DE3044356A1 - METHOD FOR PRODUCING GLYCOSE ISOMERASE - Google Patents

Description

SHIP ν. FONER STREHL SCHÖBEL-HOPF EBBINGHAUS FlNCK

-Tf- description

The invention relates to a process for the production of glycose isomerase with a Streptomyces strain.

It is known that the enzyme glycose isomerase does the conversion the D-glycose is realized in D-fructose, which is used more and more frequently in the food and beverage industry and the diet of highly developed countries. In combination with a complex of starch degrading enzymes (Öt-amylase and glycoamylase) enables glycose isomerase the production of glucose-fructose syrups by enzymatic means directly from starch.

Processes for the production of glycose isomerase have been known since 1957, when Marschall RO and Kooi ER (1957 Science, 125, No. 3249, 648) presented the possibility for a direct conversion of D-glycose into D-fructose by means of cells of the for the first time - have demonstrated bacterial strains Pseudomonas hydrophila No. 491 and 492. Microorganisms of the genus Streptomyces are most frequently used for the production of glucose isomerase, especially Str. Phaeochromogenes (Strandberg JW, Smily KD, 1971, Appl. Microbiol. _F 21, 4, 588; Tsumura M., Sato T., ibid ., 1129), Str.venezuelae (SU-PS 542 763; US-PS 3,622,463), Str.griseus (Hsu TY, Shon SC 1964, Sheng Wu Hua Usuen Yu Sheng Wu Li Hsuen Pao, 4, 342), St. wedmorensis (GB-PS 1,361,846), St. albus (JP-PS 49,981), St. flavovirens, St. achromogenes, St. achinatus (US-PS 3,616,221), St. olivocinereus (DE-PS 2,223,864; US-PS 3,622,463; Kooi ER, Smith RJ, 1972, Food Technol., 26, 9, 57), Str. Olivaceus (DE-PS 2,223,340; FR-PS 2,131,984) and others Species. In addition to the genus Streptomyces, proactinomycetes and actinomycetes are producers of other genera: Nocardia,

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Micromonospora (FR-PS 2 156 622; DE-PS 2 247 922), Actinoplanes (Scallet B.L., Shieh K., Ehrenthal I. 1974, starch, 26, 12, 405; U.S. Patent 3,834,988), Thermoactinomyces, Thermopolyspora (JP-PS 5Ο-1756Ο) and also some bacterial strains belonging to the genera Aerobacter (DE-PS 2 223 864), Acetobacter (U.S. Patent 3,694,314), Lactobacillus (Kent CA., Emery A.N. 1973, J. Appl. Chem. And Biotechnol., 23, 689), Bacillus (Skot G., Outtrup H. in 5th Int. Ferment.Symp., 4th Int. Spec. Symp., Yeasts, Berlin, Abstr. Pap. Berlin, 1976, 256), Arthrobacter (U.S. Patent 3,645,848), Flavobacterium (U.S. Patent 3,956,066) and others.

The known processes for the production of glucose isomerase have a number of disadvantages, mainly related to the fact that the strain producers seldom have one have high glycose isomerase activity with a favorable temperature and pH optimum of the enzyme. One temperature of the isomerization process lower than 60 to 65 ° C leads to the risk of microbial contamination of the colony. the A temperature increase of up to 90 ° C reduces the stability of the enzyme, causes discoloration of the syrup and changes in its viscosity. The most favorable with regard to the application on an industrial scale is a pH value im Range between 6.5 and 7.0, since at a pH value above 7.0 alkaline isomerization of the glycose with D-pepsicose formation and a discoloration of the syrup takes place by with a smaller one pH value (below 5.5) causes irreversible denaturation of the enzyme.

The object of the invention is to provide a method for the production of glucose isomerase with a Streptomyces strain with high Indicate activity at a high temperature and favorable pH optimum of the enzyme.

To achieve this object, the method according to the invention is characterized in that the enzymatic producer strain

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Streptomyces sp. No. 1339 is cultivated for 36 to 72 hours in the nutrient medium with the inducer xylose, a temperature of 24 to 36 ° C and an initial pH of the cultivation of 6.5 to 9.0 are maintained, and that the isomerization at a temperature in the range from 50 to 80 ° C and a pH value in the range from 6.0 to 9.0 in the presence of CoCl ₉ .6H "0 in a concentration of 5.1O ~ to L10" M, MgSO ₄ .7H ₂ O is carried out in a concentration of 1.1O to 1.10 M and a substrate concentration of 0.1 to 3 M.

The strain Streptomyces sp. No. 1339 - Glucose isomerase producer was isolated from Bulgarian soil. For its discovery, 874 Streptomyces strains were sieved (marked). The screening was carried out on modified synthetic nutrient medium No. 1 according to Krassilnikow, the Selection in two substrate levels using xylose and xylan was realized. Under these circumstances were 18 Streptomyces strains discovered that offered opportunities for the development and production of the enzyme glycose isomerase, from which the strain Streptomyces sp. 1339 has proven particularly promising for industrial purposes. Of the Stamm was registered with the State Institute for Control of Medicines, Boul. Vl. Saimov No. 26, September 29, 1979 deposited under no. 144 and has the following morphological and biochemical characteristics:

In a nutrient medium 1 with a nitrogen mineral source (according to G.F. Gause et al.) The colonies are usually oval, with shapeless edges, flat, with an in the Center domed dome-like center, slightly folded. The sporangia are spiral with no more than 1 to 3 ringlets. Coremias form in some nutrient media. The growth is good. The spores are elongated, with sharply chopped off Ends and an uneven surface and range in size from 0.4 to 0.9 microns in length and 0.3 to 0.6 microns in width. The ends are with well-defined wind-

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κΡ

3 04 4VS

no. In some cases the center of the spore is slightly deepened. They are formed through fragmentation. The colonies are oval, with shapeless ends, flat, with convex dome-like Center, slightly folded, in some cases radially articulated. The color of the air and substrate mycelium became after the color range of A.S. Bondarzew and according to the Tresner-Backus scale certainly.

In the case of the different nutrient media, the dependency changes of carbon and nitrogen sources that

4 2 Color of the air mycelium from light gray to dark gray (a - a). In nutrient medium 1 with nitrogen mineral source (after G.F. Gause and coworkers) the aerial mycelium is mouse gray (a) to

2
dark gray (a) and in nutrient medium 2 with an organic

2 Nitrogen source (according to G.F. Gause) - dark gray (a).

In culture media with different levels of carbon and nitrogen swelling, the aerial mycelium is gray to dark gray.

The substrate mycelium in nutrient medium 1 with a nitrogen mineral source (according to G.F. Gause and co-workers) has a light color

5 2

lemon-yellow to yellow-orange color (d - d). In nutrient medium 2 with an organic nitrogen source (according to GF Gause and co-workers) it is golden-yellow, and in the case of a halti
above.

7 7 holding cultivation it goes in maroon (m - ο)

In culture media with different carbon and nitrogen sources the substrate mycelium is from yellow to yellow-gray-green.

In a meat peptone agar medium: growth - weak, Air mycelium - white, very sparse, substrate mycelium - colorless.

In a potato glucose agar culture medium: growth - a lot

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good, aerial mycelium - gray to dark gray, substrate mycelium light brown to dark brown, yellow in the periphery of the colony. Pigment in the middle - yellow.

In a Tschapek culture medium with sucrose: growth - moderate, aerial mycelium - light blue, substrate mycelium - beige.

In a culture medium Tschapek with glucose: growth - moderate, aerial mycelium - greyish, substrate mycelium - beige.

In a nutrient medium with sucrose: growth - good, aerial mycelium - gray, substrate mycelium - yellow.

In a starch agar culture medium: growth - moderate, aerial mycelium - light gray, light ash color, substrate mycelium - yellow, lemon yellow.

In a starch-ammonia nutrient medium according to Mischustin: growth - good, aerial mycelium - gray to dark gray, substrate mycelium - yellow, light yellow.

In the synthetic nutrient medium according to NA Krassilnikow: growth weak to moderate, aerial mycelium - beige-gray _f substrate mycelium - yellow.

In the CPI according to N.A. Krassilnikov: growth - good, aerial mycelium

- milk gray, blue-gray, substrate mycelium - yellow-gray-green.

In the CPII to N.A. Krassilnikov: growth - good, aerial mycelium

- gray, separate white exudate, substrate mycelium - beige-gray.

In the CPIII to N.A. Krassilnikov: growth - good, aerial mycelium - gray, substrate mycelium - yellow, lemon yellow.

In the CPIV to N.A. Krassilnikow: growth - moderate, aerial myce-

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lium - light gray, greyish, substrate mycelium - cream colored.

In the CPV to N.A. Krassilnikow: WaGhstum - weak, aerial mycelium - white, substrate mycelium - beige, dark cream in color.

In the synthetic nutrient medium according to Wachsmann: growth - moderate, aerial mycelium - white, in some places gray, substrate mycelium - yellow to orange.

In a meat starch agar: growth - moderate, aerial mycelium

- gray, substrate mycelium - yellow.

In a peptone agar: growth - good, aerial mycelium - gray to dark gray, substrate mycelium - colorless with a shade of the aerial mycelium.

In a glycose asparagine agar: growth - moderate, aerial mycelium - light gray, substrate mycelium - yellow-cream colored.

In a glycerine-asparagine agar: growth - good, aerial mycelium - light gray and turns gray with aging, substrate mycelium - dark yellow.

In a tyrosine culture medium: growth - moderate, aerial mycelium

- gray, in some colonies to gray-yellow, substrate mycelium

- dark yellow to orange.

In a tyrosine casein nitrate agar: growth - moderate, aerial mycelium - ash gray, substrate mycerlium - yellow.

In a glycose tyrosine agar: growth - good, aerial mycelium

- cream-colored-gray to gray, substrate mycelium - yellow-brown.

In a sucrose nitrate agar: growth - moderate, aerial mycelium - gray, substrate mycelium - cream-colored-yellow.

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In a glycerine calcium malate agar: growth - good, aerial mycelium - light gray to gray, substrate mycelium - yellow-orange.

In a peptone bovine agar: growth - good, aerial mycelium gray, Substrate mycelium - colorless to cream-colored.

In an oat agar: growth - very good, aerial mycelium - gray, substrate mycelium - light yellow.

In a tomato agar: growth - good, aerial mycelium - dark gray, substrate mycelium - terracotta colored.

In a lead acetate agar: growth - weak, aerial mycelium

- greyish, substrate mycelium - yellow-brown.

In an iron peptone agar: growth - moderate, aerial mycelium

- mouse gray, substrate mycelium - colorless with a gray shade of aerial mycelium.

In a yeast-malt agar: growth - good, aerial mycelium - gray, Substrate mycelium - orange.

Tolerance of the strain in relation to NaCl - the same has a small tolerance in relation to the NaCl concentration in the nutrient medium. The maximum concentration is 4 %. The growth of the stem at this concentration is weak. The aerial mycelium is white. The substrate mycelium is yellow. A NaCl concentration greater than 1% has a negative effect on spore formation.

Peptonized defatted milk: At the beginning of the peptonization the reaction is acidic, after which, after a certain time, the same turns into an alkaline one.

Liquefies the gelatin, grows very well in a sucrose nutrient medium, but does not invert the sucrose, grows very well in a starch agar, and hydrolyzes the starch well, does not decompose the cellulose, does not reduce the nitrates to nitrites, excretes hydrogen sulfide, grows in Potatoes. Hemolysis is positive, tyrisinase is negative

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negative.

In a Pridham and Gottlieb culture medium, a very good Growth observed with the following carbon sources: glycose, fructose, levulose, xylose, maltose, cellobiose, galactose, Mannitol, arabinose, dextrin, rybose and glycerin.

Weakly absorbs the salicin.

Does not assimilate carbon sources such as lactose, sorbitol, Inositol, sucrose and raffinose.

Differences in pigmentation of the air and substrate mycelium depending on the carbon source are also observed. In a modified basic nutrient medium by Pridham and Gottlieb, the strain grows very well with the following nitrogen sources: NH ₄ Cl, (NH ₄ ) ₂ SO ₄ , (NH ₄ J ₂ HPO ₄ , NH ₄ H ₃ PO ₄ , NH ₄ NO ₃ , Na ₂ HPO ₄ and carbamide.

The strain shows moderate growth in a nutrient medium with NaNO ₃ . It does not grow in a nutrient medium with NaNO ₂ .

Very good growth of the strain is observed in nutrient media with the following amino acids, i.e. determined: aspartic acid, Asparagine, proline, cystine, tyrosine. The strain shows moderate growth in a nutrient medium with valine, Hydroxyproline, phenylalanine, leucine, alanine. It does not grow in a nutrient medium with glutamic acid.

There will be differences depending on the nitrogen source in the pigmentation of the air and substrate mycelium observed.

Streptomyces strain # 1339 is similar in some respects

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Properties of Streptomyces griseoflavus, belonging to Gray series according to Bergey's (1974) - Actinomyces griseoflavus from the Flavus Group to N.A. Krassilnikov - 197O. Of the the latter differs in a number of cultural-morphological and phjsLological-biochemical properties, described in the species characteristic of Bergey's (1974) and N.A. Krassilnikov (1970). Streptomyces griseoflavus has e.g. elongated rod-like and oval spores with a smooth surface, has a tolerance of 7 to 10% NaCl on. Weakly hydrolyzes the starch. Absorbs the sucrose and sorbitol, but not the galactose. Consequently is the Streptomyces strain No. 1339 with the closely related Streptomyces griseoflavus (Actinomyces griseoflavus) not identical and therefore we write it to the Gray series according to Bergey's (1974) and the Flavus group according to N.A. Krassilnikov (197O) as Streptomyces sp. No. 1339 to.

The strain producer is placed in 500 ml Erlenmeyer flasks containing 50 ml fermentation nutrient medium for 36 to 96 hours at a temperature of 24 to 36 ° C, initial pH the cultivation of 6.5 to 9.0 cultivated on a shaker at 180 to 320 revolutions / minute.

The isomerization of glucose to fructose with glucose isomerase from the strain Streptomyces sp. No. 1339 can be obtained by direct treatment with fresh mycelium (excreted by centrifugation at 12,000 revolutions / minute and rinsing three times with 0.05 M phosphate buffer with a pH value of 7.0) or with dried mycelium (air-dried or acetone-dried cells); with an enzymatic solution by decomposition by means of ultrasound or autolysis of the cell material and secretion of supernatants Centrifugation at 15,000 rpm; with culture centrifugate, containing extracellular isomerase or cells immobilized on a solid support.

The fructose formed in the reaction mixture is

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Stein-Karbasol method (Dische Z., Borenfreund E., 1951, J. Biol. Chem., 192, 583) and the strain activity is expressed in mg of fructose per ml of culture fluid or in international glycose isomerase units (GIU) . A GIU is the enzyme quantum which, at 70 ° C and a pH value of 7.0, IM glycose solution in 0.05 M phosphate buffer, 1.1O " ⁴ M CoCl ₂ .6H ₂ O and 1.1O ~ ² M MgSO. .7H ₂ O in one minute

Converts 1 µmol of glycose into 1 µmol of fructose.

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

The strain Streptomyces is used to produce the glucose isomerase sp. 1339 has been used, which has great biosynthetic possibilities of the enzyme (12,000 to 20,000 (GIU) / 1), which is 4 to 2O times the activity of the strain producers known from the patent literature (US Pat. No. 3,616,221, 3,834,988; GB-PS 1,376,787, 1,451,273, 1,284,218; SU-PS 534 490, 534 491, 542 763; DE-PS 2 219 713, 2 247 922,

2,018,518, 1,934,461). The enzyme obtained is characterized with a high temperature optimum (70 ⁰ C) for small Optimal concentrations of Co (1.1o ~ M) Mg (1.10 M) in the Isomerisationsgemisch; a favorable pH optimum (7.0) and a significant thermal stability between 40 and 65 ° C.

The invention is explained in more detail on the basis of the examples given below:

Example I.

The strain producer is maintained in test tubes with inclined agar, the nutrient media having the following composition exhibit:

1. Xylose culture medium: Xylose - 20 g, agar - 20 g, KNO ~ - 1.0 g, K ₃ HPO ₄ - O, 5 g, MgSO ₄ .7H ₂ O - 0.5 g, NaCl - O, 5 g, CaCO ₃ 1.0 g, FeSO ₄ - O ₇ OOl g and water up to 11.

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2. Potato-glycose-agar: potato extract of 300 g cooked Potatoes, glucose - 10 g, agar - 20 g and water up to 2 1.

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

To a well-germinated material from a 10 to 15-day culture, 1 or 2 (preferably 1) 6 ml of inoculation nutrient medium with the following composition are added to the nutrient medium: xylose - 1.0 %, tryptone - 2.0%, MgSO .. 7H ₂ O - 0.1 X ₁ K ₃ HPO ₄ - 0.25%.

Subsequently, a rinsing of the cell mass is carried out, after which the test tube on a shaker for 24 hours at 30 ⁰ C and 240 revolutions / minute is moved. An inoculation medium with the following composition is sown from this culture adapted in this way: xylose - 1.0 %, corn extract - 3.0 % (according to dry matter), MgSO ₄ -TH ₃ O - 0.1 %, K ₂ HPO ₄ - 0.25%, agar - 0.1%.

After 60 hours of cultivation under the conditions listed above, the fermentation nutrient medium with the following composition is inoculated with 5% of the inoculum: xylose 1.0 %, corn extract - 3.0% (based on dry matter), MgSO ₄ .7H ₂ O 0 , 1 %, KCl - 0.0075 %, CoCl ₂ .6H ₂ O - O, O24%.

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

When the Streptomyces sp. No. 1339 are 140 to 220 g of moist biomass per 1 liter Culture fluid produced.

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Example II.

The isomerization of glucose to fructose by means of glucose isomerase from the strain Streptomyces sp. No. 1339 is at a

Temperature of 7O ⁰ C, pH of 7.0 in the presence of

-4 CoCl ₂ .6H ₂ O in a concentration of 1.10 M ₇ MgSO ₄ .7HO in

a concentration of 1.1O ~ M and a substrate concentration of 1 M carried out.

The activity of the Streptomyces sp. No. 1339 is up to 215 mg fructose per ml of culture fluid or 12,000 to 20,000 GIU per 1 liter of culture fluid.

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Claims (2)

Claims 1. A process for the production of glucose isomerase, characterized in that the enzymatic producer strain Streptomyces sp. No. 1339 is cultivated for 36 to 72 hours in the nutrient medium with the inducer xylose, with a temperature of 24 to 36 ° C and initial pH of the cultivation of 6.5 to 9.0 are maintained, and that the isomerization at a temperature in the range of 50 ° to 80 ⁰ C and at a pH value in the range of 6.0 to 9.0 in the presence of CoCl ₂ · 6H ₃ O in concentration from 5.10 "to 1.10" M, MgSO ₄ -TH ₃ O in a concentration of -4 -1
1.10 to 1.10 M and substrate concentration from 0.1 to 3 M. is carried out. 2. The method according to claim 1, characterized in that the nutrient medium used has the following composition: 130036/0553 Xylose 1.0 to 2.0 %, Corn extract 1.5 to 4.0 % (after dry Dimensions) , MgSO ₄ .7H ₂ O 0.05 to 0.2 %, KCl 0.005 to O, O1%, .6H ₀ O 0.008 to 0.036%. 130036/0553