JP2005204559A - Gene encoding catalase for hydrogen peroxide-tolerant microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene encoding a highly active catalase, and to provide a method for producing the highly active catalase using the gene. <P>SOLUTION: The gene for the highly active catalase, isolated from a new strain Exiguobacterium oxidotolericum and having a specific base sequence, is provided. The method for producing the highly active catalase using the gene is also provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a catalase gene derived from the bacterium Exiguobacterium oxidotolericum (FERM P-18203), and a catalase that is an expression product of this gene (hereinafter sometimes referred to as “EO catalase”) It is about. More specifically, the present invention relates to a novel catalase useful for purification of industrial wastewater containing hydrogen peroxide, a gene encoding this catalase, and a genetic engineering material for expression of this gene.

  Hydrogen peroxide is normally generated during the process of oxygen metabolism in aerobic organisms, but since it acts on cells in a toxic manner (for example, a damaging effect on cell membranes, proteins, DNA, etc.), most animal and plant cells do not have this. An enzyme (catalase) for decomposing hydrogen peroxide into water and oxygen is widely distributed.

Hydrogen peroxide is also a cause of denaturation due to, for example, lipid oxidation of foods and various compounds, or a toxic pollutant in factory wastewater. For this reason, catalase for decomposing hydrogen peroxide is regarded as promising as an antioxidant and a purification agent for contaminated water / soil, etc., and a cattle liver catalase preparation is commercially available, as well as Miorococcus ( Miorococcus). Catalase derived from bacteria such as luteus (lysodeikticus) ) is also known. (Non-Patent Document 1)

In addition, a new strain, Exiguobacterium oxidotolericum, which is resistant to hydrogen peroxide, has been isolated from industrial wastewater containing high concentrations of hydrogen peroxide. It has been found to have much stronger catalase activity than that of typical microorganisms. (Patent Document 1)

On the other hand, the present inventors produce a catalase activity in which a purified enzyme derived from the same enzyme gene of Exiguobacterium oxidotolericum has a much stronger activity than that of E. coli and Bacillus subtilis catalase. I found out.

JP 2002-253215 A Murshudov GN, Melik-Adamyan WR, Grebenko AI, Barynin VV, Vagin AA, Vainshtein BK, Dauter, Z, Wilson, KS (1992) Three-dimensional structure of catalase from Micrococcuslysodeikticus at 1.5 A resolution.FEBS Lett 312, 127-131 . Claiborne, A., Malinowski, DP, Fridovich, I. (1979) Purification and characterization of hydroperoxidase II of Escherichia coli B. J Biol Chem 254, 11664-11668. Loewen PC, Switala, J. (1987) Purification and characterization of catalase-1 from Bacillus subtilis.Biochem Cell Biol 65, 939-947.

  An object of the present invention is to provide a method for cloning a catalase gene having a significantly stronger activity than conventional catalases of E. coli and Bacillus subtilis, providing the gene, and a method for producing a highly active catalase using the gene. Is to provide.

As a result of intensive studies to achieve the above problems, the present inventors have completed the present invention by using a new strain Exiguobacterium oxidotolericum isolated by the inventors. It came. This strain exhibits an activity value of about 1,000,000 U / mg protein in the purified enzyme.

That is, the gist of the present invention is as follows.
(1) A catalase gene encoding the following protein (a) or (b).
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a) and having catalase activity

(2) A catalase gene comprising the following DNA (a) or (b):
(a) DNA consisting of the base sequence of SEQ ID NO: 1.
(b) A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) and encodes a protein having catalase activity.

(3) A recombinant vector carrying the gene according to (1) or (2).
(4) A transformant transformed with the recombinant vector according to (3).
(5) The following catalase (a) or (b).
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a) and having catalase activity

(6) A method for producing the catalase, comprising culturing the transformant according to (4) in a medium to produce the catalase according to (5).

  According to the present invention, a gene for a highly active catalase was provided, and it was possible to produce a highly active catalase efficiently and in high yield using the gene.

The catalase gene derived from Exiguobacterium oxidotolericum (FERM P-18203) of the present invention (hereinafter sometimes referred to as “ ektA ”) has a base sequence of 1,476 bp shown in SEQ ID NO: 1. And has a single open reading frame (ORF) encoding ektA consisting of 491 amino acid residues. The EO catalase of the present invention is an expression product of the gene ektA and having the amino acid sequence of SEQ ID NO: 2. This strain exhibits an activity value of about 1,000,000 U / mg protein in the purified enzyme. Incidentally, the catalases of Escherichia coli and Bacillus subtilis are 8,900 U / mg protein and 170,000 U / mg protein, respectively (Non-patent document 2 and Non-patent document 3).

The gene ektA is a gene isolated and identified from Exiguobacterium oxidotolericum by the method of Examples described later. Therefore, this gene ektA can be obtained by the same method as in Examples described later.

Alternatively, this gene ektA can be obtained by screening a genomic library of Exiguobacterium oxidotolericum using a probe prepared based on the DNA sequence (SEQ ID NO: 1) provided by this application. It can also be acquired. However, as described later, in the acquisition of the catalase gene of Exiguobacterium oxidotolericum, it could not be obtained by screening a genomic library or the like.

The gene ektA of this invention is obtained by a method of preparing a peptide by chemical synthesis based on the amino acid sequence provided by this application, or a method of producing by recombinant DNA technology using the gene ektA provided by this application. Can do. For example, in the case of obtaining a gene ektA by recombinant DNA techniques, the RNA was prepared by in vitro transcription from a vector having a gene ektA of the present invention, which by performing in vitro translation as a template, it is possible to obtain a gene ektA . In addition, the gene ektA can be recombined into an appropriate expression vector by a known method, and if this recombinant vector is transformed into E. coli, Bacillus subtilis, yeast, animal or plant cells, etc., the gene ektA can be expressed in large quantities in these transformants. Can do. Furthermore, EO catalase can be prepared in large quantities as an extracellular enzyme by coexisting an ektA gene with a vector capable of secreting a protein outside the cell.

When the EO catalase of the present invention is produced by in vitro translation, the gene ektA of the present invention is recombined into a vector having an RNA polymerase promoter, such as a rabbit reticulocyte lysate or wheat germ extract containing an RNA polymerase corresponding to the promoter. What is necessary is just to add to an in vitro translation system. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Examples of vectors containing these RNA polymerase promoters include pT7Blue, pBluescript II, pZero II, and pET-23 (+).

Further, in order to express the gene ektA of the present invention a microorganism such as E. coli, replicable origin in microorganisms, a promoter, a ribosome binding site, DNA cloning site, the expression vector having a terminator, the present invention gene ektA And an expression vector is prepared by recombination, and a host cell is transformed with this expression vector, and then the obtained transformant is cultured. At this time, if a start codon and a stop codon are added before and after an arbitrary translation region, a protein fragment containing the arbitrary region can be obtained. Alternatively, it can be expressed as a fusion protein with another protein. Only the target EO catalase can be obtained by cleaving this fusion protein with an appropriate protease. Examples of E. coli expression vectors include pUC systems such as pBluescript II, pET expression system, and pGEX expression system.

When the gene ektA of the present invention is expressed in a eukaryotic cell, the gene ektA of the present invention is recombined into an eukaryotic expression vector having a promoter, a splicing region, a poly (A) addition site, etc. To introduce. Examples of expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYES2. As eukaryotic cells, mammalian cultured cells such as monkey kidney cells COS7, Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, Xenopus egg cells, etc. are generally used, but are not limited thereto. . In order to introduce an expression vector into a eukaryotic cell, a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method can be used.

  After the expression of catalase in prokaryotic cells or eukaryotic cells by the above method, the target protein is simply purified from the culture by combining known separation operations. For example, sonication, salting out and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, etc. It is.

Exiguobacterium oxidotolericum has a variety of mutants, all of which produce highly active catalase (the specific activity of the purified enzyme is over 700,000 U / mg protein). Therefore, even if there is a gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in SEQ ID NO: 2, the protein consisting of the amino acid sequence consists of the amino acid sequence of SEQ ID NO: 2. Those genes are included in the catalase gene of the present invention as long as they have high catalase activity similar to that of protein. Similarly, a catalase in which a plurality of amino acid additions, deletions and / or substitutions with other amino acids caused by the mutation of these bases is also possible as long as it has the activity of a catalase having the amino acid sequence of SEQ ID NO: 2. It is included in this invention. The mutation is obtained by, for example, a site-specific mutation method.

Further, as long as it hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of SEQ ID NO: 1 and has the activity of catalase having the amino acid sequence of SEQ ID NO: 2, it is included in this invention. The stringent condition refers to a condition in which a specific hybrid is formed and a non-specific hybrid is not formed, that is, high homology to the gene ektA of the present invention (homology is 90% or more, preferably Is 95% or more). More specifically, such conditions include hybridization at 42 to 68 ° C. in the presence of 0.5 to 1 M NaCl, and then using room temperature to 0.1 to 2 times SSC (saline sodium citrate) solution. This can be achieved by washing the filter at 68 ° C.

  Hereinafter, the present invention will be described in more detail and specifically by examples. However, the technical scope of the present invention is not limited by these examples.

(Example 1) Cloning of catalase gene ektA (1) Preparation of probe EO catalase collected from a culture of Exiguobacterium oxidotolericum was prepared using a Q-Sepharose Fast Flow anion exchange column (Pharmacia) And Sephacryl S-300 gel fractionation column (Pharmacia). Further, this purified catalase (1.6 μg) was subjected to polyacrylamide electrophoresis, blotted on a polyvinylidene fluoride membrane (Millipore), and stained with Coomassie brilliant blue. A catalase band was cut out from the membrane, and the N-terminal amino acid sequence of EO catalase was determined by an automatic protein sequencer (Applied Biosystem 491: manufactured by Perkin-Elmer).

Synthesize oligonucleotides from the N-terminal amino acid sequence determined as described above, compare the determined N-terminal amino acid sequence with the amino acid sequence of catalase from other bacteria, and synthesize oligonucleotides from highly conserved amino acid sequences. did. Then, PCR was carried out using these oligonucleotides as primers and genomic DNA of Exiguobacterium oxidotolericum as a template.

The composition of the PCR reaction solution (total volume 100 μl) is as follows.
10x PCR buffer (Takara): 10 μl
10 mM deoxynucleoside triphosphate mixture (Takara): 4 μl
Genomic DNA: 0.6 μg
Oligonucleotide primer: 100 pmol each
TaqDNA polymerase (Roshe): 2.5 U
Distilled water

PCR conditions were as follows.
(94 ° C: 2 minutes, 50 ° C: 30 seconds, 72 ° C: 2 minutes) x 1 cycle (94 ° C: 30 seconds, 50 ° C: 30 seconds, 72 ° C: 2 minutes) x 25 cycles (94 ° C: 30 seconds, 50 ℃: 30 seconds, 72 ℃: 6 minutes) x 1 cycle

By PCR as described above, the base sequence of the PCR product (731 bp DNA fragment) using primer P-N1 (SEQ ID NO: 3) and primer P-S1 (SEQ ID NO: 4) is highly homologous to Listeriaseeligeri and Deinococcus radiodurans catalase It was confirmed to show sex. For this reason, oligonucleotide P-N2 (SEQ ID NO: 5) and P-S2 (SEQ ID NO: 6) were synthesized from the determined sequence, and the PCR product (630 bp DNA fragment) using primer P-N2 and primer P-S2 : PP-630) was used as a catalase gene cloning probe.

(2) Library construction and screening 1
Chromosomal DNA of Exiguobacterium oxidotolericum was cleaved by treating with restriction enzyme Sal I at 37 ° C. for 1 hour, and subjected to agarose electrophoresis. The 3kbDNA fragment probe PP-630 hybridizes excised from the gel, using a ligation kit (Takara Co.), and ligated into the Sal I site of the plasmid pT7Blue, E. coli E. Coli DH5 [alpha was transformed with this recombinant plasmid A library was built.

  Subsequently, colony hybridization was performed using PP-630 as a probe, but no colonies showing a positive reaction were obtained.

(3) Library construction and screening 2
37 ° C. The chromosomal DNA with restriction enzyme Sau 3A I for IG di O Corynebacterium oxide Tre Re cam (Exiguobacterium oxidotolericum), cut and treated 1 hour, using a ligation kit (Takara Co.), as well as restriction enzyme BamH I Limbda DASH II vector (Stratagene) was ligated. Next, this recombinant lambda phage DNA was packaged using Gigapack III Gold (Stratagene) and infected with E. coli XL1-Blue MRA (P2) to construct a library.

Plaque hybridization was performed using PP-630 as a probe, and plaques Pla3 and Pla4 showing a positive reaction to the probe were obtained. Lambda phage DNA was recovered from this plaque, cut with a restriction enzyme Sal I at 37 ° C. for 24 hours, and subjected to agarose electrophoresis. Next, the 1 kb DNA fragment hybridized with the probe PP-630 was excised from the gel and ligated to the Sal I site of the plasmid PUC119 using a ligation kit (Takara) to transform E. coli DH5α. The transformed Escherichia coli was cultured, and plasmids P-Pla3 and P-Pla4 were extracted from the cells. The partial base sequences of the insert DNA fragments of P-Pla3 and P-Pla4 were determined using a DNA sequencer (model 310: manufactured by Perkin-Elmer). As a result, it was found that the insert sequences of P-Pla3 and P-Pla4 were cleaved at the Sau 3A I site at the same position of the catalase sequence and linked to other Sau 3A I fragments. It was judged not suitable for the cloning of the catalase gene of bacterial oxidorelicum ( Exiguobacterium oxidotolericum ).

(4) Inverse PCR
Four types of oligonucleotides P-N3 (SEQ ID NO: 7), P-N4 (SEQ ID NO: 8), P-S3 (SEQ ID NO: 9) and P-S4 (SEQ ID NO: 10) are synthesized from the internal sequence of probe PP-630. did. Oligonucleotides P-N3 and P-S3 were made to have inner base sequences with respect to P-N4 and P-S4, respectively. Then, genomic DNA of Exiguobacterium oxidotolericum was digested with the restriction enzyme Xba I at 37 ° C. for 8 hours, and self-ligation was performed using a ligation kit (manufactured by Takara). Next, PCR was performed using oligonucleotides P-N3 and P-R3 as primers and the self-ligated Xba I digestion product as a template.

The composition of the PCR reaction solution (total volume 50 μl) is as follows.
Mg ²⁺ free 10xPCR buffer (Takara): 5 μl
25 mM MgCl ₂ : 3 μl
2.5 mM deoxynucleoside triphosphate mixture (Takara): 8 μl
Ligation solution: 1 μl
Oligonucleotide primer: 50 pmol each
LA TaqDNA polymerase (Takara): 2.5 U
Distilled water

PCR conditions were as follows.
(96 ° C: 2 minutes, 55 ° C: 30 seconds, 68 ° C: 10 minutes) x 1 cycle (96 ° C: 30 seconds, 55 ° C: 30 seconds, 68 ° C: 10 minutes) x 25 cycles

  By PCR as described above, a PCR product (4.5 kbp DNA fragment: PP-4500) when using primers P-N3 and P-S3 was obtained. Further, PCR was performed in the same manner using oligonucleotides P-N4 and P-S4 as primers and PCR product PP-4500 as a template. As a result, a PCR product (4.5 kbp DNA fragment: PP-4500-) was obtained, and it was confirmed that the PCR product PP-4500 was definitely derived from the base sequence of the probe PP-630. Therefore, the catalase part (SEQ ID NO: 1) of the base sequence of this PCR product PP-4500- was determined using a DNA sequencer (model 310: manufactured by Perkin-Elmer). As a result, it was confirmed that this DNA fragment PP-4500- contains the entire 1476 bp sequence (SEQ ID NO: 1) including the ORF of 1473 bases encoding the 491 amino acid sequence (SEQ ID NO: 2).

Furthermore, for all the 491 amino acid sequence of SEQ ID NO: 2, Listeriaseeligeri kat catalase and E. coli E. Coli HPII type catalase KatE results were compared with the corresponding amino acid, as shown in FIGS. 1A and 1B, a high homology to the three-way Since the sex was recognized, a DNA fragment having the base sequence of SEQ ID NO: 1 was identified as a catalase gene and named gene ektA .

(Example 2) Expression of gene ektA in Escherichia coli From the base sequences before and after the catalase gene ektA (SEQ ID NO: 11) whose base sequence was determined in Example 1, oligonucleotides P-N5 (SEQ ID NO: 12) and P-S5 (SEQ ID NO: 13) was synthesized. Then, PCR was carried out using these oligonucleotides as primers and genomic DNA of Exiguobacterium oxidotolericum as a template.

The composition of the PCR reaction solution (total volume 50 μl) is as follows.
10x PCR buffer (Takara): 5 μl
2.5 mM deoxynucleoside triphosphate mixture (Takara): 4 μl
Genomic DNA: 0.1 μg
Oligonucleotide primer: 10 pmol each
PfuTurbo DNA polymerase (Stratagene): 2.5 U
Distilled water

PCR conditions were as follows.
(95 ° C: 2 minutes 30 seconds, 55 ° C: 30 seconds, 72 ° C: 3 minutes) x 1 cycle (95 ° C: 30 seconds, 55 ° C: 30 seconds, 72 ° C: 3 minutes) x 30 cycles (94 ° C: 30 seconds 55 ° C: 30 seconds, 72 ° C: 10 minutes) x 1 cycle

By PCR as described above, a PCR product (1546 bp DNA fragment: PP-kat) was obtained using primers P-N5 and P-S5. This PCR product was ligated to pZErO-2 vector (manufactured by Invitrogen) treated with restriction enzyme EcoR V using a ligation kit (manufactured by Takara) to transform E. coli DH5α. The transformed Escherichia coli was cultured in an LB medium containing kanamycin sulfate at a concentration of 50 mg / L for screening, and a plasmid was extracted from the cells containing the insert. Further, this plasmid was treated with restriction enzymes Hind III and Xho I to obtain a DNA fragment (1628 bp DNA fragment: pp-kat +) containing PP-kat. This DNA fragment PP-kat + using a ligation kit (Takara Co.), and ligated to the restriction enzyme Hind III and pET-23 were treated with Xho I (+) vector (Novagen) E. coli E. Coli BL21 ( DE3) was transformed.

  Subsequently, the cells were inoculated into an LB liquid medium containing ampicillin at a concentration of 50 mg / l and cultured at 37 ° C. for 12 hours. Thereafter, 1/100 volume of the bacterial cell culture solution was newly inoculated into the same liquid medium, cultured at 37 ° C. for 2 hours, IPTG was added to a concentration of 0.5 mM, and cultured for 8 hours. As a result, the obtained microbial cells were collected by centrifugation, and the microbial cells were crushed with an ultrasonic crusher to obtain an extract containing all the microbial cells.

  The obtained extract was subjected to SDS-polyacrylamide gel electrophoresis according to a conventional method. As a result, a strong band was observed at the same position as native catalase. Furthermore, when the catalase activity of the extract was measured, it showed an extremely high activity of 16000 U / mg. confirmed.

(Example 3) Highly active catalase gene by modification of the acquired gene, acquisition of its transformed Escherichia coli and production of highly active catalase Randomized plasmid DNA obtained by recombining the gene ektA obtained in Example 1 with the pZERO II vector E. coli XL1-Red strain, which can introduce mutations, was introduced by the heat shock method to obtain a plasmid having ektA with random mutations. The plasmid retaining ektA before the mutation and the plasmid retaining ektA after the mutation were introduced into E. coli XL1-Blue, respectively, at a concentration of 50 mg / l kanamycin, 50 μM The cells were cultured in an LB liquid medium supplemented with IPTG. Bacteria were collected from each culture, cell-free extracts were prepared from each, and the activity per protein amount (mg) of the catalase was measured. As a result, the activity per unit amount of catalase of the strain holding the plasmid with ektA before introducing the mutation was 50.8 U / mg-protein, whereas it had ektA after introducing the mutation. The activity per unit amount of catalase of the strain carrying the plasmid was 241.9 U / mg-protein, which was about 5 times higher. The catalase activity of these crude enzyme solutions is considerably lower than that in Example 2, but this difference is due to the difference in the expression level due to the difference in the promoter activity of the expression vector.

For amino acid sequences of SEQ ID NO: 2, shows homology with Listeriaseeligeri kat catalase and E. coli E. Coli HPII type catalase KatE amino acid sequence. For amino acid sequences of SEQ ID NO: 2, shows homology with Listeriaseeligeri kat catalase and E. coli E. Coli HPII type catalase KatE amino acid sequence.

Claims (6)

A catalase gene encoding the following protein (a) or (b).
(a) A protein consisting of the amino acid sequence of SEQ ID NO: 2.
(b) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a) and having catalase activity. A catalase gene comprising the following DNA (a) or (b):
(a) DNA consisting of the base sequence of SEQ ID NO: 1.
(b) A DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) and encodes a protein having catalase activity.   A recombinant vector carrying the gene according to claim 1 or 2.   A transformant transformed with the recombinant vector according to claim 3. The following catalase (a) or (b).
(a) a protein comprising the amino acid sequence of SEQ ID NO: 2
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence (a) and having catalase activity   A method for producing the catalase comprising culturing the transformant according to claim 4 in a medium to produce the catalase according to claim 5.

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