JPS6012037B2 - Production method of itaconic acid using immobilized microorganisms - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing itaconic acid by contacting and reacting a microorganism capable of producing itaconic acid immobilized on a gel-like carrier with a sugar solution.

Specifically, the cells or spores of a strain having the ability to produce itaconic acid selected from the Asparagillus genus Asparagillus terius and Asparagillus itaconicus obtained by culturing in advance are treated with polyacrylamide, kappaa,
This is a method in which microorganisms are entrapping immobilized on a gel carrier such as carrageenan or alginic acid, and the immobilized microorganisms are brought into contact with spears. In this method, there are very few complicated creations as in the so-called fermentation method, and the process can be managed in a state that is easy to purify. recent years,
Attempts have been made to produce useful substances using immobilized microorganisms, and a typical example is the production of ethanol from glucose using yeast.

On the other hand, it is quite difficult to obtain the desired product using complex biological reactions and without generating synthetic organisms. Additionally, there has been no attempt to produce itaconic acid by immobilizing Asparagillus cells and utilizing this biological reaction, and the present invention is the first to do so.

The present inventors have discovered that Asparagillus terius ($pergllusten), which is generally used for itaconic acid fermentation production,
Cultured microbial cells or spores of E female) are immobilized on various gels of polyacrylamide, kappa carrageenan, or calcium alginate, molded into spheres or blocks, and this molded product is used as a solid catalyst in a ventilated cylindrical reactor. discovered that itaconic acid could be obtained by using glucose or sucrose as a substrate and reacting with air or oxygen aeration.

The present invention is constituted by such novel biotechnological findings. In the present invention, microorganisms having the ability to produce itaconic acid include strains belonging to the genus Asparagillus, such as Asparagillus terius, Asparagillus itaconicus (
Persica persitaconicus) or derived bacteria thereof are suitable.

A carbon source that can be assimilated by the bacterial strain is used as a substrate,
For example, brans such as glucose, fructose or sucrose or mixtures of two or more thereof are suitable.

These culture conditions may be those suitable for each bacterial species in the case of generally known itaconic acid fermentation.

For example, for Asparagillus terius, 10% glucose, 0.27% ammonium phosphate, 0.03
% ammonium nitrate, 0.20% magnesium sulfate, 0
．． Cultivate submerged in a medium based on 10% corn steep liquor (C, S, L) at pH 5.0 and with aeration of 30 qo. A nutrient source involved in itaconic acid production is added to the medium as necessary. These microorganisms are collected, washed, and then subjected to entrapping immobilization, or the culture method is merely an example and does not limit the present invention in any way. In the case of filamentous fungi, the spores formed by solid-state culture can of course be collected and used in the present invention in the same manner as the deep-cultured fungi described above. The entrapping immobilization of the microorganisms capable of producing itaconic acid can be carried out by a commonly known method for immobilizing microbial cells, but it is especially effective to employ the gelation method described below.

1 When using polyacrylamide as a fixing agent, suspend bacterial cells or spores in physiological saline, add acrylamide monomer, N,N'-methylenebisacrylamide, beta-(B) dimethylamino, propionitrile, and persulfuric acid. Add potassium and leave at room temperature to gel.

2 Method for gelling carrageenan The cells or spores of the above-mentioned cellulose are mixed with 4% kappa carrageenan, and the slurry is injected into the gel using a syringe.
% potassium chloride solution and gel it into a spherical shape.

3 Alginic acid gelation method Add sodium alginate to a 2% suspension of bacterial cells or spores obtained in the same manner as described in the acrylamide gelation method, heat to 30 oo, make a slurry, and use a syringe. The mixture was dropped into a 0.1 molar calcium chloride solution and coagulated to form a spherical gel.

The above-mentioned gelation methods are merely examples, and in addition to these, collagen, cellulose succinate, casein succinate, methyl acrylate, methacrylic acid copolymer, etc. may also be used as gelation bases. You can get the effect. The production of itaconic acid in the present invention can be carried out either by a batch method or by a continuous catalytic reaction using a column.

That is, immobilized bacterial cells or immobilized spores are subjected to a reaction with p.
The suspension is suspended in a buffer solution adjusted to 100% H and packed into a cylindrical fluidized bed column. Fermentable sugars such as glucose and molasses are added to this at a concentration of 1% to 20%, and a glass filter etc. is added from the bottom of the column. ventilate through. If the aeration is too strong, the reaction efficiency tends to deteriorate, so try not to disturb the immobilized microorganism layer too much. The reaction temperature varies somewhat depending on the strain of bacteria to be tested, but is preferably around 30 oo. The reaction solution is replaced with fresh solution every 2 hours to continue the reaction. In addition, in the case of a continuous method, a substrate solution containing residues is continuously pumped into a column filled with immobilized microorganisms using a Verista pump, and the reaction solution is flowed out from the other side of the reaction column at the same rate as the injection rate.

Good results can be obtained by adjusting the temperature of the column as necessary to maintain the reaction at optimal conditions. The actual state of the present invention will be shown below with reference to Examples. The itaconic acid production ability of the immobilized microorganism was determined from the amount of itaconic acid produced by the reaction. Example 1 Asparagillus teriu IF preserved in wort agar
O-6123 spores were treated with 10% glucose at pH 5.0,
0.27%N is day 2P04, 0.30%NH4N03,
0.20% MgS04 7 days 20, 0.10% C. S.
The cells were cultured in a 500 Kami-Sakaguchi flask containing 100 m' of an aqueous solution having the composition of L, and the cells were separated from the culture solution after 7 hours of incubation, when the itaconic acid production activity was high, and washed with water.

Claims (1)

[Scope of Claims] 1. An immobilized microorganism characterized in that a microorganism having the ability to produce itaconic acid selected from Asparagillus terius or Asparagillus itaconicus immobilized on a gel-like carrier is brought into contact reaction with a sugar solution. Method for producing itaconic acid. 2. The method according to claim 1, wherein a carrier selected from polyacrylamide, Katsupah carrageenan and alginic acid is used. 3. The method according to claim 1, wherein one or more selected from glucose, fructose, and sucrose is used as the sugar solution. 4. The method according to claim 1 or 3, in which the immobilized microorganism is subjected to a contact reaction while continuously or intermittently replenishing the sugar solution.