JPS6058071A - Preparation of immobilized mold of microorganism or enzyme - Google Patents

Abstract

PURPOSE:To obtain an immobilized mold or enzyme having low reduction in activity, by insolubilizing a mold or enzyme with tannin or a crosslinking agent, blending it with a solution prepared by dissolving a gel substrate under heating, cooling the solution, spraying the solution and bringing it into contact with a cooling liquid to prepare small gel. CONSTITUTION:A mold of microorganism or enzyme is insolubilized with tannin or a polyfunctional crosslinking reagent, or the enzyme is adsorbed on an insolubilized carrier, (directly or after it is suspended in water, buffer solution, or hydrophilic organic solvent), it is blended with a solution prepared by dissolving carrageenan, agar, or gelatin as a gel substrate in water, buffer solution or hydrophilic organic solvent under heating, (at a temperature not to solidify the gel substrate, not to deactivate the mold or enzyme). The mixed solution is sprayed on brought into contact with a cooling liquid and processed into small gel.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing immobilized microbial cells or enzymes, and its object is to produce immobilized microbial cells or enzymes that have significantly increased reaction efficiency with a substrate. The purpose is to obtain an enzyme gel.

Conventionally, the method of comprehensively immobilizing microorganisms, enzymes, etc. with carrageenan, agar, gelatin, etc. has been difficult to reduce activity during immobilization (and in recent years, attempts have been made to apply it to the food and pharmaceutical industries as a safe method from a food hygiene perspective). It is being

Usually, microbial cells or enzymes are combined with carrageenan,
When entrapping immobilization with a gel base material such as agar or gelatin, the microorganisms or enzymes are suspended in a heated solution of the gel base material, and then dripped into the cooling liquid using a syringe or a perforated plate. Alternatively, it is formed into a spherical or thread-like gel by flowing down. that time,
Gel particle size is generally adjusted by adjusting the droplet flow rate, but liquids containing these gel base materials have a high viscosity (and the droplets are difficult to balance between the surface tension of the liquid and gravity). Because of this, it is only possible to form particles with a particle size larger than the outer diameter of the nozzle used to dispense one drop. Conventionally, particles with a particle size of about 12 mm to 12 mm can be formed. 〇Also, when performing an enzyme reaction using microorganisms or enzymes immobilized by the gel entrapment method, in order to perform the enzyme reaction efficiently, it is necessary to sufficiently permeate the substrate into the immobilization gel. extremely important.

At this time, since the gel exhibits resistance (diffusion resistance) to the movement of the substrate, the smaller the gel particle size, the higher the reaction efficiency.
Furthermore, when using immobilized microorganisms, the microorganisms in conventional gels with large particle diameters tend to proliferate near the gel surface. Engineering, pp. 3gg-3ri!, published by Tokyo Kagaku Dojinsha), etc.

In order to increase the reaction efficiency, it is desired to shorten the mass transfer distance, make the gel particles smaller, and increase the surface area of the gel accordingly.

On the other hand, when microbial cells or enzymes are immobilized using the microcapsule method, it is possible to form microparticles;
In addition to the complicated molding operation, the monomer,
Since a polymerization catalyst or an organic solvent is used, there are drawbacks such as a significant decrease in activity.

The present invention eliminates these drawbacks of the prior art.

The gel particle size of the immobilized microorganisms or enzymes is made minute, so that it is possible to easily obtain an immobilized gel with significantly increased reaction efficiency with the substrate.

Specifically, the present invention uses microbial cells, enzymes insolubilized with tannins or polyfunctional crosslinking reagents, or enzymes adsorbed onto an insolubilized carrier, as they are.

Alternatively, these can be suspended in water, a buffer solution, or a hydrophilic organic solvent, and a solution prepared by heating and dissolving carrageenan, agar, or gelatin as a gel base in water, a buffer solution, or a hydrophilic organic solvent. , an immobilized microorganism characterized in that a liquid mixed at a temperature that does not solidify the gel base material and do not kill or deactivate the microorganism cells or enzymes is sprayed into contact with a cooling liquid to form a microgel. 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 bacteria,
The enzyme may be of any type, such as yeast, mold, actinomycetes, etc., and may be of any type, for example, oxidoreductases such as alcohol dehydrogenase, glucose oxidase, lactate dehydrogenase, D-glutamyl transferase, Transferases such as glutamine transaminase and hekin kinase, hydrolytic enzymes such as leucine aminopeptidase, carboxypeptidase, and peptidase, lyase enzymes such as fumarase, aspartase, and β-tyrosinase, and isomerase enzymes such as glucose isomerase and mannose isomerase. Representative examples include gauze enzymes such as , glutathione synthase, and NAD synthase. The above-mentioned enzymes are insolubilized with tannin or a polyfunctional crosslinking reagent, or the enzymes are adsorbed onto an insolubilized carrier.

First, when insolubilizing with tannin, the amount of enzyme should be
- Add a solution containing tannin (10 times the amount cW/W), react with stirring at pH below, preferably pHJ ~ 7, and separate the resulting enzyme precipitate by conventional separation such as centrifugation or filtration. Obtaining an insolubilized enzyme using means 0 Note that the above-mentioned tannins include tannic acid.

Pyrogallol tannins such as gallic tannins or pentadol tannins, catechol tannins such as hacha tea.

Tannins (catechol polymers) etc. obtained from cacao etc. are used. These tannins may be unrefined as long as they have a tannin effect (for example, commercially available persimmon tannins may also be used. These tannins can be used alone or as a mixture of one or more tannins.

In addition, when insolubilizing with a polyfunctional crosslinking agent, add the enzyme to a solution containing a polyfunctional crosslinking agent of l-λ04bCW/V). The reaction is carried out at ~41Qc for io minutes ~/A hours, and the resulting enzyme precipitate is subjected to conventional separation means such as centrifugation and filtration to obtain an insolubilized enzyme.

In addition, as a polyfunctional crosslinking agent, polyaldehyde
, (/cyanates, etc. are suitable, such as dialdehyde starch glyoxal, malonaldehyde, succinic aldehyde, glutaraldehyde, pimeridialdehyde, hexamethylene isocyanate), p-) lylene diisocyanate, etc., and in particular Glutaraldehyde is preferable. As a means for adsorbing the enzyme to an insolubilizing carrier, 1. Ordinary adsorbents 1. For example, activated carbon, silica gel, acid clay, porous glass, etc., DEAE-Sephadex, CM-Sephadex, DEAE-cellulose. , CM-cellulose, Amberlight IR-ri! , DOWEX! Either an insolubilized carrier such as an ion exchanger such as θ is packed in a column and the enzyme is passed through the column, or the insoluble carrier is mixed with the enzyme, stirred and adsorbed, and then centrifuged, for example, if necessary.

The enzyme adsorbed on the insolubilized carrier is obtained by separation means such as filtration. Thereafter, if necessary, the polyfunctional crosslinking agent may be added to the insolubilized carrier that has adsorbed the enzyme and allowed to react.

Next, the above-mentioned microbial cells and enzymes are insolubilized with tannin or a polyfunctional cross-linking reagent, or the enzymes are adsorbed onto an insolubilized carrier, either as they are, or in water, a buffer solution, or a hydrophilic organic solvent. Add carrageenan, agar, or gelatin as a gel base to water, a buffer solution, or a hydrophilic organic solvent at about 70C.
A liquid obtained by heating and dissolving the above liquid or a liquid obtained by appropriately cooling the liquid.

, jj-jjU are mixed at a temperature at which the gel base material is not solidified and the microbial cells or enzymes are not killed or deactivated to obtain a solution in which the gel base material is dissolved.

Buffers used for this purpose include, for example, acetate buffer, pine kilvein buffer, phosphate buffer, Tris buffer, veronal buffer, etc., and examples of aqueous organic solvents include methyl alcohol, ethyl alcohol, flobyl alcohol, etc. , acetone, etc., and these organic solvents are usually used at a ratio of about 10-λθ (W/V).

And carrageenan and agar as gel base materials are usually O
The ratio is about 1j to 3.0 (W/V), and gelatin is used at about g-cos (w7v).

Next, a solution obtained by mixing and dissolving the gel base material as described above is sprayed into contact with a cooling liquid using a sprayer, preferably a pressurized sprayer, to obtain minute immobilized microbial cells or enzymes.

Cooling fluids used for this purpose include, for example, potassium chloride, ammonium chloride-aluminum chloride, bamboo solution 1, an aqueous solution containing about 1-10 mm (W/V) of these, olive oil, corn oil, palm oil, Matsukou whale head. oil, animal and vegetable oils such as fish oil, mineral oils such as spindle oil and turbine oil, and silicone oils.

Synthetic oils such as boil oil and stand oil, linoleic acid.

Unsaturated fatty acids such as lyluic acid and oleic acid are used, and liquids obtained by cooling these to below IOC are used.

In addition, when using carrageenan as the gel base material,
It is preferable to use a cooling solution of an aqueous solution of potassium salt, ammonium chloride, aluminum chloride, etc. When agar or gelatin is used as the gel base, animal or vegetable oil,
Mineral oil 1 It is preferable to use a cooling liquid such as synthetic oil or unsaturated fatty acids 0 And when obtaining immobilized microbial cells or enzymes, the spray contact means is usually sprayed using a pressurized spray nozzle. Preferably 0 then.

As the pressure nozzle, a pressurized spray nozzle such as a simple jet nozzle, an impingement jet nozzle, a swirl nozzle, or a two-fluid nozzle may be used.

A rotating disc sprayer or a sprayer using sound waves, static electricity, vibrations, etc. may also be used. And the spray pressure when pressurized spraying is ', j-A 00 "j' / ca - G
.

Preferably it is about /-/Y'''t/catΦG.

In addition, these pressurized spray conditions are affected by the types of microbial cells and enzymes used, the concentration of alginate, the type of nozzle, etc., so they should be selected accordingly. As for the size of the enzyme gel particles, the normal average particle diameter is at most goo~? θ0
In the present invention, it is desirable to adjust the average particle size to 117 microns or less, and by forming such fine particles, the reaction efficiency with the substrate can be significantly increased.

In particular, the fixed 11S microbial cells were prepared as described above (,!θ
If the particles are of the order of 0 microns or less, for example, when carrying out fermentation using the microbial cells, there is no need to grow the microorganisms by culturing them in a culture medium in advance, and the microorganisms can be used directly for fermentation. It has outstanding advantages such as:

As described above, by using microbial cells or enzymes immobilized according to the present invention, the reaction efficiency with substrates can be significantly increased, and even if the enzymes are repeatedly used on various substrates, the immobilization The present invention is of great industrial significance, as the detachment from the enzyme-containing gel is significantly suppressed.

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

Example 1 Tannic acid [manufactured by Wako Tsumugi Co., Ltd.]
Dissolved in 0-year-old M Tris buffer (pH 7,0),
Leucine aminopeptidase specimen purified using DEAE-cellulose and ammonium sulfate fractionated from Aspergillus oryzae FERM P-//Geta furrow culture.
The leucine aminopeptidase was thoroughly mixed with a solution of leucine aminopeptidase dissolved in an ojM Tris buffer (CpH 7,0) at λ0, to precipitate the leucine aminopeptidase.

The precipitate was centrifuged in a conventional manner, and then washed four times with the above-mentioned Tris buffer i&c (pH 7.0).

To the obtained insoluble enzyme (300 ml as dry matter),
Agar and water were added to give an agar concentration of /% (W/V) and dissolved by heating at 9 IC, then an agar aqueous solution cooled to ysc - 0 mb was added and mixed. Temperature during mixing:
4t oC), and then use it as an airless sprayer [co-fluid type, nozzle diameter: 0. ! Pottery, spray pressure 2, 7,
oKy/crA*G, manufactured by Burgess, UK] was sprayed under pressure into corn oil cooled to jC, and the average particle size was 100~
Leucine aminopeptidase immobilized in the form of λso micron particles was obtained (immobilized enzyme of Example 1).

Table 1 shows the results of comparing the activity of the immobilized enzyme of Example 1 with the activities of Control-1 and Control-2, which are leucine aminopeptidase immobilized by a conventional method. In addition, as a control, add 5 solutions of the above-mentioned insolubilizing enzyme (leucine aminopeptidase) J00m9 to agar with water to give an agar concentration of 71 (W/V), and mix well. Temperature at: <t.

C), the average particle diameter obtained by dropping this into corn oil cooled to SC using a syringe (caliber: λran)! It is a leucine aminopeptidase immobilized in a spherical shape.

Control-2: 3001 ng of the above insolubilizing enzyme (leucine aminopeptidase) was added to agar to give an agar concentration of /% (W/V) and dissolved by heating at 9°C.
Next, add the agar water bath solution omb cooled to 41 IC and mix well (temperature at the time of mixing (Nige OC)), and drop this into the corn oil cooled to IC using a syringe (caliber: /a) to obtain average particles. This is a leucine aminopeptidase immobilized in a spherical shape with two diameters.

Relative enzyme activity was measured using Nakadai's method using each of the above-mentioned immobilized enzymes (O1λ?) and using leucine-p-nitroanilide as a substrate [Tadanobu Nakadai, Japan Soy Sauce Research Institute Report 3. 99 (/ri 77 years)] and the obtained activity value.

Table 1 shows the relative #element activity values (嗟).

As is clear from Table 1, the immobilized enzyme of Example 1 has a significantly higher enzyme activity value than any of the control immobilized enzymes.Example 2 Pig heart Origin of lactate dehydrogenase (Boehringer M)/
DO da 0. θ! M acetate buffer (pH 7,0)
Add 5vrt of λ,, + 1 (W/V) glutaraldehyde water bath solution, and add while stirring.
After reacting for 93θ minutes and centrifuging the obtained insolubilized enzyme by a conventional method, it was added to a 0.03M acetate buffer (pHj) 3
After washing with OOMB, an insolubilized enzyme was obtained.

The obtained insolubilized enzyme! 0. ojM acetate buffer WCp
Hz, t) to the suspension and add 1-2% (W/
V) Gelatin and water were added to the gelatin concentration and dissolved by heating in GOC, and then gelatin water bath solution J-07718 cooled to 41 IC was added and mixed thoroughly (temperature at the time of mixing: 37 C). - Handy Bainter W - / 7 o (λ fluid type.

Nozzle diameter: o, 7tra, sprayed under pressure into silicone oil cooled to IC with Nippon Wagner Spraytex Co., Ltd. ↓) at a spray pressure of 1λOKp/ci”G, average particle size/J-
Obtained lactate dehydrogenase immobilized in the form of fine particles of 17 to λso microns (immobilized enzyme of Example 2) The results of comparison with enzyme activity are shown in Table 2. In addition, control-1: the above-mentioned insolubilizing enzyme (lactate dehydrogenase) tom
To a suspension of 00-0j acetate buffer (pHj, j ) j O in g, water was added to gelatin to give a gelatin concentration of 12% (W/V), and the mixture was heated with gOC. Aqueous gelatin solution dissolved and then cooled to IC! 0
Temperature when mixing: ? 7C) is a spherically immobilized lactate dehydrogenase with an average particle diameter of JNII obtained by dropping this into silicone oil cooled to JC using a syringe (caliber: C). The above insolubilizing enzyme (lactate dehydrogenase) toI
ng to 0.03 MM acid buffer (pHj, z).

ml of gelatin and water was added to the suspension to give a gelatin concentration of 12% (W/V).
An aqueous gelatin solution that was heated and dissolved in ljC and then cooled to ljC! Add the θ width and mix (temperature during mixing: 77C), drop this into silicone oil cooled to JC using a syringe (caliber: im) and fix it in a spherical shape with an average particle diameter of i, ja. It is a converted lactate dehydrogenase.

In addition, the relative enzyme activity measurement test was carried out using each of the above-mentioned immobilized enzymes (o, 2 g) and using pyruvate as a substrate using the Strusenbach method [Strusenbach, Method-in-Engineering/Timology-9゜]. 47g-2g'g (19b
Table 2 shows the relative enzyme activity value (%) compared with the control-1 specimen (enzyme activity value; ioo%). As is clear from Table 2, the immobilized enzyme of Example 2 has a significantly higher enzyme activity value than any of the control immobilized enzymes. [Manufactured by Fuji Deguison Chemical Co., Ltd.] iy
and urease (type 9. manufactured by Sigma) ioo da o,
o! M Tris buffer (pH 7,0) and mix;
Urease was adsorbed to the thyroid by stirring with IC for about 1 hour, which was then centrifuged in a conventional manner, and then washed with the above-mentioned Tonos buffer (PH 7, 17).

The enzyme soIng adsorbed on the obtained thyroid was
% (W/V) of gelatin concentration was heated and dissolved in gOC, and then mixed with gelatin water bath solution cooled to jOC - 〇mA. Temperature during mixing: II, tC) , which was used in Example 2.
Urease immobilized in the form of fine particles with an average particle size of 750 to 300 microns was obtained by spraying under pressure at a spray pressure of s and sKy/crA・G into a linoleic acid solution cooled to jC using a Handy Vainter W-/70. (Immobilized enzyme of Example 3) The activity of the immobilized enzyme of Example 3 was compared with the activity of a control, which is urease immobilized by a conventional method. The results are shown in Table 3.

In addition, as a control, goC was prepared by adding water to gelatin so that the gelatin concentration was 7λ% (W/V), containing the enzyme X (UL'7-se)
Then, mix with gelatin aqueous solution cooled to gOC (temperature at the time of mixing: da IC), and drop this into the linoleic acid solution cooled to IC using a syringe (caliber: /w). The resulting urease is immobilized in a spherical shape with an average particle size of 0.

In addition, relative enzyme activity measurement tests were carried out using each of the above-mentioned immobilized enzymes (O2 = ?) and using urea as a substrate using Lyssel's method [Lyssel F.G., The Enteams 3rd edition,!, 7 to /(/97/)), and the relative enzyme activity value (%) was shown by comparing the obtained activity value with the reference specimen (enzyme activity value = IOθ%). It is a table.

Table 3 As is clear from Table 3, the immobilized enzyme of Example 3 has a significantly higher enzyme activity value than the control immobilized enzyme.

Example 4 Glucose 2% (W/V), Peptone 1% (W/V),
yeast x*s/% (W/V), and salt i.

Culture medium containing % (W/V) CpH! , j) t, 1 to 3
After putting it in a flask and sterilizing it by a conventional method, the culture medium was injected with Zatsucharomyces luxii ATCC/0, ? g J
The seed culture solution IO was inoculated and cultured with shaking at 30C for 7 hours. Obtained cultured solution l! Centrifuge to collect bacteria,
After freeze-drying this! ? Dry yeast cells were obtained.

The obtained yeast cells! ? , o, os to Cumber carrageenan to give a carrageenan concentration of 1% (W/V)
M Tris buffer (pH 7, O) was added and dissolved by heating at 9DC, and then mixed with aqueous carrageenan solution 6001jLb cooled to Guo C. After introducing helium gas into the sealed pressure vessel, the pressure inside the pressure vessel is adjusted to lOM/C1/I@G.
Then, open the cock of the nozzle [MKBOIio 3g type, nozzle diameter: o, ttm, manufactured by Ikeuchi Co., Ltd.] and spray under pressure into a 70% (W/V) potassium chloride water bath solution cooled to -3C. to obtain immobilized yeast cells in the form of fine particles with an average particle diameter of 20-300 microns (immobilized yeast of Example 4). Alcohol productivity of the immobilized yeast of Example 4 was determined by the conventional method. Table 4 shows the results of comparing the alcohol productivity of immobilized yeast cells, Control-1 and Control-2. In addition.

Control-1: The above yeast cells, 5', 7% (W/V)
Aqueous carrageenan solution was prepared by heating and dissolving carrageenan with o, o M Tris buffer (pH 7, o) at 90C to give a carrageenan concentration of g to Mix it with o rnb (temperature during mixing: +OC), and use it in a syringe (caliber: Kowari-).
An average particle diameter of 5 trrrh obtained by dropping into a 70% (W/V) potassium chloride aqueous solution cooled to -3C using
− is a yeast cell immobilized in a spherical shape, and Control-2 = the above yeast cell! A mixture of carrageenan and O, Oj''M) Lith buffer (1) H7-17) was added to carrageenan to give a carrageenan concentration of 1% (W/V) and dissolved by heating at 9θC. Next, mix with JOOml of carrageenan aqueous solution cooled to 4tjC (temperature at mixing: yoC), and use a syringe (caliber: / mrh).
Yeast cells are immobilized in a spherical shape with an average particle diameter of 100 ml, which is obtained by dropping them into an IQ% (W/V) potassium chloride water bath solution that has been cooled using a 13C button.

In addition, the measurement of alcohol productivity was performed using glucose g% (W
/V), peptone 1% (W/V), yeast x kiss o,,
7% (W/V) and saline/g% (W/V) (pHt, a) to io omb.

! After putting it in a flask and sterilizing it by a conventional method, each of the above-mentioned immobilized yeast (wet bacterial cells) was added to the medium! Add l.

This was done by measuring the alcohol concentration of the culture solution that was incubated at 3θC for 13 days in each medium.

The results are shown in Table 4.

As is clear from Table 4, it was found that when the immobilized yeast of Example 4 was used, the amount of alcohol produced was significantly improved compared to when any of the immobilized control yeasts were used.

Applicant: Kikkoman Corporation

Claims (1)

[Scope of Claims] (1) Microbial cells, enzymes insolubilized with tannins or polyfunctional crosslinking reagents, or enzymes adsorbed to insolubilized carriers, as they are, or in water, buffer, Or, if the suspension in a hydrophilic organic solvent is heated and dissolved in water, a buffer solution, or a hydrophilic organic solvent with carrageenan, agar, or gelatin as a gel base material, the gel base material does not solidify, and A manufacturing solution for immobilized microbial cells or enzymes, an aluminum chloride aqueous solution, characterized in that the solution is mixed at a temperature that does not kill or deactivate the microbial cells or enzymes and is sprayed into contact with a cooling liquid to form a microgel. At least one selected from animal and vegetable oils, mineral oils, synthetic oils, and unsaturated fatty acids; A method for producing immobilized microbial cells or enzymes as described in the section of the patent claim 0 (3) ) o, s~t, o oKy/clt *
At a spray pressure of G. The method for producing immobilized microbial cells or enzymes according to claim 1, which comprises pressurized spraying. (4) The method for producing immobilized microbial cells or enzymes according to claim 1, wherein the gel average particle diameter of the immobilized microbial cells or enzymes is not more than too microns. (5) Method 0 for producing immobilized microbial cells or enzymes according to claim 1, wherein the polyfunctional crosslinking reagent is glutaraldehyde.