JP2009201436A - Polyol dehydrogenase composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition in which the preservation stability of a polyol dehydrogenase containing pyrroloquinolinequinone as a prosthetic group is remarkably improved, and to provide a measuring reagent for a polyol using the composition. <P>SOLUTION: The polyol dehydrogenase composition comprises the polyol dehydrogenase containing the pyrroloquinolinequinone as the prosthetic group, a cyclic amino acid, a non-reducing sugar or an amino glycoside-based antibiotic having a guanidino group, a metal compound containing a metal forming a divalent metal ion, and a surfactant. The measuring reagent for the polyol is obtained by using the composition. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to the storage stability of a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group (hereinafter also referred to as “PQQ-dependent PDH” or simply “polyol dehydrogenase”), particularly polyol dehydrogenation after lyophilization. The present invention relates to a polyol dehydrogenase composition with improved enzyme storage stability, a polyol measuring reagent containing the composition, and a polyol quantification method using the composition.

  Polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group is present in bacterial cell membranes, and it is known to extract and purify from bacteria belonging to the genus Gluconobacter. For quantification of glycerol, sorbitol and mannitol It's being used.

  Conventionally, for example, with respect to glycerol, glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) or glycerol-3-phosphate dehydrogenase (GPDH) as shown in the following formulas (1) and (2): ) Is known.

However, this method has a problem that the reaction is complicated because two kinds of enzymes are used. Further, when glycerol-3-phosphate oxidase is used, there is a problem that it is affected by dissolved oxygen. Moreover, when glycerol-3-phosphate dehydrogenase is used, it is necessary to add expensive NAD ⁺ .

  As a method using one kind of enzyme without being affected by dissolved oxygen, a method using NAD-dependent glycerol dehydrogenase is known as shown in the following formula (3).

However, since NAD-dependent glycerol dehydrogenase is not a coenzyme-linked enzyme, it is necessary to add expensive NAD ⁺ .

As a cheaper and simpler glycerol quantification method, there is a method using a polyol dehydrogenase (PQQ-dependent PDH) containing pyrroloquinoline quinone as a prosthetic group. Since this method is carried out by the reaction of the following formula (4), it is not affected by dissolved oxygen, the reaction is simple, there is no need to use a plurality of enzymes, and there is no need to add expensive NAD ^+. There is.

  However, since PQQ-dependent PDH is a membrane-bound enzyme, there is a problem that storage stability is poor. For this reason, it becomes an important subject to improve the stability of the PQQ-dependent PDH.

  Conventionally, enzyme stabilizers include proteins such as bovine serum albumin, human serum albumin and ovalbumin, sugars such as glucose, trehalose and raffinose, polyhydric alcohols such as glycerol and ethylene glycol, amino acids such as arginine and glutamic acid, Salts such as calcium ions and magnesium ions, reducing agents such as dithiothreitol and 2-mercaptoethanol are known. However, it is not known whether these stabilizers are effective against PQQ-dependent PDH.

  In Patent Document 1, a glucose dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group similar to PQQ-dependent PDH, (i) aspartic acid, glutamic acid, α-ketoglutaric acid, malic acid, By adding at least one compound selected from the group consisting of α-ketogluconic acid, α-cyclodextrin and salts thereof, and (ii) a stabilizer containing albumin, it is much more stable than before. It is disclosed that an enzyme composition is obtained.

Patent Document 2 discloses a polyol dehydrogenase composition by adding a compound having a divalent metal ion, a non-reducing sugar, and a surfactant as stabilizers to PQQ-dependent PDH. It is disclosed that it can be obtained.
JP 2001-224368 A JP 2007-259814 A

  However, the stabilizer described in Patent Document 1 cannot improve the storage stability of PQQ-dependent PDH.

  Further, in the enzyme composition described in Patent Document 2, the residual activity of the enzyme activity when stored at 37 ° C. for 1 week is around 80%, and it cannot be said that the storage stability of the enzyme is sufficient. For this reason, it is necessary to further improve the storage stability of the enzyme.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a polyol dehydrogenase composition having improved enzyme storage stability. Another object of the present invention is to provide a polyol measuring reagent containing the PQQ-dependent PDH composition capable of accurately quantifying the polyol, and a polyol quantification method using the PQQ-dependent PDH composition. .

  As a result of intensive research in view of the above-mentioned conventional problems, the present inventors have determined that aminoglycosides having cyclic amino acids, non-reducing sugars or guanidino groups with respect to polyol dehydrogenases containing pyrroloquinoline quinone as a prosthetic group. Stabilization of polyol dehydrogenase, especially the stability of polyol dehydrogenase after lyophilization is improved by adding a series of antibiotics, metal compounds containing metals that form divalent metal ions, and surfactants As a result, the present invention has been completed.

  That is, the present invention includes a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group, a cyclic amino acid, an aminoglycoside antibiotic having a non-reducing sugar or a guanidino group, and a metal that forms a divalent metal ion. A polyol dehydrogenase composition comprising a metal compound and a surfactant.

  According to the present invention, a decrease in enzyme activity over time of a polyol dehydrogenase (PQQ-dependent PDH) containing pyrroloquinoline quinone as a prosthetic group, particularly a decrease in enzyme activity over time after lyophilization is significantly suppressed. -It can be prevented and the storage stability of PQQ-dependent PDH can be significantly improved.

  Hereinafter, the present invention will be described in detail.

  According to one aspect of the present invention, a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group, a cyclic amino acid, an aminoglycoside antibiotic having a non-reducing sugar or a guanidino group, and a divalent metal ion are formed. A polyol dehydrogenase composition comprising a metal compound containing a metal and a surfactant is provided.

  In the present invention, the polyol dehydrogenase (PQQ-dependent PDH) containing pyrroloquinoline quinone as the prosthetic group may be any polyol as a substrate and may be an alcohol having two or more hydroxyl groups (including sugar alcohol). There is no particular limitation. For example, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, disaccharide-derived alcohols such as lactitol, triols such as glycerol, tetritols such as erythritol, arabitol, xylitol, Examples include pentitol such as ribitol, hexitol such as mannitol and sorbitol, and cyclitol such as inositol. Among them, glycerol (pyrroloquinoline quinone-dependent glycerol dehydrogenase), arabitol (pyrroloquinoline quinone-dependent arabitol dehydrogenase), and mannitol (pyrroloquinoline quinone-dependent mannitol dehydrogenase) are preferably used as substrates, and more preferably Glycerol is used as a substrate.

  The PQQ-dependent PDH used in the present invention can convert a polyol and an electron acceptor into a corresponding dehydrogenated product and a reduced electron acceptor. Examples of the electron acceptor to which the PQQ-dependent PDH of the present invention can be suitably used include potassium ferricyanide, phenazinium methyl sulfate and derivatives thereof, 2,6-dichlorophenolindophenol (DCIP), Wurster's blue, nitrotetrazolium blue. Etc.

  Any conventionally known enzyme can be preferably used as the PQQ-dependent PDH used in the present invention. The enzyme is known to be produced by various bacteria such as Gluconobacter and Pseudomonas. In the present invention, any PQQ-dependent PDH produced by a bacterium capable of producing these PQQ-dependent PDH (hereinafter also referred to as “PQQ-dependent PDH-producing bacterium”) can be preferably used. Among these, PQQ-dependent PDH derived from Gluconobacter genus can be preferably used. In addition, Gluconobacter oxydans NBRC 3130, 3171, 3172, 3189, 3244, 3250, 3253, 3255, 3256, 3257, 3258, 3285, 3287, 3289, 3290, 3291 due to availability. , 3292, 3293, 3294, 3462, 3990, 12467, 14819; Gluconobacter frateurii NBRC 3251, 3254, 3260, 3264, 3265, 3268, 3270, 3271, 3272, 3273, 3274, 3286, 16669 Gluconobacter cerinus NBRC 3262, 3263, 3 It is possible to use the 66,3267,3269,3275,3276 like.

  Moreover, as long as it is a PQQ-dependent PDH producing bacterium, these natural mutants or artificial mutants may be used. Artificial mutation treatment methods can be applied by methods well known to those skilled in the art, in the same manner or appropriately modified, or a combination of these methods as appropriate. As a representative strain of such a microorganism, Gluconobacter oxydans is used, and particularly Gluconobacter oxydans NBRC 3291 is preferably used.

  A specific method for obtaining PQQ-dependent PDH from the PQQ-dependent PDH-producing bacterium is not particularly limited. For example, the PQQ-dependent PDH-producing bacterium is cultured in a nutrient medium, and PQQ-dependent from the culture. The method of extracting PDH is mentioned.

  The medium for cultivating the PQQ-dependent PDH-producing bacterium is a natural medium or a synthetic medium as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance and other necessary nutrients that can be assimilated by the strain used. May be. As the carbon source, for example, glucose, glycerol, sorbitol and the like are used. As the nitrogen source, for example, nitrogen-containing natural products such as peptones, meat extracts and yeast extracts, and inorganic nitrogen-containing materials such as ammonium chloride and ammonium citrate are used. As the inorganic substance, potassium phosphate, sodium phosphate, magnesium sulfate or the like is used. In addition, specific vitamins are used as necessary. The above carbon source, nitrogen source, inorganic substance, and other necessary nutrients may be used alone or in combination of two or more.

  The culture is preferably performed by shaking culture or aeration and agitation culture. The culture temperature is 20 ° C to 50 ° C, preferably 22 ° C to 40 ° C, and most preferably 25 ° C to 35 ° C. The culture pH is 4-9, preferably 5-8. Even under conditions other than these, it is carried out if the strain to be used grows. The culture period is usually preferably 0.5 to 5 days. By the above culture, PQQ-dependent PDH is accumulated in the cells. These PQQ-dependent PDHs may be enzymes obtained by the above culture or recombinant enzymes obtained by transducing PQQ-dependent PDH genes into E. coli and the like.

  Next, the obtained PQQ-dependent PDH is extracted. As the extraction method, a commonly used extraction method can be used. For example, an ultrasonic crushing method, a French press method, an organic solvent method, a lysozyme method, or the like can be used. The method for purifying the extracted PQQ-dependent PDH is not particularly limited. For example, salting-out methods such as ammonium sulfate and sodium nitrate, metal aggregation methods using magnesium chloride and calcium chloride, denucleic acid using streptomycin and polyethyleneimine, or DEAE ( An ion exchange chromatography method such as diethylaminoethyl) -sepharose or CM (carboxymethyl) -sepharose can be used. The partially purified enzyme or purified enzyme solution obtained by these methods may be used as it is or in a chemically modified form. In the present invention, when a chemically modified form of PQQ-dependent PDH is used, the PQQ-dependent PDH derived from the culture obtained by the above method is described in, for example, JP-A-2006-271257. Such a method can be used with appropriate chemical modification. The chemical modification method is not limited to the method described in the above publication.

  The cyclic amino acid used in the present invention can improve the storage stability of PQQ-dependent PDH and can improve the storage stability of the enzyme composition of the present invention.

  Specific examples of the cyclic amino acid used in the present invention include ectoine, hydroxyectoine, proline, hydroxyproline and the like. Among these, ectoine or hydroxyectoine is preferable. These cyclic amino acids may be used alone or in combination of two or more.

  The amount of the cyclic amino acid contained in the enzyme composition of the present invention is not particularly limited as long as it can improve the storage stability of PQQ-dependent PDH, but the total mass of PQQ-dependent PDH in the enzyme composition is 100 mass. % Is preferably 10 to 500% by mass, more preferably 50 to 400% by mass, and most preferably 100 to 300%. If it is 10 mass% or more, since the effect as a stabilizer is fully exhibited, it is preferable. On the other hand, if it is 500% by mass or less, an improvement in the effect as a stabilizer commensurate with the addition is recognized, and the cyclic amino acid is dissolved when the polyol dehydrogenase composition of the present invention is redissolved with a buffer solution or the like. can do.

  The aminoglycoside antibiotic having a non-reducing sugar or guanidino group used in the present invention can improve the storage stability of PQQ-dependent PDH, and can improve the storage stability of the enzyme composition of the present invention.

  In the present invention, the “non-reducing sugar” means a saccharide having no reducing ability because it has no free aldehyde group or ketone group. Such reducing sugars may be those having the above-described properties. For example, trehalose-type microsaccharides in which reducing groups are bonded to each other, glycosides in which a reducing group of saccharides and non-saccharides are bonded, and saccharides. There are sugar alcohols reduced by hydrogenation. More specifically, trehalose-type oligosaccharides such as sucrose, trehalose and raffinose; glycosides such as alkyl glycosides, phenol glycosides and mustard oil glycosides; and sugar alcohols such as arabitol, xylitol and sorbitol Is mentioned. Of these, sugar alcohols that are substrates for PQQ-dependent PDH may not be preferred. Of these, trehalose, raffinose, and sucrose are preferable, and trehalose and raffinose are particularly preferable. These reducing sugars may be used alone or in combination of two or more.

  In the present invention, the “aminoglycoside antibiotic having a guanidino group” means a glycoside antibiotic containing an amino sugar having a guanidino group. Specific examples of such aminoglycoside antibiotics include streptomycin and dihydrostreptomycin. These aminoglycoside antibiotics having a guanidino group may be used alone or in combination of two or more.

  The amount of the aminoglycoside antibiotic having a non-reducing sugar or guanidino group contained in the enzyme composition of the present invention is not particularly limited as long as it can improve the storage stability of PQQ-dependent PDH. The total mass of the PQQ-dependent PDH is 100 mass%, preferably 1 to 200 mass%, more preferably 5 to 100 mass%. If it is 1 mass% or more, the effect as a stabilizer can fully be exhibited, and if it is 200 mass% or less, the improvement of the effect as a stabilizer suitable for addition is recognized.

  The metal compound containing a metal that forms a divalent metal ion used in the present invention can improve the storage stability of the PQQ-dependent PDH and can improve the storage stability of the composition of the present invention.

  The metal compound containing a metal that forms a divalent metal ion used in the present invention is not particularly limited as long as it can improve the storage stability of PQQ-dependent PDH and can form a divalent ion. Specific examples of the metal compound containing a metal forming the divalent metal ion include divalent metal ions such as magnesium, calcium, barium, manganese, iron, copper, cobalt, nickel, mercury, lead and zinc. The metal compound containing the metal which forms is mentioned. Among these, at least one selected from the group consisting of a compound containing magnesium and a compound containing calcium is preferable. In addition, the form of the metal compound containing a metal that forms the divalent metal ion is not particularly limited as long as it can improve the stability of the PQQ-dependent PDH. For example, the metal fluoride, chloride, Examples thereof include halides such as bromide or iodide, sulfates of the above metals, nitrates of the above metals, and phosphates of the above metals. Among these, at least one selected from the group consisting of chloride, sulfate, and nitrate is preferable. In addition, the metal compound containing the metal which forms these bivalent metal ions may be used alone, or may be used in the form of a mixture of two or more.

  The amount of the metal compound containing a metal that forms a divalent metal ion contained in the enzyme composition of the present invention is not particularly limited as long as it can improve the storage stability of PQQ-dependent PDH. The total mass of PQQ-dependent PDH is 100 mass%, preferably 1-30 mass%, more preferably 1-20 mass%. If it is 1% by mass or more, the effect as a stabilizer can be sufficiently exhibited. On the other hand, if it is 30% by mass or less, an improvement in the effect as a stabilizer commensurate with the addition is recognized. When the composition is redissolved with a buffer or the like, a metal compound containing a metal that forms a divalent metal ion can be dissolved.

  The surfactant used in the present invention can improve the storage stability of the PQQ-dependent PDH, and can improve the storage stability of the composition of the present invention.

  The type of the surfactant is not particularly limited as long as it is generally used for solubilizing membrane proteins. Specific examples include Triton (registered trademark) X-100, octyl glucoside, sodium cholate and the like, and these may be used alone or in the form of a mixture of two or more. Among these, Triton (registered trademark) X-100 is more preferable from the viewpoint of suppressing a decrease in enzyme activity.

  The amount of the surfactant contained in the enzyme composition of the present invention is not particularly limited as long as the stability of the enzyme can be improved, but preferably the total mass of PQQ-dependent PDH in the composition is 100% by mass. Is 1 to 500 mass%, more preferably 1 to 300 mass%. If it is 1 mass% or more, the effect of stabilizing the PQQ-dependent PDH can be obtained. On the other hand, if it is 500 mass% or less, the viscosity of the enzyme composition after lyophilization does not become too high, which is preferable.

  The form of the enzyme composition of the present invention is not particularly limited. For example, the enzyme composition of the present invention may be any solid form such as powder, granule, or tablet; solution such as liquid or emulsion; or semi-solid such as paste. It can be in form. Especially, since the effect of this invention is exhibited notably, it is preferable that it is a solid form.

  It is preferable that the enzyme composition of this invention contains the buffer solution of stable pH from a viewpoint of suppressing the fall of enzyme activity. As such a buffer, a known buffer can be appropriately used as long as it has a desired pH, and is not particularly limited. For example, phosphate buffer, Tris-HCl buffer, acetate buffer Solution, a GOOD buffer such as MOPS or HEPES, an amino acid buffer such as glycine-NaOH or glycylglycine-NaOH, a borate buffer, or an imidazole buffer. Among these, phosphate buffer, Tris-hydrochloric acid buffer, GOOD buffer such as MOPS, or glycylglycine-NaOH is preferable. The concentration of the buffer is not particularly limited, but is preferably 1 to 500 mM, more preferably 5 to 400 mM, and more preferably 5 to 300 mM. The pH of the buffer solution is not extremely deviated from the stable pH of the enzyme, and is preferably in the range of 4.0 to 11.0, more preferably 5.0 to 10.0.

  The enzyme composition of the present invention can contain a reducing agent such as dithiothreitol and 2-mercaptoethanol, pyrroloquinoline quinone, and the like as desired within the range not impairing the object of the present invention.

  The method for producing the enzyme composition of the present invention is not particularly limited, and an aminoglycoside antibiotic having a cyclic amino acid, a non-reducing sugar or a guanidino group is used for PQQ-dependent PDH, and a metal that forms a divalent metal ion. It can be obtained by a method comprising adding a metal compound and a surfactant.

  As a specific example, for example, a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group, a cyclic amino acid, an aminoglycoside antibiotic having a non-reducing sugar or a guanidino group, and a divalent metal ion are formed. It is a manufacturing method of a polyol dehydrogenase composition which has the step which adds the metal compound containing a metal, and surfactant as a stabilizer. By such a method, the polyol dehydrogenase composition can improve the stability of the polyol dehydrogenase.

  In the above method, the order of addition of each component is not particularly limited, and each component may be added sequentially to the PQQ-dependent PDH, or each component may be added simultaneously.

  When the enzyme composition of the present invention is in a powder form, a metal containing a metal that forms a divalent metal ion, an aminoglycoside antibiotic having a cyclic amino acid, a non-reducing sugar or a guanidino group against PQQ-dependent PDH A solution-like enzyme composition obtained by adding a compound and a surfactant can be powdered by freeze-drying or the like. At this time, the freeze-drying method is not particularly limited, and a conventionally known method can be used. In addition, the step of freeze-drying the enzyme composition may be performed immediately after the step of obtaining the enzyme composition by adding the stabilizer, or the step of obtaining the enzyme composition by adding the stabilizer and the enzyme Other steps may be performed between the lyophilization of the composition.

  The polyol dehydrogenase composition of the present invention can improve the stability of the polyol dehydrogenase, and in particular, suppresses the deactivation of the PQQ-dependent PDH during lyophilization, and the PQQ-dependent PDH after lyophilization. The storage stability of can be significantly improved. In particular, ectoine and / or hydroxyectoine is used as a cyclic amino acid, trehalose and / or raffinose as a non-reducing sugar, or streptomycin and / or dihydrostreptomycin as an aminoglycoside antibiotic having a guanidino group to form a divalent metal ion When a compound containing calcium and / or a compound containing magnesium is used as the compound containing the metal to be used, and Triton (registered trademark) (Triton) X-100 is used as the surfactant, the above-described effect is remarkably obtained.

  According to another form of this invention, the polyol measuring reagent containing the enzyme composition of the said form of this invention is provided. According to another aspect of the present invention, there is provided a method for quantifying a polyol, characterized by reacting the enzyme composition of the above aspect of the present invention with a polyol. Since the PQQ-dependent PDH contained in the enzyme composition of the present invention is excellent in quantification of polyol, it can be suitably used as a polyol measuring reagent. In addition, since PQQ-dependent PDH has PQQ as a prosthetic group, polyol can be quantified without intentionally adding PQQ to the reaction system.

  In the present invention, “polyol” means an alcohol having two or more hydroxyl groups (including sugar alcohol). Although it does not restrict | limit especially as a polyol used by this invention, Disaccharide origin alcohol, glycerol, such as ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, propylene glycol, dipropylene glycol, 1, 3- butylene glycol, or lactitol And tetritol such as erythritol, pentitol such as arabitol, xylitol, or ribitol, hexitol such as mannitol, or sorbitol, cyclitol such as inositol, and the like. Among these, glycerol is preferable.

  The polyol measuring reagent of the present invention is a reagent for quantifying polyol, and contains PQQ-dependent PDH in the polyol dehydrogenase composition. It is characterized in that PQQ-dependent PDH is used as a polyol dehydrogenase. For example, PQQ-dependent PDH contained in the enzyme composition of the present invention is used as the polyol dehydrogenase used in the polyol measurement described in Patent Publication No. 3041840, Patent Publication No. 3450911, Patent Publication No. 3494398, and the like. be able to.

  Examples of the sample containing the polyol used in the quantification method of the present invention include food, serum, plasma, and whole blood. The PQQ-dependent PDH contained in the enzyme composition of the present invention can also be used for measuring neutral fat such as serum, plasma, or whole blood. That is, the neutral fat contained in these samples is decomposed into free fatty acid and glycerol by, for example, lipoprotein lipase, and the glycerol produced here can be quantified using the PQQ-dependent PDH. . In psychiatric patients and dialysis patients, free glycerol is a problem when measuring triglycerides, but the PQQ-dependent PDH used in the present invention is used to eliminate glycerol or measure the amount of glycerol. It is possible to obtain the true triglyceride value. The PQQ-dependent PDH used in the present invention can accurately determine the polyol even if the solution contains a surfactant.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all. In the present invention, the enzyme activity of PQQ-dependent PDH was measured by the following method.

(Enzyme activity)
0.1% Triton® X-100 containing 50 μM DCIP (2,6-dichloroindophenol), 0.2 mM PMS (1-methoxy-5-methylphenazinium methyl sulfate), and 450 mM glycerol. The enzyme solution was added to 10 mM phosphate buffer (pH 7.0) containing The reaction between the enzyme and the substrate in this solution was followed by the change in absorbance of DCIP at 600 nm, and the decrease rate of the absorbance was defined as the enzyme reaction rate. Here, the enzyme activity that reduces 1 μmol of DCIP per minute was defined as 1 unit (U). The molar extinction coefficient of DCIP at pH 7.0 was 16.3 mM- ¹ .

[Examples 1 and 2 and Comparative Examples 1 to 6]
Sorbitol 2 g / 100 mL, Yeast extract 0.3 g / 100 mL, Meat extract 0.3 g / 100 mL, Corn steep liquor 0.3 g / 100 mL, Polypeptone 1 g / 100 mL, Urea 0.1 g / 100 mL, KH ₂ PO ₄ 0.1 g / 100 mL of MgSO ₄ · 7H ₂ O 0.02 g / 100 mL and CaCl ₂ · 2H ₂ O 0.1 g / 100 mL, pH 7.0 was prepared, and 100 mL of the medium was added to a 500 mL Sakaguchi flask. 80 mL was transferred and autoclaved at 121 ° C. for 20 minutes.

One platinum ear inoculum of Gluconobacter oxydans NBRC 3291 was inoculated into the above-mentioned medium as a seed fungus, and cultured at 30 ° C. for 24 hours with shaking at 140 min ⁻¹ , and this was used as a seed culture solution.

  Next, 5 L of a medium prepared with the same composition as above was transferred to an 8 L jar fermenter, autoclaved at 121 ° C. for 50 minutes, allowed to cool, and then 240 mL of the seed culture solution was transferred. This was cultured for 26 hours under the conditions of 400 rpm, aeration rate 5 L / min, 30 ° C.

  After culturing for a predetermined time, the culture solution was collected by centrifugation (8,000 rpm, 10 minutes, 4 ° C.), suspended in a buffer solution, and then disrupted by a French press. The disrupted solution was centrifuged (4,000 rpm, 10 minutes, 4 ° C.), and the resulting supernatant was ultracentrifuged (40,000 rpm, 90 minutes, 4 ° C.) to obtain a membrane fraction as a precipitate. .

  This membrane fraction was suspended in 10 mM Tris-HCl buffer (pH 8.0), Triton (registered trademark) X-100 was added to a final concentration of 1 g / 100 mL, and the mixture was stirred at 4 ° C. for 2 hours. Ultracentrifugation (40,000 rpm, 90 minutes, 4 ° C.), and the supernatant was dialyzed overnight with 10 mM phosphate buffer (pH 7.5) containing 0.1 g / 100 mL Triton® X-100, This was used as a solubilized membrane fraction.

This solubilized membrane fraction was added to FPLC (Fast Protein Liquid Chro).
A fraction of polyol dehydrogenase activity from which glucose dehydrogenase contaminated with 6 mL of ResourceQ was removed was obtained. The fraction was dialyzed overnight against 20 mM MOPS buffer (pH 7.5) containing 0.1 g / 100 mL Triton (registered trademark) X-100 to obtain an enzyme preparation having a specific activity of 17 U / mg protein. This is called PDH derived from Gluconobacter oxydans.

  Next, 1 g / 100 mL glutaraldehyde solution to a final concentration of 0.2 g / 100 mL to 40 mL of the Gluconobacter oxydans-derived PDH (specific activity 17.0 U / mg protein, protein concentration: 0.625 mg / mL) 10 mL was added and gently stirred at 20 ° C. for 60 minutes.

  Next, 50 mL of 0.5 M Tris-HCl buffer (pH 8.0) was added, and the crosslinking reaction was stopped by reacting at 20 ° C. for 30 minutes. After completion of the reaction, low molecular weight contaminants were removed by dialysis overnight with 10 mM Tris-HCl buffer (pH 8.0) containing 0.1 g / 100 mL Triton (registered trademark) X-100, and this was designated as modified PDH.

  The modified PDH thus obtained was concentrated to ultrafiltration. With respect to 100% by mass of the obtained concentrated modified PDH (specific activity 17 U / mg protein, protein concentration 2.0 mg / mL), the components described in Table 1 were respectively added so as to have the ratios described in Table 1. After freezing, freeze-drying was performed for 24 hours to obtain a powdery enzyme composition. At this time, the enzyme activity of the enzyme composition immediately after lyophilization was measured. After the obtained enzyme composition was incubated at 37 ° C. for 1 week, the enzyme activity was measured. When the enzyme activity of the enzyme composition immediately after lyophilization was defined as 100%, the residual activity (unit:%) after incubation at 37 ° C. for 1 week was calculated. The results are shown in Table 1.

From the results shown in Table 1 above, cyclic amino acids (ectoine, hydroxyectoine), non-reducing sugars (trehalose, raffinose) or aminoglycoside antibiotics having a guanidino group (dihydrostreptomycin, streptomycin) and divalent metal ions By combining a metal compound containing a metal to be formed (MgSO ₄ , CaCl ₂ ) and a surfactant (Triton (registered trademark) X-100), any one, two or three of the above were combined. Compared with the case, it was confirmed that the inactivation of the enzyme by freeze-drying can be suppressed significantly.

  From the above results, it was confirmed that the enzyme composition of the present invention can significantly improve the stability of PQQ-dependent PDH and can significantly suppress the decrease in enzyme activity due to lyophilization.

  According to the present invention, it is possible to improve the enzyme stability over time after lyophilization of a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group. It can be suitably used for quantitatively determining the polyol.

Claims (10)

A polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group,
A cyclic amino acid;
An aminoglycoside antibiotic having a non-reducing sugar or guanidino group;
A metal compound containing a metal that forms a divalent metal ion;
A surfactant,
A polyol dehydrogenase composition comprising:   The polyol dehydrogenase composition according to claim 1, wherein the polyol dehydrogenase is a glycerol dehydrogenase.   The polyol dehydrogenase composition according to claim 1 or 2, wherein the cyclic amino acid contains at least one of ectoine and hydroxyectoine.   The polyol dehydrogenase composition according to any one of claims 1 to 3, wherein the non-reducing sugar includes at least one of trehalose and raffinose, and the aminoglycoside antibiotic includes at least one of streptomycin and dihydrostreptomycin. object.   The polyol dehydrogenase composition according to any one of claims 1 to 4, wherein the metal compound is at least one selected from the group consisting of a compound containing calcium and a compound containing magnesium.   The polyol dehydrogenase composition according to any one of claims 1 to 5, wherein the surfactant is Triton (registered trademark) (Triton) X-100.   The polyol measuring reagent containing the polyol dehydrogenase composition of any one of Claims 1-6.   A method for quantifying a polyol, comprising a step of reacting the polyol dehydrogenase composition according to any one of claims 1 to 6 with a polyol.   A polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group, a cyclic amino acid, an aminoglycoside antibiotic having a non-reducing sugar or a guanidino group, a metal compound containing a metal that forms a divalent metal ion, and a surface activity And a method for producing a polyol dehydrogenase composition, comprising the step of adding an agent as a stabilizer.   A polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group, a cyclic amino acid, an aminoglycoside antibiotic having a non-reducing sugar or a guanidino group, a metal compound containing a metal that forms a divalent metal ion, and a surface activity A method for producing a polyol dehydrogenase composition, comprising: adding an agent as a stabilizer to obtain an enzyme composition; and lyophilizing the enzyme composition.