Novel flavin-dependent lactate dehydrogenase, and method for improving stability of lactate dehydrogenase

Abstract

The present invention relates to a novel flavin-dependent lactate dehydrogenase, and a method for improving the stability of lactate dehydrogenase.

Description

Novel flavin-dependent lactate dehydrogenase and method for improving the stability of lactate dehydrogenase

[0001] The present invention relates to a novel flavin-dependent lactate dehydrogenase (hereinafter referred to as LDH), a nucleic acid encoding the same, a host cell having the nucleic acid, a method for producing LDH which includes culturing the host cell, and a method for improving the stability of lactate dehydrogenase.

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

[0003] Furthermore, FMN-dependent lactate dehydrogenase (hereinafter referred to as FMN-LDH) is known as an enzyme that uses lactic acid as a substrate. The FMN-LDH derived from Saccharomyces cerevisiae (Non-Patent Document 2) known so far has a problem with stability.

[0004] Special table 2018-519507 publication

[0005] Japanese Sepsis Management Guidelines 2016, PNAS September 15, 2015, 112 (37), 11642-11647 Methods Enzymol. , 53, 238-56, 1978

[0006] Known enzymes that use lactic acid as a substrate include lactate oxidase (hereinafter referred to as LOD), NAD-dependent lactate dehydrogenase (hereinafter referred to as NAD-LDH) which uses nicotinamide dinucleotide (NAD) as a coenzyme, and FMN-LDH which uses flavin mononucleotide (FMN) as a coenzyme.

[0007] LOD has the problem of producing hydrogen peroxide as a reaction product. In particular, when considering applications where lactate sensors are attached to the skin or, in some cases, implanted subcutaneously, it is of concern that using a sensor equipped with an enzyme that can continuously produce hydrogen peroxide, a type of reactive oxygen species and a cause of oxidative stress, is undesirable. Furthermore, hydrogen peroxide produced by the action of LOD adversely affects the stabilization of the LOD itself. To avoid this resulting decrease in LOD activity, a lactate sensor incorporating catalase for the purpose of eliminating hydrogen peroxide has been proposed (Patent Document 1).

[0008] NAD-LDH catalyzes an enzyme reaction that does not produce hydrogen peroxide, so the problem of hydrogen peroxide generation does not occur. However, in the enzyme reaction system using NAD-LDH, lactic acid and pyruvic acid react reversibly, which poses the problem of not being able to obtain accurate measurements for quantitative purposes, making it difficult to use in sensors.

[0009] FMN-LDH also catalyzes an enzyme reaction that does not produce hydrogen peroxide, so there is no problem with the generation of hydrogen peroxide.Furthermore, there is no problem with a reversible reaction.Of the three enzymes mentioned above, FMN-LDH is considered to be the most promising enzyme for practical use in lactate monitoring.

[0010] However, the previously known FMN-LDH derived from Saccharomyces cerevisiae (Non-Patent Document 2) has a problem with stability. An objective of the present invention is to discover a novel flavin-dependent LDH with excellent stability and to provide a method for improving the stability of LDH.

[0011] In view of the above problems, the present inventors have searched for a novel flavin-dependent LDH with excellent stability, discovered a mutant LDH with improved stability, and completed the present invention.

[0012] The present invention encompasses the following aspects: (1) A lactate dehydrogenase having an amino acid sequence selected from the following (i) to (iii): (i) the amino acid sequence shown in SEQ ID NO: 3, (ii) an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 3, or (iii) an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 3 in a region from positions 110 to 502 of SEQ ID NO: 3 when aligned with the amino acid sequence shown in SEQ ID NO: 3, and comprising an N-terminal deletion and / or a mutation at one or more positions selected from the group consisting of positions 54, 156, 349, and 428. (2) The lactate dehydrogenase according to (1), wherein the N-terminal deletion is a deletion at positions 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 75, 2 to 83, or 2 to 91. (3) The lactate dehydrogenase according to (1) or (2) above, wherein the mutation at position 54 is A54C. (4) The lactate dehydrogenase according to any one of (1) to (3) above, wherein the mutation at position 156 is Y156F. (5) The lactate dehydrogenase according to any one of (1) to (4) above, wherein the mutation at position 349 is Y349F. (6) The lactate dehydrogenase according to any one of (1) to (5) above, wherein the mutation at position 428 is F428L. (7) A nucleic acid encoding the lactate dehydrogenase according to any one of (1) to (6) above. (8) A host cell having the nucleic acid according to (7). (9) A method for producing lactate dehydrogenase, comprising culturing the host cell according to (8). (10) A method for improving the stability of lactate dehydrogenase, which has an amino acid sequence selected from the following (i) to (iii): (i) the amino acid sequence shown in SEQ ID NO: 3; (ii) an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 3; or (iii) an amino acid sequence having 70% or more identity to the amino acid sequence shown in SEQ ID NO: 3 in the region from positions 110 to 502 of SEQ ID NO: 3 when aligned with the amino acid sequence shown in SEQ ID NO: 3, and which comprises a step of deleting the N-terminus and / or mutating at one or more positions selected from the group consisting of positions 54, 156, 349, and 428.

[0013] The present invention can provide a novel flavin-dependent LDH having excellent stability, a nucleic acid encoding the same, a host cell having the nucleic acid, a method for producing LDH comprising culturing the host cell, and a method for improving the stability of LDH.

[0014] FIG. 1 shows an alignment of the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:5.

[0015] 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.

[0016] (LDH to which the present disclosure can be applied) The LDH referred to in the present invention refers to an enzyme that catalyzes a reaction of oxidizing the hydroxyl group of lactic acid to produce pyruvic acid in the presence of an electron acceptor, similar to known wild-type or mutant LDHs. Unless otherwise specified, the substrate of LDH is lactic acid, which may be provided as a mixture of L- and D-forms, for example, a racemic form, or as the L-form.

[0017] The LDH of the present invention is (i) an amino acid sequence represented by SEQ ID NO: 3, (ii) an amino acid sequence having a high identity to the amino acid sequence represented by SEQ ID NO: 3, for example, an identity of 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, even more preferably 95% or more, 96% or more, 97% or more, 98% or more, and most preferably 99% or more, or (iii ... a region of positions 110 to 502 of SEQ ID NO: 3 when aligned with the amino acid sequence represented by SEQ ID NO: 3 The LDH of the present invention may have an amino acid sequence selected from amino acid sequences having high identity to the amino acid sequence shown in SEQ ID NO: 3, for example, 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, even more preferably 95% or more, and most preferably 96% or more, 97% or more, 98% or more, or 99% or more, and may contain an N-terminal deletion and / or a mutation at one or more positions selected from the group consisting of positions 54, 156, 349, and 428. More preferably, the LDH of the present invention has an N-terminal deletion at positions 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 75, 2 to 83, or 2 to 91 in SEQ ID NO: 3. More preferably, in the LDH of the present invention having the amino acid sequence shown in SEQ ID NO: 3, the mutation at position 54 is A54C, the mutation at position 156 is Y156F, the mutation at position 349 is Y349F, and the mutation at position 428 is F428L.

[0018] An example of a sequence having a high degree of identity to SEQ ID NO:3 is the amino acid sequence shown in SEQ ID NO:5.

[0019] The LDH of the present invention may be an LDH having the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having high identity thereto, for example, 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, even more preferably 95% or more, 96% or more, 97% or more, 98% or more, and most preferably 99% or more, and containing an N-terminal deletion and / or a mutation at position 52 in SEQ ID NO: 5. More preferably, the LDH of the present invention has an N-terminal deletion at positions 2-3, 2-4, 2-5, 2-6, 2-7, 2-73, 2-81, or 2-89 in SEQ ID NO: 5. More preferably, the mutation at position 52 in the LDH of the present invention having the amino acid sequence shown in SEQ ID NO: 5 (corresponding to position 54 in SEQ ID NO: 3 when aligned with SEQ ID NO: 3) is S52C.

[0020] As described below, the LDH of the present invention exhibits LDH activity only with the amino acid sequence from positions 110 to 502 of the amino acid sequence shown in SEQ ID NO: 3. Therefore, it is clear that the amino acid sequence from positions 110 to 502 of the amino acid sequence shown in SEQ ID NO: 3 is a region that is particularly important for the function (activity) of LDH. The LDH of the present invention may be an LDH having an amino acid sequence that, when aligned with the amino acid sequence shown in SEQ ID NO: 3, has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 3 in a region that is particularly important for the function (activity) of LDH (the region corresponding to positions 110 to 502 of SEQ ID NO: 3).

[0021] (Regarding Amino Acid Sequence Identity) Amino acid sequence identity can be calculated using programs such as maximum matching and search homology in GENETYX Ver. 11 (manufactured by Genetyx Corporation) or maximum matching and multiple alignment in DNASIS Pro (manufactured by Hitachi Solutions).

[0022] (Regarding Corresponding Positions in Amino Acid Sequences) To calculate amino acid sequence identity, an alignment of the LDH shown in SEQ ID NO: 3 with another LDH can be created to determine the positions in another LDH that correspond to specific positions in the LDH of SEQ ID NO: 3. For example, according to Figure 1 (alignment of SEQ ID NO: 3 and SEQ ID NO: 5), positions 5, 6, 7, 54, 75, 83, 91, 110, 156, 349, 428, and 502 of SEQ ID NO: 3 correspond to positions 3, 4, 5, 52, 73, 81, 89, 108, 154, 347, 426, and 500 of SEQ ID NO: 5, respectively. Amino acid sequence alignment can be performed, for example, using CLUSTALW and the Blosum62 algorithm.

[0023] In one embodiment of the present invention, the present invention relates to a nucleic acid encoding the above-mentioned LDH.

[0024] In one embodiment of the present invention, the present invention relates to a host cell comprising the above-mentioned nucleic acid. The host cell is not limited in any way as long as it is a conventional cell used in the art. For example, Escherichia coli K-12 strain and its derivatives (e.g., JM109 strain), Escherichia coli B strain and its derivatives (e.g., BL21 strain), Bacillus subtilis (e.g., Bacillus subtilis, etc.), lactic acid bacteria (e.g., Lactococcus lactis, etc.), other bacteria (e.g., Corynebacterium glutamicum, Brevibacillus choshinensis, etc.), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, etc.), filamentous fungi (e.g., Aspergillus oryzae, A. sojae, A. Niger, etc.) can be used.

[0025] In one embodiment of the present invention, the present invention relates to a method for producing LDH, which comprises culturing the host cell described above.

[0026] In one embodiment, the present invention is a method for improving the stability of LDH, comprising the step of N-terminally deleting an amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having high identity thereto, for example, 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, even more preferably 95% or more, 96% or more, 97% or more, 98% or more, and most preferably 99% or more. According to the present invention, LDH with improved stability can be produced.

[0027] In one embodiment, the present invention is a method for improving the stability of LDH, comprising the step of deleting the N-terminus and / or mutating position 52 in the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having high identity thereto, for example, 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, even more preferably 95% or more, 96% or more, 97% or more, 98% or more, and most preferably 99% or more. According to the present invention, LDH with improved stability can be produced.

[0028] In one embodiment, the present invention relates to a method for improving the stability of LDH, comprising the step of N-terminally deleting and / or mutating one or more positions selected from the group consisting of positions 54, 156, 349, and 428 in the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having a high identity thereto, for example, 70% or more, more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, even more preferably 95% or more, 96% or more, 97% or more, 98% or more, and most preferably 99% or more, in a region particularly important for the function (activity) of LDH (the region corresponding to positions 110 to 502 of SEQ ID NO: 3) when aligned with the amino acid sequence of SEQ ID NO: 3. According to the present invention, LDH with improved stability can be produced.

[0029] LDH activity can be measured using this principle of action, for example, using the following measurement system that uses phenazine methosulfate (PMS) and 2,6-dichloroindophenol (DCIP) as electron acceptors: (Reaction 1) L-lactic acid + PMS (oxidized) → pyruvic acid + PMS (reduced) (Reaction 2) PMS (reduced) + DCIP (oxidized) → PMS (oxidized) + DCIP (reduced)

[0030] Specifically, first, in (Reaction 1), PMS (reduced form) is produced as L-lactic acid is oxidized. Then, in the subsequent (Reaction 2), DCIP is reduced as PMS (reduced form) is oxidized. The degree of disappearance of this "DCIP (oxidized form)" is detected as a change in absorbance at a wavelength of 520 nm, and the enzyme activity can be determined based on this change.

[0031] Specifically, LDH activity can be measured according to the following procedure. 170 μL of 1 M potassium phosphate buffer (pH 7.5), 300 μL of 50 mM DL-lactic acid solution, 250 μL of 1.8 mM DCIP solution, and 680 μL of ultrapure water are mixed and kept at 37°C for 2 minutes or more. Next, 50 μL of 30 mM PMS solution and 50 μL of enzyme sample solution are added to start the reaction. The absorbance is measured at the start of the reaction and over time, and the decrease in absorbance at 520 nm per minute (ΔA ５２０ ) and calculate the LDH activity according to the following formula 1. In this case, 1 U of LDH activity is defined as the amount of enzyme that reduces 1 μmol of DCIP in 1 minute in the presence of 10 mM L-lactic acid at 37° C.

[0032]

[0033] In the formula, 1.5 is the volume (mL) of the reaction reagent + enzyme reagent, and 6.8 is the millimolar absorption coefficient (mM －１ cm －１ ), 0.05 is the volume of the enzyme solution (mL), 1.0 is the optical path length of the cell (cm), ΔA ５２０，ｂｌａｎｋ represents the decrease in absorbance at 520 nm per minute when the reaction was initiated by adding 10 mM potassium phosphate buffer (pH 6.0) containing 0.15% (w / v) bovine serum albumin instead of the enzyme sample solution, and Df represents the dilution factor.

[0034] (Method for Evaluating LDH Thermal Stability) The thermal stability of LDH can be evaluated by heating LDH at a predetermined temperature for a predetermined time and comparing the activity before and after heating. Specifically, a 150 mM potassium phosphate buffer (pH 7.5) containing LDH and a final concentration of 0.15% (w / v) bovine serum albumin is placed on ice and heated for 15 minutes at a predetermined temperature (e.g., 45°C, 50°C, 55°C, or 60°C), and the LDH activity is then measured. The activity of the LDH solution after heating can be calculated by setting the LDH activity of the LDH solution that was placed on ice without heat treatment to 100, and the residual activity rate (%) can be measured.

[0035] (Improvement of LDH Stability) In the present disclosure, "improved stability" includes not only "improved thermal stability in a solution state," but also "improved thermal stability in a dried state," or "improved thermal stability in a drying process," or "improved storage stability (long-term stability) in a solution state," or "improved storage stability (long-term stability) in a dried state." In other words, "improved stability" in the present disclosure means that, in a composition containing LDH in the presence of a specific stabilizer, the residual activity rate (%) of LDH maintained after heat treatment for a certain period of time under certain temperature conditions or after long-term storage is increased compared to before the introduction of the mutation.

[0036] Specifically, the residual activity rate (%) is obtained by measuring the LDH activity value (a) of the solution before heat treatment or long-term storage and the LDH activity value (b) after heat treatment or long-term storage, and then calculating ((b) / (a) × 100). The residual activity rate (%) of the LDH after heat treatment or long-term storage following mutation introduction is calculated and designated as A. The residual activity rate (%) of the LDH before mutation introduction, which is used as a comparison, is calculated by subjecting it to the same treatment and then calculating B. If A / B>1, the stability of the LDH is evaluated to have been improved.

[0037] The present disclosure will be described in more detail below with reference to examples, although the technical scope of the present disclosure is not limited to these examples in any way.

[0038] 1. Preparation of LDH Expression Plasmids Preparation of Various Recombinant Plasmids The 578 amino acids shown in SEQ ID NO: 1, which is the amino acid sequence of Pichia kudriavzevii-derived LDH (PkLDH), were compared with the 506 amino acids shown in SEQ ID NO: 2, which is Saccharomyces cerevisiae-derived LDH (ScLDH). A 1,509 bp gene (including the stop codon TAA) shown in SEQ ID NO: 4, encoding the 502 amino acids shown in SEQ ID NO: 3, in which positions 2 to 77 of SEQ ID NO: 1 were deleted, was obtained as double-stranded DNA by PCR of the gene fragment, according to a standard method. Similarly, a 1,503 bp gene (including the stop codon TAA) shown in SEQ ID NO: 6, encoding the 500 amino acids shown in SEQ ID NO: 5, was obtained as double-stranded DNA by PCR of the gene fragment, according to a standard method. These genes were inserted into the multicloning site of the plasmid pKK223-3 by a conventional method to obtain the recombinant plasmid pKK223-3-PkLDH.

[0039] 2. Preparation of Mutant PkLDH Expression Plasmids Using the obtained wild-type (SEQ ID NO: 3) or mutant PkLDH expression plasmid pKK223-3-PkLDH as a template, PCR was performed using the synthetic oligonucleotides of SEQ ID NOs: 7-27 and KOD-One PCR Master Mix (Toyobo Co., Ltd.) under the following conditions. The primer sets used to prepare the mutant PkLDH expression plasmids are summarized in Table 1. Specifically, 25 μL of KOD-One PCR Master Mix, 50 ng of pKK223-3-PkLDH, and 15 pmol of each of the above synthetic oligonucleotides were added, and the total volume was adjusted to 50 μL with sterilized water. The prepared reaction solution was incubated at 94 ° C for 2 minutes using a thermal cycler (Bio-Rad), followed by 7 to 15 cycles of "98 ° C, 10 seconds" - "55 ° C, 5 seconds" - "68 ° C, 40 seconds". The DNA thus obtained was treated with the restriction enzyme DpnI (NEW ENGLAND BIOLABS), and the remaining template DNA was cleaved. When introducing a point mutation, this reaction solution was used as is to transform E. coli JM109 and developed on LB-100 μg / mL ampicillin (hereinafter referred to as Amp) agar medium. When deleting a part of the amino acid sequence, 2.0 μL of the DpnI-treated PCR product was added to Ligation high Ver. 5.0 μL of 2 (Toyobo Co., Ltd.), 1.0 μL of 5 U / μL T4 polyucleotide kinase (Toyobo Co., Ltd.), and 7.0 μL of sterilized water were added and reacted at 16 ° C for 1 hour. The resulting mixture was used for transformation. The grown colonies were inoculated into 2.5 mL of LB-Amp medium [1% (w / v) bactotryptone, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, 100 μg / mL Amp] and cultured with shaking at 37 ° C for 20 hours to obtain a culture. This culture was centrifuged at 15,000 rpm for 5 minutes to collect the bacteria. Next, the recombinant plasmid was extracted from the bacteria using FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.) and purified to obtain DNA.

[0040]

[0041] 3. Preparation of Various LDH Crude Enzyme Solutions Escherichia coli JM109 or BL21 was used as the LDH-producing bacterium. First, a colony of transformed Escherichia coli JM109 or BL21 was cultured on an LB plate medium (containing 100 μg / mL Amp) in advance, picked with a toothpick, and cultured in a small test tube containing 4 mL of LB medium (containing 100 μg / mL Amp and 0.1 mM isopropyl-β-thiogalactopyranoside (hereinafter referred to as IPTG)) at 30°C for 20 hours with shaking at 180 rpm.

[0042] After completion of the cultivation, the culture medium was centrifuged at 7,000 rpm at 4°C for 5 minutes, the supernatant was removed, and the bacterial cells were recovered. The obtained bacterial cells were then suspended in 600 μL of 150 mM potassium phosphate buffer (pH 7.5). The above bacterial cell suspension was subjected to ultrasonic disruption using an ultrasonic homogenizer US-150E (manufactured by Nippon Seiki Seisakusho) until the suspension became translucent, and then centrifuged at 15,000 rpm at 4°C for 5 minutes to recover the supernatant. The enzyme activity was measured using the supernatant of this bacterial cell disruption solution (hereinafter referred to as crude enzyme).

[0043] 4. Verification of the Effect of Various Mutations on the Stability of PkLDH

[0049] 1. Using the crude PkLDH enzyme obtained above, mutations that improve the stability of PkLDH were searched for by comparing the residual activity (%) of LDH before and after the introduction of mutations, according to the method for evaluating LDH thermal stability described above. LDH thermal stability was evaluated by heating LDH at a predetermined temperature for a predetermined time and comparing the activity before and after heating. Specifically, 150 mM potassium phosphate buffer (pH 7.5) containing LDH and a final concentration of 0.15% (w / v) bovine serum albumin was placed on ice and heated at 55°C for 15 minutes, after which the LDH activity was measured. Furthermore, the LDH activity of an LDH solution that had been left on ice without heat treatment was measured, and the activity of the LDH solution after heating was calculated by setting the activity at 100, and the residual activity (%) was measured. Specifically, the residual activity rate (%) was calculated by measuring the LDH activity value (a) of the solution before heat treatment or long-term storage and the LDH activity value (b) after heat treatment or long-term storage, and then calculating ((b) / (a) × 100). The degree of the effect of introducing various mutations to improve LDH stability was evaluated by measuring the residual activity rate (%) of LDH after introducing various mutations and comparing it with the residual activity rate (%) before mutation introduction. Specifically, the relative value of the residual activity rate of the enzyme after mutation introduction was calculated when the residual activity rate of the comparison crude enzyme was set to 1, and values ​​greater than 1 were evaluated as having an improved stability effect. For example, if the residual activity rate of the comparison crude enzyme was 80% and the residual activity rate of the enzyme after mutation introduction was 85%, the relative value of the residual activity would be 1.06. LDH activity was measured according to the following procedure. 170 μL of 1 M potassium phosphate buffer (pH 7.5), 300 μL of 50 mM L-lactic acid solution, 250 μL of 1.8 mM DCIP solution, and 680 μL of ultrapure water were mixed and incubated at 37°C for 2 minutes or more. Next, 50 μL of 30 mM PMS solution and 50 μL of enzyme sample solution were added to start the reaction. The absorbance was measured at the start of the reaction and over time, and the decrease in absorbance at 520 nm per minute (ΔA ５２０) was determined, and the LDH activity was calculated according to the following formula 1. In this case, 1 U of LDH activity was defined as the amount of enzyme that reduces 1 μmol of DCIP in 1 minute in the presence of 10 mM L-lactic acid at 37° C.

[0044]

[0045] In the formula, 1.5 is the volume (mL) of the reaction reagent + enzyme reagent, and 6.8 is the millimolar absorption coefficient (mM －１ cm －１ ), 0.05 is the volume of the enzyme solution (mL), 1.0 is the optical path length of the cell (cm), ΔA ５２０，ｂｌａｎｋ represents the decrease in absorbance at 520 nm per minute when the reaction was initiated by adding 10 mM potassium phosphate buffer (pH 6.0) containing 0.15% (w / v) bovine serum albumin instead of the enzyme sample solution, and Df represents the dilution factor.

[0046] According to the above-mentioned method for evaluating thermal stability, the residual activity of LDH was calculated using an LDH solution (2-3 U / mL) containing 150 mM potassium phosphate buffer and 0.15% (w / v) bovine serum albumin (BSA).

[0047] Table 2 shows the relative values ​​of the residual activity rate of LDH containing an N-terminal deletion according to the present invention, with the residual activity rate of wild-type PkLDH shown in SEQ ID NO: 3 (comparative example) set to 1. Table 3 also shows the relative values ​​of the residual activity rate of LDH containing an N-terminal deletion according to the present invention, with the residual activity rate of LDH having F428L in wild-type PkLDH shown in SEQ ID NO: 3 (invention 4) set to 1.

[0048] As a result of intensive investigation focusing on the amino acids on the N-terminal side of PkLDH, as shown in Tables 2 and 3, deletions of positions 2 to 75 (ΔA2 to L75), deletions of positions 2 to 83 (ΔA2 to V83), deletions of positions 2 to 91 (ΔA2 to L91), F428L mutation and deletion of position 2 (F428L / ΔA2), F428L and deletion of positions 2 to 3 (F42 It was revealed that the thermostability of PkLDH was improved by introducing F428L and a deletion of positions 2 to 4 (F428L / ΔA2-T3), F428L and a deletion of positions 2 to 4 (F428L / ΔA2-G4), F428L and a deletion of positions 2 to 5 (F428L / ΔA2-S5), F428L and a deletion of positions 2 to 6 (F428L / ΔA2-D6), and F428L and a deletion of positions 2 to 7 (F428L / ΔA2-S7) into PkLDH. Furthermore, although the thermostability of a mutant in which positions 2 to 109 of PkLDH were deleted (ΔA2-N109) was not evaluated, LDH activity was detected.

[0049]

[0050]

[0051] 5. Verification 2 of the Effect of Accumulation of Mutations on the Improvement of PkLDH Stability Table 4 shows the relative residual activity rates of LDHs according to the present invention further containing the Y156F mutation, Y349F mutation, and F428L mutation, relative to the residual activity rate of LDH (Invention 6) containing F428L in wild-type PkLDH shown in SEQ ID NO: 3 and deletion at positions 2 and 3 (Invention 6), which is set to 1.

[0052] As a result of extensively searching for mutations that would further improve the stability of the F428L / ΔA2 to T3 obtained above, it was revealed that, as shown in Table 4, the introduction of the Y156F mutation, the Y349F mutation, and the F428L mutation resulted in higher stability than the original.

[0053]

[0054] 6. Verification 3 of the Effect of Accumulation of Mutations on the Improvement of PkLDH Stability Table 5 shows the relative residual activity rates of an LDH further containing A54C and an amino acid sequence shown in SEQ ID NO: 5 (hereinafter referred to as PkLDH-C), with the residual activity rate of an LDH (Invention 12) having Y156F, Y349F, and F428L in the wild-type PkLDH shown in SEQ ID NO: 3 and deletions at positions 2 and 3 set to 1.

[0055] Mutations that further improve the stability of the PkLDH / Y156F / Y349F / F428L / ΔA2-T3 obtained above were searched for extensively. As a result, as shown in Table 5, it was revealed that an LDH into which the A54C mutation had been introduced and an LDH having the amino acid sequence shown in SEQ ID NO: 5 have higher stability than the original LDH.

[0056] For reference, the results of an alignment of the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 5 are shown in FIG.

[0057] Subsequently, a thorough search for mutations that further improve the stability of LDH was conducted, and as a result, it was revealed that PkLDH-C into which the S52C mutation (corresponding to the A54C mutation in SEQ ID NO: 3) was introduced had higher stability than the original, as shown in Table 6.

Claims

1. The following (i) to (iii): (i) the amino acid sequence shown in SEQ ID NO: 3; (ii) an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 3, or (iii) an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 3 in the region from positions 110 to 502 of SEQ ID NO: 3 when aligned with the amino acid sequence shown in SEQ ID NO: 3 and comprising an N-terminal deletion and / or a mutation at one or more positions selected from the group consisting of positions 54, 156, 349, and 428.

2. 2. The lactate dehydrogenase according to claim 1, wherein the N-terminal deletion is at positions 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 75, 2 to 83, or 2 to 91.

3. The lactate dehydrogenase according to claim 1 or 2, wherein the mutation at position 54 is A54C.

4. The lactate dehydrogenase according to claim 1 or 2, wherein the mutation at position 156 is Y156F.

5. The lactate dehydrogenase according to claim 1 or 2, wherein the mutation at position 349 is Y349F.

6. The lactate dehydrogenase according to claim 1 or 2, wherein the mutation at position 428 is F428L.

7. A nucleic acid encoding the lactate dehydrogenase according to any one of claims 1 to 6.

8. A host cell comprising the nucleic acid of claim 7.

9. A method for producing lactate dehydrogenase, comprising culturing the host cell of claim 8.

10. The following (i) to (iii): (i) the amino acid sequence shown in SEQ ID NO: 3; (ii) an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 3, or (iii) an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 3 in the region from positions 110 to 502 of SEQ ID NO: 3 when aligned with the amino acid sequence shown in SEQ ID NO: 3 and a step of N-terminally deleting a sequence selected from the group consisting of positions 54, 156, 349, and 428, and / or mutating the sequence at one or more positions selected from the group consisting of positions 54, 156, 349, and 428.