JP2015008707A - Enzyme stabilization method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an enzyme stabilization method for an enzyme composition which comprises an enzyme as a component and utilizes a function of the enzyme.SOLUTION: This invention provides a composition in which poly-γ-L-glutamic acid coexists with an enzyme, the poly-γ-L-glutamic acid being polyamino acid in which α-amino group and γ-carboxyl group of L-glutamic acid are amide-bonded, and a method to stabilize an enzyme using the composition.

Description

  The present invention relates to a method for stabilizing enzymes.

  Enzymes are a type of protein with a catalytic function, and are widely used in various applications because of their characteristics such as their action occurring under mild conditions and high catalytic reaction selectivity. (Non-Patent Document 1). For example, analytical reagents for molecular biology, analytical reagents for biochemistry, in vitro diagnostics, liquid in vitro diagnostics, dry in vitro diagnostics processed into chips or slits, enzyme sensors and enzyme electrodes Pharmaceuticals, foods and beverages.

Needless to say, it is important to stably maintain the enzyme activity in order to make the composition containing the enzyme usable for a long period of time. Various methods have been investigated so far to stabilize the enzyme. For example, a method is known in which various amino acids, sugars, polyols, metal ions, and the like are allowed to coexist in an enzyme and stabilized. The stabilization mechanism has also been studied (Non-Patent Document 2). However, the stabilization effect varies depending on the type of enzyme, and in the current situation, the stabilizer is selected by trial and error.

Special table 2002-517204 Japanese Patent No. 4720614

Japan Patent Office Homepage Patent Map by Technology Field Chemistry 1 Enzyme Utilization Technology (Created in 1997) (http://www.jpo.go.jp/shiryou/s_sonota/map/kouuso/frame.html) Protein Nucleic Acid Enzyme Vol. 30 (10), pp 1115-1126 (1985)

  An object of the present invention is to stabilize an enzyme in a composition using the enzyme as a constituent and utilizing the function of the enzyme.

  The present inventors have found that poly-γ-L-glutamic acid (hereinafter also referred to as L-PGA) has an effect of stabilizing the enzyme, and completed the present invention.

That is, the present invention is as follows.
(Item 1) An enzyme composition comprising poly-γ-L-glutamic acid.
(Item 2) A method for stabilizing an enzyme, characterized in that poly-γ-L-glutamic acid is allowed to coexist with the enzyme.

  In order to stabilize the enzyme according to the present invention, an analytical reagent for molecular biology using the function of the enzyme, an analytical reagent for biochemical use, an in-vitro diagnostic agent, a liquid in-vitro diagnostic agent, a chip-shaped or slit-processed dry reagent Compositions such as in-vitro diagnostic agents, enzyme sensors, enzyme electrodes, pharmaceuticals, foods and beverages can be made available for a long period of time.

(Poly-γ-L-glutamic acid)
The poly-γ-L-glutamic acid used in the present invention is a polyamino acid in which an α-amino group and γ-carboxyl group of L-glutamic acid are amide-bonded, and the structure thereof is a structure represented by the following formula (1). (Patent Document 2).

(In formula (1), n represents the number of polymerizations)
Although the average molecular weight of L-PGA contained in the skin external preparation which concerns on this invention is not specifically limited, Preferably it is 1.3 million or more, More preferably, it is 2 million or more, More preferably, it is 3.5 million or more. Here, “average molecular weight” intends the number average molecular weight (Mn) calculated in terms of molecular weight of pullulan standard substance.

The method for obtaining L-PGA is not particularly limited, and L-PGA obtained by various conventionally known methods may be used. For example, L-PGA obtained using a microorganism that produces L-PGA may be used. . For example, it can be produced by culturing a microorganism belonging to Natrialba aegyptica and recovering L-PGA from the culture solution (Patent Document 2).

Various known methods can be used for the method for detecting and / or quantifying L-PGA, and are not particularly limited. For example, a commercially available L-glutamic acid measurement kit (for example, manufactured by Yamasa Shoyu Co., Ltd.) may be used. When the presence of L-PGA in the enzyme composition is detected by any of these methods, it is presumed that the enzyme composition contains L-PGA.

(Enzyme composition)
The enzyme to be stabilized in the present invention is not particularly limited in its intended purpose, type, concentration, form of presence, and the like. For example, as long as it is an enzyme as a constituent and is present in a composition utilizing the function of the enzyme, an analytical reagent for molecular biology, an analytical reagent for biochemistry, an in vitro diagnostic agent, a liquid in vitro diagnostic agent, Examples include dry in-vitro diagnostic agents processed into chips or slits, enzyme sensors, enzyme electrodes, pharmaceuticals, foods and beverages. These compositions have already been established in production technology and application technology in their respective industrial fields. Therefore, the knowledge can be applied to the present invention to stabilize at least one of the enzymes present in various compositions, and the mode is not particularly limited.

Enzymes applied to these reagents may be simple proteins or complex proteins containing non-amino acid components such as heme, vitamin derivatives, lipids, and carbohydrates. Further, it may be labeled with a labeling compound such as a dye, biotin or avidin, modified with various compounds, conjugated with an antibody, or the like.

In the following, several industrial fields are taken up and illustrated.

(Analytical reagents for molecular biology)
The enzyme used for the analytical reagent and the research reagent for molecular biology is not particularly limited, and examples thereof include peroxidase and alkaline phosphatase used for sandwich ELISA and the like. In general, peroxidase and alkaline phosphatase generate color, luminescence, or fluorescence by an enzyme reaction with a substrate, and qualitative and quantitative analysis is possible. As the chromogenic substrate, various Trinder reagents, 4-aminoantipyrine, electron carriers such as tetrazolium salts and phenazine methosulfate, and dyes such as leuco reagents can be used. As the luminescent substrate, an ECL (electrochemiluminescence) reagent or a fluorescent substance such as 10-acetyl-3,7-dihydroxyphenoxazine can be used. In the analytical reagent for molecular biology use, peroxidase and alkaline phosphatase may be in the state of an enzyme-labeled compound. For example, those conjugated with antibodies, those conjugated with antigens, dyes or biotin, or compounds labeled with avidin, streptavidin, neutravidin, etc. Included in alkaline phosphatase. The present invention can be applied to the stabilization of at least one of these enzymes.

Other enzymes used in analytical reagents and research reagents for molecular biology include reporter assays and luciferases used in the surrounding fields. So far, various luciferases isolated from beetles such as the firefly family, the mosquito family, the firefly family, and the Iriomote family are known. These luciferases may be further modified for the purpose of changing the wavelength of the emission spectrum.

Examples of other enzymes used for analysis reagents and research reagents for molecular biology include PCR, RT-PCR, and DNA polymerases, RNA polymerases, reverse transcriptases and the like used in the peripheral fields. Examples of the DNA polymerase include family A (PolI type) such as Taq and Tth, and family B (α type) such as KOD and Pfu, and either of them may be used. Examples of the RNA polymerase include those derived from phages such as T3, T7, and SP6. Examples of the reverse transcriptase include those derived from retroviruses such as M-MLV (Moloney Murine Leukemia Virus) and AMV (Avian Myeloblastosis Virus). The present invention can be applied to the stabilization of at least one of these enzymes.

In addition, ligase, nuclease, restriction enzyme, DNA modifying enzyme, sugar chain analysis and various glycosidases used in the peripheral field, protein analysis and various proteases used in the peripheral field, etc. Is mentioned. The present invention can be applied to the stabilization of at least one of these enzymes.

(Biological component measurement reagent)
It does not specifically limit as an enzyme used for a biological component measuring reagent use. The biological component measurement reagent includes an analytical reagent for biochemical use, an in vitro diagnostic agent, a liquid in vitro diagnostic agent, and the like. In a broad sense, it includes a dry in-vitro diagnostic agent processed into a chip shape or a slit shape, an enzyme sensor, an enzyme electrode, and the like. As long as these biological component measuring reagents are designed so as to be able to measure biological components, the form thereof is not limited and may be prepared at the time of measurement, or may be prepared and stored in advance. It may be a kit.

As a biological component measurement reagent, for example, a reagent used in a biological component measurement method by an enzyme method, particularly a method using an oxidase-peroxidase-oxidation-reduction color reagent (hereinafter also referred to as color former) system, that is, in a sample. Examples include reagents that use a method in which a substance to be measured is enzymatically reacted to generate hydrogen peroxide, and this is reacted with a color former in the presence of peroxidase to perform colorimetric determination. Such a biological component measurement method has already been established in the art. Therefore, the knowledge can be applied to the present invention to measure the amount or concentration of biological components in various samples, and the mode is not particularly limited. Examples thereof include reagents for measuring biological components such as uric acid (UA), creatinine (CRE), triglyceride (TG), and cholesterol (CHO).

In the enzyme composition to which the present invention is applied, the origin of the enzyme and the like are not particularly limited, and commercially available products and the like can be used. As the redox coloring reagent, any kind of dye may be used as long as it reacts with hydrogen peroxide to produce a color. For example, a combination of a hydrogen donor and a coupler (so-called Trinder reagent), a leuco body And tetrazolium salts. There are no particular restrictions on the amount used or the form of addition. All of these can be obtained as commercial products.

For example, since the reagent for measuring UA is designed to quantify hydrogen peroxide generated by the reaction of uricase using UA as a substrate by a peroxidase-chromogenic system, the reagent includes uricase and peroxidase. The present invention can be applied to the stabilization of at least one of these enzymes.
On the other hand, the CRE measuring reagent reacts creatinine generated by the reaction of creatinine amidinohydrolase using CRE as a substrate with creatine amide hydrolase to generate sarcosine, and further, sarcosine using sarcosine oxidase generates hydrogen peroxide. By designing a so-called coupling reaction, the CRE concentration can be determined by a peroxidase-color former system. This reagent thus includes creatinine amidinohydrolase, creatinamide hydrolase, sarcosine oxidase and peroxidase. The present invention can be applied to the stabilization of at least one of these enzymes.
When measuring TG, hydrogen peroxide is generated using lipoprotein lipase using TG as a substrate, and glycerol kinase and glycerol triphosphate oxidase as a conjugating enzyme. Quantification becomes possible. Thus, this reagent includes lipoprotein lipase, glycerol kinase, glycerol triphosphate oxidase and peroxidase. The present invention can be applied to the stabilization of at least one of these enzymes.

In addition to the above, a method for measuring cholesterol (including lipoprotein fractionated cholesterol such as free cholesterol, HDL cholesterol, LDL cholesterol) by conjugating cholesterol esterase and cholesterol oxidase, purine nucleotide phosphorylase and xanthine oxidase (in some cases Furthermore, a method of measuring inorganic phosphorus or inosine by conjugating uricase), a method of measuring glucose using glucose oxidase, a method of measuring ALT (GPT) by measuring pyruvic acid using pyruvate oxidase, etc. Can be illustrated.

By the way, in these methods, there are known problems that are easily affected by interfering substances coexisting in samples such as serum, for example, bioreductive substances such as ascorbic acid and bilirubin. Various countermeasures such as a so-called erasing system have been studied and overcome. For example, ascorbic acid can be eliminated by allowing ascorbate oxidase to act on the sample. Further, bilirubin can be eliminated by allowing bilirubin oxidase to act on the sample.
Furthermore, when several stages of conjugation reactions are designed, a positive error occurs due to the inclusion of the reaction intermediate in the sample. A typical method is to cause an enzyme other than the enzyme that directly acts on the measurement target substance to act on the sample to generate hydrogen peroxide, erase the reaction intermediate with catalase, and then react the enzyme that acts directly on the measurement target substance. In this method, hydrogen peroxide is generated in addition to the system, and this is reacted with a color former in the presence of peroxidase, and at the same time, the action of catalase is virtually stopped to perform colorimetric determination. In the case of CRE measurement, an enzyme other than creatinine amidinohydrolase (creatine amide hydrolase and sarcosine oxidase) is allowed to act on the sample, and hydrogen peroxide generated due to a reaction intermediate (creatine and the like) is eliminated with catalase. Creatinine amidinohydrolase is added to the reaction system, and hydrogen peroxide generated due to the CRE to be measured is reacted with a color former in the presence of peroxidase for colorimetric determination (at the same time, the action of catalase Is substantially stopped.).
Reagents employing such a method include ascorbate oxidase, bilirubin oxidase, catalase and the like. The present invention can be applied to the stabilization of at least one of these enzymes.

  As a method for measuring biological components by an enzyme method, there is also a method that utilizes redox of NAD by nicotinamide adenine dinucleotide (NAD) -dependent dehydrogenase. Since this method has already been established in the technical field, the amount or concentration of a biological component in various samples can be measured according to a known method. Although the aspect is not particularly limited, for example, in the case of a composition for measuring glucose, the concentration of glucose is obtained by utilizing the fact that NADH generated by reducing NAD by a glucose dehydrogenase reaction has absorption at 340 nm. be able to. Alternatively, the concentration of glucose can be determined by further reducing an electron acceptor such as DCPIP, returning itself to NAD, and colorimetrically determining a difference in absorbance caused by a change in the structure of DCPIP.

Also in this method, various biological components can be measured by designing a conjugate reaction. For example, in addition to glucose, reagents for measuring lactic acid measurement with lactate dehydrogenase (LDH) and biological components such as total branched chain amino acid (BCAA) can be exemplified.
When measuring lactic acid, LDH dehydrogenates L-lactic acid in the presence of the coenzyme NAD to produce pyruvic acid, and measures the increase in absorbance based on absorption of NADH produced in the process in the ultraviolet region wavelength. Can measure lactic acid. Thus, this reagent includes lactate dehydrogenase.
When measuring the total branched chain amino acid (BCAA), when leucine dehydrogenase is allowed to act on BCAA (valine, leucine, isoleucine) in a sample, when these BCAAs are converted into respective α-keto acids, NAD is converted to NADH. Produces. BCAA can be measured by measuring the amount of increase in absorbance based on the absorption of the NADH in the ultraviolet region wavelength. In the case of this measurement system, the following reaction may be further conjugated. The produced NADH reduces WST-1 which is a reducing coloring reagent through the action of diaphorase to produce formazan. BCAA can be determined by measuring the change in absorbance of the color of formazan. Thus, this reagent includes leucine dehydrogenase and diaphorase.
In addition to the above, for example, AST (GOT) is measured by measuring oxaloacetate using malate dehydrogenase, ALT (GPT) is measured by measuring pyruvate using lactate dehydrogenase The method of doing is mentioned.
The present invention can be applied to the stabilization of at least one of these enzymes.

  In addition, even if it is other than NAD, a measurement system similar to the above can be assembled by utilizing the difference in absorption of the ultraviolet part or visible part in the oxidized state and the reduced state. Examples thereof include nicotinamide adenine dinucleotide phosphate (NADP) and β-thionicotinamide adenine dinucleotide (thioNAD).

Means for carrying out the above method include a method using a liquid reagent (or kit) configured to be applicable to a general-purpose automatic analyzer (for example, Hitachi 7170 type automatic analyzer), a means such as lyophilization. Various methods such as a method using a reagent (or kit) composed of a combination of a produced dry preparation and a solution, a so-called dry system in which an enzyme or the like is supported on an appropriate carrier, and a method using a sensor A form can be illustrated.
Preferably, the analysis is performed by an automatic analyzer using a liquid reagent in which the reagent is divided into two (hereinafter also referred to as a two-reagent liquid reagent). In this method, the first type of reagent (hereinafter also referred to as the first reagent or R1) is first added to the sample and allowed to react for a certain period of time, and then the second type of reagent (hereinafter also referred to as the first reagent or R1). The target component can be quantified by adding and reacting, and measuring the change in absorbance during this period.

In the composition of the present invention, the content of L-PGA is not particularly limited. The concentration in the liquid state is preferably 0.1% or more, and more preferably 0.2% or more. Moreover, it is preferably 1% or less.

Buffer components, preservatives, salts, enzyme stabilizers, chromogen stabilizers and the like may be added to the composition of the present invention. There are no particular restrictions on the amount used or the form of addition. All of these can be obtained as commercial products.
Examples of the buffer solution component include a phosphate buffer solution, a tris (hydroxymethyl) aminomethane buffer solution, and a Good buffer solution. Examples of the good buffer include MES, Bis-Tris, ACES, BES, MOPS, PIPES, TES, HEPES, Tricine, Bicine, POPSO, TAPS, CHES, and CAPS. With these buffer components, the pH of the composition of the present invention is preferably maintained within the range of 5.0 to 9.0.
Examples of antiseptics include azides, chelating agents, antibiotics, and antibacterial agents. Antibacterial agents include 2-methyl-4-isothiazolin-3-one (MIT), 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), 1,2-benzisothiazolin-3-one ( BIT), an isothiazoline-based preservative, imidazolidinyl urea, and the like. In addition, as commercially available products, Procrine 150 (manufactured by Spelco, obtained from Sigma Aldrich Japan), Proclin 300 (manufactured by Spelco, obtained from Sigma Aldrich Japan), Actiside MBS (manufactured by Sor Japan), single As one product, Actiside B20 (N) (manufactured by So Japan), MIT (manufactured by Roche, Sigma) and the like can be mentioned.
Examples of the chelating agent include ethylenediaminetetraacetic acid and its salt.
Antibiotics include gentamicin, kanamycin, chloramphenicol and the like.
Examples of the salts include sodium chloride, potassium chloride, aluminum chloride and the like.
Enzyme stabilizers include sucrose, trehalose, cyclodextrin, gluconate, amino acids and the like.
Examples of chromogen stabilizers include chelating agents such as ethylenediaminetetraacetic acid and its salts, and cyclodextrins.

  In the present invention, “the enzyme composition is stable” means that the enzyme activity is maintained even during the heating treatment, and preferably has a residual enzyme activity after storage at 35 ° C. for 7 days. Is 10% or more, more preferably 20% or more, more preferably 40% or more, more preferably 60% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% compared to before storage. That means it is maintained. When the enzyme composition containing L-PGA satisfies this requirement, it is assumed that the enzyme composition is stabilized by containing L-PGA. The enzyme activity measurement method can be appropriately employed by those skilled in the art from known methods, for example, methods described in catalogs and package inserts (in the case of commercial products), methods described in literature, and the like.

  Hereinafter, the present invention will be specifically described by way of examples. In addition, this invention is not specifically limited by an Example.

Example 1
Residual activity after preparation of each enzyme shown in Table 1 containing 3 U / mL and 5 g / L of L-PGA and storage at 35 ° C. for 7 days (activity value after storage relative to activity value immediately after dissolution) Ratio).

In either enzyme, the enzyme activity remained after storage at 35 ° C. for 7 days.

The present invention includes an analytical reagent for molecular biology, an analytical reagent for biochemistry, an in vitro diagnostic agent, a liquid in vitro diagnostic agent, a dry in vitro diagnostic agent processed into a chip shape or a slit shape, an enzyme sensor and an enzyme electrode, It can be applied to compositions such as pharmaceuticals, foods and beverages.

Claims (2)

An enzyme composition comprising poly-γ-L-glutamic acid. A method for stabilizing an enzyme, characterized in that poly-γ-L-glutamic acid is allowed to coexist with the enzyme.