JP2527064B2 - Enzyme stabilization method, stabilizer and enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to cyclodextrin glycosyltransferase (CGTase), glucoamylase (GA), β-
The present invention relates to a method for stabilizing enzymes such as glucosidase (GD), α-amylase, β-amylase, and maltooligosaccharide-forming enzyme.

CGTase is an enzyme commonly used on an industrial scale to produce cyclodextrin from starch. Cyclodextrin is a substance that is widely used as a bulking agent in various foods, and is an essential substance for producing a clathrate compound that has a wide application in enhancing the stabilization of pharmaceuticals and deodorizing odorous substances, Therefore, stabilizing CGTase is a very significant technology in industry.

GA is an enzyme that is used routinely when glucose is produced from starch, and GA stabilization is an extremely significant technique.

Still another enzyme according to the present invention is a substance having extremely high degree of application which is indispensable for manufacturing pharmaceuticals and providing inexpensive and high-quality food, and it is extremely industrially difficult to stabilize these enzymes. It is of great significance.

[Conventional Technology] Recent technological advances in the technology of enzyme reactions are remarkable, and there is a sense of another life.

In addition, the immobilized enzyme method applying chitosan beads in recent years, in particular, performs an efficient reaction in that the amount of enzyme used is smaller than the conventional method such as an enzymatic reaction in a solution. It has been known that there is a remarkable effect in that it is possible.

Several patent applications have been made for the immobilized enzyme method using chitosan beads.For example, Japanese Patent Laid-Open No. 63-196290 discloses that after immobilizing cyclodextrin glucanotransferase on porous chitosan beads. ,
Further, a technique of performing a crosslinking treatment with a crosslinking agent is disclosed.

[Problems to be Solved by the Invention] Although many studies have been made on the efficient application method of the above-mentioned enzyme, sufficient results have not necessarily been obtained for stabilization of the enzyme itself. Enzymes lose their activity over time,
For example, in the case of CGTase, the activity was normally halved within a few days, and the activity was usually lost after several tens of days.

Even in the case of immobilized enzyme, in such a case, there was no method other than supplying the enzyme itself newly. The technical difficulty to be solved by the present invention lies in this point.

[Means for Solving the Problems] As a result of intensive research for solving the technical problems, the present inventors have found that the following general formula [I] (Here, R represents hydrogen, lower alkyl, phenylalkyl, phenylalkenyl, phenylalkynyl, phenoxyalkyl, phenoxyalkenyl, or phenoxyalkynyl. Here, phenyl includes substituted or unsubstituted). N-
We have encountered the fact that the above-mentioned problems are successfully solved by performing an enzymatic reaction in the presence of a substituted moranolin derivative or a glucose oligomer thereof.

More interestingly, the present inventors have found that, in the presence of the compound represented by the above general formula [I] or the glucose oligomer thereof, the enzyme once subjected to the enzymatic reaction is followed by the presence of [I] or the glucose oligomer thereof. The present inventors have also completed the present invention by discovering the fact that the high activity rate of the activated enzyme is maintained even in the non-activated state.

Therefore, one of the features of the present invention is a method for stabilizing an enzyme by carrying out an enzymatic reaction in the presence of [I] or a glucose oligomer thereof, and another feature is [I] or its The enzyme itself is stabilized by carrying out an enzymatic reaction in the presence of glucose oligomers.

Exemplifying the method for producing an immobilized enzyme applied in the present invention, a cross-linking agent is first reacted with chitosan beads to form cross-links on amino groups on the surface of chitosan beads. Then, the enzyme according to the present invention is immobilized on the end of the cross-linking agent.

As the cross-linking agent according to the present invention, for example, glutaraldehyde or the like can be used.

As the chitosan beads according to the present invention, for example, Chitopearl (Fuji Spinning Co., Ltd.) BCW-1000 series and the like can be used.

In order to produce the immobilized enzyme according to the present invention, for example, a suitable buffer solution (eg, pH 6.
0.1M acetic acid buffer of 0), the above chitosan beads are added, and the mixture is gently shaken, an appropriate concentration (eg, 25 m / v%) of glutaraldehyde is added, and an appropriate time (eg, 1 to 48 hours), After gently shaking, filtering and washing with water, in a suitable buffer solution (eg, 0.025M acetate buffer of pH 6.0), add a CGTase solution of an appropriate concentration (eg, 1 to 500 mg / ml), After gently shaking for an appropriate time (eg, 1 to 48 hours), it can be obtained by filtration and washing with water.

The above method is a method first established by the present inventors, whereby the immobilized enzyme method can be carried out without using the technique disclosed in JP-A-63-196290. it can.

For example, immobilized CGTase can retain 90% or more of its initial activity after 413 days at 55 ° C, and immobilized GA can give 80% or more of its initial activity after 43 days at 55 ° C. ,
It can hold 80% or more at 50 ° C for 175 days and 90% or more at 40 ° C for 133 days.

Although it is not always clear why this fact occurs, [I] is a pseudo-sugar having a structure similar to glucose, which is a constituent sugar of the substrate (eg, starch, dextrin, maltooligosaccharide, maltose sugar) of these enzymes. Since it is (pseudopiperidinose), it can be inferred that some function is exerted in relation to the enzyme.

 The constitution of the present invention will be described in more detail below.

The gist of the present invention lies in the point that the compound represented by the general formula [I] or the glucose oligomer thereof is present in the reaction of the enzyme.

In this case, the enzyme may be in a dissolved state or immobilized, but usually the immobilized enzyme is used for industrial purposes.

The compound represented by [I] according to the present invention is a known substance, for example, JP-B-56-099195 and JP-B-60-
2038 gazette, Japanese Patent Publication No. 61-2076, Japanese Patent Publication No. 62-242691
It can be obtained by the method disclosed in Japanese Patent Publication.

The glucose oligomer according to the present invention has the following general formula [II] (Wherein R is the same as above, n is an integer from 0 to 24).

The lower alkyl represented by R in the general formulas [I] and [II] according to the present invention includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.
-Butyl, tert-butyl and the like can be mentioned.

The carbon number of alkyl in phenylalkyl is 1 to
5 can be mentioned. In this case, phenyl includes substituted or unsubstituted phenyl.

The carbon number of alkenyl in phenylalkenyl is
2-5 can be mentioned. In this case, phenyl includes substituted or unsubstituted phenyl.

The number of carbon atoms of alkynyl in phenylalkynyl is
2-5 can be mentioned. In this case, phenyl includes substituted or unsubstituted phenyl.

The carbon number of alkyl in phenoxyalkyl is 1
~ 5 can be mentioned. In this case, phenyl includes substituted or unsubstituted phenyl.

The alkenyl in the phenoxyalkenyl has 2 to 5 carbon atoms. In this case, phenyl includes substituted or unsubstituted phenyl.

The alkynyl in the phenoxyalkynyl has 2 to 5 carbon atoms. In this case, phenyl includes substituted or unsubstituted phenyl.

The enzyme according to the present invention can be stabilized, for example, as follows.

In stabilizing the immobilized enzyme, the compound of the present invention is added to the immobilized enzyme obtained as described above. When the compound of the present invention is used as a substrate, the stabilization of the present invention can be carried out simply by adding the substrate itself. When the substrate is not the compound of the present invention, the enzymatic reaction can be carried out on the immobilized enzyme stabilized by the compound of the present invention.

After adding the compound of the present invention, for example, the reaction is carried out at a temperature at which an ordinary enzymatic reaction is carried out for an ordinary time.

[Examples] Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples according to the present invention.

Reference example 1 Immobilization of CGTase (1) 2.5 w / v% of 10 Fujibo Ltd. Chitopearl BCW-3010
0.1M acetate buffer containing glutaraldehyde (pH 6.0) 4
After immersion at 0 at room temperature for 24 hours, it was thoroughly washed with ion-exchanged water. Then 20 mg of protein per 1 ml of chitopearl
Corresponding CGTase [Hayashibara Biochemical Research Laboratories, Inc. Bacillus stearothermophilus
Origin] was further added, and an acetate buffer (pH 6.0) was further added so that the final concentration was 0.025 M, so that the total liquid volume was 30. Immersion was carried out for 24 hours at room temperature as it was, followed by thorough washing with deionized water to obtain immobilized CGTase. The protein adsorption amount of the obtained beads was 19.5 mg / ml (beads).

Reference Example 2 Immobilization of CGTase (2) In the same manner as in Reference Example 1, CGTase derived from Bacillus stearothermophilus was immobilized so that the amount of protein adsorbed was 5 mg / ml (beads).

Reference Example 3 Immobilization of GA After dialysis of Amano Pharmaceutical Co. glucozyme NL-3, 60-
A protein concentration of 65 mg / ml was used as the GA aqueous solution.

2.5% w / v% of 1 from Fuji Spinning Co., Ltd. Chitopearl BCW-3010
50 mM acetate buffer (pH 5.0) containing glutaraldedo 3
After soaking at room temperature for 24 hours, it was thoroughly washed with ion-exchanged water. Then, add GA aqueous solution equivalent to 100 mg of protein per 1 ml of chitopearl, and further add water to bring the total volume to 3
I tried to become. Immersion was carried out for 24 hours at room temperature as it was, followed by thorough washing with deionized water to obtain immobilized GA. The protein adsorption amount of the obtained beads was 12.8 mg / ml (beads).

Reference Example 4 Culture of Moranolin-Producing Bacteria 200 ml of a medium having a composition of starch 2%, soybean powder 1%, potassium chloride 0.05%, magnesium sulfate heptahydrate 0.05%, salt 0.2%, calcium carbonate 0.35% (pH 7.2) was used. , Transfer to a 500 ml Erlenmeyer flask and sterilize as usual, then add Streptomyces lavendulae SEN-
Strain culture of 158 strains was inoculated with several platinum ear spores and incubated at 27 ° C for 3
The culture was performed with shaking at 200 rpm for a day, and this was used as a seed culture.

Starch 8%, soybean flour 3%, yeast extract 1.5%, potassium chloride 0.05%, magnesium sulfate heptahydrate 0.05%, salt 0.1.
Medium 250 composed of 1% and calcium carbonate 0.15% (pH 7.2)
Was placed in a 420-volume jar fermenter, sterilized in the usual manner, and seed culture 2.4 was inoculated. 100 revolutions /
Min, aeration rate 125 / min, and culture was carried out at 27 ° C for 10 days. As a result of high-performance liquid chromatography quantifying the moranolin in the culture broth, about 3500 μg / ml of moranolin was contained.

Reference Example 5 Partial Purification of Moranolin-Containing Culture Solution The culture solution obtained by the method of Reference Example 4 was applied to an ultrafiltration membrane (UF Module; MU-6303-HG manufactured by Kuraray Co., Ltd.), and the passing solution was further subjected to a reverse osmosis membrane (Toyobo Co., Ltd.). The non-passage liquid concentrated by applying a hollow fiber manufactured by HR-5155 F1) was used as a partially purified product.

Reference Example 6 Glycosylation reaction of partially purified product of culture solution containing moranolin Partly purified product obtained by treating culture solution (2000) cultured in the same manner as in Reference Example 4 by the method of Reference Example 5 (150; containing about 7 kg of moranolin) A 420 ton jar fermenter and 50
The column was added to the jar fermenter of the reactor connected. Further, 70 kg of Amycor No. 6L was added and dissolved by heating. After adjusting the pH to 9.0 with 6N sodium hydroxide, water was added to bring the total volume to 280. Immobilized CGTase 30 produced by the method of Reference Example 1 was packed in a column and reacted at 55 ° C. for 48 hours while circulating at a flow rate of 3 / min to obtain a moranolin glycosyl transfer reaction solution.

Example 1 Stabilization of immobilized CGTase (transglycosylation reaction of moranolin) Moranolin 2.5 w / v%, Amycol No. 6L (manufactured by Nippon Starch Chemical Co., Ltd.) 25 w / v%, in a 100 ml aqueous solution of unadjusted pH, Reference Example 1 10 ml of the immobilized CGTase obtained in 1. was added, and rotation and shaking was continued at 55 ° C.
Beads (immobilized CG
(Tase) is filtered, washed thoroughly with water, and then immobilized as follows.
The enzyme activity of CGTase was measured.

(Method for measuring enzyme activity of immobilized CGTase) Moranolin 2.5w / v%, Amycol No.6L 25w / v%, pH
1 ml of beads (washed and immobilized CGTase after a lapse of a certain time at 55 ° C) was added to 10 ml of an unadjusted aqueous solution, and the mixture was reacted at 55 ° C for 24 hours. 3 ml of the reaction solution was added to the strongly acidic ion exchange resin Dowex 50W.
Pass it through 7 ml of x2 (H ⁺ ), wash thoroughly with water, elute with 0.5N ammonia water, concentrate the eluate to dryness under reduced pressure, dissolve in 3 ml of water, and inject 10 μ of this into high performance liquid chromatography. And analyzed to determine the concentration of unreacted moranolin. The analytical conditions for high performance liquid chromatography are: column (Nucleosil 5NH ₂ , 5 μm, 4 mm id × 25 cm), developing solvent (acetonitrile-water = 70: 30), detector (Hitachi 655A).
-30, RI detector), data processor (Hitachi D-200)
0).

When the reaction progress rate at the time of starting the rotary shaking at 55 ° C. was set to 100, the relative reaction progress rate after a certain period of time was calculated by the above formula, and this value was defined as the enzyme activity retention rate. The results are shown in Table 1. The fact that the enzyme activity of the immobilized CGTase is stabilized by the moranolin according to the present invention is clear.

Example 2 Stabilization of immobilized CGTase (sucrose transfer reaction of N-methylmoranolin) A jacketed column having a diameter of 1 cm was filled with 20 ml of the immobilized CGTase obtained in Reference Example 1, and 1.5 w of N-methylmoranolin was used.
/ v%, soluble starch 9w / v%, pH unadjusted solution were continuously passed through at a passage rate of 160 ml / day at 55 ° C, and the reaction progress rate of the passed solution was determined with time. The reaction progress rate and activity retention rate were determined in the same manner as in Example 1. As a result, it was found that almost 100% of the activity was retained even after 140 days.

Example 3 Stabilization of immobilized CGTase (moranolin, glucose oligomer of moranolin) 1 ml of the immobilized CGTase obtained in Reference Example 2 was supplemented with moranolin (2.5 mg).
w / v%) and a glucose oligomer of moranolin (mixture) (5.0 w / v%) were added, and the mixture was shaken at 55 ° C. in a total volume of 10 ml.

The glucose oligomer of moranolin refers to the following.

Moranolin 3w / v% and Amycol No.6L 30w / v% were dissolved in water to adjust the total volume to 500ml without adjusting the pH, and then 50ml of immobilized CGTase obtained in Reference Example 1 was added and reacted at 55 ° C for 48 hours. After that, the solution was filtered through a glass filter to remove the immobilized enzyme, and the filtrate was washed with a strongly acidic ion exchange resin Dowex 50W x 2 (H ⁺ ) 2
After passing through a 00 ml column and washing thoroughly with water, elute with 0.5N ammonia water 1, concentrate the eluate to dryness under reduced pressure, dissolve again in an appropriate amount of water, and use the strongly acidic ion exchange resin DIAION SA- 11A (OH ^-) subjected to a column of 100 ml, eluted with water, this effluent and eluate and concentrated to dryness under reduced pressure combined, Moranorin the basic fractions of transglycosylation reaction solution Moranorin obtained by this procedure Glucose oligomer.

Table 2 shows the residual enzyme activity retention rate (%) when the enzyme activity at the time of adjustment was set to 100. The method of measuring the enzyme activity and the like were all the same as in Example 1.

CG on Moranolin, Glucose Oligomers of Moranolin
It is clear that it has a function of retaining the activity of Tase.

Example 4 Stabilization of immobilized enzyme CGTase (N-substituted moranolin, N-
Substituted Glucosyl Moranolin) In 1 ml of immobilized CGTase obtained in Reference Example 1, N-substituted moranolin (0.5 w / v%), N-substituted glucosyl moranolin (0.
5 w / v%) and shaken in a total volume of 10 ml at 55 ° C for 14 days,
The residual enzyme activity was measured. Table 3 shows the residual enzyme activity rate when the initial enzyme activity was set to 100. The method for measuring the enzyme activity is the same as in Example 1.

It is clear that the compounds according to the invention show a good stabilizing effect.

Example 5 Stabilization of immobilized GA (moranoline isotransfer reaction solution) A reaction solution containing unreacted moranolin, unreacted Amycol No. 6L and oligoglucosylmoranolin produced by the method of Reference Example 6 was treated with sulfuric acid. The pH of the aqueous solution adjusted to 5.2 was applied to a jacketed column having a diameter of 1 cm and filled with 20 ml of immobilized GA obtained in Reference Example 3 at a passage rate of 480 ml / day, and
The reaction was continuously carried out at 0 ° C. After reacting for a certain period of time, the immobilized GA in the column was taken, the enzyme activity was measured by the additional method, and the activity retention rate (stability) was determined when the enzyme activity at the start of the reaction was 100%. The results are shown in Table 4.
(Activity measuring method of immobilized GA) A fixed amount (30-80 mg wet weight) of immobilized GA was taken on a glass filter of G-2, lightly sucked to filter water, and then immobilized GA on the glass filter. Was placed on lens paper to remove excess water, and then put in a sample bottle with a stopper to determine the wet weight of immobilized GA. Next, immobilized GA 10
It was added to ml of 5% maltose solution (0.05M acetate buffer, pH 4.6) and incubated at 37 ° C for 20 minutes. 100 of the reaction solution
μ was added to 0.05 N sodium hydroxide to stop the reaction.

Glucose was quantified using 10 μ of this reaction stop solution. The quantification of glucose was performed using Diacolor GC (manufactured by Ono Pharmaceutical Co., Ltd.).

The activity was defined as follows. The enzyme activity for producing 1 μM glucose per minute was defined as 1 unit. As a blank, 0.05N sodium hydroxide mixed with the substrate in the same volume was used instead of the reaction solution.

At this time, the activity of immobilized GA is 5.56 × OD _SA / OD _STD / quality weight of immobilized GA. Here OD _SA is the sample (immobilized GA)
Absorbance at 500 nm, OD _STD means the absorbance at 500 nm of a 1 mg / ml glucose aqueous solution. In Table 4, the value of the activity retention varies, but this is a measurement error. The stabilizing effect of the compound of the present invention on immobilized GA is clear.

Example 6 Stabilization of immobilized GA (molanorein transglycosylation reaction solution) The same experiment as in Example 5 was carried out at 40 ° C. The results are shown in Table 5.

Example 7 Stabilization with GA (Aqueous Solution) (Moranolin, N-Methylmolanolin) Moranolin and N-methylmolanolin inhibit the enzymatic activity of glucoamylase, so the stabilizing effect on glucoamylase in solution is as follows. I examined it. The enzyme is Rhizopus niveu manufactured by Seikagaku Corporation.
s) derived reagent glucoamylase was used.

5 mg of enzyme was dissolved in 1 ml of 0.1M acetate buffer (pH 5.0), and 6000 μg / molnolan and N-methylmoranolin were dissolved in the solution.
It was dissolved so that the amount became ml, put into a screw cap test tube, and incubated at 50 ° C. Each enzyme solution is
Dilute 100-fold with 0.1M acetate buffer (pH 5.0) to obtain 100μ
Take 5% maltose solution (0.05M acetate buffer, pH 4.
6) Add 400μ and incubate at 40 ℃ for 20 minutes. Take 100μ of the reaction mixture and add 0.05μ sodium hydroxide 100μ
Is added to stop the reaction. Take 100 μm from this, add 3.0 ml glucose measuring reagent Diacolor GC, let stand for 30 minutes at 37 ° C to develop color, then cool with ice,
The absorbance was measured at 0 nm to measure the amount of glucose. This glucose production amount was defined as enzyme activity in the presence of an inhibitor. The relative enzyme activity was calculated when the initial enzyme activity (glucose production) was set to 100. The results are shown in Fig. 1. The enzyme stabilizing effect of the compound of the present invention is clear.

Example 8 Stabilization of immobilized GA (moranolin, N-alkylmoranolin) 5 ml of immobilized GA prepared by the method of Reference Example 3 was added with 10 ml of 0.1 M acetate buffer (pH 5.0) to form an airtight sample bottle. I put it in. At this time, moranolin and N-alkylmoranolin were dissolved at 100 mM and incubated at 55 ° C., and the residual enzyme activity was measured by the method described in Example 5. The results are shown in FIG. The enzyme stabilizing effect of the compound of the present invention is clear.

Example 9 Stabilization of β-glucosidase by moranolin and N-methylmoranolin The stabilizing effect of moranolin and N-methylmoranolin on β-glucosidase was examined. The test sample is
(1) is β-glucosidase (300 μg / ml) 0.2M buffer solution 1
ml only, (2) is β-glucosidase (300 μg / ml) 0.2
Moranoline hydrochloride 6 mg / ml added to 1 ml of M buffer,
(3) is β-glucosidase (300 μg / ml) 0.2M buffer solution 1
6 mg / ml of N-methylmoranoline hydrochloride added to ml,
Is. These test samples were kept at 60 ° C. and the residual enzyme activity was measured with time.

The enzyme activity was measured by a method of measuring the absorbance at 400 nm using p-nitrophenyl-β-D-glucoside as a substrate.

That is, 0.014M p-nitrophenyl-β-D-glucoside 200μ, 200μ of the enzyme solution sampled over time from the sample diluted to an appropriate amount and 0.2M acetate buffer (pH 4.5) 600μ The mixture is reacted at 30 ° C for 15 minutes, 2 ml of 0.1 M disodium carbonate is added to stop the reaction, color is developed, and the color is measured at 400 nm. The absorbance at the start of the reaction was set to 100, and the relative ratio of the absorbance at each time was taken as the residual activity.

The results are shown in Fig. 3. The stabilizing effect of moranolin and N-methyl moranolin is clear.

Example 10 Stabilized immobilized CGTase Immobilized CG after carrying out glycosyl transfer reaction by the method of Reference Example 6
Immobilized CGTase 5 ml that took a part of Tase and washed thoroughly with water
Then, 10 ml of 0.1 M acetate buffer (pH 6.0) was added, and the mixture was placed in an airtight sample bottle and stored at 60 ° C., and the residual enzyme activity was measured with time.

On the other hand, the immobilized CGTase prepared by the method of Reference Example 1 was similarly stored and the residual enzyme activity was measured. The enzyme activity was measured by the method described in the literature (Y. Ezure; Agri.
c. Biol. Chem., 49, 2159 (1985)). As a result, the immobilized CGTase prepared in Reference Example 1 after storage for 11 days was about 28
Although only about 100% of the enzyme activity remained, about 91% of the enzyme activity remained in the immobilized CGTase thoroughly washed after the glycosyl transfer reaction.

Example 11 Stabilization of α-amylase (gluconor oligomer of moranolin) α-amylase (manufactured by Seikagaku Corporation) was dissolved in 0.2 M acetate buffer (pH 6.0) to 200 μg / ml, and Moranolin glucose oligomer was added at 6000 μg / ml. After incubation at 60 ° C, the residual activity was measured over time. The residual activity of α-amylase was 4% in 0.2M acetate buffer (pH 5.5) using 1% soluble starch as a substrate.
The reaction solution obtained by reacting at 0 ° C for 15 minutes was subjected to the 3,5-dinitrosalicylic acid method (Kyoto University Agricultural Chemistry Experimental Manual. 9th edition, 2nd volume,
619). The relationship between the residual ratio (%) and the time with respect to the enzyme activity at the time of initial initiation is shown in FIG. Controls were those without any glucose oligomers of moranolin.

Α of the glucose oligomer of moranolin according to the present invention
-A stabilizing effect on amylase is evident.

Example 12 Stabilization of β-amylase (moranolin) β-amylase derived from soybean was dissolved in 0.02M acetate buffer (pH 4.8) to a concentration of 500 μg / ml, and then 60
Moranolin was added to a final concentration of 00 μg / ml. After incubation at 65 ° C, the residual activity was measured over time. The control was that no moranolin was added at all.

The activity of β-amylase was measured in the same manner as in Example 11, 3,
According to the 5-dinitrosalicylic acid method. The results are shown in Fig. 5.

The stabilizing effect of moranolin according to the present invention on β-amylase is clear.

[Brief description of drawings]

FIG. 1 shows the results of Example 7. The vertical axis represents the residual activity rate (%), and the horizontal axis represents the time (days). ◯ represents control, ● represents moranolin, and Δ represents N-methylmoranolin. FIG. 2 shows the results of Example 8. The vertical axis represents the residual rate of activity (%), and the horizontal axis represents time (days). ● indicates control, ○ indicates moranolin, ▲ indicates N-methylmoranolin, ■ indicates N-ethylmoranolin, □ indicates N- (n-
Propyl) moranolin, ▼ represents N- (n-butyl) moranolin, and ∇ represents N- (n-amyl) moranolin. FIG. 3 shows the results of Example 9. The vertical axis represents the residual rate of activity (%), and the horizontal axis represents time (hours). ◯ represents control, ● represents moranolin, and Δ represents N-methylmoranolin. FIG. 4 shows the results of Example 11. The vertical axis represents the residual activity rate (%), and the horizontal axis represents time (hours). ○ represents a control, and ● represents a glucose oligomer of moranolin. FIG. 5 shows the results of Example 12. The vertical axis represents the residual activity rate (%), and the horizontal axis represents time (hours). ◯ represents control, and ● represents moranolin.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12P 19/20 C12P 19/20 19/22 19/22 (72) Inventor Shin Sugiyama Kyoto City, Kyoto Prefecture Midori Tominaga, Examiner, Nippon Shinyaku Co., Ltd. 14 Nishinoshomonguchicho, Kichijoin, Minami-ku

Claims (6)

(57) [Claims] 1. When the enzyme acts on a substrate, the following general formula [I] (Here, R represents hydrogen, lower alkyl, phenylalkyl, phenylalkenyl, phenylalkynyl, phenoxyalkyl, phenoxyalkenyl, or phenoxyalkynyl. Here, phenyl includes substituted or unsubstituted). N-
A method for stabilizing an enzyme, which comprises reacting in the presence of a substituted moranolin derivative or a glucose oligomer thereof. 2. The enzyme is one enzyme selected from the group consisting of cyclodextrin glycosyltransferase, glucoamylase, β-glucosidase, α-amylase, β-amylase, and maltooligosaccharide-forming enzyme. Stabilization method. 3. The stabilization method according to claim 1, wherein the enzyme is an immobilized enzyme. 4. The following general formula [I]: (Here, R represents hydrogen, lower alkyl, phenylalkyl, phenylalkenyl, phenylalkynyl, phenoxyalkyl, phenoxyalkenyl, or phenoxyalkynyl. Here, phenyl includes substituted or unsubstituted). N-
An enzyme stabilizer, which comprises a substituted moranolin derivative or a glucose oligomer thereof. 5. The following general formula [1] (Here, R represents hydrogen, lower alkyl, phenylalkyl, phenylalkenyl, phenylalkynyl, phenoxyalkyl, phenoxyalkenyl, or phenoxyalkynyl. Here, phenyl includes substituted or unsubstituted). N-
An immobilized enzyme, characterized in that a substituted moranolin derivative or a glucose oligomer thereof is present together with the enzyme. 6. The enzyme according to claim 5, wherein the enzyme is one selected from the group consisting of cyclodextrin glycosyltransferase, glucoamylase, β-glucosidase, α-amylase, β-amylase, and maltooligosaccharide-forming enzyme. Immobilized enzyme.