JP2002253247A - Thermally stable desulfurization enzyme and gene encoding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To facilitate the isolation of sulfur in a fossil fuel. SOLUTION: The enzyme having a function for decomposing a thiophene- based compound, the gene encoding the enzyme, the vector including the gene, and the transformant are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an enzyme having a function of decomposing thiophene compounds utilizing microorganisms, that is, benzothiophene, dibenzothiophene (hereinafter referred to as "DBT") and their substituted products, or derivatives thereof. It concerns the gene that encodes it. By utilizing the enzymes and genes of the present invention, it is possible to release sulfur in benzothiophene and DBT contained in fossil fuels such as petroleum and their substituted substances, or derivatives thereof. Sulfur, which is said to diffuse into the air due to the combustion of fossil fuels, can be easily removed from fossil fuels.

[0002]

2. Description of the Related Art As a method for desulfurization for removing sulfur from a hydrocarbon fuel such as petroleum, there are known methods such as alkali washing and solvent desulfurization. However, at present, hydrodesulfurization is mainly used. I have. Hydrodesulfurization is a method in which a sulfur compound in a petroleum fraction is reacted with hydrogen in the presence of a catalyst, and removed as hydrogen sulfide to reduce the sulfur content of the product. As the catalyst, a metal catalyst such as cobalt, molybdenum, nickel, and tungsten is used with alumina as a carrier.
In the case of a molybdenum-supported alumina catalyst, cobalt or nickel is usually added as a co-catalyst in order to improve the catalytic performance. There is no doubt that hydrodesulfurization using metal catalysts is an extremely complete process that is currently widely used worldwide. However, there are several issues in terms of the process for making petroleum products in compliance with stricter environmental regulations. The following is a brief description of an example.

[0003] Metal catalysts generally have low substrate specificity and are therefore suitable for the purpose of decomposing various kinds of sulfur compounds and reducing the sulfur content of the entire fossil fuel,
It is believed that the desulfurization effect of certain groups of sulfur compounds, ie, heterocyclic sulfur compounds such as benzothiophene and DBT, and their alkyl derivatives, may be insufficient. For example, various heterocyclic organic sulfur compounds still remain in the gas oil after desulfurization. One of the causes of the insufficient desulfurization effect of the metal catalyst is considered to be steric hindrance due to substituents around sulfur atoms in these organic sulfur compounds. Among these substituents, the effect of the presence of a methyl substituent on the reactivity of metal catalysts in hydrodesulfurization has been studied for thiophene, benzothiophene, DBT, etc. According to these results, it is clear that the desulfurization reaction generally decreases as the number of substituents increases, but the position of the substituent has a very large effect on the reactivity. A report comparing the desulfurization reactivity of methyl DBTs and showing that steric hindrance due to substituents has a very large effect on the reactivity of metal catalysts has been reported, for example, by Houalla, M., Broderick, D.
H., Sapre, AV, Nag, NK, de Beer, VH, Gates,
BC, Kwart, HJ, Catalt., 61, 523-527 (1980). In fact, various alkylated derivatives of these DBTs are known to be present in significant amounts in gas oils (eg, Kabe, T., Ishihara, A. and Tajima, H. lnd.
Eng. Chem. Res., 31, 1577-1580 (1992)).

[0004] As described above, desulfurization of an organic sulfur compound having resistance to hydrodesulfurization requires a higher reaction temperature and pressure than currently used, and the amount of hydrogen to be added is also reduced. It is expected to increase significantly. Improvements in such hydrodesulfurization processes are expected to require significant capital and operating costs. As the main sulfur compound species which contains such an organic sulfur compound having resistance to hydrodesulfurization, for example, there is gas oil, and when performing a more advanced desulfurization of gas oil (ultra deep desulfurization), as described above. A significant improvement in the hydrodesulfurization process is required.

On the other hand, the enzymatic reaction performed by living organisms proceeds under relatively mild conditions, and the rate of the enzymatic reaction itself is not inferior to the rate of a reaction using a chemical catalyst. Furthermore, it is necessary to properly cope with a wide variety of biological reactions that occur in vivo, so that there are numerous types of enzymes, and these enzymes generally show very high substrate specificity. Have been. Such a feature is expected to be utilized in a so-called biodesulfurization reaction in which sulfur in sulfur compounds contained in fossil fuels is removed using microorganisms (Monticello, DJ, Hydrocar
bon Processing 39-45 (1994)).

[0006] On the other hand, there have been many reports on a method for removing sulfur from a heterocyclic sulfur compound which is a component of petroleum by using bacteria. However, these methods are based on a ring decomposition (CC bond cleavage) type reaction and a CS bond cleavage. They are roughly classified into type reactions. Examples of bacteria having CC bond attack type desulfurization activity include, for example, Pseudomo
nas sp., Pseudomonas aeruginosa, Beijerinckia sp.,
Pseudomonas alcaligenes, Pseudomonas stutzeri, Ps
eudomonas putida, Brevibacterium sp. and the like are known. These bacteria perform a type of metabolism in which the CC bond in a heterocyclic sulfur compound represented by DBT is broken, the benzene ring is decomposed, and a sulfur cascade is released through a subsequent oxidation cascade. is there. The reaction mechanism of these carbon skeleton attack type pathways is hydroxylation of aromatic ring (DBT →
1,2-dihydroxydibenzothiophene), ring cleavage, oxidation to water-soluble products (1,2-dihydroxydibenzothiophene → trans-4 [2- (3-hydroxy) thiannaphthenyl] -2-oxo-butenoic acid, 3 -Hydroxy-2-formylbenzothiophene), which is called the Kodama pathway. This type of reaction attacks the CC bond in the benzene ring of DBT, producing a variety of water-soluble substances that can be extracted from the oil. However, due to this reaction,
Other aromatic molecules in the oil are attacked, resulting in the transfer of significant amounts of hydrocarbons to the liquid phase (Hartdegen,
FJ, Coburn, JM and Roberts, RL Chem. Eng. Pr
ogress, 80, 63-67 (1984)). This leads to a decrease in the total calorific value of petroleum, which is an industrially inefficient reaction. In addition, this type of DBT oxidative degradation bacteria gives a water-soluble thiophene compound (mainly 3-hydroxy-2-formylbenzothiophene) as an oxidation product as reported by Kodama et al., Which is removed from the liquid phase. It is also a substance that is difficult to do. In addition, the carbocyclic attack of DBT often results in DBs with alkyl or allyl substituents.
DBTs substituted at these positions do not serve as substrates for the Kodama pathway, as they occur at positions 2 and 3 of T.

[0007] There have been reported microorganisms that decompose not only crude oil and coal but also model compounds containing sulfur and selectively remove sulfur as a heteroatom to produce sulfates and hydroxylated compounds. This type of reaction is considered to be a reaction that specifically cleaves the CS bond in the sulfur compound and releases sulfur in the form of sulfate, in view of the structure of the metabolite. To date, there have been reports of sulfur attack type bio-desulfurization reaction systems as shown in Table 1.

[0008]

[Table 1]

[0009] All of the biodesulfurization processes described above utilize a microbial metabolic reaction that proceeds under a temperature condition around 30 ° C. On the other hand, it is known that the rate of a chemical reaction generally increases depending on temperature. In the desulfurization step in the petroleum refining process, fractional distillation and desulfurization are performed under high temperature and high pressure conditions. Therefore, if a bio-desulfurization step is incorporated into a petroleum refining process, it would be desirable to be able to perform a bio-desulfurization reaction at a higher temperature during cooling without cooling the petroleum fraction to near normal temperature. Reports on high temperature biodesulfurization include:

Most attempts to perform desulfurization reactions at elevated temperatures using microorganisms can be found in coal desulfurization. Coal contains various sulfur compounds. Although the main inorganic sulfur compound is pyrite, a wide variety of organic sulfur compounds are mixed, and many are known to contain thiol, sulfide, disulfide, and thiophene groups. The microorganism used was Sulfol
Obus bacteria, all of which are thermophilic. Leaching of metals from mineral sulfides (Brierley C.L. &
Murr, LE, Science 179, 448-490 (1973)) and various different sulfolobuses for removing pyrite from coal.
Examples using strains have been reported (Kargi, F. & Robinso
n, JM, Biotechnol.Bioeng, 24, 2115-2121 (1982);
Kargi, F. & Robinson, JM, Appl. Environ. Microbio
l., 44, 878-883 (1982); Kargi, F. & Cervoni, TD, B
iotechnol.Letters 5, 33-38 (1983); Kargi, F. and
Robinson, JM, Biotechnol.Bioeng., 26, 687-690 (1
984); Kargi, F. & Robinson, JM, Biotechnol. Bioe
ng. 27, 41-49 (1985); Kargi, F., Biotechnol. Lett.,
9, 478-482 (1987)). Kargi and Robinson (Kargi, F. and Robinson, JM, Appl. Environ. Microbiol., 44,
878-883 (1982)), Sulfolobus acidocaldar isolated from acidic hot springs in Yellowstone National Park, USA
Some strains of ius (hereinafter referred to as “S. acidocaldarius”) grow at 45-70 ° C., but oxidize elemental sulfur at an optimal pH of 2. Pyrite oxidation by two other S. acidocaldarius strains has also been reported (Tobita, M., Yokozek
i, M., Nishikawa, N. & Kawakami, Y., Biosci. Biote
ch. Biochem. 58, 771-772 (1994)).

[0011] Among the organic sulfur compounds contained in fossil fuels, DBT and its substitution products or derivatives thereof are known to be less susceptible to hydrodesulfurization in ordinary petroleum refining processes. High temperature decomposition of the DBT by S. acidocaldarius has also been reported (Kargi, & Robinson, JM,
Biotechnol. Bioeng, 26, 687-690 (1984); Kargi, F.,
Biotechnol. Letters 9, 478-482 (1987)).

According to these reports, when a model aromatic heterocyclic sulfur compound such as thianthrene, thioxanthene, DBT or the like is reacted with this microorganism at a high temperature, these sulfur compounds are oxidized and decomposed. The oxidation of these aromatic heterocyclic sulfur compounds by S. acidocaldarius is 70 ° C
And produces sulfate ion as a reaction product. However, this reaction is a reaction in a medium that does not contain a carbon source other than the sulfur compound, and uses the sulfur compound as a carbon source. That is, it is clear that the CC bond in the sulfur compound is decomposed. Furthermore, this S. acido
caldarius can only grow in acidic media, and the oxidative degradation of DBT requires progression under harsh acidic conditions (pH 2.5). It is considered that such severe conditions cause deterioration of petroleum products, and at the same time, require an acid-resistant material in a step relating to desulfurization, which is considered to be undesirable in the process. S. aci
When docaldarius is grown under autotrophic conditions, it obtains the necessary energy from reduced iron and sulfur compounds,
Utilizes carbon dioxide as a carbon source. However, S. acido
When grown under heterotrophic conditions, caldarius can utilize various organic compounds as carbon and energy sources. That is, it is considered that the presence of fossil fuel is utilized as a carbon source.

[0013] Finnerty et al., Pseudomonas stutzeri, Ps
It has been reported that strains belonging to eudomonas alcaligenes and Pseudomonas putida degrade DBT, benzothiophene, thioxanthene and thianthrene and convert them to water-soluble substances (Finnerty, WR, Shockiey, K., Attaway, H.
in Microbial Enhanced Oil Recovery, Zajic, JE
(Ed.), Penwell. Tuisa, Okia, 83-91 (1983)). In this case, the oxidation reaction proceeds even at 55 ° C. However, degradation products of DBT by these Pseudomonas strains
Reported 3-hydroxy-2-formylbenzothiophene (Monticello, DJ, Bakker, D., Finner
ty, WR Appl.Environ.Microbiol., 49, 756-760 (198
Five)). The oxidative activity of DBT by these Pseudomonas strains is induced by the sulfur-free aromatic hydrocarbons naphthalene and salicylic acid and is blocked by chloramphenicol. From this, these Pseudomonas
It can be seen that the degradation reaction of DBT by the strain is based on the degradation by breaking the CC bond in the aromatic ring.
In addition, valuable aromatic hydrocarbons contained in petroleum fractions other than sulfur compounds may be simultaneously decomposed, which lowers the value of fuel and the quality of petroleum fractions.

As described above, the bacteria discovered so far that can decompose DBT at high temperatures cleave the CC bond in the DBT molecule and catalyze the reaction utilized as a carbon source.
As described above, a decomposition reaction of an organic sulfur compound that specifically cleaves a CS bond but leaves a CC bond without cutting it is desirable as an actual method for desulfurizing petroleum. In other words, most biosulfurization processes utilize microorganisms that exhibit the activity of cleaving the CS bond in DBT and its alkyl-substituted or derivative molecules at high temperatures and generate desulfurization products in the form of water-soluble substances. desirable.

As described above, microorganisms that perform a DBT decomposition reaction of the CS bond cleavage type are known among bacteria of several genera. For example, ATCC53968 of Rhodococcus sp. Is a well-studied DBT-degrading strain, which adds an oxygen atom to the sulfur atom of DBT to form dibenzothiophene sulfoxide (hereinafter referred to as "D
BTO) to dibenzothiophene sulfone (hereinafter, referred to as
Generates) as "DBTO _2", followed 2- (2'-hydroxyphenyl) via a sulfinic acid salt 2-hydroxybiphenyl (hereinafter, performing a reaction for generating a) as "2-HBP". However, it has been reported that even at 37 ° C. and 43 ° C., which is slightly higher than the normal culturing temperature of 30 ° C., the cultivation of the bacterium is very slow or stops growing after 48 hours (Japanese Unexamined Patent Publication No. No. 54695). From this fact, in order to carry out the high-temperature desulfurization reaction, it is necessary to convert an organic sulfur compound, particularly DBT and a substituted product thereof, or a heterocyclic sulfur compound containing a derivative compound thereof, to a high temperature at a high temperature.
It is considered optimal to use a microorganism that can specifically cleave the -S bond.

Suzuki et al. Have reported that Paenibacillus sp. Strain A11-2 can grow under high-temperature conditions and that the organic sulfur compound C
Report that it can specifically cleave -S bonds (Suz
uki, M., Konishi, J., Ishii, Y., Okumura, K., Appl.
Environ. Microbiol., 63, 3164-3169 (1997)). According to this report, this strain can grow under a temperature condition of 30-60 ° C, and can be used with DBT or 4-methyldibenzothiophene and 4,6
CS of DBT derivatives such as -dimethyldibenzothiophene
The bond can be specifically broken. Therefore, this strain is suitable for performing a high-temperature desulfurization reaction. However, at relatively low temperatures, especially around 30 ° C., the desulfurization activity is significantly reduced.

In the biodesulfurization process, a high temperature range (hereinafter, “high temperature range” means a temperature around 50 to 60 ° C.)
Maintaining high desulfurization activity under a wide range of temperature conditions (30 to 60 ° C.) is effective in simplifying the temperature control of petroleum fractions. Furuya et al., Bacillus subti
lis WU-S2B strain (Accession No.FERM P-17041) can maintain relatively high desulfurization activity under a wide range of temperature conditions including a high temperature range, and can specifically cleave the CS bond of an organic sulfur compound. (Furuya, Nishii, Nakagawa, Kirimura, Usami,
Abstracts of Annual Meeting of the Japanese Society of Agricultural Chemistry, 384 (1999)). According to this report, this strain maintains a relatively high desulfurization activity under a wide range of temperature conditions of 30 to 60 ° C, and has a high Cs of DBT or DBT derivatives such as 2,8-dimethyl DBT and 4,6-dimethyl DBT. -S bond can be specifically cleaved.

A bacterium known to cause a CS bond-cleaving type desulfurization reaction, wherein a gene encoding an enzyme activity involved in the DBT decomposition reaction has been identified and its nucleotide sequence has been determined. For example, Rhodococcus sp.
Dsz gene of IGTS8 strain (Denome, S., Oldfleld., C., N
ash, LJ and Young, KDJBacteriol., 176: 6707-
6716, 1994; Piddington, CS, Kovacevich, BR and Rambosek, J. Appl. Environ. Microbiol., 61: 468-47.
5, 1995), the dsz gene of Sphingomonas sp. Strain AD109 (US Pat. No. 6,133,016) and Paenibacillus sp.
Tds gene (Y. Ishii, J. Konishi, H. Okada, K.
Hirasawa, T. Onaka, M. Suzuki., Biochem. Biophy. R
es. Comm., 270 (1), 81-88, 2000).

The degradation reaction of DBT by IGTS8 strain was determined by DBT.
DszC catalyzes conversion to DBTO ₂ via DBTO, DBTO ₂ to 2-
DszA catalyzing the conversion of (2'-hydroxyphenyl) benzenesulfinic acid to DszB and DszB catalyzing the conversion of 2- (2'-hydroxyphenyl) benzenesulfinic acid to 2-HBP
(Denome, S., Oldfiel
d., C., Nash, LJ and Young, KDJBacteriol.,
176: 6707-6716, 1994; Gray, KA, Pogrebinshy, O.
S., Mrachko, GT, Xi, L. Monticello, DJand Squi
res, CH Nat Biotechnol., 14: 1705-1709, 1996; Old
field, C., Pogrebinsky, O., Simmonds, J., Olson,
ES and Kulpa, CF, Microbiology, 143: 2961-297
3, 1997). The corresponding genes are dszC, dszA, dszB
is called. DszC and DszA are monooxygenases,
Both are known to require coexistence of NADH-FMN oxidoreductase activity for their oxygenation reactions (Gr.
ay, KA, Pogrebinsky, OS, Mrachko, GT, Xi, L.
Monticello, DJ and Squires, CH Nat Biotechn
ol., 14: 1705-1709, 1996; Xi, L. Squires, CH, Mon.
ticello, DJ and Chids, JD Biochem. Biophys. Re
s Commun., 230: 73-76, 1997). It has been reported that when these dsz genes are induced and expressed in Escherichia coli by temperature shift, DszA activity by cell culture reaches a maximum at 39 ° C. and decreases remarkably at 42 ° C. (Denome, S., Oldfiel
d., D., Nash, LJ and Young, KDJ Bacterio
l., 176: 6707-6716, 1994). This result indicates that the desulfurizing enzyme activity of the IGTS8 strain reaches its maximum around room temperature, decreases at higher temperatures, and shows no desulfurizing activity at 50 ° C or higher (Konish's experiment).
i, J., Ishii, Y., Onaka, T., Okumura, K. and Su
zuki, M. Appl.Environ.Microbiol., 63: 3164-3169,
1997).

Gene encoding DBT degrading enzyme of A11-2 strain
tdsA, tdsB, and tdsC also produce enzymes that degrade and desulfurize DBT by a pathway similar to the DBT degradation pathway of IGTS8 strain. tdsA, tdsB, tds
The DBT-degrading enzymes TdsA, TdsB, and TdsC encoded by C catalyze the same conversion reaction as DszA, DszB, and DszC of the IGTS8 strain, respectively. (Y. Ishii, J. Konishi, H. Okada, K. Hiras
awa, T. Onaka, M. Suzuki., Biochem. Biophy. Res. C
omm., 270 (1), 81-88, 2000) When these tds genes are expressed in Escherichia coli, they are active at 50 ° C or higher, and the optimum temperature is observed at 55 ° C, but markedly below 45 ° C. Activity decreases. Therefore, as shown by the temperature specificity of both the dsz gene and the tds gene, no gene has been previously reported that efficiently directs CST-specific DBT degradation activity at a temperature of 40 to 50 ° C. is there.

[0021]

An object of the present invention is to isolate a gene involved in a high-temperature desulfurization reaction from a microorganism having the ability to act on benzothiophene and DBT-based compounds and decompose them at a high temperature. In particular, it is to create a novel desulfurized microorganism by specifying its base sequence) and introducing these genes into a microorganism different from the one from which it was isolated, and imparting desulfurization ability. In addition, such microorganisms are actually acted on benzothiophene, DBT and their alkyl derivatives to give CS
The purpose is to establish a method of releasing sulfur by breaking the bond.

[0022]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that
Bacillus subtilis WU-S2B strain (Accession number: FERM P-1704
The inventors succeeded in isolating the genes involved in the desulfurization reaction from 1), and completed the present invention.

That is, the present invention provides the following 1 to 10. 1. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids have been deleted, substituted or added, and DBTO ₂ Protein having the function of converting to 2- (2'-hydroxyphenyl) benzenesulfinic acid

2. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 4 (b) an amino acid sequence of SEQ ID NO: 4 in which one or more amino acids are deleted, substituted or added, and 2- ( A protein that has the function of converting 2'-hydroxyphenyl) benzenesulfinic acid to 2-HBP

3. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 6; (b) an amino acid sequence of SEQ ID NO: 6 in which one or more amino acids have been deleted, substituted or added, and Protein that has the function of converting to ₂

4. A vector comprising the gene according to the above 1, 2, or 3. 5. A transformant containing the vector according to 4 above. 6. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence of SEQ ID NO: 2 wherein one or more amino acids have been deleted, substituted or added, and DBTO ₂ Protein having the function of converting to 2- (2'-hydroxyphenyl) benzenesulfinic acid

7. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 4 (b) an amino acid sequence of SEQ ID NO: 4 in which one or more amino acids are deleted, substituted or added, and 2- ( Protein having the function of converting 2'-hydroxyphenyl) benzenesulfinic acid to 2-HBP

8. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 6; (b) an amino acid sequence of SEQ ID NO: 6 in which one or more amino acids have been deleted, substituted or added, and Protein that has the function of converting to ₂

9. A protein having the following properties: (1) Action: Change DBT to DBTO ₂ (2) Optimum pH: 8.0, stable pH: 8-11 (3) Optimum temperature: 45-50 ° C, stable temperature: 53 ° C or less (4) Molecular weight: 200,000 (5) Inhibition of activity: inhibited by chelating agents and SH inhibitors (6) Coenzyme requirement: requires FMNH ₂ supplied by flavin reductase

10. A protein having the following properties: (1) Action: Converts DBTO ₂ to 2- (2'-hydroxyphenyl) benzenesulfinic acid (2) Optimum pH: 7.5, stable pH: 6-10 (3) Optimum temperature: 50 ° C, stable temperature: 45 ° C or less (4) Molecular weight: 174,000 (by gel filtration method) (5) Activity inhibition: Inhibited by chelating agents and SH inhibitors (6) Coenzyme requirement: Require FMNH ₂ supplied by flavin reductase Do

[0031]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. (1) Genes encoding thermostable desulfurase The genes of the present invention include the following three types of genes. The first gene (hereinafter, referred to as “bdsA”) comprises (a) a protein represented by the amino acid sequence of SEQ ID NO: 2,
Or (b) an amino acid sequence represented by SEQ ID NO: 2 in which one or more amino acids are deleted, substituted or added, and DBTO ₂ is converted to 2- (2′-hydroxyphenyl) benzenesulfinic acid It encodes a protein having the function of performing

The second gene (hereinafter referred to as "bdsB")
(a) a protein represented by the amino acid sequence of SEQ ID NO: 4, or 1
Alternatively, it encodes a protein consisting of an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added, and having a function of converting 2- (2′-hydroxyphenyl) benzenesulfinic acid to 2-HBP.

The third gene (hereinafter referred to as "bdsC")
(a) a protein represented by the amino acid sequence of SEQ ID NO: 6, or (b) an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 6, and a DBT Through the DBTO
Those encoding a protein having a function of converting the DBTO _2.

The bdsA, bdsB, and bdsC genes are Rhod
dszA, dszB, dszC and Sphi from ococcus sp.
dszA, dszB, dszC and Pa from ngomonas sp.
enibacillus sp.t11A, tdsB derived from the strain A11-2, shows a certain homology with tdsC, but as described below, the proteins encoded by these genes are two dszA, dszB, dszC, and tdsA, tdsB, It differs in its properties from the protein encoded by tdsC.

Among the genes of the present invention, the genes encoding the amino acid sequences of SEQ ID NOs: 2, 4 and 6 can be obtained by the methods described in Examples in the present specification. Since the nucleotide sequences of these genes have already been determined, appropriate primers are synthesized based on these sequences, and Bacillus subtilis strain WU-S2B (accession number: F
It can also be obtained by performing PCR using DNA prepared from ERM P-17041) as a template.

A gene encoding an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequences of SEQ ID NOs: 2, 4 and 6 can be obtained by a technique commonly used at the time of filing the present application, for example, Site-directed mutagenesis (Zoller et al., Nucleic Acids Res. 10
6487-6500, 1982) by modifying the gene encoding the amino acid sequence of SEQ ID NO: 2, 4, or 6. The gene of the present invention encodes an enzyme involved in the degradation of DBT, and can be used for desulfurization of petroleum.

(2) Vector containing Gene Encoding Desulfurase The vector of the present invention contains the aforementioned bdsA, bdsB or bdsC gene. Such a vector can be prepared by inserting a DNA fragment containing the bdsA, bdsB or bdsC gene of the present invention into a known vector. The vector into which the DNA fragment is inserted may be determined according to the host to be transformed. For example, the vector according to the present invention includes a plasmid vector, a phagemid vector, a cosmid vector, a yeast vector, and the like. If Escherichia coli is used as a host, the following vectors are preferably used. As a strong promoter, for example,
pUR, pGEX, pUC, pET, pT7, pB, including lac, lacUV5, trp, tac, trc, λpL, T7, rrnB, etc.
It is preferable to use vectors such as luescript, pKK, pBS, pBC, and pCAL.

(3) Transformant containing vector containing gene encoding desulfurase The transformant of the present invention contains the above vector. Cells used as hosts for the transformants may be plant cells or animal cells, but microorganisms such as Escherichia coli are preferred. Representative strains include those described in Sambrook et al., Molecular Clonin.
g 71/1 as described in Laboratory Manual 2nd ed.
8, BB4, BHB2668, BHB2690, BL21 (DE3), BNN102 (C600hf
lA), C-1a, C600 (BNN93), CES200, CES201, CJ236, CSH
18, DH1, DH5, DH5α, DP50supF, ED8654, ED8767, HB
101, HMS174, JM101, JM105, JM107, JM109, JM110, K8
02, KK2186, LE392, LG90, M5219, MBM7014.5, MC106
1, MM294, MV1184, MV1193, MZ-1, NM531, NM538, NM53
9, Q358, Q359, R594, RB791, RR1, SMR10, TAP90, TG
1, TG2, XL1-Blue, XS101, XS127, Y1089, Y1090hsdR,
YK537 and the like.

The transformant of the present invention is obtained by transforming or transducing a host cell using a technique known in the art such as an electric pulse method, a competent cell method, a conjugation transfer method, and a protoplast method. be able to. These methods are described, for example, in Sambrook et al.
It is described in.

(4) Desulfurase The desulfurase of the present invention includes the following three types of proteins. The first protein (hereinafter referred to as "BdsA")
It consists of a protein represented by the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2, and DBTO ₂ is represented by 2- (2 '-Hydroxyphenyl) benzenesulfinic acid.

Second protein (hereinafter referred to as "BdsB")
Consists of a protein represented by the amino acid sequence of SEQ ID NO: 4 and an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4, and 2- (2 ′ -Hydroxyphenyl) benzenesulfinic acid to 2-HBP.

Third protein (hereinafter referred to as "BdsC")
Includes a protein represented by the amino acid sequence of SEQ ID NO: 6, wherein one or more amino acid deletions in the amino acid sequence of SEQ ID NO: 6, wherein, substituted or added in the amino acid sequence, and the DBT to DBTO ₂ And a protein having a converting function.

The above BdsA, BdsB and BdsC were obtained from Rhodococcu
ssp. IGTS8 shows a certain homology with the desulfurization enzymes DszA, DszB, and DszC derived from the IGTS8 strain, and has the same action as the enzyme, but is clearly different in the following points. (A) DszA, DszB, and DszC cannot desulfurize 4,6-dibutyldibenzothiophene, which is a hard-to-desulfurize substance.
BdsA, BdsB and BdsC are desulfurizable. (B) DszA, DszB, and DszC show desulfurization activity in the room temperature range, whereas BdsA, BdsB, and BdsC of the present invention show desulfurization activity in the middle range between room temperature and high temperature.

The desulfurase of the present invention can be produced using the above-described gene encoding the desulfurase of the present invention. In addition, the desulfurizing enzyme represented by the amino acid sequences of SEQ ID NOs: 2, 4, and 6 is Bacillus subtilis WU-S
It can also be prepared from 2B strain (Accession number: FERM P-17041) according to a conventional method.

The properties of one protein included in the BdsA of the present invention are shown below. (1) Action: DBTO ₂ a to 2- (2'-hydroxyphenyl) benzene sulfinic acid (2) pH Characteristics: As shown in FIGS. 8 and 9, the optimum pH was 7.5, stable
The pH is 6 to 10. (3) Temperature characteristics: As shown in FIGS.
(4) Molecular weight: 174,000 (by gel filtration) (5) Activity inhibition: Inhibited by chelating agents and SH inhibitors (6) Coenzyme requirement: by flavin reductase Request FMNH ₂ supplied

The properties of one protein included in the BdsC of the present invention are shown below. (1) Action: Change DBT to DBTO ₂ (2) pH characteristics: As shown in FIGS. 4 and 5, optimal pH is 8, stable
pH is 8 to 11 (3) Temperature characteristics: As shown in FIGS.
(4) Molecular weight: 200,000 (by gel filtration) (5) Inhibition of activity: Inhibited by chelating agents and SH inhibitors (6) Coenzyme requirement: Flavin reductase Supplied by claim FMNH ₂

In the present specification, the “optimal pH” of the desulfurizing enzyme
Refers to the pH that has the most suitable activity at a particular temperature (eg, 50 ° C.). Similarly, the “optimum temperature” of desulfurization enzyme
Refers to the temperature that has the most favorable activity at a particular pH (eg, pH 7.0).

As used herein, the term “stable pH” of a desulfurizing enzyme means that the enzyme solution is allowed to stand at various pHs for a certain period of time (eg, 30 minutes) and then reach a specific pH (eg, pH 7.0). When readjusted and measuring residual enzyme activity at a particular temperature (e.g., 50 ° C.), the highest activity level was 100%.
As%, it refers to a pH range that exhibits an activity level of preferably at least 60%, more preferably at least 70%, most preferably at least 80%. Similarly, the `` stable temperature '' of the desulfurization enzyme means that after leaving the enzyme solution at various temperatures for a certain period of time (for example, 30 minutes), it is readjusted to a specific temperature (for example, 50 ° C.),
When the remaining enzyme activity is measured at (e.g., pH 7.0), the highest activity level is taken as 100%, preferably 60% or more, more preferably 70% or more, most preferably
Refers to a temperature range showing an activity level of 80% or more.

[0049]

The present invention will be described below in more detail with reference to examples. The experiments related to genetic manipulation in the examples were mainly Samb
This was performed according to the method detailed in rook et al. Example 1 Cloning of Gene Fragment Encoding a Thermostable Desulfurase Rhodococcus sp. IGTS8, a cold desulfurizing bacterium, and Paenibacillussp.
It is known to have an activity to convert DBT to 2-HBP via TO ₂ and 2- (2′-hydroxyphenyl) benzenesulfinic acid.

On the other hand, the present inventors have used the present invention.
Bacillus subtilis WU-S2B strain is also Rhodococcus sp.IGTS8
Strain and Paenibacillus sp. A11-2 by the same route as DBT.
Has the ability to change to 2-HBP (JP-A-2000-139450). Where the DBT listed above is 2-HBP
Focusing on the enzyme involved in the activity of converting to and its gene, Rhodococcus sp.IGTS8 strain and Paenibacillus sp.
In the A11-2 strain, the amino acid sequence of each enzyme and the nucleotide sequence of the gene have already been determined, and they are known to have 50 to 65% homology.

Therefore, Rhodococcus sp. IGTS8 strain and Paeni
Bacillus s having the same activity as bacillus sp. strain A11-2
ubtilis WU-S2B strain was considered to have some degree of homology, so that Rhodococcus sp.
The amino acid sequence of the enzyme of aenibacillus sp. strain A11-2 was searched for a highly homologous portion, and the following amino acid sequence was found.

Paenibacillus sp. Strain A11-2 TdsA WNVVTSLNNAEARNFG (SEQ ID NO: 7) Rhodococcus sp. Strain IGTS8 strain DszA WNVVTSLNDAEARNFG (SEQ ID NO: 8) Paenibacillus sp. (SEQ ID NO: 10)

Therefore, based on the above amino acid sequence, a sense primer and an antisense primer for PCR having the following nucleotide sequences were designed and synthesized. Sense primer: WUS2-SI1 5'-GCI GAR GCI MGI AAY TTY GG-3 '(SEQ ID NO: 11) (I represents deoxyinosine) Antisense primer: WUS2-C2 5'-CGT IGC GCC AIA AGC GGT C- 3 '(SEQ ID NO: 12) (I represents deoxyinosine)

Using the sense primer and the antisense primer, PCR was performed using DNA extracted from Bacillus subtilis strain WU-S2B as a template. Bacillus subt
Preparation of DNA from the ilisWU-S2B strain was performed as follows. The Bacillus subtilis WU-S2B strain was cultured at 50 ° C. for 18 hours in an AII medium containing DBT (the composition is shown in Table 2), and the cells were collected.

[0055]

[Table 2]

The obtained cells were added to 3.7 ml of a SET buffer (6.7%
The suspension was suspended in sucrose, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0). Add 6 mg / ml lysozyme and 4 mg / ml
Of achromopeptidase in 1 ml of TE buffer (1
mM EDTA, 10 mM Tris-HCl, pH 8.0), and add
Allowed to react for minutes. Add 0.5 ml of TE buffer (25 mM EDT) to the reaction solution.
A, 50 mM Tris-HCl, pH 8.0) and 0.3 ml SDS solution (25 mM E
Add DTA, 20% (w / v) SDS, 50 mM Tris-HCl, pH 8.0), mix with stirring, react at 37 ° C for 10 minutes, stir with a mixer for 30 seconds, then add 0.3 ml of 3N NaOH After intermittently mixing by inversion for 10 minutes, 0.5 ml of Tris buffer (2M Tris-H
Cl, pH 7.0) and mix by inverting intermittently for 3 minutes.
0.7 ml of 5 M NaCl was added and mixed with stirring to prepare a cell reaction solution. Add 7 ml of phenol reagent to the bacterial cell reaction mixture, mix with stirring, centrifuge at 12,000 rpm for 5 minutes, add 7 ml of phenol-chloroform reagent to the obtained supernatant, mix with stirring, and mix at 12,000 rpm For 5 minutes.
To the obtained supernatant, add 7 ml of chloroform reagent, mix with stirring, centrifuge at 12,000 rpm for 5 minutes,
A solution was prepared. After adding 7 ml of isopropanol to the DNA solution and leaving it to stand at 0 ° C. for 30 minutes, it was centrifuged at 15,000 rpm for 30 minutes to precipitate the DNA. 70% cold precipitated DNA
(w / v) After washing with ethanol and drying under reduced pressure, 100 μl of
The DNA solution was prepared by dissolving in a TE buffer. Bac prepared
The conditions of PCR performed using illus subtilis WU-S2B strain DNA as a template are as follows.

Reaction solution composition: 10 mM Tris-HCl 1.5 mM MgCl 2 0.2 mM dNTP Mixture 1 μM Sense primer 1 μM Antisense primer 1 μg Template DNA 2.5 U Taq DNA polymerase Annealing temperature: PCR was performed with the temperature fixed at 52 ° C. PCR cycle: 95 ° C for 3min once 95 ° C for 1min ↓ 52 ° C for 1.5min Repeat this process 25 times 72 ° C for 3min ↑ 72 ° C for 10min Once DNA amplifier: DNA Thermal Cycler Temperature cycler (manufactured by PERKIN ELMER CETUS)

As a result of performing PCR under the above conditions, about 3.2 kb
To give an amplified fragment. This 3.2kb PC
The R product was cloned into E. coli strain JM109 using the pGEM-T vector. As a result of determining the nucleotide sequence of the obtained DNA fragment, the nucleotide sequence was determined to be Rhodococcus sp. IGTS
A11-2 showed 69.2% homology with the dsz gene of 8 strains and 56.9% homology with the tds gene of Paenibacillus sp.

From the determined DNA base sequence, the amino acid sequence encoded by this fragment was deduced.
ssp. DszA, DszB and Dsz, proteins encoded by the dsz gene cloned from the IGTS8 strain
The amino acid sequence of C was compared. As a result, DszA, DszB
And DszC were confirmed to have deduced amino acid sequences showing homology of 76.3%, 67.9% and 71.3%, respectively. In addition, this deduced amino acid sequence
As a result of comparison with the amino acid sequences of TdsA, TdsB and TdsC which are proteins encoded by the tds gene of ssp.
It showed 2.7% and 48.9% homology. Rhodococcus sp.
The nucleotide sequence of the desulfurization gene and the amino acid sequence of the desulfurase of IGTS8 and Paenibacillus sp.A11-2 were found to be homologous to the amino acid sequence of the desulfurase, indicating that desulfurase was encoded in this DNA fragment cloned from Bacillus subtilis WU-S2B Therefore, it was decided to use this as a probe to clone the adjacent DNA region, that is, the entire desulfurization gene.

Example 2 Preparation of Whole DNA Library DNA was prepared from Bacillus subtilis strain WU-S2B as follows. The cells were cultured at 50 ° C. for 18 hours in an AII medium (composition is shown in Table 2) containing DBT and tunicamycin to collect the cells. The obtained cells were mixed with 1 ml of B1 buffer (50 mM EDTA,
50mM Tris-HCl, 0.5% Triton X-100, 0.2mg / ml RNase
A, pH 8.0). To this suspension, 20 μl of a 100 mg / ml lysozyme solution and 45 μl of a 20 mg / ml proteinase K solution were added and reacted at 37 ° C. for 10 minutes. To the reaction solution
0.35 ml of B2 buffer (800 mM GuHCl, 20% Tween-20, pH 5.5
) Was added, stirred and mixed, and reacted at 50 ° C. for 30 minutes.
The mixture was stirred with a mixer for 2 seconds to prepare a cell reaction solution. QIAGEN GENOMIC-TIP20 / G filled with anion exchange resin (QIGEN
(AGEN) column with 2 ml of QBT buffer (750 mM NaCl, 50
mM MOPS, 15% ethanol, 0.15% Triton X-100, pH7.0)
And the cell reaction solution was injected into the column. The column was washed with 3 ml of QC buffer (1.0 M NaCl, 50 mM MOPS, 15% ethano
l, pH 7.0) and 2 ml of QF buffer (1.25 M Na
Genomic DN with Cl, 50mM Tris-HCl, 15% ethanol, pH 8.5)
Solution A was eluted. After adding 1.4 ml of isopropanol to the genomic DNA solution to precipitate the DNA, it was wound up with a glass rod and collected. Dissolve the recovered DNA in 50 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
A DNA solution was prepared.

The whole DNA library of Bacillus subtilis WU-S2B strain was prepared as follows. Bacillus sub
tilis WU-S2B strain
After digestion with Sau3AI for 6 minutes, 7 minutes and 8 minutes, the digest was extracted with phenol-chloroform, precipitated with ethanol, centrifuged, and the recovered DNA fragment was treated with 3.5 units of alkaline phosphatase derived from calf intestine. Dephosphorylation was performed by treating at 37 ° C. for 18 hours. After alkaline phosphatase treatment, DNA was extracted by phenol-chloroform treatment, and recovered by ethanol precipitation. About 0.2 μg of the obtained DNA fragment was placed at 4 ° C. in the presence of about 2 μg of λ DASHI / BamHI arm and 2 units of T4 DNA ligase.
The reaction was performed for 18 hours. Gigapack III Gold pa
In vitro packaging was performed by reacting with ckaging Extract, and a phage library was prepared.
The titer of the resulting phage library was 10 ⁵ pfu / ml.

[Example 3] Screening of whole DNA library DN for screening phage library
The A probe was prepared as follows. DNA fragment obtained by the method described in Example 1 and Rhodococcus sp.
A region having a particularly high homology between the base sequences of the dsz gene of the GTS8 strain was selected and a sense primer (Rhodoc
447 from the 427th position from the 5 'end of dszA of occus sp. IGTS8
And the antisense primer (R
hodococcus sp. strain IGTS8 strain dszC from the 5 'end of the dszC (corresponding to the nucleotide sequence from position 1135 to position 1153) was prepared: Sense primer: WUS2-NSI1 5'-GCA TGA CAT CCG ATA CG-3' (SEQ ID NO: 13) ) Antisense primer WUS2-NC2 5'-TAG TTT GGG TGG GTT CC-3 '(SEQ ID NO: 14).

Using this primer, Bacillus subtili
s By performing PCR using DNA prepared from the WU-S2B strain as a template, the DN of the inner region of the DNA fragment obtained in Example 1 was determined.
The A fragment was amplified. Using the obtained PCR product as a template, a DSZAC probe labeled with dioxygenin (DIG) was prepared by a random prime method (multiprime method). D
The method for preparing the IG-labeled probe was according to the protocol of Boehringer Mannheim. The method for preparing a DIG-labeled probe is described below.

5 μl of the obtained PCR product was heat-denatured in boiling water for 10 minutes, and cooled on ice containing salt. To the resulting denatured DNA solution, add 2 μl of a hexanucleotide mixture (0.5 M Tris-HCl, 0.1 M MgCl 2, 1 mM Dithioerythrito
l, 2mg / ml BSA, 3.143mg / ml Random Primer, pH7.2)
2 μl of dNTP labeling mixture (1 mM dATP, 1 mM dCTP, 1 mM dGT
P, 0.65mM dTTP, 0.35mM DIG-dUTP, pH7.5), 10μl
Of sterile distilled water and 1 μl of Klenow enzyme (2 units) were added and reacted at 37 ° C. for 18 hours. Add 2 μl of the reaction
The reaction was stopped by adding a 0.2 M EDTA solution. Then 2μ
One liter of 4M LiCl and 60 μl of cold ethanol (−20 ° C.) were added, left at −70 ° C. for 30 minutes, and centrifuged at 15,000 rpm for 30 minutes to precipitate DNA. 70% cold precipitated DNA
(w / v) After washing with ethanol and drying by suction, 50 μl
Was dissolved in a TE buffer solution to prepare a DIG-labeled probe.

The desulfurization gene of Bacillus subtilis strain WU-S2B was screened for DIG-labeled DSZA prepared by the method described above.
Plaque hybridization was performed on plaques transcribed on a Hybond N + membrane using the C probe. To detect hybridizing clones, use DIG-
ELISA (Boehringer Mannheim) was used. When about 2,000 phage plaques were screened from the genomic library using the DSZAC probe, three positive plaques were detected. Single plaques were separated from these three plaques, and plaque hybridization was performed again. As a result, it was confirmed that all three plaques were positive plaques. Phage clones were prepared using the detected DSZAC probe-positive plaques, and phage DNA was extracted from those clones using the QIAGENLambda kit. Phage DNA prepared using three positive plaques
Was cut with EcoRI and NotI to prepare a restriction enzyme map shown in FIG. Furthermore, these three types of phage DNA were obtained by digestion with various restriction enzymes such as NotI and NheI.
When the DNA fragment was subjected to Southern blot analysis using a DSZAC probe, it was confirmed that the DNA fragment hybridized to an Nhel fragment of about 7.1 kb. From the results of these restriction enzyme maps and Southern blot analysis, the No. 1 phage
It was considered that the dsz gene was encoded in the NheI fragment of about 7.1 kb of DNA.

Example 4 Confirmation of Desulfurization Activity Next, in order to confirm whether the desulfurization gene is present in the NheI fragment, the DBT resolution by recombinant E. coli was tested. The NheI fragment of about 7.1 kb was inserted into the XbaI site of the plasmid vector pUC19 to prepare pU1Nh (see FIG. 1). This recombinant Escherichia coli JM109 strain having pU1Nh was transformed into an LB medium containing 0.1 mg / ml of ampicillin (Molecular Cloning Laborat
ory Manual 2nd) (LB-Amp medium) to which DBT was added as a substrate, and cultured overnight at 37 ° C.
In addition, JM109 carrying only the vector pUC19 as a control strain
The strain was cultured under similar conditions. 37 in LB-Amp medium (described above)
Pre-cultivation was performed at ℃, overnight to obtain a pre-culture solution. Next, 1/100
Add the volume of the preculture to the assay medium (LB-Amp medium supplemented with DBT as a substrate), culture at 37 ° C for 18 hours, extract the degradation products using ethyl acetate, Chromatographic analysis was performed. As a result, pU1Nh
It was confirmed that 2-HBP was produced in recombinant Escherichia coli harboring DBT when cultured in a medium containing DBT as a substrate.
In addition, recombinant Escherichia coli containing only the vector did not show such conversion activity at all. This proved that the NheI fragment of about 7.1 kb inserted into pU1Nh had a sequence capable of encoding all activities for catalyzing a series of conversion reactions from DBT to 2-HBP.

Example 5 Determination of Nucleotide Sequence of Desulfurization Gene Using phage DNA and pU1Nh prepared from a positive plaque, the nucleotide sequence of about 4.1 kb of the NheI fragment of about 7.1 kb was determined by the primer walking method. went. Sequencing of clones is performed using BigDye Terminator Cycle Sequen
ABI PRISM using the cing Kit (AppliedBiosystems)
The nucleotide sequence was determined using 310 Genetic Analyzer (Applied Biosystems) (SEQ ID NOs: 1, 3, and 5). The obtained base sequence data was obtained from GENETYX-MAC / ATSQ v3.0 and GENETYX.
Analysis was performed using X-MAC v10.0.

As a result of searching for the ORF in the determined sequence of about 4.1 kb, three ORFs having a length of 1 kb or more were found in the same direction. These ORFs were named ORF 1, 2, and 3 from the upstream side. ORFs 1, 2, and 3 encode 453, 356, and 415 amino acids, respectively. ORF 1 translation stop codon TGA and ORF
The translation initiation codon ATG of 2 partially overlaps,
5'-ATGA-3 'sequence, Rhodococcus sp.
IGTS8 in dsz operon and Paenibacillus sp. A11
-2 strain has the same structure as the nucleotide sequence in the tds operon. Rhodococ for these ORFs
Analysis of the nucleotide sequence homology with the dsz gene of cus sp. IGTS8 strain showed that ORF 1,2,3 showed 72.8%, 67.4%, 71.3% homology with dsz A, B, C of IGTS8 strain, respectively Showed sex. Analysis of nucleotide sequence homology with the tds gene of Paenibacillus sp. A11-2 strain revealed that ORF 1,2
3 are the tds A, B, and Paenibacillus sp.
It showed 61.3%, 56.8% and 56.0% homology with C. Furthermore, when the amino acid sequence of the protein encoded by the Bacillus subtilis WU-S2B strain gene was estimated based on the nucleotide sequence of the gene, the polypeptides encoded by ORFs 1, 2, and 3 were found to be Rhodococcus sp.
It showed 78.6%, 67.9%, and 72.0% homology with A, B, and C.
In addition, Tds A, B, C and 63 of Paenibacillus sp.
The homology was 7%, 52.7%, and 50.6%.

The amino acid sequence of the protein encoded by the ORF of Bacillus subtilis strain WU-S2B and Rhodococcus sp. IG
Comparison with the amino acid sequence of the protein encoded by the TS8 dsz sequence reveals several distinctive differences. First, when BdsA encoded by ORF 1 is compared with DszA, different portions of amino acids are continuously observed at the carboxyl terminal side. When BdsA and TdsA are compared, the sequence of the amino terminus of BdsA is longer than the sequence of the amino terminus of TdsA, and further, different portions of amino acids are continuously observed, particularly on the carboxyl terminus side. BdsB encoded by DszB and ORF 2
In the amino acid sequence of DszB, the amino-terminal sequence of DszB is longer and longer than the amino-terminal sequence of BdsB. When TdsB and BdsB are compared, the sequence at the carboxyl terminus of BdsB is longer than the sequence at the carboxyl terminus of TdsB. In particular, the homology of the amino acid sequence on the carboxyl terminal side is low. When the amino acid sequences of DszC and BdsC encoded by ORF 3 are compared, the full-length sizes are almost the same, but the homology of the amino-terminal amino acid sequence is low. Also, Td
When comparing sC and BdsC, the amino-terminal sequence of BdsC is longer and longer than the amino-terminal sequence of TdsC.

Example 7 (1) Purification of Flavin Reductase Required for Enzyme Activity Measurement In order to measure enzyme activity at 50 ° C., flavin reductase having activity even at this temperature is required. For this purpose, the enzyme was purified from a flavin reductase (TdsD) highly expressing strain derived from Paenibacillus sp. Strain A11-2. The high-expressing strain was cultured in LB medium containing 50 μg / ml of ampicillin at 37 ° C overnight with shaking, and 0.2 mM IPTG was added thereto, followed by further culturing for 4 hours to obtain cells. 11 g wet weight
Cells were suspended in buffer A (50 mM Tris-HCl, pH 7.0, 10% glycerol, 1 mM dithiothreitol), and disrupted by an ultrasonic homogenizer (Branson, model 450) at 4 ° C for 15 minutes. Was. The supernatant after centrifugation at 10,000 xg for 30 minutes was used as a cell-free extract. This cell-free extract was sufficiently dialyzed against buffer A, and then applied to a Q-Sepharose column equilibrated with the same buffer. After washing with the same buffer and buffer A containing 0.05 M and 0.1 M potassium chloride, elution was carried out with buffer A containing 0.15 M potassium chloride. The active fractions are collected, concentrated by ultrafiltration, and then subjected to ultrafiltration to remove the buffer in which the enzyme is dissolved using buffer A containing 1.25 M ammonium sulfate.
I made it. Phenyl-Toy obtained by equilibrating this solution with the same buffer
applied to opearl column. After washing with the same buffer solution, a linear gradient elution with ammonium sulfate was performed up to buffer solution A containing no ammonium sulfate, and the active fraction was collected and concentrated by ultrafiltration. Subsequently, the buffer in which the enzyme was dissolved was changed to buffer A containing 0.15 M sodium chloride by ultrafiltration. The enzyme solution was applied to a Superdex200 HR10 / 30 column equilibrated with the same buffer, eluted with the same buffer, and the active fraction was collected and concentrated by ultrafiltration. As a result, it was confirmed that the active fraction was electrophoretically uniform.

The enzyme activity was measured in a reaction solution containing 20 mM phosphate buffer, pH 7.0, 0.5 mM NADH, and 0.02 mM FMN. The decrease rate of NADH at 70 ° C. was 340 nm.
Was calculated by determining the rate of decrease in the absorbance of the sample. The amount of enzyme that reduces 1 μmol of NADH per minute was defined as 1 unit.

(2) Purification of BdsC The B. subtilis WU-S2B strain was added to an AII medium (see Table 2) for 0.5 minute.
Aeration and agitation culture was performed using a jar fermenter at 40 ° C for 2 days in a medium supplemented with a DBT / ethanol solution to a concentration of 4 mM to obtain 334 g of cells (wet cell weight).
The obtained cells were added to buffer A (50 mM Tris-HCl, pH 7.0,
Suspend in 10% glycerol, 1 mM dithiothreitol) and sonicate at 4 ° C (Branson, model 450).
C, crushed for 75 minutes. The supernatant after centrifugation at 10,000 xg for 60 minutes was used as a cell-free extract. This cell-free extract was sufficiently dialyzed against buffer A containing 0.1 M potassium chloride, and then DEAE-Sep equilibrated with the same buffer.
It was applied to a harose column. After washing with the same buffer, 0.2
Linear gradient elution was performed with potassium chloride up to buffer A containing 5 M potassium chloride. Active fractions were collected and concentrated by ultrafiltration. This solution was sufficiently dialyzed against buffer A containing 0.25 M potassium chloride, and then applied to a Q-Sepharose column equilibrated with the same buffer. Eluted with the same buffer, separated into fractions containing protein A and fractions containing protein C shown below, collected and concentrated by ultrafiltration, and then the enzyme was dissolved using ultrafiltration. Buffer containing 1 M ammonium sulfate
A Phenyl-To was prepared by equilibrating this solution with the same buffer.
applied to yopearl column. After washing with the same buffer solution, a linear gradient elution with ammonium sulfate was performed up to buffer solution A containing no ammonium sulfate, and the active fraction was collected and concentrated by ultrafiltration. As a result, it was confirmed that the active fraction was electrophoretically uniform.

The measurement of the enzyme activity was performed using 10 μg of the purified enzyme,
mM phosphate buffer pH 7.0, 0.01 mM FMN, 6 mM NAD
H, an enzyme reaction solution containing 0.45 units / ml TdsD and 0.3 mM DBT was shaken at 50 ° C. 1 / 10th of 12M
After the reaction was stopped by adding HCl, 8/10 volume of ethyl acetate was added, mixed well, centrifuged at 12,000 rpm for 3 minutes, and the upper layer was subjected to HPLC analysis. 1 nmo per minute
The amount of enzyme that produces 1 l of DBTO ₂ was defined as 1 unit.
In the measurement of the enzyme activity in the purification step, an appropriate amount of the enzyme sufficient to confirm the activity using the above reaction solution was used. Table 3 shows the enzyme activities at each purification step.
In addition, the activity of the above purified enzyme reaction solution was measured at various temperatures to examine the temperature dependence of the enzyme. FIG. 2 shows the temperature dependence of the BdsC enzyme activity. The residual activity of the above purified enzyme solution after being left at various temperatures for 30 minutes was measured as described above, and the temperature stability of the enzyme was examined. The temperature stability for the enzyme is shown in FIG. In addition, 100 mM of each buffer having various pH, 0.01 mM FMN, 6 mM FADH, 0.45 units / ml Tds
D, The reaction solution containing 0.3 mM DBT was shaken at 50 ° C., and the enzyme activity was measured to examine the pH dependence of the enzyme. The pH dependence is shown in FIG. Also, 100 mM of each buffer having various pH and the purified enzyme were mixed and left for 30 minutes, and then 100 mM phosphate buffer pH 7.0, 0.01
mM FMN, 6 mM NADH, 0.45 units / ml TdsD, 0.3 mM DBT
Was shaken at 50 ° C. to examine the pH stability of the enzyme. The pH stability of the enzyme is shown in FIG.

[0074]

[Table 3]

Example 8 (1) Purification of BdsA The active fraction collected at the stage of Q-Sepharose during the purification of BdsC was dialyzed against buffer A containing 0.1 M potassium chloride, and then purified. Q-Sepharo equilibrated with buffer
Applied to se column. After washing with the same buffer, 0.3 M
A linear gradient elution with potassium chloride was performed up to buffer A containing potassium chloride. Active fractions were collected and concentrated by ultrafiltration. Subsequently, the buffer in which the enzyme was dissolved was changed to buffer A containing 0.7 M ammonium sulfate using ultrafiltration. This solution was applied to a Phenyl-Toyopearl column equilibrated with the same buffer. After washing with the same buffer and buffer A containing 0.5 M, 0.4 M and 0.3 M ammonium sulfate, respectively, elution was carried out with buffer A containing 0.2 M ammonium sulfate, and the fractions were collected and concentrated by ultrafiltration. Subsequently, the buffer in which the enzyme was dissolved was changed to buffer A containing 0.15 M sodium chloride by ultrafiltration. This enzyme solution was equilibrated with the same buffer.
The mixture was applied to a Superdex200 HR10 / 30 column, eluted with the same buffer, and the active fraction was collected and concentrated by ultrafiltration.
In addition, use ultrafiltration to remove the buffer in which the enzyme is dissolved.
Buffer A containing 0.25 M sodium chloride. The enzyme solution was applied to a MonoQ HR10 / 10 column equilibrated with the same buffer, washed with the same buffer, and then subjected to linear gradient elution with sodium chloride up to buffer A containing 0.35 M sodium chloride. Active fractions were collected and concentrated by ultrafiltration. As a result, it was confirmed that the active fraction was electrophoretically uniform.

The measurement of the enzyme activity was carried out in the same manner as in the above-mentioned method for measuring the activity of BdsC, except that DBTO ₂ was added instead of DBT as a substrate. Table 4 shows the enzyme activity at each stage. FIG. 6 shows the temperature dependence of the BdsA enzyme activity, FIG. 7 shows the temperature stability, FIG. 8 shows the pH dependence, and FIG.
Are shown below.

[0077]

[Table 4]

[0078]

The present invention provides novel genes and enzymes involved in desulfurization. By utilizing these genes and enzymes, sulfur in fossil fuels can be easily released.

[0079]

[Sequence List] SEQUENCE LISTING <110> Petroleum Energy Center <120> Thermostable desulfurising enzyme and the gene encoding there <130> P01-0068 <160> 14 <170> PatentIn Ver. 2.0 <210> 1 <211> 4031 <212 > DNA <213> Bacillus subtilis <220> <221> CDS <222> (312) .. (1670) <400> 1 ctagcgaggt gcgagttccg cgcggtagcc atccgcttca ttgattttta catcgatacc 60 gccgcggtgg ccgtcagggt gtcgaagccg ggcggaggtg cacaccccgc cggttggtga 120 tcttgctcac aggtggcgct ttgacggcac tcgtccgggg gcgagaatcg gcgaaggtcg 180 ggtgggtcgt cgggaggaag tggcgtgttg ggtgcccctt tgaatcatcg ggatcctaac 240 tctgttgggg tcggttgcag tagccgagca ttggtctccg gtaactgatc cccacggagg 300 cactgctgat t atg gct gaa caa cgc caa cta cat tta gcc ggg ttt ttc 350 Met Ala Glu Gln Arg Gln Leu His Leu Ala Gly Phe Phe 1 5 10 tcg gcc ggc aac gtc act cat gcg cat ggc gcg tgg cgc cat gtc ggt 398 Ser Ala Gly Asn Val Thr His Ala His Gly Ala Trp Arg His Val Gly 15 20 25 gcg acg aac ggt ttt ctg act ggc gag ttc tac aag cag att gcg cgg 446 Ala Thr Asn Gly Phe Leu Thr Gly Glu Phe Tyr Lys Gln Ile Ala Arg 30 35 40 45 acc ctg gag cgg ggc aag ttt gat ctg ttg ttt ctt ccc gat ggc ctg 494 Thr Leu Glu Arg Gly Lys Phe Asp Leu Leu Phe Leu Pro Asp Gly Leu 50 55 60 gcc atc gaa gat agc tat ggc gat aat ttg gag acg ggg gtc ggt ctg 542 Ala Ile Glu Asp Ser Tyr Gly Asp Asn Leu Glu Thr Gly Val Gly Leu 65 70 75 ggt ggc cag ggc gcg gtg gcg ctg gag ccg acc agg gc g ag Gly Gly Gln Gly Ala Val Ala Leu Glu Pro Thr Ser Val Ile Ala Thr 80 85 90 atg gct gcg gtg acc cag cga tta ggt ctg ggc gct acg gtt tcg acg 638 Met Ala Ala Val Thr Gln Arg Leu Gly Leu Gly Ala Thr Val Ser Thr 95 100 105 acc tac tac ccg ccc tat cat gtg gct cgg gtg ttt gcc acg ctc gac 686 Thr Tyr Tyr Pro Pro Tyr His Val Ala Arg Val Phe Ala Thr Leu Asp 110 115 120 125 aat ctg tct gat ggc cgg atc tcg tgg aat gtg gtc acg tcg ctg aac 734 Asn Leu Ser Asp Gly Arg Ile Ser Trp Asn Val Val Thr Ser Leu Asn 130 135 140 gac tcc gag gca cgc aac ttc ggt gtg gat gag cac ctt gag cat gac 782 Asp Ser Glu Ala Alg Asn Phe Gly V al Asp Glu His Leu Glu His Asp 145 150 155 atc cga tac gac cgg gct gac gaa ttc ttg gag gcc gtc aag aag cta 830 Ile Arg Tyr Asp Arg Ala Asp Glu Phe Leu Glu Ala Val Lys Lys Leu 160 165 170 tgg agt tct tgg tcc gag gat gcc ttg ttg ttg gac aag gtt ggc ggt 878 Trp Ser Ser Trp Ser Glu Asp Ala Leu Leu Leu Asp Lys Val Gly Gly 175 180 185 cgg ttc gct gat ccc aag aag gtt caa tac gtc aat cat cgc gg Arg Phe Ala Asp Pro Lys Lys Val Gln Tyr Val Asn His Arg Gly Arg 190 195 200 205 tgg ttg tcg gtc cgt ggc ccg ttg cag gtt cca cgg tcc cgt cag ggt 974 Trp Leu Ser Val Arg Gly Pro Leu Gln Val Pro Arg Ser Arg Gln Gly 210 215 220 gaa ccg gtc atc ttg cag gcc ggc ttg tcg ccg cgg ggg cgt cgc ttc 1022 Glu Pro Val Ile Leu Gln Ala Gly Leu Ser Pro Arg Gly Arg Arg Phe 225 230 235 gcg ggg cgc ggg ttc agc gtc tcg ccc aac ctc gac 1070 Ala Gly Arg Trp Ala Glu Ala Val Phe Ser Val Ser Pro Asn Leu Asp 240 245 250 atc atg cgc gcg gtc tat cag gac atc aaa gct cac gtt gcg gcc gcg 1118 Ale Met Aret l Tyr Gln Asp Ile Lys Ala His Val Ala Ala Ala 255 260 265 ggg cgt gac ccg gag caa aca aag gta ttc acc gcg gtg atg ccg gtg 1166 Gly Arg Asp Pro Glu Gln Thr Lys Val Phe Thr Ala Val Met Pro Val 270 275 280 285 ctc ggt gag aca gag cag gtg gcc cgg gaa cgt ctg gaa tat ctc aat 1214 Leu Gly Glu Thr Glu Gln Val Ala Arg Glu Arg Leu Glu Tyr Leu Asn 290 295 300 tcg ctg gtg cat ccc gagatg gc tcc agc cac agc 1262 Ser Leu Val His Pro Glu Val Gly Leu Ser Thr Leu Ser Ser His Ser 305 310 315 ggc ttg aat ctc tcc aag tat ccg ctg gat acg aag ttt tcc gac atc 1310 Gly Leu Asn Leu Ser Lys Tyr Pro Leu Asp Thr Lys Phe Ser Asp Ile 320 325 330 gtc gcc gat ctc ggg gat cgt cac gtg ccg acc atg ttg cag atg ttt 1358 Val Ala Asp Leu Gly Asp Arg His Val Pro Thr Met Leu Gln Met Phe 335 340 345 tcc gcc gtg gcc ggt ggc ggt gcg gac ctg acg ctg gcc gaa ctg ggc 1406 Ser Ala Val Ala Gly Gly Gly Ala Asp Leu Thr Leu Ala Glu Leu Gly 350 355 360 365 cgc cga tac ggc acc aac gtc ggg ttt gtg ccg cag tg 1454 Arg Arg Tyr Gly Thr Asn Val Gly Phe Val Pro Gln Trp Ala Gly Thr 370 375 380 gcc gaa cag atc gct gat caa ttg att agt cac ttt gag gcc ggg gcc 1502 Ala Glu Gln Ile Ala Asp Gln Leu Ile Ser His Phe Glu Ala Gly Ala 385 390 395 gct gac gga ttc atc att tcg ccg gcg tat ctg ccg ggt att tac gag 1550 Ala Asp Gly Phe Ile Ile Ser Pro Ala Tyr Leu Pro Gly Ile Tyr Glu 400 405 410 gag ttc gtc gac gcc cac ctg ctg cag caa cgt ggt gtg ttc 1598 Glu Phe Val Asp Gln Val Val Pro Leu Leu Gln Gln Arg Gly Val Phe 415 420 425 cgt acc gag tac gaa ggg aca acg ctg cgt gag cac ctt ggg ttg gct 1646 Arg Thr Glu Tyr Gly Thr Thr Leu Arg Glu His Leu Gly Leu Ala 430 435 440 445 cac cct gaa gtc ggg agc atc cga tgaccaccac cgatgttgat catgacgttc 1700 His Pro Glu Val Gly Ser Ile Arg 450 ttgcctacag caactgcccg gttcccaatg cactactgac agcgctggag tcaaacctgt 1760 tggccggcaa cgggatttcg ctgaatgtgc tcagcggcgc gcaagcaggg ttgcatttca 1820 cctacgatca tcccgcttac acccgctttg gtggcgaaat tccacctctt atcagtgagg 1880 gcctgcgggc gccg ggacgc acccgcctgc taggcatcac ccctctggcc ggtcggcaag 1940 ggatctatgt ccgtgttgac agcccggtga catcaccgga gcagctgcgg ggccggcggg 2000 tgggtgtgtc gggtgcagcg atccgcatcc tcaccggcga gttgggcgat tatcggcact 2060 tggacccgtg gcggcaaaca ctgatcgcgc tgggtacctg ggaggcgcgg ggacttttgc 2120 agaccctgca catcgggggg ataggggtcg acgacgtcga gttggtgcgt atcgaaagcc 2180 ctggcgtcga cgtgcccgaa gagcggctgg aagcagcggc cagtgtcaaa ggcgctgacc 2240 tgtttcccga cgtggccggc caccagaggg acatcctgga tagcggcaac gttgatgcgc 2300 tgttcacctg gttgccctgg gcggcggagt tagaagatct cagcggagcg cgggtgctcg 2360 ccgatctcgg cgatgaccaa cgaagccggt atgccagtgt ctggacggtc agcgctcagc 2420 tggttgacga gcgacccgac ctggtgcaac gactcgtcga cgcggcggtc caagccagcc 2480 gttgggcaca ggcccatccc gaggaaaccg ttggcatcca tgccgccaac ctcggggtcg 2540 cgccctcggc gatcggacgc ggtttcggcg ctgatttcgc ccaacatctg atcccacggc 2600 ttgacgactc ggcactggcc gttgtcgacc aaactcaaca gttcctcctc gaccacaatc 2660 tcctcgacca tccggtggac ctcacgcagt gggcggcacc gcaattcctc actcaatcca 2720 cgactggaga ccagcaatga ccctgaccga tgacaccgcc acggcacaga acaataggaa 2780 tggcgacccc atcgaggtgg cgcgcgagct gacgcgaaag tggcaagcta ccgtcgtcga 2840 gcgcgacaag gctggtggtt cggcgacaga agagcgtgag gatctacgcg ccagtgggct 2900 gctctcggtg accgttcccc ggcacctggg cggttgggga gccgactggc ctaccgctct 2960 ggaagtggtg cgtgagatcg ccaaggtcga tggatcgctg ggccacttgt tcggatacca 3020 cctcagcacc ccagccgtta ttgatctgtg gggttcaccc gagcaaagtg aacgcctact 3080 tcgtcaactg gccgaaaaca actggtggac cgggaacgcc tccagcgaga acaacagcca 3140 catcctcgaa tggaaggtaa ctgccacccc gaccgacgac ggcgggtacc ttctcaacgg 3200 tatcaagcac ttcagcagtg gagccaaggg ctcagatctg ctcttggtgt tcggcgtgat 3260 accggagggt tccccacagc aaggcgccat cgttgccgcg gccattccca ccaccagaga 3320 aggcgttcaa acaaacgacg actggcaggc gctagggatg cggcgcaccg acagcggcac 3380 caccgaattc cacaacgtcg tagtccgtcc cgacgaggtg ctgggcaaac ccaatgccgt 3440 tgtcgaggcg ttcctggcgt ccgggcgcgg cagcctattc ggcccgatcg tgcagttggt 3500 gttcgcaacg gtgtatctgg ggatcgcgcg tggtgcgctc gaaacagcgc gcgagtacac 3560 ccgaacgcag gcacgcccct ggac gccggc tggggttact caagccgtcg aggatccgta 3620 caccattcgc agctacggcg aattcggtat cgaactacag gctggcgatg ctgcagcacg 3680 tgaggctgcg cagctgctac agaccgcgtg ggacaaaggt gacgcattaa ctccacaaga 3740 acgcggcgaa ctcgcggtac aggtagccgg agtcaaagcc atagccacca aagcgggcct 3800 ggacgtgacc agccgaattt tcgaggtcat cggtgcccgg ggaacccacc caaactatgg 3860 attcgaccgg ttctggcgca acatccgcac ccacacgctg cacgatcccg tctcttacaa 3920 gatcgctgaa gtcggaaact acgtccttaa ccagcgctac ccgatacccg gtttcacttc 3980 ctgactccag gaccgcgctg ccgggcacgc gttaatgcct gagaacggtg g 4031 <210> 2 <211> 453 <212> PRT <213> Bacillus subtilis <400> 2 Met Ala Glu Gln Arg Gln Leu His Leu Ala Gly Phe Phe Ser Ala Gly 1 5 10 15 Asn Val Thr His Ala His Gly Ala Trp Arg His Val Gly Ala Thr Asn 20 25 30 Gly Phe Leu Thr Gly Glu Phe Tyr Lys Gln Ile Ala Arg Thr Leu Glu 35 40 45 Arg Gly Lys Phe Asp Leu Leu Phe Leu Pro Asp Gly Leu Ala Ile Glu 50 55 60 Asp Ser Tyr Gly Asp Asn Leu Glu Thr Gly Val Gly Leu Gly Gly Gln 65 70 75 80 Gly Ala Val Ala Leu Glu Pro Thr Ser Va l Ile Ala Thr Met Ala Ala 85 90 95 Val Thr Gln Arg Leu Gly Leu Gly Ala Thr Val Ser Thr Thr Tyr Tyr 100 105 110 Pro Pro Tyr His Val Ala Arg Val Phe Ala Thr Leu Asp Asn Leu Ser 115 120 125 Asp Gly Arg Ile Ser Trp Asn Val Val Thr Ser Leu Asn Asp Ser Glu 130 135 140 Ala Arg Asn Phe Gly Val Asp Glu His Leu Glu His Asp Ile Arg Tyr 145 150 155 160 Asp Arg Ala Asp Glu Phe Leu Glu Ala Val Lys Lys Leu Trp Ser Ser 165 170 175 Trp Ser Glu Asp Ala Leu Leu Leu Asp Lys Val Gly Gly Arg Phe Ala 180 185 190 Asp Pro Lys Lys Val Gln Tyr Val Asn His Arg Gly Arg Trp Leu Ser 195 200 205 Val Arg Gly Pro Leu Gln Val Pro Arg Ser Arg Gln Gly Glu Pro Val 210 215 220 Ile Leu Gln Ala Gly Leu Ser Pro Arg Gly Arg Arg Phe Ala Gly Arg 225 230 235 240 Trp Ala Glu Ala Val Phe Ser Val Ser Pro Asn Leu Asp Ile Met Arg 245 250 255 Ala Val Tyr Gln Asp Ile Lys Ala His Val Ala Ala Ala Gly Arg Asp 260 265 270 Pro Glu Gln Thr Lys Val Phe Thr Ala Val Met Pro Val Leu Gly Glu 275 280 285 Thr Glu Gln Val Ala Arg Glu Arg Leu Glu Tyr Leu Asn Ser Leu Val 290 295 300 His Pro Glu Val Gly Leu Ser Thr Leu Ser Ser His Ser Gly Leu Asn 305 310 315 320 Leu Ser Lys Tyr Pro Leu Asp Thr Lys Phe Ser Asp Ile Val Ala Asp 325 330 335 Leu Gly Asp Arg His Val Pro Thr Met Leu Gln Met Phe Ser Ala Val 340 345 350 Ala Gly Gly Gly Ala Asp Leu Thr Leu Ala Glu Leu Gly Arg Arg Tyr 355 360 365 Gly Thr Asn Val Gly Phe Val Pro Gln Trp Ala Gly Thr Ala Glu Gln 370 375 380 Ile Ala Asp Gln Leu Ile Ser His Phe Glu Ala Gly Ala Ala Asp Gly 385 390 395 400 Phe Ile Ile Ser Pro Ala Tyr Leu Pro Gly Ile Tyr Glu Glu Phe Val 405 410 415 Asp Gln Val Val Pro Leu Leu Gln Gln Arg Gly Val Phe Arg Thr Glu 420 425 430 Tyr Glu Gly Thr Thr Leu Arg Glu His Leu Gly Leu Ala His Pro Glu 435 440 445 Val Gly Ser Ile Arg 450 <210> 3 <211> 4031 <212> DNA < 213> Bacillus subtilis <220> <221> CDS <222> (1670) .. (2737) <400> ggcgct ttgacggcac tcgtccgggg gcgagaatcg gcgaaggtcg 180 ggtgggtcgt cgggaggaag tggcgtgttg ggtgcccctt tgaatcatcg ggatcctaac 240 tctgttgggg tcggttgcag tagccgagca ttggtctccg gtaactgatc cccacggagg 300 cactgctgat tatggctgaa caacgccaac tacatttagc cgggtttttc tcggccggca 360 acgtcactca tgcgcatggc gcgtggcgcc atgtcggtgc gacgaacggt tttctgactg 420 gcgagttcta caagcagatt gcgcggaccc tggagcgggg caagtttgat ctgttgtttc 480 ttcccgatgg cctggccatc gaagatagct atggcgataa tttggagacg ggggtcggtc 540 tgggtggcca gggcgcggtg gcgctggagc cgaccagcgt gatagcgacg atggctgcgg 600 tgacccagcg attaggtctg ggcgctacgg tttcgacgac ctactacccg ccctatcatg 660 tggctcgggt gtttgccacg ctcgacaatc tgtctgatgg ccggatctcg tggaatgtgg 720 tcacgtcgct gaacgactcc gaggcacgca acttcggtgt ggatgagcac cttgagcatg 780 acatccgata cgaccgggct gacgaattct tggaggccgt caagaagcta tggagttctt 840 ggtccgagga tgccttgttg ttggacaagg ttggcggtcg gttcgctgat cccaagaagg 900 ttcaatacgt caatcatcgc ggccgctggt tgtcggtccg tggcccgttg caggttccac 960 ggtcccgtca gggtgaaccg gtcatcttgc a ggccggctt gtcgccgcgg gggcgtcgct 1020 tcgcggggcg ctgggctgaa gcggtattca gcgtctcgcc caacctcgac atcatgcgcg 1080 cggtctatca ggacatcaaa gctcacgttg cggccgcggg gcgtgacccg gagcaaacaa 1140 aggtattcac cgcggtgatg ccggtgctcg gtgagacaga gcaggtggcc cgggaacgtc 1200 tggaatatct caattcgctg gtgcatcccg aagtgggttt gtcgacgcta tccagccaca 1260 gcggcttgaa tctctccaag tatccgctgg atacgaagtt ttccgacatc gtcgccgatc 1320 tcggggatcg tcacgtgccg accatgttgc agatgttttc cgccgtggcc ggtggcggtg 1380 cggacctgac gctggccgaa ctgggccgcc gatacggcac caacgtcggg tttgtgccgc 1440 agtgggccgg aactgccgaa cagatcgctg atcaattgat tagtcacttt gaggccgggg 1500 ccgctgacgg attcatcatt tcgccggcgt atctgccggg tatttacgag gagttcgtcg 1560 accaggtggt tccgctgctg cagcaacgtg gtgtgttccg taccgagtac gaagggacaa 1620 cgctgcgtga gcaccttggg ttggctcacc ctgaagtcgg gagcatccg atg acc acc 1678 Met Thr Thr 1 acc gat gtt gat cat gac gtt ctt gcc tac agc aac tgc ccg gtt ccc 1726 Thr Asp Val Asp His Asp Val Leu Ala Tyr Ser Asn Cys Pro Val Pro 5 10 15 aat gca cta ctg aca gcg ctg gag tca aac ctg ttg gcc ggc aac ggg 1774 Asn Ala Leu Leu Thr Ala Leu Glu Ser Asn Leu Leu Ala Gly Asn Gly 20 25 30 35 att tcg ctg aat gtg ctc agc ggc gcg caa gca ggg ttg cat Ile Serc acc 1822 Asn Val Leu Ser Gly Ala Gln Ala Gly Leu His Phe Thr 40 45 50 tac gat cat ccc gct tac acc cgc ttt ggt ggc gaa att cca cct ctt 1870 Tyr Asp His Pro Ala Tyr Thr Arg Phe Gly Gly Glu Ile Pro Pro Leu 55 60 65 atc agt gag ggc ctg cgg gcg ccg gga cgc acc cgc ctg cta ggc atc 1918 Ile Ser Glu Gly Leu Arg Ala Pro Gly Arg Thr Arg Leu Leu Gly Ile 70 75 80 acc cct ctg gcc ggt cgg caa ggg atc gt gt gtt gac agc ccg 1966 Thr Pro Leu Ala Gly Arg Gln Gly Ile Tyr Val Arg Val Asp Ser Pro 85 90 95 gtg aca tca ccg gag cag ctg cgg ggc cgg cgg gtg ggt gtg tcg ggt 2014 Val Thr Ser Pro Glu Gln Leu Arg Gly Arg Arg Val Gly Val Ser Gly 100 105 110 115 gca gcg atc cgc atc ctc acc ggc gag ttg ggc gat tat cgg cac ttg 2062 Ala Ala Ile Arg Ile Leu Thr Gly Glu Leu Gly Asp Tyr Arg His Leu 120 125 130 gac ccg tgg cgg caa aca ctg atc gcg ctg ggt acc tgg gag gcg cgg 2110 Asp Pro Trp Arg Gln Thr Leu Ile Ala Leu Gly Thr Trp Glu Ala Arg 135 140 145 gga ctt ttg cag acc ctg cac atc ggg ggg ata ggg gtc gac gac gtc 2158 Leu Gln Thr Leu His Ile Gly Gly Ile Gly Val Asp Asp Val 150 155 160 gag ttg gtg cgt atc gaa agc cct ggc gtc gac gtg ccc gaa gag cgg 2206 Glu Leu Val Arg Ile Glu Ser Pro Gly Val Asp Val Pro Glu Glu Arg 165 170 175 ctg gaa gca gcg gcc agt gtc aaa ggc gct gac ctg ttt ccc gac gtg 2254 Leu Glu Ala Ala Ala Ser Val Lys Gly Ala Asp Leu Phe Pro Asp Val 180 185 190 195 gcc ggc cac cag agg gac atg g ggc aac gtt gat gcg ctg 2302 Ala Gly His Gln Arg Asp Ile Leu Asp Ser Gly Asn Val Asp Ala Leu 200 205 210 ttc acc tgg ttg ccc tgg gcg gcg gag tta gaa gat ctc agc gga gcg 2350 Phe Thr Trp Leu Pro Ala Glu Leu Glu Asp Leu Ser Gly Ala 215 220 225 cgg gtg ctc gcc gat ctc ggc gat gac caa cga agc cgg tat gcc agt 2398 Arg Val Leu Ala Asp Leu Gly Asp Asp Gln Arg Ser Arg Tyr Ala Ser 230 235 240 g tc tgg acg gtc agc gct cag ctg gtt gac gag cga ccc gac ctg gtg 2446 Val Trp Thr Val Ser Ala Gln Leu Val Asp Glu Arg Pro Asp Leu Val 245 250 255 caa cga ctc gtc gac gcg gcg gtc caa gcc agc cgt cag gcc 2494 Gln Arg Leu Val Asp Ala Ala Val Gln Ala Ser Arg Trp Ala Gln Ala 260 265 270 275 cat ccc gag gaa acc gtt ggc atc cat gcc gcc aac ctc ggg gtc gcg 2542 His Pro Glu Glu Thr Val Gly Ile His Ala Ala Asn Leu Gly Val Ala 280 285 290 ccc tcg gcg atc gga cgc ggt ttc ggc gct gat ttc gcc caa cat ctg 2590 Pro Ser Ala Ile Gly Arg Gly Phe Gly Ala Asp Phe Ala Gln His Leu 295 300 305 atc cca cgg gac tcg gca ctg gcc gtt gtc gac caa act caa 2638 Ile Pro Arg Leu Asp Asp Ser Ala Leu Ala Val Val Asp Gln Thr Gln 310 315 320 cag ttc ctc ctc gac cac aat ctc ctc gac cat ccg gtg gac ctc acg 2686 Leu Leu Asp His Asn Leu Leu Asp His Pro Val Asp Leu Thr 325 330 335 cag tgg gcg gca ccg caa ttc ctc act caa tcc acg act gga gac cag 2734 Gln Trp Ala Ala Pro Gln Phe Leu Thr Gln Ser Thr Thr Gly As p Gln 340 345 350 355 caa tgaccctgac cgatgacacc gccacggcac agaacaatag gaatggcgac 2787 Gln cccatcgagg tggcgcgcga gctgacgcga aagtggcaag ctaccgtcgt cgagcgcgac 2847 aaggctggtg gttcggcgac agaagagcgt gaggatctac gcgccagtgg gctgctctcg 2907 gtgaccgttc cccggcacct gggcggttgg ggagccgact ggcctaccgc tctggaagtg 2967 gtgcgtgaga tcgccaaggt cgatggatcg ctgggccact tgttcggata ccacctcagc 3027 accccagccg ttattgatct gtggggttca cccgagcaaa gtgaacgcct acttcgtcaa 3087 ctggccgaaa acaactggtg gaccgggaac gcctccagcg agaacaacag ccacatcctc 3147 gaatggaagg taactgccac cccgaccgac gacggcgggt accttctcaa cggtatcaag 3207 cacttcagca gtggagccaa gggctcagat ctgctcttgg tgttcggcgt gataccggag 3267 ggttccccac agcaaggcgc catcgttgcc gcggccattc ccaccaccag agaaggcgtt 3327 caaacaaacg acgactggca ggcgctaggg atgcggcgca ccgacagcgg caccaccgaa 3387 ttccacaacg tcgtagtccg tcccgacgag gtgctgggca aacccaatgc cgttgtcgag 3447 gcgttcctgg cgtccgggcg cggcagccta ttcggcccga tcgtgcagtt ggtgttcgca 3507 acggtgtatc tggggatcgc gcgtggtgcg ctcgaaacag cgcgcgagta ca cccgaacg 3567 caggcacgcc cctggacgcc ggctggggtt actcaagccg tcgaggatcc gtacaccatt 3627 cgcagctacg gcgaattcgg tatcgaacta caggctggcg atgctgcagc acgtgaggct 3687 gcgcagctgc tacagaccgc gtgggacaaa ggtgacgcat taactccaca agaacgcggc 3747 gaactcgcgg tacaggtagc cggagtcaaa gccatagcca ccaaagcggg cctggacgtg 3807 accagccgaa ttttcgaggt catcggtgcc cggggaaccc acccaaacta tggattcgac 3867 cggttctggc gcaacatccg cacccacacg ctgcacgatc ccgtctctta caagatcgct 3927 gaagtcggaa actacgtcct taaccagcgc tacccgatac ccggtttcac ttcctgactc 3987 caggaccgcg ctgccgggca cgcgttaatg cctgagaacg gtgg 4031 <210> 4 <211> 356 <212> PRT <213> Bacillus subtilis <400> 4 Met Thr Thr Thr Asp Val Asp His Asp Val Leu Ala Tyr Ser Asn Cys 1 5 10 15 Pro Val Pro Asn Ala Leu Leu Thr Ala Leu Glu Ser Asn Leu Leu Ala 20 25 30 Gly Asn Gly Ile Ser Leu Asn Val Leu Ser Gly Ala Gln Ala Gly Leu 35 40 45 His Phe Thr Tyr Asp His Pro Ala Tyr Thr Arg Phe Gly Gly Glu Ile 50 55 60 Pro Pro Leu Ile Ser Glu Gly Leu Arg Ala Pro Gly Arg Thr Arg Leu 65 70 75 80 Leu Gl y Ile Thr Pro Leu Ala Gly Arg Gln Gly Ile Tyr Val Arg Val 85 90 95 Asp Ser Pro Val Thr Ser Pro Glu Gln Leu Arg Gly Arg Arg Val Gly 100 105 110 Val Ser Gly Ala Ala Ile Arg Ile Leu Thr Gly Glu Leu Gly Asp Tyr 115 120 125 Arg His Leu Asp Pro Trp Arg Gln Thr Leu Ile Ala Leu Gly Thr Trp 130 135 140 Glu Ala Arg Gly Leu Leu Gln Thr Leu His Ile Gly Gly Ile Gly Val 145 150 155 160 Asp Asp Val Glu Leu Val Arg Ile Glu Ser Pro Gly Val Asp Val Pro 165 170 175 Glu Glu Arg Leu Glu Ala Ala Ala Ser Val Lys Gly Ala Asp Leu Phe 180 185 190 Pro Asp Val Ala Gly His Gln Arg Asp Ile Leu Asp Ser Gly Asn Val 195 200 205 Asp Ala Leu Phe Thr Trp Leu Pro Trp Ala Ala Glu Leu Glu Asp Leu 210 215 220 Ser Gly Ala Arg Val Leu Ala Asp Leu Gly Asp Asp Gln Arg Ser Arg 225 230 235 240 Tyr Ala Ser Val Trp Thr Val Ser Ala Gln Leu Val Asp Glu Arg Pro 245 250 255 Asp Leu Val Gln Arg Leu Val Asp Ala Ala Val Gln Ala Ser Arg Trp 260 265 270 Ala Gln Ala His Pro Glu Glu Thr Val Gly Ile His Ala Ala Asn Leu 275 280 285 Gly Val Ala Pro Ser Ala Ile Gly Arg Gly Phe Gly Ala Asp Phe Ala 290 295 300 Gln His Leu Ile Pro Arg Leu Asp Asp Ser Ala Leu Ala Val Val Asp 305 310 315 320 Gln Thr Gln Gln Phe Leu Leu Asp His Asn Leu Leu Asp His Pro Val 325 330 335 Asp Leu Thr Gln Trp Ala Ala Pro Gln Phe Leu Thr Gln Ser Thr Thr 340 345 350 Gly Asp Gln Gln 355 <210> 5 <211> 4031 <212> DNA <213> Bacillus subtilis <220> < 221> CDS <222> (2737) .. (3981) <400> 5 ctagcgaggt gcgagttccg cgcggtagcc atccgcttca ttgattttta catcgatacc 60 gccgcggtgg ccgtcagggt gtcgaagccg ggcggaggtg cacaccccgc cggttggtga 120 tcttgctcac aggtggcgct ttgacggcac tcgtccgggg gcgagaatcg gcgaaggtcg 180 ggtgggtcgt cgggaggaag tggcgtgttg ggtgcccctt tgaatcatcg ggatcctaac 240 tctgttgggg tcggttgcag tagccgagca ttggtctccg gtaactgatc cccacggagg 300 cactgctgat tatggctgaa caacgccaac tacatttagc cgggtttttc tcggccggca 360 acgtcactca tgcgcatggc gcgtggcgcc atgtcggtgc ggtgaggtgc gcgaacggt tttctgactg 420 gcgagttcgag cgcag cgg gagcagtcgcag cgg gagcagtcgcag tagct atggcgataa tttggagacg ggggtcggtc 540 tgggtggcca gggcgcggtg gcgctggagc cgaccagcgt gatagcgacg atggctgcgg 600 tgacccagcg attaggtctg ggcgctacgg tttcgacgac ctactacccg ccctatcatg 660 tggctcgggt gtttgccacg ctcgacaatc tgtctgatgg ccggatctcg tggaatgtgg 720 tcacgtcgct gaacgactcc gaggcacgca acttcggtgt ggatgagcac cttgagcatg 780 acatccgata cgaccgggct gacgaattct tggaggccgt caagaagcta tggagttctt 840 ggtccgagga tgccttgttg ttggacaagg ttggcggtcg gttcgctgat cccaagaagg 900 ttcaatacgt caatcatcgc ggccgctggt tgtcggtccg tggcccgttg caggttccac 960 ggtcccgtca gggtgaaccg gtcatcttgc aggccggctt gtcgccgcgg gggcgtcgct 1020 tcgcggggcg ctgggctgaa gcggtattca gcgtctcgcc caacctcgac atcatgcgcg 1080 cggtctatca ggacatcaaa gctcacgttg cggccgcggg gcgtgacccg gagcaaacaa 1140 aggtattcac cgcggtgatg ccggtgctcg gtgagacaga gcaggtggcc cgggaacgtc 1200 tggaatatct caattcgctg gtgcatcccg aagtgggttt gtcgacgcta tccagccaca 1260 gcggcttgaa tctctccaag tatccgctgg atacgaagtt ttccgacatc gtcgccgatc 1320 tcggggatcg tcacgtgccg accatgttgc agatgtt ttc cgccgtggcc ggtggcggtg 1380 cggacctgac gctggccgaa ctgggccgcc gatacggcac caacgtcggg tttgtgccgc 1440 agtgggccgg aactgccgaa cagatcgctg atcaattgat tagtcacttt gaggccgggg 1500 ccgctgacgg attcatcatt tcgccggcgt atctgccggg tatttacgag gagttcgtcg 1560 accaggtggt tccgctgctg cagcaacgtg gtgtgttccg taccgagtac gaagggacaa 1620 cgctgcgtga gcaccttggg ttggctcacc ctgaagtcgg gagcatccga tgaccaccac 1680 cgatgttgat catgacgttc ttgcctacag caactgcccg gttcccaatg cactactgac 1740 agcgctggag tcaaacctgt tggccggcaa cgggatttcg ctgaatgtgc tcagcggcgc 1800 gcaagcaggg ttgcatttca cctacgatca tcccgcttac acccgctttg gtggcgaaat 1860 tccacctctt atcagtgagg gcctgcgggc gccgggacgc acccgcctgc taggcatcac 1920 ccctctggcc ggtcggcaag ggatctatgt ccgtgttgac agcccggtga catcaccgga 1980 gcagctgcgg ggccggcggg tgggtgtgtc gggtgcagcg atccgcatcc tcaccggcga 2040 gttgggcgat tatcggcact tggacccgtg gcggcaaaca ctgatcgcgc tgggtacctg 2100 ggaggcgcgg ggacttttgc agaccctgca catcgggggg ataggggtcg acgacgtcga 2160 gttggtgcgt atcgaaagcc ctggcgtcga cgtgcccgaa ga gcggctgg aagcagcggc 2220 cagtgtcaaa ggcgctgacc tgtttcccga cgtggccggc caccagaggg acatcctgga 2280 tagcggcaac gttgatgcgc tgttcacctg gttgccctgg gcggcggagt tagaagatct 2340 cagcggagcg cgggtgctcg ccgatctcgg cgatgaccaa cgaagccggt atgccagtgt 2400 ctggacggtc agcgctcagc tggttgacga gcgacccgac ctggtgcaac gactcgtcga 2460 cgcggcggtc caagccagcc gttgggcaca ggcccatccc gaggaaaccg ttggcatcca 2520 tgccgccaac ctcggggtcg cgccctcggc gatcggacgc ggtttcggcg ctgatttcgc 2580 ccaacatctg atcccacggc ttgacgactc ggcactggcc gttgtcgacc aaactcaaca 2640 gttcctcctc gaccacaatc tcctcgacca tccggtggac ctcacgcagt gggcggcacc 2700 gcaattcctc actcaatcca cgactggaga ccagca atg acc ctg acc gat gac 2754 Met Thr Leu Thr Asp Asp 1 5 acc gc gag ag gac gag Ag gcag gag Ag gac gc Ag gac gc Ag gcag gac Ag Asn Asn Arg Asn Gly Asp Pro Ile Glu Val Ala 10 15 20 cgc gag ctg acg cga aag tgg caa gct acc gtc gtc gag cgc gac aag 2850 Arg Glu Leu Thr Arg Lys Trp Gln Ala Thr Val Val Glu Arg Asp Lys 25 30 35 gct ggt ggt tcg gcg aca ga a gag cgt gag gat cta cgc gcc agt ggg 2898 Ala Gly Gly Ser Ala Thr Glu Glu Arg Glu Asp Leu Arg Ala Ser Gly 40 45 50 ctg ctc tcg gtg acc gtt ccc cgg cac ctg ggc ggt tgg gga gcc gac 2946 Leu Leu Seru Val Thr Val Pro Arg His Leu Gly Gly Trp Gly Ala Asp 55 60 65 70 tgg cct acc gct ctg gaa gtg gtg cgt gag atc gcc aag gtc gat gga 2994 Trp Pro Thr Ala Leu Glu Val Val Arg Glu Ile Ala Lys Val Asp Gly 75 80 85 tcg ctg ggc cac ttg ttc gga tac cac ctc agc acc cca gcc gtt att 3042 Ser Leu Gly His Leu Phe Gly Tyr His Leu Ser Thr Pro Ala Val Ile 90 95 100 gat ctg tgg ggt tca ccc gag caa agt gaa cgc cta ctt cgt caa ctg 3090 Asp Leu Trp Gly Ser Pro Glu Gln Ser Glu Arg Leu Leu Arg Gln Leu 105 110 115 gcc gaa aac aac tgg tgg acc ggg aac gcc tcc agc gag aac aac agc 3138 Ala Glu Asn Asn Trp Trp Thr Gly Asn Ala Ser Ser Glu Asn Asn Ser 120 125 130 cac atc ctc gaa tgg aag gta act gcc acc ccg acc gac gac ggc ggg 3186 His Ile Leu Glu Trp Lys Val Thr Ala Thr Pro Thr Asp Asp Gly Gly 135 140 145 150 tac ctt ctc aa c ggt atc aag cac ttc agc agt gga gcc aag ggc tca 3234 Tyr Leu Leu Asn Gly Ile Lys His Phe Ser Ser Gly Ala Lys Gly Ser 155 160 165 gat ctg ctc ttg gtg ttc ggc gtg ata ccg gag ggt tcc Asp Leu Leu Leu Val Phe Gly Val Ile Pro Glu Gly Ser Pro Gln Gln 170 175 180 ggc gcc atc gtt gcc gcg gcc att ccc acc acc aga gaa ggc gtt caa 3330 Gly Ala Ile Val Ala Ala Ala Ile Pro Thr Thr Arg Glu Gly Val Gln 185 190 195 aca aac gac gac tgg cag gcg cta ggg atg cgg cgc acc gac agc ggc 3378 Thr Asn Asp Asp Trp Gln Ala Leu Gly Met Arg Arg Thr Asp Ser Gly 200 205 210 acc acc gaa ttc cac aac gtc gta gtc cgt ccc gac gag gtg ctg ggc 3426 Thr Thr Glu Phe His Asn Val Val Val Arg Pro Asp Glu Val Leu Gly 215 220 225 230 aaa ccc aat gcc gtt gtc gag gcg ttc ctg gcg tcc ggg cgc ggc agc 3474 Lys Pro Asn Ala Val Glu Ala Phe Leu Ala Ser Gly Arg Gly Ser 235 240 245 cta ttc ggc ccg atc gtg cag ttg gtg ttc gca acg gtg tat ctg ggg 3522 Leu Phe Gly Pro Ile Val Gln Leu Val Phe Ala Thr Val Tyr Leu Gly 250 255 260 atc gcg cgt ggt gcg ctc gaa aca gcg cgc gag tac acc cga acg cag 3570 Ile Ala Arg Gly Ala Leu Glu Thr Ala Arg Glu Tyr Thr Arg Thr Gln 265 270 275 gca cgc ccc tgg acg ccg gct ggg gtt act ca gag gat ccg 3618 Ala Arg Pro Trp Thr Pro Ala Gly Val Thr Gln Ala Val Glu Asp Pro 280 285 290 tac acc att cgc agc tac ggc gaa ttc ggt atc gaa cta cag gct ggc 3666 Tyr Thr Ile Arg Ser Tyr Gly Glu Phe Gly Ile Glu Leu Gln Ala Gly 295 300 305 310 gat gct gca gca cgt gag gct gcg cag ctg cta cag acc gcg tgg gac 3714 Asp Ala Ala Ala Ala Arg Glu Ala Ala Gln Leu Leu Gln Thr Ala Trp Asp 315 320 325 aaa ggt gca tta act cca caa gaa cgc ggc gaa ctc gcg gta cag 3762 Lys Gly Asp Ala Leu Thr Pro Gln Glu Arg Gly Glu Leu Ala Val Gln 330 335 340 gta gcc gga gtc aaa gcc ata gcc acc aaa gcg ggc ctg gac gtg gac gtg gac gtg 38g Ala Gly Val Lys Ala Ile Ala Thr Lys Ala Gly Leu Asp Val Thr 345 350 355 agc cga att ttc gag gtc atc ggt gcc cgg gga acc cac cca aac tat 3858 Ser Arg Ile Phe Glu Val Ile Gly Ala Arg Gly Thr His Pro Asn Tyr 360 365 370 gga ttc gac cgg ttc tgg cgc aac atc cgc acc cac acg ctg cac gat 3906 Gly Phe Asp Arg Phe Trp Arg Asn Ile Arg Thr His Thr Leu His Asp 375 380 385 390 ccc gtc tct tac aag atcct gaa gtc gga aac tac gtc ctt aac cag 3954 Pro Val Ser Tyr Lys Ile Ala Glu Val Gly Asn Tyr Val Leu Asn Gln 395 400 405 cgc tac ccg ata ccc ggt ttc act tcc tgactccagg accgcgctgc 4001 Arg Tyr Pro Hele 410 415 cgggcacgcg ttaatgcctg agaacggtgg 4031 <210> 6 <211> 415 <212> PRT <213> Bacillus subtilis <400> 6 Met Thr Leu Thr Asp Asp Thr Ala Thr Ala Gln Asn Asn Arg Asn Gly 1 5 10 15 Asp Pro Ile Glu Val Ala Arg Glu Leu Thr Arg Lys Trp Gln Ala Thr 20 25 30 Val Val Glu Arg Asp Lys Ala Gly Gly Ser Ala Thr Glu Glu Arg Glu 35 40 45 Asp Leu Arg Ala Ser Gly Leu Leu Ser Val Thr Val Pro Arg His Leu 50 55 60 Gly Gly Trp Gly Ala Asp Trp Pro Thr Ala Leu Glu Val Val Arg Glu 65 70 75 80 Ile Ala Lys Val Asp Gly Ser Leu Gly His Leu Phe Gly Tyr His Leu 85 90 95 Ser Thr Pro Ala Val Ile Asp Leu Tr p Gly Ser Pro Glu Gln Ser Glu 100 105 110 Arg Leu Leu Arg Gln Leu Ala Glu Asn Asn Trp Trp Thr Gly Asn Ala 115 120 125 Ser Ser Glu Asn Asn Ser His Ile Leu Glu Trp Lys Val Thr Ala Thr 130 135 140 Pro Thr Asp Asp Gly Gly Tyr Leu Leu Asn Gly Ile Lys His Phe Ser 145 150 155 160 Ser Gly Ala Lys Gly Ser Asp Leu Leu Leu Val Phe Gly Val Ile Pro 165 170 175 Glu Gly Ser Pro Gln Gln Gly Ala Ile Val Ala Ala Ala Ile Pro Thr 180 185 190 Thr Arg Glu Gly Val Gln Thr Asn Asp Asp Trp Gln Ala Leu Gly Met 195 200 205 Arg Arg Thr Asp Ser Gly Thr Thr Glu Phe His Asn Val Val Val Arg 210 215 220 Pro Asp Glu Val Leu Gly Lys Pro Asn Ala Val Val Glu Ala Phe Leu 225 230 235 240 Ala Ser Gly Arg Gly Ser Leu Phe Gly Pro Ile Val Gln Leu Val Phe 245 250 255 Ala Thr Val Tyr Leu Gly Ile Ala Arg Gly Ala Leu Glu Thr Ala Arg 260 265 270 Glu Tyr Thr Arg Thr Gln Ala Arg Pro Trp Thr Pro Ala Gly Val Thr 275 280 285 Gln Ala Val Glu Asp Pro Tyr Thr Ile Arg Ser Tyr Gly Glu Phe Gly 290 295 300 300 Ile Glu Leu Gln Ala Gly Asp Ala Ala A la Arg Glu Ala Ala Gln Leu 305 310 315 320 Leu Gln Thr Ala Trp Asp Lys Gly Asp Ala Leu Thr Pro Gln Glu Arg 325 330 335 Gly Glu Leu Ala Val Gln Val Ala Gly Val Lys Ala Ile Ala Thr Lys 340 345 350 Ala Gly Leu Asp Val Thr Ser Arg Ile Phe Glu Val Ile Gly Ala Arg 355 360 365 Gly Thr His Pro Asn Tyr Gly Phe Asp Arg Phe Trp Arg Asn Ile Arg 370 375 380 Thr His Thr Leu His Asp Pro Val Ser Tyr Lys Ile Ala Glu Val Gly 385 390 395 400 400 Asn Tyr Val Leu Asn Gln Arg Tyr Pro Ile Pro Gly Phe Thr Ser 405 410 415 <210> 7 <211> 16 <212> PRT <213> Paenibacillus sp. <400> 7 Trp Asn Val Val Thr Ser Leu Asn Asn Ala Glu Ala Arg Asn Phe Gly 1 5 10 15 <210> 8 <211> 16 <212> PRT <213> Rhodococcus sp.IGTS8 <400> 8 Trp Asn Val Val Thr Ser Leu Asn Asp Ala Glu Ala Arg Asn Phe Gly 1 5 10 15 <210> 9 <211> 18 <212> PRT <213> Paenibacillus sp. <400> 9 Gly Phe Asp Arg Phe Trp Arg Asp Ala Arg Thr His Thr Leu His Asp 15 10 15 Pro Val <210> 10 <211> 18 <212> PRT <213> Rhodococcus sp.IGTS8 <400> 10 Gly Phe Asp Arg Phe Trp A rg Asn Val Arg Thr His Ser Leu His Asp 1 5 10 15 Pro Val <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: sense primer for PCR <220 > <221> modified base <222> n = inosine <223> (3), (9) and (12) <400> 11 gcngargcnm gnaayttygg 20 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: antisense primer for PCR <220> <221> modified base <222> n = inosine <223> (4) and (11) <400> 12 cgtngcgcca naagcggtc 19 <210> 13 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: sense primer for PCR <400> 13 gcatgacatc cgatacg 17 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: antisense primer for PCR <400> 14 tagtttgggt gggttcc 17

[0080]

[Sequence Listing Free Text] SEQ ID NO: 1-nucleotide sequence containing bdsABC gene SEQ ID NO-amino acid sequence of protein BdsA encoded by bdsA gene SEQ ID NO: 3-nucleotide sequence containing bdsABC gene SEQ ID NO: encoded by bdsB gene SEQ ID NO: 5-nucleotide sequence containing bdsABC gene SEQ ID NO: 6-amino acid sequence of protein BdsC encoded by bdsC gene SEQ ID NO: 7-partial amino acid sequence of TdsA SEQ ID NO: 8-partial amino acid sequence of DszA No. 9-partial amino acid sequence of TdsC SEQ ID NO. 10-partial amino acid sequence of DszC SEQ ID NO. 11-description of artificial sequence: sense primer (3,
SEQ ID NO: 12—Description of Artificial Sequence: Antisense Primer (N = 4 and 11 N Represents Deoxyinosine) SEQ ID NO: 13—Description of Artificial Sequence: Sense Primer SEQ ID NO: 14-Description of Artificial Sequence: Antisense Primer

[Brief description of the drawings]

FIG. 1. Expression plasmid pU1Nh containing the desulfurization gene bdsABC
1 shows a restriction map of the present invention.

FIG. 2 shows the temperature dependence of BdsC enzyme activity.

FIG. 3 shows the temperature stability of BdsC enzyme activity.

FIG. 4 shows the pH dependence of BdsC enzyme activity.

FIG. 5 shows the pH stability of BdsC enzyme activity.

FIG. 6 shows the temperature dependence of BdsA enzyme activity.

FIG. 7 shows the temperature stability of BdsA enzyme activity.

FIG. 8 shows the pH dependence of BdsA enzyme activity.

FIG. 9 shows the pH stability of BdsA enzyme activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 9/14 9/02 (C12N 1/21 9/14 C12R 1:19) // ( C12N 1/21 C12N 15/00 ZNAA C12R 1:19) 5/00 A (72) Inventor Hidekazu Iwasawa 1764 Toda, Sanmu-cho, Sanmu-gun, Chiba Prefecture (72) Inventor Koji Harada 5-21- Kamisagimiya, Nakano-ku, Tokyo 42 (72) Inventor Toshiki Furuya 2-15-23, Kamirenjaku, Mitaka City, Tokyo (72) Inventor Yoshitaka Ishii 1-323 Noguchicho, Higashimurayama-shi, Tokyo 38 Third Hashiba Heights Room 303 (72) Inventor Kuni Kino Chiba 1-14-8 Mizuho, Hanamigawa-ku, Chiba-shi, Japan (72) Inventor Shoji Usami 384-9 Mizuno, Sayama-shi, Saitama (72) Inventor Kenji Maruhashi 1-6-1, Nishikubo, Shimizu-shi, Shizuoka F-term (reference) 4B024 AA03 AA17 BA08 BA11 CA04 DA06 EA04 GA11 GA19 HA01 HA03 HA12 4B050 CC03 CC08 DD02 FF09E FF10E LL05 LL10 4B065 AA19Y AA26X AB01 AC02 AC14 BA02 BB13 CA28 CA31 CA54

Claims (10)

[Claims] 1. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 2 (b) an amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and dibenzothiophene sulfone To
Protein having the function of converting to 2- (2'-hydroxyphenyl) benzenesulfinic acid 2. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 4 (b) an amino acid sequence of SEQ ID NO: 4 in which one or more amino acids are deleted, substituted or added, and 2- ( Protein having the function of converting 2'-hydroxyphenyl) benzenesulfinic acid into 2-hydroxybiphenyl 3. A gene encoding the following protein (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 6; (b) an amino acid sequence of SEQ ID NO: 6 in which one or more amino acids are deleted, substituted or added, and dibenzothiophene is Protein having the function of converting to dibenzothiophene sulfone 4. A vector comprising the gene according to claim 1, 2 or 3. 5. A transformant containing the vector according to claim 4. 6. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 2 (b) an amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been deleted, substituted or added, and dibenzothiophene sulfone To
Protein having the function of converting to 2- (2'-hydroxyphenyl) benzenesulfinic acid 7. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 4 (b) an amino acid sequence of SEQ ID NO: 4 in which one or more amino acids are deleted, substituted or added, and 2- ( Protein having the function of converting 2'-hydroxyphenyl) benzenesulfinic acid into 2-hydroxybiphenyl 8. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 6; (b) an amino acid sequence of SEQ ID NO: 6 in which one or more amino acids are deleted, substituted or added, and dibenzothiophene is Protein having the function of converting to dibenzothiophene sulfone 9. A protein having the following properties: (1) Action: dibenzothiophene is converted to dibenzothiophene sulfone (2) Optimum pH: 8.0, stable pH: 8-11 (3) Optimum temperature: 45-50 ° C, stable temperature: 53 ° C or less (4) Molecular weight : 200,000 (by gel filtration method) (5) Inhibition of activity: Inhibited by chelating agents and SH inhibitors (6) Coenzyme requirement: Requires FMNH ₂ supplied by flavin reductase 10. A protein having the following properties: (1) Action: dibenzothiophene sulfone is converted to 2- (2'-hydroxyphenyl) benzenesulfinic acid (2) Optimum pH: 7.5, stable pH: 6-10 (3) Optimum temperature: 50 ° C, stable temperature : 45 ° C or less (4) Molecular weight: 174,000 (by gel filtration) (5) Activity inhibition: Inhibited by chelating agents and SH inhibitors (6) Coenzyme requirement: FMNH ₂ supplied by flavin reductase Request