JPH0583236B2 - - Google Patents

Description

[Detailed Description of the Invention] (Field of Industrial Application) In recent years, the enzyme industry, which uses enzymes and/or bacterial cells to carry out useful reactions and to produce medicines, foods, and other products, has become increasingly popular. It is gaining importance and developing. The present invention relates to a method for immobilizing enzymes and/or bacterial cells.

(Prior Art) Conventional enzyme utilization generally involves dissolving the enzyme in the reaction solution, resulting in extremely low enzyme utilization efficiency. In order to solve this drawback, various methods for immobilizing enzymes and/or bacterial cells have been proposed in recent years. Broadly speaking, there are various types of conventional methods as follows. (a) Carrier binding method: This is a method in which an enzyme is bound to a water-insoluble carrier. (b) Crosslinking method: This is a method of insolubilizing enzyme molecules by crosslinking them with each other using a reagent having two functional groups. (c) Encapsulation method: This is a method in which the enzyme is encapsulated in a gel such as polyacrylamide gel, or the enzyme is coated with a microporous film to form microcapsules. (For example, Ichiro Chibata, ``Recent Microbial Industry to Expand Life Science'' (Sho)
57.6.1) Kagaku Kogyo Co., Ltd., P68-P91) (Problems to be solved by the invention) The various immobilization methods described above often have the following drawbacks, and at present, There is no ideal method that can be applied to the immobilization of all enzymes. That is, in (a) the carrier binding method, the binding force of the enzyme to the carrier is weak due to adsorption or ionic bonding, and the enzyme is detached from the carrier during the enzymatic reaction. With covalent bonds, it is difficult to set reaction conditions, and the active site of the enzyme protein is likely to be destroyed during the reaction. (b) In the cross-linking method, as in the case of the covalent bond described above, it is difficult to set the reaction conditions, and the active site of the enzyme protein is likely to be destroyed during the reaction. (c) In the inclusion method, it is difficult to select the polymer that forms the gel, and depending on the type of polymer, it may not be possible to obtain a gel that is stable and has appropriate strength, and the enzyme may flow out of the gel during the enzymatic reaction. Or, it is sealed in a gel so that it cannot participate in enzymatic reactions. In addition, the microencapsulation technology is complicated, and depending on the material and pore size of the microporous film, the enzyme may flow out of the microporous film during the enzyme reaction, or it may remain in the microporous film in a state where it cannot participate in the enzyme reaction. It may be confined to.

(Means for Solving the Problems) As a result of various studies to solve the above problems, the present inventors found that glucose, mannose, and mannuronic acid are used in a molar ratio of glucose:
Mannose: Mannyuronic acid = 1:0.4-0.7:4
An aqueous solution of a polysaccharide (hereinafter referred to as the polysaccharide of the present invention) that has been acetylated with a degree of acetylation of about 0 to 1.0 and has a molecular weight of 10 ³ to 10 ⁵ by the 3.5-dinitrosalicylic acid method. When an enzyme is retained in a gel obtained by contacting with metal ions, the enzyme in the gel not only efficiently participates in the enzymatic reaction, but also retains the enzyme in the gel in a gentle manner. We have found that this can be done easily and reliably under certain conditions, and have completed the present invention. The polysaccharide of the present invention can be produced by the following method, for example, as disclosed in Japanese Patent Publication No. 58-39441.

Azotobacter vinelangii
vinelandii) IFO12018 (obtained from Fermentation Research Institute (Yodogawa-ku, Osaka)) in addition to commonly used carbon sources (e.g., glucose, starch decomposition products, sucrose, etc.), as well as inorganic salts (monopotassium phosphate, monopotassium phosphate, Dipotassium phosphate, sodium chloride, calcium sulfate, magnesium sulfate, sodium acetate, etc.) trace metals (sodium molybdate, ferrous sulfate, etc.) are inoculated into a culture medium prepared by adding tap water, and cultured under aerobic conditions using a conventional method. Polysaccharides are accumulated by culturing while aerating nitrogen or nitrogen-containing gas under the atmosphere. After the cultivation is complete, solids such as bacterial bodies in the culture solution may be removed by centrifugation if it is inconvenient for the purpose of use. The accumulated polysaccharides are removed from the culture solution by separation, filtration, etc., by adding sulfuric acid, etc. to the culture solution to make it acidic, precipitate it, and inject it with caustic soda, etc. in a water-soluble solvent such as methanol, ethanol, isopropyl alcohol, etc. Neutralize and dry.

The polysaccharide of the present invention is made into an aqueous solution with an appropriate solid content concentration, and then an aqueous solution containing polyvalent metal ions is sprayed onto this aqueous solution, or the aqueous solution of the polysaccharide of the present invention is immersed in an aqueous solution containing polyvalent metal ions. By doing so, a gelled product can be produced. Enzymes and/or microbial cells can be retained in the gel by the following method. An aqueous solution containing polyvalent metal ions is sprayed onto a mixture of an aqueous solution of the polysaccharide of the present invention, an aqueous enzyme solution, and/or a dispersion of bacterial cells in water, or the above mixture is mixed into an aqueous solution containing polyvalent metal ions. Enzymes and/or
Alternatively, a gelatinous material that retains bacterial cells can be formed. By dropping the above-mentioned mixed solution into an aqueous solution containing polyvalent metal ions, a bead-shaped gelled product can be formed. Furthermore, gelled products of various shapes can be formed by roll-molding or extrusion-molding the aqueous solution of the polysaccharide of the present invention into any form such as a film or fiber, and then gelling it. Polyvalent metal ions that can be used in the present invention include calcium ions, magnesium ions, aluminum ions, iron ions, and the like. The aqueous solution containing polyvalent metal ions can be obtained by dissolving a water-soluble polyvalent metal salt such as calcium chloride, magnesium chloride, aluminum chloride, iron chloride, magnesium acetate, etc. or an alkali such as calcium hydroxide in water. The gelled product formed can also be further hardened by adding an appropriate amount of glitaraldehyde or tannin. Further, the strength of the gelled product can be improved by mixing cellulose fibers such as pulp and cotton with the above-mentioned mixed solution.

The concentration of the polysaccharide of the present invention in the above mixed solution is 1 to 20
% is appropriate; if the concentration is lower than this, the strength of the gelled product will be weakened, and if the concentration is higher than this, the viscosity will be high, making it difficult to handle.

The concentration of aqueous solution of polyvalent metal ions is 0.05M to 10M
is suitable; if this concentration is too low, the gelation rate will be too low, and if this concentration is higher, gelation will occur locally.

Various enzymes and/or bacterial cells can be immobilized according to the present invention, and examples of suitable enzymes to which the present invention is applied include urease, alcohol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase. enzyme, thiase yeast, glucose oxidase,
Glucose oxadase catalase, O-amino acid oxidase, urease, diamisoxidase, catalase, D-amino acid oxidase,
Riboxygenase, uricase, ribonuclease, hexokinase, lipase, alkaline phosphatase, acid phosphatase, nucleoedase, deoxyribonuclease, α-amylase, β-amylase, glycoamylase, glycose isomerase, cellulase, hemicellulase,
β-glycosidase, impeltase, anthomyanase, naringinase, hesperidinase,
β-glucuronidase, hyaluronidase, alkaline protease, semi-alkaline protease, acidic protease, thermolysin, collagenase, pepsin-pepsinogen, aminopeptidase, renin, trypsin, trypsin-trypsinogen, chymotrypsinogen, elastase, enterokinase, acylase, arginase, L-glutamic acid Decarboxylase, L-lysine decarboxylase, papain, and examples of suitable bacterial cells used in the present invention include bacterial cells of the above-mentioned enzymes, Aerobacter aerogenes, Bacillus subtilis,
Bacterial cells such as E. tschierrichiacoli and Streptomyces phaeochromogenes can be mentioned.

(Function) Since the present invention is constructed as described above, the immobilization method is simple and can be applied to the immobilization of all kinds of enzymes and/or bacterial cells. Since the immobilization reaction is carried out under mild conditions, the active site of the enzyme protein is not destroyed, and the enzyme and/or bacterial cells do not flow out during the enzyme reaction, resulting in high immobilization efficiency. The shape of the gelled product can be easily formed into beads, films, fibers, etc.

(Example) Example 1 0.067N sodium phosphate buffer (PH7.2) 20ml
A solution in which 400 mg of urease (manufactured by Pharmacia Japan Co., Ltd., Nata Bean) was dissolved in a solution and the polysaccharide of the present invention (glucose:mannose:mannuronic acid = 1:0.5:14, degree of acetylation 0.8, molecular weight 3 × 10 ⁴ )
Mix 40 ml of 5% aqueous solution. This mixture
Add dropwise to 0.1M calcium chloride aqueous solution to obtain bead-shaped gel. This gelled product is washed with a 0.1M calcium chloride aqueous solution to obtain 50 g (wet weight) of a gelled product containing and immobilizing urease. Add 3 g of the above gel to 100 ml of 0.05 M calcium chloride aqueous solution containing 30% urea, and heat at 20°C while stirring with a stirrer.
After holding for 15 minutes, the enzyme activity was tested and compared to the urease standard. It had an activity equivalent to 53% of that of the added enzyme, and was found to have an extremely high ability to maintain activity.

(Effects of the Invention) Since the present invention is configured as described above, enzymes and/or bacterial cells can be easily immobilized. Furthermore, it can be applied to the immobilization of all kinds of enzymes and/or microbial cells, and the activity of the immobilized enzymes and/or microbial cells is extremely maintained. Therefore, the enzyme reaction and the separation operation of the enzyme can be facilitated, the enzyme reaction can be made continuous, the enzyme utilization efficiency can be increased, and the economic cost of the enzyme reaction can be extremely reduced.

Claims (1)

[Claims] 1 It is composed of glucose, mannose and mannuronic acid, and is acetylated with a molar ratio of glucose: mannose: mannuronic acid = 1:0.4 to 0.7:4 to 17 and a degree of acetylation of about 0 to 1.0,
The molecular weight by the 3.5-dinitrosalicylic acid method is 10 ³ ~
10 An enzyme and/or fungi characterized by contacting an aqueous solution containing polyvalent metal ions with an aqueous solution containing polyvalent metal ions to gel a mixture of an aqueous solution of the polysaccharide of ⁵ , an aqueous enzyme solution, and/or an aqueous dispersion of fungal cells. Body immobilization method.