JP5514399B2 - Method for producing inorganic sulfur compound hydrolase - Google Patents

Description

  The present invention relates to a method for producing an inorganic sulfur compound hydrolase and a method for using the enzyme obtained by the production method.

  Almost all sulfur components in petroleum fuels that cause air pollution and acid rain should be removed. In this regard, due to the efforts of each oil company, Sulfur Free (sulphur content is almost zero) below the standard has been supplied to the market. On the other hand, there is a new problem that a large amount of desulfurized sulfur removed from oil is surplus. These are currently treated as waste as agricultural fertilizers mainly in China, and they are not necessarily effective solutions, and are becoming more serious with the depletion of disposal sites. That is, at present, there is a big problem that there is no technology for effective utilization or treatment of desulfurized surplus sulfur produced in large quantities with petroleum desulfurization.

Tetrathionate hydrolase, a kind of inorganic sulfur compound hydrolase, is an enzyme whose activity is detected in Acidithiobacillus genus bacteria (including some Acidiphilium genus bacteria) that are acidophilic bacteria. It was the subject of academic interest as an enzyme characteristic of Acidithiobacillus bacteria. So far, there have been reports on purification of the enzyme and examination of various properties (Non-Patent Documents 1 to 4).

An enzyme (tetrathionate hydrolase) that hydrolyzes tetrathionate, one of the reduced inorganic sulfur compounds, from Acidithiobacillus ferrooxidans (hereinafter referred to as A. ferrooxidans ), one of the iron-sulfur oxidizing bacteria that grows using sulfur as an energy source. There is a report by the present inventor about the purified product. This enzyme has been reported to detect activity only from bacteria belonging to the genus Acidithiobacillus (with the exception of one bacterium recently renamed to the genus Acidiphilium ), and the present inventor has identified the gene encoding this enzyme and its information Is registered in DDBJ, Accession No. AB259312 (Non-Patent Document 5). This gene is represented as Af-tth. This gene is 1500 bp and encodes a protein consisting of 499 amino acids. Since the sequence of the gene has been clarified, the outer membrane protein whose function was completely unknown in A. ferrooxidans and some thermophilic eosinophilic archaea ( Archaea ) has homology to Af-tth. Became clear. This makes it possible to estimate the sulfur metabolism of organisms with this gene, and to provide academically important information.

  When searching for a protein having a similar structure in the database from the primary structure of the protein estimated by the inventor identified by Af-tth, the function of the corresponding gene product is unknown, most of which is estimated by whole genome sequence analysis. It was only recognized as a protein encoded by the above structural gene or simply as an outer membrane protein.

  An enzyme is a catalyst having a three-dimensional structure and substrate specificity. For this reason, when using an enzyme, unlike the case of using an inorganic catalyst, there are various advantages such as high substrate specificity and high catalytic ability at room temperature and pressure, enabling energy-saving chemical reactions. is there. Conventionally, no attempt has been made to use an enzyme in a chemical reaction of an inorganic sulfur compound, and no attempt has been made to produce useful substances using an enzyme of an inorganic sulfur compound. Enzymatic chemistry of inorganic sulfur compounds has been studied only within the scope of academic interest as the energy metabolism of sulfur-oxidizing bacteria.

  For example, even when genetic information becomes clear, it may be difficult to express a large amount of active protein in a recombinant protein expression system. As an example of a difficult case, if the target protein has some toxicity to the host cell, the synthesis of the protein is suppressed and the expression level decreases, or the cell that synthesizes the toxic protein is killed. Thus, the host cell cannot even grow. In addition, many proteins are folded after generation, and function is expressed only after a certain three-dimensional structure is formed. Even if the target protein is expressed, it does not fold well and does not function as an enzyme. Or form an enclosure. Once such inclusion bodies are formed, even if solubilized and refolded, the amount of active protein finally obtained may be very small unless effective refolding is performed. Many. Various studies have been made and reported on methods for suppressing the formation of inclusion bodies (Non-patent Document 6, Patent Document 1). Moreover, even if the target protein is expressed as a soluble protein, the production amount is extremely reduced if it is degraded by a protease secreted by the host.

Inducible expression by recombinant Escherichia coli of Af-tth using a normal LB (Luria-Broth, L-Broth) nutrient medium hardly expressed recombinant protein, making it difficult to express in large quantities. In addition, since the recombinant protein slightly expressed in E. coli also forms inclusion bodies having no activity, it was difficult to obtain direct evidence for identifying this enzyme gene. Therefore, there is no report yet about the mass production method of tetrathionate hydrolase, and the present situation is that the enzyme has not been put into practical use.
Eur. J. Biochem. 271, 272-280 (2004) Biosci. Biotechnol. Biochem. 60, 224-227 (1996) Eur. J. Biochem. 243, 678-683 (1997) Microbiology 143, 499-504 (1997) DNA Data Bank of Japan, Accession No.AB259312 Gene 67, 31-40 (1998) JP 2005-269936 JP

  This invention makes it a subject to provide the manufacturing method which can express an inorganic sulfur compound hydrolase in large quantities. More specifically, it is an object to provide a method for producing an inorganic sulfur compound hydrolase using genetic recombination technology, and further to provide a method for using the obtained inorganic sulfur compound hydrolase. To do.

  In order to solve the above-mentioned problems, the present inventors have conducted extensive research on a method for producing an inorganic sulfur compound hydrolase. As a result, a method for culturing a host in which a gene encoding the enzyme is incorporated, and expression are obtained. Furthermore, the inventors have found that an inorganic sulfur compound hydrolase can be produced effectively by devising a method for refolding the enzyme, and thus completed the present invention.

That is, this invention consists of the following.
1. In the case of expressing and producing the inorganic sulfur compound hydrolase by culturing a host cell incorporating a gene encoding the inorganic sulfur compound hydrolase in a culture solution, the host cell is treated with tryptone, yeast extract, A method for producing an inorganic sulfur compound hydrolase, characterized in that a Terrific Broth medium containing glycerol is adjusted to pH 7.0 to 7.4 and cultured in a culture solution to which glucose is further added.
2. 2. The production method according to item 1, wherein the glucose concentration added to the Terrific Broth medium is 20 to 60 mM.
3. 3. The production method according to item 1 or 2, wherein the host is Escherichia coli.
4). When the expressed inorganic sulfur compound hydrolase is an inclusion body, the inclusion body is solubilized with a solution containing guanidine hydrochloride and then treated with a buffer solution having a pH of 1.5 to 4.5, thereby expressing the inorganic sulfur compound hydrolase. The manufacturing method in any one of the preceding clauses 1-3 which refolds and produces | generates an active type inorganic sulfur compound hydrolase.
5. 5. The production method according to any one of items 1 to 4, wherein the inorganic sulfur compound hydrolase is tetrathionate hydrolase.
6). The production method according to any one of 1 to 5 above, wherein the gene encoding the inorganic sulfur compound hydrolase is selected from the following:
1) DNA consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing;
2) Among the nucleotide sequences represented by SEQ ID NO: 1 in the sequence listing, one or more genes include a mutated nucleotide sequence such as substitution, deletion, insertion or addition, and substantially contains an inorganic sulfur compound hydrolase. DNA comprising a base sequence that can be produced;
3) DNA comprising a complementary strand of the DNA described in 1) or 2) above;
4) DNA that can hybridize with the DNA according to 1) or 2) under stringent conditions;
5) A DNA consisting of a base sequence that does not form a hybrid with the DNA described in 1) or 2) but can produce a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 due to degeneracy of the gene codon.
7). The culture solution used for the manufacturing method of any one of said clauses 1-6.
8). A sulfur component hydrolysis treatment method comprising treating a sulfur component using an inorganic sulfur compound hydrolase obtained by the production method according to any one of items 1 to 6.
9. A method for removing a sulfur component, comprising the hydrolysis treatment method according to item 8 above.

  By the method for producing an inorganic sulfur compound hydrolase of the present invention, a recombinant protein of the inorganic sulfur compound hydrolase can be expressed in a large amount, and an active enzyme by refolding can be easily obtained. As a result, further research such as analysis of the reaction mechanism of this enzyme and steric structure by crystallization can be performed. Moreover, by hydrolyzing the sulfur component using this enzyme, for example, a large amount of inorganic sulfur compounds produced with petroleum desulfurization can be treated, and further, the possibility of producing useful substances is opened.

  The present invention relates to a method for producing an inorganic sulfur compound hydrolase and a method for using the enzyme obtained by the production method. In the present invention, the inorganic sulfur compound hydrolase is not particularly limited as long as it is an enzyme capable of hydrolyzing an inorganic sulfur compound, and examples thereof include tetrathionate hydrolase. The gene encoding tetrathionate hydrolase is indicated by Af-tth, its base sequence is shown in SEQ ID NO: 1 in the sequence listing, and has already been registered in DDBJ, Accession No. AB259312 (Non-patent Document 5).

In the present invention, the gene encoding the inorganic sulfur compound hydrolase is any one selected from the following 1) to 5), and the expressed protein has a function capable of hydrolyzing the inorganic sulfur compound. That's fine.
1) DNA consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing.
2) Among the nucleotide sequences shown in SEQ ID NO: 1 in the sequence listing, one or more genes include a mutated nucleotide sequence such as substitution, deletion, insertion or addition, and substantially contain a sulfur compound hydrolase. DNA consisting of a base sequence that can be produced.
3) DNA comprising a complementary strand of the DNA described in 1) or 2) above.
4) DNA that can hybridize with the DNA according to 1) or 2) under stringent conditions.
5) A DNA consisting of a base sequence that does not form a hybrid with the DNA described in 1) or 2) but can produce a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 due to degeneracy of the gene codon.

  In the present invention, among the nucleotide sequences shown in SEQ ID NO: 1 in the sequence listing, one or more genes contain a mutated nucleotide sequence such as substitution, deletion, insertion or addition, A base sequence capable of producing an enzyme means that there may be a plurality of mutations in the gene, as long as it can produce a protein having substantially sulfur compound hydrolase activity. Moreover, even if it is DNA which consists of a complementary strand of DNA as described in said 1) or 2), it should just be able to produce the protein which has an organic sulfur compound hydrolase activity substantially.

Furthermore, stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate, pH 7.6, 5 × Denhart's solution, 10% dextran sulfate, And 20 μg / ml DNA in a solution at 42 ° C. overnight, followed by primary washing in 2 × SSC / 0.1% SDS at room temperature, followed by washing with 0.1 × SSC / 0.1% SDS at 65 ° C. It refers to the next cleaning.

  The method for producing an inorganic sulfur compound hydrolase according to the present invention is directed to a protein production method applying genetic engineering technology, and a host cell incorporating a gene encoding the inorganic sulfur compound hydrolase is cultured in a culture solution. To produce the inorganic sulfur compound hydrolase.

Here, as a method for incorporating a host cell or gene when incorporating a gene encoding an inorganic sulfur compound hydrolase into the host cell, a cell or method known per se can be used. Prokaryotic cells preferably as the host cell, e.g., streptococci (streptococci), Staphylococcus (staphylococci), E. (E. coli), Streptomyces spp (Streptomyces) and Bacillus subtilis (Bacillus subtilis), and the like, A preferred example is E. coli.

  In order to integrate the gene, a recombinant plasmid in which DNA is inserted into a vector can be used. The vector is selected according to the type of the selected host, and comprises a gene sequence intended for expression and a gene sequence carrying information on replication and control. In the present invention, those that can be used for prokaryotic cells are preferred, for example, bacterial plasmid-derived, bacteriophage-derived, transposon-derived, and combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and Phagemid etc. are mentioned.

  As a transformation method, a method known per se can be used. The transformant is cultured under conditions optimal for the culture conditions of the host. When recombinant expression of an inorganic sulfur compound hydrolase by E. coli was attempted, lysis caused mainly by cytotoxicity was observed in the LB medium commonly used in E. coli culture, and induction expression was very difficult. It was.

In the present invention, improvements were made based on TB (Terrific Broth, T-Broth) medium in order to enhance protein production by recombinant cells in recombinant host cells such as E. coli under optimal conditions. In large-scale culture and large-scale expression by scale-up, ampicillin is decomposed by acidification of the medium accompanying the growth of bacterial cells that are host cells, and Escherichia coli that does not hold the recombinant plasmid grows preferentially. The culture solution used in the production method of the present invention is a TB medium containing at least tryptone, yeast extract, and glycerol. As a result of examining the pH of the medium in order to avoid the growth of E. coli that does not hold the recombinant plasmid, pH 7.0 to 7.4 is preferred. The pH is adjusted, for example, by adding 0.9 to 1.0 M phosphate buffer to the basic medium. For the preparation of 0.9 to 1.0 M phosphate buffer, KH ₂ PO ₄ and K ₂ HPO ₄ which are generally used can be used. In this way, a phosphate buffer solution can be added to prevent a decrease in the pH of the medium.

  Furthermore, the expression of the recombinant protein in the basal state (uninduced state) can be suppressed by adding glucose to the medium. In the present invention, the concentration of glucose added can be 20 to 60 mM, and more preferably 50 to 60 mM. Under the above culture conditions, a large amount of inorganic sulfur compound hydrolase can be expressed and produced.

  In the present invention, when an inorganic sulfur compound hydrolase is expressed in prokaryotic cells, an inclusion body is formed, so that the expressed inorganic sulfur compound hydrolase is insolubilized. It can be said that TB medium is more effective than LB medium as a medium for effectively forming inclusion bodies and increasing the expression level.

  The present invention also extends to a culture solution used in the above production method. Specifically, a culture medium in which glucose is added to 20 to 60 mM, preferably 50 to 60 mM, and pH is adjusted to pH 7.0 to 7.4 to TB medium containing at least tryptone, yeast extract, and glycerol. . Furthermore, ampicillin can be added to a final concentration of 50 to 100 g / ml.

In order to obtain an active enzyme of an inorganic sulfur compound hydrolase from the formed inclusion bodies, the host cells are washed with a buffer solution, and then the cells are crushed or lysed using ultrasonic waves or the like, and a commercially available reagent such as BugBuster ^{( R)} (Takara Bio Inc.) can be used to extract inclusion bodies. An insoluble fraction can be obtained from the extract containing the inclusion bodies by centrifugation or the like. Since the obtained insoluble fraction contains a target inclusion body, a host-derived hydrophobic membrane protein and the like, it is washed with a buffer containing a surfactant to remove proteins other than the target enzyme. The insoluble fraction after washing can be subjected to the following refolding operation using the inclusion body fraction of the target enzyme. Here, the inclusion body fraction can be stored frozen in a pellet state.

  About the said inclusion body fraction, a target enzyme can be solubilized using a chemical denaturant. As the chemical denaturing agent, a guanidine hydrochloride solution of 0 to 6 M, preferably about 6 M can be used. The solubilized target enzyme can be treated with a buffer of pH 1.5 to 4.5, preferably pH 2.5 to 3.5. For example, by injecting the inclusion body fraction into a dialysis tube and treating with the above buffer, the chemical denaturant can be gently removed and the target enzyme can be refolded. Since the inorganic sulfur compound hydrolase of the present invention is a membrane protein localized in the outer membrane, an active enzyme folded normally is insolubilized as it is. Therefore, the target enzyme can be refolded more effectively by increasing the ionic strength of the buffer solution after the solubilization treatment. As a method for increasing the ionic strength of the buffer, for example, 0.1 to 0.5 M, preferably about 0.4 M ammonium sulfate can be added.

  An activated target enzyme can be obtained by centrifuging the solution containing the solubilized target enzyme and collecting the supernatant.

  The activated target enzyme obtained by the production method and refolding operation of the present invention can hydrolyze inorganic sulfur compounds. For example, by treating a large amount of inorganic sulfur compound produced with the desulfurization of petroleum with the enzyme obtained by the production method of the present invention, it is possible to treat surplus sulfur. Accordingly, the present invention extends to a method for hydrolyzing a sulfur component by using the obtained active target enzyme. Furthermore, the present invention extends to a method for removing a sulfur component including removing a hydrolyzed material.

  Hereinafter, the present invention will be described more specifically with reference to comparative examples and examples, but the present invention is not limited thereto.

(Example 1) Expression by gene recombination of tetrathionate hydrolase The gene encoding tetrathionate hydrolase is indicated by Af-tth, its base sequence is shown in SEQ ID NO: 1 in the sequence listing, and it is already DDBJ, Accession No. Also registered in .AB259312 (Non-Patent Document 5).

  Expression of tetrathionate hydrolase (hereinafter referred to as “target enzyme”) in recombinant Escherichia coli introduced with Af-tth using TB culture medium (TB basic medium supplemented with 20-60 mM glucose) Tried. A gene obtained by removing a portion encoding 32 amino acids from the N-terminal side presumed to be a signal peptide from the full length of all genes of Af-tth (Accession No. AB259312) was incorporated into pET21a plasmid and transformed into E. coli.

The TB culture solution used for the culture of the present invention was prepared as follows.
Tryptone 12 g, yeast extract 24 g, and glycerol 4 ml were diluted with 900 ml of demineralized water or tap water. The messed up solution and 0.9 ml of 0.9-1.0 M potassium phosphate buffer were autoclaved separately at 121 ° C. for 20 minutes. After sterilization and cooling to room temperature, both were aseptically mixed in a clean bench (pH 7.0-7.4). Glucose was added to a final concentration of 20 to 60 mM, and ampicillin was further added to a final concentration of 50 to 100 g / ml.
The transformed Escherichia coli was cultured for 36 hours at room temperature in TB culture medium supplemented with Isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM to induce and express the recombinant target enzyme.

When the outer membrane protein produced from the recombinant target enzyme obtained above and A. ferrooxidans was confirmed by SDS-PAGE (CBB staining) and Western blotting, a band was observed in almost 50 kDa. (FIG. 1). Note that the anti-tetrathionate hydrolase antibody prepared in Comparative Example 1 was used for Western blotting.

The molecular weight calculated from the primary sequence of the gene was 49714, which was consistent with the molecular mass of the product enzyme, 50 kDa. Of the amino acids constituting tetrathionate hydrolase, 32 amino acids from the N-terminus have a secretory signal sequence of Sec type. The 13 amino acids immediately after that were confirmed to completely match the N-terminal amino acid sequence of tetrathionate hydrolase produced from A. ferrooxidans .

(Example 2) Refolding of inclusion bodies Escherichia coli inducing and expressing the recombinant target enzyme of Example 1 was collected by centrifugation and washed with 1.0 M potassium phosphate buffer (pH 7.0). The washed cells were suspended in the same buffer, and the cells were crushed by ultrasonic waves, etc., and the cells were dissolved using BugBuster ^(TM) (Takara Bio Inc.).

The bacterial cell extract was centrifuged and the insoluble fraction was collected. When the bacterial cell extract (W), the insoluble fraction (I) and the soluble fraction (supernatant fraction: S) thus obtained were confirmed by Western blotting (?), A band was observed in the insoluble fraction. And confirmed to exist as inclusion bodies (FIG. 2).
For Western blotting, an antibody prepared using recombinant tetrathionate hydrolase expressed in Comparative Example 1 described later was used.

  The insoluble fraction includes a host-derived hydrophobic membrane protein in addition to inclusion bodies of the target enzyme. In order to remove proteins other than the target enzyme inclusion body, the protein was washed three times with 0.1 M potassium phosphate buffer (pH 7.0) containing 4% (v / v) surfactant (Triton X-100). Then, it was washed 3 times with sterilized pure water to remove the surfactant. This was used as the inclusion body fraction of the target enzyme in the following refolding experiment. The inclusion body fraction can be stored frozen in a pellet state.

6M-guanidine hydrochloride was added to the inclusion body fraction and solubilized. Inject the solubilized target enzyme solution into a dialysis tube, dialyze with 0.1 M -Ala-SO ₄ ^2- buffer (pH 3.0), 0.4 M ammonium sulfate, and gently remove guanidine hydrochloride. Enzyme refolding was performed.

Since the target enzyme is a membrane protein localized in the outer membrane, the active enzyme folded normally is insolubilized as it is. Therefore, we succeeded in solubilizing the target enzyme with the active form by increasing the ionic strength by adding 0.4 M ammonium sulfate to the dilution buffer.
Finally, the supernatant was recovered by centrifugation to secure an active recombinant target enzyme.

(Comparative Example 1) Expression of gene recombination target enzyme in LB medium
We tried to express the target enzyme in recombinant Escherichia coli introduced with Af-tth in LB medium. The procedure was the same as in Example 1 except that the medium was LB medium.
Although the expression level of the target enzyme in the LB medium was slight, an anti-tetrathionate hydrolase antibody was prepared based on this expression level.

(Experimental Example 1) Confirmation of Expression Level of Target Enzyme For the insoluble fraction obtained in Example 1 and Comparative Example 1, the expression level of the target enzyme was confirmed by SDS-PAGE.
As a result, when expressed by the method of Comparative Example 1, expression was observed only on a small scale (about 5 ml), but no expression was observed when scaled up to 100 ml or more. On the other hand, when expressed by the method of Example 1, recombinant expression was possible even when scaled up to 100 ml or more, and a large amount of recombinant target enzyme could be obtained.

  The expression level when scaled up to 100 ml or more was examined by SDS-PAGE (FIG. 3). As a result, when IPTG was induced using the culture medium of Example 1, a strong band was observed in the 50 kDa portion, confirming that the target enzyme was effectively expressed.

(Experimental Example 2) with by the target enzyme and Example 2 of the insoluble inclusion bodies obtained in the confirmation Example 1 hydrolytic resolution of the target enzyme in the target enzyme obtained by refolding, hydrolysis of a pressurized water resolution Torachion acid The effect of the reaction was confirmed by absorbance (A _{480 nm} ) (FIG. 4). As a result, no hydrolysis activity was observed in the insoluble target enzyme obtained in Example 1, but it was confirmed that the target enzyme obtained in Example 2 has hydrolytic ability.
It was confirmed by HPLC analysis that the reaction mechanism was S ₄ O ₆ ² + H ₂ O-> S ⁰ + S ₂ O ₃ ² + SO ₄ ² + 2H ⁺ .

  As described in detail above, the method for producing an inorganic sulfur compound hydrolase according to the present invention makes it possible to express a large amount of a recombinant protein of an inorganic sulfur compound hydrolase and further develop a method for obtaining an active enzyme by refolding. It was. This allows further research such as analysis of the reaction mechanism of this enzyme and steric structure by crystallization, and opens up the possibility of producing inorganic sulfur compounds and useful substances. By understanding the detailed reaction mechanism of inorganic sulfur compound hydrolase, the substrate specificity is changed by genetic engineering techniques, and sulfur (usually eight molecules of sulfur form SS bonds with each other to form an eight-membered ring). There is a possibility of hydrolysis.

  Normally, if water-insoluble sulfur can be hydrated by hydrolyzing it with an inorganic sulfur compound hydrolase, the biological affinity will be particularly enhanced, and the reactivity will be dramatically increased. The possibility of application to highly efficient biological treatment can be expected. Biological treatment methods are generally performed at ordinary temperatures and pressures, and are useful as technologies that save energy and have a low environmental impact.

  In particular, the problem of large-scale surplus of petroleum desulfurization sulfur has become serious, and the production method of the present invention may lead to a useful solution to this problem. By treating the petroleum desulfurized surplus sulfur using the inorganic sulfur compound hydrolase produced by the method of the present invention, a highly selective or energy-saving reaction is expected, and the environment, food, energy, etc. It can be expected as an effective solution to the problem.

In addition, it was revealed that the recombinant inorganic sulfur compound hydrolase obtained by the production method of the present invention is equivalent to the outer membrane protein of A. ferrooxidans. It is also possible to effectively use an organism having a gene to be encoded.

It is a figure which shows that the recombinant tetrathionate hydrolase produced by E. coli is a protein equivalent to an outer membrane protein (enzyme) produced from A. ferrooxidans . (Example 2) It is a figure which shows that the recombinant tetrathionate hydrolase produced by E. coli exists in inclusion bodies. (Example 2) It is a figure which shows the expression level of the recombinant tetrathionate hydrolase by the conventional method and the method of this invention. (Experimental example 1) It is a figure which shows the hydrolysis activity of the recombinant tetrathionate hydrolase obtained by refolding by the method of this invention. (Experimental example 2)

Claims (4)

A DNA encoding a tetrathionate hydrolase derived from Acidithiobacillus genus bacteria, wherein the tetrathionate hydrolase is expressed by culturing E. coli into which any one of the following DNAs 1) to 5) is incorporated in a culture solution In the case of production, the Escherichia coli is cultured in a culture solution containing Terrific Broth medium containing tryptone, yeast extract and glycerol adjusted to pH 7.0 to 7.4 and further supplemented with glucose. In the case of inclusion bodies, the inclusion bodies are solubilized with a solution containing guanidine hydrochloride and then refolded with tetrathionate hydrolase expressed by treatment with a buffer solution having a pH of 1.5 to 4.5. Method for producing tetrathionate hydrolase :
1) DNA consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing;
2) A base sequence encoding tetrathionate hydrolase, including a mutated base sequence in which one to a plurality of bases are substituted, deleted, inserted or added among the base sequences shown in SEQ ID NO: 1 in the sequence listing DNA comprising:
3) DNA comprising a complementary strand of the DNA described in 1) or 2) above;
4) DNA that hybridizes with the DNA according to 1) or 2) under stringent conditions;
5) Does not hybridize with the DNA described in 1) or 2) under stringent conditions, but consists of a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 due to degeneracy of the gene codon DNA. The manufacturing method of Claim 1 whose glucose concentration added to a Terrific Broth culture medium is 20-60 mM. The production method according to claim 1 or 2, wherein the buffer solution to be treated after the inclusion body is solubilized with a solution containing guanidine hydrochloride is a buffer solution having a pH of 1.5 to 4.5 containing 0.1 to 0.5 M ammonium sulfate. The production method according to any one of claims 1 to 3, wherein the treatment performed after the inclusion body is solubilized with a solution containing guanidine hydrochloride is a dialysis treatment.