CS219266B2 - Method of preparation of the glucosoizomeraze - Google Patents

Description

The invention relates to a process for the preparation of the enzyme glucose isomerase, the isomerization of which is suitable for the production of fructose and a syrup containing fructose and glucose.

For the enzymatic isomerization of glucose to fructose, glucose isomerase (D-xylose-ketol isomerase EC 5.3.1.5) is added to the glucose solution, for example corn syrup, and the reaction conditions are controlled by converting the glucose fraction into fructose, whereby the amount of glucose converted to fructose is a function of the equilibrium constant which is 1.08 at 60 ° C.

Numerous published works describe enzymatic methods for converting glucose into a syrup containing glucose and fructose by utilizing the activity of microorganisms of the genera Pseudomonas, Lactobacillus, Escherichia, Aerobacter, Arthobacter, Bacillus, Streptomyces and other microorganisms.

It has now been found that a microorganism of the genus Agrobacterium, not yet described for this purpose, is also capable of producing an isomerizing enzyme. isomerizes glucose when grown on a suitable culture medium.

The invention therefore relates to a process for the preparation of glucose isomerase particularly suitable for the production of fructose and fructose syrup from glucose, characterized in that the microorganisms of the strain Agrobacterium designated NRRL B 11291 and NRRL B 11394 are cultivated under aerobic conditions on a solid or liquid medium. comprising an assimilable carbon source, a phosphorus source, a nitrogen source, and a mineral salt at 20 to 40 ° C for 10 to 48 hours and a pH of 5.5 to 8.5.

The microorganisms used for the preparation of glucose isomerase isolated from agricultural land and marked with the above symbols have strain number 1302 - 1303 - 1304 and have the following morphological and biochemical characteristics:

Microscopic morphology:

Single, sometimes paired rods, gram-negative, 0.8 to 1.5 to 2.0 microns in size, capsule and spore-free, mobile with 4 to 6 penetrable flagella.

Macroscopic morphology:

Kdonie on agar broth (Difco): raised, non-cracked, cream-colored, transparent, external diameter 0,5 to 1 mm, smooth. Abundant growth on calcium glycerol phosphate-mannitol nitrate agar, with brown staining and background formation.

Biochemical properties:

Growth at 4 to 39 ° C on all simple laboratory media even in the absence of growth factor and in the absence of amino acids, using ammonium NH ⁺ ions, nitrated NO 5 - ions or amino acids as the sole nitrogen source.

Oxidase: positive (N. Kovacs, 1956, Nature, London, 178, 703).

Catalase: positive (M. Levine, D. Q. Andersen, 1932, J. Bact. 23, 337-347).

Nitrites produced from nitrates (C. E. ZoBell, 1932, J. Bact. 24, 273).

Non-hydrolyzed Tween 80 (G. Sierra, 1957, Antonie van Leeuwenhoek 23, 15-22).

Kasem, gelatin, cellulose, starch, agar: non-hydrolyzed (V. B. D. Skermann, A Guide to the Identification of Genera of Bacteria, 2nd Edition, The Williams and Wilkins Book Co. " Baltimore, 1967).

Produced by 3-ketoglycosides (M.J. Bernaerts, J. DeLey, 1963, Nature 197, 406).

Aniline blue-mannitol agar: growth with dye absorption (A.A. Hendrickson, J.L. Baldwin, A.J. Riker, 1934, J. Bact. 28, 597-618).

Litmus milk: gray-brown

Utilization of carbohydrates: · oxidative (R. Hugh, E. Leifson, 1953, J. Bact. 66, 24-26).

As the only source of - carbon in the culture medium described at the end by R. Y. Stanier (R. Y. Stanier et al., 1966, J. Gen. Microbiol. 43, 159--271). - the following compounds are used: D (- | -) glucose, L (+) arabinose, D (-J-) xylose, D (+) rhamnose, lactose, cellobinose, maltose, citrate, - acetate, lactate, propionate, L- aspartic acid, L-asparagine, L-histidine, L-alanine, L-arginine, D (-j-) threalose, D (-) ribose.

Carrot Root Inactivity (J.A. Lippincott, Β -B. - Lippincott, 1969, J. Gen. Microbiol. 59, 1 / 57-75).

tubers roots

1302 ——

1303- ——

1304 + -

Comparing these characteristics to those described in Bergey's Manual of Determinative Bacteriology, VIII. The microorganisms of the invention belong to the family Rhizobiaoeae, genus Agrobacterium, tuber-forming species (1304).

Strains designated 1302 and 1304 are deposited at the Northern Regional Research Center of Peoria III (Sp. St. A.), Where NRRL B 11291 and NRRL B 11394 are designated.

The microorganism cultures to which the invention also relates can be prepared under aerobic conditions by any conventional method, for example in surface cultures or preferably in submerged cultures, using stirred fermenters.

The culture medium, which may be either solid or liquid, comprises an assimilable carbon source, a phosphorus source, a nitrogen source, and a mineral salt. Cultures can grow

219 26 S at a temperature of 20 to 40 ° C, preferably 25 to 35 ° C for 10 to 48 hours, preferably 20 to 30 hours, at a pH of 5.5 to 8.5, preferably 6, 5 to 7.5. Glucose, xylose, lactose, lactate, acetate, corn steep liquor, glycerol can be used as the carbon source. Meat and casein hydrolysates, soybean hydrolysates, ammonium salts, nitrates and urea can be used as nitrogen sources.

A suitable culture medium has, for example, the following composition:

glucose 1 ° g / l (grams per liter xylose 5 g / l KH2PO4 1 g / l MgSO4.7 _H2O 1 g / l NH4Cl 2 g / l pH 7.0 to 7.2

Extraction of glycose isomerase from bacterial paste is carried out by methods known in the art used in enzymology.

To this end, the cells are disintegrated with specially adapted equipment, such as a French cell press, a Manton Gaulin Homogeniser, a rotary disintegrator, etc., and also with ultrasonic devices.

The enzyme can also be satisfactorily extracted from acetonized powders and freeze-dried formulations.

The glucose isomerase obtained from the microbial strains to which the invention relates is characterized by good heat stability.

Glucose isomerization can be carried out in a manner known per se. The glucose solution can be directly added to the culture of microorganisms, i.e. to fresh cells or to cultures from frozen cells, or from acetonated powder or freeze-dried cells.

Similarly, it is possible to use isomerase-containing preparations as isomerase extracts and concentrates, crude or purified isomerase preparations and preparations obtained from the above microorganisms.

Finally, technical and economic improvements can be achieved by immobilizing the enzyme by combining it with macromolecular compounds to form chemical bonds with the substrate or ionic type bonds or by physical immobilization.

The invention is illustrated by the following non-limiting examples.

Example 1

Prepare a culture medium of the following composition:

glucose 5 g / 1 xylose 5 g / 1 KH2PO4 13 g / 1 (NH4) 2SO4 2 g / 1 MgSOd. 7 H2O 0.4 g / l FeSO4.7 H2O 10 mg / 1 (milligrams per liter) CaC12.6 H2O 40 mg / l MHSO4.5 H2O 5 mg / l ZnSO4.7 H2O 5 mg / l CuSO4.5 H2O 1 mg / l C0Cl2.6 H2O 1 mg / l

The pH is adjusted to 7.2 with sodium hydroxide and the culture medium is divided into 100 ml aliquots into 500 ml Erlenmeyer flasks. Sterilize for 30 minutes at 110 ° C and then inoculate flasks with a culture of strain number 1304 from slanted agar containing the same culture medium with 2% agar (Difco) and incubate for 24 hours at 30 ° C, circumferentially stirred at rpm. 220 / min. The cells were then collected by centrifugation and washing with 0.1 molar buffer (Na 2 SO 3, pH 7, containing 1.10 ^-4 mol Co ⁺⁺ and 5.10 ^-3 mol Mg ⁺⁺ ). 2 g of wet cells are obtained from 100 ml of such culture broth. These cells are resuspended in 20 ml of the above buffer and the cell suspension is introduced into a French cell press so that the cells are crushed. Enzyme activity was determined in the extract thus obtained: 1 g of wet cells contained 560 enzyme units. Enzymatic activity is determined as follows:

Into a reaction mixture containing:

4.5 ml substrate solution (D-glucose, 3.67 parts by mole, Mg ⁺⁺ 5.10 ^-3 moles of CO ⁺ 10 ^-4 mol and 0.1 mol NažSO3 in demineralized water, pH 7.0) is then added 0 5 ml of crude extract. Incubate for two hours on a steam bath at 60 ° C. The reaction was quenched by cooling the mixture to 0 ° C. The optical rotation of the sample (and 2 hours) and the original sample (and ₀ ) is measured at room temperature with a Perkins-Elmer 141, Na-line (549 nanometers) optical path of the cuvette: 0.1 decimeter.

If the SNAMPROGETTI unit is defined as the proportion of enzyme that produces 1 milligram of fructose per hour under the above test conditions, then the enzyme units per gram of wet cells can be calculated from this equation of enzyme unit per gram (α _ο α ₂ hrs) X mg of determined glucose X 10 wet cells - ( _agglut .) - (« _fl . _Ukt .) X 0,1 X С X 0,5 X 2 where (ofgiuc.) = + 54,16 ° (offruct.) = - 98,35 °

O = glucose concentration in grams per ml of solution

0,1 = optical path in decimeters

Example 2

Broth cultures of strain number 1304 were prepared as described in Example 1.

10.2 g of wet cells are obtained from 500 ml of broth culture, which are suspended in 40 ml of buffer (NaaSO3 0 · 1 molar) (pH7) and added with stirring at -10 DEG C. to 200 ml of acetone. After treatment for 15 minutes, the cells were collected by filtration and washed with acetone on filter paper, collected and dried under vacuum at room temperature. This gave 1.9 g of dry cells.

The activity of the thus obtained acetonated powder is 2200 units.

Example 3

Cells of strain number 1304 were prepared as described in Example 1, and treated with acetone as described in Example 2.

The acetone treated cells were used to determine the percentage of glucose isomerization as a function of time in the reaction mixture.

The test conditions are as follows:

(a) Acetone treated cells: 10 g

(b) Substrate volume: 1 liter

c) Reaction temperature: 60 ° O

The substrate has the following composition:

glucose 60% (w / w) by volume, Mg ⁺⁺ : 5 millimoles, CO ++: 0.1 millimoles, Na 2 SO 3: 100 millimoles, pH 7.0.

The following isomerization percentages were found:

Cas, h Conversion, % 3 12.5 6 20.8 9 27.8 12 32.3 15 Dec 37.7 18 40.6 23 44.8

Example 4

Broth cultures of strains 1302 and 1303 and the corresponding cell extracts were prepared as described in Example 1 above. 1.72 g and 1.85 g of wet cells, respectively, were obtained from 100 ml of these broth cultures.

Activity per gram of wet cells is 412 units for strain number 1302 and 476 units for strain number 1303.

Claims (5)

OBJECT OF THE INVENTION Process for preparing glucose isomerase particularly suitable for producing fructose and fructose syrup from glucose, characterized in that the microorganisms of the strain Agrobacterium designated NRRL B 11291 and NRRL B 11394 are cultivated under aerobic conditions on a culture solid or liquid medium containing an assimilable carbon source, phosphorus source , a nitrogen source and a mineral salt, at a temperature of 20 to 40 ° C for 10 to 48 hours and a pH of 5.5 to 8.5, and the formed enzyme is isolated. 2. The method of claim 1, wherein the microorganisms are cultured at 25-35 [deg.] C. for 20-30 hours and at a pH of 6.5-7.5. 3. A method according to claim 1 or 2, characterized in that the glucose isomerase is extracted from the bacterial paste, the cells optionally being crushed prior to extraction. 4. The process of claim 1 wherein the glucose isomerase is extracted from acetonized powders. 5. Method according to points. 1 and 2, characterized in that the glucose isomerase is extracted from freeze-dried preparations. Severography, n. P., Plant 7 Most