JPS62278984A - Co-immobilized enzyme and microbial cell - Google Patents

Abstract

PURPOSE:To obtain a co-immobilized enzyme and microbial cell capable of simplifying a production process of isomerized sugar, by bonding amylase to a microbial cell having glucose isomerase activity. CONSTITUTION:A microbial cell belonging to Streptomyces genus, Bacillus genus, etc., and having glucose isomerase activity is bonded with an amylase produced e.g. by Aspergillus sp. K-27 strain by the reaction e.g. at 4-9pH and 20-30 deg.C in the presence of a crosslinking agent such as glutaraldehyde and an enzyme precipitant such as tannin.

Description

[Detailed Description of the Invention] 3. Field of Industrial Use The present invention relates to a co-immobilized enzyme and bacterial cells that are useful for the production of high-fructose sugar.

(Prior Art) High fructose sugar (glucose-fructose mixture) obtained by isomerizing glucose obtained by decomposing starch is used as a sweetener for various foods including soft drinks. To obtain high fructose sugar, use 1 usually.

First, starch milk with a pl+ of 6.5 to 7 containing 30 to 40% starch is prepared three times, and Ca 2 * (calcium ions) and α-amylase (usually α-amylase derived from bacteria) are added to this. In addition, a primary liquefaction reaction is performed at 85 to 90°C. next.

After heating to 120-140'C to cleave the silicosoluble starch micelles, α-amylase is added again at 85°C to perform a secondary liquefaction reaction. The temperature of the starch liquefied product containing oligosaccharides as the main component obtained in this way is adjusted to 55 to 60°C, and
After adjusting H to 4.5 to 5.0, add glucoamylase derived from Aspergillus or Rhizopus to 48
A saccharification reaction is carried out for ~72 hours to decompose it into glucose. After the saccharification reaction is completed, it is purified using activated carbon or ion exchange resin, concentrated to a glucose concentration of 40 to 45%, and adjusted to a pH of 7.5 to 8.0. When this is continuously supplied to a column packed with immobilized glucose isomerase,
Glucose isomerized to produce fructose, resulting in a glucose-fructose mixture. When the solution flowing out of the column is purified using activated carbon or ion exchange resin and concentrated, high fructose corn syrup with a fructose content of 42 to 45% is obtained. In this way, for the production of isomerized sugar syrup.

Two steps are required: (1) hydrolysis of starch, and (2) isomerization of sugar, and starch hydrolysis in particular takes a long time. For example, to further decompose starch liquefied product into glucose, as described above.

It requires 48-72 hours. This manufacturing process also has the disadvantage that the enzyme cannot be used repeatedly.

(Problem that the invention attempts to solve) The present invention solves the above-mentioned conventional drawbacks.

The purpose is to provide an enzyme/bacteria co-immobilized product that can simplify the production process of isomerized IJg.

Another object of the present invention is to provide an enzyme/microbial cell co-immobilized product that allows repeated use of an amylolytic enzyme.

(Means for Solving the Problems) The enzyme/bacteria co-immobilized product of the present invention is formed by binding an amylolytic enzyme to microbial cells having glucose isomerase activity, thereby achieving the above object.

As the microbial cells used in the enzyme/bacteria co-immobilized product of the present invention, any microorganism having glucose isomerase activity can be used. Such microorganisms include actinobacteria! [1] Actinobacteria are particularly preferably used. Examples of actinomycetes include Streptomyces phaeochromogenes (Streptomyces pheochromogenes).
aeochromogenes), Streptomyces fradiae (S.
diae) + Streptomyces olivaceus (S
trep tomycesolivaceus)
, Streptomyces californica
tow ces californicus),
Streptomyces Kanamyceticus (Stre
tom ceskanam ceticus), Streptomyces baaginia (Streptom c
es f), Streptomyces alps (
Albus).

Streptomyces olibochromogenes, Streptomyces rubigenosus
There are Streptomyces bacteria such as Ces rubogenosus). Examples of bacteria include Bacillus coagulans,
Bacillus bacteria such as Bacillus megaterium: Pseudomonas hydrophilal
a) such as Pseudomonas genus; Arthrobacter genus; Lactobacillus plebis (Lactobacillus plebis);
Lactobacillus bacteria such as Actinoplanes missouriensis (usbrevis);
es m1s-souriensis). Two or more of these may be used simultaneously. Even if the microbial cells are live bacteria.

The bacterial cells may be heat-treated at about 60 to 80°C in advance.

Any enzyme that can hydrolyze starch can be used as the starch degrading enzyme, and its origin does not matter. For example, α
-Amylase, β-amylase.

Amylases such as glucoamylase; α-glucosidase; pullulanase; and isoamylase are used. Among amylases, glucoamylase is produced by Aspergillus and Rhizopus bacteria.

In particular, Aspergillus sp. K-27 strain (Danfuji [
Amylase produced by Udansp, L27) is preferably used. The mode of decomposition of starch by the amylolytic enzymes mentioned above is 1.
For example, α-1,4 bonds are cleaved.

α-1,6 bond is cleaved. There are various types, so select two or more types as necessary to ensure that starch is effectively decomposed into glucose. The amount of starch degrading enzyme used is not particularly limited.

A crosslinking agent and an enzyme precipitant (protein flocculant) are used to bind the above bacterial cells and the starch degrading enzyme. As the crosslinking agent, glutaraldehyde, diisocyanate, hexamethylenediamine, hexamethylenetetramine, etc. are used. In particular, glutaraldehyde is preferably used. Glutaraldehyde is 1 to 10-1% in one reaction solution.
It is added to give a concentration of . Tannin is preferably used as the enzyme precipitant (protein flocculant). Since tannins promote protein adsorption, bacterial cells and enzymes can bond more effectively. Tannin is 0.5 to 4 in the 2 reaction solutions.
It is added to a concentration of 0-1%.

To obtain the enzyme/bacteria co-immobilized product of the present invention, 1. For example, first, the above-mentioned microbial cells are partially dehydrated by freeze-drying, spray-drying, etc. as necessary, and then the amylolytic enzyme-containing product is dehydrated. Add to the aqueous solution. Add a crosslinking agent and an enzyme precipitant to this, if necessary.

Stir/shake. The pH of the reaction solution is 4-9. Preferably 4
．． 5-7.5. The reaction temperature is 20 to 30°C.

The reaction time is from several minutes to several days, preferably from 0.5 to 5 hours.

The obtained enzyme/bacteria co-immobilized product can be directly used for starch decomposition and
Although it can be used for isomerization, if necessary, it may also be used as an entrapping immobilization product supported on an inert carrier or surrounded by a polymer compound.

Porous glass, porous ceramic, ion exchange resin, etc. can be used as the inert carrier.

The co-immobilized product is supported on such an inert carrier by adsorption, chemical bonding, or the like. Polymer compounds used for the preparation of entrapping immobilization products include carrageenan, gelatin, and polyacrylamide.

Examples include alginic acid and polyvinyl alcohol.

The above-mentioned co-immobilized product is wrapped (enclosed) in these polymeric compounds, and then molded using an appropriate granulation means.

The polymer compound used in the entrapping immobilization product is used in the minimum amount necessary to entrap the enzyme/bacteria co-immobilized product.

As a result, the permeability of starch and dextrin, which are substrates for enzyme reactions, can be maintained, so there is no risk of reducing the activity of the 5 starch-degrading enzymes. Furthermore, if necessary, a crosslinking agent or a protein flocculant is used to improve the physical strength of the entrapping immobilization product. As the cross-linking agent and protein flocculant, the same compounds 1 as those used in the preparation of the enzyme/bacteria co-immobilized product, such as glutaraldehyde, hexamethylene diamine, and tannin, are preferably used.

To produce isomerized sugar syrup from starch using the obtained enzyme/bacteria co-immobilized product, 1. First, the co-immobilized product, the co-immobilized product carrier, or the entrapping immobilized product of the co-immobilized product is placed in a column. Fill. Separately, add 5-50% starch according to the usual method.
, preferably in a proportion of 30 to 40%.
- A starch liquefied product with a DE of 5 to 25 is prepared by the action of amylase or the like. This is purified by activated carbon or ion exchange chromatography as needed, and then supplied to the column.

pl+ during column supply is 5-8. Preferably it is 6 to 7, and the temperature is 25 to 70°C, preferably 45 to 60°C. Flow rate when performing continuous supply (space vel
ocity) is 0.05 to 4. Oh+”, preferably 0
．． 05 to 1.0 hr-'.

If continuous supply is carried out under such conditions, high fructose corn syrup with a fructose content of 42% or more can be obtained.

(Function) As described above, the enzyme/microbial cell co-immobilized product of the present invention is obtained by binding and integrating an amylolytic enzyme to a microorganism having glucose isomerase activity.

When this is packed into a column and the starch liquefied product is supplied, the starch saccharification process and the produced glucose isomerization process are performed in the same column, completing the isomerized sugar production in one step. Since the conventional two-step process of starch saccharification and isomerization can be combined into one process, large-scale equipment is not required. Moreover,
High fructose corn syrup can be produced continuously. Furthermore, since glucose is immediately isomerized and the glucose concentration in one reaction solution is kept relatively low, there are almost no transfer products due to the action of glucoamylase, and the yield of fructose increases.

Since the enzymes used, such as amylase, are immobilized, they can be used repeatedly.

Enzyme/bacteria co-immobilized products have the advantage of high enzyme activity per i-position, and can be used as is, but enzyme/bacteria co-immobilized products may also be used as carriers or enzyme/bacteria co-immobilized products. The co-immobilized product is more convenient to handle than the above-mentioned enzyme/bacteria co-immobilized product. It can be used in place of immobilized materials.

(Example) The invention will be explained below with reference to examples.

Daiarashi dish (A) Enzyme/bacteria co-immobilized product (comprehensive immobilization'!!!IJ
) preparation of 0.2 g of Nigercose isomerase bacterial cell preparation (manufactured by Nagase Seikagaku Kogyo Co., Ltd.; freeze-dried preparation of Streptomyces sp.) and -27 (manufactured by Daikin Industries, Ltd.; production sample of Aspergillus sp. K-27) Amylase; 20.0OOAON/g) 0.5g was mixed. Add to this 1.5% glutaraldehyde and 20%
% tannin and O, IM phosphate buffer (
pH7,2) 3 n+j! 30 at 30°C.
Allowed to react for minutes. This was made into a uniform suspension using a glass homogenizer and filtered through a 0.45 μm filter.

It was washed with 0.2M phosphate buffer (ρ116.5) to obtain an enzyme/bacteria co-immobilized product.

3.5% ginan aqueous solution heated to 50°C
The above enzyme/bacteria co-immobilized product was added to the solution and thoroughly mixed. While stirring corn oil heated to 50'C, the above aqueous solution was added dropwise to form fine beads. After cooling this corn oil with ice water and adding 0.3M MCI, it was further left in an ice water bath for 3 to 4 hours. Next, remove the corn oil and remove the 0. Group phosphate buffer (p) 17.0) and 0. Hexamethylenediamine was added to the solution to give a concentration of 0.2F and 90mM, respectively, and the mixture was gently stirred at 5'C for 10 minutes. Add 25% glutaraldehyde to this
Add so that it is 0mFI.

Stir gently for 30 minutes at 5°C. Collect the hardened beads in this way with a glass filter and collect 0.3MK.
0.11 phosphate buffer containing Cl (pl+ 6.5)
The mixture was immediately washed with water to obtain an entrapping immobilized product. The glucoamylase activity and glucose isomerase activity of this entrapping immobilized product were measured by the following method, and it was found that 421.
J/g (dry entrapping immobilized product) and 280/g (dry entrapping immobilized product).

Glucoamylase activity measurement method: 25mM maltose)
Encapsulated immobilized material in 1 mft of solution (pH 6,5) =
, s>vso.

Add μl and shake at 40°C for 30 minutes. This reaction? F!
Collect 1.50μ2 and make 200μ of water! and heat in a boiling water bath for 30 seconds. The amount of glucose was determined by the glucose oxidase method, and 1M per minute was determined under the above reaction conditions.
Let the activity of the entrapping immobilizate to decompose a mole of maltose be luni-nod (tl).

Glucose isomerase activity measurement method: Q, 0.2 containing 2M glucose and 0.01M magnesium sulfate
Add 500 μl of the entrapping immobilization suspension to 500 μl of 4-phosphate buffer (Cp86.5), and react at 70°C for 60 minutes. 50 μl of this reaction solution was collected, and 50 μg of 0
．． 51 Add to perchloric acid solution. Add distilled water for 2 me
500 μp of which is used as the C test solution, and fructose is quantified by the cysteine-carbazole method. (2) Unit/t (U) refers to the activity of an entrapping immobilized product that produces 1M mol of fructose per minute under the above reaction conditions.

(B) Preparation of isomerized sugar syrup = The entrapping immobilized product obtained in section (8) was packed into a 1.5 cmφ x 13 cm0 column,
The temperature was kept at 5°C. Separately, 5 waxy corn flour: F,
The EJ liquid was liquefied using α-amylase according to a conventional method.
A phosphoric acid buffer solution (pH 6.5) and magnesium sulfate were added to this at O, IM and 0.0 M, respectively. Added so that it becomes OIM. This starch liquefied product (30%) was continuously supplied to the column at a flow rate of 0.1 hr-'. Analysis of the column effluent by high performance liquid chromatography (IIl'LC) revealed isomerized sugar syrup containing about 45% fructose. IIPLC is Toyo Soda Kogyo Co., Ltd. l 1lL
c803D, and the column used was TSKgel NI+2-60 manufactured by Toyo Soda Kogyo Co., Ltd. (particle size 5 pm;
4.6φ×250+nm).

The solvent used was 80% acetonitrile, and the flow rate was 0.8 m.
The measurement was carried out at room temperature as 1/+min. A differential refractometer was used for detection.

Grand Duke ■1 (A) Preparation of enzyme/bacteria co-immobilized product (comprehensive immobilized product):
Example 1 (8) except that 0.5 g of glucozyme (manufactured by Nagase Seikagaku Kogyo Co., Ltd.; glucoamylase agent produced by Rhizopus flexure; 20,000 AIIN/g) was used in place of -27 for dabiase. Same as section.

The glucoamylase activity of the obtained entrapping immobilization was 3 fl
U/g (dry entrapping immobilized product) and glucose isomerase activity was 270/g (dry entrapping immobilized product).

(B) Two 3gI products of isomerized high-fructose sugar When the entrapping immobilized product obtained in Example (A) was used to prepare isomerized high-fructose sugar in the same manner as in Example 1 (B), approximately 42% The production of isomerized sugar containing fructose was carried out by lif LU.

Disaster boil ■ Yu (^) Preparation of immobilized material for enzymes and bacterial cells (comprehensive immobilization ￥! IJ): XL128-C (instead of -27) for dabiase
Manufactured by Nagase Seikagaku Kogyo Co., Ltd.: Glucoamylase produced by Aspergillus bacteria; 3,000 FLS U/g)
The same as in Example 1 (A) except that 0.9 g was used.

The glucoamylase activity of the obtained entrapped immobilized product was 39υ
/g (dry entrapping immobilized product), and the glucose isomerase activity was 30 [J/g (dry entrapping immobilized product).

(B) Preparation of isomerized high-fructose sugar (2) Using the entrapping immobilized product obtained in this example (^), isomerized high-fruct sugar was prepared in the same manner as in (B) of Example 1. The production of isomerized sugar containing fructose was confirmed.

Isamu Kato ↓ (A) Preparation of immobilized material for enzymes and bacterial cells (comprehensive immobilized material):
3j4 entrapping immobilization products were prepared using 4λ as described in Example 1 (A). However, 4.5 g of glucose isomerase cells were further added to the enzyme/bacterium immobilized material so that they were simultaneously encapsulated with carrageenan.

The glucoamylase activity of the obtained entrapped immobilized product was 5.9
0/g (dry entrapping immobilized product), and glucose isomerase activity was 761/g (dry entrapping immobilized product).

(B) Preparation of isomerized sugar syrup: Liquefied waxy corn starch (30%) obtained in the same manner as in Example 1 (B) 2
ml of the entrapping immobilized product obtained in Section (8) of this Example
(2) was added and reacted at 55°C with stirring. After 48 hours, the presence of isomerized high-fructose sugar containing about 45% fructose was confirmed in the reaction solution, and after 72 hours, the presence of isomerized high-fructose sugar containing about 48% fructose was confirmed.

(Effect of the invention) The enzyme-immobilized product for bacterial cells of the present invention is thus prepared.

It is obtained by binding a starch degrading enzyme to microbial cells having glucose isomerase activity. When this is used to produce isomerized sugar, the starch saccharification step and the isomerization step can be performed in one step. Therefore, high-fructose sugar can be produced continuously without requiring large-scale equipment.

Furthermore, since the glucose concentration is kept low by sequential isomerization, there is no formation of oligosaccharides caused by the reverse reaction of glucoamylase, and a product with a high isomerization rate and high purity can be obtained. Furthermore, since the enzyme activity per unit weight of the enzyme/bacteria immobilized product is high, high fructose sugar is effectively produced. The starch-degrading enzyme used to break down starch is immobilized and can be used repeatedly. Therefore, by using the enzyme-immobilized product for bacterial cells of the present invention, high-fructose corn syrup can be produced in a short time and at low cost.

that's all

Claims (1)

[Scope of Claims] 1. An enzyme/bacteria co-immobilized product in which a starch degrading enzyme is bound to microbial cells having glucose isomerase activity. 2. The starch degrading enzyme is Aspergillus (￥Asper
2. The co-immobilized product according to claim 1, which is an enzyme derived from a bacterium of the genus P. gillus. 3. The starch degrading enzyme is Aspergillus sp. K
-27(￥Aspergillus￥ sp.K-27
2. The co-immobilized product according to claim 1 or 2, which is an enzyme derived from ). 4. The starch degrading enzyme is Rhizopus (Rhizopus).
¥) The co-immobilized product according to claim 1, which is an enzyme derived from a fungus of the genus. 5. The microorganism is Streptomyces (\Strept
The co-immobilized product according to claim 1, which is a bacterium belonging to the genus O-myces. 6. The co-immobilized product according to claim 1, wherein the microorganism is of the genus Bacillus.