JP2000245478A - Oxidoreductase gene - Google Patents

Abstract

(57) Abstract: An enzyme having a function of oxidizing NADH and reducing FMN in combination with an enzyme having a function of decomposing a thiophene compound, and a gene encoding the same. [Effect] The sulfur in the thiophene-based compound contained in fossil fuels such as petroleum can be efficiently released.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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. The present invention relates to an enzyme having a function of oxidizing NADH and reducing FMN by conjugation, and a gene encoding the enzyme. By utilizing the enzymes and genes of the present invention, benzothiophene and DBT contained in fossil fuels such as petroleum and the like, and sulfur in substituted substances or derivatives thereof can be released, so that petroleum, coal, etc. Sulfur that diffuses into the air due to the combustion of fossil fuel can be easily removed from the fossil fuel.

[0002]

2. Description of the Related Art (1) Hydrodesulfurization As a desulfurization method for removing sulfur from hydrocarbon fuel such as petroleum, there are known methods such as alkali washing and solvent desulfurization. Desulfurization is the mainstream. 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 metal-catalyzed hydrodesulfurization is a very complete process that is now 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 organic 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, B.
C., Kwart, HJ, Catalt., 61, 523-527 (1980). Indeed, it is known that various alkylated derivatives of DBT are 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.

(2) CC bond-cleaving type microbial desulfurization On the other hand, the enzymatic reaction performed by organisms proceeds under relatively mild conditions, and the rate of the enzymatic reaction itself is comparable to that of a reaction using a chemical catalyst. It has the feature of. In addition, there are numerous types of enzymes that need to respond appropriately to a wide variety of biological reactions that occur in vivo,
These enzymes are generally known to exhibit very high substrate specificity. Such a feature is expected to be utilized in a so-called biodesulfurization reaction for removing sulfur in a sulfur compound contained in a fossil fuel using a microorganism (Monticello, DJ, Hydrocarbon Proces).
sing39-45 (1994)).

[0006] On the other hand, there have been many reports on the method of removing sulfur from a heterocyclic sulfur compound which is a component of petroleum using bacteria. However, these methods are based on a ring decomposition (CC bond cleavage) type reaction and a CS bond cleavage type reaction. They are roughly divided into reactions. Bacteria having CC bond attack type desulfurization activity include mesophilic bacteria and thermophilic bacteria. Examples of the former are Pseudomonas sp., Pseudomonas
aeruginosa, Beijerinckia sp., Pseudomonas alcalig
enes, Pseudomonas stutzeri, Pseudomonasputida, Bre
vibacterium sp. and the like are known. These bacteria contain CC in heterocyclic organic sulfur compounds represented by DBT.
This is a type of metabolism in which a bond is broken, the benzene ring is decomposed, and a sulfuric acid salt is released by a subsequent oxidation reaction cascade. The reaction mechanism of these carbon skeleton attack type pathways is hydroxylation of aromatic ring (DBT →→ 1,2-dihydroxy DBT), ring cleavage, oxidation to water-soluble product (1,2-dihydroxy DBT →→ trans- 4 [2- (3-hydroxy) thiannaphthenyl] -2-oxo-butenoic acid, 3-hydroxy-2
-Formylbenzothiophene) and Ko
It is called the dama pathway. This type of reaction allows the DB
The CC bond in the benzene ring of T is attacked, producing various water-soluble substances that can be extracted from the oil. However, this reaction attacks other aromatic molecules in the oil, which results in a significant transfer of hydrocarbons to the liquid phase (Hart
degen, FJ, Coburn, JM and Roberts, RL Chem.
Eng. Progress, 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 occurs at the 2- and 3-positions of DBTs with alkyl or allyl substituents, so substituted DBTs at these positions are not substrates for the Kodama pathway.

On the other hand, it is known that the speed of a chemical reaction generally increases depending on the temperature. In the desulfurization step in the petroleum refining process, fractional distillation and desulfurization reaction 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); K
argi, 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 Ro
binson, JM, Biotechnol.Bioeng., 26, 687-690 (198
4); Kargi, F. & Robinson, JM, Biotechnol. Bioeng.
27, 41-49 (1985); Kargi, F., Biotechnol. Lett., 9,
478-482 (1987)). Kargi and Robinson (Kargi, F and Robi
nson, JM, Appl.Environ.Microbiol., 44, 878-883
According to (1982)), a strain of Sulfolobus acidocaldarius isolated from acidic hot springs in Yellowstone National Park, USA, grows at 45-70 ° C but oxidizes elemental sulfur at an optimal pH of 2. Another two species of Sulfolobus acidocalda
The oxidation of pyrite by the rius strain has also been reported (Tobita,
M., Yokozeki, M., Nishikawa, N. & Kawakami, Y., Bi
osci. Biotech. Biochem. 58, 771-772 (1994)).

[0009] 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. The DBT Sulfolobus acidocaldariu
s (hereinafter referred to as “S. acidocaldarius”) has also been reported (Kargi, & Robinson, JM, Biote
chnol. Bioeng, 26, 687-690 (1984); Kargi, F., Biotec
hnol. Letters 9, 478-482 (1987)).

According to these reports, when model aromatic heterocyclic sulfur compounds such as thianthrene, thioxanthene, and DBT are reacted with this microorganism at a high temperature, these sulfur compounds are oxidized and decomposed. Oxidation of these aromatic heterocyclic sulfur compounds by S. acidocaldarius has been observed at 70 ° C. and yields sulfate ions as reaction products. 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. acidocaldar
ius 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. acidocald
When arius is grown under autotrophic conditions, it obtains the necessary energy from reduced iron and sulfur compounds and uses carbon dioxide as a carbon source. However, S. acidocaldar
ius, when grown under heterotrophic conditions, 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.

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 e
(eds.) Penwell. Tuisa, Okia, 83-91 (1983). In this case, the oxidation reaction proceeds even at 55 ° C. But,
The degradation products of DBT by these Pseudomonas strains were
3-hydroxy-2-formylbenzothiophene reported by dama et al. (Monticello, DJ, Bakker, D., Fi
nnerty, WR Appl.Environ.Microbiol., 49, 756-76
0 (1985)). 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 Pseudomo
It can be seen that the degradation reaction of DBT by the nas 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.

(3) Microorganism desulfurization of CS bond-cleaving type Not only crude oil and coal but also model compounds containing sulfur are decomposed, and sulfur as a hetero atom is selectively removed to produce sulfates and hydroxylated compounds. Microorganisms have been reported.
This type of reaction, given the structure of its metabolites,
It is thought to be a reaction that specifically cleaves the CS bond in the sulfur compound and releases sulfur in the form of sulfate.
To date, there have been reports of biodesulfurization reaction systems using sulfur-attacking mesophilic bacteria as shown in Table 1.

[0013]

[Table 1]

On the other hand, recently, the present inventors have isolated the CS bond-cleavable thermophilic bacterium as Paenibacillus sp. Strain for the first time in the world (JP-A-10-036859). CS
As described above, a decomposition reaction of an organic sulfur compound of a type that specifically breaks a bond but leaves a CC bond without breaking it is desirable as an actual method for desulfurizing petroleum. That is, at elevated temperatures, DBT and its alkyl-substituted products,
It is most desirable as a biodesulfurization process to utilize microorganisms that exhibit the activity of cleaving CS bonds in their derivative molecules and produce desulfurized products in the form of water-soluble substances.
Once the gene involved in the high-temperature desulfurization activity of this strain is isolated, the gene can be introduced and expressed in other organisms using a genetic engineering technique such as recombinant DNA technology, and it can be used in a wide range of organisms. High temperature desulfurization ability can be provided.

(4) Oxidoreductase A bacterium which is known to cause a CS bond cleavage type desulfurization reaction, a gene encoding an enzyme activity involved in the DBT decomposition reaction has been identified, and its nucleotide sequence has been determined. Is that, to the best of our knowledge, Rhodococcus sp.
Only the dsz gene of IGTS8 strain (Denome, S., Oldflel
d., C., Nash, LJ and Young, KDJBacteriol., 17
6: 6707-6716, 1994; Piddington, CS, Kovacevich,
BR and Rambosek, J. Appl.Environ.Microbiol., 6
1: 468-475, 1995). The degradation reaction of DBT by IGTS8 strain is DB
DszC catalyzes the conversion of T to DBTO ₂ via DBTO, DBTO ₂
Catalyzed by three enzymes, DszA, which catalyzes the conversion of 2- (2'-hydroxyphenyl) benzenesulfinic acid to 2- (2'-hydroxyphenyl) benzenesulfinic acid, and DszB, which catalyzes the conversion of 2- (2'-hydroxyphenyl) benzenesulfinic acid to 2-HBP (Denome, S., Ol
dfield., C., Nash, LJ and Young, KDJBacterio
l., 176: 6707-6716, 1994; Gray, KA, Pogrebinshy,
OS, Mrachko, GT, Xi, L. Monticello, DJ and S
quires, CH Nat Biotechnol., 14: 1705-1709, 1996;
Oldfield, C., Pogrebinsky, O., Simmonds, J., Olso
n, ES and Kulpa, CF, Microbiology, 143: 2961-29
73, 1997). The corresponding genes are dszA, dszB, dsz
Called C.

DszC and DszA are monooxygenases, and it is known that their oxygenation requires coexistence of NADH-FMN oxidoreductase activity (Gray, KA, Po
grebinsky, OS, Mrachko, GT, Xi, L. Monticello,
DJ and Squires, CH Nat Biotechnol., 14: 1705-1
709, 1996; Xi, L. Squires, CH, Monticello, DJ
and Chids, JD Biochem. Biophys. Res Commun., 23
0: 73-76, 1997). This enzyme having NADH-FMN oxidoreductase activity is called DszD, and its gene sequence is Gray, KA
(US Pat. No. 5,804,433, or US Pat. No. 5,811,285).

[0017]

An object of the present invention is to isolate a bacterium having an enzyme which acts on benzothiophene and a DBT-based compound and efficiently promotes an oxidation reaction.
Isolate MN oxidoreductase, isolate the gene encoding this enzyme, identify its structure (especially the nucleotide sequence),
The purpose is to create a novel desulfurized microorganism by introducing these genes into a microorganism different from the one from which it was isolated and conferring desulfurization ability. Another object of the present invention is to establish a method for releasing sulfur by actually causing such a microorganism to act on benzothiophene, DBT and an alkyl derivative thereof, and cleaving a CS bond of these compounds.

[0018]

Means for Solving the Problems NADH-FMN oxidoreductase is widely distributed in non-desulfurized bacteria, and it is required that the optimum factor be used as a promoter of the DBT oxidation reaction. The present inventors have screened NADH-FMN oxidoreductase-producing bacteria that widely promote DBT oxidation from the natural world in order to solve the above-mentioned problems. As a result, Paenibacillus polymixa A-1 (Paenibacill
us polymyxa A-1) strain was isolated from soil, and a gene encoding an oxidoreductase that promotes the DBT oxidation reaction was successfully isolated therefrom, thereby completing the present invention.

That is, the first aspect of the present invention relates to a gene encoding an enzyme which is conjugated to a desulfurizing enzyme and is involved in a desulfurizing reaction.
The second aspect of the present invention relates to a vector containing the above gene.
The third aspect of the present invention relates to a transformant containing the above vector. A fourth aspect of the present invention relates to an enzyme conjugated with a desulfurization enzyme and involved in a desulfurization reaction. The fifth aspect of the present invention relates to a strain having an enzyme conjugated to a desulfurization enzyme and involved in a desulfurization reaction, and a transformant using the same as a host.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. (1) A gene encoding an enzyme involved in a desulfurization reaction conjugated with a desulfurization enzyme The gene of the present invention comprises (a) a protein represented by the amino acid sequence of SEQ ID NO: 2, or (b) an amino acid of SEQ ID NO: 2 It encodes a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the sequence and having a function of oxidizing NADH and reducing FMN. (a) and (b) proteins are DB
It is conjugated to an enzyme that converts TO ₂ to 2- (2′-hydroxyphenyl) benzenesulfinic acid and an enzyme that converts DBT to DBTO2 via DBTO. Although the above genes show some homology to the gene encoding Nitroreductase, these
Nitroreductases having properties similar to those of the present invention have not been reported.

Among the genes of the present invention, the gene encoding the amino acid sequence represented by SEQ ID NO: 2 can be obtained by the method described in Examples of the present specification. In addition, since the nucleotide sequences of these genes have already been determined as shown in SEQ ID NO: 1, appropriate primers are synthesized based on these sequences and, for example, Paenibacilluspol
It can also be obtained by performing PCR using DNA prepared from ymyxa A-1 strain as a template. In addition, this Paenib
The acillus polymyxa A-1 strain has been deposited with the National Institute of Advanced Industrial Science and Technology under the accession number FERM P-17274 (deposit date: March 2, 1999).

In the amino acid sequence of SEQ ID NO: 2, 1
Alternatively, a gene encoding an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added 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. 6487-6500,
1982) by modifying the gene encoding the amino acid sequence of SEQ ID NO: 2. Since the gene of the present invention encodes an enzyme involved in the degradation of DBT, it can be used for desulfurization of petroleum.

(2) Vector containing a gene encoding an enzyme involved in a desulfurization reaction conjugated with a desulfurizing enzyme The vector of the present invention contains the above-mentioned gene. Such a vector can be prepared by inserting a DNA fragment containing the 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, and if Escherichia coli is used as the host, the following vectors are preferably used.
Strong promoters such as lac, lacUV5, tr
pUR system including p, tac, trc, λpL, T7, rrnB, etc., p
GEX, pUC, pET, pT7, pBluescript, pKK
It is preferable to use a vector such as a system, pBS system, pBC system, or pCAL system.

(3) Transformant Containing a Vector Containing a Gene Encoding an Enzyme Involved in the Desulfurization Reaction Conjugated with 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 (C
600hflA), C-1a, C600 (BNN93), CES200, CES201, CJ23
6, CSH18, DH1, DH5, DH5α, DP50supF, ED8654, E
D8767, HB101, HMS174, JM101, JM105, JM107, JM10
9, JM110, K802, KK2186, LE392, LG90, M5219, MBM
7014.5, MC1061, MM294, MV1184, MV1193, MZ-1, NM5
31, NM538, NM539, Q358, Q359, R594, RB791, RR1
, SMR10, TAP90, TG1, TG2, XL1-Blue, XS101, X
S127, Y1089, Y1090hsdR, YK537 and the like.

(4) Enzyme conjugated with desulfurization enzyme and involved in desulfurization reaction Enzymes conjugated with desulfurization enzyme of the present invention and involved in desulfurization reaction include the following proteins (a) and (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 are deleted, substituted or added, and oxidizes NADH With a function to reduce FMN

The above-mentioned protein is obtained from Rhodococcus sp.IGT
Conjugated with the desulfurizing enzyme from S8 strain, there is almost no homology with the enzyme DszD, which has the function of oxidizing NADH and reducing FMN, and has the same action as the enzyme, but is clearly identifiable in the following points. Different. In DBT and DBTO ₂ oxidation reactions, specific activity as oxidoreductase (μmol of DBT that oxidizes 1 mg of protein per minute) is several times higher than DszD, and NADPH is used as a coenzyme in addition to NADH it can.

The enzyme conjugated to the desulfurization enzyme of the present invention and involved in the desulfurization reaction can be produced by using the above-mentioned gene encoding the enzyme conjugated to the desulfurization enzyme and involved in the desulfurization reaction. In addition, an enzyme conjugated with the desulfurization enzyme represented by the amino acid sequence of SEQ ID NO: 2 and involved in the desulfurization reaction can be prepared from Paenibacillus polymyxa A-1 according to a conventional method.

(5) Paenibacillus polymyxa A-1 strain and a transformant using it as a host strain Paenibacillus polymyxa A-1 have the mycological properties as described in Example 1, and Has been deposited with the National Institute of Advanced Industrial Science and Technology. Paenibac
The desulfurization enzyme gene to be introduced into the illus polymyxa A-1 strain is not particularly limited as long as it is a gene involved in the desulfurization reaction.For example, dszA, dszB derived from Rhodococcus sp.IGTS8,
dszC gene, a desulfurase gene derived from Burkhoderia sp. strain C1 (Escherichia coli containing this gene has been deposited with the National Institute of Bioscience and Human Technology as FERM P-17270), Pa
Desulfurase genes derived from enibacillus sp. strains A11-1 and A11-2 (these two strains have been deposited with the National Institute of Bioscience and Human Technology, FERM P-15751 and FERM P-15752, respectively). And the like. The method for introducing a desulfurase gene into Paenibacillus polymyxa A-1 strain is not particularly limited, and a general method for introducing a foreign gene into a microorganism can be adopted.

[0029]

The present invention will be described below in more detail with reference to examples. Experiments related to genetic engineering in the examples were mainly
atis et al. (Sambrook, J., Fritsch, E., F. and Mani
atis, T. 1989. Molecular Cloning. A Laboratory Man
ual. 2nd. Cold Spring Harbor Laboratory Press, Col
d Spring Harbor, NY.).

[Example 1] Screening of NADH-FMN oxidoreductase producing bacteria BY medium (1 g of peptone per liter, 3.0 g of sodium chloride)
g, 7.0 g of meat extract, 5.0 g of yeast extract, pH 7.0) In a 300 ml Erlenmeyer flask containing 50 ml, various test strains are subjected to rotary shaking culture at 30 ° C. for 1 or 2 days, and then the cells are centrifuged. The cells were collected and cultivated in 50 mM potassium phosphate buffer (pH 7.
0) After suspending in 5 ml and sonication, it was centrifuged and the supernatant was used as a cell-free extract. On the other hand, DBT containing DBT oxidase DszC
Degrading enzymes are described in the literature (Oshiro, T., Suzuki, K., and Izu
mi, Y., J. Ferment. Bioeng., 83, 233-237 (1997)) to prepare a cell-free extract from Rhodococcus erythropolis strain D-1.

The oxidoreductase activity of the test strain was determined by Rhodococ
cus erythropolis D-1 cell-free extract 50μl, NADH 3m
M, FMN 10 μM, DBT 0.24 mM, potassium phosphate buffer (pH 7.0) 100 mM, total volume 0.5 ml, shake reaction at 35 ° C. for 12 hours, and stop the reaction by adding 50 μl 1M hydrochloric acid , 400 μl of ethyl acetate were added. Centrifuge this,
The supernatant is injected into high performance chromatography and the resulting DBT
The sulfone was quantified. The activity of NADH-FMN oxidoreductase is
The amount of enzyme that produces 1 nmol of DBT sulfone per minute is 1 unit.
t. The oxidoreductase activity of each of the 54 test strains is shown in FIGS. As shown in the figure, Paenibacillus polym
The activity of the yxa A-1 strain was highest when NADH was used as a coenzyme. Therefore, the bacteriological properties of this strain were examined.
The results are shown below.

Bacillus; 0.5-0.7 × 2.5-6.0 μm spores; + ellipsoid + round-swelling the sporangium-catalase; + oxidase;-VP reaction; + pH of VP culture 4.99 Anaerobic growth; + Maximum growth temperature; C. Growth at pH 5.7; + Growth with 2% NaCl; + Acid production; D-glucose, L-arabinose, D-xylose, D-mannitol, D-fructose All + gas production from glucose; + hydrolytic ; Starch, gelatin, casein +, tween
80 Weak +, esculin-Citrate, propionate availability;-Growth on 0.001% lysozyme; Weak + lecithinase;-Tyrosine degradability;-NO2 production from NO3; + Indole;-Phenylalanine deaminase;-Arginine dihydrolase; ―

The A-1 strain was identified in Bergey's Manual
f Systematic Bacteriology, Volume 1-4, Baltimore W
illiams and Wilkins. We have also investigated the cellular fatty acid composition and 16S rDNA of this strain,
Both were in good agreement with those of Paenibacillus polymyxa. Particularly, 16S rDNA showed 98.7% homology.

Example 2 Cultivation, preparation of cell-free extract,
And purification of oxidoreductase Paenibacillus polymyxa A-1 strain in 5 ml of BYG medium (1% peptone, 1% glucose, 0.7% meat extract, 0.5% yeast extract,
Inoculate 18mm test tube containing 0.3% sodium chloride)
A pre-culture solution was prepared by reciprocating shaking culture at 30 ° C for days. Next, 5 ml of the preculture solution was added to a 2 L Sakaguchi flask containing 500 ml of BYG medium, and reciprocal shaking culture was performed at 30 ° C. for 2 days.

1 g of the wet cells was mixed with 2 ml of buffer 1 (50 mM Tris-HCl buffer (pH 8.0) containing 1 mM DTT and 10% glycerol (V / V)).
l, sonication and centrifugation (14,000 xg,
(4 ° C, 20 minutes), and the supernatant was used as a cell-free extract. NA
DH-FMN oxidoreductase activity was 20 mM potassium phosphate buffer, 0.5 mM NADH, 0.02 mM for the A-1 strain enzyme.
Enzyme reaction at 35 ° C using M FMN
The rate of decrease of the absorbance at 40 nm was measured using an ultraviolet spectrophotometer. Enzyme activity 1u was defined as the amount of enzyme that oxidizes 1 μmol of NADH per minute.

Example 3 Partial Amino Acid Sequence of Purified Enzyme The purified enzyme was treated with lysyl endopeptidase, trypsin and CNBr to isolate an internal peptide, and the amino acid sequence of each was determined. They are shown in FIG. 3 together with the N-terminal amino acid sequence. No homology was found between the N-terminus of the FMN-NADH oxidoreductase of Rhodococcus erythropolis IGTS8 and the N-terminus of the enzyme derived from the fungus.

Example 4 Function of the present enzyme First, NADH was immobilized, and the substrate specificity on the electron acceptor side was examined. This enzyme was used for FAD, riboflavin, and lumiflavin having an isoalloxatidine ring in addition to FMN. On the other hand, it had about 80% activity. This enzyme can also use nitrogen-containing aromatic compounds such as nitrofurazone and methyl 4-nitrobenzoate as substrates, and also showed activity on artificial electron acceptors such as DCPIP and cytochrome C.

Next, the activity of NADH and NADPH as electron donors was compared using FMN, FAD, riboflavin, and nitrofurazone. As shown in FIG. 4, in all cases, the activity was higher when NADPH was used than when NADH was used. When FMN was used as the electron acceptor, the difference in activity was 1.5 times. DszD of Rhodococcus erythropolis IGTS8 is FAD and NAD
It is known that PH is not used as a substrate at all (Gray, K.,
Pogrebinsky, O., Mrachko, G., Xi, L., Monticello,
D., and Squires, C., Natute Biotechnol., 14, 1705-
1709 (1996).).

Further, DszC
Was used for the coupling assay. The result is shown in FIG. Oxidoreductase activity was observed when NADPH was used as the electron donor in the same manner as NADH. As an electron acceptor
No activity was observed in FAD and nitrofurazone, and DszC activity was found only when FMN and riboflavin were used. In addition, when FMN was used, the activity against NADH was 1.2 times higher than NADPH, contrary to the oxidoreductase activity.

Example 5 Cloning of Gene Fragment Encoding Desulfurization-Related Enzyme FMN-NAD Purified from Paenibacillus polymyxa A-1
The amino-terminal and internal amino acid sequences of the enzyme having H redox activity were determined in Example 3. Their sequences are as follows: Amino terminal NH2-SSSRKNETLSVISERGAVKSYVKDFKM ----- COOH Internal sequence 1 --YLPTMLISVGKAQVPARPSGRFPLSDVVVKGSF --- Internal sequence 2 --FLVIESEADK--Internal sequence 3 --NAVIYDQAVEAGALPAEI-- Based on the internal sequence 1 and the internal sequence 3 among the sequences, PCR primers as shown below were prepared. Here, I represents inosine. Sense primer 5'- TAY GAY CAR GCI GTI GAR GCI GGI GC -3 'Antisense primer 5'- TGI GCY TTI CCI ACI SWD ATI ARC AT -3'

Using these sense primers and antisense primers, PCR was performed using DNA extracted from Paenibacillus polymyxa A-1 as a template. Paenibacil
Preparation of DNA from luspolymyxa A-1 strain was performed as follows. Paenibac cultured in LB medium containing DBT at 30 ° C for 24 hours
illus polymyxa A-1 strain in 30 ml of fresh 10 ml LB medium
After culturing for 24 hours, the cells were collected. 1 ml of the obtained cells
B1 buffer (50 mM EDTA, 50 mM Tris-HCl, 0.5% Triton
X-100, 0.2 mg / ml RNaseA, pH 8.0). In this suspension, 20 μl of a 100 mg / ml lysozyme solution and 20 mg / ml
Of Proteinase K solution was added and reacted at 37 ° C. for 10 minutes. Add 0.35 ml of B2 buffer (800 mM GuHCl, 2
0% Tween-20, pH 5.5), stir and mix.
The reaction was allowed to proceed for 30 minutes, and the mixture was stirred with a mixer for 5 seconds to prepare a cell reaction solution. QIAGEN G filled with anion exchange resin
ENOMIC-TIP20 / G (manufactured by QIAGEN) column was mixed with 2 ml of QBT buffer (750 mM NaCl, 50 mM MOPS, 15% ethanol, 0.15% Trit).
on X-100, pH 7.0), and the reaction mixture was injected into the column. Wash the column with 3 ml of QC buffer (1.0 M NaCl, 50m
M MOPS, 15% ethanol, pH7.0)
QF buffer (1.25M NaCl, 50mM Tris-HCl, 15% ethanol,
The genomic DNA solution was eluted at pH 8.5). 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. 50 collected DNA
A genomic DNA solution was prepared by dissolving in μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

The prepared Paenibacillus polymyxa A-1 strain DN
The conditions of PCR performed using A as a template are as follows. Reaction solution composition: 50 mM KCl 1.5 mM MgCl ₂ 0.2 mM each dNTP Mixture 0.2 μM sense primer 0.2 μM antisense primer 200 ng template DNA 2.5 U Taq DNA polymerase Annealing temperature: temperature between 49 ° C and 56 ° C at 1 ° C intervals PCR was performed with changes. PCR cycle: 95 ° C for 1 min once 95 ° C for 1 min ↓ 49-56 ° C for 2 min Repeat 30 times 72 ° C for 5 min DNA 72 ° C for 7 min Once DNA amplifier: Robocycler ^™ GRADIENT96 Temperature cycler (STRATAGENE)

As a result of performing PCR under the above conditions, it was confirmed that when the annealing temperature was 49 to 56 ° C., an amplified fragment of about 270 bp was obtained. This 270 bp PCR product is
It was cloned into E. coli strain JM109 using pCR-Script SK (+) vector. The amino acid sequence predicted from this sequence is NADH-F purified from Paenibacillus polymyxa A-1 strain.
It completely matched the internal partial amino acid sequence of MN oxidoreductase. Therefore, this nucleotide sequence was considered to belong to NADH-FMN oxidoreductase.

Example 6 Cloning of Gene Fragment Encoding Desulfurization-Related Enzyme by Reverse PCR Method Next, based on a part of the base sequence of NADH: FMN oxidoreductase identified in [Example 5], The following primers were prepared, and fragments before and after the present sequence were cloned by inverse PCR. <Inverse PCR primer> Sense primer 5'-TAG CTC GCG ATG CAG CGA TT -3 'Antisense primer 5'-GGT ACG CGC CAT TGA TTT GT -3'

The genomic DNA of Paenibacillus sp.
mHI, EcoRI, HindIII, PstI, SalI, XbaI, NruI, Dra
Inverse PCR was performed on the fragments digested with I, Ssp, and BalI.As a result, HindIII, SalI, DraI, and SspI fragments were amplified at about 1.4 kb, 1.4 kb, 1.3 kb, and 2.9 kb, respectively. . Each PCR fragment was cloned into the pCR-BluntII-TOPO vector, and the sequence was determined. The determined nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence deduced therefrom is shown in SEQ ID NO: 2. When this amino acid sequence was compared with the amino acid sequence determined in [Example 3] (see FIG. 3), they were all identical. Therefore, this gene sequence is P.
It was confirmed that the gene was a polymyxa NADH-FMN oxidoreductase gene.

A homology search was performed on the obtained sequence. As a result, NADH Dehyd of Thermus thermophilus HB8 was
Major NAD (P) H-Flavin of drogenase and Vibrio fischeri
High homology with oxidoreductase (FraseI).
Therefore, it was suggested that the gene contained in the PCR amplified fragment obtained in this experiment encodes a Reductase-like polypeptide. In addition, since the amino acid sequence was identical with the amino acid sequence determined in [Example 3], it was confirmed that the gene was NADH-FMN oxidoreductase.

[0047]

Industrial Applicability The present invention provides a novel oxidoreductase conjugated to a desulfurase and a gene encoding the same. By utilizing these enzymes and genes, sulfur in fossil fuels can be released efficiently.

[0048]

[Sequence List] SEQUENCE LISTING <110> PETEROLEUM ENERGY CENTER <120> SANKAKANGEN KOUSO IDENSHI <130> P99-0092 <160> 2 <170> PatentIn Ver. 2.0 <210> 1 <211> 645 <212> DNA <213> Paenibacillus polymyxa <220> <221> CDS <222> (1) .. (624) <400> 1 atg tcc agc tct cgt aaa aat gaa acc ttg tcc gta att agt gaa cgg 48 Met Ser Ser Ser Arg Lys Asn Glu Thr Leu Ser Val Ile Ser Glu Arg 1 5 10 15 ggg gcg gta aaa agc tat gta aag gat ttt aaa atg cca gag gaa gat 96 Gly Ala Val Lys Ser Tyr Val Lys Asp Phe Lys Met Pro Glu Glu Asp 20 25 30 ctg gaa gca atc ttg act gcc gcg gta gaa gct cct tcc gca tgg aac 144 Leu Glu Ala Ile Leu Thr Ala Ala Val Glu Ala Pro Ser Ala Trp Asn 35 40 45 ctt caa cat tgg aaa ttc ctc gtt atc gaa agc gaa gca gac aaa caa Leu Gln His Trp Lys Phe Leu Val Ile Glu Ser Glu Ala Asp Lys Gln 50 55 60 aaa ttg ttg cct atc gcc tac aac caa ggt caa gta gca gag agc tct 240 Lys Leu Leu Pro Ile Ala Tyr Asn Gln Gly Gln Val Ala Glu Ser Ser 65 70 75 80 gtt acg att gcc gta ctg ggt gat ctg gaa gcg aac cgt aat gct gtc 288 Val Thr Ile Ala Val Leu Gly Asp Leu Glu Ala Asn Arg Asn Ala Val 85 90 95 att tat gat caa gcg gta gaa gca gga gca ttg cca gcg gaa atc cgt 336 Ile Tyr Asp Gln Ala Val Glu Ala Gly Leu Pro Ala Glu Ile Arg 100 105 110 gag gca ttg gta gga tca atc aat ggc tcg tac caa aac ccg cag gta 384 Glu Ala Leu Val Gly Ser Ile Asn Gly Ser Tyr Gln Asn Pro Gln Val 115 120 125 gct cgc gat gca gcg att ctg aac tct gct ctt gca gca caa aac att 432 Ala Arg Asp Ala Ala Ile Leu Asn Ser Ala Leu Ala Ala Gln Asn Ile 130 135 140 atg ctg gct gct aaa tct ttg ggc tac gat act tgt gca atg ggc gga 480 M Ala Ala Lys Ser Leu Gly Tyr Asp Thr Cys Ala Met Gly Gly 145 150 155 160 ttt att cca gca caa ttg atc gag caa ttc aat att ccg gca cgt tac 528 Phe Ile Pro Ala Gln Leu Ile Glu Gln Phe Asn Ile Pro Ala Ar Tyr 165 170 175 ctg ccg acc atg ctt att agt gta ggt aaa gca caa gta cca gca cgt 576 Leu Pro Thr Met Leu Ile Ser Val Gly Lys Ala Gln Val Pro Ala Arg 180 185 190 cca tca gga cgt ttc cca ctg tct gac gtt gtt gta aaa ggc agc ttc 624 Pro Ser Gly Arg Phe Pro Leu Ser Asp Val Val Lys Gly Ser Phe 195 200 205 taagacgaat agtgaatata a 645 <210> 2 <211> 208 <212> PRT <213> Paenibacillus polymyxa <400> 2 Met Ser Ser Ser Arg Lys Asn Glu Thr Leu Ser Val Ile Ser Glu Arg 1 5 10 15 Gly Ala Val Lys Ser Tyr Val Lys Asp Phe Lys Met Pro Glu Glu Asp 20 25 30 Leu Glu Ala Ile Leu Thr Ala Ala Val Glu Ala Pro Ser Ala Trp Asn 35 40 45 Leu Gln His Trp Lys Phe Leu Val Ile Glu Ser Glu Ala Asp Lys Gln 50 55 60 Lys Leu Leu Pro Ile Ala Tyr Asn Gln Gly Gln Val Ala Glu Ser Ser 65 70 75 80 Val Thr Ile Ala Val Leu Gly Asp Leu Glu Ala Asn Arg Asn Ala Val 85 90 95 Ile Tyr Asp Gln Ala Val Glu Ala Gly Ala Leu Pro Ala Glu Ile Arg 100 105 110 Glu Ala Leu Val Gly Ser Ile Asn Gly Ser Tyr Gln Asn Pro Gln Val 115 120 125 Ala Arg Asp Ala Ala Ile Leu Asn Ser Ala Leu Ala Ala Gln Asn Ile 130 135 140 Met Leu Ala Ala Lys Ser Leu Gly Tyr Asp Thr Cys Ala Met Gly Gly 145 150 155 160 Phe Ile Pro Ala Gln Leu Ile Glu Gln Phe Asn Ile Pro Ala Arg Tyr 165 170 175 Leu Pro Thr Met Leu Ile Ser Val Gly Lys Ala Gln Val Pro Ala Arg 180 185 190 Pro Ser Gly Arg Phe Pro Leu Ser Asp Val Val Val Lys Gly Ser Phe 195 200 205

[Brief description of the drawings]

FIG. 1 is a diagram showing the activity of oxidoreductase in various strains.

FIG. 2 shows oxidoreductase activities in various strains.

FIG. 3 is a view showing a partial amino acid sequence of an oxidoreductase derived from strain A-1.

FIG. 4 is a diagram showing the reactivity of the A-1 strain-derived oxidoreductases NADH and NADPH.

FIG. 5 is a diagram showing the reactivity of A-1 strain-derived oxidoreductases NADH and NADPH in a coupling assay with DszC.

Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) (C12N 15/09 ZNA C12R 1:01) (C12N 1/20 C12R 1:01) (C12N 1/21 C12R 1:01 ) (C12N 9/02 C12R 1:01) (72) Inventor Kouyoshi 2-9-23-302 Oharano Nishisakayacho, Nishikyo-ku, Kyoto-shi, Kyoto F-term (reference) 4B024 AA17 BA08 CA04 DA05 EA04 GA11 HA01 4B050 CC01 DD02 LL05 4B065 AA01X AA01Y AB01 BA02 CA28 CA56

Claims (6)

[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) a protein represented by 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; A protein that has the function of oxidizing and reducing FMN 2. A vector comprising the gene according to claim 1. 3. A transformant containing the vector according to claim 2. 4. A protein represented by the following (a) or (b): (a) a protein represented by the amino acid sequence of SEQ ID NO: 2; (b) a protein represented by 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; A protein that has the function of oxidizing and reducing FMN 5. A Paenibacillus polymixa strain A-1. 6. A transformant obtained by introducing a desulfurase gene into Paenibacillus polymixa strain A-1.