JPS5911185A - Low-temperature immobilization of enzyme or mold - Google Patents

Abstract

PURPOSE:To prepare an immobilized enzyme or mold having extremely high retention power of enzymatic activity, by gelatinizing an aqueous solution of cold-water-soluble polysaccharide containing an enzyme and/or mold at low temperature. CONSTITUTION:An aqueous solution of enzyme and/or water dispersion of mold is blended at 0-30 deg.C, preferably at 0-20 deg.C with an aqueous solution of cold- water-soluble polysaccharide consisting essentially of D-galactose and 3,6-anhydro-D-galactose, containing >=10w/w sulfate group in the molecule, such as an aqueous solution of sodium salt of kappa-carrageenan or iota-carrageenan to give a mixed solution, which is dropped to an aqueous solution containing a metallic ion having >=24 atomic weight, or the aqueous solution is dropped to the mixed solution, so that the mixed solution is gelatinized.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for low-temperature immobilization of enzymes and/or bacterial cells. The enzyme industry, which uses enzymes and/or microbial cells to perform useful reactions and produce medicines, foods, and other products, has been progressing in recent years by minimizing the requirements of conventional enzymes. Thread utilization is generally reactive RF
One drawback was that the utilization efficiency of the # system was extremely low because the enzyme was dissolved during the process.

In recent years, in order to resolve this issue, efforts have been made to bind or disperse enzymes and/or bacterial cells in molecular substances, form porous gels or granules, and subject them to enzyme reactions as complexes that can be used repeatedly and continuously. Research on the immobilization of so-called enzymes and/or bacterial cells is being actively conducted. Examples of these methods include mixing an enzyme with an aqueous polyacrylamide solution and heating and gelling this together with a catalyst. Both methods of dissolving cold water-resistant carrageenan in water at 40 to 60'C result in kelization at 7M.
It has the disadvantage that the enzyme is sensitive to heat or deactivated. Another drawback of the former method is that the enzyme immobilized product is
Since it does not have porosity as it is, it is necessary to dry and crush it to increase the surface area that comes into contact with the substrate in the enzyme reaction.

Enzymes remain stable at low temperatures, and even if they have been lost, their activity can be recovered at low temperatures. It is thought that a single horse that retains its activity will have an effect greater than that of the first period.11. Perhaps because of the low temperature, there has been no effective low-temperature fixation method until now.

From this perspective, the present invention provides a method for immobilizing enzymes and/or bacterial cells at a low temperature of 0 to 30'C, preferably 0 to 20'C. As a result of repeated research on methods for carrying out this process, we found that sulfate groups were added to an enzyme solution and/or an aqueous dispersion of bacterial cells at a temperature of 0 to 30 degrees, preferably 0 to 2 degrees.
Contains W% or more in the molecule, D-lactose and 3.6-
A mixture of anhydro-D-lactose and a cold water-soluble aqueous solution containing 1 d2 atoms of anhydro-D-lactose as the main component
Dropped into an aqueous solution containing metal ions of 4 or more, or by dropping an aqueous solution containing metal ions with a one-star resistance of 24 or more into the mixed liquid, the mixed liquid is gelled. l
The present inventors have discovered that the invention can immobilize S and/or bacterial cells, and have come to realize the invention.

D-lactose and 3.6-anhydro-D-lactose are used as upper components, and the sulfate group is added to the molecule (4) at 10'W/
Examples of the cold water OI bathable polyesters containing vr% or more include the sodium salt of kappa carrageenan, the sodium salt of iota carrageenan, and the sodium salt of furseleran.

Examples of metal ions with a concentration of 24 or higher include potassium ions, rubidium ions, cesium ions, magnesium ions, potassium ions, strontium ions, barium ions, aluminum ions, manganese ions, and iron ions.

Further, as the combination of the multi-ηM phase and metal ions, for example, in the case of the sodium salt of kappa carrageenan, potassium ions and magnesium ions are preferable. The gelled carrageenan can also be further hardened by adding a suitable amount of glutaraldehyde or tannin.

Examples of enzymes suitable for use in the present invention include urease, alcohol dehydration, lactic acid dehydrogenation, malic acid dehydration enzyme, thiase enzyme, glucose oxidase, and gelcose oquinodase secamino acid. oxidase,
Lipoxygenase, uricase, ribonuclease, hexokinase, 1171°-se, alkaline phosphatase, i-electrobosphatase, nucleoedase, deoxy7 ribonuclease, α-amylase, β-amylase,
curcoamylase, curcose isomerase, cellulase, hemicellular 1,3-curconotase, invertase, antomianase, naringinase, hesperisonase, 3-curcuronitase, hyaluronitase,
Alkali 1 cow) "rochiryose, semi-asalkali brotease, sashimi [raw protease, ;-mo, trypson tribuno 7-ken, kimotrivunonoken,
Elastase, enterokinase, anorase, arginase, L-curtamic acid decarboxylase, L-11 decarboxylated fermented yarn, papain, and cells of each of the above-mentioned enzymes as 1711 of the pigeon-loving cells used in Honjamei, We present the following examples of bacterial bodies such as Aerobacter aerogenes, Azotobacter vitrifolia, Bacterium spp. will be explained in more detail.

Example 1 Slow 0.067N+1:,'#fl-t 11 um
A solution of 200 mg of urease dissolved in IO'C1 and 4% Soy Gina MV'1O1 (kappa carrageenan sodium salt manufactured by Mitsubishi Acetate Co., Ltd.) in 20 ml of $4. Aqueous solution 20m with IO
/ mix. This mixture was added dropwise to a 5% potassium chloride water bath solution at 10°C, and transferred to #6 in a Zum water bath, and 30 g of carrageenan gel (rlijt
j&) can be obtained.

Add 3g of gel to 3% bQ (H7, 0) 1
Add 00ml I and use Starcy+)'t, M! L
After holding at 20'C for 15 minutes, the enzyme activity was tested and compared with the urease standard. Attachment 7+
It was found to have an activity equivalent to 42% of that of the u enzyme, and to have an extremely high ability to maintain activity.

Example 2 0.067 N sodium phosphate Jt both solutions (H7, O)
A solution prepared by dissolving 200 mg of catalase in 20+++/ with IO'C and 4% #Aginar MV201 (Mitsubishi Acetate Co., Ltd., I4 Tacarrageenan sodium salt) at 10'C.
Mix 10+n/ of aqueous solution. This mixture is 10, 5
% aqueous solution of calcium chloride ζ dropwise into the lucium aqueous solution)
Carrageenan gel 30 containing and immobilizing purified catalase
g (1m ton@) is obtained.

Add 3 g of the above Kel to 15% hydrogen peroxide solution (pH 7°6
) Pour into 100 ml and stir with Starcy.
After holding at 0'C for 3 minutes, the enzyme activity was found to be high at 49°C.

Example 3 10 g of talcose isomerase producing bacteria (Streptomyces, Phaeochromogenes) was added to 20 mA' of physiological saline (
A suspension of 4% Soaginar MV+ot (sodium salt of kappa carrageenan from Mitsubishi Acetate Co., Ltd.) was mixed with 30 m7 of a 10° C. aqueous solution. To this mixture, add 5
Add 10 ml of % potassium chloride solution to EA. The obtained fibrous gel was washed with a 2% potassium chloride aqueous solution to obtain 50 g of bacterial cell-containing carakinan gel (7♀Shenzhen).

Add 3g of the above gel to 10g of glucose at night (PH7
,5) 100ml! After stirring with Starcy and holding mJ for 60 minutes at 60 mJ, the tTlTl property was tested and compared with that of the bacterial cells. It had an activity equivalent to 45% of the added bacterial cells.

Special Liver Determination 1 Guo Ren Ll Starification Co., Ltd.

Claims (1)

[Claims] (1) In an enzyme aqueous solution and/or an aqueous dispersion of bacterial cells,
At a temperature of 0 to 30'C, a cold-water soluble polyester containing sulfate groups in the molecule in an IOW/W% or more and containing D-caractose and 3.6-atahydro-D-caractose as a main component. The mixed liquid is added dropwise to an aqueous solution containing a metal ion with an atom size of 24 or more, or a water f6 liquid containing a metal ion with an atom w24 or more is added dropwise to the mixed liquid. A method for low-temperature immobilization of 1y element or bacterial cells, characterized by kelizing. (2) The method for low-temperature immobilization of enzymes or microbial cells according to claim (1), wherein the cold water iJ-soluble polycycle is a sodium salt of casoper carrageenan or a sodium salt of iota carrageenan. () For atoms of 24 or more, the above metal ions, potassium ions, rubidium ions, senlum ions, 7-gatium ions, calcium ions, strontium ions (1)
” A method for low-temperature immobilization of enzymes or bacterial cells.