DE3044357A1 - METHOD FOR PRODUCING GLYCOSE ISOMERASE - Google Patents

Description

SHIP ν. FONER STREHL SCHÖBEL-HOPF EBSINQHAUS FINCK

³ 304A3B7

description

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

It is known that the enzyme glycose isomerase does the conversion the D-glycose is realized in D-fructose, whichever More common use in the food and beverage industry and the dietary nutrition of highly developed countries. In combination with a complex of starch-degrading enzymes (% -amylase and glycoamylase), the glycose isomerase enables the production of glucose-fructose syrups by enzymatic means directly from starch.

Processes for the production of glucose isomerase have been known since 1957, when the possibility for a direct conversion of D-glucose to D-fructose by cells of the bacterial strain Pseudomonas hydrophyla No. 491 and 492 was demonstrated for the first time. Microorganisms of the genus Streptomyces are most frequently used in the production of glucose isomerase. An absolute prerequisite, which up to now. for the biosynthesis of the enzyme glycose isomerase has been established, the presence of various mineral salts in the culture media intended for cultivation. The nutrient media described in the literature and patents contain magnesium salts as MgSO, .7H ₂ O and cobalt as CoCl ₂ .6H ₂ O (Yamanaka K. 1961, Agr. Biol. Former., 25, 4, 272). The concentrations in which these salts are introduced into the culture medium depend on the nature of the microorganism producer and usually vary in the range from 0.02 to 0.5 Si for MgSO..7H ₂ O and from 0.005 to 0.024 % for CoCl ₂ .6H ₂ O (Takasaki Y., Ibid. 1966, 3o, 12, 1247).

In some Streptomyces species it is, though, used for activation

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ORIGINAL INSPECTED

Magnesium ions are added, cobalt absolutely necessary in order to to arrive at a glycose isomerase formation (Stradberg. G.W., Smiley K.L. 1971, Appl.Microb., 21, 588; Takasaki Y., Kosogu Y. 1969, Academic Press Inc. New York, p. 561; Tsumura N., Hagi M., Sato T. 1967, Agr. Biol. Chem., 31, 902).

Strains and mutant producers of glucose isomerase are sought which have the ability to produce sufficient Enzyme yield to produce in the absence of cobalt ions in the culture medium for cultivation. From GP-PS 1 411 763, 764, 765 and GB-PS 1,328,970 and 1,362,365 are mutants and Artrobacter species known that do not require cobalt for the biosynthesis of glycose isomerase.

By adding cobalt and magnesium ions to the glycose solutions the enzymatic activity is also strongly influenced during the isomerization. It has been proven that this Metals are cofactors of the enzyme and that glycose isomerase can be attributed to the metal enzymes (Danno G. Ibid. 1971, 35, 7, 997). It was found that in Glucose isomerase from Str. Sp. YT # 5, 4.1 cobalt atoms and 33 magnesium atoms are contained in one enzyme molecule. It \ d.rd accepted (Danno G., Ibid. 1971, 35, 7, 997), that the connection of the Mg and Co with the glycose isomerase is necessary for their conversion into active form. as well it is believed that a change in enzyme conformation occurs during this process. Some authors are of the opinion that the introduction of cobalt ions, especially together with magnesium ions, increases the thermal stability the glycose isomerase is significantly increased (Danno G., Ibid. 1971, 35, 7, 997; Sergio S., Kare L. 1975, Appl. Microbiol., 29, 6, 745).

The presence of larger amounts of Co ⁺ " ¹ " in the fructose syrups is undesirable (Jacobziner H., Raybin HW, 1961,

I 1

Arch. Pediat. 78, 200), although there are traces of Co for nutritional

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human intervention is necessary (GB-PS 1 411 763-65). The level of cobalt ions in fructose syrup is approximately 1 mM (US Pat 3 623 953), but it has also been shown that this concentration causes certain toxic effects in rats (Levy H., Levinson V., Shade Α., 1950, Arch. Biochem., 27, 34). Around to remove the cobalt ions from the glucose-fructose syrups after the isomerization process is complete they must necessarily be worked with ion exchange resins to their maximum separation. This additional The process requires the creation of industrial automated ion exchange systems. The company Mi-Car International uses fully automated ion exchange systems with two sections of the cation-anion parts (highly acidic Cation exchange resin Duolite C25-D and weakly alkaline anion resin Duolite S-56). This ion exchange system requires periodic regeneration with acids or bases and large amounts of additional pure water. About the salary of cobalt ions in the glycose-fructose syrups processed by means of the ion exchange system a test using an automatic absorption spectrophotometer is accomplished by continuously sampling the syrup stream, thereby making the cation exchange system work is regulated. During this processing, the cobalt ions must be reduced to a level of ppb at 25% S. effectively eliminated (Mi-Car Interantional Inc. Isomerized Syrup Process. 1975, MFG Information). This process complicates production and has a negative effect on effectiveness and cost price.

The object of the invention is to develop a method for the production of glucose isomerase with a parent producer, there being no cobalt ions for the biosynthesis of the enzyme and for the isomerization reaction of D-glycosis in D-fructose and should therefore also have commercial success.

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To achieve this object, the method is according to the invention characterized in that the enzymatic producer strain Streptomyces sp. No. 765 for 36 to 72 hours in the nutrient medium is cultivated with inducer xylose, with a temperature of 24 to 36 ° C and an initial pH value of the cultivation from 6.5 to 9.0 are maintained, and that the Isomerization at a temperature in the range from 50 to 90 ° C and a pH in the range of 6.0 to 9.0 in the presence

-4-2 of MgSO ₄ .7H ₂ O in a concentration of 1.10 to 1.10 IXl and a substrate concentration of 0.1 to 3 M is carried out.

The strain Streptomyces sp. No. 765 - Glucose isomerase producer was isolated from Bulgarian soil. For its discovery, 874 Streptomyces strains were sieved. the Sifting (screening) was carried out in a modified synthetic Nutrient medium No. 1 according to Krassilnikow carried out by selecting on two substrate levels with the use of Xylose and xylan was accomplished. Under these conditions, 18 strains of Streptomyces were found with opportunities for development and production of the enzyme glycose isomerase discovered, from which the strain Streptomyces sp. No. 765 has been shown to be capable of the enzyme glycose isomerase in the absence to be produced by Co in the culture medium.

Streptomyces sp. No. 765 is at the State Institute for Control of Medicines, Boul. Vl. Saimov, on September 29th 1979 and received the number 143. The strain has the following morphological and biochemical characteristics on:

In nutrient medium No. 1 with nitrogen mineral source (according to G.F. Gause and co-workers), the colonies are usually round in shape with smooth edges, curved in the middle, swollen and forming a depression like a small crater and just as strongly folded radially. The growth is good. The sporangia (spore leaves) of the young cultures are

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elongated, monopodinl arranged with 3 to 5 ringlets on the spirals, adding the same in older cultures as small Brooms (panicles) are huddled together.

The spores are elongated with rounded ends; at a magnification of over 16,000 times, ¹ , hair-like formations are observed on the surface. At a smaller magnification, the spores look smooth; the spore size varies between 0.9 to 1.2 µm (microns) in length and 0.4 to 0.6 µm in width.

The color of the air and substrate mycelium is according to the A.S. Bondarzew color scale and according to the Tresner and Backus scale has been determined. Changed in the different nutrient media depending on the carbon and nitrogen sources the color of the aerial mycelium changes from white (d), light gray to purple (a to a). In nutrient medium No. 1 with a Nitrogen mineral spring (according to G.F. Gause and colleagues) the color of the aerial mycelium is light gray to mouse gray (a ' to a) and in a nutrient medium with an organic nitrogen source (according to G.F. Gause and Mirarbeitern) it is dark-

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gray to gray-purple (a to a).

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

The substrate mycelium in nutrient medium 1 with nitrogen mineral source (according to G.F. Gause and co-workers) has a light dove-gray color to ultramarine (1 to ν). In the nutrient medium 2 with an organic nitrogen source (according to G.F. Gause and colleagues) the substrate mycelium is ultramarine (v) and with continued cultivation, it turns black (a).

In culture media with different carbon and nitrogen sources the substrate mycelium is from dark red to black.

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In meat peptone agar medium: growth - weak. Aerial mycelium - pink on the edges - brick red. Substrate mycelium

- brick red.

In a potato-glucose-agar nutrient medium: the stem grows very good, aerial mycelium - gray-blue, substrate mycelium - blue to dark blue. ;

In Tschapek nutrient medium with sucrose: growth - weak, Aerial mycelium - pink, substrate mycelium - light brique.

In Tschapek nutrient medium with glucose: growth - moderate, aerial mycelium - light blue, substrate mycelium - colorless with a shade the color of aerial mycelium.

In a nutrient medium with sucrose: growth - good, aerial mycelium - sky blue, substrate mycelium - dark blue to black.

In a starch agar culture medium: growth - good, aerial mycelium

- gray, substrate mycelium - blue.

In a starch-ammonia culture medium from Mischustin: growth

- very good, aerial mycelium - gray, substrate mycelium - wine-red to dark wine-red.

In a synthetic nutrient medium according to N.A. Krassilnikov: Growth - moderate, aerial mycelium - light ash gray, substrate mycelium - pink-purple.

In a CPI according to N.A. Krassilnikow: Growth - good, aerial mycelium - gray-blue, substrate mycelium - red-brown.

In a CPII according to N.A. Krassilnikov: growth - weak to moderate, aerial mycelium - blue, substrate mycelium - dark beige.

In a CPIV according to N.A. Krassilnikov: growth - weak, Aerial mycelium - light gray, substrate mycelium - colorless, with a shade in the color of aerial mycelium.

In a CPIII according to Krassilnikow: growth - good, aerial mycelium - gray to ultramarine, substrate mycelium - wine red.

In a CPV according to Krassilnikow: growth - good, aerial mycelium

- gray, substrate mycelium - dark purple.

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In a synthetic culture medium according to Wachsmann: growth moderate, Aerial mycelium - gray to mouse gray, substrate mycelium dark beige to red-brown.

In a meat starch agar medium: growth - weak, Aerial mycelium - white, substrate mycelium - beige.

In peptone agar: growth - good, aerial mycelium - gray, substrate mycelium - colorless, with gray shading of the aerial mycelium.

In glycose asparagine agar: growth - good, i.e. very good, Aerial mycelium - gray to ultramarine, substrate mycelium - blue-violet to dark gray.

In glycerine asparagine agar: growth - good, aerial mycelium ultramarine, Substrate mycelium - dark purple to black.

In a tyrosine medium: growth - good, aerial mycelium blue-gray, ultramarine, substrate mycelium - red-brown.

In a tyrosine-casein-nitrate agar: growth - weak, aerial mycelium - white, substrate mycelium ^ - beige.

In a glycose tyrosine agar: growth - moderate, aerial mycelium - cream-colored-gray, substrate mycelium - purple.

In a sucrose nitrate agar: growth - good, aerial mycelium - gray to ultramarine, substrate mycelium - blue to dark blue.

In a glycerine calcium malate agar: growth - good, aerial mycelium - from beige-blue to ultramarine, substrate mycelium dark purple.

In a peptone bovine agar: growth - moderate, aerial mycelium - gray, substrate mycelium - colorless with a gray shade of the aerial mycelium.

In an oat agar: growth - moderate, aerial mycelium - gray, substrate mycelium - wine-red.

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

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In a lead acetate agar: growth - weak, aerial mycelium brown, Substrate mycelium - colorless with a shade of gray from aerial mycelium.

In an iron peptone agar: growth - good, aerial mycelium gray, Substrate mycelium - colorless, with a gray shade of aerial mycelium.

In yeast-malt-agar: growth - moderate, aerial mycelium - gray, mouse gray, substrate mycelium - dark cream colored.

Tolerance of the strain to NaCl - it has a small tolerance to the concentration of sodium chloride in the nutrient medium. The maximum concentration is 4%. The growth of the stem at this concentration is weak, aerial mycelium - light blue, substrate mycelium - dark blue. A higher NaCl concentration than 2% has a negative effect on spore formation.

Coagulated defatted milk: does not liquefy the gelatin, grows well on a sucrose nutrient medium, but inventories the sucrose does not, grows very well in starch agar and also hydrolyzes the starch well. Does not decompose the cellulose, reduces nitrates to nitrites, secretes hydrogen sulfide, grows on potatoes, hemolysis - negative, tyrosinalysis positive, forms melanoids.

In a basic culture medium by Pridham and Gottlieb, a good growth found with the following carbon sources: glucose, pructose, lactose, levulose, xylose, mannose, cellobiose, Galactose, mannitol, inositol, arabiose, dextrin, ribose and glycerin. Weakly adsorbs the salicin. Does not grow in culture media with sorbitol, sucrose and raffinose. There will also be differences in the pigmentation of the Air and substrate mycelium observed depending on the carbon source.

In a modified basic medium by Pridham and Gottlieb, good growth of the strain with the following nitrogen sources is found: NH ₄ Cl, - (NH ₄ ) ₂ ^SO 4 '

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PO. and carbamide.

The strain shows moderate growth in a nutrient medium with NH4NO- and Na ^ HPO. on by placing the same in a nutrient medium nit NaNOo and NaNO “does not grow at all.

In culture media with the following amino acids: glutamic acid, aspartic acid, Alanine, valine, asparagine, very good growth of the stem is observed. The latter shows a moderate Growth in culture media with leucine, cystine, proline, hydroxyproline, phenylalanine and tyrosine. There will be differences observed in the pigmentation of the air and substrate mycelium depending on the nitrogen source.

In some characteristics, Streptomyces strain No. 765 is similar to Streptomyces coelicolor, which is the Gray series according to Bergey's - 1974 (Actinomyces coelicolor from the group Coelicolor according to N.A. Krassilnikow - 1970). The latter differs in some culture-r-morphological and physiological-biochemical properties, which are described in the Species-Characteristic - Bergey's (1974) and N.A. Krassilnikov (197O) are described. Streptomyces coelicolor has e.g. 1 to 3 ringlets on the spirals, liquefies the gelatin slow, peptonized defatted milk, tyrosinase negative. Thus, Streptomyces strain No. 765 is with not identical to the Streptomyces coelicolor (Actinomyces coelicolor), which is similar to it, and therefore we write it to the Gray series according to Bergey's (1974) and the Coelicolor group according to N.A. Krassilnikow (1970) too.

The strain producer is fermented in 50 ml nutrient medium containing 500 ml Erlenmeyer flask for 36 to 96 hours at a temperature of 24 to 36 ° C, initial pH of cultivation between 6.5 and 9.0 on a shaker Cultivated 180 to 320 revolutions / minute.

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The isomerization of glucose to fructose with glucose isomerase from the strain Streptomyces sp. No. 765 can be done using direct treatment with fresh mycelium (excreted by centrifugation at 12,000 revolutions / minute and three times Rinsing with 0.05 M phosphate buffer with a pH value of 7.0) or with dried mycelium (air-dry or acetone-dry cells), with an enzymatic solution by decomposition by means of ultrasound or autolysis of the cell material and secretion of supernatants by centrifugation at 15,000 revolutions / minute, with culture centrifugate containing extracellular isomerase, or on a solid support immobilized cells.

The fructose formed in the reaction mixture is determined by the cysteine-carbasol method (Dishe Z-, Borenfreund E., 1951, J. Biol. Erst., 192, 583) and the stock activity is expressed in mg fructose per ml of culture fluid or in international glycose Isomerase units (GIU) expressed. A GIU is the enzyme quantum that converts 1 µmol glycose into 1 pmol fructose in one minute at 7O C and a pH value of 7.0, IM glycose solution in 0.05 M phosphate buffer and 2.10 M MgSO ₄ .7H _{2 O} .

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

The strain Streptomyces sp. No. 765 produces the enzyme glycose isomerase in the absence of cobalt ions in the fermentation medium. The resulting enzyme converts the D-glycose into D-fructose in the absence of cobalt ions in isomerization mixture, which significantly simplifies the technological process will; is the use of ion exchange systems to remove the cobalt from the fructose containing syrups not necessary anymore. Compared to the strains known from the patent literature — microorganisms and mutant producers of glycose isomerase, which does not contain cobalt ions in the Require culture medium and in the isomerization mixtures

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(GB-PS 1 411 763, 1 411 764, 1 411 765, 1 328 970, 1 362 365), outperforms Streptomyces sp. No. 765 same with his Glycose isomerase activity, favorable pH optimum (7.0), a higher temperature optimum (80 ° C) of the enzyme and a substantial thermal stability between 40 and 70 ° 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 exhibited:

1. Xylose nutrient medium

Xylose 20 g

Agar 20 g

KNO ₃ 1.0 g

K ₂ HPO ₄ 0.5 g

MgSO ₄ .7H ₂ O '0.5 g

NaCl 0.5 g

CaCO ₃ I / O g

FeSO ₄ 0.001 g

Water up to 11.

2. Potato glycose agar

Potato extract from 3OO g boiled potatoes

Glycose 10.0 g

Agar 20.0 g of water up to

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

A well-germinated material takes 10 to 15 days Culture in nutrient medium 1 or 2 (preferably 1) are 6 ml

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Inoculation nutrient medium with the following composition was added: xylose 1 %, beef extract 2%, MgSO ₄ -TH ₃ OO, 1 %, K ₂ HPO ₄ O, 3%.

It is carried out a rinsing the coat weight, after which the test tube on a shaker for 24 hours at 30 ⁰ C and 240 revolutions is moved minute /. From this culture adapted in this way, an inoculation medium with the following composition is sown: xylose 1 %, maize extract 3.0 % (dry matter), MgSO..VH ₃ O 0.1%, K ₃ HPO ₄ O, 25%, agar 0.1 %.

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

When the Streptomyces sp. No. 765 is 160 to 240 g of moist biomass per 1 liter of culture liquid manufactured.

Example II.

The isomerization of glucose to fructose by means of glucose isomerase from the strain Streptomyces sp. No. 765 is at a temperature of 70 ° C, pH 7.0 in the presence of

_2

MgSO..7HO in a concentration of 2.10 M and substrate concentration Performed 1 st.

The activity of the Streptomyces sp. No. 765 is 75 to 130 mg of fructose per ml of culture fluid or 7,000 to 12,000 GIU per 1 liter of culture fluid.

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Claims (2)

PROCESS FOR THE PRODUCTION OF GLYCOSE ISOMERASE Patent pending 1. A process for the production of glucose isomerase, characterized in that the enzymatic producer strain Streptomyces sp.Nr.765, which is deposited at the State Institute for Control of Medicines, Blvd.VI, Saimov No. 26, Sofia, Bulgaria, is cultivated for 36 to 72 hours in the nutrient medium with 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 90 ° C and a pH value in the range from 6.0 to 9.0 in the presence of MgSO .. 7H ₂ O in a concentration of 1.10 ~ to 1.1O ~ M and a substrate concentration of 0.1 to 3 M will. 2. The method according to claim 1, characterized in that that the nutrient medium used has the following composition: 130027/0890 ORIGINAL INSPECTED 304A357 Xylose Corn extract Na acetate Ι, σ up to 2.0 %, 1.5 to 4.0% (with dry matter), 0.25 to 1.0 %. 130027/08 9-0