JPS60227679A - Production of immobilized enzyme - Google Patents

Abstract

PURPOSE:A specific fibroin and an enzyme is dissolved in water, the solution is foamed, then formed before the precipitation of fibroin or coagulation of the solution to facilitate the production of immobilized enzyme of high enzymatic activity and duration with a porous texture in industrial scales. CONSTITUTION:Starting silk such as bourettes are degummed to remove sericins and the product is dissolved in water. The solution is stirred to allow the crystals of fibroin to orient and air is bubbled to form a foam and an enzyme is dissolved in the foamed solution. Then, the foamed solution is subjected to solidification process, before precipitation of fibroin or coagulation of the solution to effect the production of an immobilized enzyme of high enzymatic activity, duration, and porous texture with strength enough to be practially used with industrial easiness and inexpensively.

Description

[Detailed description of the invention] The present invention relates to a method for producing immobilized enzymes.

Enzymes have high substrate specificity for various chemical reactions, mild reaction conditions, and high reaction efficiency, and are currently widely used in the food, pharmaceutical, and cosmetic industries as extremely useful catalysts. There is. However, since enzymes are generally water-soluble, it is difficult to recover the used layer, which is not only uneconomical, but also has the disadvantage that separation from reaction products is difficult.

Against this background, many proposals have been made to immobilize enzymes on various carriers. For example, methods such as immobilization of enzymes by covalent bonding or ionic bonding to glass beads carriers, and methods for entrapping enzymes in polyacrylamide gel bodies have been widely studied, and some have been put into practical use.

However, conventional enzyme-immobilized carriers have mainly been in the form of powders, beads, or membranes. The method of filling an immobilized enzyme in a column or the like and carrying out the enzymatic reaction continuously is one of the most effective means for industrial efficiency and ease of use. In order to increase the contact between the enzyme and the reaction medium, it is important to increase the amount of enzyme immobilized and to increase the surface area of the carrier itself. For this purpose, in the case of granular carriers, the particle size must be reduced. Since Kyogi, which is rumored to increase the surface area by 11, requires a large pressure to flow the reaction solution, there is a limit to reducing the particle size.

In order to solve this problem, attempts have been made to immobilize enzymes in porous materials, but the reality is that there is still no satisfactory result. For example, there is a method of chemically bonding an enzyme to various water-insoluble porous carriers having active groups, and a method of adding an enzyme force<f6 liquid to a urethane prepolymer and foaming and solidifying it to encapsulate the enzyme in a foam. Although many attempts have been made, all tL uses violent chemical reactions, which tends to cause deactivation of the enzyme, and it is difficult to obtain enzymes with high enzyme activity per unit yield or unit volume.

As a result of the inventors' research to solve the above-mentioned problems,
The present invention has been completed, and its purpose is to have a microporous fiber structure with excellent enzyme activity and durability, as well as practical strength! 8] To provide a method for industrially easily and inexpensively producing a pharyngeal enzyme. The above purpose is to apply a shearing force to a foamy water bath solution in which fibroin with oriented crystal regions and an enzyme dissolved therein is subjected to a molding process in which precipitation of fibroin in the aqueous solution or coagulation of the aqueous solution occurs. This is achieved by a method for producing an immobilized enzyme, which is characterized by forming a molded body with a pore structure.

The fibroin forming the pore structure preferably has a crystallinity of 20% or more from the viewpoint of water resistance and strength of the molded article. Here, the degree of crystallinity is calculated by measuring the X-ray wide-angle diffraction intensity of the sample and relative to the X-ray wide-angle diffraction intensity of an amorphous fibroin film formed from a fibroin aqueous solution at 4° CRH 40%. be.

The types of enzymes applicable to the present invention are not particularly limited, but include, for example, aldolase, phosphatase, asparaginase, inverdase, uricase, urokinase, glucosidase, calactosidase, ribosphrease, cholinesterase, urease, lipase,
Phospholipase, amylase, dextranase, catalase, glucose dehydrogenase, NADPH dehydrogenase, tyrosinase, lactate dehydrogenase,
Peroxidase, cytochrome C oxidase 5-1ase, amino acid oxidase, glucosyltransferase, amino acid ayatil!・Transferase, taleatin phosphokinase, glucokinase, fumarase,
Examples include phosphofructoaldolase, aspartase, citrate lyase, ribose phosphate isomerase, glutathione synthetase, and asparagine synthetase.

These enzymes may be used alone or in combination with two or more enzymes. Furthermore, instead of enzymes, bacterial cells that have produced and accumulated enzymes may be used.

The amount of enzyme used varies depending on the type of enzyme, degree of purification, purpose of use, etc., and cannot be determined unconditionally, but it is usually 0.1 to 50% by weight, preferably 1 to 40% by weight, based on fibroin per unit weight. . In the present invention, the foamy aqueous solution can be prepared by various methods, and a preferred example thereof is as follows.

Eyebrow thread, eyebrow waste, hiss, kiki, silk cloth waste, black
Aqueous solution 6- is prepared by removing sericin by scouring silk raw materials such as silk according to a conventional method and using the method described below.

That is, the above-mentioned scouring silk raw material is mixed with a copper-ethylenediamine aqueous solution, a hydroxide (1[-ammonia aqueous solution, an embodied lithium aqueous solution, a calcium, magnesium, or curved lead hydrochloride or nitrate aqueous solution, a thiocyanate aqueous solution An aqueous solution of fibroin is prepared in advance by dissolving it in a solvent capable of dissolving fibroin, such as fibroin, and desalting it by dialysis through a dialysis membrane such as cellulose glands.
As the solvent, calcium chloride, calcium nitrate, and magnesium nitrate are preferred due to their price and quality stability. The salt concentration of this solvent is usually 10 to 80% by volume, and a water-miscible solvent such as methyl alcohol, ethyl alcohol, propyl alcohol, acetone, dimethylformamide, or dimethyl sulfoxide may be added to facilitate dissolution. can.

The aqueous fibroin solution obtained by the method described above can be used after being appropriately concentrated or diluted, and is usually used in an amount of 1 to 20% by weight. The aqueous fibroin solution prepared in this manner contains lower alcohols such as methyl alcohol, ethyl alcohol, propyl alcohol, and glycerin, hydrochloric acid,
Inorganic acids such as sulfuric acid and phosphoric acid, organic acids such as citric acid and acetic acid,
Alternatively, coagulating salts such as ammonium sulfate, 1-lium sulfate, sodium chloride, sodium acetate, and sodium citrate may be allowed to coexist.

Because the fibroin is dissolved in water, a flow velocity gradient is applied to the mixture of fibroin and enzyme at the same time as foaming, or at the same time as the foam is being foamed. The shearing force can be applied by, for example, stirring or passing through pores or slits. Due to the unique properties of fibroin, the volume, fibroin concentration, molding time, etc. are adjusted to 80% by using a stirrer such as a homomixer or a disperser to mold the foamy aqueous solution subjected to shearing force.
Set the rotation speed and stirring time appropriately.

Note: By applying a 1'-shear force in this way, the orientation and crystallization of fibroin progresses over time without any subsequent operation, and the viscosity increases, causing precipitation of fibroin in the solution or It is important to mold the enzyme before it solidifies, and when mixing the enzyme with the fibroin aqueous solution, 1tR is before or after applying shear force to the fibroin aqueous solution, during the foaming process, or after the foaming process. Although not particularly limited, it is most preferably added and mixed after foaming and before molding. If the enzyme is added and mixed after foaming, it is possible to prevent the enzyme from deactivating due to the action of shearing force, and it is an enzyme-encompassing enzyme that has a very high yield of enzyme activity and has excellent affinity with polymeric substrates. When a porous body is obtained,
There is an advantage. In addition, the enzyme can be added as a powder, an aqueous solution, after dispersion in water, an organic solvent, a dispersion, etc.
It is preferable to add the fibroin in the form of an aqueous solution because it can be easily dissolved and mixed in the fibroin aqueous solution. In this case, a suitable 9-buffer solution, temperature, stabilizer, etc. can be selected depending on the enzyme.

As a method for foaming the fibroin water bath liquid, methods such as gas blowing, high-speed stirring, or addition of a foaming agent may be used alone or in combination. In this case, usually 1 to 20% by weight
It can be carried out in the presence of a thickening agent or an interfacial agent as necessary in an aqueous fibroin solution. Examples of thickeners include water-soluble cellulose derivatives such as carboxymethyl old lulose sodium salt, polysaccharides such as sodium alginate, and synthetic water-soluble polymers such as polyvinyl alcohol, polyethylene glycol, and polyacrylates.

Examples of surfactants include higher fatty alkaline soaps, alkyl sulfonic acids, anionic surfactants such as sulfuric ester salts of higher alcohols, surfactants, cationic surfactants such as higher alkyl quaternary ammonium salts, and polyethylene glycosurfactants. Examples include nonionic surfactants such as Al higher fatty acid ester and higher fatty acid ester of sorbitan.

Gas is normally blown into air, nitrogen gas, carbon dioxide gas, etc. to the extent that the fibroin aqueous solution becomes foamy, that is, 1
Blow at a rate of ~50 L/min through a capillary tube with an internal diameter of 08-5 m. High-speed stirring may be similarly performed at 100 to 2000 revolutions per minute. As the blowing agent, one that generates decomposition gas at a relatively low temperature, or one that generates gas at room temperature by adjusting the pH is used.

Examples include azobisisobutyronitrile, dinitropentamethylenetetramine-urea, each n-carbon ia, and the like.

High-speed stirring or the combined use of high-speed stirring and other methods has the advantage that shearing force can be applied to the fibroin water bath solution and foaming can be effected at the same time. When molding using the foamy aqueous solution prepared in this way, a known molding method may be used to RH the appropriate method and mold it into various forms depending on the intended use of the enzyme. For example, if a foamed mixture of fibroin and enzyme is poured as it is or poured into another appropriate container and left to stand, the entire mixture will solidify over time and a porous body will be obtained. At this time, if it is left at a low temperature or in a frozen state, it is possible to prevent the bubbles from disappearing. In the foamed mixture of fibroin and enzyme, when the fibroin solidifies, the walls of the closed cells are collapsed, so that the obtained molded product has a pore structure with high open porosity.

The solidified molded product is further washed with water or dried (normal pressure, reduced pressure,
Freezing) is also possible.

The immobilized enzyme obtained by the method of the present invention has excellent enzyme activity, durability and practical strength, and since the material is a highly hydrophilic natural protein, it can be used in foods, drugs, and cosmetics. It is very useful for bioreactors, diagnostic agents, etc. in the field.

The method of the present invention will be explained in detail in Examples below.

Example 1 1kL of fresh water was added to Marcel soap 0.5% aqueous solution 50%
was immersed in the mixture and stirred and mixed at 80°C for 3 hours to substantially completely remove sericin and oil, thoroughly washed with water, and then dried at 70°C. Next, 65% calcium chloride aqueous solution 4
kq and ethyl alcohol 1. 2. Put the refined Boulet 0 and Sky into the goo containing '6t9,
The mixture was stirred and dissolved at 80 to 85°C for 1 hour. The resulting viscous solution was diluted by adding 3.2 kQ of 80°C warm water. The fibroin concentration of the solution was 85% by weight. Further, a portion of the solution was subjected to fibroin dialysis using a dialysis device using regenerated cellulose-based hollow fibers. A water bath solution containing 15% by weight of fibroin from the dialysate was obtained.

50v of 15% by weight fibroin aqueous solution
150'Ng of carboxymethylcellulose sodium salt was dissolved in a 1Rt beaker. Next, fibroin and β-glucosidase (2 units
/~) was used to produce a porous body.

l) After adding and mixing 5 m of aqueous β-glucosidase containing 5% by weight of fibroin to the fibroin aqueous solution, nitrogen gas was blown at 50 m/min through a glass tube with an inner diameter of 0.5 mm to form a foam. Even after 48 hours at 4°C, the fibroin did not solidify, the air bubbles were reduced by half, and the bottom of the beaker became a solution.

2) After adding and mixing a β-glucosidase aqueous solution 5- of 5% by mass based on buproin to the fibroin aqueous solution,
2,0001 revolutions per propeller type stirring blade with a diameter of 8 crn
A shearing force of 1 hour was applied.

Next, nitrogen gas was blown into the mixture for 50-7 minutes through a glass tube with an inner diameter of 0.5 mm to form a foam to a volume of about 100 mm. When the fibroin and enzyme solution foamed at 4°C was allowed to stand, it solidified after about 20 minutes.

3) Transfer the fibroin aqueous solution to a glass tube with an inner diameter of 8 cm while blowing nitrogen gas for 30-7 minutes.
Stir with a 1M propeller type stirring blade at a, o o o revolutions/min, apply shear force, and leave the body at 0 for 10 minutes.
It foamed to about 0 mg. Next, the blowing of nitrogen gas was stopped, the stirring was changed to 100 revolutions/min, and an aqueous solution of β-glucosidase (5%) based on fibroin was added, and after 30 seconds, the mixture was left standing in a refrigerator at 4°C. After about 1 hour, the entire mixture solidified.

4) A porous body was obtained in the same manner as in 8) except that the amount of β-glucosidase was 2% by weight based on fibroin.

The three types of enzyme-enclosed porous materials from 2) to 4) were taken out, washed with water, and some were freeze-dried to determine the physical properties of the porous materials.
The remainder was used for measuring enzyme activity. Enzyme activity measurement was performed by adding the enzyme-enclosed porous material 50IIv into 0.02 molar concentration of Sadicin 10-, which was adjusted to pH 7.0 with a buffer solution, and reacting it at 37°C for 6 minutes with stirring. was determined by colorimetric determination. In addition, in order to evaluate the activity durability, the washing-activity measurement operation was repeated 10 times.
The 0th activity retention rate was achieved.

As shown in Table 1, all of the enzyme-enclosed porous materials of the present invention have excellent enzyme activity and durability.

Packing 1-8 enzyme-enclosed porous materials into a column with an inner diameter of 1 m,
When a 0.02 mol salicin solution was passed through the sample at a flow rate of 0.6-7 m for 7 consecutive days and the reaction rate was followed by sampling, the reaction rate was in the range of 92-98%, and the reaction rate decreased as time progressed. There was nothing to do.

Example 2 5.5 m% fibroin water bath solution 5 obtained in Example 1 (
1 with a homomixer (Sumi μ54G manufactured by Machimyo Kika Kogyo Co., Ltd.)
The mixture was subjected to bending force at 1,000 rpm for 120 seconds to form a foam, and then 5% aqueous dextranase was added to the fibroin, and the mixture was slowly stirred and mixed with a glass rod. Next, the container was immersed in dry ice-methanol and frozen.

After being left at -20°C overnight, it was freeze-dried to obtain an enzyme-enclosed fibroin porous material. Porosity 927%, crystallinity 28%
Met. When this porous material was thoroughly washed and activity was measured, the viscosity yield was 19%, and the durability test similar to that in Example 1 showed 93%, indicating that, as a comprehensive method, the retention of activity against polymeric substrates is extremely high. there were. Enzyme activity was measured using the following method.

2 ml of substrate solution (500,000 dextran 2 for average molecule
．． Put enzyme-enclosed porous material 519 in 5% pi (5,4),
After reacting at 40°C for 200 minutes, 1% 3,5-dinitrosalnaric acid alkaline aqueous solution 8- is added to stop the reaction. Then after 5 minutes in a boiling water bath, 20
The mixture was cooled on ice and measured by absorbance at 540 nm.

Example 8 The 15% by weight aqueous fibroin solution prepared in Example 1 was mixed with the homomixer used in Example 2 for 15 seconds.

4000 rotations/rotation side shearing force was applied. Next, the diameter is 3m
While stirring at 200 revolutions/min with a propeller-type stirring blade, carbon dioxide gas was blown into the foam at 100 mA/min through a glass tube with an inner diameter of 1 m, then the blowing of carbon dioxide gas was stopped, and various enzymes shown in Table 2 were mixed. The aqueous solution was added and mixed, and the mixture was left standing in a refrigerator at 4°C.

All solidified within 30 to 60 minutes.

When the activity of various enzyme-enclosed porous materials was measured, all of them had high activity and excellent durability.

Activity Measuring Method 1) Ampore produced using urease 3M % urea aqueous solution was quantified by the Nessler method.

2) An oxygen reaction was carried out at 40°C using an aqueous solution of glucoamylase (pH 6.0) containing 1% by weight of glucoamylase as a substrate, and the amount of glucose produced was fixed.

8) β- is lactosidase p-nitrophenyl-β-ri-caractobira 18- Table 2 Example 4 The fibroin water bath solution obtained in the same manner as in Example 1 was
Concentrated to 0% by weight, this 502 was pumped through 100 pores of 0.2 swφ at a speed of 501 nt/am using a diaphragm pump.
It passed through a nozzle in the hole and was circulated for 10 minutes to apply shearing force. 200 μm of carboxymethylcellulose sodium salt was dissolved.

Next, nitrogen gas is blown in to form a foam, and urease and β
- galactosidase for each fibroin
% by weight was added and mixed using a water rod, and when the mixture was left to stand at 4°C, it solidified in about 1 hour.

After washing the obtained enzyme-enclosed porous material with water, the activities of urease and β-galactosidase were measured separately in the same manner as in Example 8, and the activity yields were 40% and 42%, and the durability was 88% and 91%, respectively. Ta.

As described above, according to the method of the present invention, a complex enzyme-based porous body could be easily obtained.

Claims (7)

[Claims] (1) A foamy aqueous solution in which fibroin, whose crystalline regions have been oriented by applying shearing force, and an enzyme are dissolved is subjected to a molding process to form pores before precipitation of fibroin in the aqueous solution or coagulation of the aqueous solution occurs. A method for producing a monopodase, which is characterized by forming a molded body with a structure. (2) The method for producing an immobilized enzyme according to claim (1), wherein the fibroin has a crystallinity of 20% or more. (3) The foamy aqueous solution contained at most 50% by weight of enzyme relative to fibroin. The scope of claim No. (
The method for producing an immobilized enzyme according to item 1) or item (2). (4) Immobilization according to any one of claims (1) to (3), wherein the foamy aqueous solution is prepared by adding and mixing an enzyme aqueous solution to a foamy aqueous solution of fibroin. Enzyme production method. (5) Claim (1) wherein the foamy aqueous solution is prepared by strongly stirring an aqueous solution in which fibroin and an enzyme are dissolved, distributing fibroin crystals, and simultaneously blowing air bubbles into the foamy solution. A method for producing an immobilized enzyme according to any one of items (4) to (4). (6) The foamy aqueous solution strongly stirs the fibroin aqueous solution,
The product is prepared by adding and mixing an enzyme aqueous solution to a fibroin aqueous solution which is made foamy by orienting fibroin crystals and at the same time blowing air bubbles into the foam. Method for producing the internalized enzyme described. (7) The method for producing an immobilized enzyme according to any one of claims (1) to (6), wherein the molded body has a pore structure with a porosity of 50% or more.