JPH08116970A - High-purity glucide-relating enzyme and production of immobilized glucide-relating enzyme - Google Patents

Abstract

PURPOSE: To enable a large volume and high level purification of a glucide- relating enzyme such as α-amylase or β-amylase at a practical treatment rate by packing a column with insoluble gel prepared by treating starch or gum with epichlorohydrin. CONSTITUTION: A column is packed with at least one of starch, guar gum and cellulose which is cross-linked with epichlorohydrin and a glucide-relating enzyme in water or an aqueous ammonium sulfate solution is adsorbed to the column. The column is washed sufficiently with an aqueous ammonium sulfate solution to remove the impurities not adsorbed on the column and then the enzyme is eluted and recovered with water or a buffer solution. A gel to which the enzyme is adsorbed is mixed with gelatin and formed to give the immobilized enzyme preparation. Further, when it is mixed with Celite and formed to give an immobilized enzyme preparation of high mechanical strength.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for producing a high-purity carbohydrate-related enzyme and an immobilized carbohydrate-related enzyme. More specifically, a novel method for producing high-purity enzyme products such as α-amylase, β-amylase, glucoamylase, cyclodextrin synthase, isoamylase, pullulanase, dextranase, cellulase, pectinase, and immobilization of the enzyme The present invention relates to a method for manufacturing lumber.

[0002]

2. Description of the Related Art As a method for purifying an amylase enzyme, a method of adsorbing the enzyme to starch granules has been used for a long time. For this adsorption, a batch method is used, and the starch granules after the adsorption treatment are washed to remove impurities such as non-adsorbed proteins.

[0003] The starch-adsorbed enzyme partially purified as described above can be directly used for the production of various carbohydrates. To obtain a purified enzyme product, the enzyme-adsorbed starch granules are suspended in water or a buffer and added. Generally, a method is employed in which the adsorbed enzyme is eluted and recovered by physicochemical treatment such as warming or pH adjustment.

There is also a method in which a soluble substrate or substrate analog having a higher affinity than starch is added to an enzyme-adsorbed starch suspension and stirred to solubilize and separate the enzyme.

In order to promote the adsorption of the amylase-based enzyme, the object may be achieved by stirring gently at a temperature as low as possible for a long time such as overnight, but generally the adsorption rate is low and ammonium sulfate (hereinafter, Ammonium sulfate is sometimes abbreviated.) And a batch method of adding ethanol are also used.

A column method is also known as a method for purifying amylase utilizing its adsorptivity to a substrate. In this case, a column in which sugars such as monosaccharides, oligosaccharides and cyclodextrins are covalently immobilized on a suitable carrier such as sepharose is used. After adsorbing an enzyme solution such as amylase through this column, unnecessary proteins and the like are washed and removed, and then a free substrate or substrate analog is passed through the column to elute and collect the adsorbed amylase-based enzyme.

As a method for producing immobilized enzyme lumber, there is a method for producing immobilized isomerase, which has been put to practical use as the most efficient immobilized enzyme lumber.

[0008]

However, in the batch method, when saccharide-related enzymes are adsorbed onto starch granules, ammonium sulfate, ethanol, etc. can be added to perform the adsorption satisfactorily, but the adsorbed enzymes are eluted and recovered. Requires suction filtration. At this time, clogging is likely to occur, and a washing operation is required for large-scale processing, but there is a problem that this operation is difficult to perform.

On the other hand, the column method is also called an affinity chromatography method, in which a monosaccharide or oligosaccharide having a high affinity for an amylase enzyme is bound to an appropriate support (carrier) by a chemical reaction. Need to be prepared.

[0010] These carriers are commercially available, but are expensive and are limited to the research level, and are unsuitable for large-scale purification of practical enzymes. Cyclodextrins are also known, but they are also expensive, and the amount of enzyme bound per gram of carrier is small. Many of the conventional affinity columns have a low adsorptivity for the amylase-based enzyme, and cannot be used as a practical and efficient purification method. In addition, there is an example in which a corn liquefied starch is included in diatomaceous earth and then treated with epichlorohydrin as a substrate for α-amylase adsorption, but in this method, enzyme adsorption and immobilization are insufficient. It is difficult to apply to production of enzyme lumber for practical use, such as difficulty in molding.

Since the isomerase is an intracellular enzyme,
It is obtained by solidifying the cells with gelatin and molding them.
No practical method for immobilization of extracellular enzymes and no production of immobilized enzyme lumber has been performed.

Recently, the use of inexpensive purified enzymes and immobilized enzymes in columns for bioreactors is increasing, and there is a strong demand for the development of a method for producing large amounts of inexpensive purified enzymes and a method for producing immobilized enzymes. I have been.

The present invention combines the advantages of both the batch method and the column method as a method for practically and efficiently purifying and immobilizing a carbohydrate-related enzyme satisfying such requirements in a large amount and at a high level. The present invention provides a product of a high-purity carbohydrate-related enzyme and an immobilized lumber of the enzyme by the new method.

[0014]

DISCLOSURE OF THE INVENTION In the present invention, in order to improve a conventional method for producing a carbohydrate-related enzyme, a method for purifying the enzyme has been studied diligently, and starch or gums crosslinked with epichlorohydrin have been filled. After using a column to adsorb saccharide-related enzymes in ammonium sulfate solution, wash thoroughly with the same solution,
It has been found that these problems can be solved by removing impurities such as proteins not adsorbed to the column and then eluting and recovering the adsorbed enzyme using water or a buffer. That is, it was found that if starch or gums were treated with epichlorohydrin to give an insoluble gel and packed in a column, the water or buffer solution would not be clogged and could be used repeatedly for a long time without lowering the flow rate.

[0015] Further, if the purified enzyme is mixed with a protein such as gelatin while being purified and adsorbed on the gel and molded, an immobilized enzyme material can be obtained, and celite, diatomaceous earth,
It has been found that an immobilized enzyme lumber having high mechanical strength can be obtained by mixing with shirasu, fine rocks, etc., and appropriately molding.

Further, when the carbohydrate-related enzyme is adsorbed onto the column thus prepared, if the flow rate is lowered to such a low temperature that the column does not freeze, the adsorption rate is low, but the adsorbent is not used. In both cases, the method of the present invention can be applied.

That is, the present invention relates to a method in which a sugar-related enzyme is added to a column packed with at least one substance selected from starch crosslinked with epichlorohydrin, guar gum crosslinked with epichlorohydrin, and cellulose in a solution of water or ammonium sulfate. Adsorbing and washing with an ammonium sulfate solution to remove impurities other than the enzyme, and then eluted and recovered the adsorbed enzyme using water or a buffer to produce a high-purity saccharide-related enzyme, A saccharide-related enzyme is adsorbed on a column packed with at least one substance selected from starch crosslinked with epichlorohydrin, guar gum crosslinked with epichlorohydrin, and cellulose in a solution of water or ammonium sulfate, and washed with an ammonium sulfate solution. After removing impurities other than the enzyme by using A method for producing an immobilized carbohydrate-related enzyme, comprising mixing at least one substance selected from gelatin, wheat protein, and soybean protein; starch crosslinked with epichlorohydrin; guar gum and cellulose crosslinked with epichlorohydrin. A saccharide-related enzyme is adsorbed on a column packed with at least one substance selected from the group consisting of water or ammonium sulfate solution, and washed with an ammonium sulfate solution to remove impurities other than the enzyme. Celite, diatomaceous earth,
At least one selected from volcanic ash soil (silas), clay, silica gel, alumina, bentonite, and ceramic
Provided is a method for producing an immobilized sugar-related enzyme, which comprises mixing a carrier of a seed and at least one substance selected from gelatin, wheat protein and soybean protein, and granulating or molding the mixture. Is.

There are various types of starch that can be used in the present invention, for example, soluble starch, corn starch, potato starch and the like. Also, instead of starch, cellulose, pectin, various gums can be used,
Some do not become gels by epichlorohydrin treatment, and some do not need to be gelled. For example, cellulose can be used as it is, and pectin does not become a gel even if treated with epichlorohydrin. Therefore, a mixture obtained by mixing with gelatin and drying is used. Furthermore, if it is mixed with celite and packed into a column so as not to cause clogging even with starch granules or soluble starch, it can be used in the present invention.

For adsorption of pectinase, guar gum treated with epichlorohydrin is suitable. For dextranase, dextransucrase, etc., dextran crosslinked with epichlorohydrin can be used, and for arginase, a crosslinked substrate corresponding to the enzyme such as crosslinked alginic acid can be applied to the method of the present invention.

There are various other cross-linking methods. At least one of such cross-linked starch, gums, and cellulose is packed in a column, and clogging occurs when an enzyme solution is passed through the column. Instead, the method of the present invention can be applied as long as the target enzyme can be adsorbed.

There are various other adsorbent gels and adsorbents such as Toyopearl, Biogel P-2, and Sephadex. In any case, ammonium sulfate is necessary for the efficient adsorption of the sugar enzyme, and in the ammonium sulfate solution. After adsorption, a purified enzyme preparation is obtained by eluting with water. Also, an enzyme immobilized on chitopearl can be obtained by applying the method of the present invention.

In the production of immobilized enzyme lumber, in the present invention, the enzyme-adsorbing carrier is gelatin (for example, commercially available purified powder,
Embedded with at least one substance selected from the group consisting of Hanui Chemical Co., Ltd., wheat protein (eg, vital gluten, manufactured by Sanwa Starch Industries), and soy protein (eg, isolated soy protein, manufactured by Fuji Oil). This prevents enzyme elution, but if elution is not negligible, glutaraldehyde can be used to reinforce fixation. The method of the present invention can be applied as long as the activity can be expressed without the enzyme being eluted.

Instead of ammonium sulfate, ethanol can be used as an adsorption aid or water can be used, but the efficiency is lower than that of ammonium sulfate. The method of the present invention can be applied to any salt, magnesium chloride salt, or the like as long as it exhibits an adsorption effect, but ammonium sulfate is most suitable in terms of effect and economy. In addition, as an adsorption aid, polyethylene glycol, glycerol, sodium sulfate, urea, and even triton,
Tweens and other surfactants were tested, but none were found to be superior to ammonium sulfate, although they were somewhat effective.

For the purpose of effective elution, soluble starch,
Even when using a saccharide solution such as α-cyclodextrin, maltitol, sorbitol, or methyl glucoside, nothing could be found that is superior to pure water or a buffer solution.

The epichlorohydrin crosslinking treatment was performed as follows. That is, 1 g of starch was dissolved in 5 mL of a 5 mol (M) sodium hydroxide solution, and 2 mL of epichlorohydrin was mixed with the solution, stirred vigorously at 60 ° C., gelled, left at room temperature overnight, and ground in a mortar. The resulting gel was stored at 4 ° C. by suction filtration and washing with water.

Approximately 1 mL of the gel was filled in a 5 mL syringe, and 100 to 200 μL of an ammonium sulfate solution of the enzyme was placed on the gel and left at room temperature for 30 minutes. Gravity drop, 2 to 3 minutes each for ammonium sulfate solution 1
After washing 5 times with mL, an enzyme-adsorbed gel was obtained. Pure water 1 mL each
The enzyme was eluted at least 5 times with, the enzyme activity of each fraction was measured by the Somogyi method, and the amount of protein was measured by OD280.

The enzyme agent to which the method of the present invention can be applied includes various carbohydrate-related enzymes, such as α-amylase and β-amylase.
-Amylase, glucoamylase, cyclodextrin synthase, isoamylase, pullulanase, dextranase, cellulase, pectinase, etc., in addition to these, DFA synthase, mannosidase, galactosidase, dextran sucrase, amylo-1,6-glucosidase, Trehalase, lysozyme, chitinase, chitosanase, isomerase, arginase, heparinase, β
-Glucuronidase, sialidase, chondroitinase, sugar chain-related enzymes such as N-acetylglucosaminidase, and other glycosyltransferases, as long as they adsorb,
The method of the invention can be applied.

The concentration of ammonium sulfate may be in the range of 0 to 5.8 M, but conditions for adsorbing the target enzyme should be selected, and usually about 0.5 to 3 M is preferable. At low temperatures, adsorption is possible even with a low ammonium sulfate concentration. In order to improve the adsorption, the flow rate may be reduced. Factors such as ammonium sulphate concentration, flow rate, and temperature should be considered and operated at economic flow rates.

The elution of the enzyme can be generally carried out by flowing pure water or a buffer solution at room temperature in an amount of 5 times the volume of the gel, but depending on the enzyme, 10 times the amount may be required for the recovery. High-purity enzyme preparation can be produced by concentrating the collected liquid with a collodion bag or an ultrafiltration membrane. 40m as buffer
M acetate buffer (pH 5.2), 40 mM phosphate buffer (pH 7.0) and the like can be used.

In the immobilization of the enzyme-adsorbed gel, the finely crushed gel was immersed in a 4-30% gelatin solution which had been dissolved and cooled at room temperature, taken out, and dried at room temperature. The immobilized enzyme prepared in this manner has a high activity expression rate when the concentration of the gelatin solution is low, but is easily eluted. Therefore, 2.
It was immersed in a 5% glutaraldehyde solution (pH 7.0), degassed for 3 hours, and washed with water to obtain an immobilized enzyme which was difficult to elute.

The enzymatic agent obtained by this method has low mechanical strength and is difficult to maintain a flow rate. Therefore, for a large column, it is desirable to add celite or the like as an immobilization carrier. Then, 5 mL of 20% gelatin solution, 2.8 g of Celite, and 1 g of enzyme adsorption gel were mixed and kneaded, put into a garlic press, squeezed, made into thin noodles, and dried at room temperature to obtain an immobilized enzyme lumber. In the case of soy protein and wheat protein, even if they are put into a garlic press and extruded, the liquid only oozes out and does not become thin noodles. Crushed to obtain an immobilized enzyme sawn timber.

Instead of celite, diatomaceous earth, volcanic ash soil (silas), clay, silica gel, alumina, bentonite,
The substance such as ceramic may be any substance as long as it becomes a solid by mixing and molding with at least one substance selected from enzyme adsorbent, gelatin, soybean protein and wheat protein, and can obtain an appropriate flow rate. In addition, activated carbon, porous glass, kaolinite, hydroxyapatite, calcium phosphate, metal oxide, synthetic resin and the like can be used as a carrier in the present invention.

The amount of gelatin used for immobilizing the enzyme is arbitrary, but if a 10% to 30% gelatin solution is used, molding is easy and a stable enzyme preparation can be obtained. Other than the above, soybean protein, wheat protein and the like can be used as the immobilized adhesive. In addition, proteins such as meat protein and twain may be available.

The form of the starting enzyme to be purified or immobilized includes commercially available crude enzyme solutions and crude enzyme powders.
When using a culture solution of an enzyme-producing bacterium as a starting enzyme, pH
After adjustment, the target enzyme may be isoelectrically precipitated, concentrated, and partially purified by ammonium sulfate precipitation, and then loaded on a starch gel.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto. Less than,
Unless otherwise specified,% is shown by W / V%, and a soluble starch solution was used as a substrate for activity measurement.

[0036]

【Example】

Example 1 A commercially available crude enzyme powdered Taka-amylase was dissolved in 400 μL of a cold ammonium sulfate solution, and 200 μL of a centrifuged clear solution was filled with 1 mL of a soluble starch gel treated with epichlorohydrin in 5 mL.
And allowed to stand for 30 minutes at room temperature for adsorption.
The enzyme was adsorbed by washing with ammonium sulfate solution 5 times with 1 mL each in free fall, 2 to 3 minutes. At room temperature, when a 3M ammonium sulfate solution was used, as shown in FIG. Could be adsorbed. 1 mL of water from the washing column, 5 mL each
Pour into the column once and allow the adsorbed enzyme to fall almost 100% by gravity
I was able to collect it.

Example 2 Example 1 except that the concentration of ammonium sulfate, drop conditions and temperature conditions were different.
1 (a), (b)
The adsorption rate drops to about 90% and 80% as shown in FIG.
An adsorption rate of 0% was obtained, and at a drop speed of 10 minutes or more at a low temperature, 50% or more of the activity was adsorbed. In addition, 2
In M or 1M ammonium sulfate, as shown in FIGS. 1 (a) and 1 (b),
Although the adsorption is incomplete, the adsorption rate is increased by lowering the temperature, and further, by setting the flow rate to about 30 minutes,
Even with pure water, 50% was adsorbed.

Example 3 193 mg of commercially available porcine pancreatic amylase (manufactured by Sigma) was added to 40
1 μm of epichlorohydrin-crosslinked soluble starch gel dissolved in 0 μL of 1M ammonium sulfate solution and the whole solution was passed through 1M ammonium sulfate solution
After being loaded on the L column and allowed to stand at room temperature for 30 minutes, it was allowed to flow off by gravity flow, washed with 1 mL of 1M ammonium sulfate solution 5 times, and then 1 mL of pure water was flowed 5 times by gravity flow, as shown in FIG. Such an activity recovery curve was obtained.

Example 4 Commercially available bacterial α-amylase (Bacillus amyloliquefaciens)
The activity recovery curve shown in FIG. 3 was obtained in the same manner as in Example 3 except that 4 mg of enzyme) and a cross-linked corn starch gel were used.

Example 5 The activity recovery curve of FIG. 4 was obtained in the same manner as in Example 3 except that 60 mg of commercially available α-amylase (manufactured by Wako, Bacillus subtilis enzyme) and crosslinked potato starch gel were used.

Example 6 Commercial cyclodextrin synthase (CGTase, manufactured by Hayashibara)
The activity recovery curve of FIG. 5 was obtained in the same manner as in Example 3 except that 60 mg was used. As is clear from the figure, the value of hydrolytic activity is low, but the cyclodextrin synthetic activity is high.

Example 7 Commercially available glucoamylase (Amano Pharmaceutical Co., glucozyme) 6
An activity recovery curve as shown in FIG. 6 was obtained in the same manner as in Example 3 except that 0 mg was used. Although the recovery rate in this example is low, most activities can be adsorbed when the ammonium sulfate concentration is 2 M or more.

Example 8 The activity recovery curve of FIG. 7 was obtained in the same manner as in Example 3 except that 60 mg of commercially available isoamylase (Hayashibara) was used. In addition, pullulan was used as an activity measurement substrate.

Example 9 The activity recovery curve of FIG. 8 was obtained in the same manner as in Example 3, except that 8 mg of commercially available pullulanase (manufactured by Hayashibara) was used. In addition,
Pullulan was used as an activity measurement substrate.

Example 10 An activity recovery curve as shown in FIG. 9 was obtained in the same manner as in Example 3 except that 14 mg of a commercially available β-amylase (manufactured by Wako, Wako) was used.

Example 11 The activity curve of FIG. 10 was obtained in the same manner as in Example 3, except that 58 mg of a commercially available crude cellulase enzyme (manufactured by Amano Pharmaceutical Co., Ltd.) was used. As is clear from the figure, a considerable amount of amylolytic enzyme is mixed in the commercially available crude cellulase enzyme.

Example 12 The activity curve shown in FIG. 11 was obtained in the same manner as in Example 3 except that 1 mg of a commercially available crude cellulase enzyme (manufactured by Meiji Seika) was dissolved in 400 μL of 1 M ammonium sulfate solution and loaded on a P-cellulose 1 mL column. It was

Example 13 2 mg of commercially available pectinase (Fluka) was added to a 1M ammonium sulfate solution 4
In the same manner as in Example 3 except that the solution was dissolved in 00 μL and loaded on a 1 mL column of epichlorohydrin crosslinked guar gum.
The activity curve of FIG. 12 was obtained. The activity in this example used pectin as a substrate.

Example 14 500 μg of commercially available dextranase (manufactured by Seikagaku Corporation) was
The activity curve of FIG. 13 was obtained in the same manner as in Example 3 except that the solution was dissolved in 00 μL of a 1 M ammonium sulfate solution and loaded on a 1 mL Sephadex G-10 column. Dextran was used as a substrate for activity measurement.

Example 15 1 g of the enzyme-adsorbed gel prepared in Example 6, 5 mL of a 20% gelatin solution, and 2.8 g of celite were mixed and kneaded, put into a garlic press, squeezed, made into thin noodles, dried at room temperature, and fixed. A lubricated enzyme sawn was obtained. Drying may be allowed to stand at room temperature overnight or more, and the mixture is appropriately crushed and packed into a column, and the starch solution is added to 50 to 6 times.
The solution was gently passed at 0 ° C to obtain a cyclodextrin-containing sugar solution.

Example 16 1 g of the enzyme-adsorbing gel prepared in Example 7, 5 mL of 20% gelatin solution and 2.6 g of diatomaceous earth were mixed and kneaded, put in a garlic press, squeezed to form thin noodles, dried at room temperature and fixed. An enzyme-treated lumber was obtained.

Example 17 1 g of the enzyme-adsorbed gel prepared in Example 8, 5 mL of a 20% gelatin solution, and 2.7 g of shirasu were mixed, kneaded, put in a garlic press, squeezed, dried into thin noodles, dried at room temperature, and fixed. A lubricated enzyme sawn was obtained.

Example 18 1 g of the enzyme-adsorbed gel prepared in Example 9, 5 mL of a 20% gelatin solution and 2.8 g of silica gel were mixed, kneaded, put into a garlic press, squeezed, made into fine noodles, and dried at room temperature.
An immobilized enzyme sawn was obtained.

Example 19 1 g of the enzyme-adsorbed gel prepared in Example 10, 5 mL of a 20% gelatin solution, and 2.6 g of ceramic powder were mixed, kneaded, put into a garlic press, squeezed, made into thin noodles, and dried at room temperature. An immobilized enzyme sawn was obtained.

Example 20 1 g of each of the enzyme-adsorbed gels prepared in Examples 6 and 10, 20%
10 mL of gelatin solution and 5.4 g of ceramic powder were mixed and kneaded, put in a garlic press, squeezed, made into a thin noodle shape, and dried at room temperature to obtain an immobilized mixed enzyme lumber.

Example 21 1 g of each of the enzyme-adsorbed gels prepared in Examples 6, 9 and 10
15 mL of a 0% gelatin solution and 8.4 g of celite were mixed and kneaded, put into a garlic press, squeezed, made into a thin noodle shape, and dried at room temperature to obtain an immobilized mixed enzyme lumber.

Example 22 To 500 mg of commercially available soybean protein, 5 mL of pure water was added, dissolved in a boiling water bath and allowed to cool to room temperature. Then, 1 g of the enzyme-adsorbed gel prepared in Example 6 and 3.5 g of Celite were mixed. Kneading, 5
The mixture was spread on a wall of a 0 mL beaker to a thickness of about 1 or 2 mm, dried at room temperature for 2 days, and crushed to an appropriate size to obtain an immobilized mixed enzyme lumber. If half of the soybean protein is replaced with gelatin, it can be formed into fine noodles.

Example 23 To 500 mg of commercially available wheat protein, 5 mL of pure water was added, heated on a boiling water bath and allowed to cool to room temperature, and 1 g of the enzyme-adsorbed gel prepared in Example 6 and 2.5 g of celite were mixed. Kneading, 5
The mixture was spread on a wall of a 0 mL beaker to a thickness of about 1 or 2 mm, dried at room temperature for 2 days, and crushed to an appropriate size to obtain an immobilized mixed enzyme lumber.

[0059]

According to the method of the present invention, when a specific substance is packed in a column, a saccharide-related enzyme can be adsorbed in water or ammonium sulfate solution using an enzyme adsorption carrier capable of maintaining a flow rate, and impurities other than the enzyme can be adsorbed. Can be washed and removed with the same solution to obtain an enzyme-linked gel and an enzyme-conjugated product, and the enzyme can be eluted with pure water or a buffer to obtain a purified enzyme.

The enzyme-conjugated gel or enzyme-conjugated product can be used as it is for immobilization, mixed with various immobilization base materials, and bound with gelatin or the like to obtain an immobilized enzyme material. As described above, the method of the present invention is extremely efficient in that an enzyme agent for immobilization can be produced simultaneously with purification, and its application range is extremely wide.

Further, various sugar-related enzymes can be combined and immobilized, and a branched cyclodextrin can be directly produced from a starch solution. Furthermore, various carbohydrates can be prepared by arranging immobilized sugar chain-related enzyme materials and the like in parallel or / and in series and passing them through various substrates.

Further, even if the enzyme has a low reaction efficiency, the desired purpose can be achieved by recycling.
For example, a small amount of trehalose is reversely synthesized when immobilized trehalase is allowed to act on high-concentration glucose, but if the enzyme column and activated carbon column are connected in series and the glucose solution is recycled, trehalose accumulates and the accumulated sugar Quality can be produced by eluting with ethanol. Since the amylase is adsorbed from the cellulase enzyme agent, it can be applied to the removal of amylase-based enzyme activity contained in other enzyme agents.

[Brief description of drawings]

1 (a), 1 (b) and 1 (C) are activity recovery curves of powdered Taka-amylase, a commercially available crude enzyme, when using various concentrations of ammonium sulfate solutions.

FIG. 2 is an activity recovery curve of a commercially available porcine pancreatic amylase using a 1M ammonium sulfate solution.

FIG. 3: Commercially available bacterial α-amylase (Bacillus amyloli)
2 shows an activity recovery curve when a 1M concentration ammonium sulfate solution is used for an enzyme (quefaciens enzyme).

FIG. 4 is an activity recovery curve of a commercially available α-amylase (Bacillus subtilis enzyme) when a 1M concentration ammonium sulfate solution is used.

FIG. 5: Commercial cyclodextrin synthase (CGTas
It is an activity recovery curve when using 1M concentration ammonium sulfate solution about e).

FIG. 6 is an activity recovery curve of a commercially available glucoamylase (glucozyme) when a 1M ammonium sulfate solution is used.

FIG. 7 is an activity recovery curve of a commercially available isoamylase when a 1 M concentration ammonium sulfate solution is used.

FIG. 8 is an activity recovery curve of a commercially available pullulanase when a 1M ammonium sulfate solution is used.

FIG. 9: 1 M of commercially available β-amylase (wheat)
It is an activity recovery curve when a concentration ammonium sulfate solution is used.

FIG. 10 is an activity recovery curve for a commercially available crude cellulase enzyme using a 1M ammonium sulfate solution.

FIG. 11 is an activity recovery curve of a commercially available crude cellulase enzyme when a 1M ammonium sulfate solution is used.

FIG. 12 is an activity recovery curve of a commercially available pectinase using a 1M ammonium sulfate solution.

FIG. 13 is an activity recovery curve of a commercially available dextranase using a 1M ammonium sulfate solution.

[Explanation of symbols]

The thin line is the 280 nm UV absorption curve (measurement of protein content)
, And the thick line indicates the activity curve (measurement of the generated reducing power). In addition, → indicates the point in time when the pure water for elution started to flow.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12N 9/34 9/42 9/44 9/46 11/10 11/12

Claims (7)

[Claims] 1. A saccharide-related enzyme is adsorbed on a column packed with at least one substance selected from starch crosslinked with epichlorohydrin, guar gum crosslinked with epichlorohydrin and cellulose in a solution of water or ammonium sulfate, A method for producing a high-purity saccharide-related enzyme, comprising washing with an ammonium sulfate solution to remove impurities other than the enzyme, and eluting and recovering the adsorbed enzyme using water or a buffer. 2. The method according to claim 1, wherein the concentration of ammonium sulfate is 0 to 5.8 mol. 3. The method according to claim 1, wherein the carbohydrate-related enzyme is any of α-amylase, β-amylase, glucoamylase, cyclodextrin synthase, isoamylase, pullulanase, dextranase, cellulase and pectinase. 4. A saccharide-related enzyme is adsorbed on a column packed with at least one substance selected from starch crosslinked with epichlorohydrin, guar gum crosslinked with epichlorohydrin and cellulose in a solution of water or ammonium sulfate, After removing impurities other than the enzyme by washing with an ammonium sulfate solution, an immobilized saccharide characterized by mixing an enzyme-adsorbed gel with at least one substance selected from gelatin, wheat protein and soy protein. Method for producing related enzymes. 5. The method according to claim 4, wherein the concentration of ammonium sulfate is 0 to 5.8 mol. 6. A sugar-related enzyme is adsorbed in a solution of water or ammonium sulfate to a column packed with at least one substance selected from epichlorohydrin-crosslinked starch, epichlorohydrin-crosslinked guar gum and cellulose. After washing with ammonium sulfate solution to remove impurities other than the enzyme, at least one selected from celite, diatomaceous earth, volcanic ash soil (shirasu), clay, silica gel, alumina, bentonite, and ceramics on the sugar-related enzyme adsorbent gel. A method for producing an immobilized sugar-related enzyme, which comprises mixing a seed carrier and at least one substance selected from gelatin, wheat protein and soybean protein, and granulating or molding the mixture. 7. The method according to claim 6, wherein the concentration of ammonium sulfate is 0 to 5.8 mol.