DE1792748A1 - METHOD OF PRODUCING GLUCOSE ISOMERIZING ENZYME - Google Patents

Description

DR. MOLLER-BORe OIPL.-PHYS. DM. IviANITZ DIPL-CHEM. DR. DEUFEL DlPL-ING. FINSTERWALD dipl.-ing. ÜRÄMK0W

PATENT LAWYERS

179274a

2 p.?.?: "· 19Ä

D / Sh - A 2210

AGMCY OF INDUSTRIAL SCIENCE AND TECHNOLOGY 1, 3-cnome, Kasumigaseki, Chiyoda-ku, Tokyo,

Japan

Process for the preparation of glucose isomerizing enzyme

Priority: Japan from May 11th 1965 Mr. 27525/65

The invention relates to a method for producing glucose isomerizing enzyme (enzyme that converts glucose into Fructose) for industrial use.

The methods heretofore known for converting glucose to fructose include alkali methods, including of methods using anion exchangers, and the enzymatic ^ method. In the practical However, when applied, these 2 groups of methods have shown the following disadvantages.

The alkali methods, such as those described in U.S. Pat 2 356 664, 2 487 121 and 2 746 889 are

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cause the loss of sugar through decomposition. There are also other deficiencies, including an insufficient one Conversion ratio.

The enzymatic method is based for the transfer of Glucose in! Fructose on the activity of enzyme. The only enzyme known so far is that from bacteria produced glucose isomerizing enzyme.

S. Mitsuhashi and Mitarb, were the first to find that the enzyme preparation derived from Lactobacillus pentosus is capable, albeit to a limited extent, of converting glucose to fructose. (See J. Biol. Ghem. 204 , 1011-1018 (1953)).

Marshall and co-workers discovered an enzyme in Pseudomonas hydrohila that can convert glucose into fructose in the presence of arsenate. (See Science, 125 , 648-649 (1957)). It was then reported that the same enzyme was found in Aerobacter cloacae by S. Tsumura et al. (Agr. Biol. Chem., (Japan), £ 2, 616-625 (1961)). These bacterial-origin glucose isomerizing enzymes have all or some of the following deficiencies in the manner of preparation, the amount and the quality of the yield, and because of these deficiencies, they have not been usable for industrial production and utilization until now. Therefore, the isomerization reaction using these enzymes has not yet been put into technical practice.

The sizes are as follows:
1. In the production of a glucose isomerizing enzyme

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from bacteria, the culture medium requires the inclusion of expensive pure D-xylose.

2. The yield of enzyme is low.

3. The isomerization action resulting from the use of the enzyme is due, requires the presence of harmful arsenate and / or Ma.

4. The glucose isomerizing enzyme obtained lacks too much the stability required for industrial use the quality.

The object of the present invention is a process for the preparation of a glucose isomerizing enzyme which is free from the above-mentioned defects and has sufficient industrial applicability.

According to the invention, a new type of glucose isomerizing agent Obtained by a certain strain of a microorganism, which will be described below, in an enzyme Medium is grown that contains xylan and / or xylan Contains material.

The generation of the new enzyme is possible because the microorganism to be used for the invention is able to Assimilate xylan, d. H. Can produce xylanase, which has the ability to produce xylan or derivatives, the D-xyIo se units in xylan-containing material, can hydrolyze.

Xylan consists mainly of D-xylose units. Is xylan

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one of the main components of hemicellulose that comes in handy occurs in all land plants. D-xylose is technically made through Acid hydrolysis produced by xylan.

The xylan-containing materials which are useful for the present invention include all xylan-containing land plants, such as straw, wheat, bran, corn on the cob, corn husk, rice bran and wood, as well as derivatives thereof including the Sewage resulting from the processing of such land plant materials such as the various types of pulp waste and liquor as well as corn steep liquor. The expression "xylan-containing material", as efc is used here, refers to all materials that contain substances after hydrolysis by the ". xylanase such sub-> punch how D-xylose produce, which lead to the production of glucose isomerizing enzyme.

It has been found that a glucose isomerizing enzyme is produced in remarkably high yield when in a medium containing xylan or xylan-containing material, a microorganism is cultivated which is able to assimilate this xylan and which is glucose isomerizing Deliver enzyme.

The glucose isomerizing enzyme can, for example, by Cultivation of the microorganism to be used according to the invention can be generated in a medium containing D-xylose. the end For economic reasons, however, it may be more advantageous to grow the microorganism to be cultivated in a medium the xylan or xylan-containing material contains.

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~ 5 -

As a microorganism that assimilates xylan and the one that isomerizes glucose Enzyme generated, a strain of the genus Streptomyces is used according to the invention.

The culture for producing the glucoseisomerisierenden enzyme is used usually in an aqueous medium, usually called "at about 30 ⁰ G under aerobic conditions is carried out.

The addition of cobalt salt increases the yield of glucose isomerizing agent Enzyme in a remarkable way. Therefore, 1x 10 * "^ moles of cobalt salt are usually added to the medium.

In actual cultivation, the glucose isomerizing occurs Enzyme usually occurs in the cells. After the cultivation is finished, therefore, the cells which become rich Represent a source of enzyme, separated from the broth. The soluble enzyme can also be used as a source of enzyme will.

The conversion of glucose into fructose is carried out by letting this enzyme act on the glucose solution. This by the microorganism of the genus Streptomyces The enzyme produced is effective over a wide range of pH, temperature and glucose concentration, and therefore shows itself particularly advantageous for use in technical glucose isomerization.

If desired, the glucose isomerization can be carried out to such an extent that up to about 50 % of the initial glucose is converted to fructose.

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This leads to the production of a syrup that practically does that Invert sugar has sweetening properties.

The isomerization by the produced according to the invention. The enzyme can be carried out at a pH value between about 5.5 and about 8, preferably between about 6.8 and about 7 * 2, with 7 being the optimum. The reaction temperature can expediently be in the range between about 4- 5 ⁰ C and about 80 ⁰ C., wherein the region is preferably between about 60 ⁰ C and about 70 ⁰ C.

The following examples illustrate the invention. example 1

A pre-sterilized medium of 400 ml containing 3 % wheat bran (with a xylan content of 21%), 4 % ice mash water, 0.1% MgSO ^ -THgO and 0.3% EaCl was treated with Streptomyces albus, strain XT-No . 44- inoculated and cultivated with shaking at a temperature of 30 ⁰ O for 20 to 30 hours. The seeds obtained from this batch were transplanted into 10 liters of medium of the above composition and further cultivated with aeration with a shaking fermenter (aeration value 7.3 liters / min; 200 rpm; 30 ⁰ O). The activity of the glucose isomerizing enzyme in the cells reaches its maximum within 20 to 25 hours. The activity of the glucose isomerizing enzyme was determined with the following reaction mixture:

0.2 m phosphate buffer solution (pH 7 ^) 0.5 ml

1 m glucose solution 0.2 ml

0.1 m MgSO ^ * 7H ₂ O solution 0.1 ml

Enzyme solution 0.2-0.3

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The above mixture was made up to a total volume of 2 ml with water and ^{incubated at 70 °} C. for 1 hour. The reaction was stopped by adding 2 ml of 0.5 M perchloric acid. The fructose content was determined by the cysteine-garbazole method The amount of enzyme which produces 1 mg of fructose per hour from glucose at 70 ^° C. and the above test conditions is defined as one enzyme unit.

From 50 ml of the above Kulimrlösung the cell mass is through Separated by centrifugation, washed with water, suspended in water and sonicated at 10 kHz for 15 min. After the sound treatment -JO ml of the supernatant solution are obtained and used as an enzyme source. The enzyme activity was 623 units.

50 g of glucose, 5 _{ml of 0.1 M MgSO ^ 7H 2} O, 25 ml of 0.2 M phosphate buffer solution (pH 7.5) and water were then added to this enzyme solution. The total volume was adjusted to 100 ml with water. The mixture was incubated at 65 ° C. for 50 hours while the pH was kept between 6.8-7> 2 during the reaction. After completion of the reaction, the precipitate was removed by centrifugation and the supernatant liquid was treated with an ion exchange resin to remove the inorganic salt and protein and then evaporated in vacuo.

It became a sweet syrup consisting of 54% glucose and Get 46% fructose. This mixture became according to conventional Process separated from fructose and calcium by adding milk of lime. The fructose obtained was further decolorized and, after concentration, crystallized by reducing the pressure to remove solvent. 11 g of fructose were obtained.

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The product was examined for fructose by paper chromatography, where phenol: water (4-: 1) or pyridine: butanol: Water (4: 6: 3) were used as the solvent. An esorcinol reagent, which is a red color specific for ketosis Color generated was used as a means of identification. As a result, it was found that the Ef value corresponds to exactly Ef value of comparative fructose corresponds. The rotation of a "solution of the product was also exactly equal to the value a standard fructose solution.

Example 2

In this example pure xylan was used as the carbon source of the medium, as well as ßreptomyces flavovirens AiDCO No. 3320, Streptomyces achromogenus ATÖO Fr. 12 767, Streptomyces echinatus (NIHJ 180) and Streptomyces albus (TT-Nr. 4-) used.

These microorganisms were cultivated in a medium of 50 ml at 30 ⁰ O, which contained 1 % polypeptone, 0.3 % KgHPO ^, 0.1 % MgSO ₂ , · 7ΗοΟ and 0.5 % xylan. After inoculation, the medium was shaken. After the cultivation, the cells were collected by centrifugation and, after washing with water, the collected cells were suspended in water and sonicated at 10 kHz for 150 min. After this sonic treatment, 20 ml of supernatant liquid was obtained and used as an enzyme solution. The activities of the enzyme solutions obtained are shown in the following table;

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Table I.

Cultures

Initial residual xylan xylan (g / 50ml (g / 50 ml medium) medium)

Enzyme activity protein culture

vity of the four

(Units) cell time

(mg) (ii)

St.
flavoviruses
ATGC No. 3320 0, 25th 0.20 10 , 0 66 A. 14th St.
achromo genus
ATOC No. 12767 0 , 25 0.16 41 , 2 76 ,8th 10 St.
echinatus
NiHJ 180 0 , 25 0.19 32 ,8th 82 , 3 22nd St. albus.
(YT No. 4) 0 , 25 0.07 122 ,8th 86 34

The enzyme activity shown in Table I are the j? Rüctosemenge again, which was produced when 80 ml of 0.1 M glucose solution containing 20 ml of enzyme solution for 1 h at 70 ⁰ C allowed to react for expectant s. Although there are differences in enzyme activity, each xyianas simulating strain gave glucose isomerizing enzyme.

Example 3

A seed medium (50 ml) composed of 1 % xylose, 1% polypeptone, 0.3% K ₂ HPO ^, 0.1% MgSO ^ 7H ₂ O was placed in a flask and sterilized. Streptomyces albus (YT-No. 4) was then inoculated into the medium and cultivated at 30 ° for 3 days. This culture was then used as seed for the following experiment,

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The medium for the production of the enzyme contained 5 % rice bran,

3 % maize mash water, 0.1 % MgSO ₂ , * 7H ₂ O and was adjusted to a pH of 7> 0. 100 ml of this were placed in a 50 ml flask and sterilized. Then they were with

4 ml of the above-mentioned seeds are inoculated and ^{cultivated at 30 °} C. for 30 h inter shaking. Enzyme solution prepared from 50 ml of this culture had an enzyme activity of 700 units. The cultured cells were then collected by centrifugation. The gesa m Melten cells were then suspended in water and destroyed by 15-minute irradiation with 10 kHz. The disrupted cells were centrifuged and the obtained supernatant was used as a source of enzyme. This enzyme solution was reacted with 200 g of glucose in the same manner as in the previous example. 49.5 % of the glucose (200 g) used as substrate for the enzyme was converted to fructose. After removing the protein and purifying using activated carbon and ion exchange resin, the above reaction solution was concentrated to a water content of 25%. The yield was 195 g, measured as an anhydrous substance.

Example 4

This example illustrates the use of corn husks and corn cobs as sources of xylan.

The medium (50 ml, pH 7 »5>)> which contained 4 % corn mash water, 0.1% MgSO ₄ '7H ₂ O, 0.025 % CoCl ₂ * 6H ₂ O and 3% corn hulls or 3% corn cobs, which was added to a Sieve size Fr. was placed in a 200 ml flask

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■ and sterilized. Dana was inoculated the medium with Streptomyces albus ATCG 21132 (ϊΤ-ΪΓο. 5) and 48 h cultured with shaking "at 30 ⁰ C. The activity of glucose isomerizing enzymes as indicated in Table II.

Tabel

Xylan source enzyme activity (units)

Blank 24.5

Mai shiil s en 481.0

Corn on the cob 320.5

The reason for the production of glucose isomerizing enzyme in the blank for the xylan source is to that corn steep liquor is also a xylan-containing material.

The glucose isomerizing enzymes are generated when using corn husks and corn cobs as a source of xylan.

Units , 1 m MgSO ^ -TH ₂ O given. The total volume was made up to 200 ml with water. The mixture was inoculated at 70 ° 0. During the reaction the pH was kept between 6.8 and 7.2 with NaOH.

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After the reaction, protein was removed from the reaction solution and the solution was purified using activated carbon and ion exchange resin. The solution was then concentrated to a water content of 25 %. The yield obtained by the above procedure was 98.0 g of anhydrous substance.

Example 5

A seed medium containing 1 % xylose, 1% polypepone, 0.5 % E ₂ HPO ^ and 0.1% MgßO ^ / THgO was placed in a flask and sterilized. Then, the medium was inoculated with ßtreptomyces albus (YT-No. 4), and cultured for 48 h at 30 ⁰ C, to prepare an inoculum. In order to increase cell production, an inoculation solution from the seed stage was inoculated for inoculation with 50 liters of medium, and this was cultured. The latter was used 500 I ^- to inoculate medium, and these were cultured and finally these were used to inoculate 5000 1 medium and cultivated dieee. The composition of the media used in this treatment (excluding the seed medium) was 3% wheat bran, 4 % maize mash water, 0.1 MgßO ^ / THgO and 0.025 % CoGlo'BHoO and water. After ßterilisation this medium the initial pH value was adjusted to 6.5 Jnit Haoh, the cultivation was carried out at 30 ⁰ C at an aeration in an amount of one-third of the volume of broth per minute and 200 U / pm stirring speed. The enzyme activity reached a maximum after 24 hours. The enzyme solution prepared from 50 ml of broth had an activity of 750 units.

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After the reaction was complete, the wheat bran in the broth was removed using a sieve. The cells were collected using a filter press to recover 1000 kg of cells. The collected cells were then in Suspended in water. The muddy cells were disrupted using a high pressure homogenizer. The destroyed cells were used as a source of enzyme.

A converted starch syrup of DE = * 92.5 in a concentration of 50% (consisting of 87% glucose and 13 % disaccharide-containing oligosaccharide) was used as the substrate for isomerization. 1500 kg (calculated as solid) of the above converted starch syrup was placed in an isomerization tank of 30 nr capacity. 0.0025 m Mg (OH) o was added and after the syrup had been heated to 65 ⁰ C, the enzyme source was added. The pH was kept near 7iO using UaOH. The drawing shows the conversion ratio, the influence of temperature and the influence of pH during the isomerization process.

After 90 hours, the conversion ratio reached about 45 %. The reaction solution was then purified using activated carbon and ion exchange resin such as the conventional purification process. The solution was concentrated to a water content of 25 % . The yield of the product produced was 19600 kg. Analysis of this product found 29.6% fructose, 36 % glucose, 9.4% disaccharide-containing oligosaccharide and 25 % water.

Example 6

Using the procedure of Example 5, seeds were

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cultivation, intermediate cultivation, and main cultivation carried out successively.

The medium composition contained 3 % wheat bran, 4 % corn mash liquor, 0.1% MgSO ^ -TH ₂ O and 0.025 % CoCl ₂ * 6H ₂ 0. After the medium had been sterilized, the initial pH of the medium was increased with UaOH adjusted to 6.5 and the cultivation "at 30 ⁰ C, aeration at one third of the volume of broth per minute and stirring at 200 UOmin performed. After 24 h the cell activity reaches a maximum. the cells were collected by means of a filter press, wherein 900 kg of cells were obtained, and the collected cells were used as an enzyme source.

A starch syrup in 50% concentration with a DE value of 97 »0 (consisting of 95% glucose and 5 % disaccharide-containing oligosaccharide) was used as the substrate for isomerization. 15000 kg (calculated as

Solids content) of the above-mentioned converted starch residue syrups were placed in an isomerization tank of 30 m

Capacity given. 0.0025 m MgSO ^ 'TfLjO were added,

the syrup is heated to 65 ⁰ C, and the enzyme source is added, the pH was maintained at about 7 Vert> 0 Haoh.

After 100 h the conversion ratio was about 47 %. The reaction solution was then cleaned with activated carbon and ion exchange resin as in conventional cleaning processes. The product obtained after concentrating to a water content of 25 % weighed 19500 kg. Analysis of this product showed: 33.4 % fructose, 37.7 % glucose, 3.9 % disaccharide-containing oligosaccharide and 25 % water.

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The enzymatic process of the invention represents a new one and economical veg for converting glucose to fructose, and the end product is one containing fructose Syrup which has an invert sugar equivalent sweetness.

Claims;

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Claims (3)

Pat ent a η s ρ r ü c h e Process for the preparation of glucose isomerizing enzyme, characterized in that one a microorganism of the genus Streptomyces, which is used for Formation of glucose isomerizing enzyme is able to grow. 2. The method according to claim 1, characterized in that a microorganism of the genus ßreptomyces is used to assimilate the xylan and / or D-xylose can. 3. The method according to claim 1 and 2, characterized in that the microorganism is in a medium is grown, the xylan, xylan-containing material or contains D-xyIose. 4 ·. Method according to Claims 1 "to 3, characterized in that the cultivation in a medium is carried out, the corn steep water as xylanhaltiges Contains material. 309832/1017