JPS6098985A - Preparation of immobilized mold of microorganism for enzyme - Google Patents

Abstract

PURPOSE:To prepare an immobilized mold of microorganism or enzyme having improved mechanical strength of gel, low pollution of microorganism and reduction in enzymatic activity, by releasing silica sol containing a mold of microorganism or enzyme to a gaseous phase, so that it is instantly gelatinized. CONSTITUTION:A mold of microorganism, enzyme, enzyme insolubilzed with tannin or a polyfunctional crosslinking reagent, or enzyme adsorbed on an insolubilized carrier is suspended in water, buffer solution (e.g., buffer solution of phosphoric acid), or hydrophilic organic solvent (e.g., ethyl alcohol). The suspension is added to silica sol obtained by blending an aqueous solution of alkali silicate with an acid solution, they are blended as rapidly as possible, the mixed silica sol is adjusted to 70-210g/l SiO2 concentration and 3-10pH, released to an gaseous phase as early as possible, so that it is gelatinized in a short time, to give an immobilized mold of microorganism or enzyme having <=500mu average particle diameter.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing immobilized microbial cells or enzymes, and its purpose is to produce a spherical gel with significantly less microbial contamination and reduction in enzyme activity, and with high mechanical gel strength. An object of the present invention is to provide a method for efficiently obtaining immobilized microbial cells or enzymes by simple operations.

Conventionally, organic substances such as natural polymeric substances such as alginate, carrageenan, gelatin, and agar, and photocurable resins such as polyurethane have been used as carriers for entrapping and immobilizing microbial bodies or enzyme-containing gels. However, when these carriers are used, since the gel skeleton is an organic substance, there are drawbacks such as being easily contaminated by bacteria, and the gel having low mechanical strength and being easily deformed and swollen.

The following method is known as a method using an inorganic substance, particularly silica gel, as an immobilization carrier.

For example, in the method of British Patent No. 1 U1,76g1, hydrochloric acid is added to an aqueous sodium silicate solution to prepare a silica sol with pH/, &, and salts such as common salt are removed by dialysis to obtain a stable silica sol. The pH of the sol! After adjusting the amount to about ~g, microbial cells or enzymes are added to it to form a gel. Call number 0190 and Tokukai Sho! In the method of Gu 4t9J9-, an enzyme is adsorbed onto colloidal silica (silica sol), and then salt is added to gel it, followed by freezing and thawing operations to obtain an amorphous, crushed immobilized gel. There is.

However, these methods require time to prepare the base silica sol, the entrapping immobilization (gelation) operation is complicated, and when using these as a bioreactor, since the silica gel is amorphous, In order to use these materials in a substrate reaction container, there are drawbacks such as a sufficient filling effect cannot be obtained unless the gel is prepared in a shape suitable for the container.

On the other hand, as a method that takes gel molding into consideration, Tokukai Sho! ! -l
There is a method called KO2TATAθ, which uses silicic acid) I
A mixed aqueous solution of Jum and microbial cells or enzymes is suspended in a hydrophobic M-organic material such as toluene or cyclohexane, and in this state it is brought into contact with barium to form a gel, thereby entrapping and immobilizing the microbial cells or enzymes. This is a method for obtaining spherical silica hydrogel.

However, hydrophobic M agents (suspending agents) such as toluene and cyclohexane used in this method reduce the activity of microbial cells or enzymes, and also require an operation to separate and remove such suspending agents from the silica hydrogel. In addition to being extremely complicated, the pore structure of the resulting spherical silica gel is non-uniform, resulting in disadvantages such as weak mechanical strength of the gel.

The method of JP-A-33-313-3 involves molding a mixture of a colloidal silica suspension, a porous filler, and microbial cells using an extrusion molding machine. In addition to being complicated, the processing time is also long.
Furthermore, the resulting gel has drawbacks such as low mechanical strength.

Therefore, as a result of intensive studies to resolve these drawbacks of the conventional technology, the present inventors found that if microbial cells or enzyme-containing silica sol were released into the gas phase to instantaneously gel, it would be possible to achieve comprehensive immobilization. The present invention was completed based on the realization that it is an advantage to simultaneously and efficiently perform gel formation and gel formation.That is, the present invention is based on the invention. or an enzyme adsorbed onto an insolubilized carrier suspended in water, a buffer solution, or a hydrophilic organic solvent is added to the silica sol obtained by mixing an alkaline silicate aqueous solution and an acid solution and mixed. The concentration of Sin in the mixed silica sol was set to 70-
An immobilized microorganism characterized by setting U10f/Jl, pI (3 to IQ), and then releasing it into the gas phase as soon as possible and gelling it in an extremely short time to form a spherical silica gel. This is a method for producing bacterial cells or enzymes.

The present invention will be explained in detail below.

First, the microorganisms used in the present invention include m bacteria,
Any type of bacterial cells such as yeast, mold, actinomycetes, etc. may be used. Furthermore, any type of enzyme may be used (e.g., oxidoreductases such as alcohol dehydrogenase, glucose oxidase, lactate dehydrogenase, transferases such as D-glutamyl transferase, glutamine transaminase, hexokinase, leucine aminopeptidase, Carboxy!
Hydrolytic enzymes such as peptidase and beninlinase, lyase enzymes such as fumarase, aspartase and β-tyrosinase, isomerase enzymes such as glucose isomerase and mannose isomerase, glutathione synthase, NAD
Typical examples include glue gauze enzymes such as synthase.

The above-mentioned enzyme may be used by insolubilizing it with tannin or a polyfunctional crosslinking reagent, or by adsorbing the enzyme onto an insolubilizing carrier.

First, when insolubilizing with tannin, the amount of enzyme should be
, a solution containing -10 times the amount (W/W) of tannin is added, and the reaction is carried out with stirring at PHg or below, preferably PHJ~2, and the resulting enzyme precipitate is subjected to conventional methods such as centrifugation, The insolubilized enzyme is obtained using the following separation means.

In addition to tannic acid, the tannins used include pyrogallol tannins such as gallic tannins or pentadol tannins, and catechol tannins such as tannin substances (catechol polymers) obtained from tea, cacao, etc. These tannins may be those that have tannin action and have not been purified to the limit; for example, commercially available persimmon tannins may also be used. These can be used alone or as a mixture of one or more tannins.

When insolubilizing with a polyfunctional crosslinking agent, add the enzyme to a solution containing 71-0% (W/V) of the polyfunctional crosslinking agent. The reaction is carried out at ~aoC for 10 minutes to 16 hours, and the insolubilized enzyme is obtained from the resulting enzyme precipitate using conventional separation means such as centrifugation and r-filtration.

Suitable polyfunctional crosslinking agents include polyaldehydes, incyanates, etc., such as dialdehyde starch glyoxal, malonaldehyde, succinic aldehyde, glutaraldehyde, pimeridialdehyde,
Hexamethylene incyanate), p-toluylene diincyanate, etc., and glutaraldehyde is particularly desirable.

As a means of adsorbing the enzyme to the insolubilizing carrier,
Common adsorbents such as activated carbon, silica gel, dispersible clay,
Porous glass, etc., DEAE-7Fadex, CM-
Sephadex, DEAE-Cellulose, CM-Cellulose, Amberlite IR-Qj, Dowex! θ
Pack an insolubilizing carrier such as an ion exchanger into a column and pass the # purple through i-f, or mix the insolubilizing carrier with an enzyme,
After stirring and adsorption, the enzyme is adsorbed on the insolubilized carrier by separation means such as centrifugation or r-filtration, if necessary.After that, if necessary, the polyfunctional A cross-linking agent may be added and the reaction may be carried out.

The above-mentioned microbial cells, starch, and enzymes insolubilized with tannins or polyfunctional crosslinking reagents, or enzymes adsorbed onto insolubilized carriers, can be suspended in water, buffer solution, or II'i hydrophilic organic solvent. and use it.

Buffers used for this purpose include, for example, intoxicant buffer, pine kilguane buffer, phosphate buffer, Tris buffer, veronal buffer, etc., and examples of aqueous organic solvents include methyl alcohol, ethyl alcohol, propyl alcohol, Examples include acetone, and these organic solvents are usually used in an amount of about io to 0% (%'V).

On the other hand, a silica sol is prepared by mixing an aqueous alkali silicate solution and an acid solution.

Examples of the alkali silicate water bath solution include aqueous solutions of silicic acid, and examples of acid solutions include sulfuric acid, hydrochloric acid, and nitric acid. @-Vinegar-Flute Cave Bird Layer Jojajamuda Iireru.

Next, the above-mentioned microbial cells, enzymes, and enzymes were insolubilized with tannin or a polyfunctional crosslinking reagent, or the enzymes were adsorbed onto an insolubilized carrier into the silica sol obtained by mixing an aqueous alkali silicate solution and an acid solution. Add the suspended substance in water, a buffer solution, or a hydrophilic organic solvent, and mix the microbial cells or enzymes as soon as possible so that they are homogeneous in the mixed silica sol. The concentration is 20 to 10 iP/I, preferably FiiJo-ig.
oy7! , and the pH is set to 3 to IO1, preferably 6 to 0 g. In addition, when preparing the mixed silica sol, at least one sol selected from colloidal silica, alumina silica sol, and alumina sol is added to the mixed silica sol. This is advantageous in terms of reinforcing the silica gel structure.

Also, as mentioned above, the concentration of Sin in the mixed silica sol was set to 20
Setting the ioP/i and pHJ to 10 is extremely important in gelling the mixed silica sol in a very short time and increasing the mechanical strength of the microbial cells or enzyme-containing silica gel.

Next, the mixed silica sol obtained as described above is released into the gas phase from the outlet as soon as possible, and is left in the gas phase for a very short period of time.
Gelation is usually carried out within 5 seconds, preferably within I/'iλ seconds, to obtain spherical immobilized microbial cells or enzyme-containing silica gel.

The gas phase medium used here is usually air, but if desired, an inert gas such as nitrogen gas may be used.

Since the gelation in the gas phase is completed in an extremely short time, there is little possibility of contamination with microorganisms, especially miscellaneous bacteria (and, moreover, there is no substantial reduction in the activity of the target microorganism or enzyme. can be prevented.

In addition, since the gel obtained by the gelling operation described above has a spherical shape, the mechanical strength of the gel is increased, and when the gel is packed in a substrate contact tower and reacted with a substrate, the voids between the gels are reduced. This has the advantage that the rate can be kept constant at about tl, thereby substantially preventing a decrease in substrate reaction efficiency.

Furthermore, the particle size of the spherical immobilized microbial cells or enzyme-containing gel obtained by the present invention can be adjusted to
Particles with a particle size of about μ~1OWrIk can be obtained, but especially the average particle size! If it is a gel with fine particles of about 00μ or less,
This is advantageous because the reaction efficiency with the substrate can be significantly increased.

The device used to obtain the above-mentioned immobilized microbial cells or enzymes may be of any shape or structure as long as the object of the present invention is achieved. an inlet for an aqueous alkali silicate solution and an acid solution, an inlet for microbial cells or enzymes is provided at the bottom of the container, and a mixing blade is provided in the container;
According to the present invention, a container equipped with a discharge nozzle for discharging microorganism cells or enzyme-containing silica gel into the gas phase from the bottom of the container is preferable for carrying out the present invention efficiently. , the comprehensive immobilization 11Z of microbial cells or enzyme-containing silica sol and gel formation can be carried out simultaneously and efficiently in an extremely short period of time, and the resulting gel has significantly less microbial contamination and decrease in enzyme activity, and is moreover a mechanical gel. It is extremely strong.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 A sodium silicate aqueous solution (5i02/Na2O molar ratio = 3.g, Sin, content concentration Hig%-W/V) and <zN-sulfuric acid aqueous solution were mixed with a stirring blade and a discharge port using a metering pump. The microorganisms were fed under pressure into each container through a separate inlet port, and Disatucharomyces cerevisiae FO0-2aa was cultured and collected through another inlet port in the container. Add a solution (yeast viable cell count: /, OX / D'. / mb) suspended in M phosphate buffer (1) H4, 77) at a constant ratio and mix. 5i
OJ 1 degree: l,? oiP/L, pH near-j)
, the outlet at the bottom of the container (nozzle diameter knee 211II11
), the mixed silica sol was continuously released into the air, and the silica hydrogel containing yeast cells was collected in a saucer containing acetic acid. The immobilized yeast cells are spherical and have an average particle diameter of λ~, 7
m (r) (moisture content on dry weight basis: ECO 0%) Next, the ethanol productivity of the immobilized yeast cells was measured as follows.

The above immobilized yeast cells 6! was packed in a jacketed column (inner diameter = 3 cm, height: i6 cm) kept at 30C, and the column was filled with glucose g% (W/V) and ammonium sulfate O1λ%.
(W/V), yeast extract. , a liquid medium (p, Fi, 7..7) containing 1% (W/V) in an ascending method (SV = o, λ)
Ethanol fermentation was carried out while passing for λ days. The results are shown in Table 1.

Table 1 From Table 1, it was found that the ethanol fermentation ability of the immobilized yeast cells obtained in Example 1 was extremely excellent despite being kept for a long period of time.

Example 2 Sodium silicate aqueous solution (5i02/Na2O molar ratio: J, y, 5in2 containing *pi 2s s%-W/V
) and gN-sulfuric acid aqueous solution were pumped into a container with a stirring blade and a discharge port using a metering pump through separate inlets, and then urease (Taibuta, Sigma Co., Ltd. Add and mix a 1%-W/■ aqueous solution of silica sol (manufactured by Mimaki Co., Ltd.) at a fixed ratio (concentration of 5i02 in the mixed silica sol in the container: Co. f/II, pH: 1.2), and then open the outlet (nozzle) at the bottom of the container. The mixed silica sol is continuously released into the air from the diameter knee (m), and the gelation time is 2 (i, 0 seconds), fixed 1.
The urease-containing silica hydrogel was collected. .

The obtained immobilized urease is spherical and has an average particle size! ~4
wm [moisture content on dry basis: 4I. θ].

Example 3 A sodium silicate aqueous solution (5102/NazO molar ratio: 3.g, 5t (h concentration: 1g%) - W/VL & tN - hydrochloric acid aqueous solution was mixed with a stirring blade and a discharge port using a metering pump. They were each pumped into a container overlapping each other through separate inlets.

On the other hand, tannin awakening [OU Hikari Junyaku Co., Ltd.] Ko 0θ? λ
oooab o, ozM Tris buffer 7 (1 (pH 7,0
) dissolved in Aspergillus oryzae FER
Ammonium sulfate was fractionated from the wrinkle culture of M P-t Ig 2 and DEAE
- Insolubilized leucine aminopeptidase prepared using cellulose was mixed with 0.0 IMh Lys buffer (pH 7-0) of Yuooomb and centrifuged. An insolubilized enzyme solution was prepared by adding o, osM Tris solution (pH 7, +1 toomb) to the enzyme.

Next, add this insolubilized enzyme solution at a constant rate from another inlet in the container and mix. /A, pH: ri, ri, the mixed silica sol is continuously released into the air from the outlet (nozzle diameter: ttrrh) at the bottom of the container.Gelification time: t, z
The leucine aminopeptidase-containing M silica hydrogel was collected in a saucer containing Tris buffer (pH 2, θ). Moisture content on dry basis: 13
In addition, one immobilized gel obtained from the above procedure (approximately 0%) was 0%.
Nakadai's method using leucine-p-nitroanilide as a substrate [Tadanobu Nakadai, Japan Soy Sauce Research Institute Report J, 9ri (
(1974)], the result was a unit of lλOOθ.

Example 4 Sodium silicate water bath solution (5if2/ Na2O molar ratio: J, l, Sin, content concentration: 1g% W/
v) and N-sulfuric acid aqueous solution are each pumped into a container equipped with a stirring blade and a discharge port using a metering pump through separate inlets, and Pediococcus sulfuric acid is further injected into the container from another inlet in the container. Halophilus FERM P-6aa.

were cultured, collected, and suspended in colloidal silica sol (Sin, 30% aqueous solution, Snowtex 30, manufactured by Nissan 1tS Gaku Co., Ltd.) (PH: 7, 0, number of viable lactic acid bacteria: !0) ×/(17I7mJ,) is added at a constant rate and mixed. 5iOz concentration in the mixed silica sol in the container:
/ A g sP/ A, pH near, j), the mixed silica sol is continuously released into the air from the outlet at the bottom of the container (nozzle diameter: i, 3 mx+), gelling time: i,
3 seconds), 0.1M phosphate buffer (p) I7. θ) The silica hydrogel containing lactic acid bacteria was collected in a saucer containing the lactic acid bacteria.

The obtained immobilized yeast cells were spherical and had an average particle diameter of 2 to 3 mm (water content on a dry weight basis: soθ%).Next, the immobilized lactic acid bacteria cells were subjected to lactic acid fermentation. The above immobilized lactic acid bacteria cells 13Jf were packed into a jacketed column (inner diameter: Jcrn, height 130cm) kept at aoC, and meat extract/% (W/ V), polypeptone/% (W/V), I! Mother extract 1% (W/V), glucose 1% (W/V), thioglycolate 0.1% (
Milk powder fermentation was carried out while passing through a liquid medium (pH 7, co) containing NaCl 1/3% (W/V) and NaCl 1/3% (W/V) in an ascending method (SV=o, i) for 30 days. The results are shown in Table 2.

Table 2 From Table 2, it was found that the lactic acid fermentation ability of the immobilized lactic acid bacteria cells obtained in Example 4 was extremely excellent despite being kept for a long period of time.

Example 5 A sodium silicate aqueous solution (5if2/Na, 0 molar ratio: 3.g, Sin, content concentration: 1 g% W/V) and &N-sulfuric acid water bath solution were mixed with a stirring blade and released using a metering pump. Each is pressure-fed from a separate inlet into a container having an outlet,
Furthermore, Saccharomyces cerevisiae IFOO--g was cultured and collected from another inlet in the container, and acetate buffer (p
Hj, t) (number of viable yeast cells: / ,
Add and mix OX/0''/11b) at a constant ratio. Sin, concentration in the mixed silica sol in the container; i J
The mixed silica sol is continuously released into the air from the outlet (nozzle diameter: /, jwa) at the bottom of the container, and the flying silica sol is injected with compressed air from the side. Spray to atomize and gel time: 1
．． ! sec), and the silica hydrogel containing yeast cells was collected in a saucer containing an acetate buffer (pHj, j).

The obtained immobilized yeast cells were spherical and had an average particle diameter of aoo microns (water content on a dry weight basis: 520%).

Applicant: Kikkoman Corporation

Claims (4)

[Claims] (1) Microbial cells, enzymes, enzymes insolubilized with tannins or polyfunctional crosslinking reagents, or enzymes insoluble 111
．． The adsorbed material on the carrier is suspended in water, a buffer solution, or a hydrophilic organic solvent, and then mixed with a silica aqueous alkaline solution and R11.
1 solution and mixed to make the 5in2 concentration in the mixed silica sol 70-2/o.
An immobilized microorganism characterized by setting the pH to 7-/0, then releasing it into the gas phase as soon as possible and gelling it in a very short time to form a spherical silica gel. Method for producing bacterial cells or enzymes. (2) The method according to claim 1, characterized in that at least one sol selected from colloidal silica, alumina silica sol, and alumina sol is added to the mixed silica sol. (3) Gel 11 hours! 8. The method according to claim 7, wherein the method is within seconds. (4) Claims in which the gel average particle diameter of the immobilized microbial cells or enzymes is 500 microns or less! /
The method described in section. (5) 5in2 concentration in mixed silica sol 130~/g
Oi! 2. The method according to claim 1, characterized in that -/A and pH are adjusted to 6 to 6 g, respectively.