DK153570B - PROCEDURE FOR CREATING GLUCOSE ISOMERASE - Google Patents

Description

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DK 153570B

The invention relates to a process for the preparation of glucose isomerase by growing a strain of the genus Streptomyces at a temperature of 24-36 ° C in a nutrient medium having an initial pH between 6.5 and 9.0 and containing xylose.

It is known that by means of the enzyme glucose isomerase, D-glucose is converted to D-fructose, which is increasingly used in the food industry and in the manufacture of dietary foods in a number of developed countries. Glucose isomerase, in combination with a complex of amylolytic enzymes (α-amylase and glucoamylase), allows glucose-fructose syrups and fructose to be obtained directly from starch using enzymes.

Some methods of producing glucose isomerase have been known since 1957, when ROMarshall and ERKooi (28) for the first time 15 demonstrated that it was possible to convert D-glucose directly to D-fructose using cells belonging to the bacterial strain Pseudomonas hydrophilic N.491 and 492. For the production of glucose isomerase, mostly microorganisms are used

The Streptomyces genus and, above all, the species Streptomyces 2Q phaeochromogenes (32,36), S. Venezuela (1,23), S. griseus (22), S.

wedmorensis (2), S. albus (33), S. flavovirens, S. acromogenes, S. echinatus (34), S. olivocinereus (19,23,25), S. acromogenes, S.

olivaceus (16,17), as well as others. In addition to the Streptomyces genus, there are other active producers of the order Actinomycetales, belonging to the 25 genera: Nocardia, Micromonospora (20,21), Actinoplanes (29,30),

Thermoactinomyces, Thermopolyspora (35) as well as some other bacterial strains belonging to the genera: Aerobacter (19), Acetobacter (27), Lactobacillus (24), Bacillus (31), Arthrobacter (26), Flavobacterium (3), and others.

In the known methods of providing glucose isomerase, the producer strain rarely combines high glucose isomerase activity with a favorable temperature and pH optimum for the enzyme. A lower isomerization temperature than 60-65 ° C causes the risk of microbial contamination of the reactor in which the isomerization is carried out.

3, - Increasing the temperature to 90 ° C decreases the stability of the enzyme, causes staining of the syrup as well as any change in viscosity.

As for industrial use, the most favorable pH range is from 6.5 to 7.0, because thereby a pH of above 7.0 an alkaline isomerization of glucose to form D-psicosis and staining of 2

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the syrup, and a lower pH (below 5.5) causes an irreversible denaturation of the enzyme.

It has now been found that a novel microorganism strain ^ Streptomyces sp. N. 1339 produces glucose isomerase in high yield, while the found glucose isomerase has a high temperature optimum and a favorable pH optimum.

Accordingly, the process of the invention is characterized in that, as a microorganism, the strain Strep-tomyces sp. N. 1339 deposited with the Institute of Drug Control of the Bulgarian State, under the number 144.

Streptomyces strain sp. N.1339 has been isolated from Bulgarian soil. 874 Streptomyces strains have been studied for glucose isomerase production. The study was carried out on the modified Ig synthetic culture medium N.l after Krasilnikov, the selection being carried out at two substrate levels using xylose and xylan. Under these conditions, 18 Streptomyces strains have been found that can develop and produce the enzyme glucose isomerase. Of these Streptomyces strains, sp. N.1339 is said to have the greatest prospects in terms of industrial application. The strain has been in the collection at The State Institute for Drugs Control, bul. Vladimir Zaimov N.26 since September 29, 1979 under number 144 and has the following morphological and biochemical properties. On culture medium 1 with nitrogen mineral wedges (according to G.F.Gause et al.), The colonies 25 usually have an oval shape, with unformed edges, flat with convex dome-like centers and are easily folded radially in some cases. The sporangia are helical with no more than 1-3 turns. On some cultural media, coremias form. The growth is good. The spores have a length of 0.4-0.9 microns and a width of 0.3-0.6 microns. The ends have well-defined corners. In some cases, the grooves have a slightly concave center. They are formed by fragmentation.

The color of the aerial and substrate mycelia is determined according to A.S.Bondartsev's color scale and the Tresner-Backus scale.

With different culture medium, the color of the aerial mycelium changes from 4 2 3g light gray to dark gray (a-a) depending on the carbon and nitrogen sources. On culture medium 1 with an inorganic nitrogen source (according to GFGause et al.) The color is mouse gray (aj to dark gray (a)) and on culture medium 2 with an organic nitrogen source (according to GFGause) the color is dark gray (a). carbon and 3

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nitrogen wedge which is air mycelium gray to dark gray.

On culture medium 1 with a mineral nitrogen source (according to G.F. Gause et al.), The substrate mycelium has a light lemon-yellow color to ^ yellow-orange (d -d). On culture medium 2 with an organic nitrogen source (according to G.F.Gause et al.), The substrate mycelium is golden-yellow and after a prolonged period of cultivation becomes almost chestnut (n / -o ^).

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

1Q On culture medium meat-peptone agar. Low growth. Aerial mycelium white, very sparse. Substrate mycelium: colorless.

On culture medium potato-glucose agar. Growth very good. Aerial mycelium gray to dark gray. Substrate mycelium light brown to dark brown, on the border of the colony: yellow. Pigment in center yellow.

On culture medium Tchapek with sucrose. Medium growth. Aerial mycelium light gray. Substrate mycelium beige.

On culture medium Tchapek with glucose. Medium growth. Aerial mycelium greyish. Substrate mycelium cream.

On culture medium with sucrose. Good growth. Air mycelium gray.

2Q Substrate mycelium yellow.

On culture medium amylum agar. Medium growth. Aerial mycelium light gray, with pale ash gray color. Substrate mycelium yellow, lemon yellow.

On amylium-ammonia culture medium according to Mishustin. Good growth. Aerial mycelium gray to dark gray. Substrate mycelium yellow, pale yellow.

25 On synthetic medium according to Krassilnikov. Growth low to medium.

Aerial mycelium beige-gray. Substrate mycelium yellow.

At CPI according to N.A.Krassilnikov. Good growth. Air mycelium milk-gray, blue-gray. Substrate mycelium yellow-grayish-green.

On the CPU according to N.A.Krassilnikov. Good growth. Aerial mycelium gray, 30 white exudate is excreted. Substrate mycelium cream-gray.

On CPIII according to Krassilnikov. Good growth. Air mycelium gray. Substrate mycelium yellow, lemon colored.

At CPIV according to N.A.Krassilnikov. Medium growth. Aerial mycelium light gray, grayish. Substrate mycelium cream.

35 On CPV according to N.A.Krassilnikov. Low growth. Aerial mycelium white.

Substrate mycelium beige, dark cream.

On synthetic medium according to Vaxman. Medium growth. Aerial mycelium white, on separate parts gray. Substrate mycelium yellow to orange.

On meat amylum agar. Medium growth. Air mycelium gray.

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Substrate mycelium yellow.

On peptone agar. Good growth. Aerial mycelium gray to dark gray. Substrate mycelium colorless with a shade of aerial mycelium.

^ On glucose-asparagine agar. Medium growth. Aerial mycelium light gray.

Substrate mycelium of yellow cream.

On glycerin-asparagine agar. Good growth. Aerial mycelium light gray as it becomes gray with aging. Substrate mycelium dark yellow.

On tyrosine agar. Medium growth. Aerial mycelium gray, in some 1Q colonies greyish yellow. Substrate mycelium dark yellow to orange.

On tyrosine-casein nitrate agar. Medium growth. Aerial mycelium ash gray. Substrate mycelium yellow.

On glucose tyrosine agar. Good growth. Aerial mycelium cream-gray to gray. Substrate mycelium yellow brown.

1C On sucrose nitrate agar. Medium growth. Air mycelium gray. Sub-io stratmycelium cream-yellow.

On glycerol-calcium-malate agar. Good growth. Aerial mycelium light gray to gray. Substrate mycelium yellow orange.

On peptone beef agar. Good growth. Air mycelium gray. Substrate-20 mycelium colorless to beige.

On oat agar. Growth very good. Air mycelium gray. Substrate mycelium pale yellow.

On tomato agar. Good growth. Aerial mycelium dark gray. Substrate mycelium terracotta colored.

25 On lead acetate agar. Low growth. Aerial mycelium greyish. Substratmy celium yellow-brown.

On iron-peptone agar. Medium growth. Aerial mycelium light gray. Substrate mycelium colorless with a gray shade of aerial mycelium.

On yeast-malted agar. Good growth. Air mycelium gray. Substrate mycelium 30 orange.

Against NaCl in the medium, the tolerance of the strain is low. The maximum concentration is 4%. At this concentration, the growth of the stem is low. Aerial mycelium is white. Substrate mycelium yellow. A concentration above 1% adversely affects the degree of sporulation.

2g The strain peptonizes low-fat milk. At the beginning of the peptonization, the reaction is sour, but after some time it becomes all potassium.

It melts gelatin. It grows very well on a sucrose medium but does not invert sucrose. It grows very well on an amylase agar, 5

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and it hydrolyzes starch well.

It does not decompose cellulose and does not reduce nitrates to nitrites. It releases hydrogen sulfide. It grows on potatoes.

5 Hemolysis positive. Tyrosinase negative.

It has been found that on the basic culture medium of Pridham and Gottlieb, growth is good in the presence of the following carbon sources: glucose, fructose, levulose, xylose, maltose, cellobiose, galactose, mannitol, arabinose, dextrose, ribose and glycerol.

The IQ Tribe absorbs salicylic on a smaller scale.

It does not absorb the carbon sources lactose, sorbit, inosit, sucrose and raffinose.

There are some differences in the pigmentation of the air and substrate mycelia depending on the carbon source.

It has been found that on Pridham and Gottlieb's modified basal culture medium, the growth of the strain is good in the presence of the following nitrogen sources: NH4 Cl, (NH4) 2SO4, (NH4) 2HPO4, NH4H2PO4, NH4N0g, Na2HPO4 and carbamide.

Growth is moderate on a growth medium with NaNQg. The strain does not grow 2q on a growth medium with NaNO2.

Very good growth of the strain has been observed on growth media with the following amino acids: aspartic acid, asparagine, proline, cystine, tyrosine. Growth is moderate on a growth medium with val in, hydroxyproline, phenyl alanine, leucine and alanine. The strain does not grow on a growth medium with giutamic acid.

Depending on the source of nitrogen, some differences in the pigmentation of the air and substrate mycelia are observed.

According to some characteristics, the Streptomyces strain N. is similar to Streptomyces griseoflavus belonging to the Gray series of Bergey's 2Q (1974) and Actinomyces griseoflavus by the Flavus group of N.A.Krassilnikov (1970). The latter is distinguished from the former by a number of morphological, cultural and physiological, biochemical properties described in the species characterization of Bergey's (1974) and N.A.Krassilnikov (1970). Streptomyces griseoflavus 25, e.g. oblong rod-like and oval spores with smooth surface; it shows tolerance to NaCl from 7 to 10%. It weakly hydrolyses starch. It absorbs sucrose and sorbit and does not absorb galactose. Therefore, the Streptomyces strain N.1339 is not identical to the similar Streptomyces griseoflavus (Actinomyces griseoflavus).

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This is why it is called Streptomyces sp. N.1339. It belongs to the Gray series according to Bergey's (1974) and the Flavus group according to N.A.Krassilnikov (1970).

The producer strain can be grown in 500 ml Erlenmeyer flasks containing 50 ml of fermentation medium for from 36 to 96 hours at a temperature of 24 to 36 ° C, an initial pH of 6.5 to 9.0, on a shaker with 180 to 320 rpm.

Isomerization of glucose to fructose by means of 10 glucose isomerase from the strain Streptomyces sp. N.1339 can be performed by direct treatment with fresh mycelium (separated by centrifugation at 12,000 rpm and washed three times with 0.05M phosphate buffer of pH 7.0) or with dried mycelium (air dried or acetone dried cells), with enzyme solution (obtained after an ultrasonic disintegration or autolysis of cell material and separation of supernatant by centrifugation at 15,000 rpm), with culture centrifugate containing extracellular isomerase or with cells immobilized on a hard support.

The fructose formed in the reaction mixture is measured according to the 2Q cysteine-carbazole method (18) and the activity of the strain is expressed in mg of fructose per ml. ml of culture fluid or in international glucose isomerase units (GIU). One GIU is equal to the amount of enzyme obtained at 70 ° C and pH

7.0, 1M glucose solution in 0.05M phosphate buffer, 1x10 "4M CoCl₂ * 6H₂O and 1x10_2H MgSO4« 7H₂O in 1 minute converts 1 piol glucose to 1 / tmol 2 g fructose.

The advantages of the method of the present invention are the following:

In order to produce glucose isomerase, the strain Streptomyces sp. N.1339, capable of synthesizing a large amount of the 3o enzyme (12,000-20,000 GIU), which is from 4 to 20 times more than the activity of the known strains of literature (4-15).

The enzyme obtained is characterized by a high temperature optimum (70 ° C) at low optimal Co ++ concentrations (lx10 ”S1) and 4-4- -7

Mg concentrations (1x10 M) in the isomerization mixture. It has a favorable pH optimum (7.0) and considerable thermostability between 40 and 65 ° C.

The invention is illustrated by the following example:

EXAMPLE

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Streptomyces N.1339 is kept in test tubes with sloping agar on growth media of the following composition: 5 1. Xvlose growth medium xylose 20 g agar 20 g KN03 1.0 g K2HP04 0.5 g 10 MgS04-7H20 0.5 g

NaCl 0.5 g

CaCO 3 1.0 g

FeSO 4 0.001 g water to 1 liter.

2. Potato-glucose acar potato extract from 300 g cooked potatoes glucose 10 g agar 20 g 2Q water to 2 liters.

It is recommended that both growth media are used alternately to maintain the stem.

To a well-sprouted material from a 10-15 day old culture on growth medium 1 or 2 (1 recommended) is added 6 ml of inoculation growth medium having the following composition: xylose 1.0% tryptone 2.0% 3Q MgSO4'7H20 0.1 % K2HPO4 0.25%

Water up to 100%

The cell mass is washed out and the tube is placed on a shaker for 24 hours at 30 ° C and 240 rpm. Inoculated culture thus inoculated with inoculation medium of the following composition is seeded: xylose 1.0% corn extract 3.0% (dry weight) 8

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MgSQ4'7H2Q 0.1% K2HPG4 0.25% agar 0.1%

Water up to 100%

After 60 hours of cultivation under the above conditions, 5% of the inoculum is transferred to a fermentation growth medium of the following composition: 10 xylose 1% of the weight of the fermentation medium corn extract 3.0% (dry weight) of the weight of the fermentation medium

MgSO4'7H2O 0.1% of fermentation medium weight KC1 0.0075% weight of fermentation medium j,. CoC12®6H20 0.024% by weight of fermentation medium

Water up to 100%

The culture is carried out in 500 ml Erlenmeyer flasks containing 50 ml of fermentation medium. The cultivation is carried out for 60 hours at 30 ° C on a 2Q shaker at 240 rpm. The initial pH is 8.5.

After 60 hours of cultivation of Streptomyces sp. N.1339 is obtained from 140 to 220 g of moist biomass per liter. 1 liter of culture fluid.

The isomerization of glucose to fructose by glucose isomerase from the strain Streptomyces sp. N.1339 is carried out at a temperature of 70 ° C, a pH of 7.0 in the presence of C o Cl of 1x10 M and a substrate concentration of 1M.

The activity of the strain Streptomyces sp. N.1339 is 130-215 mg of fructose per day. 1 ml of culture fluid or 12,000-20,000 GIU per 1 liter of 30 culture liquid.

REFERENCES

1. Gratcheva, I.M., Mositchev, M.S., Gavristov, A.B., Filippov, S.A., 1977. Aut.cert.SSSR, No.542763. Bull.'Otkritie, izobre- 33 tenia "N.2.

2. Patent No. 1,361,846, England.

3. Patent No. 3,956,066, USA

4. Patent No. 3,616,221, USA

5. Patent No. 3,834,988, USA

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6. Patent No. No. 1,451,273, United Kingdom. No. 1,376,787, England 8. Patent No. 1,284,218, England

9. Patent No. 534,490, USSR

10. Patent No. 534,491, USSR

11. Patent No. 542,763, USSR

12. Patent No. 2,219,713, BRD

13. Patent No. No. 2,247,922, BRD 1Q 14. Patent No. 2,018,518, BRD

15. Patent No. 1,934,461, BRD

16. Patent No. 2,223,340, BRD

17. Patent No. 2.131.984, France 18. Dische, Z., Borenfreund, E., 1951, J. Biol. Chem., 192, 583.

. 15 19. Heady, R.E., Jacaway, W.A., Patent No.2225864, BRD

20. Horwath, R. O., Cole, G. W., 1973. Patent No.2156622, France

21. Horwath, R. O., Cole, G. W., 1973. Patent No.2247922, BRD

22. Hsu, T.Y., Shon, S.C., 1964. Sheng Wu Hua Usuen Yu Sheng Wu Li Hsuen Pao, 4, 342.

23. Iizuka, H., Ayukawa, Y., Suekane, M., Kanno, M., 1971. Patent

No.3622463, USA

24. Kent, C. A., Emery, A. N., 1973, J. Appl.Chem. and Biotechnol., 23, 689.

25. Cage, E. R., Smyth, R. J., 1972, Food Technol., 26, 9, 57.

25 26. Lee, C. K., Lawrence, E. N., Long, M. E., 1972. Patent No.

3645848, USA

27. Liyd, N.E., Lewis, L.T., Logan, R.M., Patel, D.N., 1972. Patent No.3694314, USA

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3834988, USA

31. Skot, G., Outtrup, H. In "5th Int.Ferment.Symp., 4th Int.Spec. Syrn.Yeast, Berlin, 1976, Abstr.Pap." - Berlin, 1976, 256.

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35. Takasaki, Y., 1975. Patent No.50-17560, Japan 36. Tsumura, M., Sato, T. 1965, Agr.B1o1.Chem., 29, 1129.

Claims (2)

A process for preparing glucose isomerase by growing a strain of the genus Streptomyces at a temperature of 24-36 ° C in a nutrient medium having an initial pH between 6.5 and 9.0 and containing xylose, characterized by that, as a microorganism, the strain Streptomyces sp. N. 1339 deposited with the Bulgarian State Institute for Drug Control (Institute for 10 Drugs Control), under number 144, Process according to claim 1, characterized in that the growth medium used has the following composition: xylose 1.0-2.0% by weight of the growth medium 15 corn extract 1.5-4.0% (dry weight) of the growth medium weight MgSO4 -T1 0.05-0.2% of growth medium weight KCl 0.005-0.01% of growth medium weight CoClg + HgO 0.008-0.036% of growth medium weight Water up to 100% 20 25 30 35