JPS6043957B2 - Method for producing gluconic acid using immobilized microorganisms - Google Patents

Description

Detailed Description of the Invention The present invention relates to a method for producing gluconic acid by contacting and reacting a microorganism capable of producing gluconic acid immobilized on a gel-like carrier with a sugar solution. Bacterial cells or spores of a strain of Aspadillas capable of producing gluconic acid are comprehensively immobilized on a gel carrier such as polyacrylamide, kappa galakian, alginic acid, etc. -re, j
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■■、''-■１■■・ Unlike the so-called fermentation method, there are very few complicated by-products, and the process can be managed in a state that is easy to purify.

In recent years, methods for producing useful substances using immobilized microorganisms have been attempted, and a typical example is a method for producing ethanol from glucose using yeast.
On the other hand, it is quite difficult to obtain the desired product without producing organisms with complex biological reactions.
The present invention is the first in which there has been no attempt to produce gluconic acid by immobilizing cells of the genus Aspadillas and utilizing this biological reaction.

The present inventor has discovered that Aspergillus niger, which is generally used for gluconic acid fermentation production,
The cultured cells or spores of G. ger) are immobilized on various gels of polyacrylamide, Katsupah-Galakian, or cardinium alginate, and then molded into a spherical or block shape, and this molded product is used as a solid catalyst in a ventilated cylindrical reactor. It was discovered that gluconic acid could be obtained by using glucose or sucrose as a substrate and performing the reaction while aerating air or oxygen.

The present invention is constituted by such novel biotechnological findings. In the present invention, as the microorganism capable of producing gluconic acid, strains having the ability to produce gluconic acid belonging to the genus Aspagillus are used, and in particular, Aspagillus niger G-011, ATCCl, which has a strong ability to produce gluconic acid.
Ol5 or the like can be suitably used.

The culture conditions may be those suitable for generally known gluconic acid fermentation.

For example, a medium based on 10% sugar beet or cane molasses solution with pH 3. Culture in deep water at 30°C under aeration conditions.

A nutrient source involved in gluconic acid production is added to the medium as necessary. The cultured microorganisms are collected, washed, and then subjected to entrapping immobilization, but the culture method is merely an example and does not limit the present invention in any way.

It goes without saying that spores formed by solid-state culture can be collected and used in the present invention in the same manner as the deep-cultured microbial cells described above.

The entrapping immobilization of the microorganisms capable of producing gluconic acid can be carried out by a commonly known method for immobilizing microbial cells, but it is especially effective to employ the gelling method shown here.

1 When using polyacrylamide as a fixative, add acrylamide monomer, N,N″-methylenebisacrylamide, beta (β) dimethylaminoprobionitrile, and potassium persulfate to a suspension of bacterial cells or spores in physiological saline. Leave at room temperature to gel.

2 Method for gelling carrageenan Heat the same bacterial cells or spores as above with 4% Katsupaa carrageenan, make a slurry, and inject it into a slurry using a syringe.
% potassium chloride solution and form into a spherical gel.

3 Alginic acid gelation method To a suspension of bacterial cells or spores obtained in the same manner as described in the acrylamide gelation method, add sodium alginate to a concentration of 2%, heat to 30°C, make a slurry, and use a syringe. The mixture is solidified in a 0.1M calcium chloride solution to form a spherical gel.

The above-mentioned gelation methods are merely examples, and collagen, cellulose succinate, casein succinate, methyl acrylate, methacrylic acid copolymer, etc. may also be used as the gelation base to achieve the sufficient effect of the present invention. can get.

The production of gluconic acid in the present invention can be carried out either batchwise or by continuous contact reaction using a column.

That is, immobilized bacterial cells or immobilized spores are treated with P suitable for the reaction.
A cylindrical fluidized bed column is filled with the suspension in a buffer solution adjusted to 1.0 H, and fermentable sugars such as glucose, sucrose, and molasses are added to this at a concentration of 1% to 20%, and the suspension is placed in the lower part of the column. Ventilate through a glass filter, etc. 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 is preferably around 30'C. The reaction solution is exchanged with a new solution every two cycles to continue the reaction. In the case of a continuous method, a substrate solution containing a sugar solution is continuously fed into a column filled with immobilized microorganisms using a peristaltic pump or the like, 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 examples, and the ability of immobilized microorganisms to produce gluconic acid was determined from the amount of fugluconic acid produced by the reaction. Example 1 Spores of Aspagillus niger G-011 preserved in sugar beet molasses agar were added to 10% sugar beet molasses PH5. &10
The cells were shaken and cultured in a 500 ml Sakaro flask containing 0 ml of the culture solution, and after 9 eifI, which has a high gluconic acid production activity, the bacterial cells were separated from the culture solution and washed with water.

6 y of acrylamide monomer was added to a suspension of 10 y of the washed bacterial cells in 32 ml of 0.9% saline. ．． Dissolve and mix 0.32y of N,N''-methylenebisacrylamide, add 41n1 of 5% β-dimethylaminoprobionitrile, and further add 2.5% potassium persulfate (K2S2O8)4.
Mix ml thoroughly and leave to stand at 25°C to obtain immobilized Aspagillus cells. The prepared gel was cut into blocks of 4Twt3 to 3 pentasaccharides, packed in a sterile gauze bag, and heated in a cylindrical reactor with 100ml of 20rr 1M tartrate buffer containing 6% sucrose (PH3.O
), and the reaction solution was exchanged 5 times every 24 hours. 0.3y/after each reaction for 2@
Df, l. 3'/De,l. 5y/Df, l. 7th evening g
A gluconic acid solution of /De and 1.7q/d' was obtained.
No other organic acid by-products that would interfere with purification were observed in the reaction solution. Example 2 Spores of Aspagillus niger G-011 preserved in sugar beet molasses agar were added to 32 ml of 0.9% saline so that the number of spores was 107 to 103/ml.
1 and immobilized with polyacrylamide according to Example 1 to obtain immobilized spores.

Claims (1)

[Scope of Claims] 1. A method for producing gluconic acid using an immobilized microorganism, which comprises contacting and reacting a strain of gluconic acid-producing bacteria belonging to the genus Aspagillus immobilized on a gel-like carrier with a sugar solution. 2. A method for producing gluconic acid using an immobilized microorganism according to claim 1, which uses a carrier selected from polyacrylamide, Katsupah carrageenan, and alginic acid. 3. A method for producing gluconic acid using an immobilized microorganism according to claim 1, which uses glucose or sucrose as a sugar solution. 4. Contact reaction with immobilized microorganisms while continuously or intermittently replenishing sugar solution
A method for producing gluconic acid using an immobilized microorganism as described in .