KR20080073188A - Support for immobilizing a biocatalyst comprising silica bead and use thereof - Google Patents

Abstract

A support for immobilizing a biocatalyst and an enzyme immobilized thereon are provided to be harmless to human body and possible to be reused, show increased stability, and be easy to control reactivity, pH and temperature, thereby being widely used in a food or a medicine related industry. A support for immobilizing a biocatalyst comprises silica bead of which an outer layer is crosslinked with chitosan and casein, wherein the silica bead has a pore size of 50-1,000 angstrom. An immobilized enzyme is characterized in that the support for immobilizing the biocatalyst and an enzyme such as alpha-amylase of Bacillus licheniformis are bonded by an amide bond. To degrade starch, the immobilized alpha-amylase is used.

Description

Support for immobilizing a biocatalyst comprising silica gel beads, and use thereof

1 is a graph showing the effect of pH on the starch hydrolytic activity of the immobilized enzyme and free enzyme prepared according to an embodiment of the present invention.

Figure 2 is a graph showing the effect of temperature on the starch hydrolytic activity of the immobilized enzyme and free enzyme prepared according to an embodiment of the present invention.

Figure 3 is a graph comparing the thermal stability of the immobilized enzyme and free enzyme prepared according to an embodiment of the present invention.

Figure 4 is a graph comparing the change over time of the immobilized enzyme and free enzyme prepared according to an embodiment of the present invention.

5 is a graph showing a change in activity according to the reuse of the immobilized enzyme prepared according to an embodiment of the present invention.

Figure 6 is a graph comparing the storage stability of the immobilized enzyme and free enzyme prepared according to an embodiment of the present invention measured at 4 ℃ conditions.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a carrier for immobilizing a biocatalyst using silica beads, an immobilized enzyme using the carrier, a method for preparing the immobilized enzyme, and a method of using the same.

[Private Technology]

The basic concept of enzyme immobilization is the field of enzyme utilization technology of biochemical process using enzyme, and it can be divided into technology field to improve biological activity of enzyme and technology field to stabilize and economically use the activity. The latter has been developed around the immobilization method, whereas the research has been conducted in the direction of the research.

An important purpose of immobilizing the enzyme is to increase the economics of the enzymatic reaction process because the enzyme can be easily recovered and reused, and the reaction mode can be variously applied batchwise or continuously. Therefore, in order to effectively use natural enzymes in a biochemical process, the enzymes must be immobilized. A general enzyme immobilization method is a physical adsorption method or a chemical method. The physical adsorption method mainly uses an ion-exchange method, but the ion-exchange method has the advantage of being non-toxic, but has a problem in that its binding strength is weak. In addition, the chemical method is to use a reagent to fix the enzyme by forming a covalent bond by a chemical reaction, the method has a strong cross-linking force, but because the reagent used for immobilization of the enzyme is toxic Another disadvantage is that it is difficult to use in the pharmaceutical industry.

Enzyme immobilization, which involves the enzymatic immobilization by binding an enzyme to an organic or inorganic carrier, reuses and performs continuous processing is well known. Organics (e.g. cellulose, nylon, polyacrylamide) are disadvantageous as carriers because they have poor mechanical stability and can break down the bonds with enzymes due to corrosion by solvents, changes in pH and ionic strength and invasion by microorganisms. Because it can. Therefore, an inorganic carrier to which an enzyme is adsorbed or covalently adsorbed has been proposed. The binding type depends on the conditions of use of the enzyme and the characteristics of the substrate. In other words, if the substrate has a strong salt concentration, the adsorption method cannot be applied because the adsorption of the adsorbed enzyme occurs. Therefore, covalent bonds of enzymes take precedence. The surface of the carrier should encompass specific functional groups that induce the binding of enzymes. Most carriers cannot cover functional groups and require surface pretreatment.

Immobilization by covalent bonds is a method of covalently bonding the surface of the carrier and the enzyme with a binder or a pier to surface-treat the carrier or to introduce functional groups into the enzyme, and to prevent the active enzymes of the supported enzyme from being blocked.

In order to solve the problems of the prior art, an object of the present invention is to provide a carrier for immobilizing a biocatalyst having a high activity and a method for producing the same.

Still another object of the present invention is to provide an immobilized enzyme prepared using the carrier for immobilizing the biocatalyst, a method for preparing the immobilized enzyme, and a method of using the same.

In order to achieve the above technical problem, the present invention relates to a silica bead, and a carrier for immobilizing a biocatalyst in which chitosan and casein are crosslinked to the outer surface of the silica beads and a method for preparing the same. Preferably, the present invention may be a carrier for immobilizing a biocatalyst prepared by immersing silica beads in a mixed solution of chitosan and casein, and then drying and reacting the dried beads with an epichlorohydrin solution. The concentration of chitosan and casein contained in the mixed solution may be 0.1% to 10% (w / v).

The present invention also relates to an immobilized enzyme immobilized with a biocatalyst on the carrier for immobilizing a biocatalyst and a method for preparing the same. Preferably, the biocatalyst immobilizing enzyme may be an immobilizing enzyme in which the biocatalyst immobilizing carrier and the enzyme are combined by an amide bond, and the immobilizing enzyme is the presence of a transglutaminase. It may be prepared by reacting under. The biocatalyst may preferably be an enzyme, more preferably alpha-amylase, for example heat resistant alpha-amylase.

The present invention also relates to a method of degrading starch by using an immobilized enzyme in which an enzyme is immobilized on the carrier for immobilizing the biocatalyst.

The biocatalyst immobilization carrier prepared by treating the silica beads according to the present invention with a mixed solution of chitosan-casein has the advantage that the enzyme can be immobilized using a transglutaminase. In particular, when the enzyme is widely used in food or medical-related industries such as alpha-amylase, it is possible to immobilize the enzyme by using a transglutase, a nontoxic enzyme that is harmless to humans instead of a chemical sample. Very useful as

In addition, the enzyme immobilized using a transglutase in the biocatalyst immobilization carrier is a non-toxic enzyme that is harmless to the human body instead of a chemical sample, and thus can be used in food or medicine-related industries, and can be reused. Stability is increased, responsiveness is controlled, temperature and pH control is free.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a carrier for immobilizing a biocatalyst in which chitosan and / or casein are crosslinked to an outer layer of silica beads. More specifically, it may be a carrier for immobilizing a biocatalyst prepared by immersing silica beads in a mixed solution of chitosan and casein and drying them, and reacting the dried beads with an epichlorohydrin solution.

Silica beads included in the carrier according to the present invention have excellent thermal stability. The silica beads may preferably be silica gel beads or composite silica gel beads. For example, the composite silica gel beads may be TiO ₂ composite silica gel beads, Al ₂ O ₃ composite silica gel beads, or ZrO ₂ composite silica gel beads. In order to secure a large surface area of the carrier to increase the enzymatic activity per volume and to provide sufficient pore space for the immobilization of the enzyme, the pore size of the silica beads is preferably set to 50 kPa to 1000 kPa. The particle size of the silica beads may be 10 mesh to 80 mesh, preferably 30 mesh to 70 mesh, more preferably 35 mesh to 60 mesh.

The chitosan may be a material manufactured by easily processing the chitin contained in the shellfish to the human body, and in detail, may be a material prepared by deacetylating the chitin. In order to solve the problems of the conventional immobilization enzyme, the biocatalyst immobilization carrier is a transglutaminase to form an amide bond between the carrier for biocatalyst immobilization and glutamine and lysine of the biocatalyst. In order to form, an acyl group is also required in a carrier for immobilizing a biocatalyst. The carrier for immobilization of the biocatalyst can not only be given strength and stability by the chitosan, but also acyl groups are supplied by casein, so that the carrier and the enzyme are amide bonds using transglutaminase. It can be used for the preparation of the immobilized enzyme bound.

Casein or casein salt may be used for surface treatment of the carrier, and the casein salt may be sodium caseinate (Na Caseinate). The casein sodium is an additive widely used in foods and is harmless to the human body. Since the casein improves the strength and stability of the immobilized carrier and may coat the immobilized support with high density, the casein-immobilized carrier may be a strong and stable biocatalyst immobilized carrier.

The content of the chitosan and casein contained in the mixed solution of the chitosan and casein is sufficient to provide a contact opportunity of silica beads, chitosan and casein to secure the production yield of the carrier for immobilizing the biocatalyst, and to minimize the production cost, 0.1 parts by weight to 10 parts by weight, preferably 1 part by weight to 7.5 parts by weight, and more preferably 3 parts by weight to 6 parts by weight, based on 100 parts by weight of the silica beads.

The concentration of chitosan and casein contained in the mixed solution may be 0.1% to 10% (w / v), preferably 0.5% to 5%, and most preferably 0.75% to 2.0%.

The biocatalyst immobilization carrier may be prepared by crosslinking chitosan and casein on an outer layer of the silica beads by surface treating the silica beads with chitosan and casein. As an example, the carrier for immobilizing a biocatalyst in which chitosan and casein are cross-linked to the outer layer of silica beads is dipped by immersing silica beads in a mixed solution of chitosan and casein and drying the dried beads with an epichlorohydrin solution. It can be prepared by reaction.

  The present invention also relates to an immobilized biocatalyst in which a biocatalyst is immobilized on the carrier for immobilizing the biocatalyst. The immobilized biocatalyst may be an immobilized catalyst bonded by an amide bond between the carrier for immobilizing the biocatalyst and the biocatalyst. For example, the immobilization catalyst may be an immobilization enzyme prepared by reacting the biocatalyst immobilization carrier with an enzyme in the presence of transglutaminase.

The biocatalyst may be applied to anything that can exhibit catalytic activity. For example, live or processed microbial cultures, microbial concentrates, microbial powders, microbial sludge or organism-derived enzymes or coenzyme extracts, enzymes or coenzyme concentrates, enzymes or coenzyme powders may be applied, preferably enzymes. The enzyme that can be immobilized on the biocatalyst immobilization carrier includes all kinds of enzymes, for example, a hydrolase (for example, amylase, glycosidase, protease), redox enzymes (glucosidase, catalase), isomerase ( Glucose isomerase) and transferase (dextran sucrase), and the like. Preferably the enzyme is alpha-amylase, more preferably heat-resistant alpha-amylase, for example, it may be heat-resistant alpha-amylase of Bacillus licheniformis .

The biocatalyst content is preferably less than the saturated solubility of the biocatalyst in consideration of the immobilization efficiency and a sufficient concentration. Specifically, the biocatalyst may be 100 unit to 5000 unit with respect to 1 g of the biocatalyst immobilization carrier, preferably May be from 500 units to 3000 units, more preferably from 1000 units to 2000 units, even more preferably from 1400 units to 1600 units. For example, in the case of heat-resistant alpha-amylase, 1 unit is an amount of an enzyme that produces 1 μmole of reducing sugar (glucose) in 1 minute at 60 ° C. and pH 7.0. The amount of the reducing sugar can be measured by DNS (3,5-dintrosalicylic acid reagent) quantitative method. For example, in the case of using the heat-resistant alpha-amylase of Bacillus licheniformis , 3 ml of enzyme solution containing 0.54 mg of alpha-amylase per 1 ml of solution is added to 1 g of the immobilization carrier to immobilize the enzyme. The enzyme activity of the enzyme solution was 1500 units.

The transglutase is an enzyme that forms an amide bond between glutamine and lysine. In the present invention, the transglutase may be purchased and used commercially available, it can be used for production strains or those produced by recombinant microorganisms introduced with the transglutase coding gene, preferably microorganism-derived transgluta It may be a tammy agent. As an example, the transglutamiase may be a transglutase derived from Streptoverticillium mobaraense . The transglutase derived from Streptoverticlium varibari phosphate may be obtained by extracting from the culture of the Streptoverticlium varibari phosphate and treating with cold ethyl alcohol. The transglutase may be a white to pale yellow to dark brown powder, granules, lumps or transparent to dark brown liquids, and the optimal pH of the transglutase extracted from the Streptoverticilium mobilarise is pH 6-7, optimal temperature 50-55 degreeC.

The present invention also relates to a process for preparing the immobilized catalyst.

More specifically, the present invention comprises the steps of a) preparing a carrier for biocatalytic immobilization by surface treatment of silica beads with chitosan and / or casein; And

b) immobilizing a biocatalyst to a carrier for immobilizing the biocatalyst using a transglutaminase.

a) preparation of immobilization carrier

The preparing of the biocatalyst immobilization carrier may be prepared by cross-linking chitosan and / or casein on an outer layer of the silica beads by surface treating the silica beads with the chitosan and / or casein. Preferably, the preparing of the carrier for immobilizing the biocatalyst may include a-1) preparing a mixture by immersing silica beads in a mixed solution of chitosan and / or casein; And a-2) adding the mixture to an epichlorohydrin solution to crosslink the silica beads with chitosan and / or casein.

The step of preparing a mixture by immersing the silica beads in the mixed solution of chitosan and / or casein is an example of dipping silica gel beads in the mixed solution of chitosan and / or casein and 10 to 24 hours, preferably 18 hours to It may be carried out by holding at 20 ℃, 15 ℃ to 30 ℃, preferably 20 ℃ to 25 ℃.

Preparing the mixture by immersing silica beads in the mixed solution of chitosan and / or casein may further include drying the mixture. The drying step may be reduced pressure drying, vacuum drying or heat drying, but is not limited thereto.

In the mixed solution of chitosan and / or casein, the solvent in which the chitosan and / or casein is dissolved may be an acetic acid solution, preferably an acetic acid aqueous solution or an acetic acid buffer solution. The concentration of chitosan and casein contained in the mixed solution of chitosan and / or casein may be 0.1% to 10% (w / v), preferably 0.5% to 5%, and most preferably 0.75% to 2.0%. have.

On the other hand, the content of the chitosan and casein contained in the mixed solution of the chitosan and casein provides sufficient contact opportunities of silica beads, chitosan and casein to secure the production yield of the carrier for immobilizing the biocatalyst, minimizing the manufacturing cost In order to achieve 100 parts by weight of silica beads, 0.1 parts by weight to 10 parts by weight, preferably 1 part by weight to 7.5 parts by weight, and more preferably 3 parts by weight to 6 parts by weight.

Step a-2) of reacting the mixture with an epichlorohydrin solution to form a crosslinking step adds the mixture prepared by immersing silica beads in the mixed solution of chitosan and / or casein to the epichlorohydrin solution. 30 to 90 ° C, preferably 50 to 75 ° C, more preferably 55 to 65 ° C and pH 7.5 to pH 11, preferably pH 8.5 to pH 10, more preferably pH 9.5 to pH 10 2 hours to 16 hours, preferably 4 hours to 12 hours, more preferably 6 hours to 8 hours.

The concentration of epichlorohydrin in the epichlorohydrin solution may be 0.01 M to 1 M, preferably 0.05 M to 0.5 M, more preferably 0.75 M to 1.25 M, to dissolve the epichlorohydrin. The solvent may be dimethyl sulfoxide (DMSO), and preferably may include 0.1 M NaOH in addition to DMSO. The epichlorohydrin may form a bond between a hydroxyl group (OH group) and a hydroxyl group, an amine group (NH ₂ group) and an amine group and a hydroxyl group and an amine group, and the chitosan and casein have both a hydroxyl group and an amine group. Thus, when chitosan and casein are added to the silica and reacted with epichlorohydrin, the silica may be coated by surface treatment with chitosan and casein, that is, crosslinking of the chitosan and / or casein, to coat the silica. have

Surface treatment of the silica beads with chitosan and / or casein may further comprise a step of washing after reacting with the epichlorohydrin solution. The washing may be performed using NaCl solution or distilled water, preferably distilled water. For example, after washing with distilled water, the process of recovering using filter paper may be repeated three to five times. It is not limited.

b) immobilizing the biocatalyst on the carrier

B) immobilizing the biocatalyst on the biocatalyst immobilization carrier is an amide bond between the biocatalyst immobilization carrier and glutamine and lysine of the biocatalyst using a transglutaminase. It may be a step of binding with (amide bond), it can be carried out by adding the carrier for the immobilization of the biocatalyst to the solution containing the biocatalyst and transglutase and proceed with the reaction.

An amide bond between the biocatalyst immobilization carrier and glutamine and lysine of the biocatalyst immobilization carrier is added to the solution containing the biocatalyst and the transglutase and the reaction proceeds. Bonding with amide bonds is accomplished by stirring. After the reaction is carried out, it is preferable to sufficiently wash the carrier to which the enzyme is immobilized with a suitable buffer to remove the enzyme, the excess enzyme and the transglutase, which are insufficiently bound and are easily separated. Since the preferred reaction pH of the present invention is pH 7, an additional buffer may not be used, and distilled water may be used as the buffer. Immobilizing the biocatalyst to the carrier for immobilizing the biocatalyst using the transglutaminase may include adding a diluent or a stabilizer for the purpose of titration, quality preservation, and the like. can do.

If the amount of the solution containing the biocatalyst and the transglutase is too small compared to the content of the carrier for immobilization, the carrier may be damaged by shaking or stirring during the reaction with the transglutase, and the amount of the solution is too high. The higher the immobilization reaction rate can be reduced. For example, the amount of the solution containing the biocatalyst and the transglutase and the content of the carrier for the immobilization of the biocatalyst are based on the amount of the solution containing the biocatalyst and the transglutase 10 ml of the carrier for the immobilization of the biocatalyst The content of may be 0.5 g to 3 g, preferably 1 g to 1.5 g.

The content of the biocatalyst is preferably less than the saturated solubility of the enzyme in terms of immobilization efficiency and a sufficient concentration. Specifically, the biocatalyst immobilization support may be 100 units to 5000 units, preferably 500 units to 3000 units, more preferably 1000 units to 2000 units, more preferably 1000 units to 2000 units, based on 1 g of the carrier. Even more preferably, it may be 1400 unit to 1600 unit. For example, in the case of heat-resistant alpha-amylase, 1 unit is an amount of an enzyme that produces 1 μmole of reducing sugar (glucose) in 1 minute at 60 ° C. and pH 7.0. The amount of the reducing sugar can be measured by DNS (3,5-dintrosalicylic acid reagent) quantitative method. For example, in case of using the heat-resistant alpha-amylase of Bacillus licheniformis , 1 g of the immobilized carrier is mixed with 1500 units of the alpha-amylase and 180 units of transglutase, followed by reaction at 37 ° C. for 4 hours. To prepare an immobilized enzyme.

The reaction conditions and reaction execution time of the step of immobilizing the biocatalyst on the carrier for immobilizing the biocatalyst using the transglutaminase may be modified according to the type of the biocatalyst and the transglutaminase, preferably May be performed at reaction conditions within a temperature range in which the transglutase or biocatalyst is not inactivated and a pH range in which the transglutase or the catalyst is not denatured. In order to adjust the pH range to a pH range in which transglutase or the catalyst is not denatured, a buffer may be used, and the buffer may be an acetic acid buffer, a phosphate buffer, a tris hydrochloric acid buffer, or the like.

The present invention also provides a method of using an immobilized enzyme in which a biocatalyst is immobilized on the carrier for immobilizing the biocatalyst. The immobilized enzyme is preferably the immobilized enzyme may be prepared by reacting the biocatalyst immobilization carrier and alpha-amylase in the presence of a transglutase, the alpha-amylase is preferably heat-resistant alpha-amylaseyl Can be.

The enzyme in the biocatalyst immobilization carrier of the present invention can not only maintain high activity under the same reaction conditions as the existing free enzyme, but also have an excellent effect on the reusability, storage and stability of the enzyme. The beneficial effects of producing possible immobilized enzymes are recognized. In addition, when the immobilized enzyme is used, a very industrially significant effect is recognized in terms of improving the purity of the product obtained by the enzymatic reaction, reducing the amount of enzyme used, and reducing the reaction vessel.

The method of using the immobilized enzyme may preferably be a starch decomposition method using the immobilized enzyme.

The starch digestion method of the present invention may be a conventional starch digestion method using an enzyme, and specifically, the carrier for immobilizing the biocatalyst of the present invention may be selected from malto oligosaccharide, starch, cyclodextrin, pullulan, acarbose and para-nitrite. Contact with a solution in which at least one selected from the group consisting of phenyl maltopentaoside is dissolved, preferably a solution in which malto oligosaccharides, starches or mixtures thereof are dissolved, and pH 4 to pH 8, preferably pH 5 to pH 7 and 30 ° C to 90 ° C, preferably 50 ° C to 80 ° C, more preferably 60 ° C to 75 ° C.

By the starch decomposition method, glucoTM, maltose, maltotetraose, maltotriose, pannose, acarbiocin-glucose, para-nitrophenyl glucoside, para-nitrophenyl maltoside and mixtures thereof, preferably May prepare one or more sugar compounds selected from the group consisting of glucoTM, maltose, maltotetraose, maltotriose and mixtures thereof.

The present invention will be described in more detail with reference to the following examples, but the protection scope of the present invention is not intended to be limited to the following examples.

EXAMPLE

Example 1 Preparation of Immobilized Enzyme

1-1: Chitosan-Casein Coated Silica Bead Preparation

A chitosan-casein mixed solution was prepared by dissolving 1% chitosan and 1% casein in a 1M acetic acid solution. 5 g of silica gel beads were immersed in the mixed solution and allowed to react overnight. The supernatant reacted overnight was poured out and the silica gel beads were dried under vacuum conditions. The beads were resuspended in DMSO to which 0.1 M epichlorohydrin was added and reacted at 60 ° C. for 6 hours. After the reaction, the beads which were subjected to the reaction in DMSO to which 0.1M epichlorohydrin was added were washed with distilled water.

As shown in Table 1 below, the experiment was repeated to vary the concentration of chitosan and casein, and the samples 1 to 5 were obtained.

Specifically, the chitosan-casein coated silica beads were prepared by the following method. The silica gel beads used in this example had a particle size of the carrier from 35 mesh to 60 mesh, the pore size of the carrier was 60 mm 3, and the silica gel (Davisil ^™ , grade 636, Sigma 236802, 99%) was 99%. Sigma, USA). The mixed solution (silica buffer solution) for preparing the silica gel beads is 1 M containing 1% chitosan (Sigma, Medium molecular weight, USA) and 1% sodium casein (Sigma C8654, Casein sodium salt from bovine milk, USA) It was prepared by mixing 5 g of silica gel in 100 ml distilled water to which acetic acid was added, and the chitosan and casein coated silica beads were mixed with 100 ml of the mixed solution for preparing the silica gel beads overnight, specifically 18 After the mixture was left at room temperature for 20 hours and prepared in a dry powder state by a drying (evaporation) process, 50 ml of DMSO (Dimethyl sulfoxide, pH 10) and epichlorohydrin (Sigma E1055, USA) were added thereto. It was prepared by inducing a reaction with chitosan and casein at pH 10 and 60 ° C, reacting for 6 hours, and washing three times with distilled water.

1-2: Immobilization of Alpha-amylase on the Coated Silica Beads

After adding the coated silica gel beads obtained in Example 1-1 to the alpha-amylase solution, a transglutaminase solution (pH 7.0) was added to react for 6 hours. The obtained enzyme immobilized beads were filtered and washed with distilled water. The experiment was performed on each of Samples 1 to 5 obtained in Example 1-1.

Specifically, an alpha-amylase solution and a transglutaminase solution prepared by adding alpha-amylase (7500 unit) and transglutaminase (225 unit) to PBS (phosphate buffer solution, pH 7.0) Then, PBS (phosphate buffer solution, pH 7.0) was further added so that the total volume was 10 ml, and 5 g of beads were added to the mixed solution, followed by reaction at 37 ° C. for 6 hours. After performing the reaction, the prepared enzyme immobilized beads were filtered and washed (waterman NO ₂ ) three times and the activity was measured.

In the case of the alpha-amylase, 1 unit is an amount of an enzyme that produces 1 μmole of reducing sugar (glucose) in 1 minute at 60 ° C. and pH 7.0, and the measurement of the reducing sugar is DNS (3,5-dintrosalicylic acid reagent).

The alpha-amylase was cultivated a commercial strain of Bacillus licheniformis (ATCC 1450) strain to obtain the amylase secreted by the strain during the cultivation, the transglutaminase was cultured by Streptomyces mobaraensis (ATCC 29032) strain The amylase secreted by the strain during the culture was obtained and used.

As shown in Table 1, 1% (w / v) of chitosan and casein as a result of coating the immobilized carrier at various ratios in order to find the optimal conditions for the substrate of transglutaminase, namely, casein and chitosan, which form crosslinks It was confirmed that the highest enzymatic activity was obtained when the alpha-amylase was immobilized using an immobilized carrier coated at a rate of 1% (w / v).

Example 2: Properties of Immobilized Enzymes

2-1: pH Characteristics of Immobilized Enzyme

The enzyme activity was measured according to the pH of the immobilized enzyme and the free enzyme prepared in Example 1-2 as follows.

Starch (1%) was added to PBS buffer at pH 7.0 to prepare a starch solution (soluble starch solution) to be used for the reaction of this experiment. 1 g (236 unit) of the immobilized enzyme prepared in Example 1-2 and 0.5 mL (250 unit) free amylase obtained in Example 1-2 were added to 2 ml of the prepared 1% starch solution, respectively. The reaction was carried out at 60 ° C. for 10 minutes. After the reaction was carried out, the measurement of the reducing sugar produced due to the enzyme activity was performed by adding 0.5 ml of the reaction solution after the reaction to 2 ml of DNS solution and reacting at 100 ° C. for 10 minutes, and the color development by the reducing sugar was performed at 540 nm. It examined by DNS reducing sugar quantification method to measure.

The starch hydrolysis activity of the immobilized enzyme and the free enzyme was measured under various pH conditions by the above method. The effect of pH on the enzyme activity is shown in the graph of FIG.

As shown in FIG. 1, the immobilized enzyme (●) during starch hydrolysis according to pH conditions showed the highest enzyme activity at pH 6, such as free enzyme (○). The characteristics of the immobilized enzyme of the present invention tends to be the same or inferior to the enzyme, but it is advantageous in that it can be recovered and reused as long as the enzyme activity is maintained compared to the free enzyme that can be used only in one reaction. It was confirmed that there is. The activity described in FIG. 1 is a relative activity measured when the highest activity is 100%, and it is confirmed that the immobilized enzyme shows higher residual activity for a wide range of pH than the free enzyme in the case of immobilized enzyme. It was found that the stability against was very good.

2-2: Temperature Characteristics of Immobilized Enzyme

Enzyme activity measurement according to the temperature of the immobilized enzyme and the free enzyme prepared in Example 1-2 except that the reaction was carried out at a pH of pH 7.0, a specific temperature in the range of 30 ℃ to 90 ℃ And was carried out in the same manner as in Example 2-1, the results are shown in FIG.

As shown in FIG. 2, the activity of the immobilized enzyme (●) and the free enzyme (○) during starch hydrolysis according to the temperature conditions showed the highest activity at 70 ℃. The immobilized enzyme of the present invention showed basically the same temperature characteristics as the free enzyme in the immobilized state, and the activity of the immobilized enzyme was found to be the same as that of the free enzyme or slightly lower than that of the free enzyme. Compared to the free enzyme available only in the reaction, it was confirmed that the enzyme has a beneficial effect in that it can be recovered and reused as long as the enzyme activity is maintained.

2-3: Thermal Stability of Immobilized Enzymes

The immobilized enzyme (●) prepared in Example 1-2 and the free enzyme (○) of Example 2-1 were heat treated for 1 hour in the range of 60 ° C. to 100 ° C., and then recovered to recover alpha-amylase. Residual activity was measured by the method described above in 2-1. The residual activity was measured at pH 7.0 and 60 ° C., and the measurement results of residual activity indicating thermal stability of the enzyme are shown in FIG. 3.

As shown in FIG. 3, heat treatment for one hour at each temperature showed high stability up to 80 ° C., while both immobilized enzyme and free enzyme maintained at least 90% of the original enzyme activity. In one case, it was confirmed that the temperature stability of the enzyme is lowered. In spite of this tendency, the immobilized enzyme was found to exhibit 80% or more of residual enzyme activity even when heat-treated at a temperature of 100 ° C. compared with the free enzyme, and thus showed high stability compared to the free enzyme.

2-4: Comparison of Enzyme Activity between Immobilized Enzyme and Free Enzyme

Measurement of changes in enzyme activity over time of the immobilized enzyme and free enzyme prepared in Example 1-2 was performed as follows.

2% starch was added to PBS buffer at pH 7.0 to prepare a starch solution (soluble starch solution) to be used in the reaction of this experiment. 220 units of the immobilized enzyme prepared in Example 1-2 and 220 units of the free amylase of Example 2-1 were added to 4 ml of the prepared 2% starch solution, and then the reaction was performed at 60 ° C. for 8 hours. In the meantime, the reaction solution was obtained at each time, and the enzyme activity was measured by the DNS reducing glycometry of Example 2-1. The results are shown in FIG. 4.

As shown in FIG. 4, as a result of comparing the activity of the immobilized enzyme (●) and the free enzyme (○), the immobilized enzyme and the free enzyme showed almost the same activity at 1 hour after the start of the reaction. After 2 hours, the free enzyme was able to confirm that the enzyme activity reached saturation. In contrast, the immobilized enzyme continued to increase in activity, and it was confirmed that the enzyme activity reached saturation only after 6 hours. As a result of the measurement at 6 hours, the enzyme activity of the immobilized enzyme was found to be about 2.5 times higher than that of the free enzyme.

The above results indicate that the enzyme activity continued due to the temperature stability of the enzyme increased by immobilization, and that the enzymes which were interrupted in contact with the original substrate having a high degree of polymerization among the enzymes immobilized on the immobilization carrier were reacted. It was considered that this has a form capable of reacting in contact with substrates having a shortened degree of polymerization as the length increases.

2-5: Review of Reusability of Immobilized Enzymes

In order to confirm the reusability of the immobilized enzyme prepared in Example 1-2, reusability was measured by the following method.

1 g of the immobilized enzyme prepared in Example 1-2 was added to 2 mL of the 1% starch solution prepared in Example 2-1, and then reacted at 60 ° C. for 10 minutes. After the reaction, the immobilized enzyme was recovered, washed with PBS buffer (pH 7.0), and the enzyme reaction was performed again in the same manner as above, and the same reaction was continued for 12 times. After each of the above reactions, the reduced equivalents produced using the recovered reaction solution as samples were subjected to DNS reduction quantification in the same manner as in Example 2-1 to measure enzyme activity, and the results are shown in FIG. 5.

As shown in FIG. 5, it was confirmed that the immobilized enzyme maintains at least 70% of the enzyme activity of the immobilized enzyme even when 12 times are reused. From the above results, it was confirmed that the immobilized enzyme prepared by the present invention has very good reusability of the enzyme.

2-6: Review of Stability during Storage of Immobilized Enzyme

In order to examine the stability during the storage of the immobilized enzyme prepared in 1-2 above, the activity of the immobilized enzyme and the free enzyme was measured daily at 4 ° C together with the free enzyme, and the stability during the storage was examined. It was.

In order to confirm the storage and stability of the immobilized enzyme prepared in Example 1-2, reusability was measured by the following method.

1 g of immobilized enzyme prepared in Example 1-2 and 1 ml of free enzyme of Example 2-1 were collected and stored in a container at 4 ° C., respectively, to carry out the activity of each enzyme daily. Measured under the conditions of pH 7.0 and 60 ℃ by the method described in Example 2-1, the stability during storage over time was confirmed and the results are shown in FIG.

 As shown in FIG. 6, in the case of the free enzyme, the enzyme activity was rapidly decreased as the storage period elapsed, and the enzyme activity was completely lost on the 12th day of storage. On the other hand, the immobilized enzyme showed a small decrease in the activity of the enzyme even when the storage period elapsed, and showed a high stability of maintaining 90% or more of the original activity even after 12 days. From the above results, it was confirmed that the immobilized enzyme prepared by the present invention was very excellent in storage and stability, and from the above results, it was confirmed that there was a high economic effect in practical industrial aspects.

As described above, the immobilized enzyme prepared by immobilizing the enzyme on the biocatalyst immobilization carrier of the present invention can not only maintain high activity under the same reaction conditions as the existing free enzyme, but also reusability and storage and stability of the enzyme. In addition, since it has an excellent effect, it was recognized that an advantageous effect to prepare an immobilized enzyme capable of continuous enzymatic reaction over a long period of time. In addition, the use of the immobilized enzyme is expected to have a very industrially significant effect in terms of improving the purity of the product obtained by the enzymatic reaction, reducing the amount of enzyme used, and reduction of the reaction vessel.

Claims (8)

A carrier for immobilizing a biocatalyst in which chitosan and casein are cross-linked to silica beads and outer layers of the beads. The method of claim 1, The silica bead carrier for immobilizing a biocatalyst having a pore size of 50 GPa to 1000 GPa. The method of claim 1, The carrier is a carrier for immobilizing a biocatalyst which is prepared by immersing silica beads in a mixed solution of chitosan and casein, followed by drying, and reacting the dried beads with an epichlorohydrin solution. The method of claim 3, wherein The mixed solution is a carrier for immobilizing a biocatalyst, wherein chitosan and casein are mixed in an amount of 0.1 parts by weight to 10 parts by weight with respect to 100 parts by weight of silica beads. An immobilized enzyme in which the carrier for immobilizing the biocatalyst according to any one of claims 1 to 4 and the enzyme are bonded by an amide bond. The method of claim 5, wherein Immobilized enzyme prepared by reacting the biocatalyst immobilized carrier and enzyme in the presence of transglutaminase. The method of claim 5, wherein The enzyme is an immobilized enzyme alpha-amylase of Bacillus licheniformis ( Bacillus licheniformis ). A method of degrading starch using an immobilized alpha-amylase according to claim 7.

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