JPS59118087A - Immobilized microbial cell capable of producing glucose- isomerase, its preparation, and isomerization of glucose - Google Patents

Abstract

PURPOSE:To provide the titled microbial cell having high ratio of developing enzymatic activity, by kneading cells of glucose-isomerase-producing microorganism with an aqueous solution of amynoacetalized PVA having specific molecular weight in the presence of a cobalt salt, and crosslinking the kneaded product with a polyaldehyde. CONSTITUTION:Glucose-isomerase-producing microbial cell is dried, pulverized, and kneaded with an aqueous solution of an aminoacetalized PVA having a molecular weight of about 600-6,000 and containing a cobalt salt. The kneaded product is passed through a sieve, formed in the form of a rod, dried, granulated, and crosslinked with a polyaldehyde. The cobalt salt forms a complex in the presence of hydroxyl group and is immobilized together with the microbial cells. The enzymatic activity can be kept stably, and the product can be used continuously for a long period.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized cobalt ion-containing microbial cell that increases glucose isomerase activity, a method for producing the same, and a method for isomerizing glucose using the same.

Glucose isomerase is the common name for an enzyme that reversibly converts HD-glucose (simply referred to as glucose) and D-fructose (simply referred to as fructose) to each other,
Conventionally, it has been used mainly for the purpose of producing fructose from glucose. Industrially, this enzyme is used to isomerize glucose and produce a fructose-containing sugar solution.

Generally, when performing an enzymatic reaction using the present glucose isomerase, if the present enzyme is used in a state dissolved in water, after the reaction is completed, in order to obtain an isomerized sugar solution containing glucose and fructose, Even if this enzyme is still active, even if the enzyme is denatured and removed, -
If the reaction mixture was not discarded after each reaction, 1-1 did not result. In order to avoid such uneconomical problems, many methods have been reported in which the enzyme is immobilized in advance to make it insoluble in water and used efficiently in the same way as a single-body catalyst. .

Since glucose isomerase is primarily an intracellular enzyme, it cannot be obtained from glucose isomerase-producing bacteria.In addition to methods for immobilizing the enzyme, many methods for immobilizing its producing bacteria have also been reported. Recently, it has been discovered that when used in the form of bacterial cells, there is no need for the step of separating glucose isomerase from the bacterial cells and producing N.
It is said that the use of immobilized bacterial cells is industrially more advantageous than the use of immobilized enzymes because the enzyme activity is less unstable than that of purified enzymes. Therefore, several companies have commercialized immobilized bacteria for glucose isomerization, but when used continuously for a long period of time,
There were problems with enzyme activity stability, enzyme activity expression, and hardness that could withstand long-term use.
It is known that the presence of an activator is necessary, and ume enhances activity and CO greatly affects stability, but in the glucose isomerization method using ordinary immobilized enzymes or immobilized bacteria, It was necessary to include these metal activators in a glucose solution for isomerization, and then to subject the obtained isomerization pre-liquid to deionization treatment using an ion exchanger. In particular, Co2 had to be strictly removed because it was a heavy metal ion.

In order to solve the above-mentioned problems in the use of metal activators, a method of simultaneously immobilizing bacterial cells and Mg (Japanese Patent Laid-Open No. 52
-120187, JP 52125690), simultaneous treatment of bacterial cells with poorly soluble Mg compounds and poorly soluble Co compounds (
A method of immobilization (Japanese Unexamined Patent Publication Nos. 54-44089) has been proposed, but it has not been put to practical use.

Therefore, the present inventors conducted various studies in order to obtain immobilized bacterial cells that have a high enzyme activity expression rate, stable enzyme activity, and can withstand long-term continuous use. The mixture is crushed and kneaded with an aqueous solution of low molecular weight aminoacetalized polyvinyl alcohol containing a cobalt salt. It was found that when treated, cobalt ions form a complex due to the presence of hydroxyl groups in polyvinyl alcohol that are not converted to aminoacetylation, and are immobilized together with the bacterial cells, resulting in the desired immobilized bacterial cells.

The present invention has been completed based on the above considerations,
A cobalt ion-containing immobilized box having polyaldehydonalglucose isomerase activity, which is obtained by kneading glucose isomerase-producing bacterial cells with an aqueous solution of aminoacetalized polyvinyl alcohol having a molecular weight of about 600 to 6,000 in the presence of a cobalt salt, and a method for producing the same. In addition, when producing an isomerized sugar solution containing glucose and fructose by isomerizing glucose by the action of glucose isomerase in a glucose-containing solution,
(This is a glucose isomerization method in which glucose is isomerized into fructose by bringing a glucose-containing solution into contact with immobilized bacterial cells containing ruto ions, and the first step is glucose isomerase activity. Raw 75: Stable, with excellent enzyme activity expression rate, suitable for long-term continuous use.
An object of the present invention is to provide a cobalt ion-containing immobilized box that can be obtained, a method for producing the same, and a method for producing glucose using the same.

Glucose isomerase producing bacteria I* u used in the present invention
Known glucose isomerase-producing bacteria, such as Streptomyces, ] (Tilus, Lactobacillus,
Aerono (genus Futa, earth mouth/<ri l-tg,
This is a bacterial cell obtained from a culture of glucose isomerase-producing bacteria belonging to the genus Brevibacterium, Pseudomonas genus, Actinoplanes genus, etc.

The preferred 91J of the glucose isomerase-producing bacteria is Streptomyces alius ATCC2113.
2. Streptomyces orinoxaeus NRRL358
3. Streptomyces novochromogenes ATCC
2 1 1 1 4, Streptomyces pheochromogenes NRRL 3 5 5 9, Streptomyces
Phaeochromogenes FFJRM-P221, Bacillus
Coagulans NRRL5 6 56, Bacillus licheniformis ATCC31604, Lactobacillus plebis IF"03960, etc.75=The above-mentioned bacterial cells include all of the above-mentioned glucose isomerase-producing bacteria in culture obtained by culturing them by known culture methods. * to 1
Examples include live microbial cells collected by centrifugation using 9 over-shafts, dried microbial cells obtained by heat-drying or freeze-drying the viable microbial cells, and dried crushed microbial cells obtained by pulverizing the dried microbial cells. .

In the present invention, first, 1 JBe of bacterial cells are mixed with aminoacecooled polyvinyl alcohol having a molecular weight of about 600 to 6,000 in the presence of Konolt's salt.

Cobalt salts are salts that form CO′+ in aqueous solution;
For example, it is used in the form of an aqueous solution such as COCl2.

This aqueous solution may be mixed with the aminoacetal IJ vinyl alcohol aqueous solution to attack the bacterial cells.
Alternatively, they may be added separately to the bacterial cells. The amount of conoclusite salt to be used may be determined by determining the amount of its aqueous solution so that it is in the range of 0.01 to 2 millimoles per gram of dry weight of the bacterial cells.

The aminoacetalized polyvinyl alcohol is an aminoacetalized polyvinyl alcohol having a molecular weight of about 500 to 5,000.

A polyvinyl alcohol with a polymerization degree of about 12 to 120 and a low ff that is easily soluble in water.
Polyvinyl alcohol of degree i Acyclic aldehydes having acyclic aminoaldehyde groups, such as aminoacetaldehyde, 2-aminopropionaldehyde, 3-aminopropionaldehyde, 2-aminobutyraldehyde, 4-aminobutyraldehyde, 5- Examples include aminovaleraldehyde, and aminoacetaldehyde is a preferred example. Examples of the acetal include di-lower alkyl acetals of the above-mentioned aminoaldehydes, such as dimethyl acetal and diethyl acetal. These aldehydes or acetals may be reacted in a proportion of about 2 to 20 moles with respect to polyvinyl alcohol. In this case, it is advantageous to use a strong acid such as sulfuric acid or hydrochloric acid as catalyst 17. Since this aminoacetalization reaction proceeds almost quantitatively, aminoacetal groups are introduced at a ratio of about 2 to 20 mol.

The bacterial cells are kneaded with the aminoacetalized polyvinyl alcohol aqueous solution under conditions of pH approximately 7 to 13.

The amount of aminoacetalized polyvinyl alcohol used is preferably in the range of about 2 to 200 Ing per gram of dry weight of the bacterial cells. The concentration of the aminoacetalized polybonyl alcohol aqueous solution is usually 2 to 20%. The amount of this aqueous solution, including the amount of the cobalt aqueous solution and the alkaline aqueous solution or buffer solution for pH adjustment, should be determined within the range that allows the microbial cells to be kneaded to a hardness that can be molded into rods or granules. be.

The thus obtained kneaded material is molded into rods or granules using an extrusion granulator, and the rods are dried and crushed or passed through a sizing machine to form them into granules. be able to. The thus obtained molded product is left in a wet state or dried, and then crosslinked with polyaldehyde to immobilize the bacterial cells together with cobalt ions.

Examples of the polyaldehyde include dialdehydes such as malonaldehyde, succinaldehyde, glutaraldehyde, and adipine aldehyde, with glutaraldehyde being the most preferred example. The amount of glutaraldehyde used is approximately 0.3 to 10% based on the dry polymerization of the kneaded product.
It is preferable to carry out the crosslinking treatment at a ratio of . This crosslinking treatment is
The process proceeds simply by holding the molded product in a water solution of dialdehyde. After the crosslinking treatment, excess polyaldehyde may be removed by washing with water.

The immobilized bacterial cells obtained in this way not only have an excellent expression rate of glucose isomerase activity, but also can be easily crosslinked due to the excellent reactivity of the primary amine group of aminoacetalized polyvinyl alcohol and polyaldehyde. It has excellent hardness and can withstand continuous use. Moreover, cobalt ions are present in the form of a complex with aminoacetalized polyvinyl alcohol due to the presence of residual hydroxyl groups that cannot be converted into aminoacetalized polyvinyl alcohol. be done.

To isomerize glucose using upturned immobilized bacterial cells 1
4. The immobilized bacterial cells may be packed into a jacketed column in which a water flow is circulated, and the glucose-containing liquid may be passed through and continuously circulated. This glucose-containing 7-terminal solution is prepared by dissolving glucose up to a maximum concentration of 60% in an aqueous solution of a magnesium salt such as magnesium sulfate, magnesium chloride, magnesium phosphate, etc. having a concentration of about 1 to 5mM. pH1 e.g. pf (8~
9, preferably 8.5, may be adjusted with an alkali such as an aqueous sodium hydroxide solution. The isomerization reaction is usually 40
Since the reaction is carried out at a temperature in the range of -65°C, the flow rate of the Nigelcose-containing liquid is preferably maintained so that the inside of the jacket is maintained at the above temperature so as to maintain a constant isomerization rate during the operation. For good operation, this isomerization rate is generally between 40 and 50%. Therefore, it is necessary to gradually reduce the flow rate as the enzyme activity of the immobilized bacterial cells decreases.

Next, the present invention will be specifically explained with reference to Examples and Reference Examples, but the present invention is not limited thereto.

In addition, in the Examples, the Guryuu-suimerase activity is Ayr,
Biol, Chem, 30 (12), 1247
~1253 (1966), measured based on the literature, 70℃
Under the conditions of action for 60 minutes, the glucose of 1 to 7 lac 1.
- the necessary glucose isomerase activity to convert to
The unit is (GIU).

Determination of fructose Shima J, Biol, Chem.
1. Determined by the cysteine-carbazole method described in 192 +583 (1951). Ta.

Example 1 Preparation of crushed and dried bacterial cells of glucose isomerase producing bacteria
247 ml of culture solution 1801 (x+40aGIU/g dry cup body) obtained by culturing according to the method described in 253 (1966) was added 5771ml of COCl2.6H20. After dissolving the mixture to a concentration of lVI and heating at 70°C for 1 hour, the bacterial cells were collected by centrifugation. The bacterial cells were vacuum dried at 50°C for 70 hours (drying in one step (7714G IU)
lg) was passed. This is done using a Contrabrex crusher (
Pulverized dry bacterial cells (6904GI)
610 g of U/&) was obtained.

Example 2 Preparation of immobilized bacterial cells Polyvinyl alcohol [molecular weight: approx. 12
00 (degree of polymerization 27), viscosity 19.9CP (21°C)]
5 ml of a 2.96% aqueous solution of 5M CoCl2*6H20 adjusted to pH 9.0 with 5N-NaOH aqueous solution
3 ml of aqueous solution was added and kneaded at room temperature. This kneaded product was extruded through a 20-mesh sieve, and the resulting rod-shaped molded product was loosened and vacuum-dried at 35° C. for 20 hours. This dried product was extruded through a 20 mesh sieve to form granules (5667G
IU/g, activity yield 82.1%)9. g5+ was obtained.

This molded product 9.0. ! 9 to 25 tiglutaraldehyde aqueous solution 0. (0.1M borate buffer (pH 8,4) containing
)225 tnl was added and left at room temperature for 1 hour. The treated bacterial cells were collected by filtration, washed with 51 parts of water, and vacuum dried at 35° C. for 20 hours to obtain 7.929 immobilized bacterial cells.

342.2 GIU/& (activity yield 49.6 GI) Example
3 Preparation example 2 of immobilized bacterial cells t, -+te, O-5R/ICoC12/6
Instead of adjusting the pH of the H20 aqueous solution to 9.0, adjust it to 12.0.
A granular molded product (5808 GIU/g, activity yield 84.1%) was adjusted to IO,02,! ;1 was obtained.

This molding*9.0 & 25% glutaraldehyde aqueous solution Q, 0.1 M* Utl [[Liquid 2] containing 9 inl.
251 nl was added and left at room temperature for 1 hour. The treated bacterial cells were washed with 1 1 L-1 water 51, and then vacuum dried at 35°C for 20 hours to obtain immobilized bacterial cells 7.87.9. 3803
GIU, # (activity yield 55.1%).

Example 4 Preparation of immobilized bacterial cells In Example 2, 8.0 ml of the 25 tiglutaraldehyde aqueous solution was used instead of 9 ml to prepare 8.0 ml of the immobilized bacterial cells.
Obtained 28 jq. 1760 GIU/F (activity yield 25
．． 5%).

Example 5 Preparation of immobilization box 9.0 g of the granular molded product obtained in Example 3 was mixed with 225 mA of IM borate buffer containing 25 g of tiglutaraldehyde aqueous solution and 0+m! was added and left at room temperature for 1 hour.

Filter the treated bacterial cells and add water 5! ! After washing with
After vacuum drying for 0 hours, 8.19 g of immobilized bacterial cells were obtained. 1
6I2GIU/,! li+ (activity yield 23.3H) Example 6 Preparation of immobilized bacterial cells In Example 2, IMCoCl was used instead of the 0.5 McoC12.6H20 aqueous solution. - Immobilized bacterial cells 7.87.9 were obtained using 6H20 aqueous solution. 33I1GIU/g (activity yield: 48.0).

Example 7 Preparation of immobilized bacterial cells In Example 2, 1.48% aqueous solution of aminoacetalized polyvinyl alcohol was used instead of 2.96% aqueous solution.
A granular molded product was obtained using the aqueous solution. 5008GIU,
# (activity yield 7265 yen).

This molded product was crosslinked with glutaraldehyde in the same manner as described in Example 2 to obtain immobilized bacterial cells.

4197 GIu/g (activity yield 60.8%) Example 8 Preparation of immobilized bacterial cells In ν1] 2, instead of using 5 ml of a 2.96% aqueous solution of aminoacetalized polyvinyl alcohol, 3 ml was used. Used, 0.5Mcoc1□・6H
The granules were extruded using 5 ml of 2o aqueous solution. 5
388GIU/? (Activity yield 78.0%).

To 9.0 g of this molded product, 1. (3 ml of 0.1 M poric acid solution (p) i8
．． 4) 225 rnl was added and left at room temperature for 1 hour.

One sample of treated bacterial cells was purchased, washed with 51 parts of water, and vacuum dried at 35° C. for 20 hours to obtain immobilized bacterial cells. 3814GI
U/, 9 (activity yield 55.2%).

Reference ρli 1. In Example 2, 6 ml of water was used instead of 5 ml of the aqueous solution of aminoacetalized polyvinyl alcohol and 0.5M CoCl2. - Using 3 tnl of water instead of 6H2o aqueous solution 31810, immobilized bacterial cells containing no aminoacetalized polyvinyl alcohol and cobalt ions 7.58,? I passed. a6s6GIu/, 9 (activity yield 53.0%) [Control sample I] Reference example 2 Preparation of immobilized solid not containing cobalt ions In Example 2, 0.5M CoCl2.6H20 aqueous solution 3r7+l was replaced with water. Using 3 ml, immobilized bacterial cells 7.84.9 containing no cobalt ions were obtained. 35
20 GIU/, F (Activity yield 51.0%) [Control sample If] Practical example 9 Glucose isomerization actual sister - Immobilized bacterial cells obtained in 1/15 2.481 & (4
000G I U) in 0.1M boric acid relaxation solution (pH 8,
4) using a jacketed column (12X 96 mm
), and the inside of the jacket was heated at a constant temperature of 60°C to 5N-N.
5 mIVIM adjusted to pH 8.5 with aUH aqueous solution
y SO4・7H20 40 ligose solution 11
．． It was run continuously at a flow rate of 417 IIV hours. When the total amount of glucose treated until the isomerization rate of the reaction solution decreased to 404, it was 2720.

In contrast, the immobilized bacterial cells 1.094. ! 9 (4000GI
U,/,9) and immobilized bacterial cells obtained in Reference Example 2 1.13
The total amount of glucose required to reduce the isomerization rate to 40% using 6 g (4000 G I U/jF) was determined to be 720 g and 640 g, respectively.

From this, it was found that the immobilized bacterial cells of the present invention had about 4 times the ability to isomerize glucose than the immobilized cells of the present invention.

Example Glucose Isomerization In Example 9, the immobilized bacterial cells obtained in Example 5 were
Immobilized bacterial cells 2.273.9 obtained in Example 4 instead of 19
(4000 GIU/9) was used to continuously perform glucose isomerization. The total amount of glucose treated until the glucose isomerization rate decreased to 40 g was determined to be 1720 g. From this, it can be seen that the immobilized bacterial cell lr:i of the present invention retains approximately 2.5 times the ability to isomerize glucose than when the immobilized bacterial cells obtained in Reference Examples 1 and 2 are used as a control. I found out that there is.

Example 11 Glucose isomerization and stability of immobilized bacterial cells 1.169 g of immobilized bacterial cells obtained in Example 2 (4000G
IU/l, immobilized bacterial cells obtained in Example 4 2.273.9 (
4000GIU/g), the immobilized bacterial cells obtained in Example 5 2.
481 g (4000GI uig) and Example 6
Immobilized bacterial cells 1.209.9 (4000G IT
J/p) was packed into a jacketed column (12X 96 mm) using 0.1 M borate buffer (pH 8,4), and the inside of the jacket was heated at a constant temperature of 60°C with 5 N -Na0
5mMWLg' adjusted to pH 8.5 with 11 aqueous solution
11. 40% glucose solution containing So, 7 H20.
625 at a flow rate of 41 mV. A time (total amount of treated glucose 2854.4.l was flowed, after which the residual glucose isomerase activity of each immobilized bacterial cell was measured. As a control, the immobilized cells obtained in Reference Example 1 1.094!9
The results of comparison using 0G I U/'g) were as follows.

Residual activity: Immobilized bacterial cells obtained in Example 2 28.0%
〃 4 // 45.
1 chi/75 u 46
．． 3%/76/7 3
7.5chi reference example 1 ″ 1
7.8% From this, it can be seen that the fixed bacterial cells of the present invention are the same as those of the control amyl acetalized hollyvinyl alcohol (immobilized without ion) (continuously used once from the U bacterial cells).
&d Noglucose isomerase residual activity = 1 It was found that it remained high for a long time.

4 temples d Hoshide j petitioner Toyo Jozo Co., Ltd. Representative: Ito Fujiuma

Claims (1)

[Scope of Claims] (1) Glucose produced by cross-linking with polyaldehyde a mixture obtained by kneading glucose isomerase-producing bacterial cells with an aqueous solution of aminoacetalized polyvinyl alcohol having a molecular weight of approximately 600 to 6,000 in the presence of a cobalt salt. A cobalt ion-containing immobilized bacterial cell with isomerase activity. 2) Glucose isomerase-producing microorganisms are obtained from a culture of glucose isomerase-producing microorganisms belonging to the genus Streptomyces, Bacillus, Lactobacillus, Aerobacter, Arthrobacterium, Brevibacterium, Pseudomonas or Actinoplanes. 42. The immobilized box according to claim 41, which is a bacterial cell. 3) A patent claim in which the glucose isomerase-producing bacteria is Streptomyces alpus, Streptomyces olivaceus, Streptomyces olibochromogenes, Streptomyces pheochromogenes, Bacillus coagulans, Bacillus licheniformis, or Lactobacillus plebis. The immobilized bacterial cells according to item 2. 4) The immobilized microbial cells according to claim 2, wherein the microbial cells are live microbial cells, dried microbial cells, or dried and crushed microbial cells. 5) Add cobalt salt to IJ 0.0 per 1 g of dry weight of the bacterial cells.
The immobilized bacterial cells according to claim 1, which are used in an amount of 1 to 2 mmol. 6) The immobilized bacterial cells according to claim 1, wherein the aminoacetalized polyvinyl alcohol aqueous solution has a concentration in the range of about 2 to 20%. 7) The immobilized bacterial cell according to claim 6, wherein the aminoacetalized polyvinyl alcohol is polyvinyl alcohol aminoacetalized with an acyclic aminoaldehyde or its acetal. 8), the non-fJ aminoaldehyde is aminoacetaldehyde, 2-aminopropionaldehyde, 3-aminopropionaldehyde, 2-aminobutyraldehyde, 4
8. The immobilized bacterial cell according to claim 7, wherein the -aminobutyraldehyde solution is 5-amino-ceraldehyde. 9) The immobilized bacterial cell according to claim 7, wherein the acetal is an alkyl acetal of the aminoaldehyde. IO), the immobilized bacterial cells according to claim 7, wherein the aminoacetalized polyvinyl alcohol is used in an amount of about 2 to 200 m9 per 1 g of dry weight of the bacterial cells. 11), ml with about 2 to 20 molar aminoacetalization
The fixed F. fungal cells according to claim 7, which are 12) Polyvinyl alcohol has a molecular weight of about 500-50
8. The immobilized microbial cell according to claim 7, which is a water-soluble polyvinyl alcohol having a molecular weight of 0.00 and a hardness of 60% or more. 13) The immobilized bacterial cells according to claim 1, wherein the kneading is carried out under conditions of pH approximately 7 to 13. 14) The immobilized bacterial cells according to claim 1, wherein the kneaded product is a rod-shaped or granular molded product or a dried product thereof. 15) The immobilized bacterial cell according to claim 1, wherein the polyaldehyde is glutaraldehyde. 16), Claim 1 in which glutaraldehyde is used in a range of about 0.3 to 10% based on the dry weight of the kneaded product.
Immobilized bacterial cells according to item 5. 17) The immobilized box according to claim 1, which contains cobalt ions in a form that forms a complex with the aminoacetalized polyvinyl alcohol. 18) Glucose isomerase, which is characterized in that glucose isomerase-producing bacterial cells are kneaded with an aqueous solution of aminoacetalized polyvinyl alcohol having a molecular weight of about 500 to 5,000 in the presence of a cobalt salt, and the kneaded product is subjected to a crosslinking ratio treatment with polyaldehyde. A method for producing an active cobalt ion-containing immobilized bacterial cell. 19) Request for a claim for a glucose isomerase-producing bacterial cell belonging to the genus Streptomyces, Bacillus, Lactobacillus, Aerobacter, Arthronophyta, Brevibacterium, Pseudomonas, or Actinoplanes. 20), glucose isomerase activity 1fi 75 varnish) and 7') Mrs. alps, Streptomyces
The manufacturing method according to claim 19, wherein the first horn (Ceus, Streptomyces novochromogenes, Streptomyces pheochromochenes, Bacillus coagulans,) . 21), the bacterial cell is a living bacterial cell, a dry bacterial weight, or one dried powder frame; the method for producing jacillus according to claim 19; 22), Cobalt salt per tkl g of the dried bacterial cells
．． Use 1 to 2 mmol! Claim 18
Manufacturing method described in section. 23) The method of claim 18, wherein the aminoacetalized polyvinyl alcohol aqueous solution has a concentration in the range of about 2-20%. 24) A method for producing polyvinyl alcohol in which the aminoacetalized polyvinyl alcohol is aminoacetalized with an acyclic amino acid (i) dehyde or its acetal. 25), the acyclic aldehyde is aminoacetaldehyde 2
-aminopropionaldehyde, 3-aminopropionaldehyde, 2-aminobutyraldehyde-7minovaleraldehyde Claim 24
Manufacturing method described in section. 26) The production method according to claim 24, wherein the acetal is a di-lower alkyl acetal of the aminoaldehyde. 27), about 2 to 200 m9 +l of the aminoacetalized polyvinyl alcohol per dry weight II of the bacterial cells
The manufacturing method according to claim 24, which is used in iα range. 28) The production method according to claim 24, wherein the aminoacetalization is about 2 to 20 molar. 29), polyvinyl alcohol has a molecular weight of about 500-50
25. The production method according to claim 24, wherein the polyvinyl alcohol has a saponification degree of 60% or more. 30) The manufacturing method according to claim 18, wherein the kneading is carried out at a pH of about 7 to 13. 31) The manufacturing method according to claim 18, wherein the kneaded product is a rod-shaped or granular molded product or a dried product thereof. 32) The manufacturing method according to claim 18, wherein the polyaldehyde is glutaraldehyde. 33) The manufacturing method according to claim 114, wherein glutaraldehyde is used in an amount of about 3 to 10% based on the dry weight of the kneaded product. 34) The production method according to claim 18, wherein cobalt ions are contained in a form forming a complex with the aminoacetalized polyvinyl alcohol. 35) When producing a monoisomeric high-fructose solution containing glucose and fructose by isomerizing glucose in a glucose-containing solution by the action of glucose isomerase, glucose isomerase-producing bacterial cells are grown in the presence of cobalt salts. Glucose is obtained by contacting cobalt ion-containing immobilized microbial cells with glucose isomerase activity, which are obtained by crosslinking a mixture with an aqueous solution of aminoacetalized polyvinyl alcohol having a molecular weight of about 600 to 6,000, with polyaldehyde, to obtain glucose. A method of isomerizing glucose that is characterized by isomerizing it into fructose.