DE3936802A1 - High mol. wt. forms of amine-contg. coenzyme prodn. - by reacting coenzyme with bis:oxiranyl cpd. and macro:molecule, giving regeneratable prods. for use in continuous enzymatic processes - Google Patents

Abstract

Prodn. of functional coenzymes (A), contg. amino-N and having increased mol. wt., comprises (1) reacting (A) with excess of a bisoxiranyl cpd. (C), then (2) reacting the prod. with a macromolecular cpd. (B) having functional gps. reactive with epoxy residues. Pref. the (A)-(C) reaction prod. is used in stoichiometric excess relative to (B), which is particularly amino-polyethylene glycol (A-PEG) of mol. wt. 2000-50000. Pref. in the first step, excess (C) is used (to avoid (A)-(A) coupling) and the excess of (C) is easily removed by exploting differences in solubility, although removal is not essential. Reaction is at room temp. and pH 7-11 for 1 hr. to 1 day, then the reaction prod. is reacted for a few hrs. with (B), opt. in the presence of NaBH4. An excess of (B) is used. USE/ADVANTAGE - These prods. are used as regenerable coenzymes in continuous enzymatic processes (e.g. in an enzyme membrane reactor). They retain the biological activity of the free coenzyme and can be prepd. more quickly than known high mol. wt. coenzyme derivs. and relatively simply.

Description

The invention relates to a method of manufacture of molecular weight-increased, functional Coenzyme (A) containing amine nitrogen by coupling to a functional group reacting with epoxy groups Macromolecule (B) via a bisoxiranylver bond (C) as a coupling agent.

The industrial application of cofactor-dependent Enzymes in continuous processes require that Regeneration of the cofactor. In addition to the non-enzymatic Regeneration that has low efficiency there are efforts to enzymatic rain enable in a continuous process. For this purpose, molecular weight increased cofactors like NAD, NADP, ATP needed.

Methods are therefore already known for using coenzymes Macromolecules, especially on water-soluble polymers how to couple polyethylene glycol (PEG).

P. Zappeli u. a. [Eur. J. Biochem. 54 (1975) 475-82] described a method according to which NAD reacted with 3,4-epoxybutyric acid and after reduction and Dimroth rearrangement coupled to aminohexyl Sepharose becomes.  

According to K.-L. Schmidt u. a. [J. Biochem. 67 (1976) 295-302] becomes epoxy-activated Sepharose by implementation of Sepharose 6 B with 1,4-butanol diol diglycidyl ether get that then with NAD to go directly to Sepharose coupled coenzyme is implemented.

An analog coupling of NAD to alginic acid using Diepoxyoctan is from R. W. Coughlin et al. a. [Biotechn. Bioeng. 18 (1976) 199-208] for alginic acid reacted with the bisoxiranyl compound and then is reacted with NAD.

C. W. Fuller u. a. or F. Le Goffic et al. a. [Eur. J. Biochem. 103 (1980) 421-30 and 108 (1980) 143-8] the coenzyme to a polymerizable, Acrylate-based monomer via epoxypropyl groups couple and then carry out a copolymerization.

In addition to these primarily for chromatographic purposes Process developed is also in DE-PS 28 41 414 a process for the production of increased molecular weight Coenzyme described, in which the coenzyme with an alkylating agent, especially ethyleneimine implemented and then with a macromolecule, in particular a polysaccharide or polyethylene glycol for the reaction brought and finally subjected to a Dimroth rearrangement becomes. A somewhat modified procedure is described in DE-OS 36 17 535, according to following the reaction of the coenzyme with ethyleneimine a stock transfer is made and only then Coupling to the macromolecule takes place.

The above-mentioned implementations are relatively lengthy and cumbersome and in terms of the achieved Products not completely convincing.

The aim of the invention is therefore a method for the production of molecular weight-increased coenzyme, the is less time consuming and relatively easy and too Products that are organic Reach activity at the free cofactors.

The Ver according to the invention developed for this purpose driving of the type mentioned is characterized in that the coenzyme (A) with an excess Bisoxiranylverbindung (C) brings and the formed reaction product with the macromolecule (B) implements.

Coupling of amino group-containing compounds such as Proteins, peptides and amino acids on agarose with With the help of 1,4-butanediol diglycidyl ether already in 1974 by L. Sundberg u. a. [J. Chromatogr. 90 (1974) 87-98], but here too was first Agarose implemented with the bisoxiranyl compound and that Then reaction product with the amino group-containing Connection brought to reaction.

In contrast, according to the invention, this is first Coenzyme with the bisoxiranyl compound implemented in Excess is used, so that a coupling of Coenzyme molecules largely suppressed together becomes. The excess bisoxiranyl compound can be in In view of the considerable differences in solubility of the implemented coenzyme on the one hand compared to the bis oxiranyl compound on the other hand very simply from the Reaction system are removed, but there is none urgent need to do so as not implemented Bisoxiranyl compound the further implementation with the Macromolecule does not interfere, unless a defined product is sought, but rather a molecular  weight increase of the coenzyme with regard to con continuous enzymatic reactions (e.g. in a Enzyme membrane reactor), in which the retention of the Coenzymes in the reaction space from a certain minimum size of the coupled macromolecule is reached.

The coupling of the Macromolecule is also preferably under Adding superstoichiometric amounts of macromolecule, which is a practically complete integration of the coenzyme in the molecule-enlarged product.

Preferably at a relatively low temperature, worked in particular at room temperature and at pH Values that range from 7 to 11 depending on Reactivity of the coenzyme to be coupled, lower pH values are preferred to prevent hydrolysis to avoid the macromolecule.

In practice, a coenzyme solution in phosphate buffer of the desired pH with the bisoxiranyl compound added and the reaction mixture about 1 hour to 1 day long shaken at room temperature. That in the aqueous phase remaining oxiranyl-containing coenzyme is then with the macromolecule, such as water in particular soluble macromolecule (e.g. PEG) and the Solution again at room temperature for a few hours touched. Amino-PEG is preferably used. A The addition of NaBH₄ is beneficial but not necessary.

The entire manufacturing process, including cleaning and separation operation about 1 to 1 1/2 days and leads to a practically 100% coupling of the Coenzyme with sufficient bioactivity.

Activity and absorption measurements on this product  indicate that the coupling is predominantly at the C₆ Amine nitrogen has occurred, so that additional rearrangements, as is known to be provided appear necessary.

In particular, as amine group-containing coenzymes NAD, NADH, NADP, NADPH and ATP implemented. The one with it coupled macromolecules are preferably polyethylene glycols with a molecular weight between 2000 and 50,000.

The bisoxiranyl compound corresponds in particular to that formula

usually an - especially linear - aliphatic 3- to 12-membered radical in which one or more C atoms replaced by oxygen or sulfur bridges could be.

The following are examples to illustrate the invention.

Example 1 Coupling of coenzymes to amino-PEG 20,000 at pH 11

a) Implementation of coenzymes with bisoxirane

13 µmoles of NAD (8.6 mg), NADH (9.2 mg) and NADP were used (10.2 mg), NADPH (10.8 mg) and ATP (6.6 mg) in 1 ml 0.05 M sodium phosphate buffer dissolved and with 26 µmol each Bisoxirane (4.8 µl) added. The reaction mixture was adjusted to pH 11 and overnight (approx. 20 hours) shaken at room temperature.

b) Coupling of amino-PEG to those implemented according to a Coenzymes

54 µmol (1080 mg) amino PEG 20,000 were in 10 ml 0.05 M Sodium phosphate buffer pH 11 dissolved and with the solution of the derivatized coenzyme mixed. The solution was then mixed with a spatula tip of NaBH₄ and 20 hours long stirred at room temperature. Then follows the cleaning and the determination of the yield of the product via gel chromatography on Sephadex G 25.

The products were assessed photometrically and enzymatically. The following enzymes were used:
Pig muscle lactate dehydrogenase isoenzyme 5 (1)
Pyruvate decarboxylase / alcohol dehydrogenase from yeast (2)
Yeast hexokinase / glucose-6-phosphate dehydrogenase (3)
Thermoanaerobium brochii alcohol dehydrogenase (4)

The absorption maxima give an indication of the Kopp at the adenine ring. The coupling at the N₁ point causes a maximum at 259 nm, while the coupling on C₆-amine nitrogen shows a maximum at 265-267 nm.

The yields relate to the amounts used Coenzyme.  

In further experiments, the reaction conditions varies:

Example 2 Determination of the amounts of coenzyme bound when used of different amounts of amino-PEG 20,000

Three batches, each with 2.8 mmol NADH (1986 mg) were in 10 ml of 0.05 M sodium phosphate buffer pH 11 dissolved, with 5.6 mmol of bisoxirane (1.03 ml) were added and overnight shaken. This solution was doubled Amino-PEG 20,000 (4 times the amount of functional groups), the equimolar amount (twice the number of functional Groups) and half the amount of amino-PEG (equivalent Amount of functional groups) added:

• A1: 5.6 mmol amino-PEG 20,000 (112 g) were in 800 ml 0.05 M sodium phosphate buffer pH 11 dissolved and with added to the coenzyme solution.
• A2: 2.8 mmol amino-PEG 20,000 (56 g) were in 450 ml Buffer dissolved and mixed with the coenzyme solution.
• A3: 1.4 mmol amino-PEG 20,000 (28 g) were in 300 ml Buffer dissolved and mixed with the coenzyme solution.

All three batches were then treated with 16.8 mol NaBH₄ (635.5 mg) added and overnight at room temperature shaken. The control of the reaction was carried out via gel chromatography on Sephadex G 25 (Column diameter = 1 cm, height = 30 cm). The product was dialyzed and then lyophilized.

The yields were 100% for batches A1 and A2 and 88% for batch A3, ie the functional groups on the PEG must be in excess for the reaction to proceed to completion. The absorption maxima of the approaches were:
A1 264 + 340 nm
A2 264 + 338 nm
A3 267 + 340 nm

There were no increases in absorption between 300 and 310 nm. It can be concluded from this that the N- 1 position on the adenine ring is free. The factor A₂₆₇ / A₃₄₀ 3.7 gives an indication of the coupling at the N-6 point. The factor for the approaches listed above was included 3.8.

The concentration of NADH was determined with Using an oak line with native NADH at 340 nm. This results in a concentration of 0.5 mmol NADH per 1 mmol PEG at A1 and 0.8 mmol per 1 mmol PEG at A2.

The activity of the molecular weight increased NADH was measured with enzymes 1 and 2. For this purpose for the enzyme tests equimolar amount of native and mole Weighed in NADH and the activities determined under otherwise constant conditions.

Experiments 1: lactate dehydrogenase (1)
1250 µl NADH (0.245 mmol in Tris / HCl buffer pH 7.2)
250 µl pyruvate (9.76 mol in Tris / HCl buffer pH 7.2)

The reaction was started by adding 5 ul enzyme and followed for 1 min at 340 nm.

The activity of lactate dehydrogenase was 4 times higher with native NADH than with the same amount of PEG-NADH.
Experiment 2: Coupled enzyme test to determine the activity of pyruvate decarboxylase (PDC)
490 µl NADH (0.247 mmol in citrate buffer pH 6)
500 µl Na pyruvate (39 mg in 10 ml citrate buffer pH 6)
10 µl yeast alcohol dehydrogenase (80 U)

The reaction was started by adding 50 μl PDC and the course of the reaction was observed at 340 nm.

Alcohol dehydrogenase had the same activity with PEG-NADH and native NADH.

The activity was calculated according to the following Formula:

V = test volume
v = volume of the enzyme solution
ε = molar extinction coefficient of NADH
d = layer thickness of the cuvette
Definition U: 1 unit corresponds to 1 µmol substrate turnover / min

Example 3 Observation of the time course and the pH dependence the NADH coupling

The investigation of the temporal course of the NADH coupling was with different pH values and below different amino PEG concentrations (molecular weight 20,000). In all batches 0.06 mmol) Bisoxiran used.  

Example 4

Studies on the course of the reaction and the pH dependency for the coenzymes NAD, NADP, NADPH and ATP.

In further experiments, the course of the reaction was included other coenzymes were observed analogously to Example 3. The PEG concentration became constant with these approaches held. The concentrations of the individual substances are shown in the table below.  

Enzyme tests were carried out on the products listed above:
NAD (enzyme 1) showed activity at all pH values,
NADP (enzyme 3) was active in the batches with pH 7 and 9,
NADPH (enzyme 4) and ATP (enzyme 3) show activity in all batches.

Example 5 Production of PEG-NADH with unmodified PEG

12.5 µmol of NADH (8.9 mg) was dissolved in 1 ml of 0.05 M sodium phosphate buffer pH 11 dissolved, with 25 µmol bisoxirane (4.6 µl) spiked and shaken overnight. The reaction mixture was made into a solution of 25 µmol (500 mg) PEG added to 10 ml of 0.05 M sodium phosphate buffer pH 11. The solution was mixed with a spatula tip of NaBH₄.

The yield was 58% after 2 hours; 30 hours at 64%.

Example 6 Coupling of NADH to other macromolecules

Three batches, each with 31 µmol (22 mg) NADH were in 1 ml 0.05 M sodium phosphate buffer pH 11 dissolved, with 62 µmol (11.4 µl) Bisoxiran added and shaken for 20 hours:

• A: 69.2 mg of chitosan 70,000 (2-amino-2-deoxy- (1,4) -β-D-glucopyranan; 0.39 mmol amino groups) were dissolved in acid by heating and the coenzyme solution was added.
  The yield was 63% after 24 hours.
• B: 62.7 mg of chitosan 750,000 (0.35 mmol amino groups) were dissolved by heating in acid and mixed with coenzyme solution.
  The yield was 40% for 24 hours.
• C: 59 mg chitosan 2,000,000 (0.33 mmol amino groups) were reacted with coenzyme solution as under B.
  The yield was 43% after 2 hours.

The attached graph shows a comparison between native coenzyme and molecular weight-increased coenzyme using the example of NADP.

From the Michaelis quantity plot (enzyme activity in Ab dependence on the coenzyme concentration) that the activity of alcohol dehydrogenase from Thermo anaerobium brochii (enzyme) by the invention Molecular weight increase of the coenzyme was not affected becomes.

Claims (9)

1. A process for the preparation of molecular weight-enlarged, functional amine nitrogen-containing coenzyme (A) by coupling to a macromolecule (B) having functional groups reacting with epoxy groups via a bisoxiranyl compound (C) as coupling agent, characterized in that the coenzyme (A ) with an excess of bisoxiranyl compound (C) and the reaction product formed is reacted with the macromolecule (B). 2. The method according to claim 1, characterized, that the reaction product of coenzyme (A) and bis oxiranyl compound (C) with overstoichiometric Amounts of macromolecule (B) is implemented. 3. The method according to claim 1 or 2, characterized, that the coenzyme contains an adenine ring system. 4. The method according to any one of claims 1 to 3, characterized, that as macromolecules polyethylene glycols, in particular Amino PEG, with a molecular weight of 2000 to 50,000 can be used. 5. The method according to any one of the preceding claims, characterized in that the bisoxiranyl compound of the formula corresponds in which R is an - in particular linear - aliphatic 3- to 12-membered radical in which one or more C atoms can be replaced by oxygen or sulfur bridges. 6. The method according to any one of the preceding claims, characterized, that the bisoxiranyl compound (C) by 1,4- Butanediol diglycidyl ether is formed. 7. The method according to any one of the preceding claims, characterized, that the coupling reaction in the presence of NaBH₄ be carried out. 8. The method according to any one of the preceding claims, characterized, that the product obtained by gel chromatography on Sephadex G 25. 9. The method according to any one of the preceding claims, characterized, that the coupling reactions at room temperature be performed.