DE2417642A1 - PROCESS FOR THE ENZYMATIC ISOMERIZATION OF GLUCOSE TO FRUCTOSE - Google Patents

Description

ization of glucose to fructose

The invention relates to a process for enzymatic isomerization from glucose to fructose, i.e. a process of this kind in which the properties of the enzymes that are produced by certain Microorganisms are generated when grown in a medium containing xylose, so do these microorganisms allow glucose to isomerize to fructose if they are contacted with glucose.

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

It is also known that the majority of the microorganisms which will be discussed, in spite of the fact that they are able to produce enzymes that are

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has serious disadvantages, such as in particular Difficulties in filtration after isomerization and in purification of the isomerized syrups obtained, due to the presence of colloidal substances that cause turbidity or discoloration (pigments produced by the microorganism and which are not completely removed from the mycelium when it is washed).

The above known microorganisms also have other disadvantages such as difficulty in recovery of the mycelium or mycelium at the end of an isomerization cycle, rapid degradation of the enzymatic activity of the aforementioned Mycelium, which prevents it from being isomerized by recycling it onto a plurality of successive batches Glucose syrup acts.

It has now surprisingly become a special microorganism found, namely a strain of the genus Streptomyces violaceoniger, which is not only able to produce an enzyme to produce isomerization of glucose to fructose, but also no longer the above disadvantages having.

This microorganism, its attribution properties below has been obtained from the Centralbureau voor Schimmel Cultures de Baarn (Netherlands) under the number CBS 409-73 deposited.

The invention thus relates to a method for the enzymatic isomerization of glucose to fructose, which is characterized is that the isomerization enzyme producing microorganism is a strain of Streptomyces violaceoniger, deposited at the Centralbureau voor Schimmel Cultures de Baarn (Netherlands) under the number CBS 409-73, or mutants of which begins.

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Further characteristics of the process emerge from the following Description made clear by the examples and the corresponding figures, referring to advantageous embodiments of the present invention.

In the accompanying figures, FIGS. 1, 2 and 3 relate to Example 1 and show the development on the one hand of the ρ value (curve C ^ ,, Fig. 1), on the other hand the Amount Q of reducing sugars present in the medium, expressed in g / l, (curve C ~, Fig. 2) and furthermore the

V.
ratio _1_ of the volume V ^ of the microorganism to the

lumen V _{2 of} the culture (curve C ₃ , Fig. 3) as a function of time t, expressed in hours, and

4 also relates to example 1 and shows the development of the absolute rotation _value α β, expressed in degrees (curve C-) and the true fructose content of the medium, expressed in%, (curve C ₁ -) as a function from time t, expressed in hours.

The strain of Streptomyces violaceoniger used according to the invention under aerobic conditions in submerged culture in one of the appropriate nutrients as well as xylose and carbohydrate bred containing milieu. The mycelium formed in this way is collected and can be used in glucose syrups will. . '

The culture media used to produce a mycelium of Streptomyces violaceoniger produced according to the present invention can be used, used contain 5 to 15 g of xylose per liter and 5 to 50 g of nitrogen compounds per 1, such as soy, corn water, residues of the vegetation water from potatoes and yeast extracts.

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

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5.0 <^ p " ζ 8.5, preferably 5.5 <(P _H ^ 7.5, 2-5 ° C <t <40 ° C, preferably 30 ° C <£ <35 ° C, duration 24 up to 48 hours.

It was found that this method of culture makes it possible, under these conditions, to obtain an extensive, easily filterable To obtain mycelium or an easily filterable mycelium in abundance, which has a good isomizing activity.

With regard to its use on a glucose syrup filtered the mycelium thus obtained is washed by spraying or pulverizing with water and collected in paste form. It can be kept in this form in the cold (generally at a temperature of the order of -20 C) or be dried by lyophilization, which also enables its storage, but it is used generally immediately by putting it in the one to be isomerized Syrup dispersed.

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

Temperature: 50 to 75 ° C

p ": 6 to 8

Concentration of the milieu of "glucosides": 25 to 60 %

CoCl 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.

It has been shown that the conversion yields of glucose to fructose by using the microorganism according to the invention for a mycelium amount or concentration of 1 volume

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Culture broth to 1 volume of a 50% dextrose solution within 24 hours is in the order of 45%.

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

Appearance of the spores: spherical ^ with a dimension of 0.9 bis 1.2 u and with smooth or slightly rough walls;

Appearance of the aerial mycelium or aerial mycelium: spores load-bearing and forming, gray-brownish, which liquefies into black damp spots and the branching of monopodial Has sporophores or spore carriers;

Appearance of the spirals: they are short, agglomerated, 'with 1 or2 tendrils (viticula); '

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

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

Culture on an "iron peptone agar" medium: no melanoid Pigments (which also do not occur when cultivated on a tyrosine agar medium).

Diastase activity: agar +; Starch + proteolytic activity: milk coagulation

good peptonization gelatin liquefaction

Sugar assimilated by the stem: sucrose

Inositol.

■ rhamnose

Raffinose.

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The totality of the above properties allows to confirm that the strain in question is of the genus Streptomyces violaceoniger (catalogs Waksman & Curtis and Waksman & Henrici). However, it should be noted that that the carbon assimilation spectrum differs somewhat.

Of course, the method according to the invention includes * the use of mutants of the named strain, e.g. natural or artificial mutants, the latter e.g. by irradiation with ultraviolet rays or by Treatment of the parent strain with mutagenic chemicals such as nitrosoguanidine and ethylmethylsulfonate can be.

The following examples illustrate the invention without it to restrict.

example 1

a) Production of the mycelium

525 g of corn water with 50% dry extracts, 1050 g of xylose and 15 g of magnesium sulfate are placed in a 20 liter fermentation vessel containing 15 liters of water, and everything is sterilized.

The p "value is set before the sterilization with ammonia to 6.8 and add 10 g of corn oil to prevent excessive foaming or bubbling.

After a 10-minute sterilization at 120 ° C., the medium is filled with 400 ml of a 48-hour preculture of Streptomyces violaceoniger belonging to the strain CBS 409-73 contained in an Erlenmeyer flask of 2 liters, inoculated.

The fermentation vessel is equipped with a system for mechanical . Stirring, set to 600 rpm, is provided, and the aeration is set to 8 liters of sterilized air per minute «

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The curves in FIGS. 1, 2 and 3 show the development of the p "value (FIG. 1, curve C ₁ ), the amount of reducing sugars present (FIG. 2, curve Cp) and the microorganism volume per culture. Volume (FIG. 3, curve CU) 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 it can be seen that after 24 hours of culture, the amount of Streptomyces obtained reaches a maximum and does not more increases.

The 15 l of the culture are filtered on a Büchner filter which is provided with a preliminary layer of filter earth. 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 in a dextrose solution with 500 g / kg of the solution, the traces of magnesium and cobalt salts, namely 2.4 g / l of MgSO. and 0.24 g / l CoCl ₂ , suspended.

The p “value is determined with the aid of a solution of sodium hydroxide and sodium sulfite adjusted to 6.8, then the temperature of the suspension is brought to 65 ° C and the p "with With the help of an automatic p "measuring device on the value of 6.8 held.

In Table I below, the progressive values of the absolute rotation value a _D and the true fructose content of the medium and of the ρ are summarized as a function of time.

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- 8 ~ Table I.

Isomerization of a 50% dextrose solution P _H 6.8 ± 0.2 · temperature: 65 ° C. ± 1

Time in hours ^a D. % Fructose PH 2 + 40 ° 5 8.5 6.9 ■ 4 + 29 ° 1 16.5 6.8 6th + 12 ° 2 28.0 6.9 16 - / I3 ° 1 45.5 7.0 20th - 15 ° 2 47.5 7.1

With the help of the values summarized in Table I, the corresponding curves C ₄ (a _ß ) and C ₅ (% fructose) of the diagram in FIG. 4 were created, which suggests a substantial isomerization activity, with an isomerization time of 47.5% within hours. Levulose or d-fructose are formed.

Example 2

a) Production of the mycelium

The production took place under the same conditions as in Example 1, with the difference that the corn water were replaced by 300 g of proteins that came from the flocculation of the vegetation water of potatoes, whereby the ρ value was always adjusted to 6.8 with ammonia.

After fermentation for 26 hours (stirring at 600 rpm, air supply 8 l / min.), 750 g are collected after filtration wet cells of Streptomyces violaceoniger.

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b) isomerization of glucose

The Streptomyces cake obtained above is in 15 l of a 50% by weight dextrose solution under the in Example 1 described conditions dispersed. The results obtained are summarized in Table II.

hours 52
. + 18
- 15 Tabel D. II PH ° 5
*1
* 6 6.8
7.0
6.9 time in 3 % Fructose O
5
21 0
27.5
47.5 example

This example illustrates the ease with which the strain can be filtered and the ability to be recycled.

a) Production of the mycelium

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

The corn waters were hydrolyzed by a proteolytic of casein (1% casein, i.e. 150 g, were in an ammoniacal Solution hydrolyzed by the alkaline protease for 8 hours).

After 24 hours of fermentation and practically complete disappearance of reducing substances, one obtains after Centrifugation for 10 minutes at 5000 rpm a Streptomyces volume of 0.6 ccm / 10 ml of culture.

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

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b) Isomerization with recycling of the enzyme

Seven successive isomerizations are listed 15 1 of a 50% dextrose solution.

The first isomerization is carried out with 560 g of Streptomyces cells, which 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 filtration of the the 1st operation from the reaction medium recovered Streptomyces carried out.

other Isomerizations 3, 4, 5 and 6 are on / batches of 15 1 of the syrup carried out by removing the mycelium, which after the filtration from the previous operation was recovered, 145 g of fresh cells from the fermentation vessel 1 added.

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

The results of this whole. Operations are in the following Table III summarized.

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Table III

Seven successive isomerizations performed on the same mycelium

1 1. O/ 2. V 3. Fruc 4th Fruc 5.. Fruit 6 '· ■ '% 7th Of * ·>
O Isomerization Fruc Isomerization Fruc Isomerization tose isomerization tose . Isomerization ; tose isomerization Fruc . Isomerization Fruit (O tose tose 28.5 tose ■ tose Time 42.0 Time 33.0 Time Time Time Time Time in • ' 107 14 · 20th 14th 21, o: ο Hours. Hours. Hours. 42.5 42.0 42.5 39.0 40.0 20th Hours. 27 i 38 27 38 45 • 41, 0 Hours. Hours. Hours. Hours. Hours. ■ 68 · Hours.

This procedure allows 107 1 50% glucose syrups to be added isomerize, whereby the mycelium comes from 30 1 culture broth, while without recycling it would be necessary to use the mycelium from 107 l of culture broth.

Furthermore, the figures in Table III make it possible to establish that, while the average fructose content of the seven syrups was about 42 % , the good retention of the isomerizing activity of the mycelium throughout its use was clearly demonstrated.

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Claims (5)

Claims 1.) Process for the enzymatic isomerization of glucose to fructose, characterized by the use of a strain of Streptomyces violaceoniger as an isomerization enzyme-producing microorganism which has the following taxonomic Has properties or classification properties: Appearance of the spores: spherical, with a dimension of 0.9 to 1.2 µ and with smooth or slightly rough walls; Appearance of the aerial en mycelium or aerial mycelium: bearing and forming spores, gray-brownish, which becomes. black moist spots liquefied and the branching of monopodial sporophores or spore carriers. Appearance of the spirals: they are short, agglomerated, with 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 that is pale pink that produces in the skimmed milk will; Culture on an "iron peptone agar" medium: no melanoid Pigments (which also do not occur when cultivated on a tyrosine agar medium); Diastase activity: agar +; Strength +; proteolytic activity: milk coagulation good peptonization gelatin liquefaction; sugars assimilated by the stem: sucrose Inositol Rhamnose raffinose 4098A5 / 1070 - 14 or one of the mutant tribe of this tribe. 2.) Process for the enzymatic isomerization of glucose too Fructose, characterized by the use of a stem from Streptornyces violaceoniger, registered with the Centralbureau voor Schimmel Cultures de Baarn (The Netherlands) under the number CBS 409-73, as an isomerization enzyme producing Microorganism. 3.) The method according to claim 2, characterized in that a mutant strain of the Centralbureau voor Schimmel Cultures de Baarn (Netherlands), deposited under the number CBS 409-73 used. 4.) A method for growing one of the strains of Streptomyces violaceoniger, which are used in the method according to any one of claims 1 to 3, characterized in that the strain under aerobic conditions · in a culture medium containing 5 to 15 g of xylose per 1 and 5 up to 50 g of nitrogenous substances, such as soy, corn water, residues of vegetation water from potatoes and yeast extracts per 1, is cultivated, whereby the P _H - »temperature and duration conditions are the following: 5.0 (ρ »<8.5, preferably 5.5 <p "<7.5, 25 ° C <t <40 ° C, preferably 30 ° C <t <35 ° C, duration 24 to 48 hours, the mycelium thus obtained being collected in paste form after filtration and washing. 5.) Process for the enzymatic isomerization of glucose too Fructose, characterized in that at least one of the strains according to one of Claims 1 to 3 among the following Conditions is used: AQ98A5 / 107Q Temperature: 50 to 75 ° C p _H : 6 to 8 - Concentration of the medium in glucose _: 25 to 60% of CoCl ₂ : 0 to 0.50 g / l of MgSO ₄ : 0 to 2.4 g / l 409845/1070