DK151270B - METHOD OF PREPARING GLUCOSE ISOMERASE. - Google Patents

Description

151270

The invention relates to a process for the preparation of glucose isomerase, by growing a strain of the genus Streptomyces for 36-72 hours at a temperature of 24-36 ° C in a nutrient medium having an initial pH between 6.5 and 9.0. and containing xylose.

5 It is known that the enzyme glucose isomerase converts D-glucose to D-fructose, which is increasingly used in the food industry and in diet foods in a number of developed countries. Glucose isomerase, in combination with a complex of amylolytic enzymes (α-amylase and glucoamylase), provides the possibility of obtaining glucose fructose syrups and fructose directly from starch using enzymes. Some methods of producing glucose isomerase have been known since 1957, when it was first demonstrated that it was possible to directly convert D-glucose to D-fructose using cells from the bacterial strain Pseudomonas hydrophila N.491 and N.492. To produce glucose-15 isomerase, microorganisms of the Streptomycese genus are most commonly used. It has been found that the presence of various mineral salts in the growth medium is a necessary condition for the biosynthesis of the enzyme glucose isomerase. In the culture media described in the literature and in patent publications, magnesium salts are present in the form of MgSO 2 f 2 O and 20 cobalt in the form of CoC 2 H 2 O (18). The concentrations at which these salts are added to the growth medium depend on the producer microorganism and most often range from 0.02 to 0.5% for MgSO 2 H 2 O and from 0.005 to 0.024% for CoCl 2'BHgO (14). In some Streptomyces species, cobalt is absolutely necessary for the formation of glucose isomerase, although activation is performed due to magnesium ions (13,16,17).

However, certain microorganisms are known which are capable of producing a sufficient amount of enzyme in the absence of cobalt ions in the growth medium. Cf.. ref. (2) and (9).

The addition of cobalt and magnesium ions to glucose solutions 30 during isomerization also greatly affects enzyme activity. It has been shown that these metals are cofactors of the enzyme and that glucose isomerase can be attributed as a metal enzyme (3). It has been found that 1 molecule of glucose isomerase derived from Str.sp.YT N.5 contains 4.1 cobalt atoms and 33 magnesium atoms. It is believed (3), -f * + · | · ψ 35 that the combination of Mg and Co with glucose isomerase is required for the enzyme's transformation to an active form. It is also believed that during this process, a change in the conformation of the enzyme occurs.

Some researchers assume that the introduction of cobalt ions, especially in combination with magnesium ions, greatly increases the thermostability of glucose isomerase (3.10).

-H-

The presence of higher amounts of Co in fructose syrups is undesirable (5), although traces of Co ++ are necessary for human nutrition 5 (12). The content of Co ++ ions in fructose syrup is approx. 1 mWl (1), but it has been shown that this concentration is toxic to rats. For separation of cobalt ions from glucose isomerase syrups, after the isomerization process, it is necessary to use ion exchange resins for maximum separation. It is necessary to design industrial 10 automatic ion exchange systems for this further process. Some fully automated ion exchange systems can be used which include two cation anion moieties (strong acidic cation resin Duolite C25-D and weak basic anion resin Duolite S-56). This ion exchange system requires periodic regeneration with acid and bases and requires an additional large amount of 15 purified water.

The content of cobalt ions in glucose fructose syrups processed in the ion exchange system is checked by an automatic absorption spectrophotometer using samples taken continuously from the stream. Thus, the operation of the cation system must be regulated.

20 In this machining process, Co ions must be effectively removed so that they do not exceed 5 ppm (8). This process complicates production and influences the efficiency and manufacturing cost of the product.

It has now been found that a novel microorganism strain 25 Streptomyces sp. 765 produces glucose isomerase in good yield without cobalt ions needed for the biosynthesis of the enzyme and for the isomerization of D-glucose to D-fructose. The glucose isomerase produced has an appropriate pH optimum (7.0), temperature optimum (80 ° C) and considerable thermostability. Accordingly, the process of the invention is characterized in that, as a microorganism, the strain Streptomyces sp. 765 deposited with The State Institute for Drugs Control under number 143.

Streptomyces sp. N.765 has been isolated from Bulgarian soil. 874 Streptomyces strains have been studied for glucose isomerase production. The study was conducted on the modified synthetic growth medium # 1 after Krassilnikov, 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. Among them is the tribe 3 151270

Streptomyces sp. N.765 which is capable of producing the enzyme gluco-isomerase in the absence of Co ++ in the growth medium. The strain is deposited in The State Institute for Drugs Control - bul. Vladimir Zaimov N.26, September 29, 1979 under the designation N.143, and the strain has the following 5 morphological and biochemical properties:

In growth medium 1 with a mineral nitrogen source (according to G.F.Gause and co-workers), the colonies are usually oval in shape with not-too-narrow edges, convex in the center, swollen with a crater-like cavity with strongly expressed radial folds. The growth is good. The sporangia in 10 young cultures are elongated, monopodially arranged with 3-5 twists and in older cultures they are condensed into sorghum.

The spores are oblong with rounded ends: when enlarged more than 16,000 times some hair-like formation can be observed on their surface. At a smaller magnification, the spores look smooth.

15 The size varies between 0.9 and 1.2 µm in length and the width is between 0.4 and 0.6 µm.

The color of the aerial mycelium and the substrate mycelium is determined according to the color scale of A.C.Bondartsev and the scale of Tresner and Backus.

With the various growth media, the color of the aerial mycelium changes from white 20 (d) light gray to violet (a -d), depending on carbon and nitrogen sources. On growth medium 1 with a mineral nitrogen source (according to GFGause and co-workers) the color is from light gray to mouse gray (a-a), and on a growth medium with an organic nitrogen source (according to GFGause and P 3 co-ed.) The color is light gray to gray-violet ( a -a).

25 On growth medium with different carbon and nitrogen sources, the aerial mycelium is greyish-green.

On growth medium 1 with a mineral nitrogen source (according to G.F.Gause and co. Ed.), The substrate mycelium is pigeon-colored to ultramarine (l6-v1).

30 On culture medium 2 with an organic nitrogen source (according to G.F.Gause and co-ed.) It is ultramarine (v) and after prolonged cultivation Ί it becomes black (a).

On culture medium with various carbon and nitrogen sources, the substrate mycelium is from dark red to black.

35 On meat culture medium-peptone agar. Low growth. Aerial mycelium pink and brick red along the edge. The substrate mycelium drew in stone s red.

On potato growth medium-glucose agar. Growth very good. Aerial mycelium gray-blue. Substrate mycelium blue to dark blue.

4 151270

Pa Tchapek growth medium with sucrose. Low growth. The aerial mycelium pink. The substrate mycelium bright green in stone s red.

Pi Tchapek growth medium with glucose. Medium growth. Aerial mycelium light blue. Substrate mycelium colorless with a shade of the color of the aerial mycelium.

5 On growth medium with sucrose. Good growth. Aerial mycelium sky blue.

Substrate mycelium dark blue to black.

On growth medium amylium agar. Good growth. Air mycelium gray. Substrate mycium blue.

On amylum ammonium culture according to Mishustin. Very good growth.

10 Air mycelium gray. Substrate mycelium burgundy to dark burgundy.

On synthetic medium according to N.A.Krassilnikov. Medium growth. Aerial mycelium light ash. Substrate mycelium pink-violet.

At CPI according to N.A.Krassilnikov. Good growth. Aerial mycelium gray-blue. Substrate mycelium red-brown.

15 On the CPU according to N.A.Krassilnikov. Growth low to medium. Air mycelia um blue. Substrate mycelium dark cream.

At CPI 11 according to N.A.Krassilnikov. Good growth. Aerial mycelium gray for ultramarine. Substrate mycium burgundy.

At CPIV according to N.A.Krassilnikov. Low growth. Aerial mycelium light gray.

20 Substrate mycelium colorless, with a shade of aerial mycelium color.

At CPV according to N.A.Krassilnikov. Good growth. Air mycelium gray. Substrate mycelium dark violet.

On synthetic growth medium according to Vaxman. Medium growth. Aerial mycelium gray to mouse weeping. Substrate mycelium dark cream to reddish brown.

25 On meat amylum agar. Low growth. Aerial mycelium white. Substrate mycelium lium cream.

On peptone agar. Good growth. Air mycelium gray. Substrate mycelium colorless with a gray shade of the color of the aerial mycelium.

On glucose-asparagine agar. Very good growth. Aerial mycelium gray to 30 ultramarine. Substrate mycelium blue violet to dark blue.

On glycerin-asparagine agar. Good growth. Air mycelium ultramarine. Substrate mycelium dark violet to black.

On tyrosine growth medium. Good growth. Aerial mycelium blue-gray, ultramarine. Substrate mycelium reddish brown.

35 On tyrosine-casein nitrate agar. Low growth. Aerial mycelium white. Sub stratmycelium cream colored.

On glucose tyrosine agar. Medium growth. Aerial mycelium cream gray. Substrate mycelium violet.

5 151270 On sucrose nitrate agar. Good growth. Air mycelium gray to ultramarine. Substrate mycelium blue to dark blue.

On glycerol-calcium-malate agar. Good growth. Aerial mycelium blue for ultramarine.

5 On peptone beef agar. Medium growth. Air mycelium gray.

Substrate mycelium colorless with a gray shade of the color of the aerial mycelium.

On oatmeal agar. Medium growth. Air mycelium gray. Substrate mycelium burgundy.

On tomato agar. Good growth. Air mycelium gray. Substrate mycelium 10 dark beige, terracotta color.

On lead acetate agar. Low growth. Aerial mycelium brown. Substrate mycelium colorless with a shade of the color of the aerial mycelium.

On iron-peptone agar. Good growth. Air mycelium gray. Substrate mycelium colorless with a shade of the color of the aerial mycelium.

15 On yeast malt agar. Medium growth. Aerial mycelium gray, mouse crying. Sub stratmycelium dark cream.

Strain tolerance to NaCl. It shows low tolerance to the concentration of sodium chloride in the medium. Maximum concentration is 4%. At this concentration, the growth of the stem is low. Air mycelium is light blue.

Substrate mycelium is dark blue. A concentration of more than 2% NaCl adversely affects the degree of sporulation.

It coagulates low-fat milk. It does not melt gelatin. It grows well in sucrose medium, but it does not invert sucrose. It grows well on an amylum agar and hydrolyzes starch well. It does not decompose cellulose and reduce nitrates to nitrites. It releases hydrogen sulfide. It grows on potato. Hemolysis - negative. Tyrosinase - positive, it forms melanoids.

Growth has been found on Pridham and Gottlieb's basic growth medium in the presence of the following carbon sources: glucose, fructose, lactose, levulose, xylose, mannose, cellulose, galactose, mannite, inosit, arabinose, dextrin, ribose and glycerol .

The strain absorbs salicylics on a smaller scale.

It does not grow on growth medium with sorbit, sucrose and raffinose.

Some differences can be observed in the pigmentation of the aerial mycelium and the substrate mycelium depending on the carbonium.

The growth of the strain is good on Pridham and Gottlieb's modified basal growth medium with the following nitrogen sources: NH ^Cl, (NH ^SO ^; (NH ^) 2HPO0; (NH ^HgPC ^ and carbamide).

The growth is moderate on a growth medium with (NH 2 NOg and Na 9 HPOA. The strain does not grow at all on growth medium with NaNO1 and NaNO2.

The strain grows very well on growth medium with the following 5 amino acids: glutamic acid, aspartic acid, alanine, valine, asparagine.

Growth is moderate on a growth medium with leucine, cystine, proline, hydroxyproline, phenylalanine and tyrosine. Depending on the nitrogen source, some differences in the pigmentation of the aerial mycelium and the substrate mycelium are observed.

10 According to some properties, the Streptomyces strain is similar to N.765

Streptomyces coelicolor belonging to the Gray series according to Bergey's -1974 (Actinomyces coelicolor by the Coelicolor group after N.A.Krassilnikov 1970). The latter is distinguished from the former by some morphological, cultural and physiological-biochemical properties described in the species characterization in Bergey's (1974) and N.A.

Krassilnikov (1970). For example, Streptomyces coelicolor has from 1 to 3 spiral shifts, it slowly melts gelatin and peptonizes low-fat milk. Its tyrosinase is negative. Therefore, the Streptomyces strain N.765 is not identical to the similar Streptomyces coelicolor 20 (Actinomyces coelicolor), which is why it is referred to as Streptomyces sp. No. 765. It belongs to the Gray series according to Bergey's (1974) and the Coelicolor group after 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 180 to 320 rpm minute.

The isomerization of glucose to fructose by glucose isomerase from the strain Streptomyces sp. N.765 can be performed by a direct treatment with fresh mycelium (separated by centrifugation at 12000 rpm and 3 times washing with 0.05 M phosphate buffer with pH 7.0) or with dried mycelium (air dried or acetone dried cells) , with enzyme solution (obtained after ultrasonic disintegration or autolysis of cell material and separation of supernatant by centrifugation at 15000 rpm) with medium after centrifugation 35 containing extracellular isomerase or with cells immobilized on a hard support.

The fructose produced in the reaction mixture is determined by the cysteine-carbazole method (4) and the activity of the strain is expressed in mg of fructose per ml. in culture liquid or in international glucose isomerase 7 151270 units (GIU). One GlU unit is equal to the amount of enzyme which at 70 ° C and pH 7.0, 1M glucose solution in 0.05M phosphate buffer and _ 2x10 M IVIgSO ^ ♦ 7H20 in 1 minute converts 1 pmol glucose to 1 pmol fructose .

The advantages of the method according to the invention are the following:

Strains Streptomyces sp. N765 produces the enzyme glucose isomerase in the absence of cobalt ions in the fermentation medium. The enzyme obtained converts D-glucose to D-fructose in the absence of cobalt ions in the isomerization mixture, which greatly facilitates the technological process and it is not necessary to use ion exchange systems to separate cobalt from the fructose-containing syrups. Compared to the strains known from the patent literature which produce glucose isomerase and which do not require cobalt ions in the culture medium and in isomerization mixtures (2a, b, c; 9a, b), Streptomyces sp N.765 is distinguished by its glucose isomerase activity, the favorable pH optimum of the enzyme (7.0), high temperature optimum (80 °) and the considerable thermostability of the enzyme between 40 and 70 ° C.

The process of the invention is further illustrated by the following example.

20

Example

Streptomyces sp. N. 765 can be maintained on the following media.

1. Xylose Growth Medium 25 xylose 20 g agar 20 g KN03 1.0 g K2HPO4 0.5 g

MgSO4-7H2O 0.5 g NaCl 0.5 g

CaCO3 1/0 g

FeSO 4 0.001 g water to 1 liter. 1 2. Potato-glucose agar potato extract from 300 g cooked potatoes glucose 10 g agar 20 g water to 1 liter.

8 151270

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), add 6 ml of inoculation growth medium having the following composition: xylose 1% beef extract 2%

MgSO4-7H2O 0.1% K2HPO4 0.3% 10

The cell mass is washed out and the tube is placed on a shaker for 24 hours at 30 ° C and 240 rpm. From the culture so adapted, seed growth medium of the following composition is seeded: xylose 1% 15 corn extract 3% (dry weight)

Na acetate 0.5%

The cultivation is carried out in 500 ml Erlenmeyer bottles with fermentation medium. The cultivation is carried out for 60 hours at 30 ° C on a shaker at 20 240 rpm. The initial pH is 8.5.

After 60 hours of cultivation of Streptomyces sp N.765, 160 to 240 g of moist biomass per ml are obtained. 1 liter of culture fluid.

Isomerization of glucose to fructose by glucose isomerase from the strain Streptomyces sp N.765 is carried out at a temperature of 70 ° C, pH 25 in the presence of MgSO 4 * 7H 2 O at a concentration of 2 x 10 M and a substrate concentration of 1 M.

The activity of the strain Streptomyces sp N.765 is 75-130 mg of fructose per day. 1 ml of culture fluid or 7,000 - 12,000 GIU per ml. 1 liter of culture fluid.

9 151270

REFERENCES

1. Cotter W.P., Lloyd N.E., Hinman C.W. 1971. Patent USA 3,623,953.

2. (a) CPC International Inc. 1975. British Patent 1,411.763.

b) CPC International Inc. 1975. British Patent 1,411.764.

C) CPC International Inc. 1975. British Patent 1,411.765.

3. Danno G. Agr.Biol.Chem. 1971, 35, 7, 997 4. Dische, Z., Borenfreud E., 1951, J. Biol. Chem., 192, 583.

5. Jacobziner H., Raybin H.W. 1961. Arch.Pediat. 78,200 6. Levy H., Levinson V., Shade A. 1950 Arch.Biochem., 27.34 10 7. Marschall R.O., Cage E.R. 1957. Science, 125, N.3249,648 8. Mi-Car International Inc. Isomerized Syrup process. 1975 MFG Information.

9, a) R.J.Reynolds Tobacco Co. 1973 British Patent 1,328,970 b) R.J.Reynolds Tobacco Co. 1974. British Patent 1,362,365 10. Sergio S., Kåre L., 1975. Appl. Microbiol.29,6,745 11. Somers E., 1974. J.Food Sci. 39,215 12. Schraeder H.A., Nason A.P., Tipton, I.H. 1967. J.Chronic.Des. 20,869 13. Stradberg G.W., Smiley K.L.1971 .Appl.Microbiol., 21,588 20 14. Takasaki Y. Agr.Biol.Chem. 1966, 30,12,1247 15. Takasaki Y., Kosegu Y., Kanbayashi A. 1969. Agr.Biol.Chem. 31,11,1527.

16. Takasaki Y., Kosogu Y. 1969.Academic Press Inc. NEW. p.561 17. Tsumura N., Hagi N., Sato T.1967.Agr.Biol.Chem., 31,902 25 18. Yamanaka K. 1961. Agr.Biol.Chem., 25,4,272.

Claims (2)

151270 Patent Claims. A process for the preparation of glucose isomerase, by growing a strain of the genus Streptomyces for 36-72 hours at a temperature of 24-36 ° C in a nutrient medium having an initial pH of 6.5 to 9 , 0, and containing xylose, characterized in that the strain Streptomyces sp. 765 deposited with The State Institute for Drugs Control under number 143. Process according to claim 1, characterized in that the nutrient medium used has the following composition: xylose 1.0-2.0% by weight based on the weight of nutrient medium corn extract 1.5-4.0% by weight based on the weight of nutrient medium Na acetate 0.25-1.0% by weight based on weight of nutrient medium