JPH08256768A - Activation of reaction by glucide hydrolyzing enzyme - Google Patents

Abstract

PURPOSE: To improve a reaction rate by carrying out a glucide hydrolyzing enzyme reaction in the presence of an alkyl sulfate and/or an alkyl sulfonate. CONSTITUTION: A reaction by any of various glucide hydrolyzing enzymes is carried out in the presence of an alkyl sulfate having 1-5 alkyl chain length and/or an alkyl sulfonate having 1-5 alkyl chain length. α-Amylase derived from Bacillus amyloliquefaciens and cellulase derived from Bacillus sp. KSM-635 are preferable as the glucide hydrolyzing enzymes.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for activating a glycolytic enzyme such as cellulase or amylase.

[0002]

BACKGROUND OF THE INVENTION Glycolytic enzymes include α-amylase, β-amylase, which cleaves the main chain consisting of α-1,4 bonds in starch, pullulanase, which cleaves α-1,6 bonds and their vicinity, isoforms. Starch debranching enzymes such as amylases, isopullulanases, and neopullulanases, glucoamylases that produce glucose, exo and endoglucanases that decompose cellulose composed of β-1,4 bonds, cellulases such as cellobiohydrase, and other β -Glucosidase, xylanase, pectinase, lysozyme and the like are known.

These glycolytic enzymes have been widely used for many years in the brewing industry, the textile industry, the pharmaceutical industry, the food industry and the like. In addition, among the hanging sugar degrading enzymes, by adding cellulase and amylase to clothes and dishwashing detergents and bleaching agents, the detergency can be dramatically improved mainly against sebum stains and starch stains. Have been found.

However, most of the glycolytic enzymes conventionally found in the natural world do not have sufficient enzyme productivity up to an amount that can provide an industrially usable amount of enzyme, and thus have sufficient enzyme reactivity. Difficult to use. Even if the enzyme productivity is sufficient, the use of a large amount of the enzyme required for the reaction requires a high cost, which may make it practically difficult to use. Further, as a problem when using the enzyme, there is a problem that the enzyme is inactivated when the reaction is carried out for a long time due to a problem such as thermal stability of the enzyme, which makes industrial use difficult.

[0005]

As means for solving such a problem, a method for improving the reaction rate of an enzyme in an enzyme reaction system, in addition to improving enzyme productivity and storage stability, That is, a method for activating an enzymatic reaction has also been conventionally studied.

As the method for activating the glycolytic enzyme reaction, various enzymes such as those used in combination with β-glucosidase for cellulase, glucoamylase with α-amylase and β-amylase, and starch debranching enzyme are used. Those that utilize synergistic effects, those that utilize protein engineering technology to improve the enzymatic reaction rate by specifically substituting amino acid residues, enzyme immobilization methods, specific ions or amphiphiles for enzyme reaction systems. A method utilizing an enzymatic reaction activator such as a volatile substance has been reported so far. Among them, the method using an enzyme reaction activator is expected to be very simple and cheap depending on the agent. Regarding such activators, those obtained by adding a specific nonionic activator, polyoxyethylene phosphate and carboxybetaine type amphoteric activator to the cellulase reaction system have been hitherto disclosed (Japanese Patent Laid-Open No. 60-210984).
No.), an endoglucanase, an α-amylase, or a pullulanase reaction system, which is obtained by adding a specific water-soluble polymer (JP-T 5-507615), or one which is obtained by adding actin to a specific cellulase. (FE
BS Letter, 187, 101-104 (1985)], obtained by adding chloride ion to a specific amylase [Cli
n. Biochem., 16, 224-228 (1983)], n-hexane in Twe
Enzyme activity of amylase and cellulase is enhanced by using a reverse-phase micelle system obtained by adding en20 (polyoxyethylene sorbitan monolaurate, Atlas Powder Co.) [Biotechnol. Bioeng., 29, 901-902 ( 198
7)] etc. have been reported. However, although these activation methods can increase the reaction rate of the enzyme to some extent, they still have problems such as insufficient activation power.

Therefore, it is an object of the present invention to provide an agent which fully activates the reaction rate of a glycolytic enzyme.

[0008]

Therefore, the inventors of the present invention have conducted an intensive search for a reaction activator of a glycolytic enzyme, and found that the reaction system of a glycolytic enzyme such as cellulase and amylase It was found that the enzyme activity was remarkably activated by adding the lower alkyl sulfate and the lower alkyl sulfonate, and the present invention was completed.

That is, the present invention relates to the reaction of glycolytic enzyme
Carbohydrate characterized by being carried out in the presence of one or more salts selected from the group consisting of alkyl sulfates having an alkyl chain length of 1 to 5 and alkyl sulfonates having an alkyl chain length of 1 to 5. The present invention provides a method for activating a reaction of a degrading enzyme. In other words, the method of the present invention provides a substrate (sugar) degrading activity of a carbohydrate degrading enzyme which is composed of an alkyl sulfate having an alkyl chain length of 1 to 5 and an alkyl sulfonate having an alkyl chain length of 1 to 5. It is improved by one or more salts selected from

As the alkyl sulfate and the alkyl sulfonate of the present invention, those in which the alkyl group has 1 to 5 carbon atoms are used, and the counter ion species are alkali metal salts such as sodium, potassium and lithium. Examples thereof include monovalent cations such as ammonium ions, preferably alkyl sulfates and alkyl sulfonates having 1 to 2 carbon atoms, and more preferably sodium methyl sulfate and potassium methyl sulfate. When the alkyl group has a large number of carbon atoms, an anionic micelle is likely to be formed by adding it at a high concentration, and conversely the reaction is easily suppressed, which is not preferable.

The carbohydrate-degrading enzyme to which the present invention is applied is not particularly limited as long as it is an enzyme capable of degrading carbohydrates.
For example, α-amylase, β-amylase, glucoamylase, pullulanase, isoamylase, isopullulanase, amylase such as neopullulanase, and exo and endoglucanase, cellulase such as cellobiohydrolase, and other β-glugosidase, xylanase,
Examples include pectinase, ligninase and lysozyme. Above all, the decomposition rate of α-amylase with respect to amylopectin and soluble starch and the decomposition activity of endoglucanase with respect to carboxymethyl cellulose (CMC) are remarkably improved. Among them, especially α- from Bacillus amyloliquefaciens
Amylase and Bacillus sp. KSM
Cellulase from the -635 strain is preferred. KSM-635 is FE
The strain has been deposited as RM BP-1485, the strain is described in JP-A-63-10977, and the detailed production method is described in Examples of JP-A-63-264699.

Other α-amylases include Bacillus subtilis Marbu
rg), Bacillus subtilus nut
is natto), Bacillus licheniformis (Bacillus lich)
eniformis), Bacillus cereus,
Bacteria such as Bacillus circulans, Bacillus macerans, Pseudomonass tutzeri, Klebusiella aerogenes, Streptomyces griseus, Streptomyces griseus, etc. Orize (Aspergillus oryzae),
Molds obtained from many organisms such as molds such as Aspergillus niger, seeds of grasses and legumes, digestive glands of animals such as humans and pigs can be used. In addition, cellulase is Fumicola lanuginosa, Trichoderma viridae.
(Trichoderma viride), Trichoderma race
erma reesei), Trichoderma koniji (Trichoderma k)
oningii) and the like.

The reaction activation method of the present invention can be applied to a usual saccharide-degrading enzyme-substrate reaction, and the alkyl chain length is 1 at an appropriate time such as an enzyme solution, a substrate solution or a buffer solution before the reaction.
1 to 2 selected from the group consisting of alkylsulfates of 5 to 5 and alkylsulfonates having an alkyl chain length of 1 to 5
It suffices to add one or more kinds of salts so that they are present in the reaction system in an amount of 1 to 1000 mM, and more preferably, the concentration of the alkyl sulfate and the alkyl sulfonate in the reaction system is 1 to 500 mM,
More preferably, it is added so as to be 1 to 100 mM.

[0014]

The reaction activation of the glycolytic enzyme according to the present invention is carried out by reacting various glycolytic enzymes with an alkylsulfate having an alkyl group having 1 to 5 carbon atoms and / or an alkyl group having 1 carbon atom. It is obtained by carrying out in the presence of an alkyl sulfonate of 5 to 5, and has an extremely great industrial significance.

[0015]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Examples 1 to 8 and Comparative Examples 1 to 4 Bacillus sp. KSM-635 strain (FERM BP-1485) described in Examples of JP-A-63-264699 was cultured and purified. An enzyme solution was prepared by dissolving an appropriate amount of the cellulase component thus obtained in ion-exchanged water. CMC in a 20 mM Glycine buffer solution (pH 10.0) (final concentration in the reaction system is 1.0
%) And an alkylsulfate (additive) shown in Table 1 were added to 0.9 mL of the reaction solution, and 0.1 mL of the enzyme solution was added, and the reaction was allowed to proceed at 40 ° C. for 5 minutes at a maximum of 25 minutes. The reducing sugars were quantified by the 3,5-dinitro-salicylic acid (DNS) method at each reaction time. That is, 1.0 mL of the DNS reagent was added to 1.0 mL of the reaction solution, the color was developed by heating at 100 ° C for 5 minutes, and after cooling, 4.0 mL of ion-exchanged water was added to dilute the mixture to a wavelength of 535 nm.
Colorimetrically determined by. The reaction rate of the enzyme is the rate of product increase (d [reducing sugar] / d [time]) in the range in which the amount of reducing sugar as a product increases linearly with time from the start of the reaction.
As the relative reaction rate, the reaction rate of the alkylsulfate-present system with respect to the alkylsulfate-free system is shown in percentage. Table 1 shows the results of the effect of addition of the alkyl sulfate on the cellulase reaction rate measured by the above method. In Table 1, the concentration means the concentration in the reaction system (hereinafter the same).

From Table 1, it was found that the activation of the enzyme reaction was up to 80% in the sodium methylsulfate addition system and up to 20% in the sodium ethylsulfate addition system.

Table 1 also shows the results of the effect of addition of sodium alkyl sulfonate measured according to the above method. From Table 1, it was found that up to 100% of the enzyme reaction was activated in the system containing sodium ethylsulfonate, up to 70% in the system containing sodium propylsulfonate, and up to 50% in the system containing sodium butylsulfonate.

[0019]

[Table 1]

Examples 9 to 16 and Comparative Examples 5 to 9 Bacillus amyloliq
u-faciens) -derived α-amylase (Seikagaku Corporation) was dissolved in deionized water in an appropriate amount to give an enzyme solution. 25 mM
The enzyme was added to 0.9 mL of the reaction solution containing potato-derived amylopectin (Sigma) (final concentration in the reaction system was 0.5%) and the alkyl sulfate (additive) shown in Table 2 dissolved in Tris-acetate buffer solution (pH 7.0). The solution (0.1 mL) was added, and the reaction was allowed to proceed at a maximum of 25 minutes at 40 ° C. by setting the reaction time every 5 minutes. The reducing sugars were quantified by the 3,5-dinitro-salicylic acid (DNS) method at each reaction time. That is, 1.0 mL of DNS reagent was added
Add mL, heat at 100 ° C for 5 minutes to develop color, cool, and then 4.
It was diluted by adding 0 mL of ion-exchanged water, and colorimetrically determined at a wavelength of 535 nm. The reaction rate of the enzyme was calculated as the rate of product increase in the range in which the amount of reducing sugar as a product linearly increases with time from the start of the reaction. The reaction rate of the salt presence system is shown in percentage. Table 2 shows the results of the effect of addition of the alkyl sulfate on the amylase reaction rate measured by the above method. From Table 2, activation of the enzyme reaction was found to be up to 40% in the sodium methylsulfate addition system and up to about 10% in the sodium ethylsulfate addition system.

[0021]

[Table 2]

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Claims (8)

[Claims] 1. The reaction of the glycolytic enzyme is carried out by one or more kinds selected from the group consisting of an alkyl sulfate having an alkyl chain length of 1 to 5 and an alkyl sulfonate having an alkyl chain length of 1 to 5. A method for activating a glycolytic enzyme reaction, which is carried out in the presence of salt. 2. The concentration of the alkyl sulfate and / or the alkyl sulfonate in the reaction system is 1 to 1000 mM.
The method for activating a glycolytic enzyme as described. 3. The method for activating a glycolytic enzyme according to claim 1, wherein the alkyl sulfate is sodium methyl sulfate. 4. The method for activating a glycolytic enzyme according to claim 1, wherein the glycolytic enzyme is amylase or cellulase. 5. The glycolytic enzyme is α-amylase produced by a bacterium of the genus Bacillus.
4. The method for activating the reaction of the glycolytic enzyme according to any one of 3 above. 6. The reaction activation of the glycolytic enzyme according to claim 1, wherein the glycolytic enzyme is α-amylase produced from Bacillus amyloliquefaciens. Law. 7. The method for activating a glycolytic enzyme according to any one of claims 1 to 3, wherein the glycolytic enzyme is a cellulase produced by a bacterium of the genus Bacillus. 8. The glycolytic enzyme is Bacillus sp.
The method for activating a glycolytic enzyme according to claim 7, which is a cellulase produced from a bacterium of acillus sp.) KSM-635 strain.