NL8006509A - METHOD FOR OBTAINING GLUCOSEISOMERASE. - Google Patents

Description

N / 30.025-Kp / Pf / cs

Method for obtaining glucose isomerase.

The invention relates to a method for obtaining glucose isomerase from a streptomyces strain.

The conversion of D-glucose into D-fructose with the aid of the enzyme glucose isomerase is known, and is increasingly used in a number of developed countries in the food processing industry and dietary food. Glucose isomerase, in combination with a group of amylolytic enzymes (cx-amylase and glucoamylase), provides the opportunity to obtain glucose-fructose-10 syrups and fructose directly from starch using enzymes. Some methods of obtaining glucose isomerase have been known since 1957, when the possibility of the direct conversion of D-glucose to D-fructose by cells of the bacterial strains Pseudomonos hydrophila 491 and 492 was demonstrated for the first time. Ant organisms of the genus Streptomyces are most commonly used to obtain glucose isomerase. In addition, it was found that the presence of various mineral salts in the culture medium is a necessary condition for the biosynthesis of the enzyme glucose isomerase. In the culture media described in the literature and patents, magnesium in the form of MgS0.7 .7H2 O and cobalt in the form of CoCl2-6H2 O are present (18). The concentrations at which these salts are added to the culture medium depend on the species producing microorganism and usually range from 0.02 to 0.5% for

MgSO 4 .7H 2 O and from 0.005 to 0.024% for CoCl 2 .6H 2 O (14). In some streptomyces, cobalt is absolutely required for the formation of glucose isomerase, although the activation is attributed to the magnesium ones (13, 16, 17).

It is desirable to discover glucose isomerase producing strains and mutants capable of producing a sufficient amount of enzyme in the absence of cobalt ions in the culture medium. The mutants of CPC International Ine are known. (2), as well as some Artrobot types 35 studied by R.J. Reynolds Tobacco (9), which do not require the presence of cobalt for glucose isomerase biosynthesis.

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The addition of cobalt and magnesium ions to the glucose solution during the isomerization also strongly influences the enzyme activity. These metals are proven to be cofactors of the enzyme, for which reason glucose-5 isomerase can be called a metal enzyme (3). In addition, it was found that one molecule of glucose isomerase originates from

Streptomyces YT5 contains 4.1 cobalt atoms and 33 magnesium atoms.

2+ 2+

It is believed that the combination of Mg and Co with glucose isomerase is necessary for the conversion of the enzyme to its active form. During this process, a change in the conformation of the enzyme would occur. Some authors hypothesize that the addition of cobalt ions, particularly along with magnesium ions, significantly increases the thermostability of glucose isomerase. (3, 15 10).

2+

Although traces of Co are necessary for human nutrition (12), the presence of higher levels of 2 + 2 Co in fructose syrups is undesirable (5). The content of Co ions in the fructose syrup is approximately 1 mM (1). However, it has been proven that this concentration has some toxic effects on rats. For the maximum separation of the cobalt ions of the syrup derived from glucose isomerase at the end of the isomerization process, it is necessary that the strains are grown in the presence of ion exchange resins.

For example, automatic ion exchange systems for this additional process must be installed in industry. Mi-Car International Company uses fully automated ion exchange systems consisting of cation and anion exchangers, the strong acid cation exchange resin Duolite C25-D 30 and the weakly basic anion exchange resin Duolite S-56. This ion exchange system requires periodic regeneration with acids or bases and large amounts of pure water.

The cobalt ion content in the glucose-fructose 35 syrups incorporated in the ion exchange system is monitored by means of samples drawn continuously from the product stream using an automatic absorption spectrophotometer. In this way, the operation of the cation exchange system is controlled. Thereby, TCo-40 ions must be removed so effectively that the final 8 00 6 50 9

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- 3 - concentration does not exceed 0.005 ppm. in 25% solutions of the syrups (8). This process complicates production and influences production efficiency and the basic cost of the product.

The object of the invention is to provide a method of industrial interest for obtaining glucose isomerase from a producing bacterial strain, in which cobalt ions are not necessary for the biosynthesis of the enzyme and for the isomerization of D-glucose into D-fructose to be.

The strain Streptomyces 765, producer of glucose isomerase, was isolated from Bulgarian soil. To this end, 874 strains of streptomyces have been carefully examined. This study was performed on the modified synthetic culture medium No. 1 to Krassilnikov, where selection was made at two substrate levels using xylose 15 and xylan. Under these conditions, 18 streptomyces strains capable of developing and producing the enzyme glucose isomerase were discovered. In addition, the strain was Streptomyces 765 which was able to produce 2+ the enzyme glucose isomerase in the absence of Co in the culture medium. The strain was deposited under number 143 on September 29, 1979 at the State Institute for Drug Control in Bulgaria, and had the following morphological and biochemical properties:

On culture medium 1 with a mineral nitrogen source 25 (after G.F. Gause and collaborators), the colonies were usually oval-shaped with shapeless edges, swollen in the center with a crater-like cavity with pronounced radial folds. The growth of the colony was good. The sporangias were elongated and monopodically placed in young cultures and had 3-5 turns, and were condensed to sorghum in older cultures. The tracks were elongated with rounded ends; at magnification greater than 16,000 times, some hair-like formations could be observed on the surface. At a weaker magnification, the tracks seemed smooth. Their dimensions varied between 0.9-1.2 µm in length and 0.4-0.6 µm in width.

The color of the air and substrate mycelium was determined according to the color scale of A.C. Bondartsev and the scale of Tresner and Backus. Depending on the carbon-40 and nitrogen sources, the color of the aerial mycelium changed from white (d1) to light gray to violet (a-d) on the different culture media. On the culture medium 1 with a mineral nitrogen source (after GF Gause and co-workers) the color was 5 light gray to mouse gray (a -a) and on a culture medium 5 with an organic nitrogen source (after GF Gause and co-workers) the color dark gray to gray violet (a -a). On culture media with other carbon and nitrogen sources, the aerial mycelium was greyish green.

On culture medium 1 with a mineral nitrogen source 10 (after G.F. Gause and co-workers), the substrate mycelium was pigeon colored to ultramarine (16-v1). On culture medium 2 with an organic nitrogen source (after G.F. Gause and co-workers) the color is ultramarine (v1), but turns black (a1) after a long culture. On culture media with other carbon and nitrogen sources, the substrate mycelium color is from dark red to black.

The growth and colors of the air and substrate mycelium on some substrates are listed in Table I.

Table I

Culture medium Growth Color of the color of the air mycelium substrate myce _lium _ flesh-peptone agar slightly pink, brick-red red along the edge potato-glucose- very good gray-blue blue to don-agar cherry blue

Tchapek with sucrose small pink light stone red

Tchapek with glucose moderate light blue colorless with a glow of the air mycelium sucrose good sky blue dark blue to black starch agar good gray blue starch ammonia to very good gray wine red to

Mishustin dark wine red synthetic medium moderate light ash gray roseviolet after N.A. Krassilnikov CPI to N.A. Scratch-good gray-blue red-brown silnikov 8 00 6 50 9 - 5 -

Table I (continued)

Culture medium Growth Color of the color of the aerial mycelium substrate myce-lium_ CPU to N.A. Scratch-light blue dark cream silnikov to moderate CPIII to N.A. Scratch-good gray to wine-red silnikov ultramarine CPIV after N.A. Scratch- light gray colorless with silnikov a glow from the air mycelium CPV to N.A. Scratch-good gray dark violet silnikov synthetic medium moderate gray to dark gray to Vaxman mouse gray reddish brown meat-starch agar low white cream peptone agar good gray colorless with a gray glow of the air mycelium glucose asparagine- very good gray to blue violet to agar ultramarine dark blue glycerin asparagine - good ultramarine dark violet agar to black tyrosine good blue-gray, red brown ultramarine tyrosine casein - low white cream nitrate agar glucose tyrosine - moderate cream gray violet agar saccharose nitrate - good gray to blue to don agar ultramarine kerbly glycerol -calcium- good blue to male agar ultramarine peptone beef- moderate gray colorless with agar to good a gray glow from the air mycelium oatmeal agar moderate gray wine red tomato agar good gray dark beige, terracotta lead acetate agar slightly brown colorless with a glow of the air mycelium enn fi «in o - 6 -

Table I (continued)

Culture medium Growth Color of the Color of the air mycelium substrate myce-_lium_ iron-peptone agar good gray colorless with a gray glow of the air mycelium yeast malt agar medium gray, mouse dark cream gray

The strain showed little tolerance to increasing concentrations of sodium chloride in the medium. The maximum concentration was 4%, the growth of the strain was low, the color of the aerial mycelium was light blue and the substrate mycelium was dark blue. A concentration higher than 2% NaCl had a negative influence on the extent of spore formation.

The strain coagulated fatless milk, but did not condense gelatin. The strain grew well on a sucrose medium, but did not invert sucrose. The strain grew very well on a starch agar and also hydrolyzed starch well. The strain did not decompose cellulose, but reduced nitrates to nitrites. The strain released hydrogen sulfide. The trunk grew on potatoes. Hemolysis was negative, tyrosinase was positive. The strain formed melanoids.

On a culture medium of Pridham and Gottlieb, growth was found to be good in the presence of the following carbon sources: glucose, fructose, lactose, levulose, xylose, mannose, cellulose, galactose, mannitol, inositol, arabinose, dextrin, ribose and glycerol. The strain absorbed salicin on a small scale, but did not grow on a culture medium containing sorbitol, sucrose and raffinose. Depending on the carbon source, some differences in the pigmentation of the venting of the substrate mycelium were observed.

Strain growth was good on a modified culture medium of Pridham and Gottlieb with the following nitrogen sources: NH ^ Cl, (NH ^) 2 ^ 4 / (NH ^ ^ HPO ^, (NH4) H2PO4 and carbamide). moderately on a culture medium with NH 2 NO 2 and Na 2 HPO 4 The strain did not grow at all on a culture medium with NaNO 2 and NaNO 2 Very good growth of the strain was observed on culture media with the following amino- * 800 6 50 9 v c - - 7 - Acids: glutamic acid, aspartic acid, alanine, valine and asparagine Growth was moderate on a culture medium containing leucine, cystine, proline, hydroxyproline, phenylalanine and tyrosine Depending on the nitrogen source, some differences in the pigmentation of the air - and the substrate mycelium observed.

In some properties, the Strepto-myces 765 strain resembles Streptomyces coelicolor from the Gray series after Bergey's, 1974 (Actinomyces coelicolor from the Coelicol group 10 after N.A. Krassilnikov, 1970). However, this is distinguished in some morphological-cultural and physiological-biochemical properties, as can be seen from the species description by Bergey's (1974) and N.A. Krassilnikov (1970). For example, Streptomyces coelicolor has spirals with 1 to 3 win-15 things, slowly condenses gelatin, and peptonizes fatless milk. His tyrosinase is negative. This shows that the Streptomyces strain No. 765 is not identical to the similar Streptomyces coelicolor (Actinomyces coelicolor) and is therefore referred to here as Streptomyces 765 from the 20 Gray series after Bergey's (1974) and the Coelicolor group after N.A. Krassilnikov (1970).

The producer strain was placed in 500 ml Erlenmeyer flasks containing 50 ml fermentation culture medium for 36 to 96 h at a temperature of 24 to 36 ° C in a shaker with 180-320 rpm. cultivated, while the pH at the start of the culture was between 6.5 and 9.0.

The isomerization of glucose to fructose by glucose isomerase from the strain Streptomyces 765 can be carried out by direct treatment with fresh mycelium (separated by centrifugation at 12,000 rpm and washed three times with a 0.05 M phosphate buffer at pH 7.0) , with dried mycelium (air or acetone dried cells)., with a solution of the enzyme (obtained by supersonic disintegration or autolysis of cell material followed by separation of the supernatant by centrifugation at 15,000 rpm), or with a culture centrifugate containing extracellular isomerase or immobilized cells on a hard support.

The fructose formed in the reaction mixture was determined by the cysteine carbasol method (4), the activity of the strain being expressed in mg fructose per ml of culture liquid or in international glucose isomerase units ( GIU). A GIü is equal to the amount of enzyme, which at 70 ° C and a pH of 7.0 in a 1 M solution of glucose 5 in a 0.05 M phosphate buffer containing 2.10- ^ M MgSO4.7H20 in one minute of 1 pmole of glucose in 1 jumol fructose turnover.

The method of the invention has the following advantages: The strain Streptomyces 765 produces the enzyme glucose-10 isomerase in the absence of cobalt ions in the fermentation medium. The enzyme obtained converts D-glucose into D-fructose in the absence of cobalt ions in the isomerization mixture, which considerably simplifies the technological process and does not require the use of ion exchange systems for the separation of cobalt from the fructose-containing syrups. Compared to the patent literature strains, microorganisms and mutant producers of glucose isomerase, which do not require the presence of cobalt ions in the culture medium and in the isomerization mixtures (2a, b, c; 9a, b), Streptomyces 765 exceeds those in glucose -20 isomerase activity, due to a favorable pH optimum (7.0) and a higher optimum temperature (80 ° C) of the enzyme, and due to a considerable thermostability between 40 and 70 ° C.

The invention is illustrated by the following examples: 25

Example I.

The producer strain was stored in test tubes with descending agar on culture media of the following composition: 1. Xylose culture medium: 30 xylose 20 g agar 20 g KN03 1.0 g K2HP04 0.5 g

MgSO4.7H20 0.5 g 35 NaCl 0.5 g

CaCO3 1.0 g

FeS04 0.001 g water up to 1 1 800 6 50 9 - 9 - 2. Potato glucose agar: potato extract from 300 g boiled potatoes glucose 10 g agar 20 g 5 water up to 1 1

Preferably, the strain was stored alternately on both culture media.

6 ml of seed culture medium of the following composition was added to well-matured material of a 10-15 day old culture on culture medium 1 or 2, preferably culture medium 1/10: xylose 1% beef extract 2%

MgS04.7H20 0.1% 15 K2HP04 0.3%

The cell mass was washed and the test tube was placed in a shaker at 240 rpm for 24 h at 30 ° C. was placed. From a culture thus treated, a little was spread over a seeding medium of the following composition: 20 xylose 1% corn extract 3% (dry weight)

MgS04.7H20 k2hpo4 agar 25 After a 60 h culture under the above conditions, 5% of the inoculum was added to a fermentation culture medium of the following composition: xylose 1% corn extract 3.0% (dry weight) 30 sodium acetate 0.5%

The culture was carried out in 500 ml Erlenmeyer flasks containing 50 ml of fermentation culture medium for 60 h at 30 ° C in a shaker at 240 rpm. The pH at the start of the culture was 8.5.

After cultivation of Streptomyces 765 for 60 hours, 160-240 g of moist biomass per 1 culture liquid was obtained.

Example II.

40 The isomerization of glucose to fructose using 800 6 50 9 - 10 - of glucose isomerase from the strain Streptomyces 765 was adjusted at a temperature of 70 ° C, a pH of 7.0 in the presence of MgSO 4 .7H 2 O at a concentration of 2.10 M, while the concentration of the substrate was 1 M, was performed.

The activity of the Streptomyces 765 strain was 75-130 mg fructose per ml culture fluid or 7,000-12,000 GIü per 1 culture liquid.

REFERENCES: 10 1. U.S. Patent 3,623,953.

2. a / British Patent 1,411,763. b / British Patent 1,411,764. c / British Patent 1,411,765.

3. Agr. Biol. Chem., 35, 7, 997, (1971).

4. J. Biol. Chem., 192, 583, (1951).

5. Arch. Pediat. 78, 200, (1961).

6. Arch. Biochem., 27, 34, (1950).

7. Science, 125, 3249, 648, (1957).

8. Mi-Car International Inc. Isomerized Syrup process, 1975, 20 MFG Information.

9. a / British Patent 1,328,970.

b / British Patent 1,362,365.

10. Appl. Microbiol., 29, 6, 745, (1975).

11. J. Food Sci., 39, 215, (1974).

12. J. Chronic. Dis., 20, 869, (1967).

13. Appl. Microb., 21, 588, (1971).

14. Agr-, Biol. Chem. , 30, 12, 1247, (1966).

15. Agr. Biol. Chem., 33, 11, 1527, (1969).

16. Takasaki Y., Kosogu Y. 1969. Academy Press Inc. N.Y.

30 p. 561.

Agr. Biol. Chem., 31, 902, (1967).

18. Agr. Biol. Chem., 25, 4, 272, (1961).

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Claims (2)

Method for obtaining glucose isomerase, characterized in that the enzyme-producing strain Streptomyces 765 with the registration number 143 of the State Institute for Medicines Control is grown in a culture medium with xylose as an inducer for 36-72 h, while the temperature is between 24 egg is kept> 36 ° C and the pH at the start of the culture is between 6.5 and 9.0; the isomerization temperature is between 50 and 90 ° C and the pH is between 6.0 and 9.0 in the presence of MgSO ^^^ O in concentrations from 1.10 ^ to 1.10 ^ M and at a concentration of the substrate from 0.1 to 3 M. Method according to claim 1, characterized in that the culture medium used has the following composition: xylose 1.0 - 2.0% corn extract 1.5 - 4.0% (dry weight) sodium acetate 0.25-1, 0%. 800 6 50 9