WO2018101403A1 - Protein (enzyme) for l-threonine dehydrogenation, and method for screening and method for preparing proteins having target activity - Google Patents

Abstract

The present invention relates to a protein having L-threonine dehydrogenase activity (including for L-threonine analysis), a method for screening said protein, a method for preparing said protein, a method for screening and a method for preparing proteins having a target activity, a method for preparing a novel synthetic protein having a target enzyme activity such as L-threonine dehydrogenase activity, and a method for analyzing L-threonine. Provided is a protein (enzyme) having L-threonine dehydrogenation activity suited for use in L-threonine analysis, the protein having higher affinity than in the past for NAD⁺ as well as high enzyme stability. Provided are a novel method for screening enzymes having a target activity on the basis of amino acid sequence data, irrespective of the enzyme name registered in a database, and a method for preparing enzymes screened by this method. Provided is a method for preparing proteins having a target enzyme activity, not limited to existing proteins. Provided is a method for analyzing L-threonine using newly discovered proteins having L-threonine dehydrogenation activity.

Description

L-threonine dehydrogenation protein (enzyme), screening method and preparation method of protein having target activity

The first aspect of the present invention is:
The present invention relates to a protein having L-threonine dehydrogenase activity (including L-threonine analysis), a screening method thereof, and a preparation method thereof (claims 1 to 3, 18 to 21).
The second aspect of the present invention is:
The present invention relates to a screening method and a preparation method of a protein having a target activity (claims 4 to 12 and 17).
The third aspect of the present invention is:
The present invention relates to a method for preparing a novel synthetic protein having a target enzyme activity such as L-threonine dehydrogenase activity (claims 13 to 17).
The fourth aspect of the present invention relates to a method for analyzing L-threonine (claim 22).
This application claims the priority of Japanese Patent Application No. 2016-234857 filed on Dec. 2, 2016, the entire description of which is specifically incorporated herein by reference.

L- threonine dehydrogenase (EC 1.1.1.103, TDH) is a hydroxy group of L- threonine NAD ⁺ as a coenzyme (L-Thr) and dehydrogenation of 2-amino-3-Ketobuchiru acid and NADH It is an enzyme that produces. TDH is classified into those belonging to the medium chain dehydrogenase / reductase family requiring zinc and those belonging to the short chain dehydrogenase / reductase family not requiring metal (SDR-TDH). Enzymes belonging to the medium chain dehydrogenase / reductase family are known to have substrate specificity not specific to threonine. On the other hand, the presence of an enzyme specific for threonine is known as an enzyme belonging to the short-chain dehydrogenase / reductase family. To date, there have been reported prokaryotic-derived TDH derived from Cupriavidus necator (CnTDH), Flavobacterium frigidimaris (FfTDH), and Thermoplasma volcanium (TvoTDH) (Patent Document 1, Non-Patent Document 5, Non-Patent Document 6). ).

By the way, in recent years, a method using amino acid sequence data derived from a large amount of genes including an enzyme gene has been reported as a method for developing an enzyme having a novel function. For example, with the development of gene sequence analysis techniques represented by next-generation sequencers, amino acid sequence data derived from a large amount of genes including enzyme genes is registered in a database such as PubMed. It is possible to extract similar amino acid sequences from other species by analyzing these registered large amounts of data using a database analysis tool such as BLASTp. However, not all of the extracted sequences of enzymes have the desired function. Therefore, it is necessary to further select a sequence having a target function from the extracted sequence. A great deal of effort is required to select sequences having this function. For this reason, a method for confirming the Genbank format file of each extracted sequence and selecting a sequence having the same name as the target enzyme has been developed as a simplified method (Non-patent Document 1).

It was shown that sequences having triose phosphate isomerase (TIM) 選 別 activity can be correctly selected using this technique. However, with this method, efficient sorting is difficult, and development of a simpler and more efficient selection method has been desired.

Therefore, a selection method that combines the three-dimensional structure analysis results of a large number of proteins accumulated so far has been studied, and the usefulness of the method of narrowing down the sequence based on the three-dimensional structure after selecting the sequence by the above method is reported. It was done. Specifically, this method identifies the amino acid residue at the active center (amino acid residue located within 5 cm of the substrate) from the crystal structure of the enzyme and substrate complex, and selects the sequence having that residue. It is. The superfamily of R-amine transaminases and PLP-dependent enzymes is selected and classified by this method (Non-patent Documents 2 and 3).

Patent Document 1: WO2011 / 108727

Non-Patent Document 1: Sullivan, B. J., Durani, V., Magliery, T. J., Triosephosphate isomerase by consensus design: dramatic differences in physical properties and activity of related variants. J. Mol. Biol. (2011) 413: 195-208
Non-Patent Document 2: Hohne, M., Schatzle, S., Jochens, H., Robins, K., Bornscheuer, UT Rational assignment of key motifs for function guides in silico enzyme identification. Nat. Chem. Biol. (2010) 6: 807-813
Non-Patent Document 3: Steffen-Munsberg, F., Vickers, C., Kohls, H., Land, H., Mallin, H., Nobili, A., Skalden, L., van den Bergh, T., Joosten , HJ, Berglund, P., Hohne, M., Bornscheuer, UT Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications. Biotechnol. Adv. (2015) 33: 566-604
Non-Patent Document 4: Morley, K. L., Kazlauskas, R. J. Improving enzyme properties: when are closer mutations better? Trends in Biotech. (2005) 23: 231-237
Non-Patent Document 5: Yoneda, K. Sakuraba, H. Muraoka, I. Oikawa, T. Ohshima, T. Crystal structure of UDP-galactose 4-epimerase-like L-threonine dehydrogenase belonging to the intermediate short-chain dehydrogenase-reductase superfamily. FEBS J. (2010) 277: 5124-5132
Non-Patent Document 6: Yoneda, K., Sakuraba, H., Araki, T., Ohshima, T. Crystal Structure of Binary and Ternary Complexes of Archaeal UDP-galactose 4-Epimerase-like L-Threonine Dehydrogenase from Thermoplasma volcanium. Biol. Chem. (2012) 287: 12966-12974
Non-Patent Document 7: Jackel et al. J. Mol. Biol. 2010, 399: 541-546

<Enzyme with desired activity, L-threonine dehydrogenase>
However, TDH described in Patent Literature 1, Non-Patent Literature 5 and Non-Patent Literature 6 has drawbacks such as low affinity for NAD ⁺ or low enzyme stability. In order to accurately measure L-Thr in biological components, development of a novel TDH suitable for L-Thr accuracy analysis that overcomes these drawbacks is desired.

The first object of the present invention is to have L-threonine dehydrogenation activity suitable for L-threonine analysis, having L-threonine dehydrogenation activity that has higher affinity for NAD ^{+ and} higher enzyme stability. It is in providing the protein (enzyme) which has.

By the way, as a method for finding a high-performance enzyme that can be used for measuring L-Thr with high accuracy, there are methods using search from the natural world and gene information. However, each method has a low probability of searching for a high-performance L-threonine dehydrogenase, and a new search method and a new enzyme development method are desired.

Both the method described in Non-Patent Document 1 and the methods described in Non-Patent Documents 2 and 3 are methods for finding a new sequence from a group of sequences registered with the same name as the target enzyme. Therefore, it is difficult to select a sequence having a target function from a group of sequences registered with a name different from that of the target enzyme.

Therefore, the second object of the present invention is to provide a new method for screening an enzyme having a target activity from amino acid sequence data regardless of the enzyme name registered in the database, and a method for preparing the enzyme screened by this method. It is to provide.

In addition, there is a report (Non-patent Document 4) that an amino acid residue located at least 5 mm away from the active center is important for exerting enzyme specificity and substrate selectivity. However, there are no reports of selecting amino acid sequences of enzymes with the objective function from the database using information on amino acid residues located at least 5 mm away from the substrate bound to the active center.

As described above, the conventional method for providing a protein having the target enzyme activity is to search for a protein having the target enzyme activity from the existing proteins (the amino acid sequence is known but the activity is unknown) as described above. The way to do is central. However, in this method, the search target is limited to existing proteins.

Therefore, a third object of the present invention is to provide a method for preparing a protein having a target enzyme activity, not limited to an existing protein.

Furthermore, a fourth object of the present invention is to provide a method for analyzing L-threonine using a newly discovered protein having L-threonine dehydrogenation activity.

The present invention is as follows.
[1]
The protein for L-threonine analysis according to any of (1) to (3) below.
(1) a protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, 5 to 10 in the sequence listing;
(2) having an amino acid sequence having 1 to 50 amino acid substitutions, deletions and / or additions in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, 5 to 10 in the sequence listing; A protein having threonine dehydrogenase activity,
(3) It has an amino acid sequence having 90% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, and 5 to 10 and has L-threonine dehydrogenase activity. protein.
[2]
Any one of the following proteins (1) to (3).
(1) a protein having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing;
(2) a protein having an amino acid sequence having 1 to 50 amino acid substitutions, deletions and / or additions in the amino acid sequence set forth in SEQ ID NO: 4 of the Sequence Listing, and having L-threonine dehydrogenase activity;
(3) A protein having an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing and having L-threonine dehydrogenase activity.
[3]
A method for preparing L-threonine dehydrogenase, comprising screening a protein having L-threonine dehydrogenase activity from a protein having an amino acid sequence set forth in any of SEQ ID NOs: 11 to 86 in the sequence listing.
[4]
A method for screening a protein having a target enzyme activity, comprising the following steps (A) to (D):
(A) Using a sequence alignment of amino acid sequences of proteins, a library consisting of a plurality of proteins having amino acid sequences similar to the amino acid sequences of known proteins having the target enzyme activity is created from a group of proteins having known amino acid sequences. To
(B) identifying at least two correlated residues from the amino acid sequence of a known protein having the target enzyme activity and the amino acid sequence of the protein selected in (A),
(C) From the protein library prepared in (A), a protein having the correlated residue specified in (B) is selected.
(D) It is confirmed whether or not the protein selected in (C) has the target enzyme activity.
[5]
The similar amino acid sequence in (A) has a sequence identity of 5 to 90%, preferably 10 to 70%, with the amino acid sequence of a known protein having the target enzyme activity obtained using BLASTp. The method according to [4], wherein the amino acid sequence is in the range of 15 to 50%.
[6]
The protein library created in (A) contains 10 to 100,000 proteins, preferably 100 to 50,000 proteins, more preferably 500 to 10,000 proteins [4] or [ 5].
[7]
Correlation residues in (B) are 50% or less conservative from the sequence alignment results for known proteins having the target enzyme activity and at least two proteins selected in (A). The method according to any one of [4] to [6], wherein the method is specified by selecting a pair of amino acid residues in which the type of is changed.
[8]
As for the correlation residue in (B), both of the two residues constituting the correlation residue are present at a distance of 5 mm or more from the active center of the protein in the three-dimensional structure of a known protein having the target enzyme activity. The method according to any one of [4] to [7], wherein
[9]
The method according to any one of [4] to [8], wherein the number of correlation residues selected in (B) is two.
[10]
The protein group whose amino acid sequence is known in (A) is:
The method according to any one of [4] to [8], wherein the protein is a protein registered with a protein registration organization and a protein encoded by a nucleic acid sequence registered with a nucleic acid sequence registration organization.
[11]
The method according to [10], wherein the protein registration organization is at least one selected from the group consisting of Genebank and PubMed, and the nucleic acid sequence registration organization is DDBj.
[12]
[4] A method for producing a protein having a target enzyme activity, comprising preparing a protein screened by the method according to any one of [11].
[13]
A method for producing an artificial protein having a target enzyme activity, comprising the steps (E) to (G).
(E) Create a candidate library consisting of a plurality of proteins having the target enzyme activity,
However, the protein contained in the candidate library may be a protein presumed to have the target enzyme activity.
(F) aligning the candidate library with an amino acid sequence of a known protein having the target enzyme activity, and identifying the most frequently occurring amino acid residue as a consensus residue;
(G) A protein having an amino acid sequence composed only of the consensus residue is designed, and the designed protein is prepared.
[14]
The method according to [13], wherein the candidate library in (E) comprises a plurality of proteins having the target enzyme activity screened by the method according to any of [4] to [11].
[15]
The method according to [13] or [14], wherein the consensus residue in (F) is identified using software capable of performing multiple sequence alignment.
[16]
The method according to [15], wherein the software is INTMSAlign or ClustalW.
[17]
The target enzyme activity is an enzyme group registered in BRENDA, and is selected from the enzyme activities of an enzyme to which an enzyme number (EC number) is given, according to any one of [4] to [16] Method.
[18]
A polynucleotide encoding the protein according to any one of (1) to (3) of [2].
[19]
A recombinant vector comprising a plasmid and the polynucleotide according to [18] inserted into the plasmid.
[20]
A transformant comprising a host and the polynucleotide according to [18] or the recombinant vector according to [19] contained in the host.
[21]
[A transformant according to [20] or a host transformed with a plasmid containing a polynucleotide encoding the protein according to any one of (1) to (3) of [2] is cultured, and the culture is subjected to [From [2] A method for producing L-threonine dehydrogenase, comprising collecting a protein having L-threonine dehydrogenase activity.
[22]
The protein according to [1],
The protein according to [2],
[3] A method for measuring L-threonine concentration in a subject using L-threonine dehydrogenase prepared by the method according to any one of [12] to [16] and [21].

According to the first aspect of the present invention (Claims 1 to 3, 18 to 21), L-threonine dehydrogenation activity having higher affinity for NAD ⁺ than that of the conventional product and higher enzyme stability, A protein (enzyme) having L-threonine dehydrogenation activity suitable for threonine analysis can be provided. By using the enzyme has high affinity for NAD ^+, it is possible to reduce the NAD ⁺ amount used in combination.
According to the second aspect of the present invention (claims 4 to 12 and 17), a protein having a target activity can be screened and prepared regardless of the enzyme name registered in the database.
According to the third aspect of the present invention (claims 13 to 17), a method for preparing a novel synthetic protein having a target enzyme activity such as L-threonine dehydrogenase activity can be provided. There is a possibility that a new synthetic protein can provide a protein with a new function such as higher thermal stability than an existing protein (enzyme).
According to the fourth aspect of the present invention (claim 22), a novel method for analyzing L-threonine can be provided. In this method, affinity for NAD ⁺ is or higher than the conventional enzyme, the thermal stability can be used with high L- threonine dehydrogenase, in the case of using the enzyme has high affinity for NAD ⁺ is used in combination When an enzyme that can reduce the amount of NAD ⁺ and has high thermostability is used, analysis at a high temperature and analysis for a long time are possible.

The positions of four amino acid residues predicted to be necessary for exerting L-threonine dehydrogenation activity, Val143, Leu174, Tyr188, and Met214 on the CnTDH structure are shown. FIG. 2 shows a schematic diagram relating to step (C) of the method for extracting an amino acid sequence having L-threonine dehydrogenation activity from an amino acid sequence database. A part of an output file obtained by analyzing a TDH candidate sequence library using INTMSAlign is shown. Residues shown in red are defined as consensus residues. The stability of TvTDH with respect to temperature at pH 7.0 is shown. The stability of TaTDH with respect to temperature at pH 7.0 is shown. The stability of mtTDH with respect to temperature at pH 7.0 is shown. (▽), の shows the change in residual activity when glucose is not added, (▼), when glucose is added at a final concentration of 20%. The stability of ThrDH_4fix with respect to temperature at pH 7.0 is shown. TvTDHH (sequence 1) shows TDH activity at different pH. (□), ナ ト リ ウ ム Sodium acetate buffer (pH 3.5-5.5), (○), Bis-tris-HCl buffer (pH 6.0-7.0), (△), Tris-HCl buffer (pH 7.0-8.5), ( ▽), BICIN buffer (pH 9.0-9.5), (◇), glycine-KOH buffer (pH 10.0-11.5). Figure 2 shows TDH activity at different pH of TaTDH ~ (sequence 2) ~. The notation of the buffer type is the same as in FIG. Figure 5 shows TDH activity at different pH of mtTDH (Sequence 3). The notation of the buffer type is the same as in FIG. The TDH activity at different pH of ThrDH_4fix (sequence 4) is shown. The notation of the buffer type is the same as in FIG. An enzymatic calibration curve of L-threonine using TvTDH is shown. An enzymatic calibration curve of L-threonine using TaTDH is shown. The enzymatic calibration curve of L-threonine using mtTDH is shown. The enzymatic calibration curve of L-threonine using ThrDH_4fix is shown. The enzymatic calibration curve by the endpoint method of L-threonine using ThrDH_4fix is shown.

The amino acid sequence selection methods reported so far are applicable only when the sequence extracted by BLASTp is registered with the correct enzyme name and the substrate recognition mechanism of the enzyme can be predicted from the three-dimensional structure. However, examples in which sequences extracted by BLASTp are registered with the correct enzyme names are extremely limited. For example, Patent Document 1, 0028 has many similarities when TDH (SDR-TDH) is targeted. It is described that the sequence is registered with a name such as NADmerdependent epimerase_dehydratase or UDP glucose 4-epimerase (GalE), and it cannot be inferred from the registered name that it has SDR-TDH activity. The present inventors also registered about 75% of the sequence names including 3 sequences whose TDH activity was confirmed among 85 sequences selected from the database by the method of the present invention described later under names other than L-threonine 3-dehydrogenase. It had been.

Therefore, in the present invention, a new method for selecting a sequence having a TDH function was developed with reference to the three-dimensional structure of SDR-TDH and CnTDH whose three-dimensional structure has been elucidated, and registered with a name different from the target enzyme. Provided is a method for easily selecting a sequence having a target function from a wide range of sequence groups including a sequence group.

Specifically, sorting was performed according to the following procedure. Actual operating conditions and methods are detailed in the examples.
(i) First step: CnTDH-like sequences are selected by BLASTp (including sequences not registered as TDH).
(ii) Second stage: Using the sequence extracted in the first stage, a plurality of amino acid residues that are separated from the active center by 5 mm or more but are estimated to play an important role in TDH activity (in the present invention, Predict 4 residues (Val143, Tyr188, Met214, Leu174).
(iii) Third stage: Select a total of 85 amino acid sequences having the predicted 4 residues.

(iv) Fourth stage: Select three sequences having low homology with CnTDH from 85 selected, and perform functional analysis such as substrate specificity.
(v) Fifth stage: Based on the extracted 85 sequences, create a sequence (amino acid sequence that does not exist in nature, called a consensus residue sequence) created with the most frequently occurring amino acid residues in each amino acid residue. Investigate the substrate specificity of the protein of the complete consensus sequence.

(iv) is for demonstrating that the sequence protein of 85 sequences extracted in (iii) has TDH activity, and (v) is a study in which artificial proteins designed with full consensus have the same function. In some cases, there is no phylogenetic bias in the sequences used for the complete consensus design, and the knowledge that all the proteins of the sequences used for the design have a high target function (Non-Patent Document 1, Non-Patent Document 1) Based on the literature 7), it is for showing the possibility that all 85 proteins selected by the method of the present invention have TDH activity, and 85 selected proteins have TDH activity. Alternatively, it is also shown in the Examples that the probability of having TDH activity is high.

Furthermore, among the above 85 proteins, there are proteins with novel amino acid sequences and proteins with known sequences that specifically act on L-Thr and have excellent thermal stability, Also found that these proteins can be used in an excellent method for the determination of L-threonine.

The present invention is an invention made based on the above findings.

<Method of screening for protein having target enzyme activity>
A method for screening a protein having a target enzyme activity, comprising the following steps (A) to (D):
(A) Using a sequence alignment of amino acid sequences of proteins, a library consisting of a plurality of proteins having amino acid sequences similar to the amino acid sequences of known proteins having the target enzyme activity is created from a group of proteins having known amino acid sequences. To
(B) identifying at least two correlated residues from the amino acid sequence of a known protein having the target enzyme activity and the amino acid sequence of the protein selected in (A),
(C) From the protein library prepared in (A), a protein having the correlated residue specified in (B) is selected.
(D) It is confirmed whether or not the protein selected in (C) has the target enzyme activity.

The similar amino acid sequence in (A) is, for example, an amino acid sequence having a sequence identity of 5 to 90% with an amino acid sequence of a known protein having the target enzyme activity obtained using BLASTp. Can be. From the viewpoint of being more similar, the amino acid sequence is preferably in the range of 10 to 70%, more preferably in the range of 15 to 50%. However, the range of the sequence identity can be appropriately selected so that the number of proteins constituting the protein library prepared in (A) is appropriate.

(A) There is no limit to the number of proteins that constitute the protein library created. However, if the amount is too small, a protein having the desired enzyme activity may not be obtained. On the other hand, the larger the number, the longer the operation. Considering these points, the number of proteins constituting the protein library is, for example, in the range of 10 to 100,000, preferably in the range of 100 to 50,000, more preferably in the range of 500 to 10,000. It is a range. However, these are merely examples and are not intended to be limited to these ranges.

Correlation residues in (B) have a conservative property of, for example, 50% or less based on the results of sequence alignment of a known protein having the target enzyme activity and at least two proteins selected in (A). It can be specified by selecting a pair of amino acid residues in which the type of residue is changed.
The low conservation is that the number of matching amino acid residues is 50 when aligning at least two sequences consisting of the target protein and the library sequence using sequence alignment software such as ClustalW. A pair of amino acid residues whose types of amino acid residues are changed in cooperation among amino acid residues having a relatively low conservation (for example, 50% or less). Select. Despite relatively low conservation, the cooperative changes in the types of amino acid residues suggest that these amino acid residues have an important role in the function of this protein. If this function includes enzyme activity, it is assumed that these amino acid residues can be used as Merck marks (standards) to search for proteins with the desired enzyme activity. .

The location of a correlated residue consisting of a pair of amino acid residues whose conservation is 50% or less and the type of amino acid residue is cooperatively changed is in the three-dimensional structure of a known protein having the target enzyme activity. If the protein is located at a certain distance from the active center of the protein, it is highly likely that the activity of the protein that has not been found so far can be found. Therefore, in the present invention, the correlation residue in (B) is any of the two residues constituting the correlation residue, in the three-dimensional structure of a known protein having the target enzyme activity, from the active center of the protein, For example, it can be a residue present at a position separated by 5 cm or more.

(B) There are at least two correlation residues selected, and preferably two correlation residues are selected and used in (C).

The protein group whose amino acid sequence is known in (A) is not particularly limited, but is, for example, a protein registered with a protein registration organization and a protein encoded by a nucleic acid sequence registered with a nucleic acid sequence registration organization. be able to. The protein registration authority can be at least one selected from the group consisting of Genebank and PubMed, and the nucleic acid sequence registration authority can be, for example, DDBj. However, it is not the intention limited to these.

In (C), a protein having the correlated residue specified in (B) is selected from the protein library created in (A). Based on the amino acid sequence of the protein contained in the protein library prepared in (A), a protein having the correlated residue specified in (B) can be selected.

In (D), the protein selected in (C) is synthesized, and it is confirmed whether it has the target enzyme activity. The protein synthesis method can be performed by a conventional method based on the amino acid sequence, the protein purification method can also be performed by a conventional method, and confirmation of whether or not the purified enzyme has the target enzyme activity, Depending on the target enzyme activity, it can be carried out by a conventional method.

<Method for producing protein having target enzyme activity>
The present invention includes a method for producing a protein having a target enzyme activity, comprising preparing a protein screened by the method of the present invention. That is, a protein having a target enzyme activity can be produced by preparing a protein screened by the above-described method of the present invention by a known genetic engineering method based on its amino acid sequence.

The target enzyme activity in the screening method and the method for producing a protein having the target enzyme activity of the present invention is an enzyme group registered in BRENDA, and can be appropriately selected from known enzyme activities. Further, the present invention is not limited to the known enzyme activity, and is a newly discovered protein having enzyme activity, and can be a target of the screening method of the present invention and the method for producing a protein having target enzyme activity.

Enzymes with an enzyme number (EC number) are, for example, oxidoreductases represented by EC numbers 1.-, transferases represented by EC numbers 2.-, and EC numbers 3.- Selected from the group consisting of a hydrolase, a removal addition (desorption) enzyme represented by EC number 4.-, an isomerase represented by EC number 5.-, and a synthetic enzyme represented by EC number 6.- Can be at least one enzyme.

Examples of the target enzyme activity include reactivity to dehydrogenate a hydroxy group bonded to the Cβ position of L-threonine, and high specificity and reactivity with L-threonine.

The screening method and protein production method will be described below, taking as an example the case where the target enzyme activity is TDH activity.

A method for screening a protein having a novel TDH activity,
(i) First step: A library consisting of proteins having an amino acid sequence similar to the amino acid sequence of the target enzyme is prepared with BLASTp (including sequences not registered as threonine dehydrogenase).
(ii) Second stage: At least two correlated residues are identified using the proteins in the library obtained in the first stage.
(iii) Third stage: An amino acid sequence having at least two correlation residues specified in the second stage is selected. As a specific example, TDH is shown below.

Step (A): With reference to the amino acid sequence of CnTDH, the amino acid sequences of 5000 proteins were extracted from the database with similar amino acid sequences. By using the CnTDH amino acid sequence as a query sequence and extracting the CnTDH family sequence using BLASTp, 5000 family sequences of CnTDH were finally obtained. The obtained sequence was used as a sequence library in the step (C). Although non-redundant protein sequence (nr) is used as the database, the type of database to be used is not limited as long as it can be selected on the BLASTp web browser. In addition to nr, usable databases include UniprotKB / Swiss-prot (swissprot), reference proteins (refseq_protein), and the like. BLOSUM62 is used as the alignment score matrix, but the type of score matrix to be used is not limited as long as it can be selected on the BLASTp web browser. For example, BLOSUM80 or PAM30 can also be used.

Step (B): Using the 5000 amino acid sequence of (A), four residues (Val143, Tyr188, Met214, Leu174) were predicted as correlated residues. A plurality (for example, 3 to 9) of sequences are randomly extracted from the sequence of CnTDH and the library prepared in step (A), and for example, a sequence pair consisting of all 4 to 10 sequences is prepared. This process is repeated to create a plurality of sequence pairs (10 or more). Amino acid sequence alignment is performed individually for all sequence pairs. By comparing the aligned results, correlated residues can be selected. In the example of Example, as a result of analysis, it was predicted that the pair of residues 143-174 and 188-214 on CnTDH was a correlated residue. In CnTDH, these residue pairs were conserved as V143, L174, Y188, and M214. A sequence having a residue pair similar to CnTDH can be predicted to have the same activity as CnTDH, and step (C) can be performed.

The distance between V143, L174, Y188, and M214 and the substrate L-Thr obtained based on the X-ray crystal structure (PDB ID: 3WMX) in which NAD ⁺ and L-Thr of CnTDH were bound was determined. As a result, the distances between the Cβ carbon atom of L-Thr and the Cα carbon atoms of V143, L174, Y188, and M214 were 12.7, 7.4, 11.6, and 10.6 A, respectively. V143, L174, Y188, and M214 were amino acid residues that were 5A or more away from L-Thr, which is a substrate necessary for exerting TDH activity.

Step (C): Sequence alignment is performed on individual amino acid sequences belonging to the CnTDH family extracted from BLASTp and CnTDH, and amino acids matching positions 143, 174, 188, and 214 of CnTDH are found in Val, Leu, Tyr and Met. 85 proteins having the same sequence were selected as TDH candidates. Extract amino acid sequences from the 5000 amino acid sequence library created in step (A) one by one, create sequence pairs with the amino acid sequence of CnTDH, and perform amino acid sequence alignment for the created 5000 sequence pairs. went. Next, based on the amino acid sequence of CnTDH, proteins having amino acid sequences having amino acid residues at positions 143, 174, 188 and 214 as Val, Leu, Tyr and Met were selected. By this method, the amino acid sequence of 85 proteins was finally extracted from the protein of 5000 amino acid sequences and defined as a TDH candidate sequence library (Genbank registration numbers of the extracted amino acid sequences are shown in Table 1) ). For example, software such as ClustalW can be used to perform amino acid sequence alignment.

Step (D): Compare the amino acid sequences of the 85 TDH candidate libraries with the sequences of CnTDH and select 3 candidates with low homology (50% or less sequence homology with CnTDH), and select the N-terminus of each enzyme. After the His-tag was added to the E. coli and expressed in E.coli, it was purified and examined for various properties. The lower the homology, the more different the sequence from TDH. Therefore, if the above three types have TDH activity, the selected 85 sequences are likely to have TDH activity.

ZP_09413997.1: Thermoanaerovibrio velox DSM 12556 produced based on a sequence homology of 50% or less with CnTDH from a TDH candidate sequence library of 85 proteins selected in step (C) Amino acid sequence of the protein (hereinafter referred to as TvTDH), YP_003318149.1: Thermoanaerovibrio acidaminovorans DSM 6589 derived amino acid sequence (hereinafter referred to as TaTDH), ADD93128.1: obtained from the metagenomic library After selecting the amino acid sequence of the protein (hereinafter referred to as mtTDH) and expressing the gene of this protein in Escherichia coli, the resulting proteins are purified to homogeneity and examined for substrate specificity, thermal stability, optimum pH and optimum temperature. The characteristics of the selected candidates were elucidated by clarifying various enzyme chemical properties.
As a result, the amino acid sequences of TvTDH, TaTDH, and mtTDH are as shown in SEQ ID NOs: 1 to 3 in the Sequence Listing. As shown in Table 2, all proteins have 50% or less sequence homology with CnTDH. No metal ions were required as a cofactor. Moreover, as shown in Example 7, the affinity for NAD ⁺ was higher than that of the existing enzyme, and the thermal stability of the enzyme was also excellent.

The amino acid sequence of TvoTDH has been reported as an enzyme specific for threonine (Yoneda, K. et al., J. Biol. Chem., 2012, 12966-12974). However, it is a protein having an amino acid sequence in which amino acid residues at positions 143, 174, 188 and 214 shown in the present invention are not Val, Leu, Tyr and Met. Furthermore, TvTDH, TaTDH, and mtTDH have 50% or less sequence identity. ThrDH_4fix is a novel protein itself.

<Protein>
The protein according to claims 1 and 2 was identified by the above method.
That is, the present invention includes any one of the following proteins for L-threonine analysis (1) to (3) (Claim 1, hereinafter sometimes referred to as protein A).
(1) a protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, 5 to 10 in the sequence listing;
(2) having an amino acid sequence having 1 to 50 amino acid substitutions, deletions and / or additions in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, 5 to 10 in the sequence listing; A protein having threonine dehydrogenase activity,
(3) It has an amino acid sequence having 90% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, and 5 to 10 and has L-threonine dehydrogenase activity. protein.

The present invention includes any one of the following proteins (1) to (3) (Claim 2, ThrDH — 4fix, hereinafter sometimes referred to as protein B).
(1) a protein having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing;
(2) a protein having an amino acid sequence having 1 to 50 amino acid substitutions, deletions and / or additions in the amino acid sequence set forth in SEQ ID NO: 4 of the Sequence Listing, and having L-threonine dehydrogenase activity;
(3) A protein having an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing and having L-threonine dehydrogenase activity.

Furthermore, the present invention includes a polynucleotide encoding any of the proteins (1) to (3) above, and a recombinant vector in which this polynucleotide is inserted into a plasmid. In addition, the present invention also includes a transformant comprising a host and the polynucleotide or the recombinant vector contained in the host.

The present invention includes culturing a host transformed with the transformant or a plasmid containing a polynucleotide encoding the protein B, and collecting a protein having L-threonine dehydrogenase activity from the culture. A method for producing L-threonine dehydrogenase is also included.

<Method for preparing L-threonine dehydrogenase>
In addition, among the 85 proteins selected above, proteins other than the above proteins A and B are also likely to be L-threonine dehydrogenases, and the present invention relates to SEQ ID NOs: 11 to 86 in the sequence listing. A method for preparing L-threonine dehydrogenase, comprising a step of screening a protein having L-threonine dehydrogenase activity from a protein having any one of the amino acid sequences described above.

A method for screening a protein having L-threonine dehydrogenase activity from a protein having an amino acid sequence set forth in any one of SEQ ID NOs: 11 to 86 in the sequence listing synthesizes the protein and requires the obtained protein And then confirming that it has L-threonine dehydrogenase activity. Protein synthesis, purification, and activity tests can be performed by conventional methods. The method for preparing the protein will be described later.

<Method for producing artificial protein having target enzyme activity>
The present invention includes a method for producing an artificial protein having a target enzyme activity including the following steps (E) to (G).
(E) Create a candidate library consisting of a plurality of proteins having the target enzyme activity,
However, the protein contained in the candidate library may be a protein presumed to have the target enzyme activity.
(F) aligning the candidate library with an amino acid sequence of a known protein having the target enzyme activity, and identifying the most frequently occurring amino acid residue as a consensus residue;
(G) A protein having an amino acid sequence composed only of the consensus residue is designed, and the designed protein is prepared.

The candidate library in (E) can be composed of a plurality of proteins having the target enzyme activity screened by the method of the present invention. However, the candidate library in (E) is not intended to be limited to these proteins, and can be appropriately selected from a plurality of proteins having other target enzyme activities.

Identification of the consensus residue in (F) can be performed, for example, using software capable of performing multiple sequence alignment. Such software can be, for example, INTMSAlign or ClustalW. For details of INTMSAlign, see Nakano, Shogo and Asano, Yasuhisa, Sci. Rep. 5: 8193 (2015) (Reference 1).
For details of ClustalW, see Thompson JD et al., Nucleic. Acids Res. 22: 4673-4680 (1994).

A method for producing an artificial protein will be described below as a continuation of the TDH example described above.
Following (D) above, the amino acid sequence of 85 TDH candidate libraries was compared with the sequence of CnTDH, and the sequence created with the most frequently occurring amino acid in each amino acid residue (sequence by consensus residue) And an amino acid sequence that does not exist in nature), was expressed in Escherichia coli as described above, and then purified and examined for various properties. It was revealed that an artificial protein having a sequence based on consensus residues is an enzyme that specifically acts on L-Thr.

The target enzyme activity in the method for producing an artificial protein of the present invention has reactivity to dehydrogenate a hydroxy group bonded to the Cβ position of L-threonine, and exhibits high specificity and reactivity to L-threonine. In addition, the enzyme group registered in BRENDA can be selected as appropriate from the activity of the enzyme given the enzyme number (EC number). For example, oxidoreductase represented by EC number 1.-, transferase represented by EC number 2.-, hydrolase represented by EC number 3.-, removal represented by EC number 4.- It can be at least one enzyme selected from the group consisting of an addition (elimination) enzyme, an isomerization enzyme represented by EC number 5.- and a synthesis enzyme represented by EC number 6.-.

In step (E), the same procedure as step (A-D) IV in “Screening method of the present invention” described above is performed to create, for example, the TDH candidate library shown in Table 1. Subsequently, using a software capable of performing multiple sequence alignment such as INTMSAlign and ClustalW, the step of analyzing the TDH candidate library of Table 1 and the amino acid sequence of CnTDH to identify consensus residues (step (F) below), And a step of designing an amino acid sequence composed only of the identified consensus residue (the following step (G)), and step (F) and step (G) of this method are as follows.

Process (G)
Using CnTDH and the TDH candidate library, amino acid sequence analysis was performed by software, INTMSAlign (reference document 1), the amino acid sequence of the TDH candidate library was aligned with the sequence of CnTDH, and the most frequently occurring amino acid residue was All residue numbers of CnTDH were identified. The amino acid residues identified by this method were used as consensus residues.

Process (F)
An amino acid sequence in which all amino acid residues of CnTDH were substituted with the consensus residues identified in step (D) was prepared. This amino acid sequence is the sequence (ThrDH_4fix) described in SEQ ID NO: 4 in the Sequence Listing, and as shown in Table 2, the sequence homology with CnTDH was 70% or less. Next, after synthesizing the gene of this sequence and expressing it in Escherichia coli, the resulting protein is purified, and the enzyme specificity is revealed by examining the substrate specificity, thermal stability, optimum pH and optimum temperature. The characteristics were clarified. As a result, the protein of this sequence required NAD ⁺ as a coenzyme, was specific for L-Thr, and did not require a metal ion as a cofactor for the activity of the enzyme. Moreover, as shown in Example 7, the affinity for NAD ⁺ was higher than that of the existing enzyme, and the thermal stability of the enzyme was also excellent.

In the present invention, the method for obtaining L-threonine dehydrogenase and the other enzyme is not particularly limited, and may be a protein synthesized by chemical synthesis or a recombinant protein produced by gene recombination technology. When producing a recombinant protein, a gene (DNA) encoding the protein is obtained as described later. By introducing this DNA into an appropriate expression system, the protein of the present invention (for example, L-threonine dehydrogenase) can be produced.

The method for obtaining a gene encoding the L-threonine dehydrogenase of the present invention is not particularly limited. The gene encoding the L-threonine dehydrogenase of the present invention is, for example, based on the information of the base sequence obtained based on the amino acid sequence described in the sequence listing, such as chemical synthesis, genetic engineering technique or mutagenesis. It can be made by any method known to those skilled in the art.

The above-described procedures such as probe or primer preparation, cDNA library construction, cDNA library screening, and target gene cloning are known to those skilled in the art. For example, Molecular Cloning 2nd Edition, Current Protocols In -It can be performed according to the method described in Molecular Biology etc.

The gene of the present invention can be used by inserting it into an appropriate vector. The type of vector used in the present invention is not particularly limited. For example, the vector may be a self-replicating vector (for example, a plasmid), or may be integrated into the host cell genome when introduced into the host cell. It may be replicated together with other chromosomes. Preferably, the vector used in the present invention is an expression vector. In the expression vector, the gene of the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). A promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.

Promoters that can operate in bacterial cells include the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha-amylase gene, and the Bacillus amyloliquefati. Enns-BAN amylase gene (Bacillus amyloliquefaciens BAN amylase gene), Bacillus subtilis alkaline protease gene (Bacillus subtilis alkaline protease gene) or promoters of the Bacillus pumilus-xylosidase gene (Bacillus pumilus xylosldase gene) or phage lambda, P _R or P _L promoters, lac E. coli (E. coli), such as trp or tac promoter and the like.

Examples of promoters that can operate in mammalian cells include the SV40 promoter, the MT-1 (metallothionein gene) promoter, or the adenovirus 2 major late promoter. Examples of promoters operable in insect cells include polyhedrin promoter, P10 promoter, autographa caliornica polyhedrosic basic protein promoter, baculovirus immediate early gene 1 promoter, or baculovirus 39K delayed early gene. There are promoters. Examples of a promoter operable in a yeast host cell include a promoter derived from a yeast glycolytic gene, an alcohol dehydrogenase gene promoter, a TPI1 promoter, an ADH2-4c promoter, and the like. Examples of promoters that can operate in filamentous fungal cells include the ADH3 promoter or the tpiA promoter.
Moreover, the gene of the present invention may be operably linked to an appropriate terminator as necessary. The recombinant vector containing the gene of the present invention may further have elements such as a polyadenylation signal (for example, derived from SV40 or adenovirus 5E1b region), a transcription enhancer sequence (for example, SV40 enhancer) and the like. The recombinant vector containing the gene of the present invention may further comprise a DNA sequence that allows the vector to replicate in the host cell, an example of which is the SV40 origin of replication (when the host cell is a mammalian cell). ).

The recombinant vector containing the gene of the present invention may further contain a selection marker. Selectable markers include, for example, genes that lack their complement in host cells such as dihydrofolate reductase (DHFR) or Schizosaccharomyces pombe TPI genes, or such as ampicillin, kanamycin, tetracycline, chloramphenicol, Mention may be made of drug resistance genes such as neomycin or hygromycin. Methods for ligating the gene, promoter, and, if desired, terminator and / or secretory signal sequence of the present invention and inserting them into an appropriate vector are well known to those skilled in the art.

A transformant can be produced by introducing a recombinant vector containing the gene of the present invention into an appropriate host. The host cell into which the recombinant vector containing the gene of the present invention is introduced may be any cell as long as it can express the gene of the present invention, and examples thereof include bacteria, yeast, fungi and higher eukaryotic cells.

Examples of bacterial cells include Gram-positive bacteria such as Bacillus or Streptomyces, or Gram-negative bacteria such as E. coli. Transformation of these bacteria may be performed by using competent cells by a protoplast method or a known method. Examples of mammalian cells include HEK293 cells, HeLa cells, COS cells, BHK cells, CHL cells, or CHO cells. Methods for transforming mammalian cells and expressing DNA sequences introduced into the cells are also known, and for example, electroporation method, calcium phosphate method, lipofection method and the like can be used.

Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces, and examples thereof include Saccharomyces cerevisiae or Saccharomyces kluyveri. Examples of the method for introducing a recombinant vector into a yeast host include an electroporation method, a spheroblast method, and a lithium acetate method.

Examples of other fungal cells are those belonging to filamentous fungi, such as Aspergillus, Neurospora, Fusarium, or Trichoderma. When filamentous fungi are used as host cells, transformation can be performed by integrating the DNA construct into the host chromosome to obtain a recombinant host cell. Integration of the DNA construct into the host chromosome can be performed according to known methods, for example, by homologous recombination or heterologous recombination.

When insect cells are used as the host, the recombinant gene transfer vector and baculovirus are co-introduced into the insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected with the insect cells. And protein can be expressed (for example, as described in Baculovirus Expression Vectors, A Laboratory Manua1; and Current Protocols in Molecular Biology, Bio / Technology, 6, 47 (1988), etc.).
As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects Coleoptera insects, can be used.
Insect cells include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company), New York, Spodoptera frugiperda ovarian cells (1992)], HiFive (manufactured by Invitrogen), which is an ovarian cell of Trichoplusia ni, can be used.

Examples of a method for co-introducing a recombinant gene introduction vector into an insect cell and the baculovirus for preparing a recombinant virus include a calcium phosphate method and a lipofection method.

The above transformant is cultured in an appropriate nutrient medium under conditions that allow expression of the introduced gene. In order to isolate and purify the protein of the present invention from the culture of the transformant, ordinary protein isolation and purification methods may be used. For example, when the protein of the present invention is expressed in a dissolved state in the cell, after the culture is completed, the cell is collected by centrifugation, suspended in an aqueous buffer, and then disrupted by an ultrasonic crusher or the like. A cell-free extract is obtained. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as diethylaminoethyl (DEAE) Sepharose, cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose and phenyl sepharose Using the hydrophobic chromatography method used, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. alone or in combination, the L- Threonine dehydrogenase can be obtained as a purified sample.

<Analytical method for L-threonine>
The present invention includes a method for analyzing L-threonine, which comprises measuring L-threonine concentration in a subject using L-threonine dehydrogenase. This method includes mixing a sample containing an analyte with the L-threonine dehydrogenase and the coenzyme NAD ⁺ of the present invention, and analyzing the amount of NADH after a predetermined time.
The L-threonine dehydrogenase of the present invention includes protein A, protein B, and proteins prepared by the method of the present invention.

The subject is not particularly limited, and can be, for example, human blood or food and drink.

The sample containing the analyte can be obtained by mixing the analyte with, for example, a buffer solution showing the optimum pH of L-threonine dehydrogenase. The optimum pH of L-threonine dehydrogenase is 10.0. In the analysis, a predetermined amount of L-threonine dehydrogenase is added to the sample containing the analyte. The amount of L-threonine dehydrogenase added can be determined as appropriate in consideration of the degree of purification, titer, etc. of L-threonine dehydrogenase, and can be, for example, in the range of 0.001 to 1 U / 200 μL.

The sample containing the analyte is mixed with coenzyme NAD ⁺ in addition to L-threonine dehydrogenase. The coenzyme NAD ⁺ can be a salt of NAD, for example, an alkali metal salt such as sodium or potassium. The amount of coenzyme NAD ⁺ mixed can be appropriately determined in consideration of the L-threonine concentration in the sample, the titer of L-threonine dehydrogenase, and the like, and can be, for example, in the range of 0.001 to 3 mM. NAD ⁺ is nicotinamide adenine dinucleotide, which is sometimes expressed as β-NAD ^+, and both are synonymous. NADP ⁺ is sometimes expressed as β-NADP ^+, and both are synonymous.

After mixing ⁺ L-threonine dehydrogenase and coenzyme NAD, analyzing the amount of NADH after a predetermined time. The predetermined time can be appropriately determined in consideration of the reaction temperature, the concentration of L-threonine contained in the analyte, the accuracy of analysis, and the like. Usually, it can be, for example, in the range of 5 seconds to 60 minutes, preferably in the range of 1 minute to 60 minutes.

After a predetermined time, the amount of NADH produced by L-threonine dehydrogenase is analyzed. The analysis of the amount of NADH can be performed directly, for example, by measuring the absorbance at 340 nm (A ₃₄₀ ), and a method of generating a dye with NADH or a method of generating fluorescence with NADH can also be used. Examples of a method for producing a dye by NADH include a method using an NADH-tetrazolium-based electron carrier. Examples of the electron carrier include PMS (phenazine methosulfate, + 0.08V) and Meldola Blue. Available. An example of a method for producing a dye with NADH is a method using diaphorase. In the method using diaphorase, diaphorase catalyzes the oxidation of NADH and the reduction of the dye to obtain color. As the dye, INT (2- (4-iodophenyl) -3- (4-nitrophenyl) -5-phenyltetrazolium chloride), NBT (nitroblue tetrazolium) and the like can be used. In the method using diaphorase, a fluorescent dye such as resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) can also be used as the dye.

The L-threonine analysis method of the present invention is suitable for a so-called microplate assay using a microplate. For example, a 96-hole microplate can be used as the microplate. The number of holes is not particularly limited. When using a 96-well microplate, the total reaction volume is, for example, 200 μL, and 100 mM glycine KCl-KOH buffer (pH 10.0), 2.5 mM NAD ⁺ , and deproteinized sample are added. The reaction is started by adding the enzyme of the present invention. For example, the temperature can be kept at 30 ° C. for 10-30 minutes, and the absorbance at 340 nm at the end point can be measured with a UV microplate spectrophotometer. The change in absorbance (ΔA) is obtained as the final absorbance minus the control value. The deproteinized sample can be prepared by, for example, ultrafiltration using Centricon YM-10.

In the method for analyzing L-threonine of the present invention, the amount of 2-amino-3-oxobutyric acid generated from L-threonine by L-threonine dehydrogenase is analyzed in addition to analyzing the amount of NADH as described above. Can also be implemented. As shown in Scheme 1 above, 2-amino-3-oxobutyric acid (α-amino-β-ketobutyric acid) produced from L-threonine is non-enzymatically decarboxylated to produce aminoacetone. This aminoacetone is oxidized by monoamine oxidase to become methylglyoxal, and at that time, ammonia and hydrogen peroxide are generated. L-threonine can be quantified by quantifying the generated ammonia or hydrogen peroxide by a known quantification method.

In addition to analyzing the amount of NADH or 2-amino-3-oxobutyric acid as described above, the L-threonine analysis method of the present invention can be performed using L-threonine dehydrogenase as shown in Scheme 1 above. This can also be done by analyzing the amount of H ⁺ produced with -amino-3-oxobutyric acid and NADH. Known methods can be used to analyze the amount of H ⁺ .

Hereinafter, the present invention will be described in more detail based on examples. However, the examples are illustrative of the present invention, and the present invention is not intended to be limited to the examples.

Example 1
Rapid and simple screening method for new proteins with TDH activity using amino acid sequence database Rapid and simple screening for new proteins with TDH activity using amino acid sequence database is (i) a sequence consisting of CnTDH family Library preparation, (ii) Selection of amino acid residues necessary to exert TDH activity at amino acid residues that are 5A or more away from the substrate L-Thr, (iii) From sequence library The three steps of extracting TDH candidate sequences were performed. The detailed method is as follows.

Step 1 Preparation of sequence library consisting of CnTDH family The amino acid sequence of CnTDH (FASTA format) is obtained from PubMed (Genbank ID of CnTDH sequence is 318609955), this CnTDH sequence is used as a query sequence, and BLASTp ( http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) was used to search and extract the family sequence of CnTDH. First, the CnTDH sequence was input to “Enter Query Sequence” of BLASTp. Next, select non-redundant protein sequence (nr) as database, BLASTp as Algorithm, click the “Algorithm parameters” tab on the website, Max target sequences is 5000, Expected threshold is 1.0E-3 I input it. BLOSUM62 was used as the alignment score matrix, and the search was performed with the other parameters kept at their initial values. On the screen that appeared after the search was completed, moved the scroll bar until the "Descriptions" item was visible, then clicked the "All" tab next to the "Select:" item. Next, click on the "Download" tab, select "Fasta (complete sequence)", and click on the "Continue" tab to obtain the CnTDH family sequence. By the above operation, 5000 CnTDH family sequences were finally obtained and used as a sequence library.

Step 2 Selection of correlation residues that can be amino acid residues necessary for exerting TDH activity A total of 4 (3-9) sequences were randomly extracted from the sequence of CnTDH and the library created in Step 1 Create a sequence pair consisting of ~ 10 sequences. Repeat this process to create multiple sequence pairs (10 or more). Amino acid sequence alignment is performed individually for all sequence pairs. Compare aligned results and select correlated residues. As a result of the analysis, it was predicted that the 143-174,188-214 residue pair on CnTDH was a correlated residue. In CnTDH, these residue pairs were conserved as Val143, Leu174, Tyr188, and Met214. A sequence having the same residue pair as CnTDH was predicted to have the same activity as CnTDH, and Step 3 was performed.

Consideration 1: Val143 is an amino acid that is adjacent to the amino acid residue (Tyr144) that directly dehydrogenates the hydroxy group of L-threonine, which is a substrate, and is located in the boundary region of the dimerization site of CnTDH. Residue. Therefore, we predicted that this residue plays an important role in the movement of Tyr144, and thought that this residue is likely to affect the activity of CnTDH.
Consideration 2: Tyr188 and Met214 exist in the vicinity of Thr186, which forms a hydrogen bond with the amino group of the substrate L-threonine, and the side chain of Tyr188 and Met214 are located between the side chains of Thr186. It was predicted that an environment that can produce an action and bind to the substrate L-threonine was indirectly created. Therefore, we thought that Tyr188 and Met214 affect the activity of CnTDH.
Discussion 3: Leu174 is located in the vicinity of the coenzyme NAD ^+, it interacts with nicotinic ring, is a substrate L- threonine and CnTDH is predicted that make indirect environmental capable of reacting. Therefore, Leu174 was also considered important for the expression of CnTDH activity.

Step 3 Extraction of TDH Candidate Sequence from Sequence Library The principle of the method for extracting a TDH candidate sequence from the sequence library prepared in step (i) is shown in FIG. Specifically, we first created a text file (temporary.fasta) that pairs the top array of 5000 arrays with the CnTDH array, and installed it in the local environment of a computer with CentOS 6 as the operation system. The sequence alignment of temporary.fasta was performed using clustalw2. Open temporary.aln obtained by this alignment with a text editor such as gedit, and in the aligned sequences, the positions 143, 174, 188 and 214 selected in step (ii) based on the amino acid sequence of CnTDH are It was examined whether it was conserved in Val, Leu, Tyr and Met, and was extracted and stored as a paired sequence with CnTDH only when all four residues were conserved. This operation was performed for all 5000 sequences, and finally 85 sequences shown in Table 1 were selected from the sequence library (5000 amino acid sequences) created in step (i) (TDH candidate sequence live). Defined as rally). The above work was done from the command line by creating a Python script (Seqclustering.py).

Example 2
How to artificially design L-threonine dehydrogenase from amino acid sequence database (i) Identify TDH consensus residues from TDH candidate sequence library to artificially design L-threonine dehydrogenase (TDH) from amino acid sequence database Subsequently, the present invention was completed by (ii) designing a protein of complete consensus, and expressing the designed protein in E. coli to confirm the enzyme activity. The specific procedure is shown below.

Step 1 Generation of TDH consensus sequence using TDH candidate sequence library In order to identify TDH consensus residues from TDH candidate sequence library, INTMSAlign (Reference 1) Installed) and set up the computing environment. Next, the following information was input to the INTMSAlign GUI started from the command line. First, the TDH sequence candidate library was selected and entered from the "dir ..." tab on the first line of the INTMSAlign GUI. The CnTDH array was selected and entered from the "dir ..." tab on the second line. The third line selected BLOSUM. Enter 500 for the 4th line, 8 for the 5th line, and leave the default settings for the 6th line. After that, input the name of the output file on the 7th line and click the OK tab to execute INTMSAlign. By performing the above operation, an output file of INTMSAlign having the format of FIG. 3 was obtained (FIG. 3). Subsequently, the output file was opened, and a consensus residue of TDH was determined from each amino acid sequence of the TDH candidate sequence library. The consensus residue was defined as the amino acid residue having the highest appearance frequency in the analysis of INTMSAlign (as shown in FIG. 3).

Step 2 Design Method of Complete Consensus Protein ThrDH — 4fix Based on the output file of FIG. 3, all amino acid sequences of CnTDH were substituted with consensus residues, and a complete consensus sequence was designed, and the sequence was set to ThrDH — 4fix. The complete consensus sequence was designed using a self-made Python script (seqcreator_INTMSAlign.py). A gene sequence corresponding to the designed complete consensus sequence was synthesized and expressed by the method shown in Example 6, and its properties were confirmed.

Example 3
Production and purification of the protein (TvTDH) selected from Thermoanaerovibrio velox DSM 12556 The sequence homology with CnTDH is less than 50% from the TDH candidate sequence library of the 85 proteins selected in Example 1 ZP_09413997.1 selected based on the above: The amino acid sequence of the protein produced by Thermoanaerovibrio velox DSM 12556 (TvTDH, the amino acid sequence of SEQ ID NO: 1) BackTranseq (http://www.ebi.ac. Using uk / Tools / st / emboss_backtranseq /), it was converted to a base sequence suitable for E. coli codon usage. A base sequence was prepared by introducing NdeI at the 5 ′ end of this base sequence and a cleavage sequence of a stop codon and BamHI at the 3 ′ end, and the entire base sequence was synthesized. Next, the pIDTSMART vector (pIDSMART-TvTDH) incorporating this nucleotide sequence was subcloned using JM109 as a host. The subcloned pIDSMART-TvTDH was treated with a restriction enzyme, ligated with pET15 (+) treated with the same restriction enzyme, and introduced into BL21 (DE3). In order to easily purify the expressed enzyme, the plasmid was introduced with a 6-residue histidine tag at the N-terminus.

The expression strain 発 現 [BL21 (DE3) / pET15b (+) TvTDH] prepared by the above method is inoculated into LB medium (2 ベ L) containing carbenicillin at a final concentration of 50 μg / mL, at 37 ° C for 8 hours and at 23 After culturing for 1 hour, IPTG having a final concentration of 0.5 mM was added, and further cultured at 23 ° C. for 20 hours. After centrifuging the culture broth at 7000 xg for 10 minutes, remove the LB liquid medium and suspend it in a buffer containing 80 mM 10 mM potassium phosphate (pH 7.0) and 50 mM NaCl, and then disrupt the cells using an ultrasonic homogenizer. . This disrupted solution was centrifuged at 12000 × g for 40 minutes, and the supernatant fraction was used as a crude enzyme solution. Immediately adsorb the crude enzyme solution to Ni Sepharose 6 Fast Flow column (GE Healthcare), wash with 50 mL buffer, and elute with buffer containing 10, 40, 70, 100, 300, 500mM imidazole. did. Each elution fraction was subjected to SDS-PAGE, and an elution fraction having a band around 35 kDa was recovered. The collected fraction was concentrated as necessary, and purified using Superdex75pg (GE Healthcare).

Example 4
Production and purification of protein (TaTDH) selected from Thermoanaerovibrio acidaminovorans DSM 6589 50% sequence homology with CnTDH from the TDH candidate sequence library of 85 proteins selected in Example 1 YP_003318149.1 selected on the basis of the following: The amino acid sequence of the protein derived from Thermoanaerovibrio acidaminovorans DSM 6589 (TaTDH, amino acid sequence of SEQ ID NO: 2) as in Example 3. The nucleotide sequence was adapted to the codon usage frequency of Escherichia coli. A base sequence was prepared by introducing NdeI at the 5 ′ end of this base sequence and a cleavage sequence of a stop codon and BamHI at the 3 ′ end, and the entire base sequence was synthesized. Next, the pIDTSMART vector (pIDSMART-TaTDH) incorporating this nucleotide sequence was subcloned using JM109 as a host. The subcloned pIDSMART-TaTDH was treated with a restriction enzyme, ligated with pET15 (+) treated with the same restriction enzyme, and introduced into BL21 (DE3). In order to easily purify the expressed enzyme, the plasmid was introduced with a 6-residue histidine tag at the N-terminus. The expression strain [BL21 (DE3) / pET15b (+) TaTDH] prepared by the above method is inoculated into LB medium (2 L) containing carbenicillin at a final concentration of 50 μg / mL, and at 37 ° C for 8 hours and 23 ° C. After culturing for 1 hour, IPTG having a final concentration of 0.5 mM was added, and further cultured at 23 ° C. for 20 hours. After centrifuging the culture solution at 7000 xg for 10 minutes, the LB liquid medium was removed, suspended in a buffer containing 80 mL of 10 mM potassium phosphate (pH 7.0) and 50 mM NaCl, and then disrupted with an ultrasonic homogenizer. . The disrupted solution was centrifuged at 12000 × g for 40 minutes, and the supernatant fraction was used as a crude enzyme solution. The crude enzyme solution was immediately adsorbed on a Ni Sepharose 6 Fast Flow column (manufactured by GE Healthcare), washed with 50 mL of buffer, and then with a buffer containing 10, 40, 70, 100, 300, 500 mM imidazole. Eluted. Each elution fraction was subjected to SDS-PAGE, and an elution fraction having a band around 35 kDa was recovered. The collected fractions were concentrated as necessary and purified using Superdex75pg (manufactured by GE Healthcare).

Example 5
Production and purification of the protein (mtTDH) obtained from the metagenome library ADD93128.1 from the TDH candidate sequence library of 85 proteins selected in Example 1 ： Amino acid arrangement of the protein obtained from the metagenomic library (mtTDH, The amino acid sequence of SEQ ID NO: 3 was converted to a base sequence suitable for E. coli codon usage in the same manner as in Example 4. A base sequence was prepared by introducing NdeI at the 5 ′ end of this base sequence and a cleavage sequence of a stop codon and BamHI at the 3 ′ end, and the entire base sequence was synthesized. Next, the pIDTSMART vector (pIDSMART-mtTDH) incorporating this nucleotide sequence was subcloned using JM109 as a host. The subcloned pIDSMART-mtTDH was treated with a restriction enzyme, ligated with pET15 (+) treated with the same restriction enzyme, and introduced into BL21 (DE3). In order to easily purify the expressed enzyme, the plasmid was introduced with a 6-residue histidine tag at the N-terminus. The expression strain [BL21 (DE3) / pET15b (+) mtTDH] prepared by the above method is inoculated into LB medium (2 L) containing carbenicillin at a final concentration of 50 μg / mL, and at 37 ° C for 8 hours and 23 ° C. After culturing for 1 hour, IPTG having a final concentration of 0.5 mM was added, and further cultured at 23 ° C. for 20 hours. After centrifuging the culture solution at 7000 g for 10 minutes, the LB liquid medium was removed, suspended in a buffer containing 80 mL of 10 mM potassium phosphate (pH 7.0) and 50 mM NaCl, and the cells were disrupted with an ultrasonic homogenizer. The disrupted solution was centrifuged at 12000 × g for 40 minutes, and the supernatant fraction was used as a crude enzyme solution. The crude enzyme solution was immediately adsorbed on a Ni Sepharose 6 Fast Flow column (manufactured by GE Healthcare), washed with 50 mL of buffer, and then used with a buffer containing 10, 40, 70, 100, 300, 500 mM imidazole. Eluted. Each eluted fraction was subjected to SDS-PAGE, and a fraction having a band around 35 kDa was recovered. The collected fractions were concentrated as necessary and purified using Superdex75pg (manufactured by GE Healthcare).

Example 5-2
From the TDH candidate sequence library of 85 proteins selected in Example 1, amino acid sequences of the following 6 proteins were obtained.
・ 340787735: NAD-dependent epimerase / dehydratase [Collimonas fungivorans Ter331] CfuTDH
・ 390941828: nucleoside-diphosphate-sugar epimerase [Belliella baltica DSM 15883] BbaTDH
・ 375012635: nucleoside-diphosphate-sugar epimerase [Owenweeksia hongkongensis DSM 17368] OhoTDH
・ 373952659: NAD-dependent epimerase / dehydratase [Mucilaginibacter paludis DSM 18603] MpaTDH
・ 365875058: UDP-glucose 4-epimerase related protein [Elizabethkingia anophelis Ag1] EanTDH
・ 343085198: NAD-dependent epimerase / dehydratase [Cyclobacterium marinum DSM 745] CmaTDH

The amino acid sequences of these six proteins were converted into base sequences suitable for E. coli codon usage in the same manner as in Example 4. Then, a base sequence in which a stop codon and a BamHI cleavage sequence were introduced at the 5 ′ end and 3 ′ end of the base sequence was designed, and the entire base sequence was synthesized. Next, this base sequence was incorporated into a pET15b vector using the InFusion method (pET15b-TDHs). The prepared expression plasmid was introduced into BL21 (DE3). The expression strain [BL21 (DE3) / pET15b-TDHs] prepared by the above method was inoculated into LB medium containing 5 μg / mL carbenicillin (5 mL) 、, 8 hours at 37 ° C and 1 hour at 23 ° C. After culturing, IPTG having a final concentration of 0.5 mM was added, and further cultured at 23 ° C. for 20 hours. After centrifuging the culture solution at 7000 xg for 10 minutes, remove the LB liquid medium and suspend it in a buffer containing 500 μL of 10 mM potassium phosphate (pH 7.0) and 50 mM NaCl, followed by an ultrasonic homogenizer (Bioruptor UCD-250 Cells were disrupted with (TOSHO DENKI CO., LTD.) After centrifuging the disrupted solution at 12000 × g for 10 minutes, the supernatant fraction was passed through a desalting column (PD SpinTrap G-25, GE Healthcare). The desalted product was used as a crude enzyme solution.

Example 6
Production and purification of consensus sequence protein (ThrDH_4fix ) designed from amino acid sequence database ThrDH_4fix (amino acid sequence of SEQ ID NO: 4) designed from amino acid sequence database of Example 2 was used in the same manner as in Example 4 The nucleotide sequence was adapted to the frequency. A base sequence was prepared by introducing NdeI at the 5 ′ end of this base sequence and a cleavage sequence of a stop codon and BamHI at the 3 ′ end, and the entire base sequence was synthesized. Next, the pIDTSMART vector (pIDSMART-ThrDH_4fix) incorporating this nucleotide sequence was subcloned using JM109 as a host. The subcloned pIDSMART-ThrDH_4fix was treated with a restriction enzyme, ligated with pET15 (+) treated with the same restriction enzyme, and introduced into BL21 (DE3). In order to easily purify the expressed enzyme, the plasmid was introduced with a 6-residue histidine tag at the N-terminus. The expression strain [BL21 (DE3) / pET15b (+) ThrDH_4fix] prepared by the above method is inoculated into LB medium (2 L) containing carbenicillin at a final concentration of 50 μg / mL, and at 37 ° C for 8 hours and 23 ° C. After culturing for 1 hour, IPTG having a final concentration of 0.5 mM was added, and further cultured at 23 ° C. for 20 hours. After centrifuging the culture solution at 7000 × g for 10 minutes, the LB liquid medium was removed, suspended in a buffer containing 80 mL of 10 mM potassium phosphate (pH 7.0) and 50 mM NaCl, and then disrupted with an ultrasonic homogenizer. . The disrupted solution was centrifuged at 12000 × g for 40 minutes, and the supernatant fraction was used as a crude enzyme solution. The crude enzyme solution was immediately adsorbed on a Ni Sepharose 6 Fast Flow column (manufactured by GE Healthcare), washed with 50 mL of buffer, and then used with a buffer containing 10, 40, 70, 100, 300, 500 mM imidazole. Eluted. Each eluted fraction was subjected to SDS-PAGE, and a fraction having a band around 35 kDa was recovered. The collected fractions were concentrated as necessary and purified using Superdex75pg (manufactured by GE Healthcare).

Example 7
Using the proteins purified to homogeneity in Examples 3-6, the following methods were used: (i) substrate specificity, (ii) reaction kinetic constants for L-threonine, (iii) thermal stability, and (iv) The appropriate pH was investigated and the enzyme chemistry was clarified. The L-threonine dehydrogenase (TDH) activity was initiated by adding the enzyme solution to 100 mM glycine-KOH buffer (pH 10.5) containing 2.5 mM NAD ⁺ and 100 mM L-threonine. The amount of change in absorbance at 340 nm was measured at 0 ° C. (reaction volume: 0.2 mL). The protein concentration was calculated based on the standard curve of BSA using a method of measuring absorbance at 280 nm (the protein concentration used the parameters in Table 3) or BioRad protein assay.

(I) Substrate specificity Using the proteins purified in Examples 3 to 6 (proteins of sequences 1 to 4), the activity against 20 kinds of protein constituting L-amino acids and D-threonine including L-threonine was examined. Enzyme activity was determined by incubating 100 mM glycine-KOH buffer (pH 10.0) containing 2.5 mM NAD ⁺ and 50 mM of each amino acid at 30 ° C for 3 minutes, and then adding the enzyme solution to start the reaction. The change in absorbance at 340 nm was measured with a visible spectrophotometer (DU800 spectrometer, Beckman). As a result, as shown in Table 3, all of the proteins of sequences 1 to 4 acted on L-threonine, but showed no activity on D-threonine and L-amino acids other than L-threonine. Therefore, it was revealed that the proteins (proteins of sequence 1-4) purified in Examples 3 to 6 are enzymes that specifically act on L-threonine.

(Ii) using the kinetics constants Example 3 protein purified by ~ 6 (protein sequence 1 to 4) to the sequence 1 to 4 Protein L- threonine and NAD ^+, responses to L- threonine and NAD ⁺ The rate constant was calculated by curve fitting by the nonlinear least square method. Maximum speed for L- threonine (k _cat [/ sec]) and Michaelis constant (K _{m, L-Thr)} in the same conditions as the enzymatic activity measurement, 2.5 mM NAD ⁺ in the presence, L- threonine concentration 2- It was determined by changing in the range of 100 mM. Furthermore, the Michaelis constant (K _{m, NAD +)} for NAD ⁺ is under 50 mM L-threonine coexistence was determined by the NAD ⁺ concentration varied from 5-500MyuM. As a result, as shown in Table 6, all of the enzymes of sequence 1-4 have a K _m value for NAD ⁺ that is significantly lower than CnThrDH and TvoTDH, and have high affinity with NAD ^+. It became clear that the concentration of NAD ⁺ in the threonine measurement should be less than 1/3 of the conventional level. In addition, the maximum speeds of sequences 1-4 all showed a value of 20.0 / sec or more, suggesting that they can be used for L-threonine measurement.

(Iii) Thermal stability The protein purified in Examples 3 to 6 (protein of sequence 1 to 4) was heated at 30 to 80 ° C. for 5 minutes at pH 7.0, and immediately cooled in an ice bath. The enzyme activity of the heat-treated enzyme solution was measured by the method of Example 7, and the remaining enzyme activity was determined. As a result, TvTDH and TaTDH remained at 65 ° C. or lower (FIGS. 4 and 5), and ThrDH_4fix remained at 60 ° C. or lower (FIG. 7) with 50% or more activity remaining. In addition, mtTDH decreased to 30% by heating at 40 ° C (Fig. 6, solid line), but when 20% (w / v) glucose was added, it was more than 50% active even at 40 ° C. Remained. On the other hand, when CnTDH and FfTDH were heated at 50 ° C. for 5 minutes, the enzyme activity was remarkably inactivated (Patent Document 1). Therefore, it was revealed that TvTDH, TaTDH, and ThrDH_4fix obtained in the present invention are enzymes that are superior in thermal stability to existing CnThrDH and FfTDH.

(Iv) Optimum pH
Using the proteins purified in Examples 3 to 6 (proteins of sequences 1 to 4), the pH of activity measurement was changed with the following buffer solution, and the optimum pH of the reaction was examined (FIG. 8-11). As a result, TvTDH is pH 7.5, TaTDH is pH 7.0, mtTDH is pH 8.5, ThrDH_4fix is maximum activity at pH 9.0, TvTDH and TaTDH are highly active in the neutral region, and mtTDH and ThrDH_4fix are also known TDH (FtTDH The optimum pH was 9.5 and TvoTDH was more active on the neutral side than the optimum pH 10.0). Therefore, it was revealed that the proteins of sequences 1 to 4 were used for the measurement of L-threonine in the pH range where NAD ⁺ is stable.
Buffers used: sodium acetate buffer (pH 3.5-5.5), Bis-tris-HCl buffer (pH 6.0-7.0), Tris-HCl buffer (pH 7.0-8.5), BICIN buffer (pH 9.0-9.5) ), Glycine-KOH buffer (pH 10.0-11.5)

As shown in Example 7, all the proteins having the sequences obtained in Examples 3 to 6 are specific to L-Thr and have higher affinity for NAD ⁺ used as a coenzyme than the existing enzymes. In addition, high activity was exhibited without adding metal ions to the enzyme reaction. In addition, the thermal stability was superior to the existing CnTDH. Therefore, it was estimated that the property of the protein obtained by the method of the present invention is superior to the existing TDH. Thus, L-threonine was measured by the following method using the proteins having the sequences obtained in Examples 3 to 6.

A reaction solution containing 2.5 mM NAD ⁺ and 50-500 μM L-threonine was heated at 30 ° C. for 3 minutes at the optimal reaction pH of each enzyme, and then purified TDH was added to start the measurement (reaction volume: 200 μL). ). The enzyme concentration of the added purified TDH was 0.11 (TaTDH), 0.11 (TvTDH), 0.22 (mtTDH), and 0.073 (ThrDH_4fix) mg / mL in the final concentration in the reaction solution. The measurement is carried out at 30 ° C, the absorbance at 340 nm is measured up to 180 seconds at intervals of 20 seconds after the start of the reaction, fitting by the linear least square method, the amount of change in absorbance per minute is calculated and the L-Thr concentration is calculated. The relationship between changes in absorbance was determined. As a result, as shown in FIGS. 12 to 15, the TvTDH, TaTDH, mtTDH, and ThrDH_4fix proteins have a good linear relationship up to 500 μM, and the protein of the sequence obtained by the method of the present invention is L-Thr. It became clear that it could be used for measurement.

Example 8
The concentration of the crude enzyme solution obtained in Example 5-2 was determined using the Bradford method (Quick Start ^™ Bradford 1 × Dye Reagent, Bio-rad). The specific activity of the crude enzyme solution for L-threonine was clarified. L-threonine dehydrogenase (TDH) activity was initiated by adding crude enzyme solution to 2.5 mM β-NAD ⁺ , 50 mM L-threonine, and 100 mM glycine-KOH buffer (pH 10.0). The absorbance change at 340 nm was measured at 30 ° C. The activity of the crude enzyme solution was calculated in U / mg (1U is the amount of enzyme capable of producing 1 μM NADH per minute). In addition to the crude enzyme solution prepared from the expression strain into which the plasmid of pET15b-TDHs was introduced, the L-threonine activity was also measured in the cell extract prepared from the strain into which the pET15b vector was introduced as a control. As a result of the measurement, the following specific activity values were obtained. Since both had L-threonine dehydrogenase activity more than twice the specific activity value of the bacterial cell extract, it was concluded that all proteins had TDH activity.

Example 9 (Measurement result by Endopoint method)
As shown in Example 8, it was revealed that any of the proteins having the sequences obtained in Examples 3 to 6 can be used for L-Thr concentration measurement by the initial rate method. Therefore, L-threonine was measured by the following method using the endpoint method, which can measure with higher accuracy than the initial velocity method. ThrDH_4fix was used as the enzyme used for the measurement.

A reaction solution containing 2.5 mM NAD ⁺ and 10-200 μM L-threonine was heated at 30 ° C for 30 minutes at the optimal reaction pH of ThrDH_4fix, and then the reaction was started by adding purified ThrDH_4fix (reaction solution volume: 200 μL) . The enzyme concentration of the added purified ThrDH_4fix was 0.077 mg / mL as the final concentration in the reaction solution. The reaction was carried out by heating for 60 minutes at 30 ° C. Immediately after heating, the absorbance at 340 nm was measured, and the L-threonine concentration in the sample was calculated based on the absorbance and the molar extinction coefficient of NADH at 340 nm (6300 M ^-1 cm ^-1 ). Fitting was performed by the square method, and the relationship between the L-threonine concentration with a known concentration and the L-threonine concentration in the sample calculated from the amount of NADH produced was determined. As a result, as shown in FIG. 16, ThrDH_4fix has a good linear relationship up to an L-threonine concentration of up to 200 μM, and the ThrDH_4fix sequence protein obtained by the method of the present invention is the L-threonine concentration obtained by the endpoint method. It was found that it can be used for concentration measurement.

References
[1] Nakano, S. and Asano, Y. Protein evolution analysis of S-hydroxynitrile lyase by complete sequence design utilizing the INTMSAlign software. Sci. Rep. (2015) 5: 8193
[2] Nakano, S., Okazaki, S., Tokiwa, H., Asano, Y. Binding of NAD + and L-Threonine Induces Stepwise Structural and Flexibility Changes in Cupriavidus necator L-Threonine Dehydrogenase. J. Biol. Chem. 2014) 289: 10445-10454
[3] Kazuoka, T., Takigawa, S., Arakawa, N., Hizukuri, Y., Muraoka, I., Oikawa, T., Soda, K. Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp. strain KUC-1, J. Bacteriol. 185: 4483-4489
[4] Yoneda, K., Sakuraba, H., Araki, T., Ohshima, T. Crystal structure of binary and ternary complexes of archaeal UDP-galactose 4-epimerase-like L-threonine dehydrogenase from Thermoplasma volcanium, J. Biol Chem. 287: 12966-12974

The present invention provides a method for quickly and easily screening a novel enzyme having a target function from an amino acid sequence database and a three-dimensional structure from a wide range of sequence groups including a sequence group registered under a name different from that of the target enzyme. It is useful for discovering and developing new enzymes. In addition, as shown in Reference 1, SDR-TDH to which CnTDH belongs is useful in fields requiring measurement of L-threonine concentration in biological samples such as human blood and foods. The protein can be used for the measurement of L-threonine.

SEQ ID NO: 1: amino acid sequence of L-threonine dehydrogenase (TvTDH) SEQ ID NO: 2: amino acid sequence of L-threonine dehydrogenase (TaTDH) SEQ ID NO: 3: amino acid sequence of L-threonine dehydrogenase (mtTDH) 4: amino acid sequence of L-threonine dehydrogenase (ThrDH — 4fix) SEQ ID NO: 5: amino acid sequence of L-threonine dehydrogenase (CfuTDH) SEQ ID NO: 6: amino acid sequence of L-threonine dehydrogenase (CmaTDH) SEQ ID NO: 7 Amino acid sequence of L-threonine dehydrogenase (MpaTDH) SEQ ID NO: 8: Amino acid sequence of L-threonine dehydrogenase (OhoTDH) SEQ ID NO: 9: Amino acid sequence of L-threonine dehydrogenase (EanTDH) SEQ ID NO: 10: L- Amino acid sequence of threonine dehydrogenase (BbaTDH) SEQ ID NO: 11 to 85: amino acid sequence of a protein other than SEQ ID NO: 1 to 10 in Table 1

Claims (22)

1. The protein for L-threonine analysis according to any of (1) to (3) below.
   (1) a protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, 5 to 10 in the sequence listing;
   (2) having an amino acid sequence having 1 to 50 amino acid substitutions, deletions and / or additions in the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, 5 to 10 in the sequence listing; A protein having threonine dehydrogenase activity,
   (3) It has an amino acid sequence having 90% or more identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, and 5 to 10 and has L-threonine dehydrogenase activity. protein.
2. Any one of the following proteins (1) to (3).
   (1) a protein having the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing;
   (2) a protein having an amino acid sequence having 1 to 50 amino acid substitutions, deletions and / or additions in the amino acid sequence set forth in SEQ ID NO: 4 of the Sequence Listing, and having L-threonine dehydrogenase activity;
   (3) A protein having an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing and having L-threonine dehydrogenase activity.
3. A method for preparing L-threonine dehydrogenase, comprising screening a protein having L-threonine dehydrogenase activity from a protein having an amino acid sequence set forth in any of SEQ ID NOs: 11 to 86 in the sequence listing.
4. A method for screening a protein having a target enzyme activity, comprising the following steps (A) to (D):
   (A) Using a sequence alignment of amino acid sequences of proteins, a library consisting of a plurality of proteins having amino acid sequences similar to the amino acid sequences of known proteins having the target enzyme activity is created from a group of proteins having known amino acid sequences. To
   (B) identifying at least two correlated residues from the amino acid sequence of a known protein having the target enzyme activity and the amino acid sequence of the protein selected in (A),
   (C) From the protein library prepared in (A), a protein having the correlated residue specified in (B) is selected.
   (D) It is confirmed whether or not the protein selected in (C) has the target enzyme activity.
5. The similar amino acid sequence in (A) has a sequence identity of 5 to 90%, preferably 10 to 70%, with the amino acid sequence of a known protein having the target enzyme activity obtained using BLASTp. The method according to claim 4, wherein the amino acid sequence is in the range of 15 to 50%.
6. 6. The protein library created in (A) comprises 10 to 100,000 proteins, preferably 100 to 50,000 proteins, more preferably 500 to 10,000 proteins. The method described in 1.
7. Correlation residues in (B) are 50% or less conservative from the sequence alignment results for known proteins having the target enzyme activity and at least two proteins selected in (A). The method according to any one of claims 4 to 6, wherein the identification is performed by selecting a pair of amino acid residues in which the type of the amino acid residue is changed.
8. As for the correlation residue in (B), both of the two residues constituting the correlation residue are present at a distance of 5 mm or more from the active center of the protein in the three-dimensional structure of a known protein having the target enzyme activity. The method according to any one of claims 4 to 7, which is a residue.
9. The method according to any one of claims 4 to 8, wherein the number of correlation residues selected in (B) is two.
10. The protein group whose amino acid sequence is known in (A) is:
    The method according to any one of claims 4 to 8, which is a protein registered by a protein registered with a protein registration organization and a nucleic acid sequence registered with a registration organization of nucleic acid sequences.
11. The method according to claim 10, wherein the protein registration authority is at least one selected from the group consisting of Genebank and PubMed, and the nucleic acid sequence registration authority is DDBj.
12. A method for producing a protein having a target enzyme activity, comprising preparing a protein screened by the method according to any one of claims 4 to 11.
13. A method for producing an artificial protein having a target enzyme activity, comprising the steps (E) to (G).
    (E) Create a candidate library consisting of a plurality of proteins having the target enzyme activity,
    However, the protein contained in the candidate library may be a protein presumed to have the target enzyme activity.
    (F) aligning the candidate library with an amino acid sequence of a known protein having the target enzyme activity, and identifying the most frequently occurring amino acid residue as a consensus residue;
    (G) A protein having an amino acid sequence composed only of the consensus residue is designed, and the designed protein is prepared.
14. The method according to claim 13, wherein the candidate library in (E) comprises a plurality of proteins having the target enzyme activity screened by the method according to any one of claims 4 to 11.
15. The method according to claim 13 or 14, wherein identification of consensus residues in (F) is performed using software capable of performing multiple sequence alignment.
16. The method of claim 15, wherein the software is INTMSAlign or ClustalW.
17. The method according to any one of claims 4 to 16, wherein the target enzyme activity is selected from the enzyme activities of an enzyme group registered in BRENDA and given an enzyme number (EC number).
18. A polynucleotide encoding the protein according to any one of (1) to (3) of claim 2.
19. A recombinant vector comprising a plasmid and the polynucleotide of claim 18 inserted into the plasmid.
20. 20. A transformant comprising a host and the polynucleotide of claim 18 or the recombinant vector of claim 19 contained in the host.
21. A host transformed with the transformant according to claim 20 or a plasmid containing the polynucleotide encoding the protein according to any one of (1) to (3) of claim 2 is cultured, and the culture is used to claim 2. A method for producing L-threonine dehydrogenase, comprising collecting the protein having L-threonine dehydrogenase activity described in 1.
22. The protein of claim 1,
    The protein according to claim 2,
    A method for measuring an L-threonine concentration in a subject using the L-threonine dehydrogenase prepared by the method according to any one of claims 3, 12 to 16, and 21.

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