DK146263B - PROCEDURE FOR ISOMERIZING GLUCOSE TO FRUCTOSE - Google Patents

Description

(19) DENMARK (S ')

(12) PUBLICATION <n, 146263 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Patent Application No: 3858/75 (51) InLCI.3: C12P 19/24 (22) Filing Date: Aug 27, 1975 (41) Aim. available: 28 fob 1977 (44) Submitted: 15 Aug 1983 (86) International Application No: - (30) Priority: - (71) Applicant: 'MITSUBISHI CHEMICAL INDUSTRIES LIMITED; Tokyo, JP, 'SEIKAGAKU KOGYO CO. LTD;

Tokyo, JP.

(72) Inventor: Yoshtmasa 'Fujita; JP, Akiyoshi 'Matsumoto; JP, Hachlro 'lehlkawa; JP, Tadashi 'Hishlda; JP, Hideo * Kato; JP, Hiroshi * Takamisawa; JP.

(74) Plenipotentiary: International Patent Office (54) Process for Isomerization of Glucose to Fructose

The invention relates to a method for isomerizing glucose to fructose with immobilized glucose isomerase.

It is known that glucose isomerase is an enzyme capable of converting glucose into fructose and vice versa, and it is used ® to produce fructose from glucose.

Such glucose isomerase has been found in various microorganisms, such as e.g. bacteria such as those belonging to the genera Pseudomonas, Bacillus and Lactobacillus, actinomycets such as Streptomyces ^ phaeochromogenus, Streptomyces fradie, Streptomyces roseochromogenus, O Streptomyces olivaceus, Streptomyces californicus, Streptomyces viaceus

For practical purposes, it is preferred to use glucose isomerase isolated from Streptomyees in an insoluble form which is obtained either by subjecting microorganism cells containing glucose isomerase to a heat treatment to obtain an intracellular fixation or by associating glucose isomerase isolated from the cells with a carrier. by adsorption, ion exchange, covalent bond formation or inclusion. In this connection, reference is made to Chemical Abstracts Vol. 69, 64824 d (1968) Tsumura et al .; USP 3,788,945; Applied Microbiology, April 1971, pp. 588-593, G.W.Strandberg et al .; and Biotechnology and Bioengineering Vol. XIV, pp. 509-513 (1972), G.W. Strand-berg et al.

Almost all of the glucose isomerase found in microorganism cells requires metal ions that may differ depending on the origin of the isomerase. It is known that glucose isomerase from Streptomyees requires magnesium ions and that the activity and resistance to heat are increased in the presence of cobalt ions (cf.

N. Tsumura et al. Agr. Biol. Chem. Vol. 29, 1126 (1965) and T. Takasa-ki, Agr. Biol. Chem. Vol. 30, p. 1249 (1966)).

In commercial production of fructose from glucose, magnesium and cobalt ions have been present in the reaction mixture. In the meantime this is not desirable as various heavy metals ions comprising cobalt ions are not allowed as food additives in many countries, so the use of cobalt ions in fructose production should be avoided.

It has now been found that the activity of immobilized glucose isomerase can be maintained for a long time without the use of cobaltions when adding certain amounts of water-soluble iron salt with magnesium salt to the glucose isomerization mixture.

Therefore, the process of the invention provides an efficient utilization of imrobilized glucose isomerase in converting glucose into fructose, and the process is characterized in that 0.005 - 5 mmol / liter iron ions are present together with 2 - 20 mmol / liter magnesium ions in the reaction mixture. .

The invention is explained in more detail below.

Examples of sources of iron ions which can be used in the process include water-soluble iron salts, such as e.g. inorganic iron salts such as ferrous sulfate, ferric sulfate, ferrochloride, ferric chloride, ammonium ferric sulfate, ammonium ferrous sulfate and iron nitrate, and organic iron salts such as iron tartrate and mixtures thereof. It should be noted, however, that certain organic iron salts, which are water-soluble, produce a diminished formation of iron ions if the base anion forms strong coordination bonds to iron, resulting in a so-called "masking effect".

Furthermore, among the inorganic saline iron phosphates are inconvenient, as they have poor solubility in water and provide an insoluble precipitate. Ferric chloride, ferrous sulfate, iron lactates, iron citrates and ammonium iron citrates, which are permitted as food additives according to Japanese food law, are examples of water-soluble iron salts which can be used according to the invention. As can be seen from the examples below, the water-soluble iron salt can be either divalent or trivalent.

Although the mechanism of action of iron ions in the process of the invention has not yet been elucidated, it is believed, without limiting the invention by any theory, that if iron ions are present with magnesium ions in the reaction mixture, the iron ions will attach to the enzyme to form certain complexes. that resists the deactivation of the enzyme due to chemical effects and / or heat effects. Thus, it is theoretically possible to subject glucose isomerase to pretreatment with an aqueous iron salt solution and thereby achieve the intended effect without the addition of iron salt due to the presence of iron ion near the isomerase, but in practice in such cases iron salt must be added to the reaction mixture. thereby maintaining a constant level of concentration of iron ion in the reaction mixture, as the iron ions attached to the glucose isomerase to form complexes will later dissociate and be removed from the reaction mixture with the product, especially by a continuous process.

The concentration of iron ions in the reaction mixture is preferably maintained from 0.02 to 0.5 m mole / liter.

Examples of water-soluble magnesium salts which can be used with the iron salt of the present process include magnesium sulfate, magnesium chloride, magnesium sulfite, magnesium carbonate and mixtures thereof.

The isomerization of glucose to fructose by the present process is suitable for continuous fabrication, in which an aqueous glucose solution is continuously contacted with immobilized glucose isomerase to obtain fructose. The process is particularly suitable for a continuous process in which an aqueous glucose solution is passed through a column in which glucose isomerase is adsorbed on a carrier to achieve conversion of glucose to fructose.

Carriers suitable for insolubilizing glucose isomerase include: (1) a macroporous anion exchange resin whose matrix is styrene-divinyl copolymer and whose anion exchange group is of the quaternary ammonium type. Such a material is further described in U.S. Patent No. 4,778,97o.

(2) a cross-linked agar cyanogen halide derivative in particulate form.

The latter material is prepared by treating agar with a crosslinking agent such as epichlorohydrin under alkaline conditions to introduce crosslinking, followed by high temperature heating and hot water washing to remove hot water soluble materials and subsequent treatment of the crosslinked agar with a aqueous solution of cyanogen halide, such as cyanogen bromide under alkaline conditions.

It has been found that in carrying out the process, the activity of isomerase is maintained in this manner for a long time, and the isomerization reaction of glucose to fructose is therefore satisfactory for commercial use.

Conveniently, an aqueous glucose solution can be subjected to continuous treatment according to the invention at a pH of from 5.5 to 9, a concentration of 20-70 wt% and a temperature of 40-80 ° C, and such a solution is passed through a column as described with a specific volumetric feed rate of 0.1-20 per. hour.

The process according to the invention is further illustrated by the following examples.

c 146263 5

Example 1

Glucose isomerase was extracted by a conventional ultrasound treatment from "Glucose Isomerase NAGASE", which consisted of cells of a strain of Streptomyces phaeochromogenes, intracellularly fixed by heat treatment and available from "Nagase Sangyo Kabu-shiki Kaisha, Osaka, Japan".

The activity titer defined below of such an extract was 200 units / ml.

An insoluble glucose isomerase was then prepared by mixing 75 ml of such extract with 15 ml of a wet macroporous strong basic anion exchange resin ("Diaion PHA" from Mitsubishi Chemical Industries, Limited, whose matrix was styrene-divinylbenzene copolymers and whose ion exchange group was a quaternary ammonium group) at room temperature for 6 hours to obtain adsorption of the isomerase on the anion exchange resin. The degree of adsorption was measured to more than 99%.

The insoluble glucose isomerase prepared in this way was packed in a sheathed column of 8 mm internal diameter.

A 3 molar aqueous glucose solution containing 5 m moles of magnesium sulfate and 1 m mole of ferrous sulfate (the concentrations indicated are ion concentrations) was passed through such a column maintained at a temperature of 67 ° C to obtain continuous isomerization while warming. water through the jacket.

The degree of activity retention, as defined below, after 10 and 20 days, is given in Table 1.

Examples 2-6

The procedure was similar to Example 1, with the exception that different metal salts were used instead of ferrous sulfate at the concentrations listed in Table 1. The degree of activity retention for the glucose isomerase after 10 and 20 days is also indicated in Table 1, which shows that better results are obtained by the addition of iron salts than other salts, with an extended isomerase activity being obtained.

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Table 1

Example Compound Ion Concentration Degree of activity number (m mol / liter) conservation (%) 10 days 20 days 1 Ferrochloride 1.0 70 45 2 Ferric chloride 0.5 62 37 3 Iron tartrate 1.0 68 40 4 - 33 11 5 Cobalt chloride 1.0 55 25 6 Calcium Chloride 1.0 30 13

Examples 7-13 In these examples, proceed as in Example 1, however, the isomerizations were performed at a temperature of 72 ° C using ferric sulfate at various concentrations as listed in Table 2.

The degree of activity retention after 7 and 13 days is also given in Table 2.

The lowest values of activity decrease are observed at a concentration in the range of 0.02 to 0.5 m mole / liter of iron ion.

Table 2

Example Concentration of Degree of activity number iron ion m mol / liter) preservation (%) 7 days 13 days I 7 0 13 5 i 8 0.02 40 22 9 0.05 52 35 10 0.1 62 49 I 11 0 , 25 50 33 12 0.5 38 11 13 3.0 26 9

Examples 14-16

The glucose isomerase used was extracted from a culture medium with incubated Streptomyces albus YT No. 5, deposited with "The Fermentation Research Institute", Japan (deposit number: η 146263 FERM P-463). The isomerase was insolubilized by the procedure of Example 1 and was used for continuous isomerizations as in Examples 1, 4 and 5. The degree of activity retention after 7 and 13 days, respectively, is given in Table 3.

Table 3

Example Compound Ion Concentration Degree of activity number (m mol / liter) preservation (%) 7 days 13 days 14 Ferrosulfate 1.0 75 60 15 - - 40 25 16 Cobaltochloride 1.0 62 49

Examples 17-19

Preparation of cross-linked agar particles.

1 liter of 4% agar particles, "Sepharose 4B" from Pharmacia Fine Chemicals, Sweden, was carefully mixed with 1 liter of aqueous sodium hydroxide solution (1 mol / liter) at room temperature. 30 ml of epichlorohydrin was added to the above mixture while kept at 60 ° C on a water bath and shaken gently for 2 hours, thereby causing a crosslinking reaction.

Then, the reaction mass was heated in an autoclave to a temperature of 120 ° C under 2 atmospheric pressure for 2 hours. The hot mass was washed with water at 90 ° C to remove the hot water-soluble material and left to cool to obtain cross-linked agar particles.

Activation of cross-linked agar particles.

The cross-linked agar particles were subjected to vacuum filtration to remove excess water and cooled to 4 ° C. To a mixture of 50 g of the particles thus obtained and 80 ml of a 2% aqueous cyanogen bromide solution was added dropwise a 2 N aqueous sodium hydroxide solution in such an amount that the solution was kept at a pH of 11 + 0.5 , thereby activating the cross-linked agar.

After the alkali absorption had ceased (resulting in an increase in the pH), the alkali addition was stopped and the reaction mass subjected to vacuum filtration and washed with water to obtain activated and cross-linked agar particles.

Insoluble glucose isomerase.

A mixture of 50 g of the thus obtained activated particles and the glucose isomerase extract of Example 1 with an activity titer of 25000 units was kept at a temperature of 4 ° C for 18 hours to obtain adsorption of the isomerase onto the particles to obtain insoluble glucose isomerase in particulate form.

Isomerization reaction.

The aforementioned insolubilized glucose isomerase with an activity titer of 4000 units was packed in a sheathed column (inner diameter 1.5 cm, length 12 cm) through which an aqueous glucose solution at a concentration of 3 moles / liter and containing various metal ions listed in Table 4 were directed downwards in an amount of 8.5 ml per ml. hour at a temperature of 67 ° C to obtain continuous isomerization.

Table 4 lists the nature and concentration of metal ions that led to the glucose solution and the variation in the amount of fructose formed during the periods indicated.

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The degree of activity retention and the activity titer indicated in the above description and requirements are measured as follows.

Degree of activity retention.

An aqueous substrate solution containing glucose, magnesium sulfate, cobaltochloride and phosphate buffer (pH = 7.2) at a concentration of 0.1 mol / liter, 0.05 mol / liter, 0.001 mol / liter and 0.05 mol / liter, respectively, is prepared. A predetermined amount, usually between 1 and 10 ml, of insoluble glucose isomerase not previously used for isomerization is added to 100 ml of the substrate solution and isomerization is carried out at a temperature of 70 ° C for 60 minutes with gentle stirring. A similar isomerization is carried out with an insolubilized isomerase which has been used a predetermined number of days.

The isomerase is then separated by filtration and the amount of fructose in the filtrate is determined by the cysteine-carbazole method.

The amount of fructose produced per ml of the insoluble glucose isomerase is calculated, the values obtained are denoted "A" for the isomerase not previously used and "A ,, r for the previously used. Then the degree of activity retention is calculated from the following equation: 1 x 100 ( %)

Activity titer of glucose isomerase extract.

A mixture of aqueous solutions of 0.2 ml of 1 m D-glueose, 0.2 ml of 0.05 m MgSO4.7H2O and 0.2 ml of 0.5 m phosphate buffer (pH = 7.2) is added to 0.2 ml glucose isomerase extract and dilute the solution with water to a total of 2 ml. The resulting dilute solution is kept at a temperature of 70 ° C for 60 minutes to obtain isomerization and the reaction is quenched by the addition of 2 ml of a 0.5 m aqueous perchloric acid solution. Then the amount of fructose formed is determined by the cysteine-carbazole method. The "unit" used in reference to the activity titer above is defined as the amount of fructose (mg) produced, the amount of isomerase extract (ml).