After 30 days of use of the same fiber there is an 87% conversion.

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Example 4

A tempone-glycerin solution (75: 25) is available for incorporation containing:

Polyethyleneimine-NAD +: 25.5 mg / ml (3.78 jimoli NADV ml) 3a, 20ß-hydroxysteroid dehydrogenase (from Streptomyces hydrogenans, Boehringer, Mannheim, GmbH): 12.5 mg / ml (225 U / ml; 25 ° C; cortisone as substrate)

Alcohol dehydrogenase (ADH) (from horse liver, Boehringer, Mannheim, GmbH): 30 mg / ml (81 U / ml; 25 ° C; ethanol as substrate).

Aldehyde dehydrogenase (from yeast, Sigma, St. Louis): 30 mg / ml (90 U / ml; 25 ° C; acetaldehyde as substrate).

A fiber is prepared as in example 1.

2 grams of fiber corresponding to -1 g of dry polymer are stirred at 25 ° C with 10 ml of solution in 0.1 M triethanolamine-HCl buffer pH 7.3 containing: KCl 0.3 M, ß-mercaptoethanol 5 mM, ethanol 1%, in order to remove the surface absorbed substances and to have a stabilizing medium for the incorporated activity.

At the end of the washing, due to the action of alcohol dehydrogenase and aldehyde dehydrogenase, PEI-NAD + is converted into the reduced form.

The washing waters are removed by filtration and 10 ml of the same buffer solution containing 28 mg of cortisone (A4-pregnene-17, 21 diol-3.11.20 - trione) are added.

The mixture is left under stirring at 25 ° C for 1 hour, after which the mixture is discharged. The solution is extracted three times with 2 ml of chloroform at a time. The organic extract is concentrated in vacuo up to 1 ml. The quantity of product (A4-preg-nene-17a, 20ß, 21 triol-3, ll-dione) and cortisone are determined by gas chromatography.

A 95% transformation is calculated.

The same fiber sample was used for twenty cortisone transformation cycles with negligible drop in activity.

Example 5

A glycerin buffer solution (75: 25) is available for incorporation containing:

Polyethyleneimine-NAD +: 25.5 mg / ml (3.78 [tmol NAD + /

ml) ß-hydroxysteroid dehydrogenase (from «Pseudomonas testosteroni», grade II, Sigma, St. Louis): 40 mg / ml Diaphorase (from pig heart, grade II, Boehringer, Mannheim, GmbH): 19.4 mg / ml of the commercial preparation (containing 5 6.45 mg / ml of enzyme protein).

A fiber is prepared as in example 1.

2 g of fiber corresponding to 1 g of dry polymer are washed with 0.067 M triethanolamine-HCl buffer, pH = 7.6 to remove the substances absorbed superficially and placed in 10 ml of an aqueous solution containing: triethanolamine buffer - HCl, 0.067 M, pH = 7.6 288 [ig of testosterone (17 ß-hydroxy-A4-androstene-3-one), 2.9 mg of 2,6-dichlorophenolindophenol. The mixture is left under stirring at 25 ° C and oxygen is gurgled for the reoxidation of the reduced dye 15. After 4 hours the mixture is extracted three times with 3 ml of ethyl acetate at a time. The organic extract is dehydrated with anhydrous sodium sulphate, filtered, concentrated to dryness. The solid residue is dissolved in 0.5 ml of methanol. The amount of product (A4-androstene-20 -3.17-dione) that is formed is 90% compared to the testosterone put in the test.

Example 6

The same fiber of example 5 was used.

25 1 g of fiber corresponding to 0.5 g of dry polymer were washed with 0.06 M triethanolamine-HCl buffer, pH = 7.6 to remove the surface absorbed substances.

Five solutions containing tam-30 were prepared. 0.06 M triethanolamine-HCl, pH = 7,6, 2,6-dichlorophenolindophenol 80 (JiM and testosterone (17ß-hydroxy-A4-adro-stene-3-one) 10 p, M, 20; aM, 30 jaM, 40 fiM and 50 [xM.

The gram of washed fiber was from time to time put into 5 ml of each solution and the mixtures were stirred at 35 25 ° C by measuring the absorbance at 600 nm. With the differences in optical density and testosterone concentrations, a calibration line was built. The same fiber was repeatedly used to dose testosterone in the solution that contained it in a concentration of 30 p.M. During 100 40 dosages there were no deviations from the value of the calibration line.

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