JP2022185218A - Biosensor for ketone body measurement - Google Patents

Abstract

To provide a biosensor for in-blood ketone body measurement that has excellent heat resistance and, particularly when making a sensor, shows a stable response value without depending on the temperature of dry treatment, the biosensor employing 3-hydroxybutyrate dehydrogenase.SOLUTION: A sensor for ketone body measurement contains a reaction layer containing a 3-hydroxybutyrate dehydrogenase derived from Geobacillus microorganisms and an electronic receptor.SELECTED DRAWING: None

Description

The present invention relates to a biosensor for measuring ketone bodies. Specifically, it relates to a biosensor for measuring ketone bodies using 3-hydroxybutyrate dehydrogenase derived from microorganisms belonging to the genus Geobacillus.

In the field of clinical examination, blood ketone bodies are used as a metabolic index that reflects the degree of insufficient insulin action among diabetic patients. Ketone bodies are a general term for acetoacetate, 3-hydroxybutyric acid and acetone, but most of blood ketone bodies are acetoacetate and 3-hydroxybutyric acid. Since ketone bodies are intermediate metabolites in the synthesis and decomposition of fat, they are rarely present in the blood. Used as an alternative energy source when unavailable. When the body's glucose supply is reduced for any reason, glycogen stored in the liver is broken down into glucose and used to maintain blood sugar levels. However, glycogen in the liver is depleted in about 18 to 24 hours, so when glucose is depleted, muscle (protein) and fat (fatty acid) stored in adipocytes become energy sources.

Ketone bodies are mainly produced in the liver as metabolites during the oxidation process of fatty acids, and they also serve as an indicator of whether carbohydrates are appropriately utilized as an energy source. Excessive ketone levels in diabetics also cause diabetic ketoacidosis (DKA), an acute medical condition that can be fatal if left untreated. DKA is a dangerous condition in which total ketone bodies become 7000 μmol/L or more due to diabetes (PET bottle syndrome) or dehydration, leading to dehydration, disturbance of consciousness, etc., and in severe cases, death. 3-Hydroxybutyrate dehydrogenase is used to quantify ketone bodies.

A generalized means for analyzing selected analytes in bodily fluid samples includes biosensors. For example, diabetics routinely use glucose sensors to self-monitor their blood sugar levels and take corrective action in response to the measured blood sugar levels to keep them within acceptable limits. Due to the large number of patients, the glucose sensor market is global scale, and companies in each country are engaged in the development of enzymes (Patent Document 1), mediators (Patent Document 2), coenzymes (Patent Document 3), etc. ing.

However, while gelcose sensors have been actively developed, there is room for improvement in sensors for measuring ketone bodies used as DKA progress diagnosis in diabetic patients. It is, for example, a 3-hydroxybutyrate dehydrogenase derived from microorganisms such as Rhodospirillum rubrum (Non-Patent Document 1), Pseudomonas lemoignei (Non-Patent Document 2), and Rhizobium meliloti. ) (Non-Patent Document 3), Alcaligenes faecalis (Patent Document 1), Rhodobacter sphaeroides (Patent Document 2) and the like. Most enzymes are unstable. The stability of the enzyme affects the preparation process, service life, and transportation conditions of the sensor. Enzyme development is desired.

JP-A-10-227755 Japanese Patent Publication No. 2001-520367 Japanese Unexamined Patent Application Publication No. 2017-104101 JP-A-8-70856 JP-A-11-318438

J. Biol. Chem. (1962), 237:603-607. J. Biol. Chem. (1965), 240:4023-4028. 1622589942388_0. (1999), 81(3):849-857.

The present invention was invented in view of the current state of the prior art as described above. An object of the present invention is to provide a biosensor for measuring blood ketone bodies using 3-hydroxybutyrate dehydrogenase, which has a stable response value.

In order to solve the above problems, the present inventors have made extensive studies and found that it is useful to use 3-hydroxybutyrate dehydrogenase derived from microorganisms belonging to the genus Geobacillus, and completed the present invention. reached. A representative aspect of the invention is as follows.

（ｂ）至適温度：５５℃以上
（ｃ）熱安定性：３０分処理後の残存活性率が９０％以上を示す範囲が６５℃以下
（ｄ）至適ｐＨ：６．０～９．０
（ｅ）ｐＨ安定性：２５℃で２０時間処理後の残存活性率が８０％以上を示す範囲が５．０～１０．０
（ｆ）分子量：約２８ｋＤａ（ＳＤＳ－ＰＡＧＥ）
（ｇ）Ｋｍ値：３－ヒドロキシ酪酸に対するＫｍ値が０．６ｍＭ以下
項３．
（ｂ）至適温度：６５℃以上である、項２に記載のケトン体測定用バイオセンサ。
項４．
（ｃ）熱安定性：１６時間処理後の残存活性率が９０％以上を示す範囲が５５℃以下である、項２に記載のケトン体測定用バイオセンサ。
項５．
（ｃ）熱安定性：３０分処理後の残存活性率が９０％以上を示す範囲が６５℃以下であり、かつ、１６時間処理後の残存活性率が９０％以上を示す範囲が５５℃以下である、項２に記載のケトン体測定用バイオセンサ。
項６．
（ｄ）至適ｐＨ：７．５～８．５である、項２に記載のケトン体測定用バイオセンサ。
項７．
（ｅ）ｐＨ安定性：２５℃で２０時間処理後の残存活性率が８０％以上を示す範囲が５．２～９．４である、項２に記載のケトン体測定用バイオセンサ。
項８．
３－ヒドロキシ酪酸脱水素酵素が遺伝子組換え酵素である、項１～７のいずれかに記載のケトン体測定用バイオセンサ。
項９．
ゲオバチルス属の微生物がゲオバチルス・ステアロサーモフィルス（Ｇｅｐｂａｃｉｌｌｕｓ ｓｔｅａｒｏｐｈｅｒｍｏｐｈｉｌｕｓ）である、項１～８のいずれかに記載のケトン体測定用バイオセンサ。
項１０．
３－ヒドロキシ酪酸脱水素酵素が、配列番号１との同一性が９０％以上であるアミノ酸配列からなる、項１～９のいずれかに記載のケトン体測定用バイオセンサ。
項１１．
３－ヒドロキシ酪酸脱水素酵素が、配列番号１との同一性が９５％以上であるアミノ酸配列からなる、項１０に記載のケトン体測定用バイオセンサ。
項１２．
３－ヒドロキシ酪酸脱水素酵素が、配列番号１において、１乃至数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなる、項１０に記載のケトン体測定用バイオセンサ。
項１３．
３－ヒドロキシ酪酸脱水素酵素が、配列番号１に記載のアミノ酸配列からなる、項１０に記載のケトン体測定用バイオセンサ。
項１４．
電子受容体がルテニウム化合物である、項１～１３のいずれかに記載のケトン体測定用バイオセンサ。
項１５．
ルテニウム化合物が、塩化ヘキサアミンルテニウム（III）、（ビピリジン）ルテニウム（II）、（４，４’－ジメチル－２，２’－ビピリジン）ルテニウム（II）、（４，４’－ジフェニル－２，２’－ビピリジン）ルテニウム（II）、（４，４’－ジアミノ－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、（４，４’－ジヒドロキシ－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、（４，４’－ジカルボキシ－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、（４，４’－ジブロモ－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、（５，５’－ジメチル－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、（５，５’－ジフェニル－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、（５，５’－ジアミノ－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、（５，５’－ジヒドロキシ－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、（５，５’－ジカルボキシ－２，２’－ビピリジン）ルテニウム（II）ルテニウム（II）、および、（５，５’－ジブロモ－２，２’－ビピリジン）ルテニウム（II）からなる群から選択された少なくとも一種である、項１４に記載のケトン体測定用バイオセンサ。
項１６．
電子受容体がフェリシアン化化合物である、項１～１３のいずれかに記載のケトン体測定用バイオセンサ。
項１７．
３－ヒドロキシ酪酸脱水素酵素溶液を保持する電極系を３０℃以上の温度で乾燥する工程を含む、ケトン体測定用バイオセンサの製造方法。
項１８．
４０℃以上の温度で乾燥する工程を含む、項１８に記載のケトン体測定用バイオセンサの製造方法。
項１９．
５０℃以上の温度で乾燥する工程を含む、項１８に記載のケトン体測定用バイオセンサの製造方法。
項２０．
３－ヒドロキシ酪酸脱水素酵素溶液を含む組成物のｐＨを、ｐＨ６．０～９．０にする工程を含む、ケトン体測定用バイオセンサの製造方法。 Section 1.
It has an electrode system having a working electrode and a counter electrode formed on an electrically insulating substrate, and the electrode system contains 3-hydroxybutyrate dehydrogenase and an electron acceptor derived from microorganisms belonging to the genus Geobacillus. A biosensor for measuring ketone bodies, having a configuration comprising a reaction layer containing
Section 2.
Item 2. The biosensor for measuring ketone bodies according to item 1, wherein the 3-hydroxybutyrate dehydrogenase has the following physicochemical properties (a) to (g).
(a) Action: catalyze the following reactions.

(b) Optimal temperature: 55° C. or higher (c) Thermal stability: The range in which the residual activity rate after 30 minutes of treatment is 90% or higher is 65° C. or lower (d) Optimal pH: 6.0 to 9.0
(e) pH stability: range of 5.0 to 10.0 showing a residual activity rate of 80% or more after treatment at 25°C for 20 hours
(f) molecular weight: about 28 kDa (SDS-PAGE)
(g) Km value: Km value for 3-hydroxybutyric acid is 0.6 mM or less Item 3.
Item 2. The biosensor for measuring ketone bodies according to Item 2, wherein (b) the optimum temperature is 65°C or higher.
Section 4.
(c) Thermal stability: The biosensor for measuring ketone bodies according to item 2, wherein the range in which the residual activity rate after 16 hours of treatment is 90% or more is 55°C or less.
Item 5.
(c) Thermal stability: The range in which the residual activity rate after 30-minute treatment is 90% or higher is 65°C or lower, and the range in which the residual activity rate is 90% or higher after 16-hour treatment is 55°C or lower. Item 3. The biosensor for measuring ketone bodies according to Item 2, wherein:
Item 6.
Item 2. The biosensor for measuring ketone bodies according to Item 2, wherein (d) the optimum pH is 7.5 to 8.5.
Item 7.
(e) pH stability: The biosensor for measuring ketone bodies according to item 2, wherein the range showing a residual activity rate of 80% or more after treatment at 25°C for 20 hours is 5.2 to 9.4.
Item 8.
Item 8. The biosensor for measuring ketone bodies according to any one of items 1 to 7, wherein the 3-hydroxybutyrate dehydrogenase is a recombinant enzyme.
Item 9.
Item 9. The biosensor for measuring ketone bodies according to any one of Items 1 to 8, wherein the Geobacillus microorganism is Gepbacillus stearothermophilus.
Item 10.
Item 10. The biosensor for measuring ketone bodies according to any one of items 1 to 9, wherein the 3-hydroxybutyrate dehydrogenase consists of an amino acid sequence having 90% or more identity with SEQ ID NO:1.
Item 11.
Item 11. The biosensor for measuring ketone bodies according to Item 10, wherein the 3-hydroxybutyrate dehydrogenase comprises an amino acid sequence having 95% or more identity with SEQ ID NO:1.
Item 12.
Item 11. The biosensor for measuring ketone bodies according to Item 10, wherein the 3-hydroxybutyrate dehydrogenase has an amino acid sequence in which one to several amino acids are deleted, substituted or added in SEQ ID NO:1.
Item 13.
Item 11. The biosensor for measuring ketone bodies according to Item 10, wherein the 3-hydroxybutyrate dehydrogenase consists of the amino acid sequence set forth in SEQ ID NO:1.
Item 14.
Item 14. The biosensor for measuring ketone bodies according to any one of items 1 to 13, wherein the electron acceptor is a ruthenium compound.
Item 15.
Ruthenium compounds include hexaamineruthenium chloride (III), (bipyridine)ruthenium (II), (4,4'-dimethyl-2,2'-bipyridine)ruthenium (II), (4,4'-diphenyl-2, 2'-bipyridine)ruthenium (II), (4,4'-diamino-2,2'-bipyridine)ruthenium (II) ruthenium (II), (4,4'-dihydroxy-2,2'-bipyridine)ruthenium (II) ruthenium (II), (4,4'-dicarboxy-2,2'-bipyridine) ruthenium (II) ruthenium (II), (4,4'-dibromo-2,2'-bipyridine) ruthenium ( II) Ruthenium (II), (5,5'-dimethyl-2,2'-bipyridine)ruthenium (II) Ruthenium (II), (5,5'-diphenyl-2,2'-bipyridine)ruthenium (II) Ruthenium (II), (5,5'-diamino-2,2'-bipyridine) ruthenium (II) ruthenium (II), (5,5'-dihydroxy-2,2'-bipyridine) ruthenium (II) ruthenium ( II), from (5,5′-dicarboxy-2,2′-bipyridine)ruthenium (II) ruthenium (II) and (5,5′-dibromo-2,2′-bipyridine)ruthenium (II) Item 15. The biosensor for measuring ketone bodies according to Item 14, which is at least one selected from the group consisting of:
Item 16.
Item 14. The biosensor for measuring ketone bodies according to any one of Items 1 to 13, wherein the electron acceptor is a ferricyanide compound.
Item 17.
A method for producing a biosensor for measuring ketone bodies, comprising the step of drying an electrode system holding a 3-hydroxybutyrate dehydrogenase solution at a temperature of 30° C. or higher.
Item 18.
Item 19. A method for producing a biosensor for measuring ketone bodies according to Item 18, comprising a step of drying at a temperature of 40°C or higher.
Item 19.
Item 19. A method for producing a biosensor for measuring ketone bodies according to Item 18, comprising a step of drying at a temperature of 50°C or higher.
Item 20.
A method for producing a biosensor for measuring ketone bodies, comprising the step of adjusting the pH of a composition containing a 3-hydroxybutyrate dehydrogenase solution to pH 6.0 to 9.0.

Since the biosensor for measuring ketone bodies of the present invention is excellent in thermal stability, it enables efficient production of a sensor strip that involves a step of applying a load such as heat treatment.

4 is a graph showing the effect on the electrode response value when the drying temperature is set at 30° C. to 50° C. when producing a biosensor for measuring ketone bodies using 3-hydroxybutyrate dehydrogenase derived from the genus Geobacillus. 4 is a graph showing the effect on the electrode response value when the drying temperature is set at 30° C. to 50° C. when producing a biosensor for measuring ketone bodies using 3-hydroxybutyrate dehydrogenase derived from the genus Pseudomonas.

The 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) used in the present invention oxidizes 3-hydroxybutyrate in the presence of nicotinamide dinucleotide (NAD) to produce acetoacetate and reduced NAD. Enzymes with physicochemical properties that reversibly catalyze reactions. In the present invention, this physicochemical property is referred to as 3-hydroxybutyrate dehydrogenase activity, and unless otherwise specified, "enzyme activity" or "activity" means 3-hydroxybutyrate dehydrogenase activity.

In the present invention, a ketone body is a general term for compounds in which a carbonyl group and two hydrocarbons are bonded. Ketone bodies present in the body include acetoacetic acid, 3-hydroxybutyric acid, and acetone.

The properties of the 3-hydroxybutyrate dehydrogenase preferably used in the present invention are described below.

The 3-hydroxybutyrate dehydrogenase used in the present invention catalyzes the following reactions.

The 3-hydroxybutyrate dehydrogenase used in the present invention has substrate specificity to act specifically on ketone bodies (3-hydroxybutyrate). In particular, 3-Hydroxypropionate, Lactate, Glycerate, 2-Hydroxybutyrate, L-Malate, D,L-apple It is characterized by low reactivity to acids (D, L-Malate), 2-butanol (sec-Butyl alcohol), gluconate, glycolate and the like. As a coenzyme, it has excellent specificity for NAD ⁺ and low reactivity for NADP ⁺ .

The optimum temperature for the 3-hydroxybutyrate dehydrogenase used in the present invention is preferably 55°C or higher, more preferably 60°C or higher, and even more preferably 65°C or higher.

The thermal stability of the 3-hydroxybutyrate dehydrogenase used in the present invention is evaluated by the residual activity rate after heat treatment for 30 minutes at pH 6.5. When the residual activity rate is 90% or more, Shall be thermally stable. More preferably, the residual activity rate is 95% or more. The range showing such thermal stability is preferably 50° C. or lower, more preferably 55° C. or lower, still more preferably 60° C. or lower, and particularly preferably 65° C. or lower.

The thermal stability of the 3-hydroxybutyrate dehydrogenase used in the present invention can also be evaluated by the residual activity after heat treatment at pH 6.5 for 16 hours. If the residual activity rate is 90% or more, it is considered to have thermal stability. More preferably, the residual activity rate is 95% or more. After heat treatment for 16 hours, such thermal stability range is preferably 45° C. or lower, more preferably 50° C. or lower, particularly preferably 55° C. or lower.

The optimum pH of the 3-hydroxybutyrate dehydrogenase used in the present invention is preferably within the range of pH 6.0 to 9.0. The pH range is more preferably 6.5 to 8.5, and particularly preferably 7.5 to 8.5.

The pH stability of the 3-hydroxybutyrate dehydrogenase used in the present invention is evaluated by the residual activity after treatment at 25° C. for 20 hours. shall have the nature of The pH stability range is preferably in the range of pH 5.0-10.0, more preferably in the range of pH 5.2-9.4. Since 3-hydroxybutyrate dehydrogenase is stable over a wide range of pH, it is very useful in that there are fewer restrictions on the types of buffer solutions used when producing a sensor for measuring ketone bodies. be.

The molecular weight of the 3-hydroxybutyrate dehydrogenase used in the present invention is preferably about 25-30 kDa, more preferably about 27-29 kDa, particularly preferably about 28 kDa, as determined by SDS-PAGE.

The Km value for 3-hydroxybutyrate of 3-hydroxybutyrate dehydrogenase used in the present invention is preferably 0.6 mM or less, more preferably 0.5 mM or less.

The 3-hydroxybutyrate dehydrogenase used in the present invention is less affected by enzyme activity inhibition by various coexisting substances, so that the reaction conditions used for measuring ketone bodies are less restricted. , which is advantageous in terms of versatility. It is also advantageous in terms of storage stability. Examples of coexisting substances that are not subject to such inhibition include various metal salts such as metal chlorides, preservatives, chelating agents, surfactants, and other chemical substances.

Specific examples of coexisting substances that are not inhibited by the 3-hydroxybutyrate dehydrogenase used in the present invention include metal chlorides such as manganese chloride, calcium chloride, manganese chloride, and cobalt chloride. , nickel chloride, iron (III) chloride, and the like. Other metal salts include, for example, zinc sulfate. Preferably, these metal chlorides or other metal salts exhibit a residual activity rate of 70% or more after treatment at 25° C. for 1 hour at a concentration of 2 mM. More preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more.

Examples of antiseptics include monoiodoacetic acid (MIA; Monoiodoacetate), sodium azide, sodium fluoride and the like. Preferably, these preservatives show a residual activity rate of 70% or more after treatment at 25° C. for 1 hour at a concentration of 2 mM to 20 mM. More preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more.

Examples of chelating agents include EDTA (ethylenediaminetetraacetic acid), phenanthroline, α,α'-dipyridyl and the like. The 3-hydroxybutyrate dehydrogenase used in the present invention preferably exhibits a residual activity rate of 70% or more after treatment at 25° C. for 1 hour with these chelating agents at a concentration of 1 mM to 50 mM. More preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more, shows a residual activity rate. Other chemicals include borate, hydroxylamine, iodoacetamide (IAA), and the like.

Surfactants include, for example, Triton X-100 (polyethylene glycol mono-p-isooctylphenyl ether), Tween 20 (polyoxyethylene sorbitan monolaurate), Brij 35 (polyoxyethylene lauryl ether), sodium cholate, Span 20 (sorbitan monolaurate) and the like. The 3-hydroxybutyrate dehydrogenase of the present invention preferably exhibits a residual activity of 70% or more after treatment at 25° C. for 1 hour with these surfactants at a concentration of 0.1%. More preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more. The use of an enzyme that is not inhibited even in the coexistence of such a surfactant is very useful for producing a sensor for measuring ketone bodies.

One preferred embodiment of the 3-hydroxybutyrate dehydrogenase used in the present invention is one derived from a microorganism belonging to the genus Geobacillus and having the following physicochemical properties (a) to (g).
(a) Action: catalyze the following reactions.

(b) Optimal temperature: 65°C or higher (c) Thermal stability: 65°C or lower (d) Optimal pH: 8.5
(e) pH stability: 5.2-9.4
(f) molecular weight: about 28 kDa (SDS-PAGE)
(g) Km value: Km value for 3-hydroxybutyric acid is 0.6 mM

Examples of 3-hydroxybutyrate dehydrogenase having the above-described physicochemical properties include enzymes produced by 3-hydroxybutyrate dehydrogenase-producing microorganisms, such as microorganisms belonging to the genus Geobacillus. Such microorganisms are preferably Geobacillus stearothermophilus, Geobacillus anatolicus, Geobacillus bogazici, Geobacillus thermoglycosidaus, Geobacillus gadicilus,ゲノモスプ（Ｇｅｏｂａｃｉｌｌｕｓ ｇｅｎｏｍｏｓｐ．）、ゲオバチルス・ジュラシクス（Ｇｅｏｂａｃｉｌｌｕｓ ｊｕｒａｓｓｉｃｕｓ）、ゲオバチルス・カウエ（Ｇｅｏｂａｃｉｌｌｕｓ ｋａｕｅ）、ゲオバチルス・マハディア（Ｇｅｏｂａｃｉｌｌｕｓ ｍａｈａｄｉａ）、ゲオバチルス・プロテイニフィラス（Ｇｅｏｂａｃｉｌｌｕｓ ｐｒｏｔｅｉｎｉｐｈｉｌｕｓ）、ゲオバチルス・サブテラネウス（Ｇｅｏｂａｃｉｌｌｕｓ ｓｕｂｔｅｒｒａｎｅｕｓ）、ゲオバチルス・（Ｇｅｏｂａｃｉｌｌｕｓ ｔｈｅｒｍｏｄｅｎｉｔｒｉｆｉｃａｎｓ）、ゲオバチルス・コーストフィラス（Ｇｅｏｂａｃｉｌｌｕｓ ｋａｕｓｔｏｐｈｉｌｕｓ）、ゲオバチルス・リトアニカス（Ｇｅｏｂａｃｉｌｌｕｓ ｌｉｔｕａｎｉｃｕｓ）、ゲオバチルス・サーモカテニュロータス（Ｇｅｏｂａｃｉｌｌｕｓ ｔｈｅｒｍｏｃａｔｅｎｕｌａｔｕｓ）、ゲオバチルス・サーモレオボランス（Ｇｅｏｂａｃｉｌｌｕｓ ｔｈｅｒｍｏｌｅｏｖｏｒａｎｓ）、ゲオバチルス・ブルカニ（ Ｇｅｏｂａｃｉｌｌｕｓ ｖｕｌｃａｎｉ）、ゲオバチルス・サーモパラフィニボランス（Ｇｅｏｂａｃｉｌｌｕｓ ｔｈｅｒｍｏｐａｒａｆｆｉｎｉｖｏｒａｎｓ）、ゲオバチルス・トロピカリス（Ｇｅｏｂａｃｉｌｌｕｓ ｔｒｏｐｉｃａｌｉｓ）、ゲオバチルス・ウラリカス（Ｇｅｏｂａｃｉｌｌｕｓ ｕｒａｌｉｃｕｓ）、ゲオバチルス・ウゼネンシス（Ｇｅｏｂａｃｉｌｌｕｓ ｕｚｅｎｅｎｓｉｓ）、ゲオバチルス・ユムタンジェネシス（Ｇｅｏｂａｃｉｌｌｕｓ ｙｕｍｔｈａｎｇｅｎｓｉｓ） etc. The 3-hydroxybutyrate dehydrogenase used in the present invention can be isolated by extracting and purifying from the culture solution obtained by culturing these Geobacillus microorganisms. Recombinant enzymes using recombinant technology are preferred.

The 3-hydroxybutyrate dehydrogenase used in the present invention is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence in which one or more amino acids are deleted, substituted or added in SEQ ID NO: 1. Also included are proteins consisting of and having 3-hydroxybutyrate dehydrogenase activity. In addition, the identity with the amino acid sequence set forth in SEQ ID NO: 1 is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90%. % or more, more preferably 95% or more, more preferably 98% or more. The gene encoding the 3-hydroxybutyrate dehydrogenase of the present invention can be obtained directly from the genome of a Geobacillus microorganism, or can be artificially synthesized.

The above gene is, for example, a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence in which one or more amino acids are deleted, substituted or added in SEQ ID NO: 1, and There is a DNA encoding a protein having 3-hydroxybutyrate dehydrogenase activity. Specifically, a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2 can be mentioned. Deletions, substitutions, and additions of DNA include those that do not change the basic properties or that improve the properties. Further, the identity with the nucleotide sequence set forth in SEQ ID NO: 2 is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90%. % or more, more preferably 95% or more, more preferably 98% or more.

The 3-hydroxybutyrate dehydrogenase used in the present invention is preferably isolated or purified 3-hydroxybutyrate dehydrogenase. In addition, 3-hydroxybutyrate dehydrogenase may be used in a state dissolved in the solution suitable for storage or in a freeze-dried state (for example, powder). Alternatively, it may be used in a form that exists in a solution (eg, buffer) suitable for storage or measurement of enzymatic activity.

Biosensor for measuring ketones As the electrode of the biosensor for measuring ketones of the present invention, for example, a carbon electrode, a gold electrode, a platinum electrode, or the like can be used. More specifically, the working electrode is not particularly limited, but any conductive material can be used without particular limitation as long as it is not oxidized when the electron carrier is oxidized. . As the counter electrode, commonly used conductive materials such as palladium, gold, platinum, and carbon can be used. 3-Hydroxybutyrate dehydrogenase is immobilized on this electrode. Examples of immobilization methods include a method using a cross-linking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photo-crosslinkable polymer, a conductive polymer, a redox polymer, etc. Alternatively, hexaamine ruthenium or its Together with an electron acceptor represented by a derivative, it may be immobilized in a polymer or adsorbed onto an electrode, or these may be used in combination. In addition, the measurement of the ketone body concentration may be carried out under the condition that a constant temperature is maintained by putting a buffer solution in a constant temperature cell.

Examples of electron acceptors that can be used include hexaamine ruthenium, potassium ferricyanide, phenazine methosulfate, p-benzoquinone, methylene blue, and ferrocene derivatives. When potassium ferricyanide (potassium hexacyanoferrate(III)) is used as an electron acceptor, the final concentration is preferably 0.5 to 200 mM, more preferably 1 to 100 mM, still more preferably 2 to 50 mM.

Alternatively, as an electron acceptor, hexaamineruthenium chloride (III), (bipyridine)ruthenium (II), (4,4'-dimethyl-2,2'-bipyridine)ruthenium (II), (4,4'-diphenyl -2,2′-bipyridine)ruthenium (II), (4,4′-diamino-2,2′-bipyridine)ruthenium (II) ruthenium (II), (4,4′-dihydroxy-2,2′- bipyridine) ruthenium (II) ruthenium (II), (4,4'-dicarboxy-2,2'-bipyridine) ruthenium (II) ruthenium (II), (4,4'-dibromo-2,2'-bipyridine ) ruthenium (II) ruthenium (II), (5,5'-dimethyl-2,2'-bipyridine) ruthenium (II) ruthenium (II), (5,5'-diphenyl-2,2'-bipyridine) ruthenium (II) Ruthenium (II), (5,5′-diamino-2,2′-bipyridine)ruthenium (II) Ruthenium (II), (5,5′-dihydroxy-2,2′-bipyridine)ruthenium (II) ) Ruthenium (II), (5,5′-dicarboxy-2,2′-bipyridine)ruthenium (II) Ruthenium (II), (5,5′-dibromo-2,2′-bipyridine)ruthenium (II) Ruthenium compounds such as can be suitably used. One of these may be selected and used, or two or more may be used in combination.

As a specific embodiment of the biosensor for measuring ketone bodies of the present invention, for example, an electrode on which 3-hydroxybutyrate dehydrogenase as described above is immobilized is used as a working electrode, a counter electrode (for example, a platinum electrode) and a reference electrode (for example, A suitable example is one using Ag/AgCl electrodes. After a constant voltage is applied and the current becomes steady, a sample containing ketone bodies can be added and the increase in current can be measured. The ketone body concentration in the sample can be calculated according to the calibration curve prepared with the ketone body standard solution of the standard concentration.

The biosensor for measuring ketone bodies of the present invention preferably has a reaction layer containing 3-hydroxybutyrate dehydrogenase as an enzyme in the electrode system. For example, an electrode system consisting of a working electrode, its counter electrode and a reference electrode is formed on an insulating substrate by a method such as screen printing, and a hydrophilic polymer and 3-hydroxybutyrate dehydrogenation are placed on this electrode system. It is made by forming a reaction layer containing an enzyme and an electron acceptor. When a sample solution containing ketone bodies is dropped onto the reaction layer of this biosensor for measuring ketone bodies, the reaction layer dissolves and the 3-hydroxybutyrate dehydrogenase reacts with the ketone bodies, resulting in reduction of the electron acceptor. be done. After completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the sample solution can be measured from the resulting oxidation current value.

A biosensor for measuring ketone bodies of the present invention comprises a first insulating substrate having a working electrode, a second insulating substrate having a counter electrode facing the working electrode, and a reagent containing at least 3-hydroxybutyrate dehydrogenase. An embodiment is illustrated comprising a layer and a sample supply channel formed between first and second insulating substrates. The working electrode, the counter electrode and the reagent layer are exposed in the sample supply path, and the distance between the working electrode and the counter electrode is about 50 μm to 200 μm, preferably about 40 μm to 150 μm, more preferably about 30 μm to 100 μm. preferable. Here, the area of the portion of the counter electrode exposed to the sample supply channel is equal to or less than the area of the portion of the working electrode exposed to the sample supply channel, and the counter electrode is directly above the working electrode. preferably located.

The working electrode has an area S1 exposed to the sample supply path of 0.01 to 20 mm ² , more preferably 0.1 to 2.0 mm ² , and an area S2 of the counter electrode exposed to the sample supply path. is 0.005 to 20 mm ² , more preferably 0.05 to 2.0 mm ² , and preferably S2≦S1. Here, it is more preferable to employ a configuration in which a spacer member is interposed between the first and second substrates.

In the biosensor for measuring ketone bodies of the present invention, the amount of sample liquid contained in the sample supply path by capillary action is preferably 10 nl to 500 nl, more preferably 5 to 100 nl, and particularly preferably 1 nl to 20 μl. be.

Preferably, the working electrode is formed on the first insulating substrate and the counter electrode is formed on the second insulating substrate. Furthermore, it is preferable that the first substrate and the second substrate have a structure in which the spacer member is sandwiched. As the first and second substrates, any material can be used as long as it is electrically insulating and has sufficient rigidity during storage and measurement. Examples thereof include thermoplastic resins such as polyethylene, polystyrene, polyvinyl chloride, polyamide and saturated polyester resins, and thermosetting resins such as urea resins, melamine resins, phenol resins, epoxy resins and unsaturated polyester resins. Polyethylene terephthalate is particularly preferred from the viewpoint of adhesion to the electrode.

As the spacer member, any material can be used as long as it is electrically insulating and has sufficient rigidity during storage and measurement. Examples thereof include thermoplastic resins such as polyethylene, polystyrene, polyvinyl chloride, polyamide and saturated polyester resins, and thermosetting resins such as urea resins, melamine resins, phenol resins, epoxy resins and unsaturated polyester resins.

In the biosensor for measuring ketone bodies of the present invention, the reagent layer preferably contains a hydrophilic polymer. Examples of hydrophilic polymers include, but are not limited to, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids such as polylysine, and polystyrene sulfone. Polymers of acid, gelatin and its derivatives, polyacrylic acid and its salts, polymethacrylic acid and its salts, starch and its derivatives, maleic anhydride or its salts. Carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose are preferred, and carboxymethylcellulose is particularly preferred.

The production of the biosensor for measuring ketone bodies of the present invention preferably includes a step of drying the electrode system holding the 3-hydroxybutyrate dehydrogenase solution at a temperature of 30° C. or higher. It is more preferable to include a step of drying at a temperature of 40° C. or higher, and it is even more preferable to have a step of drying at a temperature of 50° C. or higher.

Moreover, it is preferable to include a step of adjusting the pH of the composition containing the 3-hydroxybutyrate dehydrogenase solution to 6.0 to 9.0. More preferably, it includes a step of pH 7.0 to 9.0, and even more preferably a step of pH 7.5 to 8.5.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not particularly limited by the examples.

Example 1 Cloning of 3-hydroxybutyrate dehydrogenase SEQ ID NO: 1 shows the amino acid sequence of 3-hydroxybutyrate dehydrogenase derived from Geobacillus stearothermophilus (hereinafter abbreviated as "HBDH-GS"). Based on SEQ ID NO: 1, an artificial synthetic gene shown in SEQ ID NO: 2, optimized for codon usage of E. coli K-12 strain, was produced. Similarly, the amino acid sequence of 3-hydroxybutyrate dehydrogenase derived from Pseudomonas aeruginosa is shown in SEQ ID NO: 3 (hereinafter abbreviated as "HBDH-PA"). Based on SEQ ID NO:3, the artificial synthetic gene shown in SEQ ID NO:4 was produced.

Using these artificially synthesized genes as templates, various 3-hydroxybutyrate dehydrogenase genes were amplified using the primers shown in Table 1 (in Table 1, Fw represents forward primer and Rv represents reverse primer). Specifically, forward primers were designed to add an initiation codon to the 5' upstream side of genes encoding various 3-hydroxybutyrate dehydrogenases, and to connect a histidine tag after the initiation codon. The reverse primer was designed to amplify in a form containing the termination codon at the 3' end of various 3-hydroxybutyrate dehydrogenases. Primers of SEQ ID NO: 5 and SEQ ID NO: 6 were used for amplification of the gene encoding His-HBDH-GS. Similarly, the primer pair of SEQ ID NO:7 and SEQ ID NO:8 was used for amplification of the gene encoding His-HBDH-PA.

A restriction enzyme site NdeI is added to the N-terminal side of this primer, and a restriction enzyme site BamHI is added to the C-terminal side thereof. This DNA fragment was cleaved with restriction enzymes NdeI and BamHI, mixed with the vector plasmid pBluescriptII KSN+ cleaved with the same enzymes, added with a ligation reagent (Ligation High manufactured by Toyobo) in an amount equal to the volume of the mixture, and incubated to effect ligation. Carried out. In this way, recombinant plasmids pBSK-His-HBDH-GS and pBSK-His-HBDH-PA designed to express the 3-hydroxybutyrate dehydrogenase gene in large amounts were obtained.

Example 2 E. coli of the 3-hydroxybutyrate dehydrogenase gene. Expression and Purification in E. coli Using various plasmids constructed in Example 1, E. E. coli JM109 strain competent cells (Toyobo Competent High JM109) were transformed according to the protocol attached to this product to obtain transformants. A colony of each transformant obtained was inoculated into 5 mL of sterilized LB liquid medium (containing 50 μg/mL ampicillin) in a test tube, and then aerobically cultured by shaking at 30° C. for 16 hours. .

The resulting culture solution was used as a seed culture solution, inoculated into 100 mL of TB medium (containing 1 mM IPTG and 50 μg/mL ampicillin) in a 500 mL Sakaguchi flask, and cultured at 37° C. with a shaking speed of 180 rpm for 22 hours. . The cells thus obtained are collected by centrifugation, suspended in a 25 mM phosphate buffer solution (pH 7.5), disrupted with glass beads, and dehydrated with 3-hydroxybutyric acid from the resulting crude enzyme. High expression of His-HBDH-GS and HBDH-PA was observed when the enzyme activity was measured. The two types of lysates were applied to 1 mL of HisTrap HP (manufactured by Cytiva) equilibrated with a 25 mM phosphate buffer solution (pH 7.5) containing 20 mM imidazole, and eluted with a 25 mM phosphate buffer solution (pH 7.5) containing 500 mM imidazole. Thus, a highly pure 3-hydroxybutyrate dehydrogenase solution was obtained.

Example 3 Thermal stability evaluation of various 3-hydroxybutyrate dehydrogenase 5), heat-treated at 50° C. for 15 minutes, 3-hydroxybutyrate dehydrogenase activity was measured, and thermal stability was evaluated by residual activity. His-HBDH-GS had a residual rate of 99.8%, and His-HBDH-PA had a residual rate of 38.0%.

Example 4 Electrode Evaluation of 3-Hydroxybutyrate Dehydrogenase A predetermined amount of compound was dropped on an electrode strip in the following order and dried to prepare a sensor for measuring ketone bodies. Carboxymethyl cellulose (hereinafter referred to as CMC) 0.1 mg, NAD 0.0015 mg, 3-hydroxybutyrate dehydrogenase 0.5 U, hexaamine ruthenium 0.00175 mg. His-HBDH-GS and His-HBDH-PA obtained in Example 2 were used as 3-hydroxybutyrate dehydrogenase, and strips were prepared by setting the drying temperature to 30° C. or 50° C., respectively.

0, 0.2, 1.0, 5.0, 10.0 mM 3-HB dissolved in 0.9% NaCl was added dropwise to the strips obtained as described above, and from an applied voltage of 600 mV, The current value after 2 seconds was measured. This current value is proportional to the concentration of the generated electron acceptor reductant, that is, the concentration of the substrate in the sample solution. Therefore, by measuring this current value, the 3-HB concentration in the sample solution can be determined. The results are shown in Tables 2 and 3. A certain correlation was observed between the obtained response current value and the 3-HB concentration. % was obtained (Fig. 2). In contrast, the His-HBDH-GS of the present invention showed a response value when dried at 50° C., which was 116% equivalent to that when dried at 30° C. (FIG. 1).

As described above, the biosensor for measuring ketone bodies of the present invention is excellent in thermal stability, and thus enables efficient production of sensor strips involving heat treatment or the like. As a result, it is expected to be widely used not only in the fields of medicine and diagnosis, but also in measuring ketone bodies as an index in ketogenic diets (ketone diets).

Claims (20)

It has an electrode system having a working electrode and a counter electrode formed on an electrically insulating substrate, and the electrode system contains 3-hydroxybutyrate dehydrogenase and an electron acceptor derived from microorganisms belonging to the genus Geobacillus. A biosensor for measuring ketone bodies, having a configuration comprising a reaction layer containing 2. The biosensor for measuring ketone bodies according to claim 1, wherein the 3-hydroxybutyrate dehydrogenase has the following physicochemical properties (a) to (g).
(a) Action: catalyze the following reactions.

(b) Optimal temperature: 55° C. or higher (c) Thermal stability: The range in which the residual activity rate after 30 minutes of treatment is 90% or higher is 65° C. or lower (d) Optimal pH: 6.0 to 9.0
(e) pH stability: range of 5.0 to 10.0 showing a residual activity rate of 80% or more after treatment at 25°C for 20 hours
(f) molecular weight: about 28 kDa (SDS-PAGE)
(g) Km value: Km value for 3-hydroxybutyric acid is 0.6 mM or less (b) The biosensor for measuring ketone bodies according to claim 2, wherein the optimum temperature is 65°C or higher. (c) Thermal stability: The biosensor for measuring ketone bodies according to claim 2, wherein the range in which the rate of residual activity after treatment for 16 hours is 90% or more is 55°C or less. (c) Thermal stability: The range in which the residual activity rate after 30-minute treatment is 90% or higher is 65°C or lower, and the range in which the residual activity rate is 90% or higher after 16-hour treatment is 55°C or lower. The biosensor for measuring ketone bodies according to claim 2, wherein (d) The biosensor for measuring ketone bodies according to claim 2, wherein the optimum pH is 7.5 to 8.5. (e) pH stability: The biosensor for measuring ketone bodies according to claim 2, wherein the range showing a residual activity rate of 80% or more after treatment at 25°C for 20 hours is 5.2 to 9.4. The biosensor for measuring ketone bodies according to any one of claims 1 to 7, wherein the 3-hydroxybutyrate dehydrogenase is a recombinant enzyme. The biosensor for measuring ketone bodies according to any one of claims 1 to 8, wherein the microorganism belonging to the genus Geobacillus is Gepbacillus stearothermophilus. 10. The biosensor for measuring ketone bodies according to any one of claims 1 to 9, wherein the 3-hydroxybutyrate dehydrogenase comprises an amino acid sequence having 90% or more identity with SEQ ID NO:1. 11. The biosensor for measuring ketone bodies according to claim 10, wherein the 3-hydroxybutyrate dehydrogenase comprises an amino acid sequence having 95% or more identity with SEQ ID NO:1. 11. The biosensor for measuring ketone bodies according to claim 10, wherein the 3-hydroxybutyrate dehydrogenase has an amino acid sequence in which one to several amino acids are deleted, substituted or added in SEQ ID NO:1. 11. The biosensor for measuring ketone bodies according to claim 10, wherein the 3-hydroxybutyrate dehydrogenase consists of the amino acid sequence set forth in SEQ ID NO:1. The biosensor for measuring ketone bodies according to any one of claims 1 to 13, wherein the electron acceptor is a ruthenium compound. Ruthenium compounds include hexaamineruthenium chloride (III), (bipyridine)ruthenium (II), (4,4'-dimethyl-2,2'-bipyridine)ruthenium (II), (4,4'-diphenyl-2, 2'-bipyridine)ruthenium (II), (4,4'-diamino-2,2'-bipyridine)ruthenium (II) ruthenium (II), (4,4'-dihydroxy-2,2'-bipyridine)ruthenium (II) ruthenium (II), (4,4'-dicarboxy-2,2'-bipyridine) ruthenium (II) ruthenium (II), (4,4'-dibromo-2,2'-bipyridine) ruthenium ( II) Ruthenium (II), (5,5'-dimethyl-2,2'-bipyridine)ruthenium (II) Ruthenium (II), (5,5'-diphenyl-2,2'-bipyridine)ruthenium (II) Ruthenium (II), (5,5'-diamino-2,2'-bipyridine) ruthenium (II) ruthenium (II), (5,5'-dihydroxy-2,2'-bipyridine) ruthenium (II) ruthenium ( II), from (5,5′-dicarboxy-2,2′-bipyridine)ruthenium (II) ruthenium (II) and (5,5′-dibromo-2,2′-bipyridine)ruthenium (II) The biosensor for measuring ketone bodies according to claim 14, which is at least one selected from the group consisting of: The biosensor for measuring ketone bodies according to any one of claims 1 to 13, wherein the electron acceptor is a ferricyanide compound. A method for producing a biosensor for measuring ketone bodies, comprising the step of drying an electrode system holding a 3-hydroxybutyrate dehydrogenase solution at a temperature of 30° C. or higher. 19. The method for producing a biosensor for measuring ketone bodies according to claim 18, comprising a step of drying at a temperature of 40[deg.] C. or higher. 19. The method for producing a biosensor for measuring ketone bodies according to claim 18, comprising a step of drying at a temperature of 50[deg.] C. or higher. A method for producing a biosensor for measuring ketone bodies, comprising the step of adjusting the pH of a composition containing a 3-hydroxybutyrate dehydrogenase solution to pH 6.0 to 9.0.

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