JPS5849234B2 - Immobilization method of glucose isomerase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immobilizing glucose isomerase useful for isomerizing glucose to fructose.

Fructose is an isomer of glucose, and is about twice as sweet as glucose, making it the sweetest substance among sugars.

Therefore, since it can provide sweetness while minimizing calorie intake, its use in low-calorie foods to prevent obesity or beauty foods has recently attracted attention.

In addition, sugar solution made by isomerizing glucose has a sweetness that is approximately 1.2 to 1.4 times sweeter than sucrose, so it has been rapidly used in fields such as soft drinks, canned goods, and frozen desserts due to its economic efficiency. There is one.

Traditionally, D-fructose has been produced by hydrolyzing sucrose to separate W-fructose, but in recent years, the method of isomerizing glucose by allowing glucose isomerase to act on glucose has been expanded on an industrial scale. waiting.

By the way, in order to produce isomerized liquid sugar from glucose using glucose isomerase, it generally takes about 40 to
Glucose isomerase-producing bacteria were introduced into a 50% glucose solution, and the isomerization reaction was allowed to occur for a long time at a concentration of about 70°C, followed by isomerization through various steps such as filtration, decolorization, and deionization. I am getting liquid sugar.

However, the above method has a major drawback in that after the isomerization reaction is completed, the enzyme activity decreases to about half of that before the reaction, and when a new isomerization reaction is performed, the lost amount of enzyme must be compensated for.

Therefore, it is strongly desired to immobilize enzymes to enable long-term use of enzymes and continuous enzymatic reactions.

Various methods have been known for immobilizing glucose isomerase, regardless of whether it is a free enzyme or an intracellular enzyme.

For free enzymes, for example, a covalent carrier bonding method using a porous glass aminosilane derivative as a carrier, an ionic carrier bonding method using an ion exchanger such as DEAE-cellulose or Duolite as a carrier, polyacrylamide gel, collagen, etc. Alternatively, there is a comprehensive method using a composite material such as cellulose triacetate.

These methods have not yielded a satisfactory immobilized enzyme for industrial use.

Also known is a method in which glucose isomerase is immobilized within the bacterial cells by heating the bacterial cells to a temperature of 55° C. or higher and 90° C. or lower.

Although this method significantly improved the decrease in enzyme activity compared to untreated bacterial cells, the residual activity value was only 40-50% of the initial value.
Therefore, it could not be said to be industrially sufficient.

It is particularly strongly desired that an immobilized enzyme agent for glucose isomerase that satisfies industrial applications has the following properties.

(i) Enzyme activity is sufficiently maintained for long-term use.

(i1) Do not elute impurities.

4ii) Do not make any mistakes when filling the column.

4v) Be physically robust.

(■) Easy to handle.

(Vl) The enzyme agent must be safe.

For this purpose, the present inventors conducted various studies on the immobilization of intracellular enzyme glucose isomerase, and as a result, chitosan was added to the bacterial cells at a relatively high concentration and mixed, and the mixture was applied to the bacterial surface. The present invention was accomplished by discovering that the desired objective could be achieved by forming a chitosan film and further treating it with polyaldehyde.

That is, the present invention is a method for immobilizing glucose isomerase, which is characterized by mixing glucose isomerase-producing bacteria and chitosan in an amount of 8% by weight or more based on the dry matter of the bacteria, and then treating the mixture with polyaldehyde.

Chitosan is a white amorphous powder obtained by deacetylating chitin, and is a basic polysaccharide consisting of glucosamine.

The chitosan in the present invention is the above-mentioned chitosan or a solution thereof, such as an acid such as acetic acid, formic acid, phosphoric acid, or hydrochloric acid, or a solution obtained by dissolving it in an acidic solution of these acids.

The concentration of this solution is 0.05 to 1.0% by weight, preferably 0.
.

It is 1 to 0.5% by weight.

The amount of chitosan or its solution mixed with bacterial cells is such that the amount of chitosan is 8% by weight or more, preferably 10.0 to 50.0% by weight, based on the dry bacterial cells.

If the amount of chitosan added is less than 8% by weight based on the bacterial cells, there is a drawback that the chitosan film is too weak and the stability of the immobilized enzyme becomes poor.

In other words, desorption of the enzyme from the bacterial body is a serious problem, and damage caused by mechanical impact is a serious problem.

Examples of glucose isomerase-producing bacterial cells include Streptomyces phaeochromogenes (Str5p
tomyces phaeochromgenes
), Streptomyces frasiae (S, fr
adiae), Streptomyces roseochromogenas (S. roseochromogenus)
s), Streptomyces olivaceus (S, o
l jvaceus), Streptomyces californicas, S. vanu-ceus
us), Streptomyces baagini 7 (S, vi
actinomycetes such as Pseudomonas hydrohira (rg; niae),
Pseudomonas bacteria such as -ph r la), Bacillus megaterium
A wide range of microorganisms can be mentioned, including bacteria of the genus Bacillus such as Bacillus terium, bacteria of the genus Brevibacterium, bacteria of the genus Lactobacillus, and bacteria of the genus Aerobacterium.

These sonotai are grown in a medium containing corn staple liquor alone or in combination with defatted soybeans as a nitrogen source, carbohydrates such as starch and xyrose as a carbon source, and potassium phosphate, cobalt chloride, and magnesium hydrochloride as inorganic salts. It is obtained by culturing.

In particular, the bacterial cells used in the present invention are obtained by culturing the above-mentioned bacterial cells,
It is preferable to use the bacterial cells isolated from the culture solution as they are or the bacterial cells that have been heat-treated.

Further, the bacterial cells may be dispersed or suspended in a buffer solution having a pH of 5 to 9, particularly 5 to 7.0.

The concentration of bacterial cells is usually 0.1 to 50% by weight, preferably 0.1 to 50% by weight.
It is 5 to 40% by weight.

The method of the present invention first involves using a glucose i'inomerase-producing bacterium and at least 8% by weight of the bacterium. Mix the chitosan above.

Specifically, methods for mixing bacterial cells and chitosan include a method in which chitosan or a solution thereof is added to a liquid containing bacterial cells and mixed, and a method in which bacterial cells are added to a chitosan solution and mixed.

From the viewpoint of operability in producing the immobilized enzyme, it is preferable to add the microbial cells to the chitosan solution and mix well with stirring or knead to disperse the microbial cells in the chitosan solution.

If necessary, bacterial cells are separated from the mixture obtained by the above method by operations such as filtration and centrifugation.

In the method of the present invention, the glucose isomerase producing bacteria and chitosan may be mixed in the presence of an inorganic acid or an organic acid or a salt thereof.

The inorganic acid, organic acid, or salt thereof mentioned here has a pH within a range of 1 to 1, which does not inactivate glucose imerase.
Those having a pH of 4.5 to 7. 0 and has a buffering effect.

Such inorganic acids include phosphoric acid, hydrochloric acid, and sulfuric acid, while organic acids include succinic acid, tartaric acid, citric acid, acetic acid,
These include lactic acid, fumaric acid, maleic acid, and malic acid.
Salts of these acids include ammonium, sodium, potassium, and magnesium salts.

The concentration of the inorganic acid or organic acid or its salt varies depending on its type, but is generally preferably a 1 to 10% by weight solution.

Further, in the method of the present invention, the above-mentioned inorganic acid or organic acid or a salt thereof may be mixed after mixing the glucose isomerase-producing bacteria and chitosan.

In the method of the present invention, bacterial cells having a chitosan film obtained by mixing with chitosan or its solution according to the above method are treated with polyaldehyde to cause a crosslinking reaction.

Examples of the polyaldehyde used in the present invention include cryoxal, malonaldehyde, succinic aldehyde, glutaraldehyde, adipaldehyde, pimeline dialdehyde, suberine dialdehyde, malealdehyde, and the like.

Glucuraldehyde is particularly preferred.

The amount of polyaldehyde used is about 0.1 to 20% by weight, especially 0.5 to 1% by weight, based on the dry weight of the bacterial cells having a chitosan membrane.
It is 0% by weight.

Polyaldehyde is a 0.01-5% solution from the viewpoint of operability.
It is particularly preferable to use it as a 0.05 to 2.0% solution.

The reaction between polyaldehyde 1.' and chitosan is preferably carried out at room temperature or under slightly elevated temperature conditions, and a reaction time of 10 to 60 minutes is usually sufficient unless carried out at extremely low temperatures.

The initial pH during the reaction is about 7.0, and the pH at the end of the reaction is about 6.5.

Therefore, the pH of the reaction is 6.0 from the viewpoint of enzyme stability.
Be careful not to do the following.

The bacterial cells treated with polyaldehyde by the above method are washed with water and dried if necessary.

The drying method may be any method such as freeze drying, vacuum drying, and sun drying.

In the bacterial cells obtained according to the method of the present invention, a chitosan membrane is formed in which a crosslinking reaction occurs with polyaldehyde, and glucose isomerase is firmly immobilized within the bacterial cells.

This immobilized enzyme was added to a glucose solution (glucose 50
w/v%, magnesium sulfate 0.005M, cobalt chloride 0.001M, phosphate buffer solution 0.05M) and repeating the isomerization reaction at 60°C with stirring, the isomerization rate decreased slowly. It is resistant to physical impact caused by stirring, and the immobilized enzyme maintains its shape almost unchanged from the initial stage of the reaction.

In addition, the immobilized enzyme obtained according to the present invention has low swelling property in water and exhibits extremely little deformation of laνK during the isomerization reaction. Therefore, the immobilized enzyme obtained according to the method of the present invention is suitable for industrially isomerizing glucose. It can be used effectively.

The present invention will be explained below using examples.

Note that glucose isomerase activity is determined by glucose solution (
Glucose concentration 0.1M, magnesium sulfate 0. OIM
, phosphate buffer 0.05 M, PH7. 2), 11n9 glucose is isomerized in 1 minute at a reaction temperature of 70°C, and the enzyme activity to produce fructose is defined as 1 unit.

Example 1 Streptomyces pheochromocennes (S-tre
ptomyces phaechromogenes)
(Feikoken Bibori No. 221) with corn staple liquor,
After aeration culture in a medium containing xylose, potassium phosphate, magnesium sulfate, and cobalt chloride, cells were obtained by centrifugation.

20 g of bacterial cells obtained in this way (solid content 1 8.9 7
%) (activity value 8960 units/L) was suspended in a small amount of water.
Heat treatment was performed at 0°C for 10 minutes.

Centrifuge again to collect the bacteria, and add the bacteria to water 10077
Suspend in 1 liter and add 1500 ml of 0.05% chitosan solution (
The amount of chitosan (19.8% based on the bacterial cells) was added with stirring, and then 14 liters of a 25% glutaraldehyde solution was added and reacted at room temperature for 1 hour.

After the reaction was completed, the bacterial cells were separated by centrifugation, washed twice with water, and dried under reduced pressure.

The dry weight of bacterial cells is 4.

1 g (activity value 1003 units/g).

(Residual activity rate 45.9%). The isomerization reaction of glucose was repeatedly performed using the dried bacterial cells obtained by the above method, and the isomerization rate was measured.

The results are shown in Table 1. Example 2 20 g of bacterial cells (solid content 18.9%) (activity value 8960 units/g) having the ability to produce glucose isomerase, which were cultured in the same manner as in Example 1, were suspended in a small amount of water and incubated at 80°C for 2 hours.
Heat treatment was performed for a minute.

Collect the bacterial cells by centrifugation, and add the bacterial cells to 100 yy of water.
tA', then 0.1% chitosan/acetic acid solution (
PH6.5) IA (amount of chitosan per bacterial cell 26.4%)
) was added to this suspension with stirring.

Centrifuge again to collect the bacterial cells, and add the bacterial cells to water i.o.o.
mA' and glutaraldehyde to a final concentration of 0.
The mixture was added to a concentration of 2%, stirred well, and left at 30°C for 1 hour.

Next, centrifugation is performed to collect the bacterial cells, which have a diameter of 2ill! After extrusion molding from a plate with W holes, vacuum drying is performed.

The dry enzyme preparation obtained was 4.1 g (activity f: 8.90 units/g).

(Residual activity rate 44.7%). The above enzyme agent 4.17g (total activity value 3700 units) with a diameter of 1. 4 cx, length 10
It was packed in a cIrL glass double cylinder, 70°C hot water was circulated through the outer shell, and a glucose solution (glucose 40i mass %, phosphate buffer, magnesium sulfate 0.05 M, cobalt chloride 0) was added from the bottom of the column. .0 0 1M.
pH 8.5) was flowed at a flow rate of 6 ml/hour.

The generated fructose was measured over time to determine the isomerization rate.

The results are shown in FIG.

[Brief explanation of drawings]

FIG. 1 shows the relationship between operating time and isomerization rate.

Claims (1)

[Claims] 1. A method for immobilizing glucose isomerase, which comprises mixing glucose isomerase-producing bacteria and chitosan in an amount of 8% by weight or more based on the dry matter of the bacteria, and then treating with polyaldehyde.