KR20180117771A - A novel endonuclease derived from thermophilic bacteria and a use of the same - Google Patents

Abstract

The present invention relates to a novel endonuclease (L-fucose Isomerase) and a usage of the endonuclease, and more specifically, to an endonuclease obtained from Fervidobacterium islandicum AW-1 which is ultrahigh temperature bacteria capable of being grown at a high temperature, a polynucleotide encrypting the endonuclease, a recombinant vector containing the polynucleotide, host cells transformed with the recombinant vector, and a method for manufacturing the endonuclease comprising a step of cultivating the host cells. The endonuclease of the present invention is able to react at a high temperature and maintains enzyme activation even in a various range of pH to be used in genetic manipulation of enzymes deprived from Fervidobacterium islandicum AW-1 and enzyme production in a large quantity, or in production of variant enzymes for improving enzyme efficiency such that proteolytic activities are maintained at a high temperature to improve industrial applicability of enzyme deprived from Fervidobacterium islandicum AW-1, which is excellent for treating organic industrial wastes such as feathers of chickens. Therefore, the present invention is expected to be useful in an industry.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel high temperature bacterial endogenous norepinease,

The present invention relates to endo-nuclease, and more particularly to a novel endonuclease excellent in heat resistance as compared with the previously reported endo-nuclease, and a method of using the same.

In a method of industrially using a microorganism or a microorganism-derived enzyme, maintaining the activity under specific conditions may have important significance.

Specifically, maintaining enzyme activity under high temperature, acidic or basic conditions is advantageous in that it can control the state of a substance to be subjected to the enzymatic reaction, can control the reaction rate, etc., and is advantageous in terms of treatment or utilization of the reaction product after the first reaction It has great industrial significance.

For example, in general, the enzyme of marine microorganism having agarase has an optimum enzyme activity at about 20 ° C. and is almost inactivated at a temperature of 40 ° C. or higher. However, the temperature at which the agar converts from a solid gel state to a liquid sol state The agar decomposition enzyme capable of maintaining the agar resolving ability at a temperature of 40 ° C or higher has a very significant industrial significance. This is because when the agar is decomposed to produce a secondary product, the degradation efficiency of the agar can be remarkably increased by using an enzyme that maintains the agar resolving ability in a sol state in which the agar is in a liquid state.

In this respect, the thermophilic or thermophilic enzymes are of industrial interest in that they can be used in a variety of industries.

According to the existing studies, it has been found that the protein degradation activity is maintained at high temperature by using Fervidobacterium AW-1 islandicum AW-1) has been evaluated as being able to be used for decomposing organic wastes, detergents or cosmetics because it has various proteolytic activities, specifically a keratin-specific proteolytic activity or an excellent degradative activity against chicken hair.

Such an enzyme derived from peridobacterium lysylidiumum AW-1, which maintains proteolytic activity at a high temperature, can be applied to various industries as described above, and thus its industrial significance will be great.

In order to more industrially utilize these excellent proteases derived from Peruvita gamma listericum AW-1, it is necessary to develop a method for genetic tools for the corresponding enzymes.

In this connection, for the genetic control of thermophilic microorganisms or thermophilic microorganism-derived proteins such as the above-mentioned C. perfringens or methylation system (methylation system ) Related enzymes are required to be developed.

KR 10-0801929 B KR 10-2004-0102813 A KR 10-2017-0000813 A KR 10-2005-0064991 A

In order to solve the problems of the prior art as described above, the present invention provides an endo-nuclease capable of performing an internal hydrolysis reaction even at a high temperature, It is intended to provide an endonuclease.

It is another object of the present invention to provide a method of utilizing endonuclease to solve the problems of the prior art.

The present invention is based on the finding that the present invention has the heat resistance that can maintain or improve the reaction efficiency even at a temperature higher than 20 to 35, which is the general reaction temperature of conventional enzymes derived from ultrafast germs, and has enzyme activity even in a wide pH range including acidic or basic Endonuclease and a polynucleotide encoding the endonuclease.

In addition, the present invention provides a method for producing an endonuclease comprising the polynucleotide, a recombinant vector comprising the polynucleotide, a host cell transformed with the recombinant vector, and a step of culturing the host cell.

Specifically, the present invention relates to a method for producing a protein having a proteolytic activity at a high temperature, related to endonuclease derived from the islandicum AW-1.

The endonuclease may preferably be one having the amino acid sequence of SEQ ID NO: 2.

In addition, the endonuclease may preferably be one which is encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1.

The endonuclease may preferably be a Type II Restriction Endonuclease (Fis I).

In addition, the present invention relates to a protein having the amino acid sequence of SEQ ID NO: 2 and having a proteolytic activity at high temperature, Fervidobacterium < RTI ID = 0.0 > The present invention relates to a polynucleotide encoding an endonuclease derived from an islandicum AW-1.

The polynucleotide may preferably have the nucleotide sequence of SEQ ID NO: 1.

The present invention also relates to a recombinant vector comprising the polynucleotide.

The present invention also relates to a host cell transformed with said recombinant vector.

The present invention also relates to a method for producing endonuclease comprising culturing the host cell.

In the present invention, endonuclease refers to Restriction Endonuclease. The endonuclease is a generic term for nucleic acid internal hydrolases that identify specific sequences of double stranded DNA and cleave double stranded DNA. The endonuclease is classified into types I, II, and III according to the requirement of factors required for enzyme activity and the difference in the mode of cleavage. The endonuclease is widely distributed in fungi, and the kind thereof varies greatly depending on the type of enzyme and the recognition sequence.

Specifically, the type I endonuclease requires S-adenosylmethionine, ATP, and the positional relationship between the recognition base sequence and the cleavage point is not constant. The type Ⅱ endonuclease is comprised of a single subunit, in its activity is required only in Mg ^{2 +} and cleaves the DNA within or specific location recognition arrangement. The type Ⅲ endonuclease cleaves the DNA at the required Mg ^{2 +} and ATP, but, S- adenosyl methionine is not required, and is about 25 base pairs downstream from the recognition arrangement. Of these three enzymes, type II endonuclease is an essential enzyme for the analysis of recombinant DNA or DNA sequencing because it cleaves the DNA chain precisely at the position where the recognition sequence appears.

Hereinafter, the present invention will be described in more detail.

In order to solve the above-mentioned problems, the inventors of the present invention have been studying endo-nuclease activity which maintains the activity of the restriction enzyme even at a high temperature from ultratrophilic bacteria and the like, The endo-nuclease derived from the hot-springs microorganism Perbocobacterium islandicum AW-1, which retains its degradative activity, is identified, and the gene and amino acid sequence of the endonuclease are identified. But also in various pH ranges, thus completing the present invention.

The present invention relates to a pharmaceutical composition comprising FERBIDOBACTERIUM < RTI ID = 0.0 > AW-1 related to endonuclease derived from the islandicum AW-1.

The endo-nuclease derived from the PERBY Dobacterium dyslundimide AW-1 is a novel enzyme having low sequence homology with the previously reported endonuclease, and the enzyme activity is retained even at a higher temperature range than the endonuclease And the enzyme activity is maintained even in a wide pH range.

The endonuclease may have the amino acid sequence of SEQ ID NO: 2, and preferably has the amino acid sequence encoded by the polynucleotide having the nucleotide sequence of SEQ ID NO: 1.

The endonuclease may preferably be a Type II Restriction Endonuclease (Fis I).

Further, the present invention relates to a polynucleotide which encodes or encodes a heat-resistant endonuclease derived from the above-mentioned PERBY dumbelleri islelandicum AW-1.

The polynucleotide may preferably have the nucleotide sequence of SEQ ID NO: 1.

The range protected by the polynucleotide encoding the endonuclease may include, but is not limited to, a restriction enzyme site, such as a restriction enzyme site, for the production of the mutant enzyme, ) Or a sequence related to a specific promoter is linked to the polynucleotide sequence.

Specifically, according to an embodiment of the present invention, an initiation codon (ATG) associated with translation initiation or a termination codon (TAA) associated with translation termination can be added or inserted in the vector into which the initiation codon or termination codon is inserted, In one example, a polynucleotide sequence having a restriction site for binding by a vector for producing a combination vector is added, or a signal sequence in a wild-type with the initiation codon is added or removed can do.

In addition, the present invention relates to a protein having the amino acid sequence of SEQ ID NO: 2 and having a proteolytic activity at high temperature ( Fervidobacterium < RTI ID = 0.0 > can be a vector comprising a polynucleotide encoding an island nucleus AW-1) endonuclease.

The polynucleotide of the endonuclease may have the nucleotide sequence of SEQ ID NO: 1.

The vector may be a recombinant vector prepared by inserting the polynucleotide into a common expression vector, for example, pET-28a vector, and may be, for example, a vector having a cleavage map of FIG. The expression vector may be selected from suitable vectors depending on the type of the host cell.

In addition, the present invention may be a host cell transformed with the recombinant vector.

The recombinant vector has the amino acid sequence of SEQ ID NO: 2 and has a proteolytic activity at high temperature, such as Fervidobacterium < RTI ID = 0.0 > can be a vector comprising a polynucleotide encoding an island nucleus AW-1) endonuclease.

The host cell can be a prokaryote or an eukaryote, preferably a prokaryote, and can be, for example, E-coli.

The present invention also relates to a method for producing endonuclease comprising culturing the host cell.

The endonuclease production method may be a method using a recombinant vector comprising a polynucleotide encoding a heat-resistant endonuclease having the amino acid sequence of SEQ ID NO: 2 or a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, Preparing a transformant by transforming the recombinant vector into eukaryotic cells derived from a prokaryote, eukaryote or eukaryote, culturing the transformant, culturing the transformant from the cultured medium to obtain the endonuclease ≪ / RTI > The preparation of the recombinant vector and the production of the transformant can be carried out according to a conventional genetic engineering method.

The present invention relates to the use of the Fulvidobacterium AW-1 ( Fervidobacterium The novel endonuclease isolated from islandicum AW-1 is capable of performing the reaction at a high temperature of 40 or more and maintains the enzyme activity over a wide range of pH range as compared with the endonuclease, It is possible to mutate an enzyme from the PERBIDS gambotherium islandsidicum AW-1-derived enzyme under various conditions, thereby contributing to the improvement of the performance by the genetic method of the PERBIDS bacterial iselandicum AW-1-derived protease It is expected that the value of industrial use is very high.

1 is a photograph showing a result of amplification of endonuclease (fis I) according to an embodiment of the present invention by PCR.
FIG. 2 is a photograph showing the results of confirming whether a recombinant vector of endonuclease (fis I) and an endonuclease (fis I) are inserted in the recombinant vector according to an embodiment of the present invention.
FIG. 3 is a photograph showing SDS-PAGE analysis of the result of expression of endonuclease (fis I) using a recombinant vector of endonuclease (fis I) according to an embodiment of the present invention.
4 is a diagram showing a region (1661 bp) recognized and cleaved by endonuclease (fisI) among pUC19 according to an embodiment of the present invention.
FIG. 5 is a photograph showing a result of confirming the restriction enzyme activity of the endonuclease (fis I) of the present invention on a part (1661 bp) of pUC19 according to an embodiment of the present invention.
FIG. 6 is a photograph showing the heat resistance of the endo-nuclease (fis I) of the present invention, which is obtained by examining the heat-labile activity of the enzyme according to the present invention, on a portion (1661 bp) of pUC19 according to an embodiment of the present invention to be.
FIG. 7 is a photograph showing a result of confirming the enzyme activity of the endonuclease (fis I) according to the present invention on a portion (1661 bp) of pUC19 according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to production examples and examples. However, the following Preparation Examples and Examples are merely illustrative examples for the purpose of facilitating understanding of the present invention, and can be modified into various other forms, and the scope of the present invention is not limited to the following Examples.

Preparation Example : DNA , Reagents and medium

For the discovery of the gene encoding the thermophilic endonuclease, it has been reported that the gene encoding FERBIDOBACTERIUM AW-1 ( Fervidobacterium < RTI ID = 0.0 > The genomic DNA of islandicum AW-1 was used. E. coli DH5a was used as a host for gene cloning and cultured in Luria-Bertani (LB) medium (1% bacto tryptone, 0.5% yeast extract, 1% NaCl) at 37 ° C.

LB agar medium supplemented with kanamycin at a final concentration of 50 g / ml was used for the selection of E. coli transformants. Restriction enzyme and DNA ligation kit were purchased from Roche Korea. Diversity PCR for the production of mutant DNA library using error-prone PCR Random mutagenesis kit was purchased from Clontech.

Example  1: gene securing and Cloning

In order to obtain endonuclease, the genomic DNA of PERBI Dobacterium islandidum AW-1 isolated by the inventor of the present invention was amplified using nPfu-Forte DNA polymerase (Enzynomics) as a template. As a result of the amplification, a gene PCR product of 933 bp in size was confirmed (FIG. 1).

An endonuclease gene was obtained from the genomic DNA of the ultra-warm anaerobic bacterium, Peruvid's bacterium islandicum AW-1.

The primers mjaIII- AW-1F ( 5' -CAT ATG GAT AAT CTT TAC ATA GAT ATT CTA AG-3 ') and mjaIII- AW- 1R (5'-CTC GAG TTA GAT CCC GCA TAT ATT CTC C-3 ') was used. For PCR amplification, nPfu-Forte polymerase (AngioMix, Korea) was used. The amplified DNA fragment was ligated to a pTOP-Blunt V2 vector (Angemix, Korea) to prepare mjaIII-pTOPV2. The vector was inserted into E. coli And inserted into DH5? Cells to prepare a transformant. The DNA sequence inserted into the transformant was confirmed by treating with NdeI and XhoI restriction enzymes, and the results are shown in FIG.

As shown in FIG. 2, it was confirmed that the 933 bp gene was isolated from the recombinant vector (5369 bp), and the nucleotide sequence of the isolated gene was confirmed by Solgent (Korea) .

As a result of the analysis, it was confirmed that the endo-nuclease gene derived from PERBIDS Dobacterialislandimi com AW-1 was composed of 933 bp and encoded 310 amino acids.

Example  2: Recombination Endonuclease  Expression

MjaIII- pTOPV2 containing the endonuclease gene prepared in Example 1 was treated with restriction enzymes NdeI / XhoI, and a 933 bp DNA fragment thus obtained was inserted into E. coli expression vector pET-28a (Novagen) Vector pET-28a- mjaIII was prepared, and E. coli The recombinant vector was inserted into DH5a cells and transformed. Thereafter, the pET-28a- mjaIII was digested with restriction enzymes NdeI / XhoI to confirm insertion of an endonuclease gene fragment of 993 bp.

The recombinant vector pET-28a- mjaIII was incubated at 37 ° C with LB medium supplemented with antibiotic kananycin. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the final concentration of 1 mM to activate the T7 promoter .

SDS-PAGE was performed to confirm whether the L-endonuclease was expressed by the recombinant vector.

The SDS-PAGE was performed by Laemmli method using 12% polyacrylamide gel. Specifically, the cells expressing the recombinant protein were mixed with Laemmli's sample buffer, boiled for 10 minutes, injected into a gel, subjected to electrophoresis, and proteins were visualized using a coomassie reagent. The result of SDS-PAGE was shown in FIG.

As shown in FIG. 3, it was confirmed that pET-28a-FIFI plasmid, which is a recombinant vector, was inserted well into the recombinant host ( E. coli BL21 (DE3)) and the recombinant endonuclease was successfully expressed from the recombinant vector . SDS-PAGE of the isomerase showed a band at a molecular weight of 35 kDa, consistent with an estimate based on the nucleotide sequence.

Example  3: Recombination Endonuclease  Identification of enzyme heat resistance

The enzyme activity of the recombinant endonuclease produced in Example 2 was confirmed.

First, in order to confirm the restriction site of the enzyme (MjaIII recognition site), experiments were performed using a fragment designed using pUC19 (plasmid DNA pUC19) as shown in FIG.

First, a purified recombinant endonuclease (purified FisI, 25 ㎍ / ml) prepared using reaction buffer (10 mM Tris-HCl pH 7.9, 10 mM MgCl ₂ , 50 mM NaCl, 100 / / 25 μl of a mixture of 2.5 μl of the pUC19-derived DNA substrate (DNA substrate, 30 μg / μl) and 2.5 μl of the DNA substrate (30 μg / μl) was reacted at 65 for 3 hours and then electrophoresed using 1.5% agarose gel . The results of the electrophoresis are shown in FIG.

As shown in FIG. 5, it was confirmed that the DNA substrate of 1661 bp as the reaction substrate was cleaved to 955 bp, 565 bp and 141 bp, and the recombinase correctly recognized the Sau AI (Dpn II) Respectively.

The stability of the enzymatic activity of the recombinant endonuclease was confirmed by carrying out reactions at various conditions, specifically at various temperatures and pH ranges.

Specifically, in order to confirm the heat resistance, the above-mentioned experiment was carried out by increasing the temperature at intervals of 10 DEG C in the temperature range of 35 DEG C to 85 DEG C (Fig. 6A). Further, in order to further confirm the heat resistance, the temperature was increased at intervals of 10 DEG C in the temperature range of 30 DEG C to 100 DEG C and the enzyme of the recombinant endonuclease was exposed (cultured) for 1 hour, Experiments were performed (Fig. 6B).

As shown in FIG. 6A, the enzyme of the recombinant endonuclease was found to exhibit the most excellent enzyme activity at 65 ° C. and 75 ° C., the enzyme activity was lost at 85 ° C., and the optimum temperature range of the enzyme was 65 Lt; 0 > C to 75 < 0 > C.

As shown in FIG. 6 b, when the enzyme of the recombinant endonuclease was exposed to various temperature ranges, the enzyme activity was maintained up to 70 ° C., but the enzyme activity was lost above 80 ° C., Recombinant endonucleases were found to be heat resistant at temperatures below 80 ° C.

Also, as shown in FIG. 7, the enzyme of the recombinant endonuclease was found to exhibit excellent enzyme activity in all the pH range in which the experiment was conducted, that is, in the range of pH 5 to pH 10, It was confirmed that the enzyme activity was maintained in the pH range from slightly acidic to basic.

<110> Industry University Cooperation Foundation Silla University <120> A NOVEL ENDONUCLEASE DERIVED FROM THERMOPHILIC BACTERIA AND A USE          OF THE SAME <130> CDP20170090 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 933 <212> DNA <213> FisI gene from AW-1 <400> 1 atggagaatc tttacataga tattctaaga cgcaaaggat ggaacgtttc ggatgcaaaa 60 gatgttgtag agaaattttt gggaacgatt cttaaaacta acaagacata caactttttt 120 gttgactggg acaaggtgaa agaattcgtt gaaaaacata ggatagaaat aaatattctc 180 agtacgctta ttcattcagc ggactttgat agcgatttga ggaagatact tagaaagtat 240 cctgaagttc ttgcggttat acctatgctt atagccgttc gagagaattc tatctcagtg 300 cttcttgata ctgatcataa tgaactgcgt atcgatgagt atgatttcac cgtgcgcacg 360 ttaaacgaac aggaaataaa ttatttcgtt gagttttttg aaaagacggg gttaaagagt 420 ttttttctca acactgccga caagagtttg tatgactatc ttgtaggtgt tgaggttgga 480 ctggatacta atgccagaaa gaatagaagt gggtttttcc ttgaaaaata tttaaaccct 540 attgttaaaa gtatagccga aaaatatgga tacaatcttc tggttcagaa aaagttctca 600 caagttcctg gtgtaaatat cagtgaactt ttcaacagga aagcggatta catacttttc 660 aaagatgata gcagttatgt tcaaagatta ataaatatcg aggtcaactt ctacaatgtt 720 acaggctcga aacctcagga aattatagat tcgtatattg aaaggcaaaa tgagctaaaa 780 aaatatggat ttgagtttat actgataaca gatggacctg cttggaatgg acagaggaat 840 cagttgttca aagcttttga aaacatttct tatgtcttga atatccattt tgtccaacaa 900 ggagtcttgg aggagattat atgcgggatc taa 933 <210> 2 <211> 310 <212> PRT <213> Fisl form AW-1 <400> 2 Met Glu Asn Leu Tyr Ile Asp Ile Leu Arg Arg Lys Gly Trp Asn Val   1 5 10 15 Ser Asp Ala Lys Asp Val Val Glu Lys Phe Leu Gly Thr Ile Leu Lys              20 25 30 Thr Asn Lys Thr Tyr Asn Phe Phe Val Asp Trp Asp Lys Val Lys Glu          35 40 45 Phe Val Glu Lys His Arg Ile Glu Ile Asn Ile Leu Ser Thr Leu Ile      50 55 60 His Ser Ala Asp Phe Asp Ser Asp Leu Arg Lys Ile Leu Arg Lys Tyr  65 70 75 80 Pro Glu Val Leu Ala Val Ile Pro Met Leu Ile Ala Val Arg Glu Asn                  85 90 95 Ser Ile Ser Val Leu Leu Asp Thr Asp His Asn Glu Leu Arg Ile Asp             100 105 110 Glu Tyr Asp Phe Thr Val Arg Thr Leu Asn Glu Gln Glu Ile Asn Tyr         115 120 125 Phe Val Glu Phe Phe Glu Lys Thr Gly Leu Lys Ser Phe Phe Leu Asn     130 135 140 Thr Ala Asp Lys Ser Leu Tyr Asp Tyr Leu Val Gly Val Glu Val Gly 145 150 155 160 Leu Asp Thr Asn Ala Arg Lys Asn Arg Ser Gly Phe Phe Leu Glu Lys                 165 170 175 Tyr Leu Asn Pro Ile Val Lys Ser Ile Ala Glu Lys Tyr Gly Tyr Asn             180 185 190 Leu Leu Val Gln Lys Lys Phe Ser Gln Val Pro Gly Val Asn Ile Ser         195 200 205 Glu Leu Phe Asn Arg Lys Ala Asp Tyr Ile Leu Phe Lys Asp Asp Ser     210 215 220 Ser Tyr Val Gln Arg Leu Ile Asn Ile Glu Val Asn Phe Tyr Asn Val 225 230 235 240 Thr Gly Ser Lys Pro Glu Glu Ile Ile Asp Ser Tyr Ile Glu Arg Gln                 245 250 255 Asn Glu Leu Lys Lys Tyr Gly Phe Glu Phe Ile Leu Ile Thr Asp Gly             260 265 270 Pro Ala Trp Asn Gly Gln Arg Asn Gln Leu Phe Lys Ala Phe Glu Asn         275 280 285 Ile Ser Tyr Val Leu Asn Ile His Phe Val Gln Gln Gly Val Leu Glu     290 295 300 Glu Ile Ile Cys Gly Ile 305 310

Claims (9)

Endonuclease derived from Fervidobacterium islandicum AW-1 having proteolytic activity at high temperature. The method according to claim 1,
Wherein the endonuclease has an amino acid sequence of SEQ ID NO: 2. The method according to claim 1,
Wherein said endonuclease is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein the endonuclease is a type 2 restriction endonuclease (Fis I). 2, which has the amino acid sequence of SEQ ID NO: 2 and has a proteolytic activity at high temperature, Fervidobacterium &lt; RTI ID = 0.0 &gt; Polynucleotides encoding endonuclease derived from islandicum AW-1. 6. The method of claim 5,
Wherein the polynucleotide has the nucleotide sequence of SEQ ID NO: 1. A recombinant vector comprising the polynucleotide of claim 5. A host cell transformed with the recombinant vector of claim 7. 9. A method for producing endonuclease, comprising culturing the host cell of claim 8.

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