JPS5836956B2 - Immobilized enzyme composition - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized enzyme composition in which a non-proteolytic enzyme is bonded to granular gelatin, and more specifically, the present invention relates to an immobilized enzyme composition in which a non-proteolytic enzyme is bonded to granular gelatin, and more specifically, a non-proteolytic enzyme, a water-soluble polyfunctional crosslinking agent, and granular gelatin are simultaneously bonded to a non-proteolytic enzyme. The present invention relates to an immobilized enzyme assembly in which enzymes are unevenly distributed at high density on the surface of gelatin particles obtained by reaction, and which has both high enzymatic activity and high strength properties.

Immobilized enzymes have been used in various fields in recent years because the enzyme can be used effectively when used as a catalyst. A particulate form is preferable from the viewpoint of ease of recovery and applicability to a packed bed reactor, and various proposals have been made so far.

For example, glucose isomerase is immobilized on granular ion exchange resin (Yokote et al, Die
StArke, 2 7, 302 ('75)),
Method for immobilizing glucoamylase on a porous ceramic carrier [D.
, Lee et al.
) 2 7, 384 ('75)) or a method in which an aqueous solution containing an enzyme or an enzyme-containing bacterial cell and a water-soluble monomer is polymerized and granulated, for example, a method of immobilizing aminoacylase on an acrylamide gel (T. Mori
et al., Enzymologia, 4 3,
2 1 3 ('72)) or a method of immobilizing enzymes or enzyme-containing microbial cells and then molding them into granules (Japanese Unexamined Patent Application Publication No. 1983-1983)
No. 92277, JP-A No. 49-92278), and further,
After suspending the enzyme in an aqueous gelatin solution, this suspension is dropped into an organic solvent that is immiscible or difficult to miscible with water to form particles, and then treated with a polyfunctional crosslinking agent to form particles. Method for obtaining immobilized enzymes (Special Publication 1972-
No. 18794).

However, in the particulate immobilized enzymes obtained by these methods, the enzyme is uniformly distributed and immobilized throughout the inside and outside of the particles, so it is difficult for the substrate to diffuse into the particles or the product to be released from the inside of the particles. Due to the diffusion resistance, the apparent reaction rate decreases and the efficiency of the enzyme decreases, while the local product concentration increases inside the particles, which may cause side reactions.

Further, particularly when a polymeric substrate is used, there is a drawback that the reaction rate is slow because the diffusion rate of the substrate into the inside of the particle is low.

Further, Japanese Patent Application Laid-Open No. 53-41489 discloses a method for immobilizing enzymes on hardened gelatin, which comprises the following four steps, and an enzyme assembly obtained by this method.

Step 1: Harden the protein (gelatin) with formaldehyde.

(Insolubilized gelatin) Step 2 Two dry heat treatments.

If necessary, grind to appropriate particle size.

Step 3: Mix with the enzyme-containing aqueous solution and absorb the enzyme into the swollen hardened gelatin particles.

Step 4: The swollen particles are added to a multifunctional crosslinking agent solution in the presence of an enzyme and a gelatin precipitation solution (organic solvent) to precipitate and simultaneously crosslink and insolubilize the gelatin and enzyme.

According to this method, enzymes are evenly absorbed into gelatin and then a cross-linking reaction takes place, so the enzymatic activity of the internally occluded enzymes depends on the density of the permeable network structure of the gelatin particles. If cross-linking is carried out to the point where the desired strength is obtained, the internal enzyme will not be utilized and the apparent enzyme activity will be low.

Conversely, when the crosslinking rate is lowered, an enzyme composition with low physical strength and unusable is obtained, and an immobilized enzyme composition that is satisfactory in terms of both enzyme activity and carrier strength has not yet been obtained.

The present inventors have researched a high-performance enzyme composition that simultaneously solves the conflicting requirements of improving enzyme activity and increasing physical strength, and have completed the present invention. .

The present invention is an enzyme composition obtained by simultaneously adding an enzyme solution and a polyfunctional crosslinking agent to pulverized or molded granular gelatin, which is a dry product of natural water-soluble gelatin that is not subjected to hardening treatment, and reacting the same. be.

According to the 2') method, gelatin particles begin to swell from the surface and at the same time the crosslinking reaction proceeds from the surface, so a dense network structure is formed on the surface before the enzyme penetrates inside the gelatin particles. It is difficult for the enzyme to penetrate inside, and the enzyme itself causes a cross-linking reaction with gelatin and becomes insolubilized.
It will be unevenly distributed on the surface of the gelatin particles at high density.

Therefore, the immobilized enzyme maintains an effective enzyme concentration without reducing the apparent reaction rate due to diffusion resistance of the substrate inside the particles, has high catalytic efficiency, and has sufficient strength for use in a packed bed reactor. It is a composition.

In the immobilized enzyme of the present invention, since the enzyme is densely packed on the surface, the gelatin can be highly cross-linked without any negative effect on the enzyme action, and its physical strength can be increased. We succeeded in simultaneously satisfying the contradictory demands of increasing the chemical strength, and the enzyme was packed into a large-scale packed bed reactor and had both the enzyme activity and strength to withstand continuous use.

The present invention requires that the enzyme be unevenly distributed on the surface of the gelatin particles.

In other words, the enzyme concentration on the surface of granular gelatin is higher than that inside the gelatin particles.

Therefore, even if highly cross-linked, enzyme activity is not impaired.

This can be confirmed by microphotographs or other enzymatic chemical testing methods, and can be easily produced by the method described below.

The granular gelatin according to the present invention is obtained by extracting animal skin, bone, etc. with hot water, purifying it, and then drying it, and its shape is crushed, spherical, pelleted, etc., and is not a fine powder. .

Generally, in the present invention, crushed particles are preferred, but spherical or pellet shaped particles can also be used if necessary.

The particle size of the particulate gelatin is usually between 0.1 and 2 mTrL, with preferred particle sizes ranging between 0.2 and 1 mTrL.

When the particle size exceeds 2.0 mm, the effective surface area of the particles becomes small and the activity thereof decreases.

On the other hand, if the particle size is less than 0.1, the pressure loss will become large when used in a packed bed reactor, which is not preferable.

Enzymes used in the present invention can be selected from the group consisting of non-proteolytic enzymes, such as glucoamylase, glucose isomerase α-amylase, β-amylase, invertase, glucose oxidase,
Examples include catalase.

These enzymes are either crude enzymes recovered from the culture solution or purified using conventional purification methods.

The quantitative ratio of granular gelatin to enzyme can vary widely depending on the activity unit of the enzyme used, but usually the quantitative ratio is between 100:l and 2:1, more preferably 20:1.
~5:l.

If the ratio is less than 100:1, the amount of enzyme will be small and the activity of the granular immobilized enzyme will be low; on the other hand, if it exceeds 2:1, the amount of enzyme will be too large, and some enzymes will not be able to bind to the surface layer of the granular gelatin. This is not preferred because it causes loss of enzymes.

The water-soluble multifunctional crosslinking agent that can be used in the present invention contains two or more functional groups in its molecule that can react with the functional groups of amino acid residues in proteins.

In the present invention, the general formula OHC-(CH2)x-CH
Dialdehydes represented by O (however, X is O~5
), for example, polyaldehydes such as glutaraldehyde, polyacrolein, and dialdehyde starch, and cyanogenides such as cyanogen bromide.

Although the ratio of enzyme to water-soluble polyfunctional crosslinking agent can vary within a wide range, a preferred range is a ratio of enzyme to water-soluble polyfunctional crosslinking agent of 1:0.01 to 1:2. The range is more preferably 0.05 to 1:1.

When the ratio is less than 1:0.01, binding of enzyme to gelatin and crosslinking of gelatin will not occur sufficiently, enzyme and gelatin will be eluted during enzyme reaction, and the strength will be too low.

Further, if the ratio exceeds 1:2, the number of bonding points of the enzyme to gelatin becomes too large, and the enzyme activity decreases, which is not preferable.

When particulate gelatin and an enzyme are reacted via a water-soluble polyfunctional crosslinking agent, an aqueous solution consisting of the enzyme and the water-soluble polyfunctional crosslinking agent dissolved in an aqueous medium having a pH of 4 to 9, preferably 5 to 7. (hereinafter simply referred to as an aqueous solution) and granular gelatin are combined and stirred.

In this case, the ratio of gelatin to aqueous solution is 1:0.2 to 1.
:3, preferably in the range of 1:0.5 to 1:2.5; if the ratio is less than 1:0.2, the amount of water relative to the gelatin is insufficient and the enzyme cannot be bonded evenly to the surface layer of the gelatin particles.

On the other hand, if the ratio exceeds 1:3, the amount of water to gelatin is too large, and gelatin particles will swell excessively due to water absorption, reducing the strength of the immobilized enzyme particles.

Mixing and stirring of the gelatin particles and the aqueous solution can be done manually or mechanically.The stirring time varies depending on the amount of ingredients used, temperature, etc., but usually 5 to 60 minutes of stirring will result in good particle fixation. A converted enzyme composition is obtained.

When carrying out the present invention, the gelatin particles may be swollen in advance with water in an amount of about twice the weight of gelatin or less.

In the present invention, the temperature at which the immobilization treatment is performed is within a range that does not reduce the stability of the enzyme used, and is generally 5 to 40°C, preferably 10 to 30°C.

EXAMPLES Hereinafter, the present invention will be further explained in detail with reference to Examples, but the present invention is not limited only to these Examples.

Example 1 10L of particulate gelatin (manufactured by Nitta Gelatin) mainly consisting of particle size range of 0.3 to 0.8 mm. Glucoamylase aqueous solution (manufactured by Nagase Sangyo, originating from Aspergillus niger, purified with isopropanol, activity 62000 u/'i)
? A mixed aqueous solution of 7 rul (containing enzyme 22) and 2771 l of glutaraldehyde (manufactured by Nagase Sangyo, 50 w/w % aqueous solution) was added, and the mixture was stirred in a mortar at room temperature for 5 minutes.

After that, the obtained particulate immobilized material was left to stand for 30 minutes.
After washing by adding to ooml of water and stirring for 10 minutes, the mixture was filtered.

After repeating this washing operation 5 times, it was dried with air at 40° C. for 5 hours.

In the obtained particulate immobilized product (io.sy), most of the enzyme was present on the particle surface and exhibited an activity of 1800 units/g.

Note that 1 unit of activity here is 0. 20% dextrin in 0 5 M acetate buffer (pH 4.5) [manufactured by Nichiden Chemical Co., Ltd., Amicol 1 (D.E, 1 6.0)]
) It is defined as the amount of enzyme that produces 10 or of glucose per minute at 60°C using a solution as a substrate.

Example 2 Glucoamylase aqueous solution (isopropanol purified, containing enzyme 502 of 62000 u/P) 1 was added to particulate gelatin 2502 mainly consisting of particle size range 0.3 to 0.8
50ml and glutaraldehyde (50w/w% aqueous solution)
Add the mixed aqueous solution with 40771l,
Dar (manufactured by Jitsugyo Shokai PNV-11 type 100 r.p.m.
) for io minutes, and then allowed to stand for 1 hour.

The obtained wet immobilized product was washed with water (5 lxs times), filtered, and dried with air at 45° C. for 10 hours.

In the obtained particulate immobilized product, the enzyme is unevenly distributed on the surface, and 280
2 for 21 00 units/? activity.

Example 3 Particulate immobilized product 25' obtained in Example 2? After swelling with water for 2 hours, a jacketing force ram (2.5cm.φ
X30cr! 'LH).

600℃ hot water was circulated through the jacket, and 3
5w/W% dextrin [Nippon Chemical Amicoret 1
(D.E. 1 6.0) was used for continuous reaction.

The results are shown in Table 1. Example 4 Particulate gelatin 202 mainly having a particle size range of 0.3 to 0.8 mm contains an enzyme 52 with an activity of 2200 units/t extracted and purified from glucose isomerase aqueous solution (manufactured by Nagase Sangyo, glucose isomerase Nagase (registered trademark)). ) 14ml and glutaraldehyde aqueous solution (50w/w%
) and 3 ml of the aqueous solution were mixed and stirred vigorously for 5 minutes.

After stopping stirring and allowing the mixture to stand for 40 minutes, the obtained particulate wet immobilized product was washed with water (IJX 5 times), filtered, and dried with air at 60° C. for 10 hours.

A particulate immobilized product of 21.6P was obtained with an activity of 340 units/2.

It should be noted that 1 unit of activity refers to an IM aqueous solution of anhydrous glucose (manufactured by Wako Pure Chemical Industries, Ltd.) (5×10
” mol/J) as a substrate, 60°C, pH
8.0 refers to the amount of enzyme that produces 1 micromole of fructose per minute.

Example 5 A catalase aqueous solution (manufactured by Nagase Sangyo, activity 25
A mixture of 7 ml of glutaraldehyde aqueous solution and 1.5 ml of a glutaraldehyde aqueous solution was mixed, stirred vigorously for 5 minutes, and then allowed to stand for 40 minutes.

The obtained particulate wet immobilized product was washed with water (0.5J x 5
2 times), filtered and freeze-dried.

The obtained particulate immobilized product was 10.7f and its activity was 85
It was 0 units/2.

Note that 1 unit of activity here means 1 micromol H2 per minute at 25°C using 10-2M H202 in 10-2M phosphoric acid buffer (pH 7.0) as a substrate.
This refers to the amount of enzyme that decomposes 02.

Example 6 A β-amylase aqueous solution (manufactured by Tokyo Kasei, activity 3
A mixed aqueous solution of 400 units/2 (enzyme 0.82), an aqueous glutaraldehyde solution, and Q,7 rul were mixed and stirred vigorously for 5 minutes, and then allowed to stand for 30 minutes.

The obtained immobilized product was washed with water (0.3J×5 times), and p
After evaporation, it was dried by blowing air at 40° C. for 10 hours.

The particulate immobilized product obtained was 5.29, and its activity was 48
degree/2.

The unit of activity here refers to reducing sugar converted to 10 μg of glucose per minute at 40°C using 0.56% soluble starch as a substrate in 0.05M acetate buffer (pH 5.0). This refers to the amount of enzymes that should be taken into account.

Comparative example 1 22 glucoamylase (manufactured by Nagase Sangyo, activity 1 62
000 units/2) in an aqueous gelatin solution of IO weight %
The mixture was mixed with 1rLl and dissolved at 45°C, and the pH was adjusted to 7.

While stirring 500 ml of butyl acetate, the previously obtained enzyme-gelatin mixture was added dropwise while keeping the temperature at 50°C.

The resulting mixture was cooled to 10'C, 5 ml of a 125% by weight aqueous glutaraldehyde solution was added, stirred for 20 minutes, and then 1 liter of ethanol was added to dehydrate the particles.

Thereafter, the particles were separated and divided into four equal parts, 200 ml of aqueous glutaraldehyde solutions of various concentrations were added to each part, and the mixture was stirred for 30 minutes, filtered, washed with 1.1 ml of water, and freeze-dried.

The obtained immobilized product had glucoamylase uniformly distributed within the particles.

Its activity and strength are shown in Table 2. When the concentration of glutaraldehyde was low, the activity was high but the strength was low (could not withstand packing into the column).

Increasing the concentration of glutamaldehyde increased the strength but significantly decreased the activity.

Example 7 In Example 1, Miyagi Chemical ■ granular gelatin (strength 9
1 bloom), the washing operation was performed three times (for 30 minutes each time) with 200 ml of water, and the same process was performed except that drying was not performed.

As a result, the specific activity was 1800 units/2 dry matter, solid content 36
Even when I compressed it with my fingers at %, it didn't touch.

This still wet immobilized enzyme 102 was mixed with a 10% dextrin [manufactured by Nippon Chemical Co., Ltd., 7 mil/I6i] aqueous solution (
Stir at 50'C in 100ml (pH 4.5) (100ml).
r. p, m)L, the immobilized enzyme was sampled at regular intervals, and after thorough washing with water, the specific activity was measured and expressed in terms of dry weight.

As a result, the specific activity (units/half substance) was 180 on the first day.
0, 1750 on the 2nd day, 1750 on the 5th day, and 1700 on the 10th day.

Comparative Example 2 600 Tll of water was added to the same gelatin 1007 as in Example 7, and after dissolving at 60°C, 36% formalin aqueous solution 15
ml was added and stirred, and after 2 minutes it became a soft gel.

This was roughly crushed and dried at 115°C for 15 hours, and after being crushed, it was sieved into particle size categories of 0.3 to 0.8.

10 f of this hardened gelatin was mixed with 28 ml of the same glucoamylase aqueous solution as in Example 1, and after absorption, 2 ml of a 25% aqueous solution of glutardialdehyde and 40 ml of acetone were added.
1 was added thereto, and a crosslinking reaction was carried out at 30°G for 60 minutes with gentle stirring.

This preparation was worked up as in Example 7.

The obtained immobilized product had a specific activity of 2500. QIIL rank/? The solid content was 27%, and it was easily crushed when compressed with fingers.

When this immobilized product 102 was subjected to a stability test in the same manner as in Example 7, the specific activity (unit: 12 - dry matter) was found to be 12 on the first day.
00 on the second day, 400 on the fifth day, and unmeasurable due to shape deformation on the 10th day.

Claims (1)

[Claims] 1. An immobilized enzyme composition comprising a non-proteolytic enzyme and granular gelatin treated with a polyfunctional crosslinking agent, wherein the enzyme is unevenly distributed on the surface of the granular gelatin. .