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

Abstract

The purpose of the present invention is to provide: a device, a system, and a program for evaluating the state of the interstitial fluid, blood, urine, tears, perspiration, or saliva of a human or non-human organism, a food or drink product, a brewed product, or the like; and an evaluation method using the same.　The present invention provides a device, a system, and a program which are for evaluating the state of a sample and which comprise an action unit that is for subjecting the sample to the action of lactate dehydrogenase and a sensor that senses the state of the sample subjected to the action of the lactate dehydrogenase; a method for evaluating the state of the sample by using the same; and an enzyme used in the same.

Description

Technical Field

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

Background Art

[0002] Blood lactate concentration and sweat lactate concentration are known as markers reflecting fatigue and physical condition. Lactate is a major metabolite and is recognized as being important not only for health management indicators but also for health assessment including critically ill and / or surgical patients. The lactate value in body fluids can be an indicator for various pathological conditions 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] Regarding the purpose of physical condition management not limited to humans in a disease state, lactate monitoring is also performed as an indicator for monitoring the appropriateness of training by athletes and people who are highly interested in daily exercise (Non-Patent Document 2).

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

[0005] As an enzyme using lactate as a substrate, lactate oxidase (hereinafter, LOD) is known. LOD has a problem that its activity is lost during storage after drying. Therefore, Patent Document 4 proposes a method of drying in combination with a specific stabilizer 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 value in food can be an indicator for quality control and the like. In the manufacturing sites of various fermented food and beverages produced through the lactic acid fermentation process, such as Japanese sake, wine, whiskey, cheese, yogurt, sauerkraut, kudzu mochi, pickles, miso, soy sauce, lactic acid fermentation yeast extract, etc., in order to stably produce products of good quality, it is preferable to conduct appropriate lactic acid fermentation process management. In this process management, the amount of lactic acid can be an indicator for manufacturing process management.

[0011] Furthermore, the production of lactic acid progresses not only in fermented food and beverages subjected to lactic acid fermentation, but also in, for example, lactic acid fermentation compositions obtained by fermenting plant-based materials, which are cosmetic raw materials, with lactic acid bacteria, and lactic acid compositions produced as finished products. In the manufacturing process management and quality control of such products, the amount of lactic acid can also be an indicator.

[0012] Also, when calculating the decomposition rate when decomposing oligomers or polymers containing lactic acid, such as polylactic acid, the amount of monomeric lactic acid can be an indicator.

[0013] The measurement of the amount of lactic acid carried out for the above purposes is preferably able to be carried out frequently and simply in daily life or in the manufacturing process of each food and beverage, more preferably can be repeatedly carried out in a simpler process, and even more preferably can be automatically and continuously monitored.

[0014] Currently, there is no device for measuring lactic acid that can simply and continuously monitor the amount of lactic acid in a sample. The actual situation is that samples (for example, blood collected from a patient's body by puncture) that are likely to contain lactic acid obtained through some pretreatment from the sample are measured by measuring them each time by some method.

[0015] Invasive sampling methods such as puncture in the current measurement methods are painful and stressful for biological specimens, which hinders frequent sampling. Also, in the manufacturing process management of food and beverages where there are no issues such as pain and stress, since lactic acid has to be measured each time by manually sampling from the lactic acid-containing composition during manufacturing and the measurement data has to be manually recorded each time, such frequent sampling operations in the current lactic acid measurement technology are cumbersome.

[0016] As a technical proposal for continuously monitoring a human's state, posture, etc. over a certain period of time, for example, a system for proposing a training program or estimating a human's physical condition based on some biological data obtained by a plurality of sensors attached to a human's body, clothing, training machine, etc., and input information such as the age and gender of the human, and an information processing device for starting an application program conforming to various data have been proposed (Patent Documents 1 to 3). And in a small number of such proposals, those including the term "lactic acid measurement" can also be found. However, among those proposals, no method has been disclosed that can specifically measure the amount of lactic acid as a practical indicator and thereby monitor the state of the specimen.

[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] As enzymes using lactic acid as a substrate, lactate oxidase (hereinafter referred to as LOD), nicotinamide dinucleotide (NAD)-dependent lactate dehydrogenase (hereinafter referred to as NAD-LDH) using NAD as a coenzyme, and flavin mononucleotide (FMN)-dependent lactate dehydrogenase (hereinafter referred to as FMN-LDH) using FMN as a coenzyme are known. A blood lactic acid measuring device using LOD is commercially available. Each time measurement is performed, the stored sensor (the blood lactic acid measuring electrode part containing LOD) is taken out and inserted into a dedicated measuring device, and the blood squeezed out from a human specimen by puncture is sucked into the sensor, and the measured value displayed on the measuring device is read. Thereby, the amount of lactic acid in the specimen can be known singly, and by repeating this, the amount of lactic acid in the specimen can be measured each time.

[0020] However, as described above, when it is desired to stably, accurately and preferably continuously and simply measure lactic acid in a lactic acid-containing composition including interstitial fluid, blood, sweat, food and drink products, and chemical products of organisms over a long period of time, known measuring enzymes cannot fully cope with it.

[0021] LOD has a problem that its thermal stability is not sufficient. Even when manufacturing an LOD product, the degree of loss of activity during the drying process or during storage after drying is large. As a proposal for that problem, a method of drying in combination with a specific stabilizer during the drying process has also been proposed (Patent Document 4), but the effect of improving its stability is still insufficient. It should also be considered that the stability of LOD in the liquid state during an actual enzyme reaction is even lower than that in the powder state. For example, when assuming a scenario where a sensor using LOD is used in close contact with a human body for a long time, or a scenario where lactic acid is monitored for a long time in a manufacturing process of food and drink products that can be placed at the fermentation temperature of a lactic acid bacterium fermentation process, from around room temperature to around 37°C which is around the human body temperature, there is a concern that existing LOD cannot exhibit sufficient practicality from the viewpoint of the stability of the enzyme.

[0022] Furthermore, LOD also has the problem of generating hydrogen peroxide as a reaction product. In particular, when assuming a usage method in which a lactate sensor is adhered to the skin or, in some cases, embedded subcutaneously, there is a concern that it may not be preferable to employ a sensor equipped with an enzyme that can continuously generate hydrogen peroxide, which is one of the reactive oxygen species and also a causative substance of oxidative stress. Furthermore, the hydrogen peroxide generated by the action of LOD also has an adverse effect on the stabilization of LOD itself, and as a means of avoiding the resulting decrease in LOD activity, a lactate sensor in which catalase coexists for the purpose of hydrogen peroxide elimination has also been proposed (Patent Document 5).

[0023] Since NAD-LDH catalyzes an enzyme reaction that does not generate hydrogen peroxide, the problem of hydrogen peroxide generation does not occur. However, in the enzyme reaction system using NAD-LDH, since lactate and pyruvic acid react reversibly, there is a problem that accurate measurement values cannot be obtained for the purpose of quantification, and it is difficult to adopt it for a sensor.

[0024] FMN-LDH also catalyzes an enzyme reaction that does not generate hydrogen peroxide, so the problem of hydrogen peroxide generation does not occur. Also, since there is no problem of reversible reaction, among the above three enzymes, it is considered to be the most promising as a practical enzyme for lactate monitoring purposes. However, the FMN-LDH derived from Saccharomyces cerevisiae known so far still has problems in stability (Non-Patent Document 3).

[0025] That is, as technical problems in satisfying the market needs of realizing simple and preferably continuous lactate monitoring in the evaluation of the state of a biological sample, the production management and quality control of food and drink, etc., there are problems in the above sampling method, the problem of the complexity of the recording process associated with data processing for each single measurement, and furthermore, problems in the performance of the enzyme used for measuring lactate. An object of the present invention is to provide a device for monitoring or evaluating the state of a sample, a method for monitoring or evaluating the state of a sample, and FMN-LDH used therefor, which can solve at least a part 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 causes the device to perform a measurement process for sensing and measuring, by a sensor, the state of a specimen on which lactate dehydrogenase has acted in an action part and converting it into a signal, and a transfer process for transferring the signal obtained by the measurement process from an output part to a data processing unit, and causes the device to execute the processes, A program that causes the data processing unit to execute data processing for performing predetermined processing on the signal obtained by the measurement process. (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 lactate-containing composition including body fluids, interstitial fluids, blood, urine, tears, sweat, saliva, skin, meat, eyeballs, corneas, gastric juices, food and drinks, brewed products, chemical products, water, and soil of humans and non-human organisms. (7) The method according to (5) or (6), wherein the state of the specimen is a physical state with respect to an exercise load, a disease state, a brewing state of a brewed product accompanied by a change in the amount of lactic acid, a ripening / after-ripening degree of a food and drink accompanied by a change in the amount of lactic acid, a lactic acid content ratio in the production of a chemical product accompanied by a change in the amount of lactic acid, or the amount of lactic acid in water or soil accompanied by a change in the amount of lactic acid. (8) A device for monitoring the state of a specimen, the device comprising an action part for acting on the specimen with (A) flavin-dependent lactate dehydrogenase that maintains about 20% or more of the initial activity when allowed to elapse for 10 days at 37°C in a solution, (B) flavin-dependent lactate dehydrogenase that maintains about 20% or more of the initial activity when allowed to elapse for 3 days or more at 37°C in a solution, or (C) flavin-dependent lactate dehydrogenase that maintains about 20% or more of the initial activity when allowed to elapse for 15 hours or more at 37°C in a solution. The device including the action part. (9) A system, wherein the device according to (8) further includes an output part, and the output part 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: Effect 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 description of the drawings]

[0029] [Figure 1] Figure 1 is a schematic diagram of the sensor chip 10 according to an embodiment of the present invention, and (b) to (d) are schematic diagrams showing the members constituting the sensor chip 10. [Figure 2] Figure 2 is a diagram showing the residual activity rates of PkLDH and ScLDH after heat treatment at each temperature. [Figure 3] Figures 3A and 3B are diagrams showing the residual activity rates of PkLDH, ScLDH, CaLDH, and OgLDH after storage at 37°C for each time. [Figure 4] Figure 4 is a schematic diagram showing an example of the device and system of the embodiment of the present invention. [Figure 5] Figure 5 is a flowchart showing an example of the program of the embodiment of the present invention. [Figure 6] Figure 6 is a schematic diagram showing an example of the device and system using the transmitter of the embodiment of the present invention. [Figure 7] Figure 7 is a diagram showing the stable pH of PkLDH evaluated in Example 4. [Figure 8] Figure 8 is a diagram showing the optimal pH of PkLDH evaluated in Example 4. [Figure 9] Figure 9 is a diagram showing the relationship between the lactic acid concentration and the activity of PkLDH evaluated in Example 4. [Figure 10] Figure 10 is a diagram showing the relationship between the lactic acid concentration and the response current value evaluated in Example 8. [Figure 11-1] Figure 11-1 shows the alignment of the amino acid sequences of the lactate dehydrogenase of the present invention. [Figure 11-2] Figure 11-2 is a continuation of Figure 11-1. [Figure 12] Figure 12 is a diagram showing the residual activity rates of PkLDH and the N-terminal deletion mutant of PkLDH treated at 55°C for 15 minutes evaluated in Example 11. [Figure 13] Figure 13 is a diagram showing the residual activity rates of PkLDH and the single substitution mutant treated at 55°C for 15 minutes evaluated in Example 11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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.Or, it is lactate dehydrogenase having the amino acid sequence at positions 112 to 503 in the amino acid sequence represented by 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 therewith. Or, it is lactate dehydrogenase having the amino acid sequence at positions 102 to 499 in the amino acid sequence represented by 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 therewith. In addition, with respect to the amino acid sequence at positions 97 to 502 in SEQ ID NO: 4, the amino acid sequence at positions 1 to 96 in SEQ ID NO: 4, the amino acid sequence at positions 1 to 99 in SEQ ID NO: 7, the amino acid sequence at positions 1 to 98 in SEQ ID NO: 10, the amino acid sequence at 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 therewith can also be ligated. Similarly, with respect to the amino acid sequence at positions 100 to 505 in SEQ ID NO: 7, the amino acid sequence at positions 99 to 503 in SEQ ID NO: 10, or the amino acid sequence at positions 89 to 499 in SEQ ID NO: 12, the amino acid sequence at positions 1 to 96 in SEQ ID NO: 4, the amino acid sequence at positions 1 to 99 in SEQ ID NO: 7, the amino acid sequence at positions 1 to 98 in SEQ ID NO: 10, the amino acid sequence at 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 therewith can also be ligated. Further, with respect to the region containing the amino acid sequence at positions 1 to 96 in SEQ ID NO: 4, the amino acid sequence at positions 1 to 99 in SEQ ID NO: 7, the amino acid sequence at positions 1 to 98 in SEQ ID NO: 10, or the amino acid sequence at positions 1 to 88 in SEQ ID NO: 12, as shown in FIG. 11-1, the sequence identity is low, and it can be said that the importance is low 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, as amino acids that are particularly important for the lactate dehydrogenase of the present invention to retain its activity, there are histidine at position 361 and arginine at position 364 in the lactate dehydrogenase represented by SEQ ID NO: 4. By using the alignment of the amino acid sequences of each lactate dehydrogenase shown in FIGS. 11-1 and 11-2, it is also possible to clarify the positions corresponding to these important amino acid positions in lactate dehydrogenases other than SEQ ID NO: 4 (the positions indicated by the arrows in FIG. 11-2). In addition, FIGS. 11-1 and 11-2 show, in addition to the lactate dehydrogenases represented by SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12, among the 558 amino acids of the amino acid sequence of lactate dehydrogenase derived from Ogataea polymorpha, 503 amino acids represented by SEQ ID NO: 46 (LDH-1) with positions 2 to 51 removed, among the 558 amino acids of the amino acid sequence of lactate dehydrogenase derived from Candida californica, 506 amino acids represented by SEQ ID NO: 47 (LDH-2) with positions 2 to 86 removed, 502 amino acids represented by SEQ ID NO: 48 (LDH-3) of the amino acid sequence of lactate dehydrogenase derived from Chaetomium globosum, and 499 amino acids represented by SEQ ID NO: 49 (LDH-4) of 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 working part for allowing lactate dehydrogenase to act only needs to be composed of a material suitable for the action, and refers to a member for allowing an enzyme composed of a material such as metal, plastic, cloth, liquid, paper, or nylon to act. Further, the sensor for sensing the state of the sample may be integrated with the member for allowing the enzyme to act, may be separated therefrom, or may be directly or indirectly connected.

[0037] The sample and the lactate dehydrogenase of the present invention are arranged in the working part. Further, if necessary, an electron acceptor and / or a reagent for indicating a change in the electron acceptor (mediator) can be arranged in the working part. The sensor including the working part (which may also be expressed as a working electrode) has a working electrode containing the LDH of the present invention, a reference electrode, and a counter electrode. As the working electrode, a carbon electrode, a gold electrode, a platinum electrode, etc. are used, and the LDH of the present invention is immobilized on this electrode. Further, or separately from this, 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. As the immobilization method, there are a method using a cross-linking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photo-crosslinkable polymer, a conductive polymer, a redox polymer, etc. Alternatively, it may be fixed in a polymer or adsorbed and fixed on an electrode together with an electron mediator typified by ferrocene or its derivative, or these may be used in combination. Typically, after immobilizing the LDH of the present invention on a carbon electrode using glutaraldehyde, it is treated with a reagent having an amino group to block the glutaraldehyde. A cross-linking reagent such as poly(ethylene glycol) diglycidyl ether can also be used instead of glutaraldehyde.

[0038] As the sensor that can be used in the device of the present invention, any sensor that can sense the state of the sample on which lactate dehydrogenase has acted can be used without limitation.

[0039] [Sensor chip] As a sensor, a sensor chip may be used. FIG. 1(a) is a schematic diagram of a sensor chip 10 according to an embodiment of the present invention, and FIGS. 1(b) to 1(d) are schematic diagrams showing members constituting the sensor chip 10. The sensor chip 10 includes two or more electrodes disposed on a base material 11. The base material 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 base material 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 in the wiring direction from each electrode. The working electrode 1, the counter electrode 3, and the reference electrode 5 are arranged spaced apart from each other. Also, the working electrode 1, the counter electrode 3, and the reference electrode 5 are preferably formed integrally with the wiring portion 7 and the terminal 9. Further, the counter electrode 3 and the reference electrode 5 may be integrated.

[0040] As shown in FIGS. 1(a) and 1(c), a spacer 13 is disposed at an end of the base material 11 parallel to the wiring portion 7, and a cover 15 that covers the working electrode 1, the counter electrode 3, the reference electrode 5, and the spacer 13 is disposed. The spacer 13 and the cover 15 are made of an insulating material. The spacer 13 has a thickness substantially equal to that of the working electrode 1, the counter electrode 3, and the reference electrode 5, and preferably adheres to the working electrode 1, the counter electrode 3, and the reference electrode 5. Also, the spacer 13 and the cover 15 may be integrally formed. The cover 15 is a protective layer that prevents the wiring portion 7 from being exposed to the outside air and deteriorating, or from short-circuiting due to infiltration of the measurement sample.

[0041] As shown in FIGS. 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 lactic acid and lactate dehydrogenase. In one embodiment, the lactate dehydrogenase of the present invention may be applied, adsorbed, or immobilized on these electrodes. Preferably, the lactate dehydrogenase of the present invention is applied, adsorbed, or immobilized on the working electrode. In another embodiment, a mediator may also be applied, adsorbed, or immobilized on the electrode together with lactate dehydrogenase. As the electrode, a carbon electrode, a metal electrode such as platinum, gold, silver, nickel, and palladium, etc. can be used. In the case of a carbon electrode, materials such as pyrolytic graphite carbon (PG), glassy carbon (GC), carbon paste, and plastic foam carbon (PFC), etc. can be mentioned. The measurement system may be a two-electrode system or a three-electrode system. For example, an enzyme can be immobilized on the working electrode. Examples of the reference electrode include a standard hydrogen electrode, a reversible hydrogen electrode, a silver-silver chloride electrode (Ag / AgCl), a palladium-hydrogen electrode, and a saturated calomel electrode, etc. From the viewpoints of stability and reproducibility, it is preferable to use Ag / AgCl.

[0042] Furthermore, in order to reduce the amount of solution required for measurement, a printed electrode can also be used. In this case, the electrode is preferably formed on a substrate 11 composed of an insulating substrate. Specifically, it is desirable that the electrode is formed on the substrate 11 by photolithography technology or printing technologies such as screen printing, gravure printing, and flexographic printing. Also, examples of the material of the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, etc. However, it is more preferable to use a material with strong resistance to various solvents and chemicals.

[0043] For example, in an operating unit containing potassium ferricyanide as an electron acceptor, when lactate dehydrogenase acts on lactate contained in a sample, potassium ferricyanide changes to potassium ferrocyanide. Since potassium ferricyanide shows absorption at a wavelength of 420 nm, the state of lactate in the sample can be detected by measuring the absorbance at 420 nm with a spectrophotometer. Also, when lactate dehydrogenase acts on lactate and then potassium ferricyanide acts, the redox reaction can be measured electrochemically with a sensor. Specifically, as a sensor for electrochemical measurement, for example, a Lactate Pro 2 model LT-1730 sensor manufactured by Arkray, Inc. can be used. As other electron acceptors, 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 their derivatives, etc. can be used. Note that the lactate dehydrogenase of the present invention does not use oxygen as an electron acceptor.

[0044] For example, the measurement of lactate concentration can be performed as follows. A buffer solution is placed in a thermostatic cell and maintained at a constant temperature. An electrode with LDH and an electron acceptor (e.g., quinone-added polymer) immobilized thereon is used as a working electrode, and a counter electrode (e.g., a platinum electrode) and a reference electrode (e.g., an Ag / AgCl electrode) are used. A certain voltage is applied to the carbon electrode, and after the current becomes steady, a sample containing L-lactate is added and the increase in current is measured. The lactate concentration in the sample can be calculated according to a calibration curve prepared with a lactate solution of a standard concentration.

[0045] As a specific example, the 4U LDH of the present invention is immobilized on a glassy carbon (GC) electrode, and the response current value with respect to the lactic acid concentration is measured. In an electrolytic cell, 1.8 ml of 50 mM potassium phosphate buffer (pH 7.5) and 0.2 ml of an aqueous solution of 1 M potassium hexacyanoferrate(III) (potassium ferricyanide) are added. The GC electrode is connected to a potentiostat BAS100B / W (manufactured by BAS), the solution is stirred at 37°C, and +500 mV is applied with respect to a silver / silver chloride (saturated KCl) reference electrode. A 1 M L-lactic acid solution is added to these systems so that the final concentrations become 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 mM, and the steady-state current value is measured each time it is added. This current value is plotted against the known lactic acid concentrations (1, 2, 3, 4, 5, 10, 20, 30, 40, 50 mM) to create a calibration curve. Thereby, it becomes possible to quantify lactic acid using the enzyme-immobilized electrode using the FMN-LDH of the present invention.

[0046] In one embodiment, a solution of FMN-LDH of 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 present invention is applied or immobilized on the lactic acid sensor chip of the present invention.

[0047] In one embodiment, a continuous lactic acid monitoring device having the lactic acid sensor of the present invention is provided. Also, in one embodiment, a method for continuously monitoring lactic acid for 14 days using the LDH of the present invention is provided. The measurement period can be set according to 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, 14 days, etc.

[0048] In one embodiment, continuous lactic acid monitoring can be performed with or without recalibration. In one embodiment, 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 has sufficient thermal stability. Therefore, simple and preferably continuous lactate monitoring can be realized.

[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. In addition, the step of measuring lactic acid in step ii) may be a single measurement or a continuous measurement. 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 FIG. 4, the device for evaluating the state of the specimen in the present invention may further include an output unit. The output unit can output the signal from the sensor to the outside. Also, the output unit may be connected to the data processing unit to form a system. That is, the system includes the device and the data processing unit. The data processing unit can process the signal from the sensor.

[0052] In this specification, the "data processing unit" not only means a device such as a single computer, but also includes the concept of a network for information processing in a form in which other devices connected by a communication line such as a local network or the Internet are connected.

[0053] The connection between the output unit and the data processing unit means that the signal from the sensor can be output from the output unit and input to the data processing unit by electrical or electronic means. For example, as a physical connection method for connection, in addition to wired connection, it can also be connected by wireless communication such as wireless LAN.

[0054] The data processing unit may include a control unit. The control unit can be, for example, a CPU (Central Processing Unit). The program described later can 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 incorporates a battery 105. When the remaining battery level of this battery 105 becomes insufficient, the transmitter is configured to be discarded. The remaining battery level of the battery 105 is monitored through a measuring device 106. And when the device is replaced, the measuring device 106 checks whether the remaining amount of the battery 105 of the transmitter is sufficient until the next device replacement with respect to the remaining battery level of the battery 105 of the transmitter. And if the remaining amount is not sufficient, the user is instructed to replace the transmitter and the transmitter is made unusable.

[0060] According to the system of the present invention, it is possible to evaluate the state of a specimen, for example, the physical state with respect to an exercise load, a disease state, the brewing state of a fermented product accompanied by a change in lactic acid amount, the aging / ripening degree of a food or drink accompanied by a change in lactic acid amount, the lactic acid content ratio in the production of a processed product accompanied by a change in lactic acid amount, the state of water or soil accompanied by a change in lactic acid amount, etc.

[0061] The lactic acid dehydrogenase used in the device of the present invention is (A) a flavin-dependent lactic acid 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 the initial activity when allowed to elapse for 10 days at about 30, 35°C or 37°C in a solution; (B) a flavin-dependent lactic acid 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 the initial activity when allowed to elapse for 3 days or more at about 30, 35°C or 37°C in a solution, or (C) a flavin-dependent lactic acid 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 the initial activity when allowed to elapse for 15 hours or more at about 30, 35°C or 37°C in a solution. Further, the lactic acid dehydrogenase used in the device of the present invention may be a flavin-dependent lactic acid dehydrogenase that maintains 70% or more activity even starting from 15 hours after being put into use.

[0062] The above device may further include an output unit, and the output unit may be connected to the data processing unit to form a system.

[0063] According to the device and system of the present invention, evaluation or monitoring can be performed non-invasively on the specimen, without the sampling step and can be directly measured, that is, it is not a disposable measurement such as inserting a test strip into a sensor and pressing a button, but can be used continuously for a long time while being pasted or inserted, which is very advantageous.

[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 program can be application software that causes a predetermined information processing device to execute a predetermined process in order to operate a predetermined device. Further, the program of the present invention can also be a mobile application (app).

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

[0067] The device includes an acting part, a sensor, and an output part. The acting part, the sensor, and the output part are as described above. That is, the acting part is configured to be able to act lactic dehydrogenase on the specimen. The sensor is arranged so as to be able to sense the state of the specimen on which lactic dehydrogenase has been acted by the acting part. The output part 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 can be a program for freshness management 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 so that the sensor senses and measures the state of the beef on which lactate dehydrogenase has been allowed to act on the acting part and converts it into a signal.

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

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

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

[0081] Note that in this embodiment, in addition to the lactic acid value, by monitoring the temperature and humidity, the expiration date of beef can be predicted. Also, by inputting the meat part as a parameter, the value of the meat can be calculated and used as an indicator for the selling price. Further, in this embodiment, a proposal for the aging degree of the meat can be made according to the dish. In this embodiment, the acting part can be directly attached to the beef, and the state 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 addition, in this embodiment, in addition to the lactic acid value, by monitoring the temperature, humidity, and pH, the expiration date of beef can be predicted. Further, by inputting the meat part as a parameter, the value of the meat can be calculated and used as an indicator for the selling price. Also, in this embodiment, it is possible to propose the degree of aging of the meat according to the dish. In this embodiment, the acting part can be directly attached to the beef, and the state of the meat can be sensed by the sensor.

[0089] Conventionally, the aging management of beef has mainly been carried out by temperature control. Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactic acid monitoring can be realized. Therefore, the freshness management using lactic acid as an index can be achieved by the program of this embodiment. Therefore, the transportation conditions and storage conditions of beef can be made appropriate. By using the program of this embodiment, it also becomes an index objectively showing the value of beef and can support the judgment of consumers.

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

[0091] In the measurement process S01, the program causes the system to execute a process so that the sensor senses and measures the state of fish such as tuna on which lactate dehydrogenase has acted on the acting part and converts it into a signal.

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

[0093] In the data process S03, the program causes the system to execute a process so that a predetermined process is performed on the signal obtained in the measurement process for the data processing unit. The predetermined process of this embodiment is a process for converting the signal 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 management of fish such as tuna has mainly been carried out by controlling factors such as temperature, aging time, the temperature of the thawing brine, and the water temperature. Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized. Therefore, by means of the program of the present embodiment, aging management using lactate as an index can be carried out. As a result, the aging conditions of fish such as tuna can be optimized. By using the program of the present embodiment, it can also serve as an index objectively indicating the value of fish such as tuna, and can assist the judgment of consumers and wholesalers.

[0104] (Other Embodiments of Programs for Food and Beverages) The program of the present embodiment can be, as a program for food and beverages other than the above, for example, malolactic fermentation management of wine, bread such as sake and bagels, fermentation management of kudzu starch jelly, lactic acid fermentation management of whiskey, fermentation management of tea, programs for food safety indicators, etc. According to the program of the present embodiment, for the fermentation management of brewed wines such as wine and sake, which has relied on experience and intuition, appropriate temperature control and fermentation periods can be obtained by using, in addition to the lactate value, indicators such as pH value and dissolved oxygen value.

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

[0106] (Embodiment 1 of the Program for Animals) The program of the present embodiment can be a program for the management of racehorses. That is, in the present embodiment, the specimen is a racehorse.

[0107] In the measurement process S01, the program causes the system to execute a process so as to sense, measure, and convert into a signal the state of a racehorse in which lactate dehydrogenase has been allowed to act on the action part by a sensor.

[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 cattle health management, shipping inspection management, or beef quality control of beef cattle. That is, in this embodiment, the specimen is a cow.

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

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

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

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

[0125] In this embodiment, the action part is the ear, lactate dehydrogenase can be made to act, and the state of the cow can be sensed and measured by a sensor. Blood vessels pass through the ear, and by charging and arranging the action part and the sensor in the ear tag, it is possible to measure the blood of the ear as a specific specimen. Sweat or skin can also be used as a specimen. Also, in this embodiment, it is possible to use the stomach as the action part. For example, by measuring the amount of lactic acid in the blood, administration of lactic acid bacteria or lactic acid-containing grains can be suppressed 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 monitoring of lactate levels, and therefore the program of this embodiment can be used for health management of cattle, assessment management at the time of shipping, or control of meat quality 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 state of the dairy cow on which lactate dehydrogenase has been acted on in the action part is sensed and measured by the sensor and converted 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 so as to transfer the signal obtained in the measurement process from the output unit to the data processing unit.

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

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

[0141] When there is lactic acid in the soil, the water absorption of plants is promoted. Therefore, the water absorption of plants changes according to the amount of lactic acid in the soil. Therefore, in the present embodiment, the state of water absorption of potted plants can be evaluated.

[0142] Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized. Therefore, the program of the present embodiment can propose the addition of nutrients and the timing of watering for the plants in the flowerpot. Furthermore, when the pH is low, it is possible to suppress the growth of miscellaneous bacteria in plants. Therefore, by measuring the pH together with the amount of lactic acid, it is possible to suppress the growth of miscellaneous bacteria.

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

[0144] In measurement process S01, the program causes the system to execute a process so that a sensor senses and measures the state of soil on which lactate dehydrogenase has acted on the working part, and converts it into a signal.

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

[0146] In data processing S03, the program causes the system to execute a process so that the data processing unit performs a predetermined process on the signal obtained in the measurement process. The predetermined process in this embodiment is a process for converting the signal into a data format suitable for the indices of soil management and hydroponic cultivation management.

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

[0148] When there is lactic acid in the soil and hydroponic water, the water absorption of plants is promoted. Therefore, the water absorption of plants changes according to the amount of lactic acid in the soil and hydroponic water. Therefore, in this embodiment, it is possible to evaluate the state of water absorption of plants existing in the soil and hydroponic water.

[0149] Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactic acid monitoring can be realized. Therefore, the program of this embodiment can propose the addition of nutrients to plants, the timing of watering, and the timing of top dressing. Furthermore, when the pH is low, it becomes possible to suppress the propagation of miscellaneous bacteria in plants. Therefore, by measuring the pH together with the amount of lactic acid, it becomes possible to suppress the propagation of miscellaneous bacteria.

[0150] Conventionally, plant management has mainly been data-independent management, such as top-dressing according to time such as seasons or adding water and nutrients when the condition of the plants deteriorates. Since the lactate dehydrogenase of the present invention has sufficient thermal stability, simple and continuous lactate monitoring can be realized. When the lactate value contained in the soil becomes low, a system for calculating the addition amount of saccharides and lactate that can be assimilated by lactic acid bacteria can be provided in order to enhance the antibacterial property in the soil. Conversely, when the lactate value becomes high, a system for calculating the type and addition amount of alkaline fertilizers is provided. In addition, by measuring pH, electrical conductivity, sunlight amount, temperature, humidity, and amino acid amount, an accurate plant breeding program can be constructed and provided. Further, by analyzing the component values (for example, sugar content, acidity) of the final product such as tomatoes, an objective evaluation can be made from the perspective invisible to consumers, such as how the final product and the plant have grown over time, which can assist the judgment of consumers and wholesalers.

[0151] The present invention also relates to lactate dehydrogenase having an amino acid sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 12, and a nucleic acid encoding the same. In certain embodiments, the present invention provides a DNA having a nucleotide sequence having 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 with the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, or SEQ ID NO: 13, and encoding a protein having lactate activity.

[0152] (LDH gene) To obtain the gene encoding LDH, commonly used gene cloning methods are generally employed. For example, chromosomal DNA or mRNA can be extracted from microbial cells or various cells having the ability to produce LDH by conventional methods, such as those described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Furthermore, cDNA can be synthesized using mRNA as a template. Using the chromosomal DNA or cDNA thus obtained, a library of chromosomal DNA or cDNA can be prepared. Next, based on the amino acid sequence of the above LDH, an appropriate probe DNA is synthesized and used to select the LDH gene from the chromosomal DNA or cDNA library, or based on the above amino acid sequence, an appropriate primer DNA is prepared, and by an appropriate polymerase chain reaction (PCR method) such as the 5’RACE method or 3’RACE method, DNA containing the target gene fragment encoding LDH is amplified, and these DNA fragments are ligated to obtain DNA containing the full length of the target LDH gene.

[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 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 when allowed to elapse for 10 days at about 30 to 37°C in a solution; (B) 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 allowed to elapse for 3 days or more at about 30 to 37°C in a solution, or (C) 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 allowed to elapse for 15 hours or more at about 30 to 37°C in a solution. Further, the lactate dehydrogenase of the present invention may be a flavin-dependent lactate dehydrogenase that maintains 70% or more of its activity starting 15 hours after use. Examples of the buffer material (buffer solution) that can be used in the reaction solution of LDH include, for example, boric acid buffer containing boric acid and / or its salts, Tris-HCl buffer, phosphate buffer containing phosphoric acid and / or its salts, such as potassium phosphate buffer or sodium phosphate buffer, organic acid buffer and / or organic acid buffer containing its salts, such as tricarboxylic acid buffer containing tricarboxylic acid and / or its salts, such as citric acid buffer containing citric acid and / or its salts, monocarboxylic acid buffer and / or monocarboxylic acid buffer containing its salts, such as acetic acid buffer containing acetic acid and / or its salts, and other buffers. In addition, examples of the buffer 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-(N-morpholino)ethanesulfonic 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), tricine (N-tris(hydroxymethyl)methylglycine) and / or Good buffers containing their salts and the like. The temperature for reacting LDH may be 20 to 60 °C, preferably within the range of 30 to 55 °C.The pH for reacting LDH may be within the range of 3 to 10, preferably within the range of 6 to 10.

Examples

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

[0157] In the present invention, the evaluation of the stability of FMN-LDH after heat treatment and the evaluation of the stability test 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 encoding 506 amino acids of the amino acid sequence of lactate dehydrogenase derived from Saccharomyces cereviciae (ScLDH), with the amino acids at positions 2 to 85 removed, was obtained as cDNA by PCR of gene fragments, a conventional method. Subsequently, the 578 amino acids shown in SEQ ID NO: 3, which is the amino acid sequence of lactate dehydrogenase derived from Pichia kudriavzevii (PkLDH), were compared with ScLDH, and a 1509 bp gene (including the stop codon TAA) shown in SEQ ID NO: 5 encoding 502 amino acids of the amino acid sequence shown in SEQ ID NO: 4 with positions 2 to 77 removed was obtained as cDNA by PCR of gene fragments, a conventional method. A DNA construct was prepared by inserting the target gene, the ScLDH gene or the PkLDH gene, into the multiple cloning site of plasmid pKK223-3 by a conventional method. Specifically, at the In-Fusion Cloning Site in the multiple cloning site of pKK223-3, the ScLDH gene or the PkLDH gene was ligated using the In-Fusion HD Cloning Kit (manufactured by Clontech) according to the protocol attached to the kit to obtain expression plasmids (pKK223-3-ScLDH and pKK223-3-PkLDH). Furthermore, Escherichia coli JM109 was transformed using these plasmids, and Escherichia coli JM109(pKK223-3-ScLDH) strain and Escherichia coli JM109(pKK223-3-PkLDH) strain were inoculated into 3 ml of LB-amp medium [1% (w / v) Bacto-Tryptone, 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 respective cultures.

[0159] These cultures were collected by centrifugation at 10,000×g for 1 minute to obtain cells. From these cells, recombinant plasmids pKK223-3-ScLDH and pKK223-3-PkLDH were extracted and purified using the GenElute Plasmid Miniprep Kit (manufactured by Sigma-Aldrich), and 2.5 μg of recombinant plasmid pKK223-3-ScLDH and pKK223-3-PkLDH DNA were obtained.

[0160] (2) Production of LDH E. coli BL21(pKK223-3-ScLDH) strain transformed with pKK223-3-ScLDH was cultured at 25°C for 24 hours 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) strain transformed with pKK223-3-PkLDH was cultured at 37°C for 24 hours in 3 ml of LB-amp medium supplemented with IPTG to a final concentration of 0.1 mM. Each of the obtained cultured cells was washed with 10 mM potassium phosphate buffer (pH 7.5), then suspended in the same buffer and subjected to ultrasonic disruption treatment, and centrifuged at 20,000×g for 10 minutes to prepare 0.6 ml of a crude enzyme solution containing ScLDH or PkLDH.

[0161] (3) Measurement of LDH activity Using the above-mentioned crude enzyme solution containing ScLDH or PkLDH, the oxidative activity against L-lactic acid was measured by the method shown in the following activity measurement method. 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 PkLDH of the present invention can be measured using the following measurement system that utilizes this principle of action and uses, for example, potassium ferricyanide as an electron acceptor.

[0162] (Reaction) L-lactic acid + potassium ferricyanide → pyruvic acid + potassium ferrocyanide

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

[0164] Specifically, the LDH activity can be measured according to the following procedure. Mix 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, and incubate at 30 °C for 5 minutes. Then, add 0.05 mL of the enzyme sample solution to start the reaction. Measure the absorbance at the start of the reaction and over time, determine the decrease amount per minute (ΔA420) of the absorbance at 420 nm with the progress of the enzyme reaction, and calculate the LDH activity according to the following formula. At this time, the LDH activity is defined as 1 U for 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, 1.01 is the millimolar molar absorption coefficient (cm 2 / μmol) under the conditions of this activity measurement, 0.05 is the volume (mL) of the enzyme solution, 1.0 is the optical path length (cm) of the cell, ΔA420blank is the decrease amount per minute of the absorbance at 420 nm when the reaction is started by adding 10 mM phosphate buffer (pH 7.5) instead of the enzyme sample solution, and df represents the dilution factor.

[0167] Example 2 (Temperature stability) The crude enzyme solution was diluted to a 100 mM potassium phosphate buffer (pH 6.0) containing 0.07% BSA as the final concentration, and its temperature stability was examined. Specifically, the LDH enzyme solution prepared in (2) of Example 1 was diluted to 6 U / ml, treated at each temperature (35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C) for 10 minutes, then the LDH activity was measured, and the residual activity rate was measured in comparison with the LDH activity before treatment. Also, for comparison, the same test was conducted for ScLDH. The results are shown in Figure 2. From the results, it was found that ScLDH is unstable at 45°C, while PkLDH is stable even after heat treatment at 55°C.

[0168] Subsequently, the long-term stability at 37°C was examined. Specifically, the LDH enzyme solution was diluted to 6 U / ml with the buffer in the same manner as in the temperature stability test and stored for each time. Then, the residual activity rate after each elapsed time was measured. Also, for comparison, the same test was conducted for ScLDH. The results are shown in Figure 3 (A and B). Figure 3A is a graph of the residual rate after each elapsed time with the residual activity rate at the time when the elapsed time is zero hour set as 100%. From this, it was found that PkLDH heated to a certain extent (in this example, 37°C) has an improved residual activity rate until a certain period during storage (Figure 3A). Figure 3B is a graph of the residual activity rate after each elapsed time with the activity rate at the reference time set as 100% when 15 hours from the start of storage is taken as the reference time. PkLDH retained 99% of its activity even after 245 hours elapsed from the start of storage (230 hours after the reference time), and from 15 hours after the start, the residual activity rate became constant and did not inactivate even after storage at 37°C for about 10 days, showing very high stability (Figure 3B). On the other hand, for ScLDH, the residual activity rate began to decrease significantly from the start of storage, and the activity residual rate was 62% after 21 hours (6 hours after the reference time) and 0% after 65 hours (50 hours after the reference time), indicating instability. Also, there was no significant improvement in stability even when the enzyme amount of ScLDH was evaluated at 20 U / ml.

[0169] Example 3 (Purification of Enzyme) 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) Using the purified PkLDH enzyme solution (10 U / mL) obtained in Example 3, the pH stability was examined. Using 100 mM potassium phosphate buffer (pH 6.0 - pH 7.5), after heat treatment at 55 °C for 15 minutes, the enzyme was maintained in each buffer, and the activity was measured according to the activity measurement method described in (3) of Example 1 except using each of these enzymes. Regarding the condition with the highest activity among the activity values after treatment as 100%, the relative values were determined for the activity values under other conditions. The results are shown in Fig. 7. As a result, it was most stable at pH 6.5, and then had a relative activity of 92% at pH 6.0.

[0171] (Optimal activity pH) Using the purified PkLDH enzyme solution (10 U / mL) obtained in Example 3, the optimal activity pH was examined. Using 100 mM potassium phosphate buffer (pH 6.0 - pH 7.5), at each pH, an enzyme reaction was carried out at a temperature of 30 °C, and the relative activities (%) were compared. The results are shown in Fig. 8. As a result, for the FMN-dependent lactate dehydrogenase of the present invention, the highest activity was shown in the range of pH 6.5 - 7.5 for the optimal activity pH. It is considered that there is a possibility that good activity is also maintained at pH 8.0.

[0172] (Quantification of L-lactic acid) Using the purified PkLDH enzyme solution (17 U / ml) obtained in Example 3, 0.2 - 5 mM of L-lactic acid was measured. The results are shown in Fig. 9. As a result, it was shown that the change amount of absorbance per minute at 420 nm improved depending on the lactic acid concentration, and L-lactic acid could be quantified.

[0173] (Oxidase activity) With reference to the activity measurement method described in International Publication No. 2015 / 020200, PkLDH was used as the enzyme, L-lactic acid with a final concentration of 10 mM was used as the substrate, and the activity measurement was carried out with the measurement pH set to 7.5. The oxidase activity was defined as the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute in the presence of a substrate at a concentration of 10 mM as 1 unit (U). The amount of enzyme used was an amount showing 600 U / ml as dehydrogenase activity. As a result, the oxidase activity of PkLDH was not detected. Therefore, PkLDH was an enzyme that does not use oxygen as an electron acceptor.

[0174] Example 5 (Quantification of L-lactic acid using a printed electrode) Using the purified PkLDH enzyme solution obtained in Example 3, the quantification of L-lactic acid by printed electrode measurement was carried out. Specifically, SCREEN-PRINTED ELECTRODES (manufactured by DropSens, product number DRP-110) on which a carbon working electrode and a silver reference electrode are printed were connected to an ALS electrochemical analyzer 814D (manufactured by BAS) using a dedicated connector (manufactured by DropSens, DRP-CAC). 5 μl of the PkLDH enzyme solution, 20 μl of 100 mM potassium phosphate buffer (pH 7.5) containing 1.5 M potassium chloride, and 25 μl of an aqueous potassium ferricyanide solution were placed on the electrode. Then, a voltage of +400 mV (v.s. Ag / AgCl) was applied, and 5 μL of a lactic acid solution at a predetermined concentration was placed on the electrode for each reaction, and the current value after 120 seconds was measured. As a result, lactic acid could also be quantified in electrochemical measurement.

[0175] Example 6 (4) Preparation of recombinant plasmids pKK223-3-CaLDH DNA and pKK223-3-OgLDH DNA and production of each LDH Of the 571 amino acids represented by SEQ ID NO: 6, which is the amino acid sequence of Candida inconspicua-derived lactate dehydrogenase (CaLDH), the 505 amino acids represented by SEQ ID NO: 7 with positions 2 to 67 removed were encoded by a 1518 bp gene (including the stop codon TAA) represented by SEQ ID NO: 8 and obtained as cDNA by PCR of a gene fragment, which is a conventional method. Similarly, of the 558 amino acids represented by SEQ ID NO: 9, which is the amino acid sequence of Ogataea parapolymorpha-derived lactate dehydrogenase (OgLDH), the 503 amino acids represented by SEQ ID NO: 10 with positions 2 to 56 removed were encoded by a 1512 bp gene (including the stop codon TAA) represented by SEQ ID NO: 11 and obtained as cDNA by PCR of a gene fragment, which is a conventional method. In the same manner as in Example 1, a DNA construct was prepared by inserting the target gene, the CaLDH gene or the OgLDH gene, into the multiple cloning site of plasmid pKK223-3 by a conventional method, and expression plasmids (pKK223-3-CaLDH and pKK223-3-OgLDH) were obtained. Escherichia coli BL21(pKK223-3-CaLDH) strain transfected with pKK223-3-CaLDH and Escherichia coli BL21(pKK223-3-OgLDH) strain transfected with pKK223-3-OgLDH were cultured at 30 °C for 24 hours in 3 ml of LB-amp medium supplemented with IPTG to a final concentration of 0.1 mM. Each of the obtained cultured cells was washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the same buffer, subjected to ultrasonic disruption treatment, and centrifuged at 20,000×g for 10 minutes to prepare 0.6 ml of a 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) with amino acids at positions 2 to 96 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 16 were used; to prepare a mutant (PkLDH-98) with amino acids at positions 2 to 97 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 17 were used; to prepare a mutant (PkLDH-99) with amino acids at positions 2 to 98 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 18 were used; to prepare a mutant (PkLDH-100) with amino acids at positions 2 to 99 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 19 were used; to prepare a mutant (PkLDH-101) with amino acids at positions 2 to 100 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 20 were used; to prepare a mutant (PkLDH-102) with amino acids at positions 2 to 101 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 21 were used; to prepare a mutant (PkLDH-103) with amino acids at positions 2 to 102 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 22 were used; to prepare a mutant (PkLDH-104) with amino acids at positions 2 to 103 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 23 were used; to prepare a mutant (PkLDH-76) with amino acids at positions 2 to 75 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 50 were used; to prepare a mutant (PkLDH-84) with amino acids at positions 2 to 83 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 51 were used; to prepare a mutant (PkLDH-92) with amino acids at positions 2 to 91 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 52 were used; to prepare a mutant (PkLDH-110) with amino acids at positions 2 to 109 deleted in SEQ ID NO: 4, synthetic oligonucleotides of SEQ ID NO: 14 and 53 were used. PCR was performed using each of these synthetic oligonucleotides to obtain DNA constructs encoding PkLDH-76 to PkLDH-110.

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

[0185] The E. coli BL21 strain transfected with the plasmids encoding the obtained various PkLDH mutants was cultured at 30 °C for 24 hours in 3 ml of LB-amp medium supplemented with IPTG to a final concentration of 1 mM. Each of the obtained cultured bacterial cells was washed with 10 mM potassium phosphate buffer (pH 7.5), suspended in the same buffer, and subjected to ultrasonic disruption treatment. After centrifugation at 20,000×g for 10 minutes, 0.6 ml of a crude enzyme solution containing various PkLDH mutants was prepared. For the 13 mutants (PkLDH-76 to PkLDH-110) with the N-terminus deleted, the activity of the crude enzyme solution was 0.3 to 6 times compared to the PkLDH wild type. For the 9 mutants (PkLDH-3 / F428L to PkLDH-11 / F428L) with the N-terminus deleted, the activity of the crude enzyme solution was 2 to 11 times compared to PkLDH / F428L.

[0186] Example 11 (Thermal 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. Subsequently, the crude enzyme solutions (PkLDH / F161L, PkLDH / F187L, PkLDH / F428L) prepared in Example 10 were diluted to a 150 mM potassium phosphate buffer (pH 7.5) containing 0.15% BSA as the final concentration, and the temperature stability was examined. Specifically, the monosubstituted mutant LDH enzyme solution prepared in Example 10 was diluted to 6 U / ml, treated at 55°C for 15 minutes, then the LDH activity was measured, and the residual activity rate was measured in comparison with the LDH activity before treatment. Also, for comparison, the same test was conducted for PkLDH. The results are shown in Fig. 13. It was found that both PkLDH / F161L, PkLDH / F187L, and PkLDH / F428L had a higher residual activity rate and higher thermal stability than PkLDH. Also, the crude enzyme solutions (PkLDH-97 / F428L / L194A, PkLDH-97 / F428L / L222Y, PkLDH-97 / F428L / A274S, PkLDH-97 / F428L / L277S, PkLDH-97 / F428L / F313A, PkLDH-97 / F428L / I314S) prepared in Example 10 were used and treated at 50°C for 15 minutes. Subsequently, as a reagent for measuring LDH activity, the system using the above DCIP and PMS was used. When 5 μL of the enzyme solution after heat treatment and 145 μL of the reagent for measuring LDH activity were added and incubated at 37°C for 5 minutes, clearly the color of the activity measurement reagent changed to yellow. Therefore, it was found that these mutants had higher stability compared to ScLDH, which was completely inactivated by heat treatment at 50°C. Note that the same result was obtained when the activity measurement was carried out in the same manner using 1-methoxyphenazine methosulfate instead of PMS.

[0187] Next, in the same manner as in Example 2, the long-term stability at 37 °C was examined. 100 mM potassium phosphate buffer (pH 6.0) containing 0.07% BSA as the final concentration, various LDH mutants (PkLDH-96, PkLDH-97, PkLDH-104, PkLDH / L386R / T461R / D464R, PkLDH / F428L) were diluted to approximately 20 U / ml and stored at each time. As a result, PkLDH-96 retained 95% activity even after 89 hours from the start of storage, PkLDH-97 retained 114% activity after 43 hours from the start of storage, 114% after 72 hours, and 111% even after 171 hours, PkLDH-104 retained 97% activity after 43 hours from the start of storage, 99% after 72 hours, and 88% even after 171 hours, PkLDH / L386R / T461R / D464R retained 96% activity even after 89 hours from the start of storage, and PkLDH / F428L retained 99% activity after 43 hours, 107% after 72 hours, and 94% even after 171 hours, and was very stable compared to ScLDH. That is, it can be said that these mutants maintained 70% or more of the initial activity when at least 3 days or more had passed at 37 °C. Also, regarding PkLDH-98, PkLDH-99, PkLDH-100, PkLDH-101, PkLDH-102, PkLDH-103, PkLDH / F161L, and PkLDH / F187L, since they have high thermal stability at 55 °C compared to PkLDH, there is a high probability that they are also very stable compared to ScLDH in terms of long-term stability at 37 °C.

[0188] Example 12 (Quantification of L-lactic acid by PkLDH-immobilized electrode) Quantification of L-lactic acid was performed using the electrode immobilized with the purified PkLDH enzyme solution obtained in Example 3. Specifically, 12 U of PkLDH was applied and dried on the working electrode of SCREEN-PRINTED ELECTRODES (manufactured by DropSens, product number DRP-C110) on which a carbon working electrode was printed. Subsequently, 3 μL of 2% poly(ethylene glycol) diglycidyl ether (Mn: 6000, manufactured by Sigma) was applied and reacted at 4 °C for 22 hours. It was washed with ultrapure water to obtain a PkLDH-immobilized electrode. Using a dedicated connector (manufactured by DropSens, DRP-CAC), it was connected to an ALS electrochemical analyzer 814D (manufactured by BAS), 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 of Bindschedler's Green Leuco Base (manufactured by Tokyo Chemical Industry). +200 mV (vs Ag / AgCl) was applied, and the response current values when an L-lactic acid solution was added at regular intervals were recorded. The results are shown in Figure 8. When L-lactic acid of 1 to 7 mM was added, it was shown that the response current increased depending on the lactic acid concentration, and L-lactic acid could be quantified.

Industrial Applicability

[0189] According to the device for evaluating the state of a specimen, which includes a working part for allowing FMN-LDH to act on the specimen of the present invention and a sensor for sensing the state of the specimen on which FMN-LDH has acted, and the method for evaluating the state of a specimen using the same, lactic acid in a lactic acid-containing composition including interstitial fluid, blood, urine, tears, sweat, saliva, skin, meat, eyeball, cornea, gastric juice, food and drink, brewed products, chemical products, water, and soil of humans and non-human organisms can be measured stably, accurately, and simply over a long period. Therefore, it is useful for the health management of humans and animals, or for the manufacturing process management and quality control of food and drink, brewed products, chemical products, etc.

Explanation of Symbols

[0190] 1 Working electrode 3 Counter electrode 5 Reference electrode 7 Wiring part 9 Terminal 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. A device for monitoring the state of a specimen, the device comprising an acting part for acting on the specimen with (A) flavin-dependent lactate dehydrogenase that maintains at least about 20% of its initial activity when incubated in solution at 37°C for 10 days, (B) flavin-dependent lactate dehydrogenase that maintains at least about 20% of its initial activity when incubated in solution at 37°C for 3 days or more, or (C) flavin-dependent lactate dehydrogenase that maintains at least about 20% of its initial activity when incubated in solution at 37°C for 15 hours or more, wherein the flavin-dependent lactate dehydrogenase comprises the amino acid sequence at positions 110 to 502 in the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 90% or more identity thereto, the amino acid sequence at positions 113 to 505 in the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 90% or more identity thereto, the amino acid sequence at positions 112 to 503 in the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having 90% or more identity thereto, or the amino acid sequence at positions 102 to 499 in the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 90% or more identity thereto, a device.

2. A system, wherein the device according to claim 1 further comprises an output part, and the output part is connected to a data processing unit.

3. A method for monitoring the state of a specimen, using the device according to claim 1 or the system according to claim 2.

4. A device for evaluating the state of a specimen, comprising an acting part for acting on the specimen with flavin-dependent lactate dehydrogenase, and a sensor for sensing the state of the specimen on which the flavin-dependent lactate dehydrogenase has acted, the sensor being directly or indirectly arranged so as to be able to sense the state of the specimen at the acting part, wherein the state of the specimen is the physical state in response to an exercise load, a disease state, the brewing state of a fermented product accompanied by a change in the amount of lactic acid, the aging and / or after-ripening degree of a food or beverage accompanied by a change in the amount of lactic acid, the lactic acid content ratio in the production of a chemical product accompanied by a change in the amount of lactic acid, or the amount of lactic acid in water or soil accompanied by a change in the amount of lactic acid, a device according to claim 1.

5. The device according to claim 4, further comprising an output part for outputting a signal from the sensor.

6. The device according to claim 5, and ​ ​ A data processing unit connected to the output unit of the device and for processing signals from the sensor A system comprising the same. **Claim 7** A program for evaluating the state of a specimen by a system including a device and a data processing unit, wherein the device is the device according to claim 1, the program causes the device to perform measurement processing for sensing and measuring, by a sensor, the state of a specimen in which flavin-dependent lactate dehydrogenase is allowed to act on the acting part and converting it into a signal, transfer processing for transferring the signal obtained by the measurement processing from the output unit to the data processing unit and causing the sensor to be directly or indirectly arranged so as to be able to sense the state of the specimen in the acting part, the program causes the data processing unit to execute data processing for performing predetermined processing on the signal obtained by the measurement processing, wherein the state of the specimen to be evaluated is the physical state against exercise load, disease state, brewing state of a fermented product accompanied by a change in lactic acid amount, ripening and / or after-ripening degree of a food or drink accompanied by a change in lactic acid amount, lactic acid content ratio in the production of a processed product accompanied by a change in lactic acid amount, lactic acid amount in water and soil accompanied by a change in lactic acid amount. A program. **Claim 8** A method for providing data for evaluating the state of a specimen by using the device according to claim 4 or 5 or the system according to claim 6, wherein the state of the specimen to be evaluated is the physical state against exercise load, disease state, brewing state of a fermented product accompanied by a change in lactic acid amount, ripening and / or after-ripening degree of a food or drink accompanied by a change in lactic acid amount, lactic acid content ratio in the production of a processed product accompanied by a change in lactic acid amount, lactic acid amount in water and soil accompanied by a change in lactic acid amount. A method. **Claim 9** The specimen is a lactic acid-containing composition including body fluids, interstitial fluids, blood, urine, tears, sweat, saliva, skin, meat, eyeballs, corneas, gastric juice, foods and drinks, fermented products, processed products, water, and soil of humans and non-human organisms, wherein when the specimen is skin, meat, eyeballs, and corneas, the method includes a step of measuring lactic acid derived therefrom. The method according to claim 8. ​

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