WO2021167011A1 - 検体の状態を評価するためのデバイス、それを含むシステム、検体の状態を評価する方法及びそれに用いる乳酸デヒドロゲナーゼ - Google Patents

検体の状態を評価するためのデバイス、それを含むシステム、検体の状態を評価する方法及びそれに用いる乳酸デヒドロゲナーゼ Download PDF

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WO2021167011A1
WO2021167011A1 PCT/JP2021/006153 JP2021006153W WO2021167011A1 WO 2021167011 A1 WO2021167011 A1 WO 2021167011A1 JP 2021006153 W JP2021006153 W JP 2021006153W WO 2021167011 A1 WO2021167011 A1 WO 2021167011A1
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amino acid
sample
acid sequence
seq
lactate dehydrogenase
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English (en)
French (fr)
Japanese (ja)
Inventor
陽介 鉞
千晶 稲本
美月 中村
英一郎 菅
敦 一柳
遥香 平口
遼太 鈴木
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Kikkoman Corp
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Kikkoman Corp
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Priority to US17/801,033 priority Critical patent/US20230213499A1/en
Priority to EP21756429.3A priority patent/EP4108678A4/en
Priority to JP2022501977A priority patent/JP7714520B2/ja
Priority to KR1020227032145A priority patent/KR20220143091A/ko
Publication of WO2021167011A1 publication Critical patent/WO2021167011A1/ja
Anticipated expiration legal-status Critical
Priority to JP2025119817A priority patent/JP2025160264A/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q3/00—Condition responsive control processes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/50—Other enzymatic activities
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/05—Oxidoreductases acting on the CH-OH group of donors (1.1) with a quinone or similar compound as acceptor (1.1.5)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)

Definitions

  • the present invention uses a device for evaluating the condition of a sample, including a sensor using a flavin-dependent lactate dehydrogenase using a flavin compound as a coenzyme, a system in which the device further includes an output unit, the device or the system.
  • the present invention relates to a method for evaluating the condition of a sample.
  • the present invention also relates to flavin-dependent lactate dehydrogenase which can be suitably used for the devices, systems and methods for evaluating the state of a sample using them.
  • Blood lactate concentration and sweat lactate concentration are known as markers that reflect fatigue and physical condition. Lactic acid is a major metabolite and is recognized to be important not only for health care indicators but also for health assessments involving serious illness and / or surgical patients, and lactic acid levels in body fluids are circulatory insufficiency, liver. It can be an index for various pathological conditions such as disorders. Lactate monitoring can be used to detect sepsis, hypoxia, and the presence of cancerous tissue (Non-Patent Document 1).
  • Lactate monitoring is also used as an index for monitoring the appropriateness of training conducted by athletes and those who are highly interested in exercising on a daily basis, not only for the purpose of physical condition management for sick people. (Non-Patent Document 2).
  • Patent Documents 1 to 3 propose predetermined information processing devices as technical proposals for continuously monitoring a human condition, posture, etc. for a certain period of time (Patent Documents 1 to 3).
  • Lactic acid oxidase (hereinafter, LOD) is known as an enzyme using lactic acid as a substrate. LOD has a problem that its activity is lost during storage after drying. Therefore, Patent Document 4 proposes a method of drying in combination with a specific stabilizer in the drying step.
  • Patent Document 5 also proposes a lactic acid sensor in which catalase coexists for the purpose of eliminating hydrogen peroxide as a means for avoiding a decrease in LOD activity due to hydrogen peroxide.
  • FMN-LDH FMN-dependent lactate dehydrogenase
  • JP-A-2019-150649 Japanese Unexamined Patent Publication No. 2017-100039 International Publication No. 17/163521 Pamphlet Patent No. 5593689 Special Table 2018-519507
  • the lactic acid level in food can be an index for quality control, etc.
  • Good quality at the production site of various fermented foods and drinks produced through the lactic acid fermentation process such as sake, wine, whiskey, cheese, yogurt, sauerkraut, waste rice cake, pickles, miso, soy sauce, and lactic acid fermented yeast extract.
  • appropriate lactic acid fermentation process control is performed, and in the process control, the amount of lactic acid can be an index of production process control.
  • lactic acid is not limited to foods and drinks fermented with lactic acid, but also in, for example, a lactic acid fermentation composition obtained by fermenting a plant material which is a raw material for cosmetics with lactic acid bacteria, and a lactic acid composition produced as a chemical product. It is progressing, and the amount of lactic acid can be an index in the manufacturing process control and quality control of such products.
  • the amount of lactic acid in the monomer can be used as an index when calculating the decomposition rate.
  • the measurement of the amount of lactic acid performed for the above purpose can be carried out frequently and easily in daily life or in the manufacturing process of foods and drinks every time, and more preferably it can be repeated in the simplest possible process. It is even more preferable to be able to monitor automatically and continuously.
  • the infiltration sampling method such as puncture in the current measurement method causes pain and stress for the biological sample, and hinders frequent sampling. Also, in the manufacturing process control of foods and drinks that do not have the problem of pain and stress, lactic acid is manually sampled from the lactic acid-containing composition being manufactured each time, and then the lactic acid is measured and the measurement data is manually recorded each time. Such frequent sampling operations in current lactate measurement techniques are cumbersome because they must be.
  • a technical proposal for continuously monitoring the human condition and posture for a certain period of time for example, a plurality of sensors are attached to the human body, clothing, training machine, etc., and some biological origin obtained by these sensors is obtained.
  • a system for proposing training programs based on data and input information such as the age and gender of the person, estimating the physical condition of the person, an information processing device for activating an application program based on various data, etc. It has been proposed (Patent Documents 1 to 3). And, in a small number of such proposals, some include the word lactate measurement. However, in those proposals, no method is disclosed that can specifically measure the amount of lactic acid as an actual index and thereby monitor the state of the sample.
  • Patent Document 1 it can be attached to a training device for riding a bicycle, and heart rate information acquired from a human (driver) during training time by a heart rate monitor can be obtained as a resting heart rate of the driver or a heart rate during training.
  • a system has been proposed that can analyze based on information such as age and gender and bicycle running data, and output a message for bringing the current exercise state of the driver closer to the ideal state.
  • the blood flow in each of the first part where the blood flow does not fluctuate according to the physical condition of a human and the second part where the blood flow fluctuates according to the physical condition is measured by a laser speckle camera, a laser Doppler blood flow system, etc.
  • a system for estimating such factors has been proposed.
  • human body temperature, acceleration, heart rate data, GPS data, altitude data, etc. are used by using one or more wearable sensors attached to human underwear or jacket, hat or glasses, earplugs, headphones, or the like.
  • An information processing device that can monitor the posture of the person wearing these, estimate the movements such as sleeping, waking up, walking, etc., and if there is a sign of altitude sickness during mountain climbing, etc. has been proposed.
  • Examples of enzymes using lactic acid as a substrate include lactate oxidase (hereinafter, LOD), NAD-dependent lactate dehydrogenase (hereinafter, NAD-LDH) using nicotinamide dinucleotide (NAD) as a coenzyme, and flavin mononucleotide (FMN). ) Is a coenzyme, and FMN-dependent lactate dehydrogenase (hereinafter, FMN-LDH) is known.
  • LOD lactate oxidase
  • NAD-LDH NAD-dependent lactate dehydrogenase
  • NAD-LDH nicotinamide dinucleotide
  • FMN flavin mononucleotide
  • FMN-LDH FMN-dependent lactate dehydrogenase
  • Blood lactic acid measuring devices using LOD are commercially available, and each time a measurement is performed, the stored sensor (the electrode part for measuring blood lactic acid including LOD) is taken out
  • the blood squeezed from the sample is sucked into the sensor, and the measured value displayed on the measuring device is read.
  • the amount of lactic acid in the sample can be known in a single-shot manner, and by repeating this, the amount of lactic acid in the sample can be measured each time.
  • lactic acid in the lactic acid-containing composition containing biological interstitial fluid, blood and sweat, food and drink and chemical products is measured stably, accurately and preferably continuously and easily over a long period of time. If you want to do this, known measurement enzymes are not sufficient.
  • LOD has a problem of insufficient thermal stability, and even when manufacturing a LOD product, the degree of activity being lost during the drying process or storage after drying is large.
  • Patent Document 4 a method of drying in combination with a specific stabilizer in the drying step has also been proposed (Patent Document 4), but the effect of improving the stability is still insufficient. It should also be considered that the stability of LOD in the liquid state during the actual enzymatic reaction is even lower than the stability in the powder state.
  • lactic acid is monitored for a long period of time in a scene where a sensor using LOD is used in close contact with the human body for a long period of time, or in a manufacturing process of foods and drinks that can be placed at a fermentation temperature in a lactic acid bacteria fermentation process.
  • a sensor using LOD is used in close contact with the human body for a long period of time, or in a manufacturing process of foods and drinks that can be placed at a fermentation temperature in a lactic acid bacteria fermentation process.
  • the existing LOD cannot exhibit sufficient practicality from the viewpoint of enzyme stability from around room temperature to around 37 ° C., which is near human body temperature.
  • LOD also has a problem of producing hydrogen peroxide as a reaction product.
  • hydrogen peroxide which is one of the active oxygen species and the causative agent of oxidative stress.
  • hydrogen peroxide generated by the action of LOD also has an adverse effect on the stabilization of LOD itself, and as a means for avoiding a decrease in LOD activity due to this, a lactic acid sensor in which catalase coexists for the purpose of eliminating hydrogen peroxide has also been proposed. (Patent Document 5).
  • NAD-LDH catalyzes an enzymatic reaction that does not generate hydrogen peroxide
  • the problem of hydrogen peroxide production does not occur.
  • lactic acid and pyruvic acid react reversibly, there is a problem that an accurate measured value cannot be obtained for the use of quantification, and the sensor. It is difficult to adopt for.
  • FMN-LDH also catalyzes an enzymatic reaction that does not produce hydrogen peroxide, so the problem of hydrogen peroxide production does not arise.
  • FMN-LDH derived from Saccharomyces cerevisiae known so far. Then, there is still a problem in stability (Non-Patent Document 3).
  • An object of the present invention is to provide a device for monitoring or evaluating the state of a sample, a method for monitoring or evaluating the state of a sample, and an FMN-LDH used therefor, which can solve at least a part of the above problems. And.
  • the present inventor has conceived a device for evaluating the state of a sample having a configuration capable of easily measuring lactic acid in a sample non-invasively when measuring the amount of lactic acid in the sample. Then, a method for evaluating the state of a sample using such a device, a system used for the method, and an FMN-LDH suitable for measuring lactate were found, and the present invention was completed. Twice
  • the present invention includes the following aspects.
  • a device for evaluating the condition of a sample The action part for allowing lactate dehydrogenase to act on the sample, A device including a sensor for detecting the state of a sample on which lactate dehydrogenase has been acted, and a sensor arranged so as to be able to detect the state of the sample at the action site.
  • the device according to (1) further including an output unit for outputting a signal from a sensor.
  • the device described and A system that is connected to the output of a device and includes a data processing unit for processing signals from sensors.
  • the device is The action part for allowing lactate dehydrogenase to act on the sample, A sensor for detecting the state of a sample on which lactate dehydrogenase has acted, and a sensor arranged so as to be able to detect the state of the sample at the action site. Includes an output section for outputting signals from the sensor A data processing unit is connected to the output of the device and is configured to process the signal from the sensor.
  • the program tells the device A measurement process for detecting and measuring the state of a sample on which lactate dehydrogenase is applied at the action site and converting it into a signal, A transfer process for transferring the signal obtained in the measurement process from the output unit to the data processing unit is executed.
  • (5) A method for evaluating the condition of a sample using the device according to (1) or (2) or the system according to (3).
  • Lactic acid containing human and non-human organisms such as body fluids, interstitial fluid, blood, urine, tears, sweat, saliva, skin, flesh, eyeballs, cornea, gastric fluid, food and drink, brews, chemicals, water, and soil.
  • the method according to (5) which is a containing composition.
  • the condition of the sample is physical condition with respect to exercise load, disease condition, brewing condition of brewed product with change of lactic acid amount, aging / ripening degree of food and drink with change of lactic acid amount, chemical product manufacturing with change of lactic acid amount.
  • the method according to (5) or (6) which is the lactic acid content ratio of lactic acid, the amount of lactic acid in water or soil accompanied by a change in the amount of lactic acid.
  • a device for monitoring the condition of a sample which is a device for the sample.
  • Flavin-dependent lactate dehydrogenase which maintains about 20% or more of its initial activity when allowed to pass at 37 ° C. for 10 days in solution.
  • Flavin-dependent lactate dehydrogenase that maintains about 20% or more of the initial activity when allowed to pass at 37 ° C. for 3 days or more in the solution (B), or 15 at 37 ° C. in the solution (C).
  • a device comprising an action part for acting flavin-dependent lactate dehydrogenase, which maintains about 20% or more of the initial activity when the time has passed.
  • amino acid sequence at positions 110 to 502 in the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 70% or more identity with it the amino acid sequence at positions 113 to 505 in the amino acid sequence shown in SEQ ID NO: 7 or 70% or more thereof.
  • a lactate dehydrogenase comprising the amino acid sequence at positions 102 to 499 in the sequence or the amino acid sequence represented by SEQ ID NO: 12 or an amino acid sequence having 70% or more identity with the amino acid sequence.
  • (14) (13) A host cell having the nucleic acid described.
  • (15) (14) A method for producing lactate dehydrogenase, which comprises culturing the host cell according to the above.
  • (16) A method for evaluating the condition of a sample A method comprising i) contacting a sample with lactate dehydrogenase according to (11) or (12), and ii) measuring lactate.
  • FIG. 1A is a schematic view of a sensor chip 10 according to an embodiment of the present invention
  • FIGS. 1B to 1D are schematic views showing members constituting the sensor chip 10.
  • FIG. 2 is a diagram showing the residual activity rates of PkLDH and ScLDH after heat treatment at each temperature.
  • 3A and 3B are diagrams showing the residual activity rates of PkLDH, ScLDH, CalDH and OgLDH after storage at 37 ° C. for each time.
  • FIG. 4 is a schematic diagram showing an example of a device and a system according to an embodiment of the present invention.
  • FIG. 5 is a flow chart showing an example of the program according to the embodiment of the present invention.
  • FIG. 6 is a schematic view showing an example of a device and a system using the transmitter according to the embodiment of the present invention.
  • FIG. 7 is a diagram showing the stable pH of PkLDH evaluated in Example 4.
  • FIG. 8 is a diagram showing the optimum pH of PkLDH evaluated in Example 4.
  • FIG. 9 is a diagram showing the relationship between the lactic acid concentration evaluated in Example 4 and the activity of PkLDH.
  • FIG. 10 is a diagram showing the relationship between the lactic acid concentration evaluated in Example 8 and the response current value.
  • FIG. 11-1 shows the alignment of the amino acid sequences of the lactate dehydrogenase of the present invention.
  • FIG. 11-2 is a continuation of FIG. 11-1.
  • FIG. 11-1 shows the alignment of the amino acid sequences of the lactate dehydrogenase of the present invention.
  • FIG. 11-2 is a continuation of FIG. 11-1.
  • FIG. 11-1 shows the alignment of the amino acid sequences of the lactate dehydrogenase of the
  • FIG. 12 is a diagram showing the residual activity rates of PkLDH and N-terminal deleted mutants of PkLDH treated at 55 ° C. for 15 minutes evaluated in Example 11.
  • FIG. 13 is a diagram showing the residual activity rates of PkLDH and monosubstituted mutants treated in Example 11 at 55 ° C. for 15 minutes.
  • the sample referred to in the present invention may be a target for measuring whether or not it contains lactic acid, and is a body fluid of human and non-human organisms such as interstitial fluid, blood, urine, tears, sweat, saliva, skin, and meat. , Eyeball, corneal membrane, gastric fluid, or a lactic acid-containing composition containing non-lactic acid-fermented, lactic-fermented or lactic-fermented foods and drinks, brewed products, and chemical products. Furthermore, it may be present in an environment such as water containing lactic acid or soil.
  • the lactate dehydrogenase (LDH) referred to in the present invention may be an enzyme that catalyzes a reversible reaction of reducing pyruvate ions to lactate ions using NADH, which is a reducing agent, and may be NAD-dependent lactate dehydrogenase and FMN-dependent lactate.
  • Dehydrogenase is known, but preferably FMN-dependent lactate dehydrogenase, more preferably the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12 or 70% or more, 75% or more, 80 thereof.
  • Amino acid sequences at positions 1 to 88 in SEQ ID NO: 12, or amino acid sequences having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity with them are linked. You can also do it.
  • Amino acid sequence of, amino acid sequence of positions 1 to 99 in SEQ ID NO: 7, amino acid sequence of positions 1 to 98 in SEQ ID NO: 10, amino acid sequence of positions 1 to 88 in SEQ ID NO: 12 or 70% or more, 75% or more with them, Amino acid sequences having 80% or more, 85% or more, 90% or more, or 95% or more identity can also be linked.
  • the region containing the region has low sequence identity and can be said to be of low importance in the lactate dehydrogenase of the present invention. Therefore, among the full-length amino acid sequences, the amino acid sequence at positions 97 to 502 in SEQ ID NO: 4, the amino acid sequence at positions 100 to 505 in SEQ ID NO: 7, the amino acid sequence at positions 99 to 503 in SEQ ID NO: 10, and 89 in SEQ ID NO: 12.
  • lactate dehydrogenase containing an amino acid sequence at position ⁇ 499 or an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity with the amino acid sequence.
  • the amino acid sequence of positions 1 to 96 in No. 4 the amino acid sequence of positions 1 to 99 in SEQ ID NO: 7, the amino acid sequence of positions 1 to 98 in SEQ ID NO: 10, or the amino acid sequence of positions 1 to 88 in SEQ ID NO: 12.
  • the sequence identity may be 45% or more, 50% or more, 60% or 70% or more, respectively, or this region may be deleted.
  • amino acids may be deleted from the N-terminal sequence. ..
  • amino acids at positions 2 to 10 in SEQ ID NO: 4 may be deleted. If deleted, methionine can be added at the beginning of the sequence, as appropriate.
  • Amino acid sequence identities or similarities can be found in GENETYX Ver. It can be calculated by a program such as maximum matching or search homology of 11 (manufactured by Genetics) or a program of maximum matching or multiple alignment of DNASIS Pro (manufactured by Hitachi Solutions).
  • the amino acid sequence identity when two or more LDHs are aligned, the positions of amino acids that are the same in the two or more LDHs can be examined. Based on this information, the same region in the amino acid sequence can be determined. It is also possible to investigate the positions of amino acids that are similar in two or more LDHs. For example, CLUSTALW can be used to align a plurality of amino acid sequences.
  • Blossum62 is used as an algorithm, and amino acids judged to be similar when a plurality of amino acid sequences are aligned may be referred to as similar amino acids.
  • amino acid substitutions can be due to substitutions between such similar amino acids.
  • the amino acid sequence in the homology region of lactate dehydrogenase of the present invention is 75% or more, for example, 80% or more, 81% or more, 82% or more, 83% or more, 84% with the amino acid sequence of the homology region in SEQ ID NO: 4.
  • the device for evaluating the state of the sample in the present invention includes an action part for causing lactate dehydrogenase to act on the sample, and a sensor for detecting the state of the sample on which lactate dehydrogenase is allowed to act.
  • the sensor is arranged so that it can sense the state of the sample at the action site.
  • the working part of the sensor may be included by a gel, preferably a biocompatible gel.
  • the lactic acid in the sample can be detected by bringing the gel into contact with the sample, collecting water containing lactic acid through the gel by the effect of osmotic pressure or the like, and contacting the sensor with lactic acid.
  • the biocompatible component (that is, the material of the gel constituting the gel structure) is particularly limited as long as it has cell adhesion, biocompatibility, high transparency, and hydrophilicity.
  • synthetic molecules and biomolecules can be used.
  • the synthetic molecule described above include hydrophilic acrylic molecules such as polyethylene glycol and acrylamide.
  • polyethylene glycol dimethacrylate is a stretchable polymer, and is preferable because it can improve not only affinity for cells and biological tissues but also stretchability, viscoelasticity, and toughness.
  • biomolecules include polysaccharides such as sodium alginate, protein materials such as gelatin and silk, and extracellular matrix such as collagen. Among these, a material containing a large amount of protein is preferable.
  • the biocompatible gel material of the present embodiment it is preferable to use silk fibroin gel as one of the components having biocompatibility.
  • a silk fibroin gel containing a large amount of protein By using a silk fibroin gel containing a large amount of protein, cytotoxicity can be reduced so that the surface of the gel material is highly biocompatible and the promotion of cell adhesion is observed.
  • the synthetic molecule and biomolecule described above may be a polymer or a small molecule.
  • the molecular weight (Mw) of the polymer is not particularly limited as long as the polymer can form a gel structure, and for example, a polymer having a molecular weight of about 5,000 to 1,000,000 Da is used. Can be done.
  • the action part for acting lactate dehydrogenase is a member that acts on an enzyme made of a material such as metal, plastic, cloth, liquid, paper, or nylon, as long as it is made of a material suitable for the action.
  • the sensor that senses the state of the sample may be integrated with a member on which the enzyme acts, may be separated from the sensor, or may be directly or indirectly connected.
  • a sample and the lactate dehydrogenase of the present invention are placed in the action section.
  • an electron acceptor and / or a reagent for showing a change in the electron acceptor (mediator) can be arranged in the action part.
  • a sensor that includes a working part (sometimes referred to as a working electrode) has a working electrode, a reference electrode, and a counter electrode that include the LDH of the present invention.
  • a working electrode a carbon electrode, a gold electrode, a platinum electrode, or the like is used, and the LDH of the present invention is immobilized on the electrode. Further, or separately, an electron mediator may be immobilized on the working electrode.
  • the counter electrode can be a platinum electrode, a conventional electrode such as Pt / C, or the like.
  • the reference electrode can be a conventional electrode such as an Ag / AgCl electrode.
  • 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 photocrosslinkable polymer, a conductive polymer, an oxidation-reduced polymer, etc., or a ferrocene or a derivative thereof. It may be fixed in a polymer or adsorbed and fixed on an electrode together with a representative electronic mediator, or these may be used in combination.
  • the LDH of the present invention is immobilized on a carbon electrode with glutaraldehyde and then treated with a reagent having an amine group to block glutaraldehyde.
  • Cross-linking reagents such as poly (ethylene glycol) diglycidyl ether can also be used instead of glutaraldehyde.
  • the sensor that can be used in the device of the present invention can be used without limitation as long as it can detect the state of the sample on which lactate dehydrogenase is acted.
  • a sensor chip may be used as the sensor.
  • 1 (a) is a schematic view of a sensor chip 10 according to an embodiment of the present invention
  • FIGS. 1 (b) to 1 (d) are schematic views showing members constituting the sensor chip 10.
  • the sensor chip 10 includes two or more electrodes arranged on the base material 11.
  • the base material 11 is made of an insulating material.
  • the working electrode 1, the counter electrode 3 and the reference electrode 5 are arranged on the base material 11.
  • Each electrode is electrically connected to the wiring portion 7, and the wiring portion 7 is electrically connected to a terminal 9 located on the opposite side of the wiring direction from each electrode.
  • the working pole 1, the counter electrode 3 and the reference pole 5 are arranged apart from each other. Further, it is preferable that the working electrode 1, the counter electrode 3 and the reference electrode 5 are integrally formed with the wiring portion 7 and the terminal 9. Further, the counter electrode 3 and the reference electrode 5 may be integrated.
  • a spacer 13 is arranged at the end of the base material 11 parallel to the wiring portion 7, and the working electrode 1, the counter electrode 3, the reference electrode 5 and the spacer 13 are arranged.
  • a cover 15 for covering the above is arranged.
  • the spacer 13 and the cover 15 are made of an insulating material. It is preferable that the spacer 13 has a thickness substantially equal to that of the working pole 1, the counter electrode 3 and the reference pole 5, and is in close contact with the working pole 1, the counter electrode 3 and the reference pole 5. Further, the spacer 13 and the cover 15 may be integrally formed.
  • the cover 15 is a protective layer that prevents the wiring portion 7 from being deteriorated by being exposed to the outside air and short-circuiting due to the penetration of the measurement sample.
  • the reaction layer 19 is arranged on the working electrode 1, the counter electrode 3 and the reference electrode 5.
  • the reaction layer 19 provides a field for the reaction of lactic acid with lactate dehydrogenase.
  • the lactate dehydrogenase of the present invention may be applied, adsorbed, or immobilized on these electrodes.
  • the lactate dehydrogenase of the present invention is applied, adsorbed or immobilized on the working electrode.
  • the mediator as well as lactate dehydrogenase may be applied, adsorbed, or immobilized on the electrode.
  • a carbon electrode, a metal electrode such as platinum, gold, silver, nickel and palladium can be used.
  • examples of the material include pyrolytic graphite carbon (PG), glassy carbon (GC), carbon paste and plastic foamed carbon (PFC).
  • the measurement system may be a two-electrode system or a three-electrode system, for example, the enzyme can be immobilized on the working electrode.
  • examples of the reference electrode include a standard hydrogen electrode, a reversible hydrogen electrode, a silver-silver chloride electrode (Ag / AgCl), a palladium / hydrogen electrode, a saturated calomel electrode, and the like. From the viewpoint of stability and reproducibility, Ag / AgCl is used. It is preferable to use it.
  • a printed electrode can be used to reduce the amount of solution required for measurement.
  • the electrodes are preferably formed on the base material 11 made of an insulating substrate. Specifically, it is desirable that electrodes are formed on the base material 11 by a photolithography technique or a printing technique such as screen printing, gravure printing, or flexographic printing. Examples of the material of the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene and polyester, but it is more preferable to use a material having strong resistance to various solvents and chemicals.
  • potassium ferricyanide when lactate dehydrogenase is allowed to act on lactic acid contained in a sample in an action part containing potassium ferricyanide as an electron acceptor, potassium ferricyanide changes to potassium ferrocyanide. Since potassium ferricyanide absorbs at a wavelength of 420 nm, the state of lactic acid in the sample can be sensed by measuring the absorbance at a wavelength of 420 nm with a spectrophotometer. It is also possible to electrochemically measure the redox reaction when lactate dehydrogenase is allowed to act on lactic acid and potassium ferricyanide is allowed to act on the lactic acid.
  • a sensor for performing an electrochemical measurement for example, a sensor of Arkray's Lactate Pro 2 model number LT-1730 can be used.
  • Other electron acceptors include quinones, phenazines (eg, phenazinemethsulfate), viologens, cytochromes (eg, cytochrome b, cytochrome c), phenoxazines, phenothiazines, ferricianides, ferredoxins, etc.
  • Ferredoxin, osmium complex, ruthenium complex, phenylene diamines and derivatives thereof and the like can be used.
  • the lactate dehydrogenase of the present invention does not use oxygen as an electron acceptor.
  • the lactic acid concentration can be measured as follows. Put the buffer solution in the constant temperature cell and keep it at a constant temperature.
  • As the working electrode an electrode on which LDH and an electron acceptor (for example, a quinone-added polymer) are immobilized is used, and a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode) are used.
  • a constant voltage is applied to the carbon electrode, and after the current becomes steady, a sample containing L-lactic acid is added and the increase in the current is measured.
  • the lactic acid concentration in the sample can be calculated according to the calibration curve prepared with the standard concentration lactic acid solution.
  • the 4U LDH of the present invention is immobilized on a glassy carbon (GC) electrode, and the response current value with respect to the lactic acid concentration is measured.
  • a glassy carbon (GC) electrode To the electrolytic cell, 1.8 ml of 50 mM potassium phosphate buffer (pH 7.5) and 0.2 ml of a 1 M potassium hexacyanoferrate (III) aqueous solution (potassium ferricyanide) are added.
  • a GC electrode is connected to a potentiostat BAS100B / W (manufactured by BAS), the solution is agitated at 37 ° C., and +500 mV is applied to the silver-silver chloride (saturated KCl) reference electrode.
  • a 1 M L-lactic acid solution is added to these systems so that the final concentration is 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 mM, and the steady-state current value is measured for each addition.
  • This current value is plotted against known lactate concentrations (1, 2, 3, 4, 5, 10, 20, 30, 40, 50 mM) to create a calibration curve. This makes it possible to quantify lactic acid using an enzyme-immobilized electrode using the FMN-LDH of the present invention.
  • the lactic acid sensor chip of the present invention has 0.01U to 1000U, 0.1U to 1000U, more preferably 0.5U to 700U, more preferably 0.5U to 500U, more preferably 1U to the present invention.
  • a 300 U, more preferably 1 U to 100 U FMN-LDH solution is applied or immobilized.
  • a continuous lactate monitoring device having the lactate sensor of the present invention is provided. Also, in certain embodiments, a method of continuously monitoring lactic acid for 14 days using the LDH of the present invention is provided. The measurement period is 5 seconds, 1 minute, 5 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 15 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 14 days, etc. Can be set together.
  • continuous lactate monitoring can be performed with or without recalibration.
  • recalibration can be performed every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.
  • the lactate dehydrogenase of the present invention has sufficient thermal stability. Therefore, simple and preferably continuous lactate monitoring can be realized.
  • the present invention also relates to a method for measuring lactate, which comprises i) contacting a sample with lactate dehydrogenase and ii) measuring lactate.
  • the lactate dehydrogenase in step i) is an FMN-dependent lactate dehydrogenase, more preferably the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12 or 70% or more, 75% thereof.
  • lactate dehydrogenase having an amino acid sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity.
  • the lactate dehydrogenase in step i) is 0.01U to 1000U, 0.1U to 1000U, more preferably 0.5U to 700U, more preferably 0.5U to 500U, and more preferably 1U to 300U. , More preferably 1U-100U lactate dehydrogenase. Further, the lactate dehydrogenase of step i) has an initial activity of about 20% or more, 30% or more, 40% or more when allowed to pass at about 30, 35 ° C. or 37 ° C. for 10 days in the solution (A).
  • Flavin-dependent lactate dehydrogenase maintaining 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more; at about 30, 35 ° C or 37 ° C in the solution (B).
  • the initial activity is maintained at about 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more.
  • step i) 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more may be flavin-dependent lactate dehydrogenase.
  • the temperature at which the lactate dehydrogenase in step i) is brought into contact with the sample may be 20 to 60 ° C, preferably 30 to 55 ° C, and preferably 30 to 40 ° C. It should be.
  • the pH at the time of contacting the lactate dehydrogenase in step i) with the sample may be in the range of 3 to 10, preferably in the range of 5 to 9, preferably in the range of 6 to 8. All you need is.
  • the step of measuring lactic acid in step ii) may be a single measurement or a continuous measurement.
  • the measurement period of step ii) is 5 seconds, 1 minute, 5 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 15 hours, 24 hours, 2 days, 5 days, 7 days, 10 days or 14 days. But it may be.
  • the device for evaluating the state of the sample in the present invention can further include an output unit.
  • the output unit can output the signal from the sensor to the outside.
  • the output unit may be connected to a data processing unit to form a system. That is, the system includes a device and a data processing unit.
  • the data processing unit can process the signal from the sensor.
  • the "data processing unit” not only means a device such as a single computer, but also performs information processing in the form of connecting other devices connected by a communication line such as a local network or the Internet. Includes the concept of networks for.
  • connection between the output unit and the data processing unit means that the signal from the sensor is output from the output unit and connected by an electrical or electronic method so that it can be input to the data processing unit.
  • a wireless communication such as a wireless LAN can also be used for connection.
  • the data processing unit can include a control unit.
  • the control unit can be, for example, a CPU (Central Processing Unit).
  • the program described later can be read into the control unit.
  • the data processing unit can include a storage unit.
  • the "storage unit” refers to a memory, a hard disk, or the like that can write the input information so that it can be read.
  • the storage unit can be arranged inside a predetermined device. Further, the storage unit can exist inside another device connected by a communication line such as a local network or the Internet. By including the storage unit, the signal measured by the sensor can be stored, and the result processed by the data processing unit can be stored.
  • the data processing unit has a display unit for displaying the result processed by the data processing unit, can be connected to the display unit, or can be transferred to the display unit.
  • a means for displaying a known display such as a display, a printing device, an audio output device, and output to the outside by a communication line such as a local network or the Internet can be used. That is, the display unit not only physically has the display device in the data processing unit, but also transfers the result to the other information processing device by transferring the data (result) to the other information processing device. Including displaying.
  • the data processing unit can be equipped with a transmitter, and can be wirelessly transmitted and received to a separate measuring device equipped with a display unit and a storage unit.
  • the transmitter measures the current value measured by the device with the measuring unit 100, and sends the current value to the control unit 101.
  • the control unit 101 measures the temperature in the vicinity of the device by the temperature sensor 102, corrects the temperature, and then calculates the lactic acid concentration from the current value.
  • the control unit 101 executes the calculation of the lactic acid concentration at predetermined sampling time intervals.
  • control unit 101 performs integrated averaging of the calculated lactic acid concentration at predetermined recording time intervals and records it in the storage unit 103.
  • the control unit 101 transmits the value stored in the storage unit 103 to the measuring device 106 via the communication unit 104 in response to an instruction from the measuring device 106.
  • the transmitter in this embodiment has a built-in battery 105.
  • the transmitter is configured to be discarded when the remaining battery level of the battery 105 becomes insufficient.
  • the battery level of the battery 105 is monitored through the measuring device 106. Then, when the device is replaced, the measuring device 106 confirms whether or not the remaining battery 105 is sufficient for the next device replacement with respect to the remaining battery level of the transmitter battery 105. Then, if the remaining amount is insufficient, the user is instructed to replace the transmitter and the transmitter cannot be used.
  • the state of the sample for example, the physical state with respect to exercise load, the disease state, the brewing state of the brewed product with a change in the amount of lactic acid, the degree of aging / ripening of foods and drinks with a change in the amount of lactic acid, It is possible to evaluate the lactic acid content ratio in the production of chemical products accompanied by changes in the amount of lactic acid, the condition of water and soil accompanied by changes in the amount of lactic acid, and the like.
  • the lactate dehydrogenase used in the device of the present invention is about 20% or more, 30% or more, 40% or more of the initial activity when allowed to elapse at about 30, 35 ° C. or 37 ° C. for 10 days in the solution (A). , 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more flavin-dependent lactate dehydrogenase; in the solution (B), about 30, 35 ° C. or 37 ° C. About 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more of the initial activity when 3 days or more have passed.
  • the above device may further include an output unit, and the output unit may be connected to a data processing unit to form a system.
  • evaluation or monitoring can be performed non-invasively to the sample and can be measured directly without sampling steps, i.e. by inserting a test strip into the sensor. It is very advantageous in that it can be used for a long period of time with it stuck and inserted, instead of a disposable measurement like pressing a button.
  • the program of the present invention is a program for evaluating the state of a sample by a system including a device and a data processing unit.
  • the program can be application software that causes a predetermined information processing device to execute a predetermined process in order to operate a predetermined device.
  • the program of the present invention can also be a mobile application (app).
  • the program of the present invention is a program for operating the system as shown in FIG. 4 described above.
  • the system includes devices and data processing units.
  • the device includes an action unit, a sensor, and an output unit.
  • the working unit, the sensor, and the output unit are as described above. That is, the action part is configured so that lactate dehydrogenase can act on the sample.
  • the sensor is configured to be capable of sensing the state of the lactate dehydrogenase-treated specimen at the site of action.
  • the output unit is configured to be able to output a signal from the sensor.
  • the data processing unit is connected to the output section of the device and is configured to process the signal from the sensor.
  • the data processing unit can include a control unit, a storage unit, and a display unit.
  • the control unit, the storage unit, and the display unit are as described above.
  • the program of the present invention causes the system to execute the measurement process S01, the transfer process S02, and the data process S03.
  • the program of the present invention can further cause the system to execute the result display process S04.
  • the program causes the system to execute a process so as to detect and measure the state of the sample on which lactate dehydrogenase is acted on the acting part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • this program can be used as a program for the following purposes.
  • the program of the present invention can be used as a program for food and beverage applications. Specifically, it is as follows.
  • the program of this embodiment can be a program for managing the freshness of beef. That is, in this embodiment, the sample is beef.
  • the program causes the system to execute a process so as to detect and measure the state of beef on which lactate dehydrogenase is acted on the acting part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined processing of the present embodiment is a processing for making a data format suitable for controlling the freshness of beef.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for beef freshness management can be displayed on the display unit.
  • the expiration date of beef can be predicted by monitoring the temperature and humidity in addition to the lactic acid level.
  • the value of the meat can be calculated and used as an index of the selling price.
  • the action part is directly attached to the beef, and the state of the meat can be sensed by a sensor.
  • beef freshness management has been mainly controlled by temperature. Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized. Therefore, according to the program of the present embodiment, freshness management using lactic acid as an index can be performed. Therefore, the transportation conditions and storage conditions of beef can be appropriately adjusted. By using the program of this embodiment, it can be used as an index that objectively shows the value of beef and can support the judgment of consumers.
  • the program of this embodiment can be a program for the management and control index of beef aging. That is, in this embodiment, the sample is beef.
  • the program causes the system to execute a process so as to detect and measure the state of beef on which lactate dehydrogenase is acted on the acting part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined processing of the present embodiment is a processing for making a data format suitable for the management and control index of beef aging.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • the display can display data in a data format suitable for beef aging management and control indicators.
  • the expiration date of beef can be predicted by monitoring the temperature, humidity, and pH in addition to the lactic acid level.
  • the value of the meat can be calculated and used as an index of the selling price.
  • the action part is directly attached to the beef, and the state of the meat can be sensed by a sensor.
  • beef aging management has been mainly controlled by temperature. Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized. Therefore, according to the program of the present embodiment, freshness management using lactic acid as an index can be performed. Therefore, the transportation conditions and storage conditions of beef can be appropriately adjusted. By using the program of this embodiment, it can be used as an index that objectively shows the value of beef and can support the judgment of consumers.
  • the program of this embodiment can be a program for managing the freshness of fish such as tuna. That is, in the present embodiment, the sample is a fish such as tuna.
  • the program causes the system to execute a process so as to detect and measure the state of fish such as tuna on which lactate dehydrogenase is acted on the action part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined processing of the present embodiment is a processing for making a data format suitable for managing the freshness of fish such as tuna.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for managing the freshness of fish such as tuna can be displayed on the display unit.
  • the degree of freshness of fish such as tuna and the expiration date can be predicted by monitoring the temperature, humidity, and K value in addition to the lactic acid value.
  • the value of the fish can be calculated and used as an index of the selling price.
  • the action part can be directly attached to the eyes of the fish, and the state of the fish such as tuna can be detected by the sensor.
  • the transportation conditions and storage conditions for fish such as tuna can be appropriately set.
  • it can be used as an index that objectively shows the value of fish such as tuna, and can support the judgment of consumers and wholesalers.
  • the program of the present embodiment can be a program for management and control index of ripening of fish such as tuna. That is, in the present embodiment, the sample is a fish such as tuna.
  • the program causes the system to execute a process so as to detect and measure the state of fish such as tuna on which lactate dehydrogenase is acted on the action part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined processing of the present embodiment is a processing for making a data format suitable for the management and control index of ripening of fish such as tuna.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for the management and control index of ripening of fish such as tuna can be displayed on the display unit.
  • the action part can be directly attached to the eyes of the fish, and the state of the fish such as tuna can be detected by the sensor.
  • the aging management of fish such as tuna has been mainly controlled by the temperature, aging time, temperature of hot salt water to be thawed, and water temperature. Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized. Therefore, according to the program of the present embodiment, aging management using lactic acid as an index can be performed. Therefore, the aging conditions for fish such as tuna can be appropriately set. By using the program of this embodiment, it can be used as an index that objectively shows the value of fish such as tuna, and can support the judgment of consumers and wholesalers.
  • the program of this embodiment is a program for foods and beverages other than the above, for example, malolactic fermentation management of wine, fermentation management of bread such as sake and bagels, fermentation management of waste rice cake, lactic fermentation management of whiskey, and fermentation of tea. It can be a program for management, food safety indicators, etc. According to the program of this embodiment, fermentation management of brewed liquor such as wine and sake, which relies on experience and intuition, is appropriate by using pH value, dissolved oxygen value, etc. as indicators in addition to lactic acid value. It is possible to obtain various temperature control and fermentation period.
  • the program of the present invention can be used as a program for animal use. Specifically, it is as follows.
  • the program of this embodiment can be a program for managing racehorses. That is, in this embodiment, the sample is a racehorse.
  • the program causes the system to execute a process so as to detect and measure the state of the racehorse on which lactate dehydrogenase is acted on the action part by the sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined process of the present embodiment is a process for converting the data format suitable for the management of racehorses.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for managing racehorses can be displayed on the display unit.
  • a training program is set up using blood lactate as an index. It is known that the amount of lactic acid changes according to the intensity of exercise, and if the balance between the amount of oxygen demand and the amount of supply during exercise is maintained, blood lactic acid does not accumulate, and if the balance of supply and demand is lost, it increases. (From the JRA breeding ground diary, training load using lactic acid as an index). Therefore, in the present embodiment, the appropriateness of the training load of the racehorse can be evaluated. It is also possible to propose a horse training method using the obtained lactic acid level.
  • the program of the present embodiment can perform training management and health management of racehorses.
  • the horse is trained every time. It is also possible to reduce the burden on the trainer and the stress on the horse by collecting blood from the horse and measuring and analyzing it.
  • the program of the present embodiment can be a program for measuring the degree of fatigue of a racehorse. That is, in this embodiment, the sample is a racehorse.
  • the program causes the system to execute a process so as to detect and measure the state of the racehorse on which lactate dehydrogenase is acted on the action part by the sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined process of the present embodiment is a process for converting the data format suitable for measuring the fatigue level of a racehorse.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for measuring the degree of fatigue of a racehorse can be displayed on the display unit.
  • the amount of lactic acid changes according to the degree of fatigue of the racehorse, so that the degree of fatigue of the racehorse can be predicted from the change in the amount of lactic acid.
  • lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized. Therefore, since the program of the present embodiment can obtain an index indicating the muscle fatigue degree of the racehorse, even a beginner can judge the muscle fatigue degree of the racehorse.
  • the program of the present embodiment can be a program for cattle health management, shipping judgment management, or beef cattle meat quality control. That is, in this embodiment, the sample is a cow.
  • the program causes the system to execute a process so as to detect and measure the state of the cow on which lactate dehydrogenase is acted on the acting part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined processing of the present embodiment is a processing for converting into a data format suitable for cattle health management, shipping judgment management, or beef cattle quality control.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for cattle health management, shipping time determination management, or beef cattle quality control can be displayed on the display unit.
  • lactate dehydrogenase is allowed to act on the action part as an ear, and the state of the cow can be sensed and measured by a sensor.
  • Blood vessels pass through the ear, and by arranging an action part and a sensor in the ear tag, it is possible to measure the blood of the ear as a specific sample. Sweat and skin can also be used as samples.
  • the stomach it is also possible to use the stomach as an action part. For example, by measuring the amount of lactic acid in the blood, it is possible to suppress the administration of lactic acid bacteria or grains containing lactic acid before acute acidosis occurs.
  • the swallowing sensor can be used for health management, shipping judgment management, or beef cattle quality control. That is, lactate dehydrogenase can be allowed to act on the action part as a digestive organ such as the stomach, and the state of cattle can be sensed and measured by a sensor.
  • the amount of lactic acid assimilated by the microorganisms present in the stomach changes as a result according to the amount of lactic acid in the cow, and volatile fatty acids are generated according to the amount of lactic acid assimilated.
  • lactate dehydrogenase of the present invention Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized. Therefore, according to the program of the present embodiment, it is possible to manage the health of cattle, manage the determination at the time of shipment, or control the meat quality of beef cattle.
  • the program of this embodiment can be a program for dairy cow health care. That is, in this embodiment, the sample is a dairy cow.
  • the program causes the system to execute a process so as to detect and measure the state of the dairy cow on which lactate dehydrogenase is acted on the acting part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined process of the present embodiment is a process for converting the data format suitable for the health management of dairy cows.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for dairy cow health management can be displayed on the display unit.
  • lactate dehydrogenase is allowed to act on the action part as an ear, and the state of the dairy cow can be sensed and measured by a sensor.
  • Blood vessels pass through the ear, and by arranging an action part and a sensor in the ear tag, it is possible to measure the blood of the ear as a specific sample. Sweat and skin can also be used as samples.
  • the stomach it is also possible to use the stomach as an action part. For example, by measuring the amount of lactic acid in the blood, it is possible to suppress the administration of lactic acid bacteria or grains containing lactic acid before acute acidosis occurs.
  • the program of this embodiment can manage the health of dairy cows.
  • the value of the obtained dairy products can be evaluated from the viewpoint of the health condition of the final product and dairy cows, supporting the judgment of consumers and wholesalers. It becomes.
  • the program of the present invention can be used as a program for plant use. Specifically, it is as follows.
  • the program of this embodiment can be a program for plant management. That is, in this embodiment, the sample is a plant or soil. Specifically, the sample may be leaves, roots, sap, fruits, seeds, etc. of a plant, and may be a potted plant.
  • the program causes the system to execute a process so as to detect and measure the state of the plant on which lactate dehydrogenase is acted on the acting part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined processing of the present embodiment is a processing for making a data format suitable for plant management.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for plant management can be displayed on the display unit.
  • Lactic acid in the soil promotes water absorption of plants. Therefore, the water absorption of plants changes according to the amount of lactic acid in the soil. Therefore, in the present embodiment, the state of water absorption of the plant can be evaluated.
  • the program of the present embodiment can propose the timing of adding nutrients and watering the plants in the flowerpot. Furthermore, when the pH is low, it becomes possible to suppress the growth of various germs in the plant. Therefore, by measuring the pH together with the amount of lactic acid, it becomes possible to suppress the growth of various germs.
  • the program of this embodiment can be a program for indicators of soil management and hydroponics management. That is, in this embodiment, the sample is soil or water.
  • the program causes the system to execute a process so as to detect and measure the state of the soil on which lactate dehydrogenase is acted on the acting part by a sensor and convert it into a signal.
  • the program causes the system to execute the process so that the signal obtained in the measurement process is transferred from the output unit to the data processing unit.
  • the program causes the system to execute the processing so that the data processing unit performs a predetermined processing on the signal obtained in the measurement processing.
  • the predetermined treatment of the present embodiment is a treatment for making a data format suitable for an index of soil management and hydroponics management.
  • the program causes the system to execute the process so that the predetermined display unit displays the result processed by the data processing unit.
  • data in a data format suitable for the index of soil management and hydroponics management can be displayed on the display unit.
  • Lactic acid in soil and hydroponic water promotes water absorption of plants. Therefore, the water absorption of plants changes depending on the amount of lactic acid in the soil and hydroponic water. Therefore, in the present embodiment, it is possible to evaluate the water absorption state of plants existing in soil and hydroponic water.
  • the program of the present embodiment can propose the timing of addition of nutrients to plants, the timing of watering, and the timing of topdressing. Furthermore, when the pH is low, it becomes possible to suppress the growth of various germs in the plant. Therefore, by measuring the pH together with the amount of lactic acid, it becomes possible to suppress the growth of various germs.
  • the mainstream of plant management is not based on data, such as adding fertilizer according to the time such as the season and adding water or nutrients when the condition of the plant deteriorates.
  • the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized.
  • the lactic acid level contained in the soil becomes low, it is possible to provide a system for calculating the amount of saccharides and lactic acid added that can be assimilated by lactic acid bacteria in order to enhance the antibacterial property in the soil.
  • a system for calculating the type and amount of alkaline fertilizer added is provided.
  • the present invention has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12. It also relates to lactate dehydrogenase, which has a 95% or higher amino acid sequence, and the nucleic acid encoding it.
  • the present invention relates to the nucleotide sequences set forth in SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 13 and 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% , 84%, 85%, 86%, 87%, 88%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.
  • Provided is a DNA encoding a protein having a base sequence having sequence identity and having lactic acid activity.
  • LDH gene To obtain the gene encoding LDH, a commonly used gene cloning method is usually used. For example, chromosomal DNA or mRNA can be extracted from microbial cells having LDH-producing ability or various cells by a conventional method, for example, the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. Using the chromosomal DNA or cDNA thus obtained, a library of chromosomal DNA or cDNA can be prepared.
  • a method of synthesizing an appropriate probe DNA based on the above-mentioned LDH amino acid sequence and selecting the LDH gene from a library of chromosomal DNA or cDNA using this, or a method of selecting an appropriate primer DNA based on the above-mentioned amino acid sequence is amplified by an appropriate polymerase chain reaction (PCR method) such as the 5'RACE method or the 3'RACE method, and these DNA fragments are ligated.
  • PCR method polymerase chain reaction
  • a DNA containing the full length of the LDH gene of interest can be obtained.
  • LDH genes may be linked or inserted into various vectors, or may be integrated into a chromosome or genome.
  • kits such as TA Cloning Kit (Invitrogen) and In-Fusion HD Cloning Kit (Clontech); pUC119 (Takarabio), pUC18 (Takarabio), Commercially available plasmid vector DNAs such as pBR322 (Takara Bio), pBluescript SK + (Stratagene), pYES2 / CT (Invitrogen); and commercially available bacterial plunger vector DNAs such as ⁇ EMBL3 (Stratagene) can be used.
  • the recombinant DNA is used to transform a host organism such as Escherichia coli, preferably Escherichia coli JM109 strain (Takara Bio Inc.) or Escherichia coli DH5 ⁇ strain (Takara Bio Inc.).
  • Recombinant DNA contained in the obtained transformant is purified using QIAGEN plasmid Mini Kit (Qiagen) or the like.
  • Qiagen QIAGEN plasmid Mini Kit
  • it is preferable to use a host organism transformed with recombinant DNA containing the LDH gene for example, Escherichia coli, yeast, mold cells, filamentous fungi, etc., as the host for producing LDH.
  • the present invention also relates to a host cell having the above nucleic acid and a method for producing lactate dehydrogenase, which comprises culturing the host cell.
  • this strain may be cultured by a usual solid culture method, but it is possible. It is preferable to use the liquid culture method for culturing.
  • a medium for culturing the above strain for example, one or more nitrogen sources such as yeast extract, tripton, peptone, meat extract, corn steep liquor or leachate of soybean or wheat bran, sodium chloride, and phosphoric acid first.
  • inorganic salts such as potassium, dipotassium phosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferrous sulfate or manganese sulfate, and if necessary, add sugar raw materials, vitamins, etc. as appropriate. Is used. It is appropriate to adjust the initial pH of the medium to pH 7-9. Any conditions can be used for culturing, and for example, a culturing temperature of 20 to 42 ° C., preferably a culturing temperature of about 30 ° C. for 4 to 24 hours, and more preferably a culturing temperature of about 30 ° C.
  • LDH can be collected from the culture by using a usual enzyme collection means.
  • the cells are subjected to ultrasonic destruction treatment, grinding treatment, etc. by a conventional method, or this enzyme is extracted using a lytic enzyme such as lysozyme, or the bacterium is shaken or left to stand in the presence of toluene or the like. This enzyme can be excreted from the cells. Then, this solution is filtered, centrifuged, etc.
  • nucleic acid is removed with streptomycin sulfate, protamine sulfate, manganese sulfate, etc., and then ammonium sulfate, alcohol, acetone, etc. are added thereto. Fractionation is performed and the precipitate is collected to obtain the crude enzyme of LDH.
  • an LDH purified enzyme preparation from the above crude enzyme of LDH, for example, a gel filtration method using Sephadex, Superdex or Ultrogel; an adsorption elution method using an ion exchanger; electrophoresis using a polyacrylamide gel or the like. Method; Adsorption elution method using hydroxyapatite; Precipitation method such as sucrose density gradient centrifugation method; Affinity chromatography method; Fractionation method using molecular sieving membrane or hollow yarn membrane, etc. are appropriately selected, or a combination thereof is carried out. By doing so, a purified LDH enzyme preparation can be obtained. In this way, LDH with the desired improved stability can be obtained.
  • the lactate dehydrogenase of the present invention has about 20% or more, 30% or more, 40% or more, 50% or more, 60% of the initial activity when allowed to elapse at about 30 to 37 ° C. for 10 days in the solution (A).
  • flavin-dependent lactate dehydrogenase maintaining 70% or more, 80% or more, 90% or more or 95% or more; when allowed to pass at about 30 to 37 ° C. for 3 days or more in the solution (B).
  • Flavin-dependent lactate dehydrogenase which maintains about 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more of the initial activity, Alternatively, when allowed to elapse at about 30 to 37 ° C. for 15 hours or more in the solution (C), about 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more of the initial activity. , 80% or more, 90% or more, or 95% or more may be flavin-dependent lactate dehydrogenase.
  • the lactate dehydrogenase of the present invention may be a flavin-dependent lactate dehydrogenase that maintains 70% or more activity even after 15 hours from the time of use.
  • the buffer material (buffer solution) that can be used in the reaction solution of LDH include a borate buffer containing boric acid and / or a salt thereof, a Tris hydrochloric acid buffer, a phosphoric acid containing phosphoric acid and / or a salt thereof.
  • a buffer such as a potassium phosphate buffer or a sodium phosphate buffer, an organic acid buffer and / or an organic acid buffer containing a salt thereof, such as a tricarboxylic acid buffer and / or a tricarboxylic acid buffer containing a salt thereof, for example.
  • a citrate buffer containing citrate and / or a salt thereof, a monocarboxylic acid buffer and a monocarboxylic acid buffer containing a salt thereof, for example, a buffer such as an acetate buffer and an acetate buffer containing a salt thereof. can be mentioned.
  • ACES N- (2-acetamide) -2-aminoethanesulfonic acid
  • BES N, N-bis (2-hydroxyethyl) -2) -Aminoethanesulfonic acid
  • Bicin N, N-bis (2-hydroxyethyl) glycine
  • Bis-Tris bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane
  • CHES N-cyclohexyl-2-) Amino ethane sulfonic acid
  • EPPS 4-- (2-hydroxyethyl) -1-piperazin propane sulfonic acid
  • HEPES (4-2-hydroxyethyl-1-piperazine ethane sulfonic acid
  • HEPPSO N- (hydroxyethyl)) Piperazin-N'-2-hydroxypropanesulfonic acid
  • MES 2- (N-morpholino) ethanesulfonic acid
  • MES 2- (N-morpholino
  • Example 1 (1) Preparation of recombinant plasmid pKK223-3-ScLDH DNA and pKK223-3-PkLDH DNA Biochem. J. As described in 258, 255-259 (1989), the 506 amino acids shown in SEQ ID NO: 1 from which the amino acids at positions 2 to 85 have been removed from the 591 amino acids which are the amino acid sequences of the lactic acid dehydrogenase (ScLDH) derived from Saccharomyces cerevisiae. The gene of 1521 bp (including the stop codon TAA) represented by SEQ ID NO: 2 encoding the above was obtained as cDNA by PCR of a gene fragment which is a conventional method.
  • ScLDH lactic acid dehydrogenase
  • SEQ ID NO: 3 which is the amino acid sequence of Lactate dehydrogenase (PkLDH) derived from Pichia kudriavzevii, was compared with ScLDH, and the sequence encoding the 502 amino acid represented by SEQ ID NO: 4 from which positions 2 to 77 were removed.
  • the gene of 1509 bp (including the stop codon TAA) represented by No. 5 was obtained as cDNA by PCR of a gene fragment which is a conventional method.
  • a DNA construct was prepared by inserting the ScLDH gene or PkLDH gene, which is the target gene, into the multi-cloning site of the plasmid pKK223-3 by a conventional method.
  • the ScLDH gene or PkLDH gene was attached to the kit using the In-Fusion HD Cloning Kit (manufactured by Clontech) at the In-Fusion Cloning Site at the multi-cloning site of pKK223-3.
  • In-Fusion HD Cloning Kit manufactured by Clontech
  • expression plasmids pKK223-3-ScLDH and pKK223-3-PkLDH were obtained.
  • Escherichia coli JM109 was transformed with these plasmids, and Escherichia coli JM109 (pKK223-3-ScLDH) strain and Escherichia coli JM109 (pKK223-3-PkLDH) strain were added to 3 ml of LB-amp medium [1% (w / v). ) Bacttrypton, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, 50 ⁇ g / ml ampicillin] was inoculated and cultured with shaking at 37 ° C. for 16 hours. Got
  • Each of the obtained cultured cells was washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the buffer, ultrasonically crushed, and centrifuged at 20,000 ⁇ g for 10 minutes. Then, 0.6 ml of a crude enzyme solution containing ScLDH or PkLDH was prepared.
  • LDH activity Using the crude enzyme solution containing ScLDH or PkLDH described above, the oxidation activity for L-lactic acid was measured by the method shown in the activity measurement method below.
  • the LDH of the present invention catalyzes the reaction of oxidizing L-lactic acid to produce pyruvic acid. This may be referred to as LDH activity for convenience.
  • the LDH activity of PkLDH of the present invention can be measured by utilizing this principle of action, for example, using the following measurement system using potassium ferricyanide as an electron acceptor.
  • the degree of disappearance of the above "potassium ferricyanide” can be detected as the amount of change in absorbance at a wavelength of 420 nm, and the enzyme activity can be determined based on this amount of change.
  • LDH activity can be measured according to the following procedure. 1M potassium phosphate buffer (pH 7.5) 0.15 mL, 0.1 M L-lactic acid solution 0.15 mL, 30 mM potassium ferricyanide solution 0.075 mL, ultrapure water 1.075 mL are mixed and kept warm at 30 ° C for 5 minutes. do. Then 0.05 mL of the enzyme sample solution is added and the reaction is started. The absorbance at the start of the reaction and over time is measured, the amount of decrease in absorbance at 420 nm ( ⁇ A420) with the progress of the enzymatic reaction per minute is determined, and the LDH activity is calculated according to the following formula. At this time, LDH activity defines 1 U as the amount of enzyme that reduces 1 ⁇ mol of potassium ferricyanide per minute in the presence of L-lactic acid at a concentration of 10 mM at 30 ° C.
  • 1.5 is the liquid volume of the reaction reagent + enzyme reagent (mL)
  • 1.01 is the mmol molecular absorption coefficient (cm 2 / ⁇ mol) under the present activity measurement conditions
  • 0.05 is the liquid volume of the enzyme solution.
  • ML 1.0 is the optical path length (cm) of the cell
  • ⁇ A420blank is the absorbance at 420 nm per minute when the reaction was started by adding 10 mM phosphate buffer (pH 7.5) instead of the enzyme sample solution.
  • the amount of decrease in, df represents a dilution factor.
  • Example 2 Temporal stability
  • the above crude enzyme solution was diluted to a 100 mM potassium phosphate buffer solution (pH 6.0) containing 0.07% BSA as the final concentration, and the temperature stability was examined.
  • the LDH enzyme solution prepared in (2) of Example 1 was diluted to 6 U / ml, and each temperature (35 ° C, 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C, 65). After treatment at (° C.) for 10 minutes, LDH activity was measured, and the residual activity rate was measured as compared with LDH activity before treatment.
  • a similar test was also performed on ScLDH for comparison. The results are shown in FIG. From the results, it was found that ScLDH is unstable at 45 ° C., but PkLDH is stable even after heat treatment at 55 ° C.
  • FIG. 3A is a graph of the residual activity rate after each time elapsed, where the residual activity rate when the elapsed time is zero time is 100%. As a result, it was found that the residual activity rate of PkLDH heated to a certain extent (37 ° C. in this example) was improved by a certain period during storage (FIG.
  • FIG. 3B is a graph of the residual activity rate after each time elapses, where the activity rate at the reference time is 100% when 15 hours from the start of storage is set as the reference time.
  • PkLDH retains 99% activity even after 245 hours have passed from the start of storage (230 hours after the reference time), and after 15 hours, the residual activity rate becomes constant and is about 10 days at 37 ° C. It was not deactivated even when stored and was very stable (Fig. 3B).
  • the residual activity rate of ScLDH began to decrease remarkably from the start of storage, the residual activity rate after 21 hours (6 hours after the reference time) was 62%, and the activity after 65 hours (50 hours after the reference time).
  • the survival rate was 0%, which was unstable. Further, even if the amount of ScLDH enzyme was evaluated to be 20 U / ml, the stability was not significantly improved.
  • Example 3 Purification of enzyme
  • the crude enzyme solution of PkLDH obtained in (2) of Example 1 was subjected to 1 ml of Q Sepharose Fast Flow resin (manufactured by GE Healthcare) equilibrated with 20 mM potassium phosphate buffer (pH 7.5), and the resin was applied. Was adsorbed on. Proteins that did not adsorb to the resin were eluted with the same buffer. Subsequently, it was washed with 20 mM potassium phosphate buffer (pH 7.5) containing 50 mM sodium chloride and 20 mM potassium phosphate buffer (pH 7.5) containing 200 mM sodium chloride.
  • the adsorbed PkLDH was eluted with a 20 mM potassium phosphate buffer (pH 7.5) containing 500 mM sodium chloride.
  • the obtained crude enzyme solution of PkLDH was applied to a HiRoad 26/10 Q Sepharose HP column (manufactured by GE Healthcare) equilibrated with a 20 mM potassium phosphate buffer solution (pH 7.5) containing 150 mM sodium chloride, and one column was provided. A fraction containing the protein of interest was collected by running a volume of buffer solution.
  • the recovered enzyme solution was dialyzed against 10 mM potassium phosphate buffer (pH 7.5), adsorbed on a Hiscreen Capto Q column (manufactured by GE Healthcare) equilibrated with the buffer, and then 500 mM NaCl / 10 mM phosphorus. PkLDH adsorbed on the resin was eluted and recovered by gradually increasing the NaCl concentration with a gradient to potassium phosphate buffer (pH 7.5). The obtained fraction was analyzed by SDS-PAGE and confirmed to be purified to a purity free of other contaminating proteins, and used as a purified preparation of PkLDH. The thermal stability was equivalent to that of the crude enzyme solution.
  • Example 4 The pH stability was examined using the purified PkLDH enzyme solution (10 U / mL) obtained in Example 3. After heat treatment at 55 ° C. for 15 minutes using 100 mM potassium phosphate buffer (pH 6.0-pH 7.5), the enzyme was maintained in each buffer, and the other enzymes were used in Example 1 ( The activity was measured according to the activity measuring method described in 3). The condition with the highest activity in the activity value after the treatment was set to 100%, and the relative value was determined for the activity value under the other conditions. The results are shown in FIG. As a result, it was the most stable at pH 6.5, followed by 92% relative activity (%) at pH 6.0.
  • the optimum active pH was examined using the purified PkLDH enzyme solution (10 U / mL) obtained in Example 3. Using 100 mM potassium phosphate buffer (pH 6.0-pH 7.5), an enzymatic reaction was carried out at a temperature of 30 ° C. at each pH, and the relative activity (%) was compared. The results are shown in FIG. As a result, the optimum active pH of the FMN-dependent lactate dehydrogenase of the present invention was shown to be the highest in the pH range of 6.5 to 7.5. It is considered that there is a possibility that good activity is maintained even at pH 8.0.
  • Example 5 Quantification of L-lactic acid by printed electrode
  • SCREEN-PRINTED ELECTRODES manufactured by DropSens, product number DRP-110
  • DRP-CAC dedicated connector
  • lactic acid can also be quantified by electrochemical measurement.
  • Example 6 (4) Preparation of recombinant plasmid pKK223-3-CaLDH DNA and pKK223-3-OgLDH DNA and production of each LDH
  • SEQ ID NO: 6 which is the amino acid sequence of lactate dehydrogenase (CaLDH) derived from Candida inconspirua
  • the 505 amino acids shown in SEQ ID NO: 7 with the 2-67 positions removed are encoded by 1518 bp shown in SEQ ID NO: 8.
  • Gene including stop codon TAA was obtained as cDNA by PCR of the gene fragment, which is a conventional method.
  • SEQ ID NO: 9 which is the amino acid sequence of lactate dehydrogenase (OgLDH) derived from Ogataea parapolymerase
  • SEQ ID NO: 10 the 503 amino acids shown in SEQ ID NO: 10 from which positions 2 to 56 have been removed are encoded by SEQ ID NO: 11.
  • the 1512 bp gene (including the stop codon TAA) shown in (1) was obtained as a cDNA by PCR of a gene fragment, which is a conventional method.
  • Example 2 In the same manner as in Example 1, a DNA construct in which the target gene CaLDH gene or OgLDH gene was inserted into the multicloning site of the plasmid pKK223-3 by a conventional method was prepared, and expression plasmids (pKK223-3-CaLDH and pKK223) were prepared. -3-OgLDH) was obtained. Escherichia coli BL21 (pKK223-3-CALDH) strain transduced with pKK223-3-CaLDH and Escherichia coli BL21 (pKK223-3-OgLDH) strain transduced with pKK223-3-OgLDH so as to have a final concentration of 0.1 mM.
  • Example 7 Temporal stability Similar to Example 2, long-term stability at 37 ° C. was examined. However, 100 mM potassium phosphate buffer (pH 6.0) containing 0.07% BSA as the final concentration, CaldH or OgLDH was diluted to about 20 U / ml and stored for each time. Then, the residual activity rate after each time elapsed was measured. The results are shown in FIG. 3 (A). CalDH retains 72% activity 140 hours after the start of storage, and OgLDH retains 77% activity 140 hours after the start of storage, which is very stable compared to ScLDH. Met.
  • Example 8 Preparation of recombinant plasmid pKK223-3-ThrLDH DNA and production of each LDH. It is shown by SEQ ID NO: 13, which encodes the 499 amino acid represented by SEQ ID NO: 12, which is the amino acid sequence of lactate dehydrogenase (ThLDH) derived from Thermothelomyces thermophilus.
  • SEQ ID NO: 13 which encodes the 499 amino acid represented by SEQ ID NO: 12, which is the amino acid sequence of lactate dehydrogenase (ThLDH) derived from Thermothelomyces thermophilus.
  • the 1500 bp gene (including the stop codon TAA) was obtained as a DNA by PCR of the gene fragment, which is a conventional method.
  • a DNA construct was prepared by inserting the ThLDH gene, which is the target gene, into the multicloning site of the plasmid pKK223-3 by a conventional method, and an expression plasmid (pKK223-3-ThhLDH) was obtained.
  • Escherichia coli BL21 (pKK223-3-ThhLDH) strain transduced with pKK223-3-ThhLDH was cultured at 30 ° C. for 24 hours in 3 ml of LB-amp medium supplemented with IPTG so as to have a final concentration of 1 mM.
  • Each of the obtained cultured cells was washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the buffer, ultrasonically crushed, and centrifuged at 20,000 ⁇ g for 10 minutes. Then, 0.6 ml of a crude enzyme solution containing ThLDH was prepared.
  • Example 9 (Temperature stability) Similar to Example 2, long-term stability at 37 ° C. was examined. A 100 mM potassium phosphate buffer (pH 6.0) containing 0.07% BSA as a final concentration and ThLDH were diluted to about 20 U / ml and stored for each time. Then, the residual activity rate after each time elapsed was measured. ThLDH retained 74% activity even after 89 hours from the start of storage, and was very stable as compared with ScLDH. That is, it can be said that ThLDH maintains 70% or more of the initial activity when it is allowed to pass at 37 ° C. for at least 3 days or more.
  • Example 10 Preparation of PkLDH mutant A mutant in which the N-terminal region of PkLDH was further deleted was constructed. First, in order to prepare a mutant (PkLDH-96) in which the amino acids at positions 2 to 95 in SEQ ID NO: 4 have been deleted, the recombinant plasmid pKK223-3-PkLDH obtained in Example 1 was used as a template for the sequence. The PCR reaction was carried out under the following conditions using the synthetic oligonucleotides Nos. 14 and 15 and KOD One PCR Master Mix (manufactured by Toyobo Co., Ltd.).
  • Escherichia coli JM109 was transformed with the reaction solution and developed on LB-amp agar medium.
  • the recombinant plasmid was extracted and purified by the same method as in Example 1 to obtain 2.5 ⁇ g of DNA.
  • the base sequence of the DNA encoding PkLDH-96 in the plasmid was determined using a multicapillary DNA analysis system Applied Biosystems 3130xl Genetic Analyzer (manufactured by Life Technologies), and as a result, a DNA construct encoding PkLDH-96 was determined. Obtained.
  • PkLDH-103 a synthetic oligonucleotide of SEQ ID NOs: 14 and 22, and a variant (PkLDH-104) lacking the amino acids at positions 2 to 103 in SEQ ID NO: 4 are prepared.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 24, 25, 26, and 27 using pKK223-3-PkLDH as a template.
  • a PCR reaction was carried out under the following conditions using the recombinant plasmid pKK223-3-PkLDH as a template and the synthetic oligonucleotides of SEQ ID NOs: 24 and 25, KOD One PCR Master Mix (manufactured by Toyobo Co., Ltd.). ..
  • the obtained PCR product was treated with the restriction enzyme DpnI, the remaining template DNA was cleaved, and then Escherichia coli JM109 was transformed and developed on an LB-amp agar medium.
  • the recombinant plasmid was extracted and purified by the same method as in Example 1 to obtain 2.5 ⁇ g of DNA.
  • the nucleotide sequence of the DNA encoding the PkLDH mutant in the plasmid was determined using a multicapillary DNA analysis system Applied Biosystems 3130xl Genetic Analyzer (manufactured by Life Technologies).
  • a PCR reaction was carried out under the same conditions using the recombinant plasmid pKK223-3-PkLDH / L386R as a template and the synthetic oligonucleotides of SEQ ID NOs: 26 and 27 and KOD One PCR Master Mix (manufactured by Toyobo Co., Ltd.). , 2.5 ⁇ g of DNA was obtained.
  • the base sequence of the DNA encoding the PkLDH mutant in the plasmid was determined using a multicapillary DNA analysis system Applied Biosystems 3130xl Genetic Analyzer (manufactured by Life Technologies), and leucine at position 386 of the amino acid sequence shown in SEQ ID NO: 4.
  • L386R / T461R / D464R means that leucine at position 386 of the amino acid sequence of SEQ ID NO: 4 is replaced with arginine, threonine at position 461 is replaced with arginine, and aspartic acid at position 464 is replaced with arginine.
  • the "/" symbol means that it has all of its respective substitutions.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 28 and 29 using pKK223-3-PkLDH as a template, and PkLDH / which is a mutant in which phenylalanine at position 161 of the amino acid sequence shown in SEQ ID NO: 4 was replaced with leucine.
  • a DNA construct encoding F161L was obtained.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 30 and 31 using pKK223-3-PkLDH as a template, and PkLDH / which is a mutant in which phenylalanine at position 187 of the amino acid sequence shown in SEQ ID NO: 4 was replaced with leucine.
  • a DNA construct encoding F187L was obtained. Further, PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 32 and 33 using pKK223-3-PkLDH as a template, and PkLDH / which is a mutant in which phenylalanine at position 428 of the amino acid sequence shown in SEQ ID NO: 4 was replaced with leucine. A DNA construct encoding F428L was obtained.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 32 and 33 using pKK223-3-PkLDH-97 as a template, and phenylalanine at the position corresponding to position 428 of the amino acid sequence shown in SEQ ID NO: 4 in PkLDH-97 was obtained.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 34 and 35 using PkLDH-97 / F428L as a template, and leucine at the position corresponding to the 194th position of the amino acid sequence shown in SEQ ID NO: 4 in PkLDH-97 / F428L was obtained.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 36 and 37 using PkLDH-97 / F428L as a template, and leucine at the position corresponding to position 222 of the amino acid sequence shown in SEQ ID NO: 4 in PkLDH-97 / F428L was obtained.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 38 and 39 using PkLDH-97 / F428L as a template, and the alanine at the position corresponding to the 274th position of the amino acid sequence shown in SEQ ID NO: 4 in PkLDH-97 / F428L was obtained.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 40 and 41 using PkLDH-97 / F428L as a template, and leucine at the position corresponding to the 277th position of the amino acid sequence shown in SEQ ID NO: 4 in PkLDH-97 / F428L was obtained.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 42 and 43 using PkLDH-97 / F428L as a template, and phenylalanine at the position corresponding to the 313th position of the amino acid sequence shown in SEQ ID NO: 4 in PkLDH-97 / F428L was obtained.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 44 and 45 using PkLDH-97 / F428L as a template, and isoleucine at the position corresponding to position 314 of the amino acid sequence shown in SEQ ID NO: 4 in PkLDH-97 / F428L was obtained.
  • PkLDH / F428L was prepared in order to prepare a mutant (PkLDH-3 / F428L) in which the amino acid at position 2 in SEQ ID NO: 4 was deleted.
  • PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 14 and 54 as a template, and as a result, a DNA construct encoding PkLDH-3 / F428L was obtained.
  • the obtained Escherichia coli BL21 strain transduced with plasmids encoding various PkLDH mutants was cultured at 30 ° C. for 24 hours in 3 ml of LB-amp medium supplemented with IPTG so as to have a final concentration of 1 mM.
  • Each of the obtained cultured cells was washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the buffer, ultrasonically crushed, and centrifuged at 20,000 ⁇ g for 10 minutes. To prepare 0.6 ml of a crude enzyme solution containing various PkLDH variants.
  • the activity of the crude enzyme solution was 0.3 to 6 times higher than that of the wild type PkLDH for 13 types of mutants (PkLDH-76 to PkLDH-110) from which the N-terminal was deleted.
  • the activity of the crude enzyme solution was 2 to 11 times higher than that of PkLDH / F428L for 9 types of mutants (PkLDH-3 / F428L to PkLDH-11 / F428L) from which the N-terminal was deleted.
  • Example 11 Temporal stability
  • the crude enzyme solution (PkLDH-96 to PkLDH-104) prepared in Example 10 was diluted to a 150 mM potassium phosphate buffer solution (pH 7.5) containing 0.15% BSA as the final concentration to improve the temperature stability.
  • the LDH enzyme solution which is the N-terminal deleted mutant prepared in Example 10 was diluted to 6 U / ml, treated at 55 ° C. for 15 minutes, and then the LDH activity was measured to measure the LDH before treatment. The residual activity rate was measured in comparison with the activity.
  • a similar test was also performed on PkLDH for comparison. The results are shown in FIG.
  • PkLDH-96 to PkLDH-104 had a higher residual activity rate and higher thermal stability than PkLDH.
  • other N-terminal deleted mutants PkLDH-76, PkLDH-84, PkLDH-92, PkLDH-110, PkLDH-3 / F428L, PkLDH-4 / F428L, PkLDH-5 / F428L, PkLDH-6 / F428L, PkLDH-7 / F428L, PkLDH-8 / F428L, PkLDH-9 / F428L, PkLDH-10 / F428L, PkLDH-11 / F428L) were similarly treated at 50 ° C.
  • Example 10 the crude enzyme solution prepared in Example 10 (PkLDH-97 / F428L / L194A, PkLDH-97 / F428L / L222Y, PkLDH-97 / F428L / A274S, PkLDH-97 / F428L / L277S, PkLDH-97 / F428L / F313A, PkLDH-97 / F428L / I314S) was used for treatment at 50 ° C. for 15 minutes.
  • PkLDH-104 The activity of PkLDH-104 was maintained at 111% even after the lapse, 97% after the lapse of 43 hours from the start of storage, 99% after the lapse of 72 hours, and 88% after the lapse of 171 hours.
  • PkLDH / L386R / T461R / D464R retained 96% activity even after 89 hours from the start of storage, and PkLDH / F428L was 99% after 43 hours, 107% after 72 hours, and after 171 hours. Also retained 94% activity and was very stable compared to ScLDH. That is, it can be said that these mutants maintain 70% or more of the initial activity when allowed to pass at 37 ° C. for at least 3 days or more.
  • PkLDH-98, PkLDH-99, PkLDH-100, PkLDH-101, PkLDH-102, PkLDH-103, PkLDH / F161L and PkLDH / F187L also have higher thermal stability at 55 ° C. than PkLDH. Therefore, it is highly probable that the long-term stability at 37 ° C. is also very stable as compared with ScLDH.
  • Example 12 Quantification of L-lactic acid by PkLDH-immobilized electrode
  • L-lactic acid was quantified using an electrode on which the purified PkLDH enzyme solution obtained in Example 3 was immobilized.
  • 12 U of PkLDH was applied and dried on the working electrode of SCREEN-PRINTED ELECTRODES (manufactured by DropSens, product number DRP-C110) on which the working electrode of carbon was printed.
  • 3 ⁇ L of 2% poly (ethylene glycol) diglycidyl ether (Mn: 6000, manufactured by Sigma) was applied and reacted at 4 ° C. for 22 hours. It was washed with ultrapure water to obtain a PkLDH-immobilized electrode.
  • DRP-CAC dedicated connector
  • BAS ALS electrochemical analyzer 814D
  • platinum electrode 0.1 mg / ml Lindschedler' Three electrodes were immersed in 10 ml of PBS (pH 7.4) containing s Green Leuco Base (manufactured by Tokyo Kasei Co., Ltd.). +200 mV (vs Ag / AgCl) was applied, and the response current value when the L-lactic acid solution was added at regular intervals was recorded. The result is shown in FIG. It was shown that when 1 to 7 mM L-lactic acid was added, the response current increased in a lactic acid concentration-dependent manner, and L-lactic acid could be quantified.
  • DRP-CAC dedicated connector
  • a device for evaluating the state of a sample including an action part for allowing FMN-LDH to act on the sample of the present invention and a sensor for detecting the state of the sample on which FMN-LDH is acted, and a sample using the device.
  • interstitial fluid blood, urine, tears, sweat, saliva, skin, flesh, eyeballs, cornea, gastric fluid, food and drink, brews, chemicals. Since lactic acid in a lactic acid-containing composition containing water and soil can be measured stably, accurately and easily over a long period of time, it is possible to manage the health of humans and animals, or to manufacture foods and drinks, brewed products, chemical products, etc. It is useful in control, quality control, etc.

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WO2022258733A1 (en) 2021-06-09 2022-12-15 Directsens Gmbh Novel production method of flavocytochrome b2
WO2023013498A1 (ja) * 2021-08-03 2023-02-09 キッコーマン株式会社 新規フラビン依存性乳酸デヒドロゲナーゼ及び乳酸デヒドロゲナーゼの安定性を向上させる方法
TWI814233B (zh) * 2022-01-28 2023-09-01 住華科技股份有限公司 硼酸檢測試片及硼酸檢測方法

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EP4223884A4 (en) * 2020-09-29 2024-12-11 Kikkoman Corporation METHOD FOR IMPROVING THE STABILITY OF A COMPOSITION CONTAINING FLAVIN-DEPENDENT LACTATE DEHYDROGENASE
WO2022258733A1 (en) 2021-06-09 2022-12-15 Directsens Gmbh Novel production method of flavocytochrome b2
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TWI814233B (zh) * 2022-01-28 2023-09-01 住華科技股份有限公司 硼酸檢測試片及硼酸檢測方法

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