KR100811545B1 - [-]- Degenerate Primers for Searching [NiFe]-Hydrogenase using Polymerase Chain Reaction - Google Patents

Abstract

The present invention provides a [nickel-iron] -hydrogenase gene amplification degenerate primer consisting of a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer consisting of a nucleotide sequence shown in SEQ ID NO: 2, and (1) hydrogen production. Separating genomic DNA from the strain; (2) performing the polymerase chain reaction using the genomic DNA as a template and the degenerate primer, and (3) confirming the amplification of the [nick-iron] -hydrogenase gene. -Iron] -methods of searching for hydrogenase genes.

 [Nickel-iron] -hydrogenase, degenerate primers, polymerase chain reaction

Description

Degenerate Primers for Searching [NiFe] -Hydrogenase using Polymerase Chain Reaction}

Figure 1 is an illustration of the alignment of secretory signal sequences located at the amino terminus (N-teminus) of a small subunit of 30 [nickel-iron] -hydrogenases.

2 Escherichia It is agarose gel electrophoresis picture obtained by using the gegen DNA of coli as a template and the degenerate polymerase chain reaction method using the degenerate primer according to the present invention.

3 Hydrogenovibrio Agarose gel electrophoresis picture obtained by degenerate polymerase chain reaction using gelatin DNA of marinus as a template and degenerate primer according to the present invention.

4 is Ralstonia It is agarose gel electrophoresis picture obtained by degenerate polymerase chain reaction using genomic DNA of eutropha as a template and degenerate primer according to the present invention.

The present invention relates to a degenerate primer for polymerase chain reaction to search for a [nickel-iron] -hydrogenase gene, and more specifically, a degenerate primer for amplifying a [nickel-iron] -hydrogenase gene and using the same. The present invention relates to a method for searching for a [nickel-iron] -hydrogenase gene from a hydrogen producing strain.

At present, we are focusing on hydrogen as an energy source for overcoming environmental problems and energy crisis caused by fossil fuels. Since hydrogen is produced from the constituents of water and organic substances, there is little fear of depletion of raw materials, clean energy generated only per unit weight, and there is a great possibility of practical use of technologies such as fuel cells. Photosynthetic microbes that use sunlight as energy are active environmental treatment technologies that not only generate hydrogen but also reduce carbon dioxide in the air. Research into hydrogen evolution by microorganisms has already begun since the late 1800s, and basic research is being carried out using algae and bacteria. This biological hydrogen production has many advantages. It is environmentally friendly, can be recycled and used for food waste and sewage sludge treatment.

Common algae receive light to fix carbon dioxide in the air by photosynthesis, and simultaneously decompose water to generate oxygen and hydrogen at the same time. The reason algae can generate hydrogen is because they have hydrogenases that help them produce hydrogen. Hydrogenases play a major role in biological hydrogen production. Hydrogenase catalyzes the reversible oxidation of hydrogen molecules (H2 ↔ 2H + + 2e-) and plays an important role in the energy metabolism of microorganisms. However, there are many challenges that must be solved with the current technology for commercialization. Among them, anoxic conditions are essential for hydrogen production because hydrogenases involved in the reaction are very sensitive to oxygen generated at the same time and thus inhibit hydrogen generation, which becomes an inefficient process. Therefore, in order to increase the efficiency of hydrogen production, many researchers are interested in hydrogenase and carry out improvement studies. For this purpose, it is necessary to develop a technology for detecting and searching for hydrogenases.

Hydrogenases are [iron] -hydrogenases, [nickel-iron] -hydrogenases, and metal ions, depending on the composition of the hydrogen-activating site. free hydrogenase). Among these, [nickel-iron] -hydrogenase has a merit of having high affinity for a substrate even though its activity is lower than that of [iron] -hydrogenase. Nevertheless, [nickel-iron] -hydrogenase is little known compared to [iron] -hydrogenase.

The inventors recognize the utility of hydrogen energy and obtain the gene of [nick-iron] -hydrogenase from various hydrogen producing strains, the carboxyl terminus of the gene large subunit of the known [nick-iron] -hydrogenase A new [nickel-iron] -hydrogenase amplification primer was prepared by aligning the conserved region sequence and secretion sequence of the enzyme. The genomic DNA of the hydrogen-producing strain was used as a template, and the polymerase chain reaction was carried out using the primer. , [Nickel-Iron] -hydrogenase can be explored to complete the present invention.

In one aspect, the present invention provides a degenerate primer for amplifying a [Nickel-iron] -hydrogenase gene comprising a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer consisting of a nucleotide sequence shown in SEQ ID NO: 2 .

In another aspect, the method comprises the steps of: (1) separating genomic DNA from a hydrogen producing strain; (2) performing the polymerase chain reaction using the genomic DNA as a template and the degenerate primer, and (3) confirming the amplification of the [nick-iron] -hydrogenase gene. -Iron]-provides a method for searching for hydrogenase genes.

"[Nickel-iron] -hydrogenase" has the advantage of having a high affinity for the substrate even if the activity is lower than the [iron] -hydrogenase, and has a three-dimensional structure containing nickel as well as iron. [Nickel-iron] -hydrogenases consist of two subunits: a large subunit and a small subunit. In general, large subunits mainly contain conserved sequences of about 1800bp in size, and small subunits mainly contain variable sequences of about 1100bp in size. Among these small subunits, there is a secretion signal sequence that signals the position between the outer membrane and the inner membrane, ie periplasm, and the small subunit and the large subunit bind before secretion occurs. The bound [nickel-iron] -hydrogenase is transported to the membrane via the twin-arginine translocation pathway.

The present inventors align the conserved segment sequence and secretory sequence of the carboxyl terminus of a large subunit of the known [nickel-iron] -hydrogenase genes, and thus a novel [nick-iron] -hydrogenase amplification primers. In this case, the amplification generally means that the [nickel-iron] -hydrogenase gene is mass-produced by the polymerase chain reaction method.

Polymerase chain reaction (PCR) is one of gene detection methods that amplify specific deoxyribonucleic acid (DNA) regions by enzymes in vitro. Prior to the polymerase chain reaction, deoxyribonucleic acid detection required cloning of genes using recombinant gene technology of human genome deoxyribonucleic acid consisting of 3 billion base pairs. For this reason, much knowledge of E. coli or phage was required, and a lot of time and effort was hindering the clinical dissemination of detection. Polymerase chain reaction transformed this complex step into a few hours of enzymatic reactions, and became the center of detection for highly applicable deoxyribonucleic acid, and in all life sciences requiring a molecular biological approach. Has become an essential skill.

Meanwhile, “degeneracy” refers to a phenomenon in which one type of amino acid corresponds to a plurality of codons. Degeneracy may not determine the nucleotide sequence of a gene encoding a specific amino acid sequence, but it is possible to synthesize oligonucleotides containing all possible nucleotide sequences. For example, if you consider a tripeptide of Pro-Cys-Arg, codons corresponding to this amino acid sequence have four types of proline, two types of cysteine, and six types of arginine. 4x2x6 = 48 types. In fact, when synthesizing a dinerate oligonucleotide by a combination of 48 types of codons, the amidite is mixed at a position corresponding to a plurality of nucleotides. Therefore, the combination of degenerate oligonucleotides may be larger than the combination of codons (64 types), but one of these oligonucleotide mixtures is completely identical to the gene encoding the tripeptide. If the number of combinations is not so large and a degenerate oligonucleotide of suitable length can be designed, this oligonucleotide will act as a relatively specific primer. When polymerase chain reaction is carried out using such a degenerate oligonucleotide as a primer, DNA fragments of genes encoding the protein may be obtained based on amino acid sequence information. The primer designed for this purpose is called degenerate primer and the polymerase chain reaction is called degenerate polymerase chain reaction. This method is a technique for cloning the gene from a known amino acid sequence, but is now widely used when searching for a group of genes based on primary structure (base sequence) information of abundantly accumulated nucleic acids.

As described above, the present invention aligns the amino acid sequence of the conserved region sequence of the carboxyl terminus of the large subunit of the gene of the [Nickel-iron] -hydrogenase and the amino acid sequence of the secretory sequence. Based on the base sequences encoding them were prepared. The present invention provides a primer for amplifying a [Nickel-iron] -hydrogenase gene comprising a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer consisting of a nucleotide sequence shown in SEQ ID NO: 2.

Forward Primer:

5'- ATG CGX CGX AGY TTY CTX AAR TAY TGY AGY -3 '(SEQ ID NO: 1)

Reverse primer:

5'- RTG XGT XGA RCA XGC XAG RCA XGG RTC -3 '(SEQ ID NO: 2)

Where X is Inosine, R is A or G, Y is C or T

The primer can be used to determine whether the [nickel-iron] -hydrogenase gene is present in any strain. Accordingly, the present invention comprises the steps of (1) separating genomic DNA from the hydrogen production strain; (2) performing the polymerase chain reaction using the genomic DNA as a template and the forward primer and the reverse primer according to claim 1, and (3) confirming the amplification of the [nickel-iron] -hydrogenase gene. Provided are methods for identifying [nickel-iron] -hydrogenase genes, including steps.

Step (1) of the production method is a step of separating genomic DNA from the hydrogen producing strain. Here, "hydrogen producing strain" includes both strains known to produce hydrogen, as well as strains that are expected to produce hydrogen. Since the method of separating genomic DNA from the hydrogen producing strain is similar to the method of separating genomic DNA from conventional cells, the genomic DNA can be easily separated from the hydrogen producing strain using a commercially available genomic DNA separation kit. Can be.

Step (2) of the preparation method is a polymerase chain reaction using a genomic DNA isolated as a template, using a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer consisting of a nucleotide sequence shown in SEQ ID NO: 2 It is a step to carry out. Here, a forward primer is included in a base sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1 in the nucleotide sequence of the genomic DNA, and a reverse primer is provided in a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 in the nucleotide sequence of the genomic DNA, respectively. After hybridization, amplification occurs. In this step, the polymerase chain reaction is preferably performed under conditions where the annealing temperature of DNA double helix is 40 seconds at 55 ° C. and the extension temperature of DNA strands is 3 minutes at 72 ° C. .

On the other hand, the amplification process in step (2) occurs only when the [nickel-iron] -hydrogenase gene is present in the nucleotide sequence of genomic DNA. Therefore, if a band corresponding to the molecular weight of the [nickel-iron] -hydrogenase gene is observed by electrophoresis of the polymerase chain reaction product obtained through step (2), that is, the [nickel- If the iron] -hydrogenase gene is amplified, the hydrogen producing strain selected in step (1) expresses the [nickel-iron] -hydrogenase gene and can be confirmed to produce hydrogen.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Design and Preparation of Degenerate Primer

After searching the database for the [nickel-iron] -hydrogenase genes of microorganisms known to produce hydrogen, the secretion signal sequence of small subunits of 30 kinds of [nickel-iron] -hydrogenases and the large subunit Degenerate primers were designed and constructed to align conservative sequences at the terminal ends to detect new [nickel-iron] -hydrogenase genes (FIG. 1).

Forward Primer:

5'- ATG CGX CGX AGY TTY CTX AAR TAY TGY AGY -3 '(SEQ ID NO: 1)

Reverse primer:

5'- RTG XGT XGA RCA XGC XAG RCA XGG RTC -3 '(SEQ ID NO: 2)

Where X is Inosine, R is A or G, Y is C or T

Example 2 Detection of [nickel-iron] -hydrogenase genes from various hydrogen producing strains using degenerate primers

Esherichia Coli 's chromosome as a template, consisting of Taq DNA polymerase (Bioneer), 10x reaction buffer with MgCl2 (Bioneer), and degenerate primers, with an annealing temperature of 55 For 40 seconds at the ℃, the extension temperature of the DNA strands (extension temperature) was subjected to the polymerase chain reaction under the conditions of 3 minutes at 72 ℃. As a result of the polymerase chain reaction, a band of [nick-iron] -hydrogenase of E. coli having a size of about 2900 bp was successfully obtained (FIG. 2). As a result, it was confirmed that the sequence of the [nickel-iron] -hydrogenase gene of E. coli in the database.

Hydrogenovibrio was used in the same manner to verify the successful application of degenerate primers for other hydrogen-producing microorganisms. marinus and Ralstonia Experiments were performed using eutropha strains. After culture for each strain, to separate the chromosomes according to the method and procedure of the Wizard Genomic DNA Purification Kit (Promega) E. coli In the same manner as the previous experiment with the strain, the polymerase chain reaction was performed using the two chromosomes as templates. As a result of the polymerase chain reaction, the same [nick-iron] -hydrogenase gene bands of about 2900 bp in size were successfully detected (FIGS. 3 and 4). In addition, by sequencing the obtained gene sequence into the E. coli vector, it was possible to reveal the [nick-iron] -hydrogenase gene sequence of the two strains.

The degenerate primer of the present invention can be used to search for a gene of a new [nickel-iron] -hydrogenase from a variety of hydrogen producing strains as well as a gene of a known [nickel-iron] -hydrogenase. The obtained hydrogenase can be used for hydrogenation enzyme improvement research using protein engineering technology and can increase the efficiency of biological hydrogen production based on this. It will also be used as an electrocatalyst in the development of biofuel cells and biosensors. Instead of the chemical reactions that are now commonly used in fuel cells, the use of hydrases could lead to simpler processes and clean fuel cells without contamination. In addition, it may be used in biocorrosion induced by anaerobic microorganisms.

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Claims (3)

A primer for amplifying a [Nickel-iron] -hydrogenase gene comprising a forward primer consisting of a nucleotide sequence shown in SEQ ID NO: 1 and a reverse primer consisting of a nucleotide sequence shown in SEQ ID NO: 2. (1) separating genomic DNA from the hydrogen producing strain; (2) performing the polymerase chain reaction using the genomic DNA as a template and the forward primer and the reverse primer according to claim 1, and (3) the molecular weight of [nickel-iron] -hydrogenase in electrophoresis. Method of identifying the [nickel-iron] -hydrogenase gene comprising the step of confirming the amplification of the [nickel-iron] -hydrogenase gene by checking the band. The method of claim 2, In the polymerase chain reaction of step (2), the DNA double helix was released at an annealing temperature of 55 ° C. for 40 seconds, and the DNA strand extension temperature was 72 ° C. for 3 minutes. A method for identifying a [nickel-iron] -hydrogenase gene characterized by the above.

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