FR2473550A1 - Glucose isomerase prepn. by fermentation - using Streptomyces N 1339 and isomerising in presence of cobalt and magnesium ions - Google Patents

Abstract

Prepn. of glucose isomerase by fermentation of Streptomyces sp. N 1339 for 36-72 h at 24-36 deg. C in a culture medium contg. xylose as inducer, the initial pH being 6.5 to 9. Isomerisation of glucose takes place at 50-80 deg. C and pH 6 to 9 in presence of CoCl2.6H2O at 0.1-3 M. The organism has 4 to 20 times the activity of known organisms. The enzyme is quite stable at 40-65 deg. C and has an optimum pH of 7.0. During isomerisation, optimum low concns. of 10 power minus 4 M Co2+ and 10 power minus 2 M Mg2+ are advised.

Description

 The invention relates to a method for obtaining glucose isomerase, or glucose isomerase, from a strain of Streptomyces.

 It is known that the enzyme called glucose-isomerase transforms D-glucose into D-fructose, which is finding increasing applications in the food processing industry and in the dietetic diet in a number of developed nations. Glucose isomerase, in combination with a complex of amylolytic enzymes (o (-amylase and gluco-amylase) makes it possible to obtain glucose-fructose and fructose syrups directly from starch, using 'enzymes.

Certain methods for obtaining glucose-isomerase have been known since 1957, when RO Marshall and ER Kooi (29) demonstrated for the first time the possibility of direct transformation of D-glucose into D-fructose by means of cells belonging to the strain. bacterial Pseudomonas hydrophila Nr. 491 and 492. To obtain glucose-isomerase, microorganisms of the genus Streptomyces and especially the species Str. phaeochromogenes (34,38), Str. venezuelae (I, 24), Str. griseus (23), Str. wedmorensis (3), Str. albus (35), Str. flavovirens, Str.achromogenes, Str.achinatus (36), Str.olivocinerous (20,24,26), Str. achromogenes, Str. olivaceus (17,18), and others, are the most widely used. Besides the genus Streptomyces, there are other active producers such as proactinomyces and actinomyces belonging to other genera: Nocardia, Micromonospora (21,22), Actinoplanes (30,31), Thermoactinomyces, Thermopolyspora (37), and also certain bacterial strains belonging to the genera
Aerobacter (20), Acetobacter (28), Lactobacillus (25),
Bacillus (33), Arthrobacter (27), Flavobacterium (4), and others.

The already known methods for obtaining glucose-isomerase have a certain number of drawbacks mainly due to the fact that the producing strains rarely combine a high degree of glucose-isomerase activity with a favorable temperature and pH optimum of 1 'enzyme. The temperature of the isomerization process, below 60 - 650C, involves a danger of microbial pollution of the colony. Increasing the temperature to 900C decreases the stability of the enzyme, causes the syrup to color and changes in viscosity. For industrial application, the most favorable pH is 6.5 to 7.0 , because when the pH is higher than 7.0 an alkaline isomerization of glucose occurs with the formation of dyspsis and coloring of the syrup, and a lower pH (less than 5.5) causes an irreversible denaturation of the enzyme.

 The objective of the present invention is to provide a process for obtaining glucose-isomerase from a strain of Streptomyces which exhibits great activity with a high temperature optimum and a favorable pH of the enzyme.

The strain of Streptomyces sp. Nr. 1339, which is a producer of glucose-isomerase, was isolated from
Bulgaria. 874 strains of Streptomyces were sorted for its discovery. The sorting was carried out on the synthetic culture medium modified Nr. 1 according to Krassilnikov, the selection being carried out on two levels of substrate using xylose and xylan. Under these conditions, 18 strains of Streptomyces have been discovered which can develop and produce the enzyme called glucose isomerase. Among these strains of
Streptomyces, the species Nr. 1339 has proven to be the most promising for industrial purposes. The strain was deposited in the collection of the State Institute for Drugs Control, bul.

Vladimir Zaimov Nr. 26, September 29, 1979 under number 144, and has the following morphological and biochemical characteristics.

On culture medium 1 with a mineral source of nitrogen (after G.F. Gause and his collaborators), the colonies usually have an oval shape, with shapeless edges, flat with a convex dome-shaped center and slightly folded. The sporangia are spiral with no more than 1 to 3 turns. On some culture media, coremias are formed. The growth is good. The spores are elongated with clearly cut ends and an uneven surface. Their size is 0.4 to 0.9 micron in length and 0.3 to 0.6 micron in width. The ends have well-defined angles. In some cases, the spores are slightly concave in the middle. They are formed by fragmentation. The colonies are oval, with shapeless edges, flat, with a convex dome-shaped center, slightly folded, in some cases segmented radially.

The coloration of the aerial mycelium and of the substrate mycelium is determined according to the color scale of A.S.

Bondartsev and the Tresner and Backus scale.

 With the different culture media, the color of the aerial mycelium changes from light gray to dark gray (a4 - a2) depending on the sources of carbon and nitrogen. On culture medium 1 with a mineral source of nitrogen (after G.F.

Gause et al.), The color is mouse gray (a4) to dark gray (a2), and on culture medium 2 with an organic source of nitrogen (after G.F. Gause) the color is dark gray (a2). On culture media containing different sources of nitrogen carbon, the aerial mycelium is gray to dark gray.

On medium 1 with a mineral nitrogen source (from
GF Gause et al.), The substrate mycelium has a light lemon to orange-yellow coloration (d5 - d2). On culture medium 2 with an organic source of nitrogen (according to GF Gause and his collaborators), the substrate mycelium is golden yellow, and after prolonged culture, it becomes almost chestnut (m7 - o7).

 On culture media containing different sources of carbon and nitrogen, the substrate mycelium is yellow to greyish green yellow.

On meat-peptone-agar culture medium: weak growth, very sparse white aerial mycelium, colorless substrating mycelium.

 On potato-glucose-agar culture medium very good growth, gray to dark gray aerial mycelium, light brown to dark brown substrate mycelium, yellow on the edge of the colony, pigment in the yellow center.

On Tchapek culture medium with medium growth sucrose, light gray aerial mycelium, beige substrate mycelium.

 On Tchapek culture medium with glucose: medium growth, greyish aerial mycelium, cream-colored substrate mycelium.

 On culture medium with sucrose: good growth, gray aerial mycelium, yellow substrate mycelium.

 On starch-agar culture medium: medium growth, light gray aerial mycelium, pale ashy coloration, mycelium yellow to lemon.

On starch-ammonia culture medium, according to
Mishustin: good growth, gray to dark gray aerial mycelium, yellow to light yellow substrate mycelium.

 On synthetic culture medium according to Krassilnikov: low to medium growth, beige gray aerial mycelium, yellow substrate mycelium.

 On CPI according to N.A. Krassilnikov: good growth, milky gray, blue gray aerial mycelium, greenish yellowish green mycelium.

On CPII according to N.A. Krassilnikov: good growth, gray aerial mycelium, a white exudate separates, creamy gray substrate mycelium.

On CPIII according to N.A. Krassilnikov: good growth, gray aerial mycelium, yellow to lemon yellow substrate mycelium.

 On CPIV according to N.A. Krassilnikov: medium growth, light gray to greyish aerial mycelium, cream-colored substrate mycelium.

 On CPV according to N.A. Krassilnikov: weak growth, white aerial mycelium, beige substrate mycelium, dark cream color.

 On medium Vaxman synthetic growth medium, white, gray aerial mycelium on separate parts, yellow to orange substrating mycelium.

 On meat-starch-agar culture medium: medium growth, gray aerial mycelium, yellow substrate mycelium.

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

 On glucose-asparagine-agar: medium growth, light gray aerial mycelium, yellow to cream colored mycelium substrate.

 On glycerin-asparagine-agar: good growth, light gray aerial mycelium, becoming gray during aging, dark yellow substrating mycelium.

On tyrosine agar: medium growth, gray aerial mycelium, in some greyish yellow colonies, dark yellow to orange mycelium substrate.

 On tyrosine-casein-nitrate-agar: medium growth, ashy gray aerial mycelium, yellow substrate mycelium.

 On glucose-tyrosine-agar: good growth, creamy gray to gray aerial mycelium, yellowish brown mycelium.

On sucrose-nitrate-agar: medium growth, gray aerial mycelium, creamy yellow mycelium.

On glycerol-calcium-malate-agar: good growth, light gray to gray aerial mycelium, yellow to orange substrate mycelium.

On peptone-beef-agar: good growth, gray aerial mycelium, colorless to beige substrate mycelium.

 On oats-agar: very good growth, gray aerial mycelium, light yellow substrating mycelium.

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

 On lead acetate agar: weak growth, greyish aerial mycelium, dark yellow brown mycelium substrate.

On iron-peptone-agar: medium growth, mouse gray aerial mycelium, colorless substrating mycelium with a gray shade of aerial mycelium.

On yeast-agar malt: good growth, gray aerial mycelium, orange substrate mycelium.

 Tolerance of the strain vis-à-vis NaCl. The strain has a low tolerance for the concentration of sodium chloride in the medium. The maximum concentration is 4%. At this concentration, growth is weak. The aerial mycelium is white. The substrate mycelium is yellow. A concentration above 1% has a negative effect on the degree of sporulation.

 The strain peptonizes the skim milk. At the start of peptonization, the reaction is acidic, and after a while it becomes alkaline.

The strain dilutes the gelatin. It grows very well on a sucrose medium, but it does not invert sucrose. It grows very well on a starch-agar medium and it hydrolyzes starch well.

 It does not break down cellulose and does not reduce nitrates to nitrites. It releases hydrogen sulfide acid. It grows on potatoes. The hemolysis test is positive and the tyrosinase test negative.

It was found that on a 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 strain absorbs salicin to a small extent.

 It does not absorb carbon sources such as lactose, sorbite, inosite, sucrose and raffinose.

There are certain differences in pigmentation of the aerial mycelium and the substrate mycelium depending on the carbon source.

 It was found that on a modified basic culture medium of Pridham and Gottlieb, the growth of the strain is good in the presence of the following nitrogen sources: NH4C1, (NH4) 2SO4, (NH4) 2HPO4, NH4H2Po4 NH4NO3, Na2HPO4 and carbamide .

Growth is moderate on a culture medium containing sodium nitrate. The strain does not grow on a culture medium containing sodium nitrite.

 A very good growth of the strain was observed on culture media containing the following amino acids: asparaginic acid, asparagine, proline, cystine, tyrosine. Growth is moderate on a culture medium containing valine, hydroxyproline, phenylalanine, leucine and alanine. The strain does not grow on a culture medium containing glutamic acid.

 Depending on the nitrogen source, there are certain differences in pigmentation of the aerial mycelium and of the substrate mycelium.

 According to certain characteristics, the Streptomyces strain resembles Streptomyces griseoflavus, belonging to the Gray series after Bergey (1974) - Actinomyces griseoflavus from the Flavus group after N.A. Krassilnikov - 1970.

The latter is distinguished by a number of morphological, cultural, physiological and biochemical properties which are described in the characterization of the species by Bergey (1974) and by N.A. Krassilnikov (1970).

Streptomyces griseoflavus, for example, has spores in elongated, rod-shaped sticks with a smooth surface; it has a tolerance towards sodium chloride, from 7 to 10%. It weakly hydrolyzes the starch. It absorbs sucrose and sorbite and does not absorb galactose. Therefore, the strain of Streptomyces Nr.

1339 is not identical to the similar strain of Streptomyces griseoflavus (Actinomyces griseoflavus). This is why it is called Streptomyces sp. Nr. 1339, belonging to the Gray series after Bergey (1974) and the Flavus group after N.A. Krassilnikov (1970).

We cultivate the producing strain in flasks
500 ml Erlenmeyer flask containing 50 ml of fermentation culture medium, for 36 to 96 hours at a temperature of 24 to 360C, with an initial culture pH of 6.5 to 9.0, on a shaker rotating at 180 - 320 revolutions per minute.

The isomerization of glucose to fructose by means of the glucose-isomerase of the Streptomyces sp. Nr. 1339 can be done by direct treatment with fresh mycelium (separated by centrifugation at 1200 rpm and washed three times with 0.05 M phosphate buffer at pH 7.0) or with dry mycelium (cells air dried or acetone dried), with an enzyme solution (obtained after ultrasonic disintegration or autolysis of the cellular material and separation of the supernatant liquid by centrifugation at 15,000 rpm) 1 the result of the centrifugation of the culture containing extracellular isomerase or cells immobilized on a hard support.

The amount of fructose formed in the reaction mixture is determined using the cysteine-carbazole method (19), and the activity of the strain is expressed in milligrams of fructose per ml of culture fluid or in international units of glucose isomerase. (GIU). A
GIU is equal to the quantity of enzymes which, at 700C and in a 1 M glucose solution of pH 7.0 in a 0.05 M phate phos buffer, containing 1.10 4 M Cal2. 6 H2O and 1.10 M
MgSO4. 7 H2O, transforms in one minute 1 + mole of glucose into lp, mole of fructose.

The advantages of the process according to the invention are as follows:
To obtain glucose isomerase, the strain of Streptomyces sp. Nr. 1339, which has a great capacity for bio-synthesis of the enzyme (12,000 to 20,000
GIU), that is to say which is 4 to 20 times greater than the activity of the producing strains described in previous patents (5 - 6). The enzyme obtained is characterized by a high temperature optimum (700C) at a low optimal concentration of Co ++ (1.10-4 M) and Mg + (1.10 2 M) in the isomerization mixture. It has a favorable pH optimum (7.0) and considerable thermostability between 40 and 650C.

The invention is illustrated by the following examples
Example 1.

The producing strain is kept in test tubes with inclined agar on culture media having the following composition 1. Xylose-based culture medium
Xylose 20 g
Agar 20 9
KNO3 1.0 g
K2HPO4 0.5 g
MgSO4.7H2O 0.5 g
NaCl 0.5 g CaCO3 1.0 g FeS04 0.001 g water in sufficient quantity to make 1 1.

2. Potato-glucose-agar
Potato extract obtained from 300 g of boiled potatoes
Glucose 10 g
Agar 20 g water in sufficient quantity to make 2 1.

It is recommended, to maintain the strain, to alternate the two culture media above.

To a well-seeded material resulting from a culture of 10 to 15 days on a culture medium 1 or 2 (1 is recommended), 6 ml of inoculation culture medium having the following composition is added.
Xylose 1.0%
Tryptone 2.0%
MgS04.7H20 0.1%
K2HPO4 0.25%
The cell mass is washed and the test tube is placed on a shaking device for 24 hours at 300 ° C. and at 240 rpm. From the culture thus adapted, an inoculation medium having the next composition
Xylose 1.0%
Corn extract 3.0% (dry weight) MgS04.7H20 0.1%
K2HPO4 0.25% agar 0.1%
After 60 hours of culture under the above conditions, 5% of the inoculum is introduced into a fermentation culture medium having the following composition
Xylose 1%
Corn extract 3.0% (dry weight Ci) MgS04.7H20 0.1%
KC1 0.0075% CoC12.6H20 0.024%
The culture is carried out in 500 ml Erlenmeyer flasks containing 50 ml of fermentation culture medium. The culture is carried out for 60 hours at 300C on a shaker at 240 rpm. The initial pH of the culture is 8.5.

After 60 hours of culture of Streptomyces sp. Nr.

1339, 140 to 220 g of wet biomass are obtained per 1 1 of culture liquid.

Example 2.

 The isomerization of the glucose into fructose is carried out by means of glucose-isomerase of the strain Streptomyces sp. Nr.

1339, at a temperature of 700C, a pH of 7.0, in the presence of CoC12.6H20 at a concentration of 1.10 4M, in the presence of MgSO4.7H20 at a concentration of 1.10 2M and a substrate concentration of 1 M.

The activity of the Streptomyces sp. Nr. 1339 is 130 to 215 mg of fructose per ml of culture fluid, or 12,000 to 20,000 G.I.U. per liter of culture fluid.

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Claims (2)

1. Method for obtaining glucose-isomerase, characterized in that it consists in cultivating the enzyme-producing strain Streptomyces sp. Nr. 1339 filed under Nr. 144 at the State Institute for Drugs Control, for 36 to 72 hours in a culture medium using xylose as an inoculation medium, the temperature being maintained between 24 and 360C, the initial pH of the culture being from 6.5 to 9.0, the isomerization temperature being from 50 to 800C, the pH being from 6.0 to 9.0, in the presence of CoC12.6H2O and with a substrate concentration of 0.1 to 3 M.  2. Method according to claim 1, characterized in that the culture medium which is used has the following composition Xylose 1.0 - 2.0% Corn extract 1.5 - 4.0% (dry weight) MgSO4.7H2O 0.05 - 0.2% KC1 0.005 - 0.01% CoC12.6H2O 0.008 - 0.036%.