Device for evaluating state of specimen, system including the same, method for evaluating state of specimen, and lactate dehydrogenase used therein

Abstract

To provide devices, systems, and programs for evaluating a state of interstitial fluid, blood, urine, tears, sweat, saliva, food and beverages, brewed products, or the like of humans and non-human organisms, and to provide evaluation methods using the same.SOLUTION: The present invention provides: devices, systems, and programs for evaluating a state of a specimen, including an acting portion for applying lactate dehydrogenase to the specimen and a sensor for detecting the state of the specimen after the application of lactate dehydrogenase; a method for evaluating the state of the specimen using the same; and an enzyme used therein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a device for assessing the state of a specimen, the device including a sensor using flavin-dependent lactate dehydrogenase that uses a flavin compound as a coenzyme, a system in which the device further includes an output section, and a method for assessing the state of a specimen using the device or system. The present invention also relates to a flavin-dependent lactate dehydrogenase that can be suitably used in the device, system, and method for assessing the state of a specimen using them. [Background technology]

[0002] Blood lactate and sweat lactate concentrations are known markers of fatigue and physical condition. Lactate is a major metabolic product and is recognized as an important indicator not only for health management but also for health assessment, including in critically ill and / or surgical patients. Lactate levels in body fluids can be an indicator of various pathologies, such as circulatory failure and liver damage. Lactate monitoring can be used to detect sepsis, hypoxia, and the presence of cancerous tissue (Non-Patent Document 1).

[0003] Lactate monitoring is also used for the purpose of health management, not limited to people with illnesses, as an indicator for monitoring the appropriateness of training by athletes and people who are interested in exercising on a daily basis (Non-Patent Document 2).

[0004] Patent Documents 1 to 3 propose a specific information processing device as a technical proposal for continuously monitoring the state, posture, etc. of a person for a certain period of time (Patent Documents 1 to 3).

[0005] Lactate oxidase (hereinafter referred to as LOD) is known as an enzyme that uses lactic acid as a substrate. LOD has the problem of losing its activity during storage after drying. For this reason, Patent Document 4 proposes a method of drying LOD in combination with a specific stabilizer during the drying process.

[0006] Patent Document 5 also proposes a lactate sensor in which catalase is coexisted to eliminate hydrogen peroxide as a means of avoiding a decrease in LOD activity due to hydrogen peroxide.

[0007] Furthermore, FMN-dependent lactate dehydrogenase (hereinafter referred to as FMN-LDH) is known as an enzyme that uses lactic acid as a substrate. Non-Patent Document 3 describes that the FMN-LDH derived from Saccharomyces cerevisiae that has been known so far has a problem with stability. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 2019-150649 [Patent Document 2] Japanese Patent Application Laid-Open No. 2017-100039 [Patent Document 3] International Publication No. 17 / 163521 Brochure [Patent Document 4] Patent No. 5593689 [Patent Document 5] Special Publication No. 2018-519507 [Non-patent literature]

[0009] [Non-Patent Document 1] Japanese Sepsis Clinical Practice Guidelines 2016, PNAS September 15, 2015, 112 (37), 11642-11647 [Non-patent document 2] Sports Performance Research, 3, 31-48, 2011 [Non-patent document 3] Methods Enzymol.,53,238-56,1978 Summary of the Invention [Problem to be solved by the invention]

[0010] The lactic acid level in foods can be an index for quality control, etc. At production sites of various fermented foods and drinks produced through a lactic acid fermentation process, such as sake, wine, whiskey, cheese, yogurt, sauerkraut, kuzumochi, pickles, miso, soy sauce, and lactic acid-fermented yeast extract, it is preferable to carry out appropriate lactic acid fermentation process control in order to stably produce high-quality products, and in that process control, the amount of lactic acid can be an index for production process control.

[0011] Furthermore, lactic acid production is not limited to lactic acid fermented foods and beverages; it also occurs in lactic acid fermented compositions obtained by fermenting plant materials used as cosmetic raw materials with lactic acid bacteria, and lactic acid compositions produced as chemical products. The amount of lactic acid can also be used as an indicator for the manufacturing process management and quality control of such products.

[0012] Furthermore, when an oligomer or polymer containing lactic acid, such as polylactic acid, is decomposed, the amount of lactic acid in the monomer can be used as an index for calculating the decomposition rate.

[0013] It is preferable that the measurement of lactic acid levels for the above-mentioned purposes can be carried out frequently and easily in daily life or during each production process of food and beverages, etc., and it is even more preferable that it can be carried out repeatedly using as simple a process as possible, and it is even more preferable that it can be monitored automatically and continuously.

[0014] Currently, there is no lactate measurement device that can easily and continuously monitor the amount of lactate in a sample, and the current practice is to measure samples that may contain lactate obtained from the sample through some kind of pretreatment (for example, blood taken from a patient's body by puncture) using some method each time.

[0015] Invasive sampling methods such as puncture in current measurement methods cause pain and stress to biological specimens, which hinders frequent sampling. Furthermore, even in the production process management of foods and beverages, which do not involve pain or stress, samples must be manually taken from the lactic acid-containing composition during production each time, and the lactic acid must be measured and the measurement data must be manually recorded each time. Therefore, such frequent sampling work in current lactate measurement techniques is cumbersome.

[0016] As technical proposals for continuously monitoring a person's condition, posture, etc. for a certain period of time, for example, systems have been proposed in which multiple sensors are attached to the person's body, clothing, training machines, etc., and training programs are proposed or a person's physical condition is estimated based on some biological data acquired by the sensors, or input information such as the person's age and gender, and information processing devices that launch application programs based on various data have been proposed (Patent Documents 1 to 3). A few of these proposals also include the term "lactic acid measurement." However, none of these proposals disclose a method for specifically measuring the amount of lactate as a real indicator and thereby monitoring the condition of a sample.

[0017] For example, Patent Document 1 proposes a system that can be attached to a bicycle training machine, and that analyzes heart rate information obtained from a human (rider) during training using a heart rate monitor based on the driver's resting heart rate, heart rate during training, age and gender, and bicycle riding data, and outputs a message to bring the driver's current exercise state closer to an ideal state. Patent Document 2 proposes a system that uses non-contact camera technology such as a laser speckle camera or laser Doppler blood flow system to measure blood flow in a first region where blood flow does not vary depending on the human physical condition and a second region where blood flow varies depending on the physical condition, and estimates a person's drowsiness, drunkenness, hunger level, motion sickness, stress level, depression level, etc. based on other individual information that can be additionally input and measured as needed. Patent Document 3 proposes an information processing device that uses one or more wearable sensors attached to a person's underwear, jacket, hat, glasses, earplugs, headphones, etc. to acquire a person's body temperature, acceleration, heart rate data, GPS data, altitude data, etc., and can estimate the posture of the person wearing these sensors, their movements such as whether they are asleep, awake, or walking, and monitor whether there are any signs of altitude sickness if they are climbing a mountain. However, neither proposal discloses that the acquisition of lactate data can be easily and continuously realized in the same way as heart rate measurement, blood flow measurement, and GPS data acquisition, which are already technically realized for easy and continuous data acquisition, thereby enabling lactate monitoring and evaluation of a person's condition, nor does it propose any technical solutions to make this possible.

[0018] Technical challenges in realizing simple and preferably continuous lactate monitoring include the performance issues of the enzymes used in enzymatic methods for measuring lactate.

[0019] Known enzymes that use lactate as a substrate include lactate oxidase (LOD), NAD-dependent lactate dehydrogenase (NAD-LDH), which uses nicotinamide dinucleotide (NAD) as a coenzyme, and FMN-dependent lactate dehydrogenase (FMN-LDH), which uses flavin mononucleotide (FMN) as a coenzyme. Blood lactate measurement devices using LOD are commercially available. For each measurement, a stored sensor (the electrode part for measuring blood lactate, including the LOD) is removed and inserted into a dedicated measuring device. Blood expressed from a human sample is drawn into the sensor by puncturing, and the measured value displayed on the measuring device is read. This allows the amount of lactate in the sample to be determined in a single measurement. Repeating this process allows the amount of lactate in the sample to be measured on a case-by-case basis.

[0020] However, as mentioned above, if one wishes to measure lactic acid in lactic acid-containing compositions, including interstitial fluid of living organisms, blood, sweat, food and beverages, and chemical products, stably, accurately, and preferably continuously and easily over a long period of time, known measurement enzymes are not sufficient.

[0021] LOD has the problem of insufficient thermal stability, and when producing LOD products, a significant degree of activity is lost during the drying process or storage after drying. To address this issue, a drying method using a specific stabilizer during the drying process has been proposed (Patent Document 4), but the stability improvement effect of this method is still insufficient. It should also be noted that the stability of LOD in a liquid state during an actual enzyme reaction is even lower than its stability in a powder state. For example, when considering situations in which LOD-based sensors are used in close contact with the human body for long periods of time, or situations in which lactic acid is monitored over long periods of time in food and beverage manufacturing processes that may be exposed to fermentation temperatures during lactic acid bacteria fermentation, there are concerns that existing LODs may not be sufficiently practical in terms of enzyme stability at temperatures from around room temperature to around 37°C, which is close to human body temperature.

[0022] Furthermore, LOD has the problem of generating hydrogen peroxide as a reaction product. In particular, when considering the use of lactate sensors that are attached to the skin or, in some cases, implanted subcutaneously, there are concerns that it is not desirable to use a sensor equipped with an enzyme that can continuously generate hydrogen peroxide, a type of reactive oxygen species that causes oxidative stress. Furthermore, hydrogen peroxide generated by the action of LOD adversely affects the stabilization of LOD itself. As a means of avoiding the resulting decrease in LOD activity, a lactate sensor that coexists with catalase to eliminate hydrogen peroxide has been proposed (Patent Document 5).

[0023] NAD-LDH catalyzes an enzyme reaction that does not produce hydrogen peroxide, so the problem of hydrogen peroxide generation does not occur. However, in the enzyme reaction system catalyzed by NAD-LDH, lactate and pyruvate react reversibly, which poses the problem of not being able to obtain accurate measurements for quantitative purposes, making it difficult to use in sensors.

[0024] FMN-LDH also catalyzes an enzymatic reaction that does not produce hydrogen peroxide, so there is no problem with the generation of hydrogen peroxide.Furthermore, there is no problem with reversible reactions.Of the three enzymes mentioned above, FMN-LDH is thought to be the most promising enzyme for practical use in lactate monitoring.However, the previously known FMN-LDH derived from Saccharomyces cerevisiae still has stability issues (Non-Patent Document 3).

[0025] Specifically, technical challenges in meeting market needs for simple and preferably continuous lactate monitoring in assessing the state of biological specimens, and in the production and quality control of foods and beverages, include the above-mentioned challenges with the sampling method, the cumbersome recording process associated with data processing for each measurement, and the performance of the enzyme used to measure lactate. The present invention aims to provide a device for monitoring or assessing the state of a specimen, a method for monitoring or assessing the state of a specimen, and an FMN-LDH for use therein, which can solve at least some of the above problems. [Means for solving the problem]

[0026] In view of the above problems, the present inventor conceived a device for evaluating the state of a sample that is configured to easily measure the amount of lactate in the sample non-invasively when measuring the amount of lactate in the sample, and discovered a method for evaluating the state of a sample using such a device, a system to be used therefor, and FMN-LDH that is suitably applicable to lactate measurement, thereby completing the present invention.

[0027] The present invention includes the following aspects. (1) 1. A device for assessing the condition of a specimen, comprising: an action part for allowing lactate dehydrogenase to act on the sample; a sensor for sensing the state of a specimen on which lactate dehydrogenase has been acted, the sensor being arranged so as to be able to sense the state of the specimen in the action section; Including, the device. (2) The device according to (1), further comprising an output section for outputting a signal from the sensor. (3) (2) The device according to (2), a data processing unit connected to the output of the device for processing signals from the sensor; Including, the system. (4) A program for evaluating a state of a specimen by a system including a device and a data processing unit, The device an action part for allowing lactate dehydrogenase to act on the sample; a sensor for sensing the state of the specimen on which lactate dehydrogenase has been acted, the sensor being positioned so as to be able to sense the state of the specimen at the action portion; an output unit for outputting a signal from the sensor; Including, a data processing unit connected to the output of the device and configured to process signals from the sensor; The program tells the device: a measurement process for sensing and measuring the state of the sample on which lactate dehydrogenase has acted in the action portion using a sensor and converting the state into a signal; a transfer process for transferring the signal obtained by the measurement process from the output unit to a data processing unit; A program that causes the data processing unit to execute data processing for performing predetermined processing on the signal obtained in the measurement processing. (5) A method for evaluating the state of a specimen using the device according to (1) or (2) or the system according to (3). (6) The method according to (5), wherein the specimen is a lactic acid-containing composition including body fluids of human and non-human organisms, interstitial fluid, blood, urine, tears, sweat, saliva, skin, meat, eyeballs, cornea, gastric juice, food and beverages, brewed products, chemical products, water, and soil. (7) The method according to (5) or (6), wherein the condition of the sample is a physical condition in response to exercise load, a disease state, a brewing condition of a brewed product accompanied by a change in the amount of lactic acid, a maturation / ripening degree of a food or beverage accompanied by a change in the amount of lactic acid, a lactic acid content in a chemical product produced accompanied by a change in the amount of lactic acid, or a lactic acid amount in water or soil accompanied by a change in the amount of lactic acid. (8) 1. A device for monitoring the condition of a specimen, the device comprising: (A) A flavin-dependent lactate dehydrogenase that maintains at least about 20% of its initial activity after 10 days at 37°C in solution. (B) a flavin-dependent lactate dehydrogenase that maintains at least about 20% of its initial activity after being left in solution at 37°C for at least 3 days; or (C) Flavin-dependent lactate dehydrogenase that maintains approximately 20% or more of its initial activity after being left in solution at 37°C for 15 hours or more. A device comprising an action portion for causing the action. (9) (8) A system in which the device described above further includes an output unit, and the output unit is connected to a data processing unit. (10) A method for monitoring the state of a specimen using the device according to (8) or the system according to (9). (11) Lactate dehydrogenase comprising the amino acid sequence of 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 thereto, the amino acid sequence of positions 113 to 505 in the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 70% or more identity thereto, an amino acid sequence or an amino acid sequence having 70% or more identity thereto, the amino acid sequence of positions 112 to 503 in the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 70% or more identity thereto, or the amino acid sequence of positions 102 to 499 in the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 70% or more identity thereto. (12) The lactate dehydrogenase according to (11), having the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12, or an amino acid sequence having an identity of 70% or more thereto. (13) A nucleic acid encoding the lactate dehydrogenase according to (11) or (12). (14) (13) A host cell containing the nucleic acid according to (13). (15) A method for producing lactate dehydrogenase, comprising culturing the host cell according to (14). (16) 1. A method for assessing the condition of a specimen, comprising: i) contacting a sample with the lactate dehydrogenase according to (11) or (12); and ii) measuring lactate A method comprising: [Effects of the Invention]

[0028] According to the present invention, it is possible to provide a device for evaluating the state of a specimen, a method for evaluating the state of a specimen, and FMN-LDH for use therein. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1(a) is a schematic diagram of a sensor chip 10 according to one embodiment of the present invention, and (b) to (d) are schematic diagrams showing components that constitute the sensor chip 10. [Figure 2] FIG. 2 is a diagram showing the remaining activity rates of PkLDH and ScLDH after heat treatment at each temperature. [Figure 3] 3A and 3B are diagrams showing the remaining activity rates of PkLDH, ScLDH, CaLDH, and OgLDH after storage at 37° C. for various periods of time. [Figure 4] FIG. 4 is a schematic diagram showing an example of a device and a system according to an embodiment of the present invention. [Figure 5] FIG. 5 is a flow diagram showing an example of a program according to an embodiment of the present invention. [Figure 6] FIG. 6 is a schematic diagram showing an example of a device and a system using the transmitter according to the embodiment of the present invention. [Figure 7] FIG. 7 shows the stable pH of PkLDH evaluated in Example 4. [Figure 8] FIG. 8 shows the optimum pH of PkLDH evaluated in Example 4. [Figure 9] FIG. 9 is a diagram showing the relationship between lactate concentration and PkLDH activity evaluated in Example 4. [Figure 10] FIG. 10 is a diagram showing the relationship between the lactic acid concentration and the response current value evaluated in Example 8. [Figure 11-1] FIG. 11-1 shows an alignment of the amino acid sequences of the lactate dehydrogenases of the present invention. [Figure 11-2] Figure 11-2 is a continuation of Figure 11-1. [Figure 12] FIG. 12 shows the remaining activity of PkLDH and N-terminal deleted mutants of PkLDH treated at 55° C. for 15 minutes, as evaluated in Example 11. [Figure 13] FIG. 13 shows the remaining activity of PkLDH and single-substitution mutants treated at 55° C. for 15 minutes, as evaluated in Example 11. DETAILED DESCRIPTION OF THE INVENTION

[0030] Hereinafter, embodiments of the present invention will be described in detail. Note that the following embodiments are forms for realizing the present invention, and are not intended to limit the scope of the present invention.

[0031] The specimen referred to in the present invention may be any object for which the presence or absence of lactic acid is to be measured, and may be a body fluid of a human or non-human organism, such as interstitial fluid, blood, urine, tears, sweat, saliva, skin, meat, eyeballs, cornea, or gastric juice, or a lactic acid-containing composition including a food or drink, brewed product, or chemical product that has not undergone lactic acid fermentation or has undergone lactic acid fermentation. Furthermore, it may be an environmental substance such as water or soil that contains lactic acid.

[0032] The lactate dehydrogenase (LDH) referred to in the present invention may be any enzyme that catalyzes the reversible reaction of reducing pyruvate ions to lactate ions using the reducing agent NADH. Known examples include NAD-dependent lactate dehydrogenase and FMN-dependent lactate dehydrogenase. The lactate dehydrogenase is preferably an FMN-dependent lactate dehydrogenase, more preferably an LDH having an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12, 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 thereto. The lactate dehydrogenase is even more preferably an LDH having the amino acid sequence of positions 97 to 502 in the amino acid sequence represented by SEQ ID NO: 4, 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 thereto. Alternatively, the lactate dehydrogenase may have the amino acid sequence of positions 100 to 505 in the amino acid sequence shown in SEQ ID NO: 7, 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 thereto. Alternatively, the lactate dehydrogenase may have the amino acid sequence of positions 99 to 503 in the amino acid sequence shown in SEQ ID NO: 10, 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 thereto. Alternatively, the lactate dehydrogenase may have the amino acid sequence of positions 89 to 499 in the amino acid sequence shown in SEQ ID NO: 12, 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 thereto. More preferably, the lactate dehydrogenase has the amino acid sequence of positions 110 to 502 in the amino acid sequence shown in SEQ ID NO: 4 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 thereto. Alternatively, the lactate dehydrogenase has the amino acid sequence of positions 113 to 505 in the amino acid sequence shown in SEQ ID NO: 7 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 thereto.Alternatively, it is a lactate dehydrogenase having the amino acid sequence of positions 112 to 503 in the amino acid sequence shown in SEQ ID NO: 10, 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 thereto. Alternatively, it is a lactate dehydrogenase having the amino acid sequence of positions 102 to 499 in the amino acid sequence shown in SEQ ID NO: 12, 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 thereto. The amino acid sequence of positions 97 to 502 in SEQ ID NO: 4 can also be linked to the amino acid sequence of positions 1 to 96 in SEQ ID 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, the amino acid sequence of positions 1 to 88 in SEQ ID NO: 12, 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 thereto. Similarly, the amino acid sequence of positions 1 to 96 in SEQ ID 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, the amino acid sequence of positions 1 to 88 in SEQ ID NO: 12, 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 thereto can be linked to the amino acid sequence of positions 100 to 505 in SEQ ID NO: 7, the amino acid sequence of positions 99 to 503 in SEQ ID NO: 10, or the amino acid sequence of positions 89 to 499 in SEQ ID NO: 12. Furthermore, as shown in Figure 11-1, the region containing the amino acid sequence of positions 1 to 96 in SEQ ID 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 has low sequence identity and can be said to be of little importance in the lactate dehydrogenase of the present invention.Therefore, lactate dehydrogenase containing, of the full-length amino acid sequence, the amino acid sequence of positions 97 to 502 in SEQ ID NO: 4, the amino acid sequence of positions 100 to 505 in SEQ ID NO: 7, the amino acid sequence of positions 99 to 503 in SEQ ID NO: 10, or the amino acid sequence of positions 89 to 499 in SEQ ID NO: 12, 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 thereto, is preferred. With regard to the region containing the amino acid sequence of positions 1 to 96 in SEQ ID 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 more, or 70% or more, respectively, or this region may be deleted. Alternatively, a partial deletion may be made, for example, 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 amino acids may be deleted from the N-terminal sequence. For example, amino acids 2 to 10 in SEQ ID NO: 4 may be deleted. When a deletion is made, methionine can be added to the beginning of the sequence as appropriate.

[0033] (Regarding homologous regions) Amino acid sequence identity or similarity can be calculated using programs such as maximum matching and search homology in GENETYX Ver. 11 (Genetyx) or programs such as maximum matching and multiple alignment in DNASIS Pro (Hitachi Solutions). To calculate amino acid sequence identity, two or more LDHs can be aligned and the positions of identical amino acids in the two or more LDHs can be determined. Based on this information, identical regions in the amino acid sequences can be determined. It is also possible to examine the positions of similar amino acids in two or more LDHs. For example, multiple amino acid sequences can be aligned using CLUSTALW. In this case, the algorithm Blosum62 is used, and amino acids that are determined to be similar when multiple amino acid sequences are aligned are sometimes referred to as similar amino acids. In the mutants of the present invention, amino acid substitutions may be due to substitutions between such similar amino acids. Such alignments allow for the examination of regions where the amino acid sequences are identical and positions occupied by similar amino acids for multiple amino acid sequences. Based on this information, regions of homology (conserved regions) in the amino acid sequences can be determined. For example, based on lactate dehydrogenase shown in SEQ ID NO: 4, positions 138 to 140, 150 to 154, 185 to 194, 217 to 222, 243 to 245, 271 to 278, 280 to 283, 355 to 367, 398 to 401, 403 to 406, 425 to 433, 447 to 450, and 455 to 457 may correspond to homologous regions. Furthermore, for example, based on lactate dehydrogenase shown in SEQ ID NO: 4, positions 138 to 140, 150 to 154, 185 to 188, 191 to 194, 217 to 219, 243 to 245, 271 to 275, 280 to 283, 355 to 366, 398 to 400, 405 to 410, 425 to 431, 447 to 450, and 455 to 457 may correspond to homologous regions. Furthermore, the amino acid sequence in the homologous region of the lactate dehydrogenase of the present invention has a sequence identity of 75% or more, for example, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, for example, 99% or more, to the amino acid sequence of the homologous region in SEQ ID NO: 4. Furthermore, amino acids that are particularly important for the lactate dehydrogenase of the present invention to retain its activity include histidine at position 361 and arginine at position 364 in the lactate dehydrogenase shown in SEQ ID NO: 4. By using the alignment of the amino acid sequences of each lactate dehydrogenase shown in Figures 11-1 and 11-2, it is also possible to identify positions corresponding to these important amino acid positions in lactate dehydrogenases other than SEQ ID NO: 4 (positions indicated by arrows in Figure 11-2). 11-1 and 11-2 show alignments including not only the lactate dehydrogenase shown in SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12, but also the 503 amino acid sequence (LDH-1) shown in SEQ ID NO: 46, which is the 558 amino acid sequence of lactate dehydrogenase derived from Ogataea polymorpha, with positions 2 to 51 deleted; the 506 amino acid sequence (LDH-2) shown in SEQ ID NO: 47, which is the 558 amino acid sequence of lactate dehydrogenase derived from Candida californica, with positions 2 to 86 deleted; the 502 amino acid sequence (LDH-3) shown in SEQ ID NO: 48, which is the amino acid sequence of lactate dehydrogenase derived from Chaetomium globosum; and the 499 amino acid sequence (LDH-4) shown in SEQ ID NO: 49, which is the amino acid sequence of lactate dehydrogenase derived from Madurella mycetomatis.

[0034] First, the device and system of the present invention will be described.

[0035] As shown in FIG. 4, the device for evaluating the state of a specimen of the present invention includes an action unit for causing lactate dehydrogenase to act on the specimen, and a sensor for detecting the state of the specimen after the action of lactate dehydrogenase. The sensor is positioned so as to be able to detect the state of the specimen in the action unit. The action unit of the sensor may also be encapsulated in a gel, preferably a biocompatible gel. In this case, the gel is brought into contact with the specimen, and water containing lactic acid is collected through the gel due to the effect of osmotic pressure, etc., and the lactic acid in the specimen can be detected by contacting the sensor. The biocompatible component (i.e., the gel material that constitutes the gel structure) is not particularly limited as long as it has cell adhesiveness, biocompatibility, high transparency, and hydrophilicity, and both synthetic molecules and biological molecules can be used. Examples of the synthetic molecules include hydrophilic acrylic molecules such as polyethylene glycol and acrylamide. Among these, polyethylene glycol dimethacrylate is preferred because it is a stretchable polymer and can improve not only affinity for cells and biological tissues but also stretchability, viscoelasticity, and robustness. Examples of biomolecules include polysaccharides such as sodium alginate, protein materials such as gelatin and silk, and extracellular matrices such as collagen. Among these, materials containing a large amount of protein are preferred. Therefore, as the biocompatible gel material of this embodiment, it is preferable to use silk fibroin gel as one of the biocompatible components. By using silk fibroin gel containing a large amount of protein, the surface of the gel material is highly biocompatible and can be made less cytotoxic, to the extent that enhanced cell adhesion is observed. The synthetic molecules and biomolecules described above may be either high molecular weight or low molecular weight. The molecular weight (Mw) of the polymer is not particularly limited as long as the polymer can form a gel structure. For example, a polymer with a molecular weight of about 5,000 to 1,000,000 Da can be used.

[0036] The action part for causing lactate dehydrogenase to act may be made of a material suitable for the action, such as metal, plastic, cloth, liquid, paper, nylon, etc. The sensor for detecting the state of the specimen may be integrated with or separated from the enzyme action part, and may be directly or indirectly connected thereto.

[0037] The working part contains a sample and lactate dehydrogenase of the present invention. If necessary, the working part can also contain an electron acceptor and / or a reagent for indicating changes in the electron acceptor (mediator). A sensor containing a working part (sometimes referred to as a working electrode) has a working electrode containing the LDH of the present invention, a reference electrode, and a counter electrode. The working electrode can be a carbon electrode, a gold electrode, a platinum electrode, or the like, on which the LDH of the present invention is immobilized. Additionally or separately, an electron mediator may be immobilized on the working electrode. The counter electrode can be a conventional electrode such as a platinum electrode or Pt / C. The reference electrode can be a conventional electrode such as an Ag / AgCl electrode. Immobilization methods include using a crosslinking reagent, encapsulation in a polymer matrix, coating with a dialysis membrane, photocrosslinkable polymers, conductive polymers, and redox polymers. Alternatively, the LDH may be immobilized in a polymer or adsorbed onto an electrode together with an electron mediator, such as ferrocene or its derivatives, or these may be used in combination. Typically, the LDH of the present invention is immobilized on a carbon electrode using glutaraldehyde, and then the electrode is treated with a reagent having an amine group to block the glutaraldehyde. Crosslinking reagents such as poly(ethylene glycol) diglycidyl ether can also be used instead of glutaraldehyde.

[0038] The sensor that can be used in the device of the present invention is not limited as long as it can sense the state of a sample that has been acted upon by lactate dehydrogenase.

[0039] [Sensor chip] A sensor chip may be used as the sensor. FIG. 1(a) is a schematic diagram of a sensor chip 10 according to one embodiment of the present invention, and FIGS. 1(b) to 1(d) are schematic diagrams showing components constituting the sensor chip 10. The sensor chip 10 includes two or more electrodes disposed on a substrate 11. The substrate 11 is made of an insulating material. In FIGS. 1(a) and 1(b), as an example, a working electrode 1, a counter electrode 3, and a reference electrode 5 are disposed on the substrate 11. Each electrode is electrically connected to a wiring portion 7, and the wiring portion 7 is electrically connected to a terminal 9 located on the opposite side of the electrodes in the wiring direction. The working electrode 1, the counter electrode 3, and the reference electrode 5 are disposed spaced apart from one another. Preferably, 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. Alternatively, the counter electrode 3 and the reference electrode 5 may be integrally formed.

[0040] As shown in FIGS. 1(a) and 1(c), a spacer 13 is disposed on the end of the substrate 11 parallel to the wiring portion 7, and a cover 15 is disposed to cover the working electrode 1, counter electrode 3, reference electrode 5, and spacer 13. The spacer 13 and cover 15 are made of an insulating material. The spacer 13 preferably has approximately the same thickness as the working electrode 1, counter electrode 3, and reference electrode 5, and is in close contact with the working electrode 1, counter electrode 3, and reference electrode 5. Alternatively, the spacer 13 and cover 15 may be integrally formed. The cover 15 is a protective layer that prevents deterioration of the wiring portion 7 due to exposure to the outside air and prevents short circuits due to seepage of the measurement sample.

[0041] As shown in Figures 1(a) and 1(d), a reaction layer 19 is disposed on the working electrode 1, counter electrode 3, and reference electrode 5. The reaction layer 19 provides a site for the reaction between lactate and lactate dehydrogenase. In one embodiment, the lactate dehydrogenase of the present invention may be coated, adsorbed, or immobilized on these electrodes. Preferably, the lactate dehydrogenase of the present invention is coated, adsorbed, or immobilized on the working electrode. In another embodiment, a mediator may be coated, adsorbed, or immobilized on the electrode together with lactate dehydrogenase. Examples of electrodes that can be used include carbon electrodes and metal electrodes such as platinum, gold, silver, nickel, and palladium. For carbon electrodes, examples of materials include pyrolytic graphitic carbon (PG), glassy carbon (GC), carbon paste, and plastic-formed carbon (PFC). The measurement system may be a two-electrode or three-electrode system, and the enzyme may be immobilized on the working electrode, for example. Examples of reference electrodes include a standard hydrogen electrode, a reversible hydrogen electrode, a silver-silver chloride electrode (Ag / AgCl), a palladium-hydrogen electrode, and a saturated calomel electrode. From the viewpoints of stability and reproducibility, it is preferable to use Ag / AgCl.

[0042] Furthermore, printed electrodes can be used to reduce the amount of solution required for measurement. In this case, the electrodes are preferably formed on a base material 11 made of an insulating substrate. Specifically, it is desirable to form the electrodes on the base material 11 by photolithography or a printing technique such as screen printing, gravure printing, or flexographic printing. In addition, examples of materials for the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, but it is more preferable to use a material that is highly resistant to various solvents and chemicals.

[0043] For example, when lactate dehydrogenase acts on lactic acid in a sample in an application area containing potassium ferricyanide as an electron acceptor, the potassium ferricyanide is converted to potassium ferrocyanide. Potassium ferricyanide absorbs light at a wavelength of 420 nm, so the state of lactic acid in the sample can be detected by measuring the absorbance at 420 nm using a spectrophotometer. Furthermore, the oxidation-reduction reaction that occurs when lactate dehydrogenase acts on lactic acid and then potassium ferricyanide is added can be measured electrochemically using a sensor. A specific example of a sensor that can be used for electrochemical measurements is the Lactate Pro 2 sensor, model LT-1730, manufactured by Arkray. Other electron acceptors that can be used include quinones, phenazines (e.g., phenazine methosulfate), viologens, cytochromes (e.g., cytochrome b, cytochrome c), phenoxazines, phenothiazines, ferricyanides, ferredoxins, ferrocene, osmium complexes, ruthenium complexes, phenylenediamines and derivatives thereof, etc. The lactate dehydrogenase of the present invention does not use oxygen as an electron acceptor.

[0044] For example, lactate concentration can be measured as follows: A buffer solution is placed in a thermostatic cell and maintained at a constant temperature. An electrode with immobilized LDH and an electron acceptor (e.g., a quinone-added polymer) is used as the working electrode, and a counter electrode (e.g., a platinum electrode) and a reference electrode (e.g., 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 current is measured. The lactate concentration in the sample can be calculated according to a calibration curve created using lactate solutions of standard concentrations.

[0045] As a specific example, 4U LDH of the present invention was immobilized on a glassy carbon (GC) electrode, and the response current versus lactate concentration was measured. 1.8 ml of 50 mM potassium phosphate buffer (pH 7.5) and 0.2 ml of 1 M potassium hexacyanoferrate (III) (potassium ferricyanide) aqueous solution were added to an electrolysis cell. The GC electrode was connected to a potentiostat BAS100B / W (manufactured by BAS), the solution was stirred at 37°C, and +500 mV was applied relative to a silver-silver chloride (saturated KCl) reference electrode. 1 M L-lactic acid solution was added to the system to final concentrations of 1, 2, 3, 4, 5, 10, 20, 30, 40, and 50 mM, and the steady-state current was measured after each addition. This current was plotted against known lactate concentrations (1, 2, 3, 4, 5, 10, 20, 30, 40, and 50 mM) to create a calibration curve. This makes it possible to quantify lactic acid using an enzyme-immobilized electrode that uses the FMN-LDH of the present invention.

[0046] In one embodiment, 0.01 U to 1000 U, 0.1 U to 1000 U, more preferably 0.5 U to 700 U, more preferably 0.5 U to 500 U, more preferably 1 U to 300 U, more preferably 1 U to 100 U of the FMN-LDH solution of the present invention is applied to or immobilized on the lactate sensor chip of the present invention.

[0047] In one embodiment, a continuous lactate monitoring device is provided that includes a lactate sensor of the present invention. Also, in another embodiment, a method for continuously monitoring lactate for 14 days using the LDH of the present invention is provided. The measurement period can be set to suit the purpose, such as 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.

[0048] In some embodiments, continuous lactate monitoring can be performed with or without recalibration, for example, recalibration can be performed every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0049] The lactate dehydrogenase of the present invention is sufficiently thermostable, which allows for convenient and preferably continuous lactate monitoring.

[0050] The present invention also relates to a method for measuring lactate, comprising: i) contacting a sample with lactate dehydrogenase; and ii) measuring lactate. Specifically, the lactate dehydrogenase used in step i) is an FMN-dependent lactate dehydrogenase, more preferably a lactate dehydrogenase having an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12, 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 thereto. More specifically, the lactate dehydrogenase used in step i) is 0.01 U to 1000 U, 0.1 U to 1000 U, more preferably 0.5 U to 700 U, more preferably 0.5 U to 500 U, more preferably 1 U to 300 U, or more preferably 1 U to 100 U. Furthermore, the lactate dehydrogenase in step i) is a flavin-dependent lactate dehydrogenase that 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 its initial activity when left in a solution at about 30, 35°C, or 37°C for 10 days; or a flavin-dependent lactate dehydrogenase that 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 its initial activity when left in a solution at about 30, 35°C, or 37°C for 3 days or more. The flavin-dependent lactate dehydrogenase may be a flavin-dependent lactate dehydrogenase that 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 its initial activity after being left in solution (C) at about 30, 35, or 37°C for 15 hours or more. The temperature at which the lactate dehydrogenase is contacted with the sample in step i) may be 20 to 60°C, preferably 30 to 55°C, and more preferably 30 to 40°C. The pH at which the lactate dehydrogenase is contacted with the sample in step i) may be 3 to 10, preferably 5 to 9, and more preferably 6 to 8. Furthermore, the step of measuring lactic acid in step ii) may be a single measurement or continuous measurement, and the measurement period in step ii) may be 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.

[0051] As shown in Figure 4, the device for evaluating the state of a specimen according to the present invention may further include an output unit. The output unit can output a signal from the sensor to the outside. The output unit may also be connected to a data processing unit to form a system. That is, the system includes the device and a data processing unit. The data processing unit can process the signal from the sensor.

[0052] In this specification, the term "data processing unit" does not only refer to a single device such as a computer, but also includes the concept of a network for processing information in a form that links other devices connected by communication lines such as a local network or the Internet.

[0053] The connection between the output unit and the data processing unit means an electrical or electronic connection so that a signal from the sensor can be output from the output unit and input to the data processing unit. For example, the physical connection method can be a wired connection or a wireless connection such as a wireless LAN.

[0054] The data processing unit may include a control unit, which may be, for example, a central processing unit (CPU), and the programs described below may be loaded into the control unit.

[0055] The data processing unit may include a storage unit. In this specification, the term "storage unit" refers to a device, such as a memory or a hard disk, that can write input information so that it can be read. The storage unit may be located inside a specific device. Alternatively, the storage unit may exist inside another device connected via a communication line such as a local network or the Internet. By including a storage unit, signals measured by the sensors can be stored, and the results processed by the data processing unit can also be stored.

[0056] The data processing unit has a display unit for displaying the results processed by the data processing unit, is connected to the display unit, or can transfer the results to the display unit. The display unit can be any known display means, such as a display, a printer, an audio output device, or an output to the outside via a communication line such as a local network or the Internet. In other words, the display unit not only means that the data processing unit physically has a display device, but also means that the data processing unit can transfer data (results) to another information processing device and display the results on that other information processing device.

[0057] The data processing unit can also be equipped with a transmitter, allowing wireless transmission and reception to and from a separate measuring device equipped with a display and memory unit. The transmitter measures the current value measured by the device using a measuring unit 100 and sends the current value to a control unit 101. The control unit 101 measures the temperature near the device using a temperature sensor 102, corrects for temperature, and then calculates the lactate concentration from the current value. The control unit 101 performs this calculation of the lactate concentration at predetermined sampling time intervals.

[0058] The control unit 101 then performs an integrated average of the calculated lactate concentration at predetermined recording time intervals and records the average in the memory unit 103. The control unit 101 transmits the value stored in the memory unit 103 to the measuring device 106 via the communication unit 104 in response to an instruction from the measuring device 106.

[0059] The transmitter in this embodiment has a built-in battery 105. When the remaining battery power of this battery 105 becomes insufficient, the transmitter is configured to be discarded. The remaining battery power of battery 105 is monitored through measuring device 106. When a device is replaced, measuring device 106 checks whether the remaining battery power of battery 105 in the transmitter is sufficient until the next device replacement. If the remaining power is insufficient, the measuring device 106 instructs the user to replace the transmitter and makes the transmitter unusable.

[0060] The system of the present invention can evaluate the condition of a specimen, such as the physical condition in response to exercise load, disease state, the brewing condition of a brewed product accompanied by a change in lactic acid content, the degree of maturation and ripening of a food or beverage accompanied by a change in lactic acid content, the lactic acid content ratio in chemical product manufacturing accompanied by a change in lactic acid content, and the condition of water or soil accompanied by a change in lactic acid content.

[0061] The lactate dehydrogenase used in the device of the present invention is (A) a flavin-dependent lactate dehydrogenase that 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 its initial activity when left in a solution at about 30, 35°C, or 37°C for 10 days; or (B) a flavin-dependent lactate dehydrogenase that maintains about 20% or more, 30% or more of its initial activity when left in a solution at about 30, 35°C, or 37°C for 3 days or more. Alternatively, the lactate dehydrogenase may be a flavin-dependent lactate dehydrogenase that maintains at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of its initial activity after being left in solution (C) at about 30° C., 35° C., or 37° C. for 15 hours or more. The lactate dehydrogenase used in the device of the present invention may be a flavin-dependent lactate dehydrogenase that maintains at least 70% of its initial activity even after 15 hours from the time of use.

[0062] The device may further include an output section which may be connected to a data processing unit to form a system.

[0063] The device and system of the present invention are highly advantageous in that evaluation or monitoring can be performed non-invasively on the sample, allowing direct measurement without a sampling step, i.e., they can be left attached and inserted for long-term use, rather than being a disposable measurement where a test strip is inserted into a sensor and a button is pressed.

[0064] Next, the program of the present invention will be described. The program of the present invention is a program for evaluating the state of a specimen by a system including a device and a data processing unit.

[0065] In this specification, the term "program" refers to 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 may also be a mobile application (app).

[0066] The program of the present invention is a program for operating the system as shown in Figure 4. The system includes a device and a data processing unit.

[0067] The device includes an acting unit, a sensor, and an output unit. The acting unit, sensor, and output unit are as described above. That is, the acting unit is configured to act on the specimen with lactate dehydrogenase. The sensor is configured to be positioned so as to be able to sense the state of the specimen acted on by lactate dehydrogenase in the acting unit. The output unit is configured to be able to output a signal from the sensor.

[0068] The data processing unit is connected to the output of the device and configured to process signals from the sensor. The data processing unit may 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.

[0069] The program of the present invention causes the system to execute a measurement process S01, a transfer process S02, and a data process S03. The program of the present invention can also cause the system to execute a result display process S04.

[0070] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the sample that has been acted on by lactate dehydrogenase in the action portion, and converts it into a signal.

[0071] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0072] In data processing S03, the program causes the system to execute processing so that the data processing unit performs predetermined processing on the signals obtained in the measurement processing.

[0073] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit.

[0074] Specifically, this program can be used as a program for the following purposes:

[0075] The program of the present invention can be used as a program for food and beverage applications. Specifically, it is as follows.

[0076] (Embodiment 1 of a program for food and beverages) The program of this embodiment may be a program for managing the freshness of beef. That is, in this embodiment, the specimen is beef.

[0077] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the beef on which lactate dehydrogenase has acted in the action portion, and converts the state into a signal.

[0078] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0079] In data processing S03, the program causes the system to execute a predetermined process on the signal obtained by the measurement process in the data processing unit. The predetermined process in this embodiment is a process for converting the data into a data format suitable for beef freshness management.

[0080] In the result display process S04, the program causes the system to execute processing so that a predetermined display unit displays the results processed by the data processing unit. As a result, data in a data format suitable for beef freshness management can be displayed on the display unit.

[0081] In this embodiment, the expiration date of beef can be predicted by monitoring temperature and humidity in addition to the lactic acid level. Furthermore, by inputting the part of the meat as a parameter, the value of the meat can be calculated and used as an indicator of the selling price. Furthermore, in this embodiment, it is possible to suggest the maturity of the meat according to the dish. In this embodiment, the operating part is directly attached to the beef, and the condition of the meat can be sensed by the sensor.

[0082] Conventionally, beef freshness management has mainly been based on temperature. The lactate dehydrogenase of the present invention has sufficient thermostability, making it possible to easily and continuously monitor lactic acid. Therefore, the program of this embodiment allows freshness management using lactic acid as an indicator. This allows appropriate transportation and storage conditions for beef. Use of the program of this embodiment also serves as an objective indicator of the value of beef, supporting consumer decision-making.

[0083] (Embodiment 2 of the program for food and beverages) The program of this embodiment can be a program for managing and controlling indicators of beef aging, i.e., in this embodiment, the specimen is beef.

[0084] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the beef on which lactate dehydrogenase has acted in the action portion, and converts the state into a signal.

[0085] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0086] In data processing S03, the program causes the system to execute a predetermined process on the signals obtained in the measurement process in the data processing unit. The predetermined process in this embodiment is a process for converting the data into a data format suitable for managing and controlling the aging of beef.

[0087] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit. As a result, data in a data format suitable for managing and controlling indicators for beef aging can be displayed on the display unit.

[0088] In this embodiment, the expiration date of beef can be predicted by monitoring temperature, humidity, and pH in addition to the lactic acid value. Furthermore, by inputting the part of the meat as a parameter, the value of the meat can be calculated and used as an indicator of the selling price. Furthermore, in this embodiment, it is possible to suggest the maturity of the meat according to the dish. In this embodiment, the operating part is directly attached to the beef, and the condition of the meat can be sensed by the sensor.

[0089] Conventionally, beef aging control has mainly been based on temperature. The lactate dehydrogenase of the present invention has sufficient thermostability, allowing for simple and continuous monitoring of lactic acid. Therefore, the program of this embodiment allows for freshness control using lactic acid as an indicator. This allows for appropriate transportation and storage conditions for beef. Use of the program of this embodiment also provides an objective indicator of the value of beef, supporting consumer decision-making.

[0090] (Embodiment 3 of the program for food and beverages) The program of this embodiment may be a program for managing the freshness of fish such as tuna, etc. That is, in this embodiment, the specimen is a fish such as tuna.

[0091] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the fish, such as tuna, on which lactate dehydrogenase has been made to act in the action portion, and converts the state into a signal.

[0092] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0093] In data processing S03, the program causes the system to execute a predetermined process on the signal obtained in the measurement process in the data processing unit. The predetermined process in this embodiment is a process for converting the data into a data format suitable for freshness management of fish such as tuna.

[0094] In the result display process S04, the program causes the system to execute processing so that a predetermined display unit displays the results processed by the data processing unit. As a result, data in a data format suitable for managing the freshness of fish such as tuna can be displayed on the display unit.

[0095] In this embodiment, the freshness and expiration date of fish such as tuna can be predicted by monitoring temperature, humidity, and K value in addition to the lactic acid value. In this embodiment, the value of the fish can be calculated by inputting the type of fish as a parameter and used as an indicator of the selling price. In addition, this embodiment can suggest the freshness of the fish according to the dish. In this embodiment, for example, the operating part can be attached directly to the eye of the fish, and the condition of the fish such as tuna can be sensed by the sensor.

[0096] Traditionally, the freshness of fish such as tuna has mainly been controlled by temperature and K value (see "Sea - Nature and Culture," Tokai University Bulletin of the Faculty of Marine Science, Vol. 4, No. 2, pp. 31-46 (2006)). However, the aforementioned literature states that even when a fish is judged to be high in freshness due to a low K value, its freshness may be low as a result of acid denaturation caused by an increase in the amount of lactic acid in the muscle. The lactate dehydrogenase of the present invention has sufficient thermostability, allowing for simple and continuous monitoring of lactic acid. Therefore, the program of this embodiment enables freshness control using lactic acid as an indicator. It is also possible to control the amount of lactic acid in combination with the K value. This allows for appropriate transportation and storage conditions for fish such as tuna. The program of this embodiment can also serve as an objective indicator of the value of fish such as tuna, thereby assisting consumers and wholesalers in making decisions.

[0097] (Embodiment 4 of the program for food and beverages) The program of this embodiment can be a program for managing and controlling the aging of fish such as tuna, etc. That is, in this embodiment, the specimen is a fish such as tuna.

[0098] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the fish, such as tuna, on which lactate dehydrogenase has been made to act in the action portion, and converts the state into a signal.

[0099] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0100] In data processing S03, the program causes the system to execute a predetermined process on the signal obtained in the measurement process in the data processing unit. The predetermined process in this embodiment is a process for converting the data into a data format suitable for managing and controlling the maturation of fish such as tuna.

[0101] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit. As a result, data in a data format suitable for managing and controlling the maturation of fish such as tuna can be displayed on the display unit.

[0102] In this embodiment, the appropriate aging state of fish such as tuna can be predicted by monitoring temperature and humidity in addition to lactic acid levels. In this embodiment, the value of the aged fish can be calculated by inputting the type of fish as a parameter, and this can be used as an indicator of the selling price. In addition, this embodiment can suggest the aging level of the fish according to the dish. In this embodiment, for example, the operating part can be attached directly to the eye of the fish, and the condition of the fish such as tuna can be sensed by the sensor.

[0103] Conventionally, the aging control of fish such as tuna has mainly been controlled by the temperature, aging time, and the temperature of the warm saltwater used for thawing. The lactate dehydrogenase of the present invention has sufficient thermostability, allowing for simple and continuous monitoring of lactic acid. Therefore, the program of this embodiment allows for aging control using lactic acid as an indicator. This allows for appropriate aging conditions for fish such as tuna. Use of the program of this embodiment also serves as an objective indicator of the value of fish such as tuna, thereby assisting consumers and wholesalers in making decisions.

[0104] Other Embodiments of Programs for Food and Beverages The program of this embodiment can be a program for foods and beverages other than those mentioned above, for example, a program for managing malolactic fermentation of wine, fermentation management of bread such as sake and bagels, kuzumochi (kuzu mochi), lactic acid fermentation management of whiskey, fermentation management of tea, food safety indicators, etc. According to the program of this embodiment, fermentation management of brewed alcoholic beverages such as wine and sake, which previously relied on experience and intuition, can be performed using indicators such as pH value and dissolved oxygen value in addition to lactic acid value, thereby achieving appropriate temperature control and fermentation period.

[0105] The program of the present invention can be used as a program for animal use. Specifically, it is as follows.

[0106] (Embodiment 1 of the program for animals) The program of this embodiment may be a program for managing racehorses, that is, in this embodiment, the specimen is a racehorse.

[0107] In the measurement process S01, the program causes the system to execute a process such that the condition of the racehorse on which lactate dehydrogenase has been made to act in the action portion is sensed and measured by the sensor, and converted into a signal.

[0108] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0109] In data processing S03, the program causes the system to execute a predetermined process on the signals obtained in the measurement process. In this embodiment, the predetermined process is a process for converting the data into a data format suitable for managing racehorses.

[0110] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit, thereby enabling the display unit to display data in a data format suitable for managing racehorses.

[0111] In the training of racehorses, training programs are designed using blood lactate as an indicator. It is known that the amount of lactate changes depending on the exercise intensity, and if the balance between oxygen demand and supply during exercise is maintained, blood lactate does not accumulate, but rises when the balance of demand and supply is disrupted (JRA training facility diary, training load using lactate as an indicator). Therefore, this embodiment can evaluate the suitability of the training load for a racehorse. It is also possible to propose a horse training method using the obtained lactate value.

[0112] The lactate dehydrogenase of the present invention is sufficiently thermostable, allowing for simple and continuous lactate monitoring. Therefore, the program of this embodiment can be used to manage the training and health of racehorses. By attaching the sensor directly to the horse or to the surface of tack, such as a saddle, that comes into contact with the skin, and monitoring lactate in the blood, skin, interstitial fluid, sweat, etc., it is possible to reduce the burden on the trainer and the stress on the horse, which would otherwise be required to take blood samples from the horse for measurement and analysis after each training session.

[0113] (Embodiment 2 of the program for animals) The program of this embodiment may be a program for measuring the fatigue level of a racehorse, that is, in this embodiment, the subject is a racehorse.

[0114] In the measurement process S01, the program causes the system to execute a process such that the condition of the racehorse on which lactate dehydrogenase has been made to act in the action portion is sensed and measured by the sensor, and converted into a signal.

[0115] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0116] In data processing S03, the program causes the system to execute a predetermined process on the signals obtained in the measurement process. In this embodiment, the predetermined process is a process for converting the data into a format suitable for measuring the fatigue level of a racehorse.

[0117] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit, thereby enabling the display unit to display data in a data format suitable for measuring the fatigue level of a racehorse.

[0118] In this embodiment, the amount of lactic acid changes depending on the fatigue level of the racehorse, so the fatigue level of the racehorse can be predicted from the change in the amount of lactic acid.

[0119] The lactate dehydrogenase of the present invention has sufficient thermostability, allowing for simple and continuous monitoring of lactate levels. Therefore, the program of this embodiment can provide an index of muscle fatigue in a racehorse, allowing even beginners to determine the muscle fatigue level of a racehorse.

[0120] (Embodiment 3 of the program for animals) The program of this embodiment can be a program for managing the health of cattle, managing the evaluation at the time of shipping, or controlling the meat quality of beef cattle. That is, in this embodiment, the specimen is a cattle.

[0121] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the cow on which lactate dehydrogenase has been acted in the action portion, and converts the state into a signal.

[0122] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0123] In data processing S03, the program causes the system to execute a predetermined process on the signals obtained in the measurement process in the data processing unit. The predetermined process in this embodiment is a process for converting the data into a data format suitable for cattle health management, shipping assessment management, or beef quality control.

[0124] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit. As a result, data in a data format suitable for cattle health management, shipping assessment management, or beef cattle meat quality control can be displayed on the display unit.

[0125] In this embodiment, the ear is used as the acting part, lactate dehydrogenase is activated, and a sensor is used to sense and measure the condition of the cow. Blood vessels run through the ear, and by installing the acting part and sensor in an ear tag, it is possible to measure ear blood as a specific sample. Sweat or skin can also be used as a sample. In this embodiment, the stomach can also be used as the acting part. For example, by measuring the amount of lactic acid in the blood, it is possible to prevent the administration of lactic acid bacteria or grains containing lactic acid before acute acidosis occurs.

[0126] In this embodiment, the swallowable sensor can also be used for health management, shipping assessment management, or meat quality control of beef cattle. That is, the active part is a digestive organ such as the stomach, and lactate dehydrogenase is activated, allowing the sensor to sense and measure the condition of the cattle. In this embodiment, the amount of lactate assimilation by microorganisms present in the stomach changes depending on the amount of lactate in the cattle, resulting in the generation of volatile fatty acids depending on the amount of lactate assimilation. The cattle can then absorb these volatile fatty acids and use them as nutrients (carbon sources). Therefore, the efficiency of nutrient absorption can be predicted from changes in the amount of lactate, allowing for prediction of the cattle's health status, shipping assessment management, or meat quality control of beef cattle.

[0127] The lactate dehydrogenase of the present invention has sufficient thermostability, allowing for simple and continuous lactate monitoring, and therefore the program of this embodiment can be used for cattle health management, shipping assessment management, and meat quality control of beef cattle.

[0128] (Embodiment 4 of the program for animals) The program of this embodiment can be a program for managing the health of dairy cows, that is, in this embodiment, the specimen is a dairy cow.

[0129] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the dairy cow on which lactate dehydrogenase has been acted on in the action part, and converts the state into a signal.

[0130] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0131] In data processing S03, the program causes the system to execute a predetermined process on the signals obtained in the measurement process in the data processing unit. The predetermined process in this embodiment is a process for converting the data into a data format suitable for health management of dairy cows.

[0132] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit. As a result, data in a data format suitable for managing the health of dairy cows can be displayed on the display unit.

[0133] In this embodiment, the ear is used as the acting part, lactate dehydrogenase is activated, and a sensor is used to sense and measure the condition of the dairy cow. Blood vessels run through the ear, and by installing the acting part and sensor in an ear tag, it is possible to measure ear blood as a specific sample. Sweat or skin can also be used as a sample. In this embodiment, the stomach can also be used as the acting part. For example, by measuring the amount of lactic acid in the blood, it is possible to prevent the administration of lactic acid bacteria or grains containing lactic acid before acute acidosis occurs.

[0134] The lactate dehydrogenase of the present invention has sufficient thermostability, allowing for simple and continuous lactate monitoring. Therefore, the program of this embodiment can be used to manage the health of dairy cows. Furthermore, by analyzing the component values ​​of milk, the value of the resulting dairy product can be evaluated from the perspective of the final product and the health of the dairy cow, which can assist consumers and wholesalers in making decisions.

[0135] The program of the present invention can be used as a program for plant applications. Specifically, it is as follows.

[0136] (Embodiment 1 of the program for plants) The program of this embodiment may be a program for plant management. That is, in this embodiment, the specimen is a plant or soil. Specific examples of specimens include plant leaves, roots, sap, fruit, seeds, etc., and may also be potted plants.

[0137] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the plant on which lactate dehydrogenase has acted in the action portion, and converts the state into a signal.

[0138] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0139] In data processing S03, the program causes the system to execute a predetermined process on the signals obtained in the measurement process in the data processing unit. The predetermined process in this embodiment is a process for converting the data into a data format suitable for plant management.

[0140] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit, thereby enabling the display unit to display data in a data format suitable for plant management.

[0141] The presence of lactic acid in soil promotes water absorption by plants. Therefore, the water absorption of plants changes depending on the amount of lactic acid in the soil. Therefore, in this embodiment, the water absorption state of plants can be evaluated.

[0142] The lactate dehydrogenase of the present invention has sufficient thermostability, allowing for simple and continuous monitoring of lactate levels. Therefore, the program of this embodiment can suggest the timing of adding nutrients and watering potted plants. Furthermore, a low pH can suppress bacterial growth in plants. Therefore, measuring pH along with the amount of lactic acid can suppress bacterial growth.

[0143] (Embodiment 2 of the program for plants) The program of this embodiment can be a program for indexing soil management and hydroponic cultivation management. That is, in this embodiment, the specimen is soil or water.

[0144] In the measurement process S01, the program causes the system to execute a process such that the sensor senses and measures the state of the soil on which lactate dehydrogenase has been acted in the action portion, and converts the state into a signal.

[0145] In the transfer process S02, the program causes the system to execute a process to transfer the signal obtained in the measurement process from the output section to the data processing unit.

[0146] In data processing S03, the program causes the system to execute a predetermined process on the signals obtained in the measurement process using the data processing unit. The predetermined process in this embodiment is a process for converting the data into a data format suitable for use as an index for soil management and hydroponic cultivation management.

[0147] In the result display process S04, the program causes the system to execute processing so that the results processed by the data processing unit are displayed on a predetermined display unit. As a result, data in a data format suitable for indicators of soil management and hydroponic cultivation management can be displayed on the display unit.

[0148] The presence of lactic acid in soil and hydroponic water promotes water absorption by plants. Therefore, the water absorption of plants changes depending on the amount of lactic acid in the soil and hydroponic water. Therefore, in this embodiment, the state of water absorption of plants in soil and hydroponic water can be evaluated.

[0149] The lactate dehydrogenase of the present invention has sufficient thermostability, allowing for simple and continuous monitoring of lactate levels. Therefore, the program of this embodiment can suggest the timing of adding nutrients to plants, watering, and applying top dressing. Furthermore, a low pH can suppress the growth of unwanted bacteria in plants. Therefore, measuring the pH along with the amount of lactic acid makes it possible to suppress the growth of unwanted bacteria.

[0150] Traditionally, plant management has relied on non-data-based methods, such as applying fertilizer according to the season or adding water or nutrients when the plant's condition deteriorates. The lactate dehydrogenase of the present invention is sufficiently thermostable, enabling simple and continuous lactic acid monitoring. A system can be provided that calculates the amount of sugars and lactic acid that can be assimilated by lactic acid bacteria to be added to enhance the soil's antibacterial properties when the lactic acid level in the soil decreases. Conversely, a system can be provided that calculates the type and amount of alkaline fertilizer to be added when the lactic acid level increases. Additionally, by measuring pH, electrical conductivity, sunlight, temperature, humidity, and amino acid content, accurate plant breeding programs can be developed and provided. Furthermore, analyzing the component values ​​(e.g., sugar content, acidity) of final products such as tomatoes allows for an objective evaluation of the value of the resulting final product from a perspective invisible to consumers, such as the cultivation history of the final product and plant, thereby supporting the decision-making of consumers and wholesalers.

[0151] The present invention also relates to lactate dehydrogenases and nucleic acids encoding same, which have an amino acid sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identical to the amino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:12. In one embodiment, the present invention provides a DNA having a nucleotide sequence that has 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 sequence identity to the nucleotide sequence set forth in SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, or SEQ ID NO:13, and encoding a protein having lactic acid activity.

[0152] (LDH gene) To obtain a gene encoding LDH, a commonly used gene cloning method is used. For example, chromosomal DNA or mRNA can be extracted from microbial cells or various cells capable of producing LDH by standard methods, such as the method described in *Current Protocols in Molecular Biology* (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. The chromosomal DNA or cDNA thus obtained can be used to prepare a chromosomal DNA or cDNA library. Next, an appropriate probe DNA is synthesized based on the amino acid sequence of the LDH, and this is used to select the LDH gene from a chromosomal DNA or cDNA library. Alternatively, appropriate primer DNA is prepared based on the amino acid sequence, and DNA containing the desired gene fragment encoding LDH is amplified by an appropriate polymerase chain reaction (PCR) such as the 5'RACE method or the 3'RACE method. These DNA fragments are then linked to obtain DNA containing the full-length LDH gene of interest.

[0153] These LDH genes may be ligated or inserted into various vectors, or integrated into the chromosome or genome. When using vectors, commercially available kits such as the TA Cloning Kit (Invitrogen) or the In-Fusion HD Cloning Kit (Clontech) can be used for cloning into the vector; commercially available plasmid vector DNAs such as pUC119 (Takara Bio), pUC18 (Takara Bio), pBR322 (Takara Bio), pBluescript SK+ (Stratagene), and pYES2 / CT (Invitrogen); and commercially available bacteriophage vector DNAs such as λEMBL3 (Stratagene). The recombinant DNA is used to transform a host organism, such as Escherichia coli, preferably the JM109 strain (Takara Bio) or the DH5α strain (Takara Bio). The recombinant DNA contained in the resulting transformant is purified using a QIAGEN Plasmid Mini Kit (Qiagen) or similar. For mass production, it is preferred to use a host organism transformed with recombinant DNA containing the LDH gene, such as Escherichia coli, yeast, fungal cells, or filamentous fungi, as the host for producing LDH.

[0154] The present invention also relates to a host cell containing the nucleic acid and a method for producing lactate dehydrogenase, which comprises culturing the host cell. To produce LDH using the strain capable of producing stable LDH obtained as described above, the strain may be cultured by a conventional solid culture method, but it is preferable to use a liquid culture method whenever possible. The culture medium for culturing the above-mentioned strains may contain, for example, one or more nitrogen sources such as yeast extract, tryptone, peptone, meat extract, corn steep liquor, or soybean or wheat bran infusion, to which one or more inorganic salts such as sodium chloride, potassium diphosphate, potassium diphosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, or manganese sulfate have been added, and which may further contain carbohydrate raw materials, vitamins, etc. as needed. The initial pH of the medium is preferably adjusted to pH 7 to 9. The culture can be carried out under any conditions, for example, at a culture temperature of 20 to 42°C, preferably around 30°C, for 4 to 24 hours, more preferably around 30°C, for 8 to 16 hours, using submerged culture with aeration and agitation, shaking culture, static culture, or the like. After the culture is completed, LDH can be collected from the culture by conventional enzyme collection methods. For example, the cells can be subjected to ultrasonic disruption, grinding, or the like, or the enzyme can be extracted using a lytic enzyme such as lysozyme, or the cells can be lysed by shaking or standing in the presence of toluene, etc., to excrete the enzyme from the cells. The solution can then be filtered, centrifuged, or the like to remove solids, and nucleic acids can be removed, if necessary, with streptomycin sulfate, protamine sulfate, manganese sulfate, or the like. After this, ammonium sulfate, alcohol, acetone, or the like is added to fractionate the solution, and the precipitate is collected to obtain crude LDH enzyme.

[0155] To obtain a purified LDH enzyme preparation from the above-mentioned crude LDH enzyme, a purified LDH enzyme preparation can be obtained by appropriately selecting or combining any of the following methods: gel filtration using Sephadex, Superdex, Ultrogel, or the like; adsorption-elution using an ion exchanger; electrophoresis using polyacrylamide gel, or the like; adsorption-elution using hydroxyapatite; sedimentation methods such as sucrose density gradient centrifugation; affinity chromatography; fractionation using a molecular sieve membrane or hollow fiber membrane, or the like. In this way, LDH with the desired improved stability can be obtained. The lactate dehydrogenase of the present invention is (A) a flavin-dependent lactate dehydrogenase that 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 its initial activity when left in a solution at about 30 to 37°C for 10 days; (B) a flavin-dependent lactate dehydrogenase that maintains about 20% or more, 30% or more, 40% or more, or 95% or more of its initial activity when left in a solution at about 30 to 37°C for 3 days or more; The lactate dehydrogenase of the present invention may be a flavin-dependent lactate dehydrogenase that maintains at least 50%, 60%, 70%, 80%, 90%, or 95% of its initial activity, or a flavin-dependent lactate dehydrogenase that maintains at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of its initial activity after 15 hours or more at about 30 to 37° C. in solution (C). Furthermore, the lactate dehydrogenase of the present invention may be a flavin-dependent lactate dehydrogenase that maintains at least 70% of its initial activity even after 15 hours from the time of use. Examples of buffer materials (buffer solutions) that can be used in the LDH reaction solution include borate buffers containing boric acid and / or salts thereof, Tris-HCl buffers, phosphate buffers containing phosphoric acid and / or salts thereof, such as potassium phosphate buffers or sodium phosphate buffers, organic acid buffers containing organic acid buffers and / or salts thereof, such as tricarboxylic acid buffers containing tricarboxylic acid buffers and / or salts thereof, citrate buffers containing citric acid and / or salts thereof, monocarboxylic acid buffers containing monocarboxylic acid buffers and / or salts thereof, and acetate buffers containing acetate buffers and / or salts thereof. Furthermore, examples of buffers that can be used in the kit of the present invention include ACES (N-(2-acetamido)-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-aminoethanesulfonic acid), EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), HEPPSO (N-(hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid), MES (2-( Examples of suitable buffers include Good's buffers containing N-morpholinoethanesulfonic acid, MOPS (3-(N-morpholino)propanesulfonic acid), MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid)), TAPS (N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), and tricine (N-tris(hydroxymethyl)methylglycine), and / or salts thereof. The temperature at which LDH is reacted may be 20 to 60°C, preferably 30 to 55°C.The pH at which LDH is reacted may be within the range of 3 to 10, and preferably within the range of 6 to 10. [Example]

[0156] The present invention will be described in more detail below with reference to examples. However, the following examples are intended for illustrative purposes only and are not intended to limit the technical scope of the present invention in any way. Unless otherwise specified, reagents are commercially available or can be obtained or prepared according to conventional techniques or procedures described in known literature in the art.

[0157] In the present invention, evaluation of the stability of FMN-LDH after heat treatment and evaluation of stability under various storage conditions were carried out according to the methods of the following test examples unless otherwise specified.

[0158] Example 1 (1) Preparation of recombinant plasmids pKK223-3-ScLDH DNA and pKK223-3-PkLDH DNA As described in Biochem. J. 258, 255-259 (1989), a 1521-bp gene (including the stop codon TAA) shown in SEQ ID NO: 2 was obtained as cDNA by standard PCR of the gene fragment, encoding the 506 amino acids shown in SEQ ID NO: 1, in which the amino acids at positions 2 to 85 of the 591 amino acid sequence of lactate dehydrogenase (ScLDH) from Saccharomyces cerevisiae were deleted. Subsequently, ScLDH was compared with the 578 amino acids shown in SEQ ID NO: 3, in which the amino acid sequence of lactate dehydrogenase (PkLDH) from Pichia kudriavzevii was deleted, and a 1509-bp gene (including the stop codon TAA) shown in SEQ ID NO: 5 was obtained as cDNA by standard PCR of the gene fragment. The 578 amino acids shown in SEQ ID NO: 3, in which the amino acid sequence of lactate dehydrogenase (PkLDH) from Pichia kudriavzevii was deleted, encoding the 502 amino acids shown in SEQ ID NO: 4, in which the amino acids at positions 2 to 77 were deleted. A DNA construct was prepared by inserting the target gene, ScLDH gene or PkLDH gene, into the multicloning site of the plasmid pKK223-3 by standard methods. Specifically, the ScLDH gene or PkLDH gene was ligated into the In-Fusion Cloning Site in the multicloning site of pKK223-3 using the In-Fusion HD Cloning Kit (Clontech) according to the protocol attached to the kit to obtain expression plasmids (pKK223-3-ScLDH and pKK223-3-PkLDH). Furthermore, E. coli JM109 was transformed with these plasmids, and the E. coli JM109(pKK223-3-ScLDH) and E. coli JM109(pKK223-3-PkLDH) strains were inoculated into 3 ml of LB-amp medium [1% (w / v) bactotryptone, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, 50 μg / ml ampicillin] and cultured with shaking at 37°C for 16 hours to obtain cultures.

[0159] These cultures were centrifuged at 10,000 × g for 1 minute to collect bacterial cells, from which the recombinant plasmids pKK223-3-ScLDH and pKK223-3-PkLDH were extracted and purified using a GenElute Plasmid Miniprep Kit (Sigma-Aldrich), yielding 2.5 μg of recombinant plasmid DNA pKK223-3-ScLDH and pKK223-3-PkLDH.

[0160] (2) LDH production E. coli BL21 (pKK223-3-ScLDH) strains transduced with pKK223-3-ScLDH were cultured for 24 hours at 25°C in 3 ml of LB-amp medium supplemented with IPTG to a final concentration of 0.1 mM. Similarly, E. coli BL21 (pKK223-3-PkLDH) strains transduced with pKK223-3-PkLDH were cultured for 24 hours at 37°C in 3 ml of LB-amp medium supplemented with IPTG to a final concentration of 0.1 mM. Each culture was washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the same buffer, sonicated, and centrifuged at 20,000 × g for 10 minutes to prepare 0.6 ml of crude enzyme solution containing ScLDH or PkLDH.

[0161] (3) LDH activity measurement The oxidation activity of L-lactic acid was measured using the crude enzyme solution containing the above-mentioned ScLDH or PkLDH according to the method described in the activity measurement method below. The LDH of the present invention catalyzes the reaction of oxidizing L-lactic acid to produce pyruvic acid. For convenience, this may be referred to as LDH activity. The LDH activity of the 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 the electron acceptor.

[0162] (Reaction) L-lactic acid + potassium ferricyanide → Pyruvate + Potassium Ferrocyanide

[0163] The degree of disappearance of the above-mentioned "potassium ferricyanide" is detected as a change in absorbance at a wavelength of 420 nm, and the enzyme activity can be determined based on this change.

[0164] Specifically, LDH activity can be measured according to the following procedure. 0.15 mL of 1 M potassium phosphate buffer (pH 7.5), 0.15 mL of 0.1 M L-lactic acid solution, 0.075 mL of 30 mM potassium ferricyanide solution, and 1.075 mL of ultrapure water are mixed and incubated at 30°C for 5 minutes. Next, 0.05 mL of enzyme sample solution is added to initiate the reaction. Absorbance is measured at the start of the reaction and over time. The decrease in absorbance at 420 nm per minute (ΔA420) associated with the progress of the enzyme reaction is determined, and LDH activity is calculated according to the following formula: In this case, 1 U of LDH activity is defined as the amount of enzyme that reduces 1 μmol of potassium ferricyanide per minute in the presence of 10 mM L-lactic acid at 30°C.

[0165]

number

[0166] In the formula, 1.5 is the volume (mL) of the reaction reagent + enzyme reagent, and 1.01 is the millimolar extinction coefficient (cm) under the activity measurement conditions. 2 / μmol), 0.05 is the volume of the enzyme solution (mL), 1.0 is the optical path length of the cell (cm), ΔA420blank is the decrease in absorbance at 420 nm per minute when 10 mM phosphate buffer (pH 7.5) is added instead of the enzyme sample solution to start the reaction, and df is the dilution factor.

[0167] Example 2 (temperature stability) The crude enzyme solution was diluted to a final concentration of 100 mM potassium phosphate buffer (pH 6.0) containing 0.07% BSA, and the temperature stability was examined. Specifically, the LDH enzyme solution prepared in Example 1(2) was diluted to 6 U / ml and treated at various temperatures (35°C, 40°C, 45°C, 50°C, 55°C, 60°C, and 65°C) for 10 minutes, after which the LDH activity was measured and compared with the LDH activity before treatment to determine the residual activity. For comparison, a similar test was also performed on ScLDH. The results are shown in Figure 2. The results showed that ScLDH was unstable at 45°C, but PkLDH was stable even after heat treatment at 55°C.

[0168] Next, long-term stability at 37°C was examined. Specifically, the LDH enzyme solution was diluted with buffer to 6 U / ml, as in the temperature stability test, and stored for various periods. The remaining activity was then measured after each period. For comparison, a similar test was also performed on ScLDH. The results are shown in Figure 3 (A and B). Figure 3A is a graph of the remaining activity after each period, with the remaining activity at zero hours taken as 100%. This indicates that PkLDH heated to a certain temperature (37°C in this example) exhibits an improved remaining activity over a certain period of storage (Figure 3A). Figure 3B is a graph of the remaining activity after each period, with the activity at the reference time taken as 100% (15 hours after the start of storage). PkLDH retained 99% of its activity even after 245 hours of storage (230 hours after the baseline time). After 15 hours, the residual activity leveled off. It remained stable even after approximately 10 days of storage at 37°C (Figure 3B). On the other hand, ScLDH exhibited a significant decrease in residual activity after storage, reaching 62% after 21 hours (6 hours after the baseline time) and 0% after 65 hours (50 hours after the baseline time), demonstrating its instability. Furthermore, evaluation of ScLDH at a 20 U / ml enzyme concentration did not significantly improve its stability.

[0169] Example 3 (Enzyme purification) The crude PkLDH enzyme solution obtained in Example 1(2) was applied to 1 ml of Q Sepharose Fast Flow resin (GE Healthcare) equilibrated with 20 mM potassium phosphate buffer (pH 7.5) and allowed to adsorb to the resin. Proteins not adsorbed to the resin were eluted with the same buffer. The column was then 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 then eluted with 20 mM potassium phosphate buffer (pH 7.5) containing 500 mM sodium chloride. The resulting crude PkLDH enzyme solution was applied to a HiLoad 26 / 10 Q Sepharose HP column (GE Healthcare) equilibrated with 20 mM potassium phosphate buffer (pH 7.5) containing 150 mM sodium chloride, and a column volume of buffer was passed through to collect fractions containing the target protein. The recovered enzyme solution was then dialyzed against 10 mM potassium phosphate buffer (pH 7.5) and adsorbed onto a Hiscreen Capto Q column (GE Healthcare) equilibrated with the same buffer. The NaCl concentration was then gradually increased using a gradient up to 500 mM NaCl / 10 mM potassium phosphate buffer (pH 7.5), allowing the PkLDH adsorbed to the resin to be eluted and recovered. The obtained fraction was analyzed by SDS-PAGE and confirmed to be purified to a degree that it was free of other contaminating proteins, and was used as a purified PkLDH preparation.The thermal stability of the purified PkLDH was equivalent to that of the crude enzyme solution.

[0170] Example 4 (pH stability) The pH stability of the purified PkLDH enzyme solution (10 U / mL) obtained in Example 3 was examined. 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 activity was measured according to the activity measurement method described in Example 1(3), except for using each enzyme. The condition that yielded the highest activity after treatment was designated as 100%, and relative values ​​were calculated for activity values ​​under other conditions. The results are shown in Figure 7. The enzyme was most stable at pH 6.5, followed by pH 6.0, where the relative activity (%) was 92%.

[0171] (Optimal active pH) The optimal activity pH was determined 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), the enzyme reaction was carried out at 30°C at each pH, ​​and the relative activity (%) was compared. The results are shown in Figure 8. As a result, the optimal activity pH of the FMN-dependent lactate dehydrogenase of the present invention was found to be highest in the range of pH 6.5 to 7.5. It is possible that good activity may be maintained even at pH 8.0.

[0172] (Quantitative determination of L-lactic acid) L-lactic acid concentrations of 0.2 to 5 mM were measured using the purified PkLDH enzyme solution (17 U / ml) obtained in Example 3. The results are shown in Figure 9. As a result, the change in absorbance per minute at 420 nm increased in a lactate concentration-dependent manner, demonstrating that L-lactic acid could be quantified.

[0173] (oxidase activity) Following the activity measurement method described in International Publication WO 2015 / 020200, activity measurements were performed using PkLDH as the enzyme and L-lactic acid at a final concentration of 10 mM as the substrate at a pH of 7.5. Oxidase activity was defined as the amount of enzyme required to generate 1 μmol of hydrogen peroxide per minute in the presence of a 10 mM substrate concentration (1 unit (U)). The amount of enzyme used was an amount that exhibited 600 U / ml of dehydrogenase activity. As a result, no oxidase activity of PkLDH was detected. Therefore, PkLDH was an enzyme that does not use oxygen as an electron acceptor.

[0174] Example 5 (Quantitative determination of L-lactic acid using a printed electrode) The purified PkLDH enzyme solution obtained in Example 3 was used to quantify L-lactic acid by printed electrode measurement. Specifically, screen-printed electrons (DropSens, product number DRP-110) with a printed carbon working electrode and a printed silver reference electrode were connected to an ALS Electrochemical Analyzer 814D (BAS) using a dedicated connector (DropSens, product number DRP-CAC). 5 μL of PkLDH enzyme solution, 20 μL of 100 mM potassium phosphate buffer (pH 7.5) containing 1.5 M potassium chloride, and 25 μL of potassium ferricyanide aqueous solution were placed on the electrodes. A voltage of +400 mV (vs. Ag / AgCl) was then applied, and 5 μL of lactic acid solution of a predetermined concentration was placed on each electrode to allow the reaction. The current value was measured after 120 seconds. As a result, lactic acid could also be quantified by electrochemical measurement.

[0175] Example 6 (4) Preparation of recombinant plasmids pKK223-3-CaLDH DNA and pKK223-3-OgLDH DNA and production of each LDH A 1518-bp gene (including the stop codon TAA) shown in SEQ ID NO:8 was obtained as cDNA by standard PCR of the gene fragment, which encodes the 505 amino acids shown in SEQ ID NO:7, obtained by deleting positions 2 to 67 of the 571 amino acids shown in SEQ ID NO:6, which is the amino acid sequence of lactate dehydrogenase (CaLDH) derived from Candida inconspicua. Similarly, a 1512-bp gene (including the stop codon TAA) shown in SEQ ID NO:11 was obtained as cDNA by standard PCR of the gene fragment, which encodes the 503 amino acids shown in SEQ ID NO:10, obtained by deleting positions 2 to 56 of the 558 amino acids shown in SEQ ID NO:9, which is the amino acid sequence of lactate dehydrogenase (OgLDH) derived from Ogataea parapolymorpha. In the same manner as in Example 1, a DNA construct was prepared by inserting the target gene, CaLDH gene or OgLDH gene, into the multicloning site of the plasmid pKK223-3 by a standard method to obtain expression plasmids (pKK223-3-CaLDH and pKK223-3-OgLDH). E. coli BL21 (pKK223-3-CaLDH) strain transduced with pKK223-3-CaLDH and E. coli BL21 (pKK223-3-OgLDH) strain transduced with pKK223-3-OgLDH were cultured in 3 ml of LB-amp medium supplemented with IPTG to a final concentration of 0.1 mM at 30°C for 24 hours. The resulting cultured cells were washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the same buffer, sonicated, and centrifuged at 20,000 × g for 10 minutes to prepare 0.6 ml of crude enzyme solution containing CaLDH or OgLDH.

[0176] Example 7 (temperature stability) Long-term stability at 37°C was investigated in the same manner as in Example 2. However, CaLDH or OgLDH was diluted to a final concentration of approximately 20 U / ml in 100 mM potassium phosphate buffer (pH 6.0) containing 0.07% BSA and stored for various periods. The residual activity was then measured after each period. The results are shown in Figure 3(A). CaLDH retained 72% of its activity even after 140 hours of storage, and OgLDH retained 77% of its activity even after 140 hours of storage, demonstrating that they were significantly more stable than ScLDH.

[0177] (pH stability) CaLDH and OgLDH were purified using the same method as PkLDH. The pH stability of the purified enzyme solution (10 U / mL) was examined. Both CaLDH and OgLDH were most stable at pH 6.5, and showed a relative activity (%) of 80% or higher in the pH range of 6.0 to 7.0.

[0178] (oxidase activity) When the oxidase activity of CaLDH and OgLDH was measured, no oxidase activity was detected, indicating that CaLDH and OgLDH are enzymes that do not use oxygen as an electron acceptor.

[0179] Example 8 (5) Preparation of recombinant plasmid pKK223-3-ThLDH DNA and production of each LDH A 1500-bp gene (including the stop codon TAA) shown in SEQ ID NO: 13, which encodes the 499 amino acids shown in SEQ ID NO: 12, which is the amino acid sequence of Thermothelomyces thermophilus-derived lactate dehydrogenase (ThLDH), was obtained as cDNA by PCR of the gene fragment, a standard method. As in Example 1, a DNA construct was prepared in which the target gene, the ThLDH gene, was inserted into the multicloning site of the plasmid pKK223-3 by standard methods, and an expression plasmid (pKK223-3-ThLDH) was obtained. E. coli BL21 (pKK223-3-ThLDH) strain transformed with pKK223-3-ThLDH was cultured for 24 hours at 30°C in 3 ml of LB-amp medium supplemented with IPTG to a final concentration of 1 mM. Each cultured cell was washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the same buffer, and sonicated. The cells were then centrifuged at 20,000 × g for 10 minutes to prepare 0.6 ml of a crude enzyme solution containing ThLDH.

[0180] Example 9 (temperature stability) Long-term stability at 37°C was investigated in the same manner as in Example 2. ThLDH was diluted to a final concentration of approximately 20 U / ml with 100 mM potassium phosphate buffer (pH 6.0) containing 0.07% BSA and stored for various periods. The residual activity was then measured after each period. ThLDH retained 74% of its activity even after 89 hours of storage, demonstrating its superior stability compared to ScLDH. In other words, ThLDH maintained 70% or more of its initial activity after at least 3 days at 37°C.

[0181] Example 10 (6) Construction of PkLDH mutants A mutant with a further deletion of the N-terminal region of PkLDH was constructed. First, to prepare a mutant (PkLDH-96) in which amino acids 2 to 95 of SEQ ID NO: 4 were deleted, PCR was performed using the recombinant plasmid pKK223-3-PkLDH obtained in Example 1 as a template, the synthetic oligonucleotides of SEQ ID NOs: 14 and 15, and KOD One PCR Master Mix (Toyobo Co., Ltd.) under the following conditions: 10 μl of KOD One PCR Master Mix, 20 ng of the template pKK223-3-PkLDH, and 6 pmol of each of the synthetic oligonucleotides were added, and the total volume was adjusted to 20 μl with sterile water. The prepared reaction solution was subjected to seven cycles of "98°C, 10 seconds," "55°C, 5 seconds," and "68°C, 35 seconds" using a thermal cycler (Bio-Rad).

[0182] The resulting solution containing the PCR product was mixed with 1 μl of the restriction enzyme DpnI (NEW ENGLAND BIOLABS) and treated at 37°C for 30 minutes to cleave the remaining template DNA. Subsequently, 2 μl of the resulting DpnI-treated solution, 7 μl of sterile water, 5 μl of Ligation High (Toyobo), 1 μl of T4 Polynucleotide Kinase (Toyobo), and 7 μl of ion-exchanged water were mixed and reacted at 16°C for 1 hour. Escherichia coli JM109 was transformed with the reaction mixture and developed on LB-amp agar medium. The recombinant plasmid was extracted and purified as described in Example 1 to yield 2.5 μg of DNA. The nucleotide sequence of the DNA encoding PkLDH-96 in the plasmid was determined using a multi-capillary DNA analysis system, Applied Biosystems 3130xl Genetic Analyzer (Life Technologies). As a result, a DNA construct encoding PkLDH-96 was obtained.

[0183] Similarly, to prepare a mutant (PkLDH-97) in which amino acids at positions 2 to 96 in SEQ ID NO: 4 were deleted, synthetic oligonucleotides of SEQ ID NOs: 14 and 16 were used. To prepare a mutant (PkLDH-98) in which amino acids at positions 2 to 97 in SEQ ID NO: 4 were deleted, synthetic oligonucleotides of SEQ ID NOs: 14 and 17 were used. To prepare a mutant (PkLDH-99) in which amino acids at positions 2 to 98 in SEQ ID NO: 4 were deleted, synthetic oligonucleotides of SEQ ID NOs: 14 and 18 were used. To prepare a mutant (PkLDH-100) in which the amino acid at position 99 has been deleted, synthetic oligonucleotides of SEQ ID NOs: 14 and 19 were used. To prepare a mutant (PkLDH-101) in which the amino acids at positions 2 to 100 in SEQ ID NO: 4 have been deleted, synthetic oligonucleotides of SEQ ID NOs: 14 and 20 were used. To prepare a mutant (PkLDH-102) in which the amino acids at positions 2 to 101 in SEQ ID NO: 4 have been deleted, synthetic oligonucleotides of SEQ ID NOs: 14 and 21 were used. To prepare a mutant (PkLDH-103) in which amino acids 2 to 103 of SEQ ID NO: 4 were deleted, synthetic oligonucleotides of SEQ ID NO: 14 and 22 were used. To prepare a mutant (PkLDH-104) in which amino acids 2 to 75 of SEQ ID NO: 4 were deleted, synthetic oligonucleotides of SEQ ID NO: 14 and 23 were used. To prepare a mutant (PkLDH-76) in which amino acids 2 to 75 of SEQ ID NO: 4 were deleted, synthetic oligonucleotides of SEQ ID NO: 14 and 50 were used. To prepare a mutant (PkLDH-8) in which amino acids 2 to 83 of SEQ ID NO: 4 were deleted, synthetic oligonucleotides of SEQ ID NO: 14 and 50 were used. PCR was performed using synthetic oligonucleotides of SEQ ID NOs: 14 and 51 to prepare PkLDH-92, a mutant in which amino acids 2 to 91 of SEQ ID NO: 4 were deleted, and synthetic oligonucleotides of SEQ ID NOs: 14 and 52 to prepare PkLDH-110, a mutant in which amino acids 2 to 109 of SEQ ID NO: 4 were deleted, and synthetic oligonucleotides of SEQ ID NOs: 14 and 53 to prepare PkLDH-110, respectively, to obtain DNA constructs encoding PkLDH-76 to PkLDH-110.

[0184] PCR was also performed using pKK223-3-PkLDH as a template and the synthetic oligonucleotides SEQ ID NOS: 24, 25, 26, and 27. Specifically, PCR was performed using the recombinant plasmid pKK223-3-PkLDH as a template, the synthetic oligonucleotides SEQ ID NOS: 24 and 25, and KOD One PCR Master Mix (Toyobo Co., Ltd.) under the following conditions: 10 μl of KOD One PCR Master Mix, 20 ng of the template pKK223-3-PkLDH, and 6 pmol of each of the synthetic oligonucleotides were added, and the total volume was adjusted to 20 μl with sterile water. The prepared reaction solution was cycled 15 times using a thermal cycler (Bio-Rad) under the following conditions: 98°C, 10 seconds; 55°C, 5 seconds; 68°C, 35 seconds. The resulting PCR product was treated with the restriction enzyme DpnI to cleave the remaining template DNA, and then transformed into Escherichia coli JM109 and developed on LB-amp agar medium. The recombinant plasmid was extracted and purified as in Example 1, yielding 2.5 μg of DNA. The nucleotide sequence of the DNA encoding the PkLDH mutant in the plasmid was determined using a multi-capillary DNA analysis system, Applied Biosystems 3130xl Genetic Analyzer (Life Technologies). Similarly, PCR was performed under the same conditions using the recombinant plasmid pKK223-3-PkLDH / L386R as a template, synthetic oligonucleotides of SEQ ID NOs: 26 and 27, and KOD One PCR Master Mix (Toyobo Co., Ltd.), to yield 2.5 μg of DNA. The base sequence of the DNA encoding the PkLDH mutant in the plasmid was determined using a multi-capillary DNA analysis system, Applied Biosystems 3130xl Genetic Analyzer (Life Technologies), and a DNA construct encoding PkLDH / L386R / T461R / D464R was obtained, which is a mutant in which the leucine at position 386 of the amino acid sequence set forth in SEQ ID NO: 4 was replaced with arginine, the threonine at position 461 was replaced with arginine, and the aspartic acid at position 464 was replaced with arginine.For example, "L386R / T461R / D464R" means that the leucine at position 386 of the amino acid sequence of SEQ ID NO: 4 is replaced with arginine, the threonine at position 461 is replaced with arginine, and the aspartic acid at position 464 is replaced with arginine, and the " / " symbol means that all of the respective substitutions are present. In addition, PCR was performed using pKK223-3-PkLDH as a template and the synthetic oligonucleotides of SEQ ID NOs: 28 and 29 to obtain a DNA construct encoding PkLDH / F161L, a mutant in which phenylalanine at position 161 of the amino acid sequence of SEQ ID NO: 4 was replaced with leucine. In addition, PCR was performed using pKK223-3-PkLDH as a template and the synthetic oligonucleotides of SEQ ID NOs: 30 and 31 to obtain a DNA construct encoding PkLDH / F187L, a mutant in which phenylalanine at position 187 of the amino acid sequence of SEQ ID NO: 4 was replaced with leucine. In addition, PCR was performed using pKK223-3-PkLDH as a template and the synthetic oligonucleotides of SEQ ID NOs: 32 and 33 to obtain a DNA construct encoding PkLDH / F428L, a mutant in which phenylalanine at position 428 of the amino acid sequence of SEQ ID NO: 4 was replaced with leucine. Furthermore, PCR was performed using pKK223-3-PkLDH-97 as a template and the synthetic oligonucleotides of SEQ ID NOs: 32 and 33 to obtain a DNA construct encoding PkLDH-97 / F428L, a mutant in which phenylalanine at the position corresponding to position 428 of the amino acid sequence of SEQ ID NO: 4 in PkLDH-97 was replaced with leucine. In addition, PCR was performed using PkLDH-97 / F428L as a template and the synthetic oligonucleotides of SEQ ID NOs: 34 and 35 to obtain a DNA construct encoding PkLDH-97 / F428L / L194A, a mutant in which the leucine at the position corresponding to position 194 of the amino acid sequence of SEQ ID NO: 4 in PkLDH-97 / F428L was replaced with alanine. In addition, PCR was performed using PkLDH-97 / F428L as a template and the synthetic oligonucleotides of SEQ ID NOs: 36 and 37 to obtain a DNA construct encoding PkLDH-97 / F428L / L222Y, a mutant in which the leucine at the position corresponding to position 222 of the amino acid sequence of SEQ ID NO: 4 in PkLDH-97 / F428L was replaced with tyrosine. In addition, PCR was performed using PkLDH-97 / F428L as a template and the synthetic oligonucleotides of SEQ ID NOs: 38 and 39 to obtain a DNA construct encoding PkLDH-97 / F428L / A274S, a mutant in which alanine at the position corresponding to position 274 of the amino acid sequence of SEQ ID NO: 4 in PkLDH-97 / F428L was replaced with serine. In addition, PCR was performed using PkLDH-97 / F428L as a template and the synthetic oligonucleotides of SEQ ID NOs: 40 and 41 to obtain a DNA construct encoding PkLDH-97 / F428L / L277S, a mutant in which the leucine at the position corresponding to position 277 of the amino acid sequence of SEQ ID NO: 4 in PkLDH-97 / F428L was replaced with serine. Furthermore, PCR was performed using PkLDH-97 / F428L as a template and the synthetic oligonucleotides of SEQ ID NOs: 42 and 43 to obtain a DNA construct encoding PkLDH-97 / F428L / F313A, a mutant in which the phenylalanine at the position corresponding to position 313 of the amino acid sequence of SEQ ID NO: 4 in PkLDH-97 / F428L was replaced with alanine. Furthermore, PCR was performed using PkLDH-97 / F428L as a template and the synthetic oligonucleotides of SEQ ID NOs: 44 and 45 to obtain a DNA construct encoding PkLDH-97 / F428L / I314S, a mutant in which the isoleucine at the position corresponding to position 314 of the amino acid sequence of SEQ ID NO: 4 in PkLDH-97 / F428L was replaced with serine. Furthermore, to create a mutant (PkLDH-3 / F428L) in which the amino acid at position 2 in SEQ ID NO: 4 was deleted, similar to the case when the DNA construct encoding PkLDH-97 was prepared, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 54, resulting in a DNA construct encoding PkLDH-3 / F428L. Furthermore, to prepare a mutant (PkLDH-4 / F428L) in which the amino acids at positions 2 and 3 in SEQ ID NO: 4 were deleted, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 55, resulting in a DNA construct encoding PkLDH-4 / F428L. Furthermore, to produce a mutant (PkLDH-5 / F428L) in which amino acids 2 to 4 in SEQ ID NO: 4 were deleted, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 56, resulting in a DNA construct encoding PkLDH-4 / F428L. Furthermore, to prepare a mutant (PkLDH-6 / F428L) in which amino acids 2 to 5 in SEQ ID NO: 4 were deleted, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 57, resulting in a DNA construct encoding PkLDH-6 / F428L. Furthermore, to prepare a mutant (PkLDH-7 / F428L) in which amino acids 2 to 6 in SEQ ID NO: 4 were deleted, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 58, resulting in a DNA construct encoding PkLDH-7 / F428L. Furthermore, to produce a mutant (PkLDH-8 / F428L) in which amino acids 2 to 7 in SEQ ID NO: 4 were deleted, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 59, resulting in a DNA construct encoding PkLDH-8 / F428L. Furthermore, to produce a mutant (PkLDH-9 / F428L) in which amino acids 2 to 8 in SEQ ID NO: 4 were deleted, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 60, resulting in a DNA construct encoding PkLDH-9 / F428L. Furthermore, to prepare a mutant (PkLDH-10 / F428L) in which amino acids 2 to 9 in SEQ ID NO: 4 were deleted, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 61, resulting in a DNA construct encoding PkLDH-10 / F428L. Furthermore, to produce a mutant (PkLDH-11 / F428L) in which amino acids 2 to 10 in SEQ ID NO: 4 were deleted, PCR was performed using PkLDH / F428L as a template and synthetic oligonucleotides of SEQ ID NOs: 14 and 62, resulting in a DNA construct encoding PkLDH-11 / F428L.

[0185] E. coli BL21 strains transfected with the resulting plasmids encoding the PkLDH mutants were cultured in 3 ml of LB-amp medium supplemented with IPTG to a final concentration of 1 mM at 30°C for 24 hours. Each culture was washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the same buffer, sonicated, and centrifuged at 20,000 × g for 10 minutes to prepare 0.6 ml of crude enzyme solutions containing the PkLDH mutants. The crude enzyme activities of the 13 N-terminally deleted mutants (PkLDH-76 to PkLDH-110) ranged from 0.3 to 6 times higher than that of wild-type PkLDH. The crude enzyme activities of the nine N-terminally deleted mutants (PkLDH-3 / F428L to PkLDH-11 / F428L) ranged from 2 to 11 times higher than that of PkLDH / F428L.

[0186] Example 11 (temperature stability) The crude enzyme solutions (PkLDH-96 to PkLDH-104) prepared in Example 10 were diluted to a final concentration of 150 mM potassium phosphate buffer (pH 7.5) containing 0.15% BSA, and the temperature stability was examined. Specifically, the N-terminal deletion mutant LDH enzyme solutions prepared in Example 10 were diluted to 6 U / ml and treated at 55°C for 15 minutes, after which the LDH activity was measured and compared with the LDH activity before treatment to determine the residual activity. For comparison, a similar test was also performed on PkLDH. The results are shown in Figure 12. PkLDH-96 to PkLDH-104 all had higher residual activity and higher thermal stability than PkLDH. Other N-terminal deletion 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, and PkLDH-11 / F428L) were treated in the same manner at 50°C for 15 minutes. Next, a reagent for measuring LDH activity was prepared: 2,6-dichloroindophenol (DCIP) at a final concentration of 0.09 mM, phenazine methosulfate (PMS) at a final concentration of 0.5 mM, L-lactic acid at a final concentration of 10 mM, and potassium phosphate buffer (pH 7.5) at a final concentration of 100 mM. 5 μL of the heat-treated enzyme solution and 145 μL of the LDH activity measurement reagent were added to a 96-well plate and incubated at 37°C for 5 minutes. The color of the activity measurement reagent clearly changed to yellow. Therefore, these mutants were found to be more stable than ScLDH, which was completely inactivated by heat treatment at 50°C. Next, the crude enzyme solutions (PkLDH / F161L, PkLDH / F187L, and PkLDH / F428L) prepared in Example 10 were diluted to a final concentration of 150 mM potassium phosphate buffer (pH 7.5) containing 0.15% BSA, and their thermal stability was examined. Specifically, the single-substitution mutant LDH enzyme solutions prepared in Example 10 were diluted to 6 U / ml and treated at 55°C for 15 minutes, after which LDH activity was measured and compared with the LDH activity before treatment to determine the residual activity. For comparison, a similar test was also performed on PkLDH. The results are shown in Figure 13. PkLDH / F161L, PkLDH / F187L, and PkLDH / F428L all had higher residual activity and higher thermal stability than PkLDH. In addition, 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 treated at 50 ° C for 15 minutes. Subsequently, the above-mentioned DCIP and PMS system was used as the LDH activity measurement reagent, and 5 μL of the heat-treated enzyme solution and 145 μL of the LDH activity measurement reagent were added and incubated at 37 ° C for 5 minutes. The color of the activity measurement reagent clearly changed to yellow. Therefore, it was found that these mutants are more stable than ScLDH, which is completely inactivated by heat treatment at 50 ° C. Similar results were obtained when the activity was measured using 1-methoxyphenazine methosulfate instead of PMS.

[0187] Next, long-term stability at 37°C was examined in the same manner as in Example 2. Various LDH mutants (PkLDH-96, PkLDH-97, PkLDH-104, PkLDH / L386R / T461R / D464R, PkLDH / F428L) were diluted to approximately 20 U / ml in 100 mM potassium phosphate buffer (pH 6.0) containing 0.07% BSA as a final concentration, and stored for various periods. As a result, PkLDH-96 retained 95% of its activity even after 89 hours of storage, PkLDH-97 retained 114% of its activity after 43 hours, 114% after 72 hours, and 111% after 171 days of storage, PkLDH-104 retained 97% of its activity after 43 hours, 99% after 72 hours, and 88% after 171 days of storage, PkLDH / L386R / T461R / D464R retained 96% of its activity after 89 hours of storage, and PkLDH / F428L retained 99% of its activity after 43 hours, 107% after 72 hours, and 94% after 171 days of storage, demonstrating its excellent stability compared to ScLDH. In other words, these mutants can be said to maintain 70% or more of their initial activity after at least 3 days at 37° C. Furthermore, 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, and are therefore likely to be much more stable than ScLDH in terms of long-term stability at 37° C.

[0188] Example 12 (Quantitative determination of L-lactic acid using a PkLDH-immobilized electrode) L-lactic acid was quantified using an electrode onto which the purified PkLDH enzyme solution obtained in Example 3 was immobilized. Specifically, 12 U of PkLDH was applied to the working electrode of SCREEN-PRINTED ELECTRODES (DropSens, product number DRP-C110), which has a carbon working electrode printed thereon, and then dried. Subsequently, 3 μL of 2% poly(ethylene glycol) diglycidyl ether (Mn: 6000, Sigma) was applied, and the reaction was carried out at 4°C for 22 hours. The electrode was then washed with ultrapure water to obtain a PkLDH-immobilized electrode. The electrodes were connected to an ALS Electrochemical Analyzer 814D (BAS) using a dedicated connector (DropSens, DRP-CAC), and further connected to a silver-silver chloride reference electrode and a platinum electrode. The three electrodes were immersed in 10 ml of PBS (pH 7.4) containing 0.1 mg / ml Bindschedler's Green Leuco Base (Tokyo Chemical Industry Co., Ltd.). A voltage of +200 mV (vs. Ag / AgCl) was applied, and the response current was recorded upon the addition of L-lactate solution at regular intervals. The results are shown in Figure 8. The addition of 1 to 7 mM L-lactate demonstrated a lactate concentration-dependent increase in the response current, indicating that L-lactate was quantified. [Industrial Applicability]

[0189] The device for evaluating the state of a specimen of the present invention, which includes an action part for acting on the specimen with FMN-LDH and a sensor for sensing the state of the specimen after acting with FMN-LDH, and the method for evaluating the state of a specimen using the same, can stably, accurately and simply measure lactic acid in lactic acid-containing compositions including interstitial fluid, blood, urine, tears, sweat, saliva, skin, meat, eyeballs, cornea, gastric juice, food and beverages, brewed products, chemical products, water and soil of human and non-human organisms over a long period of time, and is therefore useful for health management of humans and animals, or for manufacturing process management and quality control of food and beverages, brewed products, chemical products and the like. [Explanation of symbols]

[0190] 1 Working electrode 3. Opposite 5 Reference pole 7 Wiring section 9 terminals 10 Sensor Chip 11 Foundation 13 Spacer 15 Cover 19 Reaction layer 100 Measuring part 101 Control section 102 Temperature Sensor 103 Storage section 104 Communications Department 105 Batteries 106 Measuring Equipment

Claims

1. 1. A device for assessing the condition of a specimen, comprising: an action part for allowing lactate dehydrogenase to act on the sample; a sensor for sensing the state of a specimen on which lactate dehydrogenase has been acted, the sensor being arranged so as to be able to sense the state of the specimen in the action section; Including, the device.

2. The device of claim 1 further comprising an output for outputting a signal from the sensor.

3. A device according to claim 2; a data processing unit connected to the output of the device for processing signals from the sensor; Including, the system.

4. A program for evaluating a state of a specimen by a system including a device and a data processing unit, The device an action part for allowing lactate dehydrogenase to act on the sample; a sensor for sensing the state of the specimen on which lactate dehydrogenase has been acted, the sensor being positioned so as to be able to sense the state of the specimen at the action portion; an output unit for outputting a signal from the sensor; Including, a data processing unit connected to the output of the device and configured to process signals from the sensor; The program tells the device: a measurement process for sensing and measuring the state of the sample on which lactate dehydrogenase has acted in the action portion using a sensor and converting the state into a signal; a transfer process for transferring the signal obtained by the measurement process from an output unit to a data processing unit; Execute A program that causes the data processing unit to execute data processing for performing predetermined processing on the signal obtained in the measurement processing.

5. A method for assessing the condition of a specimen, using the device of claim 1 or 2 or the system of claim 3.

6. The method of claim 5, wherein the specimen is a lactic acid-containing composition including body fluids of human and non-human organisms, interstitial fluid, blood, urine, tears, sweat, saliva, skin, meat, eyeballs, cornea, gastric juice, food and beverages, brewed products, chemical products, water, and soil.

7. The method according to claim 5 or 6, wherein the condition of the specimen is a physical condition in response to exercise load, a disease state, a brewing condition of a brewed product accompanied by a change in the amount of lactic acid, a degree of maturation or ripening of a food or beverage accompanied by a change in the amount of lactic acid, a lactic acid content in a chemical product produced accompanied by a change in the amount of lactic acid, or an amount of lactic acid in water or soil accompanied by a change in the amount of lactic acid.

8. 1. A device for monitoring the condition of a specimen, the device comprising: (A) a flavin-dependent lactate dehydrogenase that maintains at least about 20% of its initial activity after being left in solution at 37°C for 10 days; (B) a flavin-dependent lactate dehydrogenase that maintains about 20% or more of its initial activity after being left in solution at 37°C for 3 days or more; or (C) A flavin-dependent lactate dehydrogenase that maintains about 20% or more of its initial activity after being left in solution at 37°C for 15 hours or more. A device comprising an action portion for causing the action.

9. 9. A system in which the device of claim 8 further comprises an output, the output being connected to a data processing unit.

10. 10. A method for monitoring the condition of a specimen using the device of claim 8 or the system of claim 9.

11. A lactate dehydrogenase comprising the amino acid sequence of 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 thereto, the amino acid sequence of positions 113 to 505 in the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 70% or more identity thereto, an amino acid sequence or an amino acid sequence having 70% or more identity thereto, the amino acid sequence of positions 112 to 503 in the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 70% or more identity thereto, or the amino acid sequence of positions 102 to 499 in the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 70% or more identity thereto.

12. The lactate dehydrogenase according to claim 11, which has the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12, or an amino acid sequence having an identity of 70% or more thereto.

13. A nucleic acid encoding the lactate dehydrogenase according to claim 11 or 12.

14. A host cell comprising the nucleic acid of claim 13.

15. A method for producing lactate dehydrogenase, comprising culturing the host cell of claim 14.

16. 1. A method for assessing the condition of a specimen, comprising: i) contacting a sample with the lactate dehydrogenase of claim 11 or 12; and ii) Measuring lactate A method comprising:

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