WO1997004100A2

WO1997004100A2 - L-ascorbic acid synthesis by l-galactonatoxidoreductase

Info

Publication number: WO1997004100A2
Application number: PCT/DE1996/001339
Authority: WO
Inventors: Josef Wissler
Original assignee: Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date: 1995-07-17
Filing date: 1996-07-17
Publication date: 1997-02-06
Also published as: AU6731796A; DE19628882A1

Abstract

An enzyme suitable for oxidising L-galactano-η-lactone into L-ascorbic acid is disclosed, as well as a process for producing the same and its use.

Description

L-galactonate oxidoreductase ascorbic acid synthesis

The invention relates to an enzyme suitable for the oxidation of L-galactano-γ-lactone to L-ascorbic acid, a process for its preparation and its use.

L-ascorbic acid (vitamin C) is the best known of all vitamins and a valuable essential component of food. It is an ubiquitous active ingredient that is probably necessary for all living organisms to maintain their metabolic function. Humans have lost the ability to biosynthesize L-ascorbic acid in the course of evolution and are therefore dependent on their intake with food. Important reactions in which L-ascorbic acid plays a role are, for example, the hydroxylation of proline in the biosynthesis of procollagen, the conversion of vitamin D3 or the hydroxylation of dopamine in the synthesis of noradrenaline. Next is the effect of L-ascorbic acid Radical scavengers (ie the neutralization of free radicals that occur in the cell and the interruption of radical-induced chain reactions) are known. In addition, L-ascorbic acid is added to many foods as an antioxidant. From these varied fields of application of L-ascorbic acid, it follows that there is a great need for this substance. For cost reasons, a way must therefore be sought to produce L-ascorbic acid cheaply and easily, for example by enzymatic means.

The object of the present invention is therefore to provide an enzyme which provides L-ascorbic acid as the reaction product. Another object is to provide a method for producing this enzyme.

These tasks are solved by an enzyme according to claim 1, a DNA sequence according to claim 6 or a method according to claim 7. Advantageous refinements result from the subclaims. The inventors have found that it is possible to oxidize L-galactano-γ-lactone to L-ascorbic acid by means of the enzyme according to the invention using an electron acceptor. The electron acceptor is preferably cytochrome-c. The enzyme according to the invention is a reaction-selective protein biocatalyst for the redox reaction on galactano-γ-lactone. The biocatalytic effect of the enzyme according to the invention on the reaction in the sense of the stoichiometry of Fig. 1 can be measured photometrically. The change in optical density at 550 nm that occurs when reduced cytochrome-c is formed is recorded.

Fig. 1: Conversion of galactano-γ-lactone to L-ascorbic acid by the enzyme according to the invention

The enzyme according to the invention is characterized in that it has a hydrodynamic equivalent of molecular weight of about 56,000 Da and, inter alia, contains the following amino acid sequence:

-V-Q-Q-L-V-D-A-I-Q-E-Y-G-L-

Other sequence sections of the enzyme according to the invention include :

The amino acids used in these amino acid sequences are abbreviated with the usual one-letter code.

The amino acid sequences should be understood such that one or more exchanges or deletions can also be present.

The invention further relates to the following nucleotide sequence sections of L-galactano-1,4-lactone: ferricytochrome c-oxidoreductase

DNA sequences should also fall under the invention,

that of the nucleotides indicated above by a

or several base pairs are modified or one

with the DNA sequences hydriding DNA or a

DNA converted with the DNA sequences via the degenerate genetic code.

Further, the enzyme of the present invention is as follows

Characterized properties: - Oxidation of L-galactano-γ-lactone using cytochrome c as an electron acceptor;

- Biocatalysis of said reaction in that cytochrome c is preferred as the electron acceptor;

and the following physico-chemical properties:

- Typical protein properties and protein reactions (folin and biuret reactions);

- Melting point: about 200 ° C (decomposition below

Exclusion of air and oxygen);

- The native protein consists of only one peptide unit (protomer);

- electrophoretic migration at pH 7.40 in

Acrylamide matrices are anodic;

- soluble in aqueous media including up to 5

% Detergents at pH 4.0 to 10.0;

- it is insoluble in chloroform, benzene, xylene

and other apolar, non-aqueous and with

Water immiscible solvents;

- it denatures in chloroform, benzene and xylene;

Destruction of conformational structure and bioactivity; - it has a typical protein absorption spectrum

in the visible and ultraviolet frequency range; - It adsorbs reversibly on anion and cation exchangers, calcium phosphate gel, hydroxylapatite and hydrophobic phases of low substitution and can be natively subjected to volume distribution chromatography.

As a protein, the enzyme is a macromolecule and, like all biological macromolecules, a polymer. The distinctive property of any polymer is its construction from a repetition of a single or multiple structural units called monomers. In the case of proteins, the monomers are a group of about 20 amino acids. For the enzyme of the invention, these amino acids are described in detail above. These are linked to one another in the protein by a "peptide bond" (R-CO-NH-X) in a defined number and sequence (sequence). They are also reflected in the stated hydrodynamic equivalent of the molecular mass ("molecular weight"). Since you can build 400 (= 20 ² ) different structures by linking only 2 amino acids from a kit of 20 different amino acids, this results in the extremely large number of 20 ¹³ ~ 8.2 • 10 ¹⁶ different with just 13 amino acids Links. For comparison, it should be mentioned that according to the current state of the art only about 10 ⁴ enzymes and about 5.10 ⁴ proteins are known at all. The requirement of the determined sequence of the 13 amino acids shown above as part of the protomer characterizes the enzyme of the invention as an absolute novelty from an extraordinarily large number of at least 8.2 • 10 ¹⁶ possible chemical compounds with protein nature. It results from a single, specific combination of the amino acid building that is common for all proteins. set from the extremely large number of at least 8.2 • 10 ¹⁶ possible substances.

This is sufficient to justify the special substance properties of the new enzyme of the invention disclosed here for the first time. Characterized by the new amino acid sequence as a distinguishing feature of at least 8.2 • 10 ¹⁶ other possible substances with protein nature, it is understandable that the novel protein substance according to the invention also has the new, special properties mentioned, in particular in the form of the catalytic reaction and stereospecificity.

The enzyme is therefore also free from other biological and chemical effects, which are known as properties of other protein substances, built up and known from the same amino acid kit. This applies in particular to the following properties or effects: In addition to the activity determination mentioned, the enzyme can also be physico-chemically produced by the specific, poly- or monoclonal anti-enzyme antibody serum produced according to the invention with the molecularly uniform enzyme protein or its anti-enzyme immunoglobulin fractions according to the various customary immunochemical mixtures and immunobiological methods (eg immunodiffusion, immunoelectrophoresis, RIA, ELISA, etc.) can be determined quantitatively. The enzyme is kept ready for use in the forms customary for proteins; these are sterile, concentrated, buffered solutions of the enzyme in the range of 0.01-10 mg / ml, in particular 0.5-10 mg / ml, preferably 5 mg / ml. All common mixtures in the pH range between 5.0 and 10.0 can be used as buffers. be used, especially between 6 - 9; preferably a 50 mmol / 1 potassium phosphate buffer pH 7.8 is used which contains 10 mmol / 1 glutathione or 10 mmol / 1 2-mercaptoethanol, 10% (v / v) glycerol and 4 mmol / 1 EGTA and 1% (w / v ) Contains CHAPS. The temperature of the dissolved, ready-to-use enzyme according to the invention can be between -180 ° C and + 50 ° C, in particular between -40 ° C and + 40 ° C; storage preferably takes place at -30 ° C. and the dissolved enzyme intended for consumption is kept sterile at 20 ° C. In a particularly preferred form, the enzyme is kept at 0 ° C. in crystal suspension for long-term storage in the specified buffer, which is 70% ammonium sulfate.

The invention will now be further described with reference to Figures 2A, 2B and 2C, of which:

Bioprocess for the synthesis of L-ascorbic acid (vitamin C) from residues (pectin, D-galacturonic acid, L-galactonate) [Fig. 2A] and / or (residual) biomasses (galactose, milk sugar, lactulose, raffinose, stacchyose, melibiosis, mucus, agar, gums, carrageenans, galactans, etc. [Fig. 2B, C] using the enzyme according to the invention.

The enzyme and its fragments have an economically interesting field of application: - As a biocatalyst for L-ascorbic acid synthesis from residues as shown in Fig. 2.

for the analytical determination of chiral L-galactonate in the pH range between 4 and 12, in particular between pH 8 and 11, preferably at pH 7.8,

- for the analytical determination of chiral D-galacturonic acids and L-galactonate,

as a test pack for the determination of chiral L-galactonate and D-galacturonic acid.

The invention also relates to a process for producing and obtaining the enzyme.

In the method according to the invention, the enzyme is obtained from cells, tissues, their cultures or culture supernatant solutions after it has been separated from other accompanying proteins. Pro and eukaryotes come into consideration as cells. It has been found by the inventors that the enzyme according to the invention can be obtained from cells, tissues and their cultures and culture supernatants after separation from other proteins and substances in molecularly uniform, crystallizable and biologically selective acting and also in biologically inactive (denatured) form, in particular thereby that plants and fungi, their cell cultures and culture supernatants, in particular flowers and seedlings, preferably peas, beans, soybeans, corn and cereals, are used as the starting material.

In addition, the enzyme according to the invention can be obtained by using hybridomas of the enzyme-producing cells, in particular plants and fungi, and their cultures and culture supernatants as starting material.

It is also possible to use genetic biological recombinants, their cultures and culture supernatants as starting material, which are a natural, chemically synthesized or isolated from natural and other cells, organelles and tissues, their cultures and culture supernatants, with at least 3 • 3 = 9 bases or Parts and homologous sequences thereof encoding the partial amino acid sequence given according to claim 1.

Furthermore, the enzyme can be obtained by carrying out chemical protein synthesis of the given partial amino acid sequence or parts and homooogenic sequences thereof and the given isolation methods. Cells and tissues of plants, fungi and their cultures and culture supernatants, preferably flowers, seedlings and micelles, are particularly suitable as a natural enzyme source. If cell cultures are used, it is preferred to carry out the culture in a synthetic, serum-free culture medium which corresponds to the prior art. It is particularly advantageous to use cauliflower, bean, pea, soybean, corn and cereal seedlings, and their cells as an enzyme source; not only do they contain the enzyme in sufficient quantities, it can also be separated relatively easily from the many different accompanying protein substances and accompanying enzymes and isolated in a molecularly uniform, crystallizable, ready-to-use state.

In a preferred embodiment, hybridomas of the enzyme-producing cells, in particular the cauliflower, bean, pea, soybean, corn and cereal seedling cells, which can be obtained by conventional methods according to the prior art, are used; genetic biological recombinants which contain a natural, chemically synthesized or one from natural and other cells, organelles and tissues isolated from their cultures and culture supernatants, the given amino acid sequence with at least 3 * 3 = bases or parts and homologous sequences thereof are particularly preferred ; as cells for recombinants, use is made in particular of those which, using residual biomass (whey,

Wood sugar, vegetable oils, mineral oils, glucose, hexoses etc. can be extracted. Various methods of protein chemistry and gene biology which are customary today can be used for their production (for example oligonucleotide synthesis, monoclonal antibodies, polyme lawn chain reaction, antisense bioprocess technology, etc.).

Part of the accompanying proteins of the cell or tissue extract, which is produced by homogenizing the cells, is separated off by centrifugation, preferably by differential centrifugation. The mitochondria obtained are expediently taken up in a 20 mmol / 1 Tris / HCl buffer pH 7.8, which contains 10% (v / v) glycerol, 5 mmol / 1 glutathione, 4 mmol / l EGTA and 400 mmol / 1 sucrose contains. After solubilization of the membrane enzymes and / or octyl glucoside, in particular with at least 1% (w / v) Triton X-100 and subsequent centrifugation of insoluble membrane particles, the protein mass in the supernatant can be separated directly into further components by an anion exchange reaction, DEAE-Sepharose ^R ( fast flow) is preferred as the anion exchange substance. The enzyme fraction eluted here can then be used to obtain the enzyme

Hydroxylapatite are chromatographed. For reasons which are explained below, however, it is preferred in the process according to the invention to separate at least a part, in particular the majority, of the accompanying foreign proteins from the enzyme by targeted separation processes before chromatography on hydroxylapatite.

The use of hydroxylapatite is essential for the structure-friendly extraction of the enzyme. For technical and economic reasons, it is associated with considerable difficulties to chromatograph larger protein volumes on hydroxylapatite columns. On the one hand, hydroxyapatite tends very much with larger protein volumes severe constipation and is therefore unusable. On the other hand, hydroxyapatite is expensive, which prevents its use on a larger scale. For these reasons, it is preferred in the process according to the invention to separate a large part of the accompanying foreign proteins from the crude protein fraction before the chromatography on hydroxylapatite by suitable process steps, in order to thereby decisively reduce the volume of protein which has to be applied to the hydroxylapatite column.

In general, cleaning processes for protein bodies (proteins) and other natural products consist of a sequence of combined separation processes, which take advantage of molecular sizes, charge, shape, affinity, structural stability and molecular surface texture differences between the natural product and the accompanying foreign substances for the separation. Accordingly, numerous combinations of different separation processes for the purification of a protein can be developed. For the handling properties, technical feasibility, automatability and economy of a cleaning process as well as for the quality of the natural product sought, it is not only the type of separation steps that are important, but in particular their optimized design and their sensible combination in a cleaning sequence within the framework of structural stability and other structural parameters of the substance sought. This also means that even the use of similar separation principles (e.g. molecular sieve filtration, dialysis, ion exchange adsorption, etc.), but in different combinations, for the manageability and economy of the cleaning process can be decisive. In certain cases, the use or omission of a single technique (e.g. hydroxylapatite chromatography, affinity chromatography, etc.) at a specific point in the cleaning sequence, or within a limited partial sequence, is of crucial importance for the quality of the natural product sought as well as for the manageability and economy of its Cleaning process. These general relationships and basic principles of natural product cleaning are clearly demonstrated, for example, by the well-known fact that a dialysis, ultrafiltration and lyophilization step is not sensible in an economically reasonable and technically manageable natural product cleaning process before the total initial volume or the initial concentration of the accompanying foreign components of the natural product extract is not was reduced by other process steps.

In a preferred embodiment of the method according to the invention, a part of the accompanying foreign proteins by Positivadsorption to an anion exchanger, particularly DEAE-Sepharose ^® Fast Flow, followed by fractional elution and separated by Positivadsorption on hydrophobic phases and subsequent fractional elution of the enzyme solution. The method according to the invention enables the enzyme to be obtained in its native, enzymatically active conformation.

The invention also includes a further method, which is characterized in that the equivalent chemically and / or biologically synthesized oligonucleotide or antisense nucleotide sequences in vivo or in vitro, which encode the partial sequences given above, with at least 6 bases (or parts and homologous sequences thereof) of these structures by in vitro or in vivo methods, in particular by the polymerase chain reaction (PCR) and / or the antisense technology, used for the production and analysis of products, for example for chiral L-galactonate, age, degree of differentiation and maturation as well as origin, production process, type of food / fruit / vegetables / mushrooms / cells / Tissues, etc. A particularly preferred method of manufacture and recovery is explained in detail below using cauliflower as an example of the enzyme source.

EXAMPLE

Preparation of the cauliflower The leaves are first removed from the cauliflower with a kitchen knife. The cauliflower fruit obtained in this way is cut into small pieces using a scalpel so that it can then be picked up by the homogenizer.

Note: All work is carried out at 4 ° C unless otherwise noted. All buffers are supplemented with glutathione or 2-mercaptoethanol and used immediately before use desired pH value. In all chromatographic methods, the elution process should be registered by measuring the optical density at 280 nm.

Isolation of the mitochondria

The finely chopped, pre-chilled cauliflower fruit is poured with 1.3 times the amount of cold buffer A and with a homogenizer under constant Cooling homogenized (temperature measurement). After homogenization, the homogenate is filtered through four layers of gauze. The filtrate is subjected to centrifugation (600 xg, 200 min, 4 ° C). After centrifugation of the supernatant again (19,000 × g, 20 min, 4 ° C.), the precipitate containing the mitochondria is suspended in 1/4 of the buffer B corresponding to the amount of homogenization used and frozen at −30 ° C.

1st step: Preparation of the crude extract:

Solubilization of the proteins from the mitchondrial membrane: The mitochondrial suspension is thawed with gentle stirring at 37 ° C and 1% (w / v) of Triton X-100 is added. After an incubation of 45 min on the roller mix at 4 ° C, centrifugation is carried out (105,000 x g, 45 min, 4 ° C). The supernatant should be almost clear or slightly opalescent. This is used in the following as a crude extract for the chromatographic purification steps of the enzyme.

Step 2: Perform anion exchange chromatography on DEAE-Sepharose fast flow:

The column filled with the DEAE-Sepharose * fast flow is equilibrated with buffer C (column volume: 500 ml, column length: 30 cm). The optical density at 280 nm should be registered.

The filtered crude extract is applied to the column at a linear flow rate of 90 cm / h and rinsed with 800 ml of buffer C. Then will an increasing linear gradient of 0 - 62.5% buffer D was applied to 6 column volumes and washed out. The enzyme is eluted in about 30% buffer D in the first 1/3 of the gradients. Here it is enriched about 3.4 times compared to the crude extract.

Step 3: Perform the hydrophobic interaction chromatography on Phenyl-Sepharose ^® High Performance 35/100:

A 90% ammonium sulfate solution, which was adjusted to pH 7.8 with a concentrated ammonia solution, is slowly added dropwise to the active fractions at 20% with stirring and the mixture is stirred for a further 30 minutes while keeping the pH value (7.8) constant. The precipitate that may be formed is centrifuged off sharply (> 10,000 × g, 1 h, 4 ° C.) and separated from the solution by decanting. The clear supernatant obtained, which contains the enzyme according to the invention, is applied to the column equilibrated with buffer E and washed out with 1.5 column volumes of buffer E. An increasing gradient of 0-100% buffer F is then applied to 14 column volumes, which corresponds to a decreasing gradient of the ionic strength and at the same time an increasing gradient of the detergent concentration. The enzyme elutes in the middle of the gradient, whereby the enzyme activity can be found in a very narrow range. Enrichments of a factor of 2 and yields of 50-55% can be achieved.

Step 4: fractionated PEG precipitation

The active fractions are collected and 7% (w / v) PEG 6000 is added. After an incubation of The precipitate is centrifuged off for 30 min with gentle shaking on the roller mix at approx. 4 ° C (13500 rpm, JA 14 rotor, 20 min, 4 ° C). The supernatant is mixed with further PEG6000 so that the final concentration of PEG6000 is 30% (w / v). After incubation and centrifugation under the same conditions, the precipitate is taken up in buffer G. After centrifuging off insoluble particles, the clear supernatant is used for the next cleaning step. Enrichments of factor 1.5 and yields of 80-85% can be achieved.

5th step: Chromatography on Bio-Gel ^® HTP-hydroxyapatite:

With the active, clear supernatant of step 4, a run is carried out on a Bio-Gel ^® HTP-hydroxyapatite column (0: 25 mm; L: 204 mm). For this purpose, the column is first equilibrated with buffer G and then the active protein solution is applied at a linear flow rate of 48 cm / h. The column is then washed with two column volumes of buffer G and eluted with a linear gradient from 0-100% buffer H to 9 column volumes, the enzyme being found in the middle region. In this way, enrichments of factor 2 and yields of 55-60% can be achieved. The active fractions are collected and frozen at -30 ° C.

Step 6: Ion exchange chromatography on MONO-Q ^®

The active fractions of step 5 are thawed and diluted 1:10 with buffer I so that the Can adsorb enzyme positively. The diluted protein solution is then applied at a linear flow rate of 152.8 cm / h to the column equilibrated with buffer J (0: 10 mm; L: 100 mm) and washed out with five column volumes of buffer J. A linear gradient of 0-50% buffer K is then applied to 25 column volumes, the enzyme being eluted at approximately 25%. Enrichments of a factor of 25-30 and yields of 80-85% can be achieved. The active fractions are collected and ultrafiltered to concentrate the proteins to the desired volume. The concentrated protein solution is frozen in small portions at -30 ° C for use in step 7.

Step 7: Molecular sieve chromatography on Superose ^® 12 HR 10/30

The stationary phase, which is said to have a good separation performance in the range of 60,000, can be used as a packed column of 10 × 30 mm. The concentrated protein solution obtained in step 6 is applied to the Superose column, which was previously equilibrated with buffer L, at a linear flow rate of 30 cm / h. The enzyme can be found at an elution volume of approximately 12.5 ml.

The active fractions are concentrated as described in step 6. Step 8: Rechromatography on Superose ^® 12:

The concentrated protein solution obtained in step 7 is to re-chromatography on Superose ^® 12 equilibrated in buffer L column (0: 3.2 mm L: 30 mm)

REPLACEMENT BUTT (RULE 26) applied. The enzyme can elute in the maximum of the protein distribution with an elution volume of 1.33 ml. The activity peak is completely congruent with the protein peak.

Claims

claims

1. Enzyme, suitable for the oxidation of L-galactono-γ-lactone to L-ascorbic acid, due to the fact that the enzyme has a hydrodynamic equivalent of a molecular mass of about 56,000 Da and contains the following amino acid sequence:

-V-Q-Q-L-V-D-A-I-Q-E-Y-G-L-

2. enzyme according to claim 1,

characterized in that the native enzyme consists of only one peptide unit (protomer).

3. enzyme according to claim 1 or 2,

characterized in that it has a melting point of about 200 ° C (decomposition with exclusion of air and oxygen).

4. enzyme according to any one of claims 1 to 3,

characterized in that the enzyme is L-galactono-1,4-lactone: ferricytochrome-c-oxidoreductase.

5. Enzyme according to one of claims 1 to 4,

characterized in that the enzyme further has the following sequence segments:

DNA encoding the enzyme of claim 1, the DNA comprising:

(a) the following DNA sequences

or a DNA different therefrom by one or more base pairs, (b) one which hybridises with the DNA of (a)

DNA, or

(c) a DNA related to the DNA of (a) or (b) via the degenerate genetic code.

7. The method for producing the enzyme according to any one of claims 1 to 5, by a) working up cells, tissues, genetic biological recombinants, their cultures and

Culture supernatants after separation from other proteins or substances in a manner known per se, or b) chemical protein biosynthesis using a synthesizer. 8. Use of the enzyme or its fragments according to one of claims 1 to 5

for L-ascorbic acid synthesis

9. Use of the enzyme or its fragments according to one of claims 1 to 5

for the analytical determination of chiral L-galactonate or chiral D-galacturonic acids.

10. Use of the DNA sequences according to claim 6 as cDNA for analysis and synthesis of the corresponding molecular biological equivalence structures and associated functions.