JPH0351398B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for stabilizing enzymes such as, for example, alpha-1,6-glucosidase, amylase or cellulase. [Conventional technology] Enzymes efficiently promote reactions at medium temperature, neutrality, and near normal pressure, but each enzyme has its own optimal pH and temperature. In other words, the enzyme is stable near the optimum pH, and the optimum temperature indicates the stability limit temperature of the enzyme on the high temperature side. [Problems to be Solved by the Invention] However, when trying to apply enzymes industrially, they often have to be used at locations outside of the optimum pH. In addition, in order to use enzymes efficiently and prevent bacterial contamination during the reaction,
It is often necessary to use it at as high a temperature as possible. For this reason, many studies have been conducted on methods for stabilizing enzymes, particularly methods for improving thermostability, but no universally applicable methods are known yet. [Means for solving the problems] The present invention utilizes α-
In order to produce glucose from starch in a high yield by using 1,6-glucosidase in combination with glucoamylase, we have investigated various methods for stabilizing α-1,6-glucosidase.
It was found to be extremely effective at very low concentrations of 0.01 mol or less, and within a narrow concentration range. It was also recognized that this effect is applicable not only to α-1,6-glucosidase but also to the stabilization of various enzymes such as amylase, cellulase, glucose isomerase, and protease. The present invention has been made based on this knowledge. That is, the present invention relates to a method for thermostabilizing enzymes, which is characterized by carrying out the enzyme reaction in the presence of an aluminum salt. [Effects of the Invention] Although the present invention can be applied to many enzymes, stabilization of α-1,6-glucosidase will be explained in detail as a specific example. α-1,6-glucosidase is a general term for enzymes that hydrolyze α-1,6-glucosidic bonds in starch or its derivatives or (and) α-1,6-glucosidic bonds in pullulan, and its substrates. There are enzymes called pullulanase or isoamylase due to their specificity. α-1,6-glucosidase is used in combination with β-amylase to produce maltose from starch in good yields, and recently it has been used in combination with glucoamylase in the production of glucose from starch using an enzymatic method. It is used to increase the yield of glucose. α-1,6-glucosidase is produced by Bacillus sp.
It is known that it is produced by various bacteria such as the genus Clepsilla and Pseudomonas, and actinobacteria such as the genus Streptomyces, but many α-
1,6-glucosidase has an optimum pH of 5 to 7 and an optimum temperature of around 50°C. On the other hand, the optimum pH of glucoamylase is around 4.5 and the temperature is 60°C, and their operating conditions do not match. Therefore, as a result of investigating methods for stabilizing α-1,6-glucosidase produced by bacteria such as Klebsiella, Pseudomonas, and Bacillus, which are currently produced industrially, we found that aluminum chloride, aluminum sulfate, It has been found that when a water-soluble aluminum salt such as ammonium aluminum sulfate or aluminum lactate is added in a very dilute concentration and in a very narrow concentration range, it has a significant stabilizing effect on the enzyme. And, the pH, which is the optimal action condition for glucoamylase, is
4.5. It was found that it can act for a long time even at a temperature of 60℃. For example, using pullulan as a substrate,
When reacting at pH 4.5 and 60°C, pullulanase produced by many Bacillus and Klebsiella bacteria is inactivated within 1 hour, but when approximately 3 x 10 ^-3 mol of aluminum lactate is present. remained active for at least 3 days. The present invention also exhibits the same stabilizing effect on amylase and cellulase as it does on α-1,6-glucosidase. The present invention can be applied to amylase produced by various microorganisms such as filamentous fungi, bacteria, and yeast.
For example, the present invention can be suitably practiced on glucoamylase and enzymes that produce maltooligosaccharides such as maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose. Furthermore, the present invention can be applied to cellulases produced by various microorganisms such as Aspergillus, Trichoderma, Penicillium, Acremonium, and Sporotrichum. Examples of aluminum salts used in the present invention include inorganic acid salts such as aluminum chloride, aluminum sulfate, ammonium aluminum sulfate, aluminum nitrate, and potassium aluminum sulfate, as well as organic acid salts such as aluminum acetate and aluminum lactate. , water-soluble salts. However, aluminum salts are strongly acidic (PH 2 to 4), and many enzymes such as α-1,6-glucosidase are immediately deactivated, so it is necessary to raise the PH to at least 4 or higher. However, there is a problem in that when the pH is raised, the aluminum salt decomposes and precipitates water-insoluble aluminum hydroxide. Therefore, as a result of investigating aluminum solutions that can maintain water solubility at a stable pH for the enzyme, we found that sugar, glycerin, etc. It has been recognized that it is appropriate to adjust the pH by adjusting the pH in the presence of either sodium lactate, sodium lactate, or the like, or to adjust the pH of a solution of aluminum lactate. Particularly when using aluminum lactate, highly concentrated solutions can be prepared. Either aluminum salt was added to the reaction solution at 1
When present in the range of ×10 ^-4 to 1 ×10 ^-2 mol,
It thermally stabilizes the enzyme significantly compared to the case without additives. The table below shows the reducing sugar produced when pullulanase produced by Bacillus subtilis is reacted with approximately 5% pullulan and the indicated amount of aluminum lactate at pH 4.5 and temperature 60°C for 20 hours, expressed as glucose. This is what I did. Aluminum lactate is
Effective at 0.5×10 ^-4 molar concentration or higher, approximately 1×
A peak with an optimum concentration of 10 ^-3 to 7×10 ^-3 mol is shown. The aluminum salt may be added during the reaction or may be added to the enzyme agent in advance. The latter case is more effective when preserving enzymes for long periods of time.

[Table] The present invention can be applied not only to α-1,6-glucosidase but also to various enzymes such as many amylases, cellulases, proteases, lipases, and glucose isomerase. The details of the present invention will be explained below using Examples with respect to typical enzymes, but the present invention is not limited to these Examples. Example 1 Klebsiella pneumonia
Pneumoniae) produced by pullulanase (UK,
Add pullulan 10%, acetate buffer (PH4.5) 0.1M, and aluminum chloride to 3 x 10 ^-4 M to 0.3 units of ABM (product name: Pulluzyme), make the total volume 2ml with water, and hold at 60°C. I reacted with As a control, a sample to which aluminum chloride was not added was simultaneously reacted. Then, a fixed amount was taken over time, and the resulting reducing sugar (maltotriose) was quantified as glycose by the Somogyi-Nelson method. The results obtained are shown in Table 1.

[Table] As is clear from the table, this enzyme is inactivated within 1 hour under conditions of pH 4.5 and 60°C in the absence of aluminum chloride, but when ^aluminum chloride is When present, the stability of the enzyme was significantly improved and it showed high degradability. Here, α-1,6-glucosidase activity was measured as follows. Add an appropriate amount of enzyme solution to 0.5 ml of 2% pullulan solution (PH 0.5) dissolved in 0.1 M acetate buffer, make up to 1 ml with distilled water, and react at 40°C. Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 micromole of glucose per minute was defined as 1 unit. Example 2 Corn steep liquor 10%, lactose 2%,
Dipotassium phosphate 0.3%, magnesium sulfate (heptahydrate)
0.1%, sodium nitrate 0.25%, Tween 60 0.3
%, copper sulfate 5×10 ^-5 M, manganese chloride 2.5×
10 ^-6 M, calcium chloride 1 x 10 ^-3 M, zinc sulfate 1
^Bacillus subtilis ^TU (FERM-
BP684 was inoculated and cultured with shaking at 30°C for 4 days. After culturing, ammonium sulfate was added to the supernatant obtained by centrifugation to 70% saturation to precipitate the enzyme, which was recovered by centrifugation, dialyzed against distilled water, and used as an enzyme solution. Add 0.05 unit of this enzyme, 5% pullulan, 0.01 M acetate buffer (PH4.5), 1 x 10 ^-2 M calcium chloride, 1 x 10 ^-4 - 1 x 10 ^-2 M aluminum lactate, and add 60 units of this enzyme in a total volume of 1 ml. The reaction was carried out at ℃. At 42 and 66 hours of the reaction, the produced ring sugar was quantified as glucose. The results obtained are shown in Table 2.

[Table] As is clear from the table, when sodium lactate is used, it is effective over a relatively wide concentration range, but
The optimum temperature was found to be around 4×10 ^-3 M. Example 3 Corn steep liquor 10%, lactose 2%,
Dipotassium phosphate 0.3%, magnesium sulfate (heptahydrate)
0.1%, sodium nitrate 0.25%, Tween 60 0.3
%, copper sulfate 5×10 ^-5 M, manganese chloride 2.5×
10 ^-6 M, calcium chloride 1 x 10 ^-3 M, zinc sulfate 1
^Bacillus subtilis ^TU (FERM-
BP684 was inoculated and cultured with shaking at 30°C for 4 days. After culturing, the supernatant obtained by centrifugation was used as an enzyme solution. 0.23 units of the enzyme was added to pullulan 5%, acetate buffer (PH4.5) 0.01M, calcium chloride 1x
10 ^-3 M, aluminum chloride 1 x 10 ^-4 ~ 5 x 10 ^-4 M
, and the reaction was carried out at 60°C in a total volume of 1 ml. The amount of reducing sugar produced 48 hours after the reaction was determined as glucose. The results were as shown in Table 3. As is clear from the table, the presence of aluminum chloride significantly improved the stability of pullulanase.

[Table] Example 4 In this example, aluminum ammonium sulfate was used instead of aluminum chloride. The enzyme used in the reaction is the same as that used in Example 3. The results obtained are shown in Table 4.

[Table] Example 5 The enzyme prepared in Example 3 was added to ammonium sulfate at 70% saturation to precipitate the enzyme, collected by centrifugation, and dialyzed against distilled water, which was used as an enzyme solution. Add 0.05 units of this enzyme, 5% pullulan, 0.01 M acetate buffer (PH4.5), 1 x 10 ^-2 M calcium chloride, 0.5 x 10 ^-3 to 5 x 10 ^-3 M aluminum lactate, and add 60 The reaction was carried out at ℃. At 42 and 66 hours of the reaction, the reducing sugars produced were quantified as glucose. The results obtained are shown in Table 5.

[Table] As is clear from the table, when sodium lactate was used, it was effective over a relatively wide concentration range. Example 6 This example describes the results of a test using Pseudomonas amyloderamosa isoamylase as α-1,6-glucosidase. This enzyme is a branched long chain of amylopectin (α-
1,6-glucosidic bonds), but the α-1,6-glucosidic bonds of pullulan cannot be broken down. Also, this enzyme's optimal
PH is 3-4, optimal temperature is 52℃ (PH3.5, 10 minutes reaction)
, and rapidly deactivates at 60°C [K,
Yokobayasi et al., Bio-chimica et Biophysica
Acta 212, 458 (1970)]. A purified sample of this enzyme can be obtained from Seikagaku Kogyo Co., Ltd. To test the stability of aluminum salts on this enzyme, in combination with plant β-amylase,
A test was conducted on the production of maltose from starch. In this case, a sufficient amount of β-amylase was added to the substrate in order to eliminate the influence of β-amylase. Soluble starch 5%, acetate buffer (PH5.0) 0.01M,
Barley β-amylase (manufactured by Finschjuger, Finland (trade name: Spezyme BBA) 4.2 units,
100 units of the Pseudomonas isoamylase;
Ammonium aluminum sulfate or aluminum lactate 3 x 10 ^-4 - 5 x 10 ^-4 M, total volume 1 ml 60℃
I reacted with As a control, the reaction was carried out simultaneously without the addition of aluminum salt. A fixed amount was taken over time, and the reducing sugar (maltose) produced was quantified by the Somogyi-Nelson method. The results obtained are shown in Table 6. As is clear from the table, when aluminum salt was present, the activity was maintained for a longer period of time and maltose was obtained in higher yield than when aluminum salt was not present.

[Table] Example 7 Approximately 5% soluble starch, acetate buffer (PH5)
Aluminum lactate was added in the amount shown in Table 7 to 0.01M glucoamylase 0.27 unit (sold by Novo Japan) and reacted at 60°C in a total volume of 1 ml. As a control, a sample to which no aluminum salt was added was simultaneously reacted. Glucose produced at 140 hours of reaction was quantified by the Somogyi-Nelson method. The results obtained were as shown in Table 7. As is clear from the table, in the absence of aluminum lactate, the reaction hardly progresses after 43 hours, whereas in the presence of sodium lactate, this enzyme retains its activity for a long time and degrades at a high rate. Indicated.

[Table] Here, amylase activity was measured as follows. Add an appropriate amount of enzyme solution to 0.5 ml of 2% soluble starch solution (PH5.0) dissolved in 0.1 M acetate buffer, make up to 1 ml with distilled water, and react at 40°C. Under these conditions, the amount of enzyme that produced a reducing power equivalent to 1 micromole of glucose per minute was defined as 1 unit. Example 8 In Example 7, the results of saccharification of the reaction at the optimum pH of glucoamylase of 4.5 and a temperature of 60° C. for 66 hours are described. The results obtained were as shown in Table 8.

[Table] As is clear from the table, when aluminum lactate was present, a higher glucose yield was shown than when aluminum lactate was not present. Example 9 Approximately 5% soluble starch, Tris buffer (PH5.5)
0.05M, glucoamylase 0.14 unit (sold by Novo Japan) was added with 3×10 ^-4 M of aluminum ammonium sulfate, and reacted at 60°C. As a control, a sample to which no aluminum salt was added was simultaneously reacted. The results obtained are shown in Table 9.

[Table] As is clear from the table, when aluminum ammonium sulfate was added, compared to the case without addition,
It was found that the activity could be maintained stably for a long period of time. Example 10 In Example 7, the results of saccharification of the reaction at 4.5, the optimum pH of glucoamylase, and a temperature of 60° C. for 66 hours are described. The results obtained were as shown in Table 10.

[Table] As is clear from the table, when aluminum lactate was present, a higher glucose yield was shown than when aluminum lactate was not present. Example 11 Approximately 5% soluble starch, Tris buffer (PH5.5)
0.05M, glucoamylase 0.14 unit (sold by Novo Japan) was added with 3×10 ^-4 M of aluminum ammonium sulfate, and reacted at 60°C. As a control, a sample to which no aluminum salt was added was simultaneously reacted. The results obtained are shown in Table 11.

[Table] As is clear from the table, when aluminum ammonium sulfate was added, compared to the case without addition,
It was found that the activity could be maintained stably for a long period of time. Example 12 A liquefied starch saccharification test was conducted using the enzyme prepared in Example 3 in combination with glucoamylase. 30% liquefied starch with DE approx. 12, glucoamylase (manufactured by Finschuger, trade name Spezyme GA)
200) 0.03% (/solid content), α-1,6-glucosidase 0.4 unit (/g substrate), calcium chloride
0.01M, aluminum ammonium sulfate 5x
The saccharification reaction was carried out at 60°C and pH 4.5 under a composition of 10 ^-4 M. The results obtained were as shown in Table 12. As is clear from the table, the addition of aluminum salt increased the stability of α-1,6-glucosidase, resulting in a high yield of glucose.

[Table] Example 13 In this example, maltose-producing amylase produced by Bacillus megaterium (FERM-P7978)
126973), we will describe the results of investigating the effects of aluminum salts. This enzyme is an enzyme that hydrolyzes starch into maltose units from its non-reducing end to produce α-maltose. About 5% soluble starch, 0.01M phosphate buffer (PH6.0), and aluminum lactate were added to 0.075 units of this enzyme.
13 The amounts listed in the table were added to make a total volume of 1 ml, and the reaction was carried out at 60°C. A fixed amount was taken over time, and the maltose produced was quantified. The results obtained were as shown in Table 13.

[Table] As is clear from the table, when aluminum salt was added to the reaction solution, a higher maltose yield was shown than when no aluminum salt was added. Example 14 This example describes the results of investigating the effect of aluminum salt on the maltotriose-producing enzyme produced by Bacillus subtilis FERM-P5854. To 0.065 units of this enzyme, add 5% soluble starch, 0.01M phosphate buffer (PH6), 0.01M calcium chloride, and aluminum lactate in the amounts listed in Table 17, make a total volume of 1ml with distilled water, and react at 50°C. Ta. The results of quantifying the reducing sugar produced at 45 hours as glucose are shown in Table 14. As is clear from the table, the enzyme was stabilized in the presence of aluminum lactate.

[Table] Example 15 Trichoderuma cellulase (manufactured and sold by Kinki Yakult Co., Ltd., R-10) 1 mg, carboxymethylcellulose (CMC) 5%, acetate buffer (PH4.5) 0.05M, aluminum lactate. No.
15 Add the amount listed in the table, make the total volume 1 ml with distilled water, and heat at 55℃.
I reacted with A certain amount was taken at 42 and 66 hours of the reaction, and the produced sugar was quantified as glucose. As a control, a sample to which aluminum lactate was not added was simultaneously reacted. The results obtained are shown in Table 15.

[Table] As is clear from the table, when aluminum lactate was not present, no increase in the amount of reducing sugar produced was observed after 42 hours, but when aluminum lactate was present, the enzyme stability was improved, and an improvement in the saccharification rate was observed. Example 16 In Example 15, aluminum ammonium sulfate was used instead of aluminum lactate, and 55
The reaction was carried out at ℃. The results obtained are shown in Table 16.

[Table] As is clear from the table, the presence of an extremely small amount of aluminum ammonium sulfate of about 3 to 5 x 10 ^-4 M stabilized the enzyme, and a significant increase in reducing sugars was observed. Example 17 In Example 15, 5% Avicel was used instead of carboxymethylcellulose as the substrate, and the reaction was carried out at 55°C. The results obtained are shown in Table 17.

[Table] As is clear from the table, when no aluminum salt is present, no reducing sugar is produced after 67 hours, but when approximately 1×10 ^-3 M aluminum lactate is present, no reducing sugars are produced after 67 hours. Generation of reducing sugars was also observed thereafter.

Claims (1)

[Scope of Claims] 1. A method for thermostabilizing an enzyme, characterized in that the enzyme reaction is carried out in the presence of a water-soluble aluminum salt at a molar concentration of 1×10 ⁻⁴ to 1×10 ⁻² . 2. The method for thermostabilizing an enzyme according to claim 1, wherein the enzyme involved in the reaction is α-1,6-glucosidase. 3. The method for thermostabilizing an enzyme according to claim 1, wherein the enzyme involved in the reaction is amylase. 4. The method for thermostabilizing an enzyme according to claim 1, wherein the enzyme involved in the reaction is cellulase. 5. The method for thermally stabilizing an enzyme according to claim 1, 2, 3 or 4, wherein the water-soluble aluminum salt is an inorganic acid salt. 6. The method for thermally stabilizing an enzyme according to claim 1, 2, 3 or 4, wherein the water-soluble aluminum salt is an organic acid salt.