KR0143596B1 - Method for preparing L-Solvos by two stage reaction system - Google Patents

Abstract

The present invention relates to a method for preparing L-solvos by a two-step reaction system using two strains, and to prepare sorbitol from glucose and fructose by immobilization of Zymomonas mobilis strain after membrane permeation treatment. Doing; And converting the sorbitol prepared above into L-solvos by the immobilized Gluconobacter suboxydans strain after the membrane permeation treatment, and thus more stably and efficiently. L-Solvos can be prepared.

Description

Method for preparing L-Solvos by two stage reaction system

1 is a schematic of a two stage reaction system used to prepare L-Solvos according to the present invention.

The present invention relates to a method for preparing L-solvo, and more particularly, L-sol by a two-step reaction system using Zymomonas mobilis and Gluconobacter suboxydans strains. A method of manufacturing a boss.

L-Solvos is an intermediate of vitamin C, and is generally synthesized by the following two processes during the Reichstein process of chemically synthesizing vitamin C [Reichstein Gussner, Helv. Chim. Acta., 17, 311 (1934). First, when 50% glucose solution is reacted with nickel as a catalyst at a temperature of 50 to 150 ° C. under a pressure of 50 to 180 Hg / cm 2 to obtain D-sorbitol, the next step is acetobacter xylinum (Acetobacter xylinum), glucono L-sorbose is prepared from the D-sorbitol using acetic acid bacteria such as Bacter suboxydans and the like, which is usually converted to 96-99% within 24 hours by normal acetic acid. [Liebster et al., Chem. List, 50, 395 (1956). There has also been reported a method used by immobilizing acetic acid bacteria in the preparation of L-Solvos. [Pomortseva Krasilnikova, Khim. Farm. Zh., 6, 721 (1983); Donova Koshcheenko, Prik. Biokhim. Mikrobiol., 26, 214 (1990); ibid., 24, 368 (1988); Soetaert et al., Med. Fac. Landbouww. Rijksuniv. Gent., 54, 1511 (1989); Tanaka et al., J. Ferment. Technol., 60, 431 (1982); ibid., Appl. Microbiol. Biotechnol., 19, 85 (1984); and Koseva et al., J. Biotechnol., 19, 301 (1991).

It has also been reported to use Zymomonas mobilis instead of chemical synthesis for the preparation of sorbitol [Chun Rogers, Appl. Microbiol. Technol., 29, 19 (1988); and Jung Chun, Kyung Hee J. Genet. m 1, 35 (1989). As another method, the conversion of sorbitol to sorbose using the acetic acid bacterium may be achieved by converting the cells treated with toluene or cetyl-3-methyl ammonium to carrageenan for at least 75 days at a level of 295 g / 1. Stabilization methods have also been developed [Rehr et al., Appl. Microbiol. Biotechnol., 35, 144 (1991); Nains et al., Anal. Biochem., 196, 234 (1991); Prabhune et al., Enzyme Microb. Technol., 14, 161 (1992); and Kim et al., KIST Report BSG 70180-313-2].

In the method of combining the above two processes into one, that is, to prepare sorbitol in one step, the next step is to convert sorbitol to sorbose using acetic acid bacteria such as acetobacter xylium or Cluconobacter suboxydans. In this case, since the gluconate produced in step 1 has stronger affinity for sorbitol dehydrogenase of acetic acid than sorbitol, and inhibits sorbitol from being used as a substrate, the concentration of gluconic acid in the reactant may be 5% or more. In this case, there is a problem that sorbitol is not converted to sorbose at all.

Therefore, the present inventors have repeatedly studied to avoid the above inhibitory effect of gluconic acid and convert sorbitol to sorbose more effectively. As a result, the reaction system is separated into two stages and the cell is permeated to improve cell membrane permeability. After the immobilization, it was possible to develop a process for producing sorbose with increased stability.

Accordingly, it is an object of the present invention to provide a more effective and stable method for preparing L-Solvos by a two-step reaction system using Zymonas mobilis and Glucono suboxydans strains.

In order to achieve the above object of the present invention, the present invention comprises the steps of preparing sorbitol from glucose and fructose by immobilized Zymonas mobilis strain after membrane permeation treatment; And converting the sorbitol prepared above into L-sorbose by the immobilized gloconobacter suboxydans strain after membrane permeation treatment, thereby providing an efficient method for producing L-solvos by a two-stage reaction system. do.

That is, the present invention synthesizes sorbitol and converts the sorbitol to sorbose using a gloconobacter suboxydans to produce sorbose, wherein the sorbitol dehydrogenase ability of the gluconobacter suboxydans with gluconic acid is produced. In order to overcome the deterioration of the reaction system, the reaction system was separated into two stages: a sorbitol synthesis step using Zymomonas mobilis and a conversion of sorbitol to sorbita using Gluconobacter suboxydans, and the glucobacter suboxy The conversion of sorbitol to sorbose is enhanced by minimizing the effects of gluconic acid on sorbitol dehydrogenase by immobilizing Dans with soda alginate.

Hereinafter, the present invention will be described in detail with reference to FIG. 1.

First, as a first step of the reaction system according to the present invention, Zymonas mobilis ZM (ATCC 31821) cells are treated with 0.01 to 2.0 (w / v), preferably 0.07 to 1.0% cetyl-3-methyl ammonium bromide. After permeation of the cell membrane, 0.5 to 5.0%, preferably 1.0 to 4.0% sodium alginate solution was added to the treated cells to obtain an optimal cell concentration, and the mixture was added dropwise to 2% calcium chloride solution to obtain immobilized beads. Manufacture. The beads were filled in the first reactor 1, which is a packed reactor as shown in FIG. 1, to which a 2% aqueous solution of glucose and fructose, respectively, as a reactor was carried out at a rate of 0.1 ml per minute from the reactor tank 3. To supply.

On the other hand, Gluconobacter suboxydans KCTC 2111 cells were treated with acetic acid buffer solution (100 mM, pH 5.8) containing 1 to 20%, preferably 5 to 15% toluene, to permeate the cell membrane, and then To the optimum cell concentration by adding 0.5-5.0%, preferably 1.0-4.0% alginate soda solution to the membrane-treated or untreated Gluconobacter suboxydans KCTC 211 cells, the mixture was subjected to 2% calcium chloride solution. Immobilized beads are prepared by dropwise addition. The beads were filled in a second reactor 2, which is a fluidized bed reactor as shown in FIG. 1, and supplied with compressed air at 5 vvM from the compressed air tank 7 through an air pressure regulator 5, respectively. A 100 mM acetic acid buffer solution (pH 5.5) containing 2% sorbitol and gluconic acid is supplied.

Subsequently, after connecting the first reactor and the second reactor as shown in FIG. 1, the reaction solution was fed to the second reactor at a rate of 0.1 ml / min from the uppermost part of the first reactor, and the effluent of the first reactor and the first reactor were 2 React the reactants in the reactor. The reaction product of the second reactor is recovered to the reaction product reservoir 6 at a rate of 0.1 ml / min to carry out the analysis. Sorbitol produced by Zymonas mobilis in one step in the two-step reaction system according to the present invention was converted to 75% by Glunobacter suboxydans after membrane permeation and immobilization in two steps.

In the reaction system according to the present invention, each microorganism treated with cell membrane permeation with cetyl-3-methyl ammonium bromide and toluene, respectively, is stabilized by treatment with 0.05-2.0%, preferably 0.1-1.0% glutaraldehyde solution. It can be applied to the immobilization process. The concentration in the beads of the cells immobilized through the immobilization treatment is 20 to 300 mg / ml, preferably 30 to 250 mg / ml, which is optimal in the beads of Zymomonas mobilis used in the packed first reactor. The cell concentration is 100 mg / ml, and the optimum cell concentration in the beads of the Gluconobacter suboxydans used in the fluidized bed second reactor is preferably 80 mg / ml. In addition, the amount of beads supplied to each reactor is preferably adjusted to 50% of the operation volume in the case of Zymonas mobilis, and 20% of the operation volume in the case of Gluconobacter suboxydans.

In the reaction system, the reaction temperature at the time of reacting the effluent of the first reactor with the reactant of the second reactor is adjusted to 20 to 50 ° C., preferably 25 to 45 ° C., using the temperature control bath 4. In addition, the hydrogen ion concentration (pH) in the reaction is maintained at 4.0 to 8.0, preferably 4.2 to 7.5 using acetic acid buffer solution, dissolved oxygen concentration in the reactor is 5 to 60 5, preferably 8 to saturation concentration Keep at 50%.

For quantitative analysis of glucose and fructose, sorbitol, sorbose and gluconic acid in the reaction substrate, a μ-Spherogel (300 mm x 6.5 mm) column from Beckman, USA was used. The A-27 (250 mm x 4.6 mm) column from Biorad was performed using HPLC and RI detectors from Beckman, USA.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are intended to illustrate preferred embodiments of the present invention and are not intended to limit the scope thereof.

EXAMPLE

Example 1 Synthesis of Sorbitol by Zymonas Mobilis

Step 1: Culture and Transmembrane Treatment of Cells

Zymonas mobilis ZM4 obtained by stationary cultivation at 30 ° C. for 48 hours in a medium (pH 5.0) containing 10% glucose, 0.1% ammonium sulfate, 0.1% magnesium sulfate, 0.1% dibasic potassium phosphate and 0.5% yeast extract (ATCC 31821) The cells were washed with 100 mM acetic acid buffer (pH 5.5), added to a 0.2% cetyl-3-methyl ammonium bromide solution, and stirred at 4 ° C for 10 minutes.

Step 2: Stabilization and Immobilization of Cells

The cell membranes subjected to membrane permeation in step 1 were recovered by centrifugation, and then treated with 0.3% glutaraldehyde solution. The treated cells were washed twice with 100 mM acetic acid buffer (pH 5.5) and then dispersed in the same buffer solution. To the cell solution obtained, 3% alginate solution was added at a rate of 10 ml per 1 g of cells to increase the cell concentration to 100 mg / g. Stir at 4 ° C. for 15 h while maintaining in ml. The mixture was then added dropwise to 2% calcium chloride solution using a syringe at a rate of 1.5 kgf / cm 2 to prepare immobilized beads. The beads obtained were left standing in fresh calcium chloride solution for 30 minutes, washed with acetic acid buffer solution containing 0.2% calcium chloride (pH 5.5) and stored at 4 ° C until use.

Step 3: Synthesis of Sorbitol

The cell beads prepared in step 2 were charged to a 40 mm x 300 mm sized reactor in an amount of 50% of the working volume, and then 2% solution of glucose and fructose in acetic acid buffer (pH 5.5) as the reactor mass. Was fed to the reactor at a rate of 0.1 ml / min. Analysis of the reactants showed that the composition of the reactants consisted of only sorbitol and gluconic acid, and the conversion to sorbitol was 100%.

Example 2 Solvose Production by Gluconobacter Suboxydans

Step 1: Culture and Transmembrane Treatment of Cells

Gluconobacter suboxydans KCTC 2111 cells obtained by shaking culture at 30 ° C. for 48 hours in a medium containing 5% sorbitol, 1% bactopeptone and 0.5% yeast extract at 30 ° C. were treated with 100 mM acetic acid buffer (pH 5.5). After washing, the cells were recovered by centrifugation, and the washed cells were added to 10% acetic acid buffer solution containing toluene and stirred at 4 ° C. for 10 minutes.

Step 2: Stabilization and Immobilization of Cells

Gluconobacter suboxydans KCTC 2111 cells incubated in step 1 were treated with toluene in a membrane and treated in the same manner as in step 2 of the above example except that the cell concentration in the beads was maintained at 80 mg / ml. Immobilized cell beads were prepared.

Step 3: Conversion of Sorbitol to Solvos by Gluconobacter Suboxydans

The cell beads prepared in step 2 were charged to a 60 mm x 270 mm fluidized bed reactor in an amount of 20% of the operating volume, followed by sorbitol in acetic acid buffer (pH 5.5) as a reaction substrate while supplying air at 5 vvm. And 2% solution of each gluconic acid was fed to the reactor at a rate of 0.1 ml / min. Analysis of the reactants showed that the composition of the reactants consisted of only L-sorbose and ketogluconic acid, and the conversion to solvose was 100%.

Example 3 Synthesis of L-Solvos by Two Stage Reaction System

After connecting the reactors of Example 1 and Example 2 together with the first degree, the 2% solution of fructose and glucose was supplied to the first reactor at a rate of 0.1 ml / min to the bottom of the reactor and the Zymomo in the first reactor. The sorbitol synthesis reaction was carried out by the Nas Mobilelis ATCC 31821 strain, and the reaction solution was extracted at the rate of 0.1 ml / min from the uppermost part of the first reactor and fed to the lower part of the second reactor, thereby providing the gluconobacter sub in the second reactor. The sorbose conversion reaction with the Oxydans KCTC 2111 strain was performed. The reaction product was then recovered from the top of the second reactor at a rate of ml / min. Analysis of the recovered reaction product found that 75% of the sorbitol produced in the first reactor was converted to L-Solvos. The two stage reaction system remained stable for up to 300 hours continuously.

As described above, according to the two-stage reaction system according to the present invention, it is possible to prepare L-solvos more stably and efficiently from glucose and fructose.

Claims (11)

Preparing sorbitol from glucose and fructose by immobilization of Zymomonas mobilis strain after immobilization; And converting the sorbitol prepared above into L-solvos by the immobilized Gluconobacter suboxydans strain after the membrane permeation treatment. Way. The membrane permeation treatment of Zymomonas mobilis and Glunobacter suboxydans is performed using 0.01 to 2.0% cetyl-3-methylammonium bromide and 1 to 20% toluene solution, respectively. How to. The method of claim 2, wherein the concentration of cetyl-3-methyl ammonium bromide solution is 0.07 to 1.0 5 and the concentration of the toluene solution is 5 to 15%. The method of claim 1, wherein the immobilization of the microorganism is carried out using a 0.5 to 5.0% sodium alginate solution. The method of claim 4, wherein the concentration of the soda alginate solution is 1.0 to 4.0%. The method of claim 1, wherein the reaction is carried out while maintaining a pH of 4.0 to 8.0 at a temperature of 20 to 50 ℃ and a dissolved oxygen concentration of 5 to 60% of the saturation concentration. The method of claim 6, wherein the reaction temperature is 25 to 45 ℃, dissolved oxygen concentration is 8 to 50% of the saturation concentration, pH is 4.2 to 7.5. The method of claim 4, wherein the concentration of the immobilized cells is characterized in that 20 to 300 mg / ㎖. 9. The method according to claim 8, wherein the concentration of the immobilized cells is 30 to 250 mg / ml. The method of claim 2, wherein the membrane-treated microorganisms are stabilized by treatment with 0.05 to 2.0% glutaraldehyde solution. The method of claim 10, wherein the concentration of glutaraldehyde solution is 0.1 to 1.0%.