JPH0327198B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an improvement in a method for immobilizing enzymes or microbial cells as a carrier, and is applicable to foods, medicines,
It can be used in a wide range of fields such as brewing, agriculture, chemical industry, environmental purification, and energy industry.

[Conventional technology and problems to be solved]

Recently, methods for immobilizing enzymes and microbial cells are
Rapid progress has been made, and various techniques have been proposed, including adsorption methods, covalent bonding methods, inclusion methods, and crosslinking methods.

Therefore, if we look at the inclusion method as a typical example of the above immobilization method, when enzymes and bacterial cells are included in polysaccharide gels such as polyacrylamide gels, κ-carrageenan, alginate, agar, etc., there is a correlation with the size of the gel lattice. Even if enzymes and microorganisms are immobilized, if the gel does not have sufficient physical strength or has a large degree of swelling, the enzyme will elute during the enzyme reaction, resulting in a decrease in enzyme retention activity. Conversely, if the gel strength is weak, it may not be able to withstand large-scale use, or the immobilized enzyme or immobilized microorganisms may swell during continuous reactions, causing clogging. Even if it can withstand physical strength, it has the drawbacks of low enzyme retention activity and extremely low productivity, and is still not sufficient as an industrially usable immobilized enzyme or immobilized microorganism. Can not.

The purpose of the present invention is to solve the above-mentioned drawbacks inherent in the conventional technology of comprehensive immobilization of enzymes and microorganisms, and to
Immobilized enzymes that can be mass-produced and are industrially usable.
The aim is to provide immobilized microorganisms.

[Means adopted to achieve the purpose]

That is, in the present invention, by reacting chitosan with an aliphatic aldehyde, a part of the amino group in chitosan and the aldehyde form a Schiff base, which is represented by the following general formula. (In the formula, R is a hydrogen atom and a group represented by -(CH ₂ ) _o CH ₃ , and n in the formula is an integer of 0 to 2.) Focusing on producing a chitosan-aldehyde gel, this Through trial and error experiments, we discovered that when chitosan-aldehyde gel, enzymes or microorganisms are added, and glutaraldehyde is added, they coagulate and efficiently form a hard, resin-like gel. This method is used to immobilize enzymes or microorganisms, and the immobilized enzymes and microorganisms obtained in this way form a resin-like hard gel product and do not gel like those produced by conventional methods. It has no drawbacks such as being unable to withstand large-scale use due to its low strength, or clogging due to swelling of the immobilized enzyme or immobilized bacterial cells during continuous reactions, and also has good thermal stability. Even when exposed to heat of 80 to 100°C, there was no change in the shape of the gel.

Next, the present inventors deacetylated chitin, which is an organic skeletal material found in crabs, shrimps, etc., which is abundantly available in nature, to obtain chitosan, and further treated this with aldehyde to create a chitosan-aldehyde gel. It was also confirmed that enzymes or microbial cells could be immobilized more economically by doing so.

Chitosan-aldehyde gels can also be obtained by treating chitosan with a suitable aliphatic aldehyde. Examples of such aliphatic aldehydes include formaldehyde, acetaldehyde, propylaldehyde, and n-butyraldehyde.

Next, as enzymes for immobilization in the present invention, α-amylase, β-amylase, glucoamylase, isoamylase, α-galactosidase, β-galactosidase, invertase, glucose isomerase, galactose isomerase, chymotrypsin, acid protease, neutral protease, alkaline protease, pepsin,
A wide variety of enzymes are available, such as papain, urokinase, cellulases, hemicellulases, lipases, aminoacylases, penicillin amidases, and the like.

Hereinafter, for convenience, the present invention will be explained in more detail using β-galactosidase as an example.

In the method of the present invention, chitosan-aldehyde gel is
The pH was adjusted to 5 to 6, β-galactosidase was added thereto, and 0.5 to 2% of the total amount of glutaraldehyde was added to obtain a resinous and hard gel-like immobilized enzyme product.

The gel-like immobilized enzyme product thus obtained is
Even after drying into a powdered product, the activity of the enzyme did not decrease at all.

In addition, when the immobilized enzyme product is packed in a column, it does not clog and is a hard resin-like gel, so the reaction rate is good and the solution of the substrate lactose can flow at high speed. The productivity of lactose hydrolysis is also extremely high, and is sufficiently satisfactory even when carried out on an industrial scale.

Moreover, since the enzyme is encapsulated on the surface or inside the gel, the amount of enzyme immobilized is larger than in the conventional enzyme immobilization method using aldehyde crosslinking of chitosan.

Furthermore, as shown in FIG. 1, no decrease in enzyme activity was observed even after 20 days of continuous use.

Next, as the microbial cells that can be adopted in the present invention, a wide range of microorganisms can be mentioned, such as bacteria, actinomycetes, molds, and yeasts.

Therefore, a supplementary explanation will be provided regarding the case where Streptomyces phaeochromogenes, which contains glucose isomerase, is used as a target example.

As mentioned above, chitosan-formaldehyde gel, similar to the case of enzymes, exhibits remarkable aggregation power against actinomycete cells due to entrapment and cross-linking. Then, after adjusting the pH of the chitosan-formaldehyde gel to 8, microbial cells are added, and then 0.5 to 2% of the total amount of glutaraldehyde is added to cause flocculation and precipitation, resulting in a resin-like product of immobilized microbial cells. Obtained.

The gel-like immobilized bacterial cell product thus obtained is
As in the case of the immobilized enzyme, no decrease in activity was observed as a dried powder product.

In addition, columns filled with immobilized bacterial cells are also available.
As with the case of immobilized enzymes, there is no clogging, and since it is a resin-like gel, the reaction rate is fast and the glucose solution can be flowed at high speed. Moreover, fructose productivity is improved by the isomerization reaction of glucose. is extremely high and can be satisfied on an industrial scale.

Moreover, since the enzyme is encapsulated on the surface or inside the gel, the amount of enzyme immobilized is larger than in the conventional enzyme immobilization method using aldehyde crosslinking of chitosan.

Furthermore, the optimal temperature for isomerization reaction is 80℃,
Even at this temperature, there was no change in the shape of the gel of the present invention. As shown in FIG. 1, no extreme decrease in bacterial isomerase activity was observed even after continuous use for 20 days or more.

The above-mentioned treatments are effective for both live and dead actinomycete cells, have a high retention of intracellular enzymes, and are hard resin-like gels that prevent clogging when packed in a column. As a result, a good bacterial cell-treated product was obtained. Furthermore, the method of the present invention is effective not only for actinomycetes but also for other microorganisms such as bacteria, molds, and yeasts.

Hereinafter, the effects of the present invention will be described with examples,
This will be explained more specifically.

In addition, β-galactosidase activity is determined by the international β-galactosidase activity.
Using a galactosidase unit, prepare a lactose solution (lactose concentration 180mM, sodium acetate buffer concentration 0.1M, PH
In 4.2), the amount of enzyme that hydrolyzes lactose into glucose and galactose in 1 minute at a reaction temperature of 40°C is defined as 1 unit. Further, the immobilization yield is expressed as the ratio of the enzyme activity (Unit) of immobilized β-galactosidase to the enzyme activity (Unit) of added free β-galactosidase.

{Immobilization yield} = [enzyme activity of immobilized β-galactosidase (Unitsun)] / [enzyme activity of added free β-galactosidase (Unitsun)] × 100 In addition, Streptomyces phaeochromogenes bacterial cells The glucose isomerase activity is determined by using the international glucose isomerase unit, and the enzyme activity to isomerize 1 μmol of glucose in a glucose solution (glucose concentration 0.8M, magnesium sulfate solution 0.01M, pH 7.5) at a reaction temperature of 60°C for 1 minute is 1. Unit. The immobilization yield is expressed as the ratio of the enzyme activity (Units) of the immobilized bacterial cells to the enzyme activity (Units) of the added free bacterial cells.

{Immobilization yield} = [Glucose isomerase activity (Units) of immobilized bacterial cells] / [Glucose isomerase activity (Units) of added free bacterial cells] × 100 Example 1 0.5 g of chitosan was added to 10% acetic acid (15 ml) dissolved in
Further methanol (20 ml) was added for dilution. This is combined with formaldehyde (25 glucosamine per residue)
molar equivalent) was added and left at room temperature overnight. The resulting hard gel was pulverized with a mixer, thoroughly washed with distilled water, and further filtered to adjust the pH to 7 to produce a fine gel. Then distilled water 25
After suspending the gel in ml and adjusting the pH to 5, β-galactosidase (15 mg, Aspergillus origin) was added.
Further, by adding glutaraldehyde to a total amount of 1%, immobilization was carried out by entrapping-crosslinking.

Thus, the immobilization yield of the immobilized enzyme product obtained by the method of this example was 67.0%.

Example 2 0.5g of chitosan was dissolved in 10% acetic acid (15ml),
Further dilute by adding methanol (20ml). Propylaldehyde (25 molar equivalents per glucosamine residue) was added to this and left at room temperature overnight. The resulting hard gel was pulverized with a mixer, thoroughly washed with distilled water, filtered, and adjusted to pH 7 to produce a fine gel. Next, after suspending the gel in 25 ml of distilled water and adjusting the pH to 5, β-galactosidase (15 mg, originating from Aspergillus) was added, and glutaraldehyde was added to a total amount of 1% to immobilize by entrapment-crosslinking. did.

Thus, the immobilization yield of the immobilized enzyme product obtained by the method of this example was 64.0%.

Example 3 30 mg of glucose isomerase-producing bacterial cells (8500 Units/g) obtained by culturing Streptomyces pheochromogenes (Feikoken Bibori No. 221)
(dry matter) was suspended in 2 ml of distilled water. As in Example 1, 5 ml of 0.1M phosphate buffer was added to the chitosan-formaldehyde gel prepared in advance to adjust the pH to 8, and then the bacterial cell suspension (PH 7) was added. Next, glutaraldehyde was added to make up 1% of the total amount, and after being allowed to stand for 1 hour, the mixture was washed and filtered to obtain a resin-like immobilized bacterial cell product.

Thus, the immobilization yield of the immobilized product produced by the method of this example was 53.1%.

Example 4 In the same manner as in Example 3, 30 mg (dry matter) of glucose isomerase-producing bacterial cells (8500 Units/g) obtained by culturing Streptomyces phaeochromogenes was suspended in 2 ml of distilled water. Next, as in Example 2, 5 ml of 0.1M phosphate buffer was added to the chitosan-propyl aldedo gel prepared in advance, and the pH was adjusted to 8.
After adjusting to pH 7, a bacterial cell suspension (PH7) was added. Next, glutaraldehyde was added to make up 1% of the total amount, and the mixture was left to stand for 1 hour, followed by washing and filtration to obtain a resin-like immobilized bacterial cell product.

Thus, the immobilization yield of the immobilized product produced by the method of this example was 60.0%.

[Brief explanation of drawings]

FIG. 1 is a graph showing the course of enzyme activity when a column was filled with immobilized β-galactosidase product A and immobilized glucose isomerase-producing bacterial cell product B and continuous reactions were performed.

Claims (1)

[Scope of Claims] 1. A method for immobilizing enzymes or microbial cells, which comprises adding chitosan-aldehyde gel to enzymes or microbial cells, and then cross-linking with glutaraldehyde to form a gel-like resin and aggregate it. 2 Chitosan-aldehyde gel has the following general formula in which a Schiff base is formed by some amino groups of chitosan and aldehyde: (wherein R is a hydrogen atom and a group represented by -( _CH2 ) _oCH3 , and n is an integer of 0 to ₂ ) according to claim 1. , enzyme or microorganism immobilization methods. 3. The method according to claim 1 or 2, wherein the water content of the chitosan-aldehyde gel obtained by reacting 1 part by weight of chitosan with 1 to 50 parts by weight of aldehyde is equivalent to 100 to 1000 times the weight of chitosan itself. Method for immobilizing enzymes or microbial cells. 4 As chitosan-aldehyde gel, 80-100
The method for immobilizing enzymes or microorganisms according to any one of claims 1 to 3, wherein an enzyme is used that is stable even when exposed to heat at a temperature of 0.degree. C. and does not change shape. 5. The method for immobilizing enzymes or microbial cells according to any one of claims 1 to 4, which comprises enclosing enzymes or microbial cells on the surface and inside of the chitosan-aldehyde gel, and then cross-linking and immobilizing them with glutaraldehyde. .