CH648592A5 - METHOD FOR PRODUCING GLUCOSE ISOMERASE AND THE USE THEREOF FOR ISOMERIZING GLUCOSE TO FRUCTOSE. - Google Patents

Description

The invention relates to a process for the production of glucose isomerase using a Streptomyces strain and the use of the glucose isomerase obtained for the isomerization of glucose to fructose.

It is known that the enzyme glucose isomerase causes the conversion of D-glucose to D-fructose, which is increasingly used in the food and beverage industry and the dietary nutrition of highly developed countries. In combination with a complex of the starch-degrading enzymes (ct-amylase and glucoamylase), the glucose isomerase enables the production of glucose-fructose syrups by enzymatic means directly from starch.

Processes for the production of glucose isomerase have been known since 1957 when the possibility for a direct conversion of D-glucose into D-fructose by cells of the bacterial strain Pseudomonas hydrophyla Nos. 491 and 492 was shown for the first time. Microorganisms of the genus Streptomyces are most commonly used in the production of glucose isomerase. An unconditional prerequisite that has so far been established for the biosynthesis of the enzyme glucose isomerase is the presence of different mineral salts in the nutrient media intended for cultivation. The culture media described in literature and patents usually contain magnesium salts as MgS04-7H2Û and cobalt as CoCh-óHiO (18). The concentrations in which these salts are introduced into the culture medium depend on the nature of the microorganism producer and mostly vary in the range from 0.02 to 0.5% for MgS04-7H20 and from 0.005 to 0.024% for CoCh-óH ^ O (14).

With some Streptomyces species that already contain magnesium ions for activation, the presence of cobalt is absolutely necessary for glucose isomerase formation to take place (13, 16, 17).

Strains and mutant producers of glucose isomerase are sought which have the ability to ensure a sufficient enzyme yield in the absence of cobalt ions in the culture medium. These are mutants of CIC International Inc. (2) and known as Artrobacter species by R.J. Reynolds Tobacco researches (9) that do not require the presence of cobalt for the biosynthesis of glucose isomerase.

By adding cobalt and magnesium ions to glucose solutions during the isomerization, the enzyme

matical activity equally influenced. It has been proven that these metals are cofactors of the enzyme and that glucose isomerase can be considered to belong to the metal enzymes (3). It was found that in glucose isomerase from Str. Sp. YT No. 5 4.1 cobalt atoms and 33 magnesium atoms are contained in one enzyme molecule. It is believed (3) that the combination of Mg ++ and Co ++ with glucose isomerase is necessary for their conversion to the active form. It is also assumed that a change in the enzyme configuration occurs during this process. Some authors believe that the introduction of cobalt ions, especially together with magnesium ions, significantly increases the thermostability of glucose isomerase (3, 10).

The presence of large amounts of Co ++ in fructose syrups is undesirable (5), despite the fact that trace amounts of Co ++ are necessary for human nutrition (2). The cobalt ion content in the fructose syrup is approximately 1 mM (1), but this concentration has been shown to produce certain toxic effects in rats (6). In order to remove the cobalt ions from the glucose-fructose syrups after the end of the isomerization process, they must be treated with ion exchange resins until their maximum separation. This additional step requires the provision of industrial automated ion exchange systems. The company Mi-Car International uses fully automated ion exchange systems with two departments for cation-anion parts (strongly acidic cation exchange resin Duolite C25-D and weakly alkaline anion resin Duolite S-56). This ion exchange system requires periodic regeneration with acids or bases and large amounts of additional pure water. In order to control the cobalt ion content in the glucose-fructose syrups processed by the ion exchange system, a test is carried out using an automatic absorption spectrophotometer by taking samples of the syrup flow continuously, thereby regulating the work of the cation exchange system. In this process, cobalt ions up to a level of 5 ppb at 25% S must be removed effectively (8). This process complicates the production and has a negative impact on the effectiveness of the process and the cost price.

The object of the invention is to provide a process for the production of glucose isomerase using a parent producer, wherein no cobalt ions are required for the biosynthesis of the enzyme and for the isomerization of D-glucose in D-fructose. The process is also said to be of economic importance.

The strain Streptomyces sp. No. 765 - producer of glucose isomerase was isolated from Bulgarian soil. For its discovery, 874 Streptomyces strains were screened. The screening was carried out in a modified synthetic nutrient medium No. 1 according to Krassilnikow by selecting two substrate levels using xylose and xylan. Under these conditions, 18 strains of Streptomyces with opportunities for the development and production of the enzyme glucose isomerase were discovered, of which the strain Streptomyces sp. No. 765 proved capable of producing the enzyme glucose isomerase in the absence of Co ++ in the culture medium.

The strain is in the State Institute for Control of Medicines, Boul. Vladimir Zaimov 26, Sofia 1000, Bulgaria, on September 29, 1979 and received No. 143. It has the following morphological and biochemical properties:

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In the No. 1 nutrient medium with a nitrogen mineral source (according to GF Gause and co-workers), the colonies usually have a round shape with smooth edges, they are curved in the middle, swollen and form a depression that looks like a small crater, and they are also strongly folded radially. The growth is good. The sporangia (spore container) of the young cultures are extensive, monopodially arranged with 3 to 5 rings on the spirals, whereby in older cultures they are pressed together in the form of small brooms (panicles).

The spores are elongated and have rounded ends; at a magnification of more than 16,000 times, hair-like formations are observed on the surface. With a small enlargement, the spores look smooth; the spore size varies between 0.9-1.2 microns in length and 0.4-0.6 microns in width.

The color of the air and substrate mycelium was determined after the A.S. Bondarzew color scale and determined on the Tresner and Backus scale. With the different nutrient media depending on the carbon and nitrogen sources, the color of the aerial mycelium changes from white (d1), light gray to violet (a5 - d3). In the nutrient medium No. 1 with a nitrogen mineral source (according to GF Gause and co-workers), the color of the aerial mycelium is light gray to mouse gray (a5 - d3), and in a nutrient medium with an organic nitrogen source (according to GF Gause and co-workers) the color is dark gray to gray violet (a2 - a3).

The aerial mycelium is colored gray-green in nutrient media with different carbon and nitrogen sources.

The substrate mycelium in the nutrient medium 1 with a nitrogen mineral source (according to G.F. Gause and co-workers) has a color from heal dove gray to ultramarine (16 -V1). In nutrient medium 2 with an organic nitrogen source (according to G.F. Gause and co-workers), the substrate mycelium is ultramarine (V1), and if cultivation continues, the color changes to black (a1).

In nutrient media with different carbon and nitrogen sources, the substrate mycelium is colored dark red to black.

In a meat peptone agar medium. Growth-weak. Air mycelium - pink, around the edges - brick red. Substrate mycelium - brick red.

In a potato-glucose agar culture medium. The trunk grows very well. Air mycelium - colored gray-blue. Substrate mycelium - colored blue to dark blue.

In the Tschapek nutrient medium with sucrose. Growth-weak. Aerial mycelium - pink. Substrate mycelium - light goat-colored.

In the Tschapek culture medium with glucose. Growth - moderate. Aerial mycelium - light blue. Substrate mycelium - colorless, with a shade in the color of aerial mycelium.

In a nutrient medium with sucrose. Growth - good. Air mycelium - sky blue color. Substrate mycelium - dark blue to black colored.

In a starch agar medium. Growth - good. Aerial mycelium - gray. Substrate mycelium - blue.

In a starch-ammonia medium from Mischu-stin. Growth - very good. Aerial mycelium - gray. Substrate mycelium - wine red to dark wine red.

In a synthetic culture medium according to N.A. Krassil-nikow. Growth - moderate. Aerial mycelium - light ash gray. Substrate mycelium - pink-purple.

In a CPI according to N.A. Krassilnikow. Growth - good. Aerial mycelium - gray-blue. Substrate mycelium - red-brown. -This medium contains in g / 1: glucose: 20.0; Potassium nitrate: 1.0; K2HP04: 0.5; Magnesium sulfate: 0.5; Sodium chloride: 0.5: calcium carbonate: 1.0 and iron carbonate: 0.001.

In a CPII according to N.A. Krassilnikow. Growth -

weak to moderate. Aerial mycelium - blue. Substrate mycelium

- dark beige. - This medium contains in g / 1: glucose: 10.0; Sodium citrate: 2.2; K2HPO4: 5.65; KH2PO4: 2.38; Ammonium sulfate: 1.0; Magnesium sulfate: 1.0; Zinc sulfate: 0.015; Copper sulfate: 0.064; Iron sulfate: 0.01 and MnCh: 0.0072.

In a CPIII to Krassilnikow. Growth - good. Air mycelium - gray to ultramarine colored. Substrate mycelium - wine red. - This medium contains in g / 1: glucose: 10.0; Ammonium phosphate: 2.0; Sodium chloride: 2.0; K2HPO4: 1.0; MgS04-7Hi0: 0.5; Calcium chloride: 0.2; FeS04-7 H2O: 0.02 and ZnS04-7H20: 0.01.

In a CPIV according to N.A. Krassilnikow. Growth-weak. Aerial mycelium - light gray. Substrate mycelium - colorless, with a shade of the color of aerial mycelium. -This medium contains in g / 1: sucrose: 20.0; Potassium nitrate: 0.1; Magnesium sulfate: 0.05; HCl: 0.07; KH2PO4: 0.1 and calcium nitrate: 0.25.

In a CPV to Krassilnikow. Growth - good. Air mycelium - colored gray. Substrate mycelium - dark purple colored. - This medium contains in g / 1: starch: 20.0; Potassium nitrate: 0.1; MgS04-7 H2O: 0.05; Sodium chloride: 0.5; KH2PO4: 0.01 and K2HPO4: 1.0.

In a synthetic culture medium according to Wachsmann. Growth - moderate. Aerial mycelium - gray to mouse gray. Substrate mycelium - dark beige to red-brown.

In a meat starch agar medium. Growth-weak. Aerial mycelium - white. Substrate mycelium - beige.

In peptone agar. Growth - good. Aerial mycelium - gray. Substrate mycelium - colorless, with a gray shade of aerial mycelium.

In glucose-asparagine agar. Growth - good or very good. Aerial mycelium - gray to ultramarine. Substrate mycelium -blue-violet to dark gray.

In glycerin-asparagine agar. Growth - good. Air mycelium - ultramarine colored. Substrate mycelium - dark purple to black.

In a tyrosine culture medium. Growth - good. Aerial mycelium - blue-gray to ultramarine. Sustrate mycelium - reddish brown.

In a tyrosine casein nitrate agar. Growth-weak. Aerial mycelium - white. Substrate mycelium - beige (cream-colored).

In a glucose tyrosine agar. Growth - moderate. Aerial mycelium - cream to gray. Substrate mycelium violet.

In a sucrose nitrate agar. Growth - good. Aerial mycelium - gray to ultrmarine. Sustrate mycelium - blue to dark blue.

In a glycerin calcium malate agar. Growth - good. Air mycelium from blue to ultramarine colored. Substrate mycelium - dark purple.

In a peptone-beef agar. Growth - moderate. Aerial mycelium - gray. Substrate mycelium - colorless, with a gray shade of aerial mycelium.

In an oat agar. Growth - moderate. Aerial mycelium

- Gray. Substrate mycelium - burgundy.

In a tomato agar. Growth - good. Aerial mycelium - gray. Substrate mycelium - dark beige, terracotta colored.

In a lead acetate agar. Growth - weak. Aerial mycelium -brown. Substrate mycelium - colorless, with a shade of gray in aerial mycelium.

In an iron peptone agar. Growth - good. Aerial mycelium - gray. Substrate mycelium - colorless, with a gray shade of aerial mycelium.

In yeast-malt agar. Growth - moderate. Aerial mycelium - gray to mouse gray. Substrate mycelium - dark cream colored.

Tolerance of the strain towards NaCl - it shows the same

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a low tolerance with regard to the concentration of sodium chloride in the nutrient medium. The maximum concentration is 4%. The growth of the strain at this concentration is weak. Aerial mycelium - light blue. Substrate mycelium - dark blue. A larger NaCl concentration, which is more than 2%, has a negative effect on spore formation.

Coagulated defatted milk. Gelatin is not liquefied. Grows well on a sucrose medium, but sucrose is not inverted. Grows very well in starch agar and also hydrolyzes starch well. Cellulose is not broken down. Reduces nitrate to nitrite. Secretes hydrogen sulfide. Grows on potatoes. Hemolysis - negative, tyrosinase - positive; forms melanoids.

Good growth with the following carbon sources is found in a basic nutrient medium from Pridham and Gottlieb; Glucose, fructose, lactose, levulose, xylose, mannose, cellobiose, galactose, mannitol, inositol, arabiosis, dextrin, ribose and glycerin.

Adsorbs salicin weakly.

Does not grow in nutrient media with sorbitol, sucrose and raffinose. Differences in pigmentation of air and substrate mycelium depending on the carbon source are also observed.

A good growth of the strain with the following nitrogen sources is found in a modified basic medium from Pridham and Gottlieb: NH-iCl; (NH.O2SO4; (NH.O2HPO4; NH4H2PO4 and carbamide.

The strain shows moderate growth in a nutrient medium with NH4NO3 and Na2HPC> 4; the strain does not grow at all in a nutrient medium containing NaNCh and NaNCh.

Very good growth of the strain is observed in nutrient media with the following amino acids, glutamic acid, aspartic acid, alanine, valine and asparagine. The latter shows moderate growth in culture media with leucine, cystine, proline, hydroxyproline, phenylalanine and tyrosine. Differences in pigmentation of air and substrate mycelium depending on the nitrogen source are observed.

Some traits of Streptomyces strain No. 765 are similar to those of Streptomyces coelicolor, which belongs to the Gray series after Bergey's - 1974 (Actinomyces coelicolor from the group Coelicolor after N.A. Krassilnikow - 1970). The latter differs in some culture-morphological and physiological-biochemical properties, which in the species characteristic - Bergey's (1974) and N.A. Krassilnikow (1970) - are described. Streptomyces coelicolor has e.g. 1 to 3 rings on the spirals, gelatin liquefies only slowly, peptonised defatted milk, tyrosinase - negative. Hence the strain Streptomyces sp. No. 765 is not identical to its similar Streptomyces coelicolor (Actinomyces coelicolor) and it is therefore classified according to the Gray series according to Bergey's (1974) and the Coelicolor group according to N.A. Attributed to Krassilnikow (1970).

The parent producer, e.g. contained in 50 ml fermentation nutrient medium in a 500 ml Erlenmeyer flask, is kept for 36 to 72 hours at a temperature of 24 to 36 ° C and an initial pH between 6.5 and 9.0, preferably cultivated on a shaking machine at 180-320 rpm.

The isomerization of glucose to fructose with GlucoSe isomerase from the strain Streptomyces sp. No. 765 can be treated directly with fresh mycelium (excreted by centrifugation at 12,000 rpm and rinsed three times with 0.05 M phosphate buffer with a pH of 7.0) or with dried mycelium (air-dry or acetone-dry cells ) be performed; also with an enzymatic solution, prepared by ultrasonic decomposition or autolysis of the cell material and separation of the supernatant liquid by centrifugation at 15,000 rpm, with a culture centrifuge containing extracellular isomerase, or immobilized cells on a solid support.

The fructose formed in the reaction mixture is determined by the cysteine-carbasol method (4), and the parent activity is expressed in mg fructose per ml culture fluid or in international glucose isomerase units (GIU). A GIU is the amount of enzyme which, at 70 ° C. and a pH of 7.0, IM converts glucose solution in 0.05 M phosphate buffer and 2.10 "2 M MgS04-7H2Ü in one minute 1 mol mol glucose into 1 mol mol fructose .

The advantages of the method according to the invention are as follows:

The strain Streptomyces sp. No. 765 produces the enzyme glucose isomerase in the absence of cobalt ions in the fermentation medium. The resulting enzyme converts the D-glucose into D-fructose in the absence of cobalt ions in the isomerization mixture, which considerably simplifies the technological process; the use of ion exchange systems for the elimination of cobalt from the syrups containing fructose is no longer necessary. Compared to known microorganisms and mutants which are suitable for the production of glucose isomerase and where no cobalt ions need to be present in the culture medium and in the isomerization mixture (2a, b, c; 9a, b), Streptomyces sp. 765 the known microorganisms in relation to its glucose isomerase activity, its favorable pH optimum (7.0), furthermore in relation to the higher temperature optimum (80 °) of the enzyme and an essential thermostability between 40 and 70 ° C.

1.Xylose culture medium xylose 20 g agar 20 g KNOj 1.0 g K2HPO4 0.5 g MgS04-7H20 0.5 g NaCl 0.5 g CaCOs 1.0 g FeSÜ4 0.001 g water up to 1 liter

2. Potato-glucose agar

Potato extract from 300 g of boiled potatoes

Glucose 10.0g

Agar 20.0 g

Water up to 1 liter.

It is advisable to use both culture media alternately when maintaining the strain.

example 1

6 ml of inoculation culture medium of the following composition are added to a well-germinated material of a 10-15 day culture in culture medium 1 or 2 (preferably 1): xylose - 1%, bovine extract - 2%,

MgSÛ4-7H20 -0.1 ° / o, K2HPO4 - 0.3%.

The cell mass is rinsed off, after which the test tube is agitated on a shaker for 24 hours at 30 ° C. and 240 rpm. With this culture prepared in this way, an inoculation medium of the following composition is inoculated: xylose - 10 °,

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Maize extract - 3.0% (dry matter), MgSOWHsO - 0.1%, KaH P04 - 0.25%, agar - 0.1%.

The cultivation is carried out in a 500 ml Erlenmeyer flask containing 50 ml fermentation nutrient medium for 60 hours at a temperature of 30 ° C. on a 5 shaker at 240 rpm. The initial pH of the cultivation is 8.5.

In the 60-hour cultivation of the Streptomyces sp. 765, 160 to 240 g of moist biomass are obtained per 1 liter of culture liquid. io

Example 2

The isomerization of the glucose to fructose by means of glucose isomerase from the strain Streptomyces sp. No. 765 is carried out at a temperature of 70 ° C., a pH value of 7.0 in the presence of MgSO4-7H20 at a concentration of 1.10-2 M and a substrate concentration of 1 M. The activity of the strain Streptomyces sp. No. 765 is 75-130 mg fructose per ml culture liquid or 7000-12 000 GIU per 1 culture liquid. 20th

literature

1. Cotter W.P., Lloyd N.E., Hinman

C.W. 1971. Patent 3,623,953,25

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2. a) CPC International Inc. 1975.

British Patent 1,411,763.

b) CPC International Inc. 1975.

British Patent 1 411 764. 30

c) CPC International Inc. 1975.

British Patent 1,411,765.

3. Danno G. Ibid. 1971,35,7,997.

4. Dishe Z., Borenfreund E. 1951. J.

Biol. Chem., 192, 583.

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5. Jacobziner H., Raybin H.W.

1961. Arch. Pediat. 78, 200.

6. Levy H., Levinson V., Shade A.

1950. Arch. Biochem., 27, 34.

7. Marshal R.O., Kooi E.R. 1957.

Science, 125, No. 3249, 648.

8. Mi-Car International Inc.

Isomerized Syrup Process. 1975.

MFG information.

9. a) RJ. Reynolds Tobacco Co.

1973. British Patent 1,328,970.

b) RJ. Reynolds Tobacco Co.

1974. British Patent 1,362,365.

10. Sargio S., Kare L. 1975. Appi.

Microbiol., 29, 6, 745.

11. Somers E. 1974. J. Food Sei., 39,

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12. Schraeder H.A., Nason A.P.,

Tipton I.H. 1967. J. Chronic Dis.,

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13. Stradberg G.W., Smiley K.L.

1971. App. Microb., 21, 588.

14. Takasaki Y. Ibid. 1966,30,12,

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Kanbayasi A. 1969. Agr. Biol.

Chem., 33, 11, 1527.

16. Takasaki Y., Kosogu Y. 1969.

Academic Press Inc. New York.

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17. Tsumura N., Hagi M., Sato T.

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Claims (3)

648 592 PATENT CLAIMS 1. A process for the preparation of glucose isomerase, characterized in that the enzyme-producing strain Streptomyces sp. No. 765 for 36 to 72 hours in a nutrient medium containing the inducer xylose at a temperature of 24 to 36 ° C and an initial pH of 6.5 to 9.0. 2. The method according to claim 1, characterized in that the nutrient medium contains 1.0 to 2.0% xylose, 1.5 to 4.0% corn extract, based on the dry matter and 0.25-1.0% sodium acetate . 3. Use of the glucose isomerase obtained by the process according to claim 1 for the isomerization of glucose to fructose, characterized in that the isomerization of the glucose to fructose at a temperature of 50 to 90 ° C and a pH of 6.0 to 9.0 in the presence of MgSC> 4 • 7HîO with a concentration of 1.10 ~ 4 to 1.10-2M and a substrate concentration of 0.1 to 3M.