JPH0231954B2 - KOSONOANTEIKAZAIOYOBIKOSOHOZONHOHO - Google Patents

Description

[Detailed description of the invention]

[Object of the Invention] The present invention relates to an enzyme stabilizer and an enzyme preservation method. More specifically, the present invention relates to an enzyme stabilizer added to prevent a decrease in enzyme activity and maintain enzyme activity over a long period of time, and a method for preserving enzymes stabilized thereby. The goal is to keep unstable enzymes stable for long periods of time so that they can be used effectively. [Industrial Application Fields] Enzyme-containing compositions are used in many fields as catalysts, such as enzyme reagents, enzymes for clinical analysis, and industrial enzymes for food. However, these enzyme-containing compositions may be deactivated during storage or during freeze-drying, and it is necessary to stabilize the enzyme and prevent its deactivation in industrial fields that use enzymes. . [Prior Art] Conventionally, as stabilizers for enzyme compositions, proteins without enzymatic activity such as bovine serum argumin, human serum argumin, and egg white argumin, and saccharides such as sucrose, glucose, lactose, and water-soluble polysaccharides have been used. , salts such as ammonium sulfate, glycerol, organic solvents, etc. have been used. [Problems to be solved by the invention] Even if the enzyme stabilizer described above is used alone, a satisfactory stabilizing effect cannot be obtained for some enzymes. There were cases where enzyme activity decreased. Particularly regarding alcohol oxidase and catalase, there was a problem in that the enzyme activity significantly decreased during freeze-drying and subsequent stability tests. We conducted research on stabilizers that can keep the activities of these two types of enzymes as stable as possible, leading to the present invention. [Means for Solving the Problem] Therefore, the present inventors conducted extensive research on stabilizers that can keep the activities of these two enzymes as stable as possible even after freeze-drying, and found that they We have completed the present invention by discovering that a specific combination of enzyme stabilizers dramatically improves stability compared to when conventional stabilizers are used alone. That is, the present invention provides an enzyme stabilizer comprising two or more types of sucrose (A) and a compound (B) selected from the group consisting of lactose, inositol, sorbose, asparagine, and threonine, and this stabilizer. The present invention provides a method for preserving an enzyme, which comprises freeze-drying a composition comprising the enzyme and the enzyme. The enzyme stabilizer of the present invention is preferably a combination of sucrose and lactose, sucrose and lactose and inositol, sucrose and threonine, sucrose and sorbose, sucrose and asparagine, sucrose and inositol,
In particular, combinations of sucrose, lactose, and sucrose, lactose, and inositol are preferred. The mixing ratio of these sucrose (A) and the selected compound (B) is preferably 1:1 to 10 by weight, and the aqueous composition preferably has a sucrose content of 1 to 10% by weight. . In addition, in the case of three types of compositions,
For example, in the case of sucrose (A) / lactose (B) / inositol (B), sucrose: lactose: inositol = 1:1 ~
10: is preferably 0.001 to 1, more preferably 1:
1-10: 0.01-0.1, sucrose content 1-10% by weight
It is an aqueous composition of. The proportion of enzyme added to the stabilizer is preferably 0.1 to 5% by weight, particularly preferably 1 to 3% by weight in the stabilizer solution. The enzyme of the present invention is
For example, alcohol oxidase, glucose oxidase, cholesterol oxidase, glycerol oxidase, xanthine oxidase,
Oxidases such as ascorbate oxidase, catalase, cellulase, lipase, amylase, protease, DNA ligase, DNA polymerase, adenosine triphosphatase, ribonuclease, lactase, phosphatase, polynucleotide kinase, β-1,4-glucanase, etc. I get punched. In particular, the stabilizer of the present invention is useful against alcohol oxidase and catalase. Freeze-drying involves, for example, freezing an aqueous composition containing a stabilizer and an enzyme at -20 to -80°C;
0.15 Torr using Japan Vacuum Technology Co., Ltd. DF-01G
Dry under reduced pressure for 20 to 24 hours. The enzyme-containing composition stabilized according to the present invention comprises:
No deactivation was observed during freeze-drying, and the original enzyme activity was maintained even if the freeze-dried product was stored for a long period of time. [Example] Next, the details of the present invention will be explained using an example. In the following examples, alcohol oxidase (referred to as AOD) and catalase (CAT) containing sucrose and lactose, or sucrose and lactose, and inositol were added.
An example of freeze-drying a solution containing 30% of enzymes and examining changes in enzyme activity over time is shown. The alcohol oxidase and catalase used here are derived from Lipomyces methanosilviensis, which grows using methanol as the sole carbon source.
This strain has already been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology under Microbial Accession No. 5489 on April 15, 1980, and its mycological properties have been published in the Japanese Patent Publication No. 57-28551. As stated in the publication. Further, the properties of the AOD are as described in Japanese Patent Application No. 1983-9868. Example 1 and Comparative Example 1 (Study of various stabilizers) Each substance 6% and 6% sucrose were added to the enzyme solution of alcohol oxidase 620V and catalase 6500V/0.5ml 10mM NaPB (PH7) obtained from Lipomyces methanosylviensis described above. solution containing
Added 0.5 ml and lyophilized. The mixture was left at 37°C for one week and the residual activity was measured. The conclusion is shown in Table 1.

[Table] From the table above, sugar, threonine, sorbose,
A synergistic effect was confirmed by the combined use of asparagine and inositol. Example 2 and Comparative Example 2 (Stabilization of alcohol oxidase and catalase) Sucrose and lactose were added to the partially purified alcohol oxidase and catalase enzyme fractions from Lipomyces methanosylviensis at various concentrations and ratios shown in Table 2. and freeze-dried. Similarly, sucrose alone is also shown. The activity of alcohol oxidase and catalase before freeze-drying were each set as 100%, and the test results regarding changes in activity immediately after freeze-drying and when stored at 5°C are shown in Table 2.

[Table] The results in the table above confirm that the synergistic effect of sucrose and lactose on alcohol oxidase and catalase is significant. Example 3 The partially purified alcohol oxidase and catalase enzyme fractions from Lipomyces sylviensis were mixed with sucrose and sucrose at various concentrations and ratios shown in Table 3.
Lactose and inositol were added and freeze-dried. Table 3 shows the change in activity immediately after freeze-drying and when stored at 5°C, assuming that the enzyme activity before freeze-drying is 100%.

[Table] The results in the table above show that the effects of addition of sucrose, lactose, and inositol on alcohol oxidase and catalase are significant, and it was confirmed that the addition of a small amount of inositol further improves stability. As shown in the above Examples, the enzyme activity of the enzyme-containing composition was stably maintained by the enzyme stabilizer of the present invention.

Claims (1)

[Scope of Claims] 1. An enzyme stabilizer comprising two or more of sucrose (A) and a compound (B) selected from the group consisting of lactose, inositol, sorbose, asparagine, and threonine. 2 The weight ratio of sucrose (A) and the selected compound (B) is 1:1 to 10, and the sucrose content is 1.0 to 10.0%.
The enzyme stabilizer according to claim 1, which is (W/V). 3. The enzyme stabilizer according to claim 1, wherein the selected compound (B) is lactose and inositol. 4. A method for preserving enzymes, which comprises freeze-drying a composition comprising two or more of sucrose (A) and a compound (B) selected from the group consisting of lactose, inositol, sorbose, asparagine, and threonine, and an enzyme. 5. The enzyme preservation method according to claim 4, wherein the enzymes are alcohol oxidase and catalase.