DE2417642B2 - Process for the enzymatic isomerization of glucose to fructose - Google Patents

Description

The invention relates to a process for the enzymatic isomerization of glucose to fructose, i. H. a Process of this kind in which the properties of enzymes produced by certain microorganisms when grown in a medium containing xylose, so will these microorganisms allow glucose to isomerize to fructose when contacted with glucose.

It is known that certain microorganisms belonging to the families Pseudomonas, Bacillus, Lactobacillus and Streptomyces are capable of producing the above isomerization enzymes.

It is also known that the majority of the microorganisms which will be discussed, in spite of serious to the fact that they are capable of producing enzymes that isomerize glucose Has disadvantages, such as in particular difficulties in the filtration after isomerization and in the purification of the isomerized syrups obtained, due to the presence of colloidal substances that Cloudiness or discoloration (pigments that are and are not secreted by the microorganism be completely removed from the mycelium during its washing) (Agr. Biol. Chem, 30, 1966, 1247-53).

The above known microorganisms also have other disadvantages such as difficulties in Recovery of the macelium or mycelium at the end of an isomerization cycle, rapid degradation of the enzymatic activity of said mycelium, which prevents it from being recycled to a A plurality of successive batches of glucose syrup to be isolated acts.

A special microorganism has now been found in a surprising manner, namely a Strain of the genus Streptomyces violaceoniger that is not only capable of isomerizing an enzyme from glucose to fructose, but also no longer the above disadvantages having.

This microorganism, the assignment properties of which will be described below, was discovered in Centralbureau voor Schimmel Cultures de Baarn (Niederlange) deposited under the number CBS 409'73. bo

The invention thus relates to a process for the enzymatic isomerization of glucose to fructose, which is characterized in that one isomerizing enzyme-producing microorganism Strain of Streptomyces violaceoniger, deposited with the Centralbureau voor Schimmel Cultures de Baarn (Netherlands) under the number CBS 409-73, or mutants thereof.

Further characteristics of the process emerge from the description below, which is followed by the Examples and the corresponding figures relating to advantageous embodiments of the present invention relate to be illustrated. .

In the figures, FIGS. 1, 2 and 3 relate to Example 1 and show the development of the pH value (curve Ci, FIG. 1) on the one hand and the amount Q of reducing sugars present in the medium, expressed in g / L (curve C ₂ , Fig. 2) and furthermore des

Ratio - ± of the volume VJ of the microorganism

'1

mus to the volume V2 of the culture (curve Cy ₁ Fig. 3) as a function of the time t, expressed in hours, and

the F i g. 4 also relates to Example 1 and shows the development of the absolute rotation value expressed in degrees (curve G) and the true fructose content of the medium, expressed in%, (curve Cs) as a function of time t, expressed in hours.

The Streptomyces violaceoniger strain used according to the invention is produced under aerobic conditions in submerged culture in one containing the appropriate nutrients such as xylose and carbohydrate Bred milieu. The mycellium thus formed is collected and can be used in glucose syrup will

The culture media used for the production of a mycelium of Streptomyces violaceoniger, which according to the present invention can be used contain 5 to 15 g xylose per 1 as well 5 to 50 g of nitrogen compounds per liter, such as. B. Soy, Corn water, residues of the vegetation water from potatoes and yeast extracts.

The pH, temperature and duration conditions of this culture or breeding are incidentally:

5.0 <pH <8.5, preferably 5.5 <pH <7.5,
25 ° C <f <40 ° C, preferably 30 ^c C <f <35 ° C,
Duration 24 to 48 hours.

It turns out that this method of culture makes it easy to grow under these conditions to obtain filterable copious mycelium or an easily filterable mycelium in the upper flow, the one has good isolation activity.

With a view to its use in a glucose syrup, the mycelium obtained in this way is filtered and washed Spray or pulverize with water and collect it in paste form. It can be in this form in the cold (generally at a temperature of the order of -20 ° C) or through Lyophilization can be dried, which also allows its storage, however it is generally used immediately by dispersing it in the syrup to be isomerized.

The filtered mycellium is washed and under the following general isomerization conditions used:

Temperature: 50 to 75 ° C pH: 6 to 8 Concentration of the milieu of glucose: 25 to 60% CoCI ₂ : 0.0 to 0.50 g / l MgSO ₄ : 0.0 to 2.4 g / l

The length of time depends on the conditions and the amount of mycelium used.

from glucose to fructose by using the microorganism according to the invention for an amount of mycelium or concentration of 1 volume of culture broth to 1 volume of a 50% dextrose solution within 24 Hours on the order of 45% lit-gt

The classification properties or the taxonomic properties of the strain according to the invention from Streptomyces violaceoniger are as follows:

Appearance of the spores: spherical, with a dimension of 0.9 to 1.2 μ and with smooth or little rough maintenance;

Appearance of the aerial mycelium or aerial mycelium: bearing and forming spores, gray-brownish, the liquefies to black damp spots and the branches of monopodial sporophores or Having spore carriers;

Appearance of the spirals: they are short, agglomerated, with 1 or 2 tendrils (viticula);

Appearance of the vegetative mycelium: it is whitish to yellowish (to gem-colored).

It is possible to extract a soluble pigment, the pale pink and the one in the skimmed milk is produced.

Culture on an "iron peptone agar" medium: no melanoid pigments (which are also present in the culture tyrosine agar medium).

Diastase activity: agar +; Strength +
Proteolytic Activity:

Milk coagulation

good peptonization

Gelatin liquefaction

Sugars assimilated by the stem:
Sucrose
Inositol
Rhamnose
Raffinose

All of the above properties make it possible to confirm that the one in question Strain of the genus Streptomyces violaceoniger (catalogs Waksman & Curtis and Waksman & Henrici) It should be noted, however, that the spectrum of carbon assimilation is somewhat different.

The inventive method includes the use of mutants of said strain, which z. B. can be natural or artificial mutants, the latter e.g. B. by exposure to ultraviolet rays or by treating the parent strain with mutagenic chemicals, such as. B. nitrosoguanidine and Ethanesulfonic acid ethyl ester can be obtained.

The following examples illustrate the invention

example 1
a) Production of the mycelium

In a fermentation vessel of 201, the 151 water contains 525 g of corn water with 50% dry extracts, 1050 g of xylose and 15 g of magnesium sulfate one, with everything being sterilized.

The pH is adjusted to 6.8 with ammonia before the sterilization and 10 g of corn oil are added to a to prevent excessive foaming or bubbling.

After "; iner 10 minute sterilization at 120 ⁰ C, the medium was 400 ml of a preculture of Streptomyces violaceoniger 48stiindigen, from the strain CBS 409-73 flask Erlenmeyer-contained in a 21, aneeimDft.

The fermentation vessel is equipped with a system for mechanical stirring, set at 600 rpm, provided, and the ventilation is on 81 sterilized air set per minute

The curves in FIGS. 1, 2 and 3 show the development of the pH value (FIG. 1, curve C ₁ ), the amount of reducing sugars present _{(FIG. 2, curve C 2} ) and the microorganism volume per Culture volume (Fig. 3, curve C ₃ ) as a function of time, the latter variable being measured by the height of the sediment after centrifugation in a graduated measuring cylinder or test tube under standard conditions, ie 5000 rpm, for 10 minutes .

From Fig. 3 shows that after 24 hours of culture the Streptomyces amount obtained reaches a maximum and no longer increases

The 151 of the culture are placed on a Büchner filter, which is provided with a preliminary layer of filter earth, filtered. The collected Streptomyces is washed with drinking water.

In this way, 800 g of moist cells are collected without difficulty.

b) glucose isomerization

The 800 g of Streptomyces obtained above are suspended in a dextrose solution at 500 g / kg of the solution containing traces of magnesium and cobalt salts, namely 2.4 g / l of MgSO ₄ and 0.24 g / l of CoCl _2.

The pH is adjusted to 6.8 using a solution of sodium hydroxide and sodium sulfite, then the temperature of the suspension is brought to 65 ° C and the pH with the help of an automatic pH meter kept at the value of 6.8.

Table I below summarizes the progressive values of the absolute rotation value * _o and of the true fructose content of the medium and of the pH as a function of time.

Time in hours CD % Fructose pH 2 + 40 ° 5 8.5 6.9 4th + 29 ° 1 16.5 6.8 6th + 12 ° 2 28.0 6.9 16 -13 ° 1 45.5 7.0 20th -15 ° 2 47.5 7.1

With the help of the values summarized in table 1, the corresponding curves G («o) and Cs (% fructose) of the diagram in FIG -Fructose are formed.

Example! 2
a) Production of the mycelium

The production was carried out under the same conditions as in Example 1, with the difference that the Corn water has been replaced by 300 g of proteins that help flocculate the vegetation water of potatoes originated, the pH was always adjusted to 6.8 with ammonia.

After fermentation for 26 hours (stirring at 600 rpm, ₁ air supply 8 l / min.), One collects after the

Filtration 750 g of wet cells from Streptomyces violaceoniger.

b) isomerization of glucose

The Streptomyces cake obtained above is dissolved in a 50 weight percent dextrose solution dispersed under the conditions described in Example 1. The results obtained are shown in Table II summarized.

Table II 52 ° 5
+18 ⁰ I.
-15 ° 6 % Fructose pH Time in hours 0
27.5
46.5 6.8
7.0
6.9 0
5
21

Example 3

This example illustrates the ease of filtration of the strain and the possibility of removing it recycle.

a) Production of the mycelium

Two fermentation vessels of 15 1 are under the general conditions described in Example 1 prepared.

The corn waters were treated with a proteolytic hydrolyzate of casein (1% casein, ie 150g, were 25

30th

in an ammoniacal solution hydrolyzed by the alkaline protease for 8 hours).

After 24 hours of fermentation and practically complete disappearance of reducing substances after centrifuging for 10 minutes at 5000 rpm, a Streptimyces volume of 0.6 ecm / 10 ml of culture.

Filtration of this culture media yields 580 and 560 g of moist Streptomyces cells, respectively.

b) Isomerization with recycling of the enzyme

Seven successive isomerizations are carried out on 151 of a 50% strength dextrose solution the same conditions as example Ib. The first Isomerization is performed with 560 g of Streptomyces cells, the come from the second fermentation vessel, carried out.

The second isomerization is carried out with a new batch of 15 1 50% glucose syrup with the after the filtration of the reaction medium from the 1st operation recovered Streptomyces carried out.

The isomerizations 3, 4, 5 and 6 are carried out on others Batches of 151 of the syrup carried out by one the mycelium recovered after filtration from the previous operation, 145 g of fresh cells from fermentation vessel 1 were added.

A 7th and final isomerization is carried out without adding fresh Streptomyces cells.

The results of all these operations are in the Table III below.

Table III

Seven successive isomerizations performed on the same mycelium

1. 2. '3. 4. 5. 6. 7.

Isomerization isomerization isomerization isomerization isomerization isomerization isomerization

% Fructose

Time

% Fructose

Time

% Fructose

Time

% Fructose

Time% fructose

Time

% Fructose

Time

% Fructose

14 42.0
Hours.

20 33.0
Hours.

27 39.0
Hours.

14 28.5
Hours.

38 42.5
Hours.

42.0 38 42.5 45 40.0

Hrs . Hrs.

This procedure makes it possible to isomerize 107150% glucose syrups, whereby the mycelium 301 Culture broth originates, while without recycling it would be necessary to use the mycelium 1071 culture broth originated.

Furthermore, the numbers in Table III allow note that while the average fructose content of the seven syrups was about 42%, the good one Maintaining the isomerizing activity of the mycelium throughout its use has been clearly demonstrated became.

Ultraviolet radiation mutation

It is allowed a culture of Streptomyces violaceoniger CBS 409-73 on a slant agar at 20 21.0
Hours.

68 41.0
Hours.

Base of corn flour of 60 g / l at a pH 7

sporulate.

The spores are collected by looking at the surface of the culture in the presence of a small one Scrape off amount of sterile tryptone saline and suspend it in 10 ml of the diluent

Bring tryptone salt

The suspension thus obtained is placed in a sterile crystallizer and allowed to stir slowly Subjected to the help of a magnetic stirrer. The suspension is irradiated using an ultraviolet lamp (e.g. Philips »125W black light), which is attached at a distance of 20 cm. Every 10 minutes within 1 hour, 0.1 to 0.2 ml is withdrawn the suspension that is on the surface of a

Agar-agar milieus containing 0.5% xylose and 1% yeast extract, housed in Petri dishes, applies. After culture at 30 °, the isolated colonies are placed on inclined agar-agar on the base of cornmeal.

Starting from inclined agar cultures prepared in this way take 10 ml of a broth containing 1% dextrose and 1% yeast extract in a conical vial of Contains 50 ml. Cultivation is carried out with stirring at 30.degree. C. for 2 days. With the help of 5 ml of the thus obtained Pre-culture is inoculated with 50 ml of a production broth containing 1.2% xylose and 1% yeast extract in one Contains conical vials of 500 ml.

Cultivation is carried out with stirring at 30 ° for 3 days. 5 ml of the obtained in this way are then withdrawn Culture which is placed in a test tube containing 25 ml of a solution kept at pH 7.5 and 36 g / l dextrose and contains 5 g / l magnesium sulfate. The sample tubes are then placed in a water bath at 65 ° C for 7 Hours placed.

After cooling, the microorganism cells are separated off by filtration or centrifugation and the fructose formed in the solution is determined by means of a colorimetric technique with resorcinol an auto analyzer.

The following table shows the fructose concentrations obtained:

Fructose Strain A-6 Fructose g / l A-7 g / l CBS 409/73 3 A-8 1 CBS 409/73 3.4 A-9 2.6 CBS 409/73 3.2 A-10 3 Strain A-I 2.3 A-Il 3.5 A-2 3.8 A-12 4.8 A-3 4.5 A-13 4.3 AA 3.5 2.7 A-5 4.0 2.9

A variant of the production of the mutants of the strain violaceoniger CBS 409-73 consists in one Irradiation with ultraviolet light, whereby the spore suspension was prepared as above, but incubating for 16 to 24 hours to stimulate germination of the spores.

Mutation with the help of nitrosomethylguanidine

To a manufactured as indicated above

nitrosomethylguanidine is added to the spore suspension such an amount as to obtain a concentration of 1 to 3 mg / ml. The contact is left at 30.degree for 30 to 120 minutes. Every 30 minutes, 0.5 ml of the suspension is withdrawn, which is poured into 0.5 ml of a sterile

! ■ j Glykokoü solution of 1% introduces. After 1 hour Contact is isolated on the surface of an agar medium containing 0.5% xylose and 1% yeast extract, contained in petri dishes. After a culture at 30 ° C., the isolated colonies are applied inclined agar-agar based on corn flour.

Starting from the inclined agar samples produced in this way, 10 ml of a boullin are inoculated 1% dextrose and 1% yeast extract in a conical vial of 50 ml. Cultures under Stir at 30 ° for 2 days. With the help of 5 ml of the preculture obtained in this way, 50 ml of one is inoculated Production broth containing 1.2% xylose and 1% yeast extract in a conical bottle of 500 ml. Cultivation is carried out with stirring at 30 ° for 3 days.

j »One then takes 5 ml of the culture thus obtained, the is introduced into a test tube containing 25 ml of a solution kept at pH 7.5 and 36 g / l of dextrose and Contains 5 g / l magnesium sulphate. The test tubes are then placed in a water bath at 65 ° for 7 hours.

placed.

After cooling, the microorganism cells are separated off by filtration and centrifugation and one determines the fructose formed in the solution by a colorimetric technique with resorcinol on a Auto analyzer.

For this purpose 2 sheets of drawings

Claims (1)

Claim: Process for the enzymatic isomerization of glucose to fructose at a temperature of; 50 to 75 ° C, a pH value of 6 to 8, a concentration of glucose from 25 to 60%, of CcCb from 0 to 0.50 g / l and of MgSO ₄ from 0 to 2.4 g / l, characterized by the use of a strain of Streptomyces violaceoniger with the depository number CBS 409-73 or a mutant strain of this strain.