JPS6363200B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing fructose or fructose and glucose syrup using an isomerase obtained from a microorganism belonging to the genus Agrobacterium. In the enzymatic isomerization reaction of glucose to fructose, glucose isomerase (D-xylose-ketol isomerase EC5.3.1.5.) is added to a glucose solution (e.g. corn syrup),
The reaction conditions are controlled so that glucose is converted to fructose. In this case, the amount of glucose converted to fructose is the equilibrium constant (at 60℃
1.08). According to various documents, Pseudomonas, Lactobacillus, Escherichia, Aerobacter,
Arthrobacter, Bacillus, Streptomyces
By utilizing the activity of various microorganisms such as
Enzymatic methods for converting glucose into syrup containing glucose and fructose are disclosed. In contrast, the present inventors have discovered that a microorganism belonging to the genus Agrobacterium, which has not been disclosed to date, can also produce an isomerase enzyme that isomerizes glucose when grown in an appropriate culture medium. , led to the present invention. These microorganisms (respectively,
1302, 1303, 1304) have the following morphological and biochemical characteristics. Microscopic morphology Sometimes paired. Rod-shaped, 0.8μ x 1.5~2.0μ. Gram negative. No transparent membrane. No baozi. Motile with 4 to 6 pericytes. Macroscopic morphology Colony on agar medium (Difco): descending form. No damage to edges. Cream. Transparent. External diameter 0.5-1mm. smooth. It grows well on calcium, glycerophosphate, mannitol, nitrate, and agar media, turning brown and forming a halo. Biochemical properties In any experimental medium, without the need for any special growth factors and in the absence of amino acids, but using NH ₄ ⁺ , NO ₃ ^- or amino acids as the sole nitrogen source, Grows between 4°C and 39°C. Oxidase: Plus (N. Kovacs, Nature)”
178, 703 (1956 London) Catalase: Plus (M.Levine, DQAnderson, “Journal of Bacteriology (J.Bact.)”
23, 337-347 (1937)) Formation of nitrite from nitrate: Plus (CEZoBell, “Journal of
Bacteriology” 24, 273 (1932)) Hydrolysis of Tween 80: Minus (G.Sierra, “Antonie van
Leeuwenhokk” 23, 15-22 (1957)) Hydrolysis of casein, gelatin, cellulose, starch, agar: Minus (VBDSkermann, “Guide to the Identification of
The Genera Bacteria (Guide to the
Identification of the Genera of Bacteria)
2nd edition, published by Williams and Wilkins Book.
Baltimore, (1967)) Formation of 3-ketoglycosides: Plus (MJBernaerts, J.De Ley, “Nature” 197, 406 (1963)) Aniline blue mannitol agar: Grows by absorbing dye (AA
Hendrickson, JLBaldwin, AJRiker, “Journal of Bacteriology” 28, 597.
~618 (1934)) Litmus milk: utilization of gray-brown carbohydrates: oxidized form (R.Hugh, E.Leifson, “Journal of Bacteriology” 66, 24-26
(1953)) Compounds that can be used as carbon sources in culture media (RYStanier, et al. “J. Gen. Microbiol.”)
43, 159-271 (1966)): D(+) glucose, L(+) arabinose,
D(+) xylose, D(+) trehalose, D
(-)ribose, L(+)rhamnose, lactose, cellobiose, maltose, citrate,
Acetate, lactate, propionate, L-alpartic acid, L-asparagine, L-histidine, L-alanine, L-arginine Infectivity in carrot root discs (JA
Lippincott, BBLippincott, “Journal of General Microbiology” 59, (1),
57-75 (1969)):

[Table] These properties are described in “Bergey's Manual of Determinative Bacteriology”.
Bacteriology), 8th edition, it was determined that the microorganisms involved in the present invention belong to the family Rhizobiaceae, the genus Agrobacterium, and the species tumefaciens (1304). Strains marked 1302 and 1304 were sent to the Northern Regional Research Center (USA, USA).
NRRL B11291 and NRRL B11394, respectively. The microorganisms used in the present invention can be cultured according to conventional methods, such as a surface culture method or a submerged culture method preferably using a stirring culture machine, under aerobic conditions. The medium (which may be either solid or liquid) contains an assimilable carbon source, phosphorus source, nitrogen source, and inorganic salts. Cultivation is carried out at a temperature of 20°C to 40°C, preferably 25°C to 35°C, a pH of 5.5 to 8.5, preferably 6.5 to 7.5, and a culture time of 10 to 48 hours, preferably 20 to 30 hours. Carbon sources include glucose, xylose, lactose, lactate, acetate, corn steep liquor,
Glycerin can be used. Nitrogen sources include meat and casein hydrolysates, soybean hydrolysates,
Ammonium salts, nitrates and urea can be used. A suitable medium has, for example, the following composition. _Composition : _Glucose 10 _{( g} / ₎ It can be implemented in accordance with the law. For this purpose, special equipment such as a French pressure cell press, Manton Gaulin homogenizer, rotary disk integrator, etc. or an ultrasonic device is used to destroy the bacterial cells. Enzymes are equally well extracted by the acetone powder method or the freeze-drying method. The glucose isomerase extracted from the strain according to the present invention is characterized by good stability against heat. Glucose isomerization can be carried out according to conventional methods. That is, the glucose solution may be added directly to a culture of microorganisms (ie, live microbial cells), or may be added to frozen microbial cells, acetone powder, or lyophilized microbial cells. It is likewise possible to use isomerase-containing preparations such as extracts and concentrates thereof, crude or purified isomerase preparations, and the like. Furthermore, technical and economical improvements can be made by immobilizing the enzyme by bonding it to a macromolecular compound through chemical or ionic bonds, or by immobilizing it by physical methods. It is believed that the following examples will clarify the details of other manufacturing methods according to the invention, but these examples are not intended to limit the invention. Example 1 A culture medium having the following composition was prepared. Composition Glucose 5g/ Xylose 5 〃 KH ₂ PO ₄ 13 〃 (NH ₄ ) ₂ SO ₄ 〃2 MgSO ₄・7H ₂ O 0.4〃 FeSO ₄・7H ₂ O 10mg/ CaCl ₂ .6H ₂ O 40 〃 MgSO ₄・5H ₂ O 5 〃 ZnSO ₄・7H ₂ O 5 〃 CuSO ₄・5H ₂ O 1 〃 CoCl ₂・6H ₂ O 1 〃 Adjust the pH to 7.2 with NaOH, and add 100 ml of this culture medium to an Erlenmeyer flask (500 ml). Dispensed in portions. After sterilization at 110°C for 30 minutes, the flask was inoculated with strain 1304 grown in a slant medium of the same composition (but containing 2% agar (DIFCO)) and incubated at 30°C for 24 hours with shaking at 220 rpm. did. Then, the bacterial cells were collected by centrifugation and washed with a buffer solution (containing 0.1 mol of Na ₂ SO ₃ , pH 7, 1×10 ⁻⁴ mol of Co ²⁺ and 5×10 ⁻³ mol of Mg ²⁺ ). 2 g of moist bacterial cells were obtained from 100 ml of this culture medium. These bacterial cells were placed in 20 ml of the above buffer solution, and the bacterial cell slurry was sent to a French pressure cell press to destroy the bacterial cells. When the enzyme activity of the extract thus obtained was measured, it was found to contain 560 enzyme units per gram of wet bacterial cells. Enzyme activity was measured as follows. Substrate solution (3.67 mol D-glucose, 5 x 10 ^-3 mol Mg ²⁺ , 1 x 10 ^-4 mol Co ²⁺ and
Contains 0.1 mol of Na ₂ SO ₃ . 0.5 ml of crude extract was added to 4.5 ml of the reaction solution (PH7.0). Culture was carried out at 60°C for 2 hours on a steam bath. The reaction was then stopped by cooling the mixture to 0°C. The optical power of the sample (α ₂ h) and the optical rotation of the original sample (α ₀ ) were measured using a Perkins Elmer polarimeter 141 (Na line (549 nanometers), optical path of the cell 0.1 dm). If the amount of enzyme that produces 1 mg of fructose in 1 hour under the above conditions is defined as 1 SNAMPROGETTI unit, the unit of enzyme per 1 g of wet bacterial cells is expressed by the following formula. Unit per gram of wet bacterial cells = (α ₀ α ₂ hours) × (amount of glucose used (mg)) × 10 / (α glucose -
αfructose)×0.1×C×0.5×2 Where, (αglucose) = +54.16° (αfructose) = -98.35°C = Concentration of glucose per ml of solution (g) 0.1 = Optical path (dm) Implementation Example 2 A culture of strain 1304 was prepared in the same manner as in Example 1. 10.2 g of wet bacterial cells were obtained from 500 ml of culture.
This was suspended in 40 ml of buffer (Na ₂ SO ₄ 0.1 molar PH 7) and added to 200 ml of acetone with stirring at -10°C. After 15 minutes of treatment, the bacterial cells were collected on paper, washed with acetone, and dried under reduced pressure at room temperature. In this way, 1.9 g of dried bacterial cells were obtained. The activity per gram of the acetone powder obtained was 2200 units. Example 3 Cells of bacterial strain 1304 were prepared in the same manner as in Example 1, and treated with acetone in the same manner as in Example 2. The cells treated with acetone were then used to measure the rate of glucose isomerization versus reaction time in the reaction mixture. The test conditions are as follows. (a) Amount of bacterial cells treated with acetone: 10 g (b) Volume of substrate: 1 (c) Reaction temperature: 60°C The substrate has the following composition. Composition Glucose 60% (volume/volume) Mg ²⁺ 5 mmol Co ²⁺ 0.1 mmol Na ₂ SO ₃ 100 mmol PH 7.0 The isomerization rate is as follows. Reaction time (hour) rate of change (%) 3 12.5 6 20.8 9 27.8 12 32.3 15 37.7 18 40.6 23 44.8 Example 4 Cultures and bacterial cell extracts of strains 1302 and 1303 were prepared in the same manner as in Example 1. 1.72 g of wet bacterial cells from 100 ml of culture
and 1.85g were obtained. The activity per gram of wet bacterial cells was 412 units for strain 1302 and 476 units for strain 1303.

Claims (1)

[Claims] 1. A method for producing fructose or fructose-containing syrup, which comprises bringing glucose into contact with a processed product of a microorganism belonging to the genus Agrobacterium.