KR101835940B1 - A mutant Soluble Methane Monooxygenase derived from Methylosinus trichosporium OB3b with improved activity - Google Patents
A mutant Soluble Methane Monooxygenase derived from Methylosinus trichosporium OB3b with improved activity Download PDFInfo
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Abstract
The present invention uses quantum and molecular mechanics techniques and partial mutation techniques to generate methylosinus tricosporium OB3b trichosporium (SMMO ) derived from OB3b, more specifically, by screening based on quantum mechanics and molecular mechanics to find a residue that affects enzyme activity, and the enzyme activity is determined by mutation of the detected residue (SMMO), a nucleic acid molecule encoding the same, a vector comprising the nucleic acid molecule, a transformant containing the vector, a mutant of the methan oxidase (sMMO), and an improved methan oxidase (sMMO ). ≪ / RTI >
Description
The present invention relates to the use of Methylosinus tricosporium OB3b ( Methylosinus < RTI ID = 0.0 > trichosporium OB3b) derived from methane it relates to improving the enzymatic activity of the oxidase (Soluble Methane Monooxygenase. Hereinafter 'sMMO'"), and more particularly, to enzyme activity improved methane oxidase (sMMO), encoding it by mutation The present invention relates to a nucleic acid molecule, a vector containing the nucleic acid molecule, a transformant containing the vector, a mutant of the methanotrophicin (sMMO) and an improved methan oxidase (sMMO).
Methanotrophic bacteria refers to a group of bacteria that grows by using methane as a carbon source and an energy source. Methylomonas, Methanomonas, Methylococcus, Methylosinus, Methylobacter, Methylomicrobium and Methyl ocystis, and the like. The methanotrophic bacteria include methane monooxygenase (hereinafter referred to as MMO) which oxidizes methane to methanol and methanol dehydrogenase (hereinafter referred to as MDH) which oxidizes methanol to formaldehyde To oxidize methane gas to convert it to carbon dioxide. Specifically, the methane oxidizing bacteria oxidize methane to methanol and oxidize the methanol again to formaldehyde. Then, formaldehyde is oxidized to formic acid, which ultimately converts to less toxic carbon dioxide. Although the methanotrophic bacteria do not utilize an organic compound having a carbon-carbon bond as a growth material, many alkanes and aromatics can also be oxidized by the enzymatic action of MMO. On the other hand, much research has been carried out on the recycling of methane generated at the waste disposal site, and research is underway to convert methane to methanol using methane oxidizing bacteria. However, since the enzyme has disadvantages such as instability and low enzyme activity, it is essential to improve the properties of the enzyme for more efficient utilization.
[Prior Patent Literature]
Korean Patent Publication No. 1020040045562
The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a process for producing a methane oxidase (sMMO), which acts on the bioconversion of methanol from methane, It is to improve.
A second object of the present invention is to provide a recombinant expression vector containing the improved methane oxidase (sMMO) gene.
A third object of the present invention is to provide all the transformation strains comprising the recombinant methanogens that have been transformed with the improved genes.
A fourth object of the present invention is to provide a recombinant methane oxidase (sMMO) using recombinant methanogens bacteria transformed with an improved enzyme.
A fifth object of the present invention is to provide a residue which affects the activity of methanotrophin (sMMO) using the enzyme.
In order to accomplish the above object, the present invention provides a mutant methane oxidase (sMMO) in which the 213 th amino acid threonine of methan oxidase (sMMO) is substituted with arginine.
In one embodiment of the invention, the enzyme is selected from the group consisting of methylosinus tricosporium OB3b trichosporium OB3b ), but methane oxidase (sMMO) derived from other methane oxidizing bacteria or produced by a genetic engineering method is also included in the scope of the present invention.
In another embodiment of the present invention, it is preferable that the mutant enzyme has an amino acid sequence of SEQ ID NO: 3, but a mutant body which obtains the effect to be achieved by the present invention through one or more substitutions, deletions, inversions, And are included in the scope of the present invention.
The present invention also provides a gene encoding the enzyme of the present invention.
In one embodiment of the present invention, it is preferable that the gene has a nucleotide sequence of SEQ ID NO: 4, but mutants that obtain the effect to be achieved by the present invention through one or more substitutions, deletions, inversions, And are included in the scope of the invention.
The present invention also provides a recombinant vector comprising the gene of the present invention.
The present invention also provides a method for producing a transformant by transforming the recombinant vector of the present invention into a host cell.
The present invention provides a method for producing the mutant enzyme of the present invention comprising the step of transforming the recombinant vector of the present invention into a microorganism to produce a transformant and expressing the enzyme of the present invention.
The present invention can also be used to improve the productivity of methanol from methane using the mutant of the present invention.
The present invention also provides a method for producing methanol by treating the above mutant methane oxidase (sMMO) of the present invention to a substrate.
In one embodiment of the present invention, the substrate is preferably an alkane or an aromatic compound, but is not limited thereto.
Hereinafter, the present invention will be described.
The present invention provides an underlying technology for identifying the determinants of the activity of methane oxidase (sMMO) by presenting important residues that can regulate the enzyme activity of the methane oxidase (sMMO) of the present invention.
The present invention also provides highly active methane oxidase (sMMO) using the mutant methane oxidase (sMMO) of the present invention, which can be used to efficiently produce methanol.
Hereinafter, the present invention will be described in detail.
SEQ ID NO: 4 shows the nucleotide sequence of the mutated methionine oxidase (sMMO) gene of the present invention, and SEQ ID NO: 3 shows the amino acid sequence that the gene encodes. As described above, there may be mutations such as deletion, substitution, addition, and the like for several amino acids, as long as the polypeptide having the amino acid sequence has methane oxidase (sMMO) activity. In addition, the gene of the present invention includes, in addition to the base sequence encoding the amino acid sequence represented by SEQ ID NO: 4, a hemiacetate coding for the same polypeptide that differs only in the axial codon. Variations such as deletion, substitution, and addition can be introduced by site mutation introduction methods (Current Protocols in Molecular Biology, Vol. 1, p. 811, 1994).
The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used for preparing the recombinant vector. For example, when bacteria such as Escherichia coli and methanotrophic bacteria are used as a host, the recombinant vector of the present invention can autonomously replicate itself in a host, and can also be used as a promoter, DNA containing methane oxidase (sMMO) It is preferable that it has a structure necessary for expression of a transcription termination sequence and the like. As the expression vector used in the present invention, pT2ML was used, but any expression vector satisfying the above requirements can be used.
As the promoter, any promoter that can be expressed in a host can be used, and a promoter derived from methane oxidizing bacteria, E. coli, or the like can be used. As a method for introducing the recombinant DNA into bacteria, a conjugation method and the like can be used.
The recombinant vector may also contain fragments having various functions for inhibiting, amplifying, or inducing expression, a gene for selecting a transformant, a gene for resistance to antibiotics, or a gene encoding a signal for secretion outside the cells May be additionally provided.
The production of the mutated methane oxidase (sMMO) according to the present invention is carried out as follows. A transformant obtained by transforming a host with a recombinant vector having a gene encoding a mutated enzyme is cultured and methan oxidase (sMMO), which is a gene product, is produced and accumulated in the culture (culture cell or culture supernatant) Loses.
As a method for culturing the transformant of the present invention, a conventional method used for culturing a host may be used.
In addition, the promoter induces the expression of the naturally-occurring microorganism when the transformed microorganism is cultured using the wild-type host promoter.
Further, cultivation of a transformant using a microorganism as a host, production of methan oxidase (sMMO) by the transformant, accumulation in the microorganism, and recovery of methan oxidase (sMMO) from the microorganism are limited to the above methods It is not.
In the present invention, the gene of methane oxidase (sMMO) was cloned from methylolenosine tricosporium OB3b in order to secure a highly active enzyme and to present a residue that plays an important role in enzyme activity. By presenting moieties that play an important role in high activity, we can provide an underlying technology for the identification of highly active determinants. It was possible to increase the activity of converting a methane to methanol using a mutant enzyme obtained by mutating residues at positions that play an important role in enzyme activity.
The activity value of MTsMMO T213R, which is a mutant in which the important residues are substituted using the methoxylin oxidase (sMMO) derived from the methylosinus tricosporium OB3b obtained in the present invention, against the naphthalene substrate is 399 nmoles / min / mg.
In the present invention, several methane oxidase (sMMO) mutants showing high activity were obtained. This will be useful for economical production of methanol from methane by overcoming the low enzyme activity problems of conventional methane oxidase (sMMO).
The present invention relates to the use of Methylosinus tricosporium OB3b trichosporium (SMMO ) derived from OB3b- derived methionine oxidase (sMMO), thereby increasing the ability of the substrate to bind to the enzyme and increasing the activity of the enzyme. In addition, methanol can be efficiently produced using the above mutant of methane oxidase (sMMO) and the improved methane oxidase (sMMO).
Figure 1 is a schematic diagram of quantitative screening based on activation energy.
Figure 2 is a computer analysis of the active sites to which a substrate of methane oxidase (sMMO) derived from methylolenosine tricosporium OB3b, analyzed on a molecular mechanics basis, binds.
Hereinafter, the present invention will be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.
Example One: Methyl rosiners Tricosporium OB3b Culture conditions of
Table 1 shows the composition of the NMS medium used for the culture of methane-oxidizing bacteria. Methylosynergic tricosporium OB3b strain was cultured on NMS medium containing 1M H 2 So 4 and 1M NaOH, supplemented with 1.0 μmol / L CuSO 4 .
Table 1 shows the composition of NMS medium and trace element solution
Example 2: Methane oxidation enzyme ( sMMO ) Gene Cloning
Based on the nucleotide sequence of the gene coding for methane oxidase (sMMO) of methylrogenase tricosporium OB3b using pTJS176 (Smith TJ and Murrell JC, 2011) as a template, the primer P1-5'-ATG GCG ATC AGT CTC (SEQ ID NO: 1) P4 R-5'-CCC CGA TCC GCT CGC CAA ATT CTG A-3 '(SEQ ID NO: 2) was prepared and PCR was performed. The PCR product was digested with restriction enzymes BamHI and NdeI, and cloned into pT2ML (Smith TJ and Murrell JC, 2011) with the same restriction enzymes. The cloned plasmid is transformed into E. coli XL1 blue, followed by sequencing. The confirmed plasmid is transformed into Escherichia coli S17-1 and used as a conjugation host.
Example 3: Recombinant For transformation Conjugation Way
Recombinant Escherichia coli S17-1 prepared in Example 1 and Example 2 was cultured overnight in 10 ml of LB medium, and 50 ml of a culture solution (OD600 = 0.5) of methylrogenase tricosporium OB3b were mixed, followed by centrifugation at 5000 xg for 10 minutes do. The separated pellet is resuspended in 50 ml of sterile NMS medium and filtered through a nitrocellulose filter (0.2 μM pore size) to collect the cells. Transfer the collected nitrocellulose filter to a NMS solid medium containing 0.02% proteose peptone (w / v), add 50% air / methane, seal and incubate at 30 ° C for 24 hours. After incubation, add nitrocellulose filter to 10 ml NMS medium, suspend and centrifuge to collect only the cells, then resuspend in 1 ml NMS liquid medium. 100 μl of NMS medium and 20 μg / ml of streptomycin are plated on NMS agar medium. The resuspended cells are plated out and 50% air / methane is injected. The cells are cultured at 30 ° C. for 1.5-2 weeks until clear colony appears. The selected colonies are cultured in NMS solid medium containing 20 μg / ml gentamycin, streptomycin, spectinomycin, and nalidixic acid. Transformation was confirmed by colony PCR using the P1 and P4 primers used in Example 1.
Example 4: with high activity Methane oxidation enzyme ( sMMO )of Mutant making
Example 4-1: Highly active Methane oxidation enzyme ( sMMO )of Mutant For production QM / MM based screening
Quantum mechanics / molecular mechanics methods of Discovery Studio 4.5 and Materials Studio 8.0 (Accelrys Inc. San Diego, Calif., USA) according to the flow chart of Figure 1 were used to determine residues inside and outside the active site The activation energy of the substituted mutant, E a (Kcal / mol) were calculated and screened. Activation energies (Kcal / mol) of the mutants in which the residues of the substrate binding sites of the enzyme were substituted were compared with the activation energies of wild enzymes. The mutation and screening of the whole protein as well as the active site can be carried out through a QM-based quasi-silico screening system in a very fast and quantitatively first shell (residue within 5A based on methane bound to the active site) and a second shell And residues within 5-7 A based on the methane left over) were screened. When the activation energy is low, the energy barrier of the enzyme reaction is lowered and the turnover rate of the enzyme is improved. As shown in FIG. 2, the activation energy (Kcal / mol) of the mutant in which the residues of the substrate binding site of the enzyme was substituted with the alanine residue was compared with the activation energy of the wild enzyme. As shown in Table 2, the Q150A, C151A, F188A, T213A, I217A, and V239A mutant enzymes had lower activation energies than the wild-type enzymes, and Q150A, C151A, F188A, T213A, I217A and V239A residues were selected as the target residues for intensive mutagenesis .
Example 4-2: Highly active Methane oxidation enzyme ( sMMO )of Mutant For production QM / MM based screening ( Insilico Saturated mutation)
The activation energy (Kcal / mol) of the saturated mutants in which the residues selected in Example 4-1 were replaced with several other residues were compared with the binding energies of wild-type enzymes. As shown in Table 3, Q150A, C151A, C151R, F188G, T213R, and V239I mutants had lower activation energies than wild-type enzymes, and Q150A, C151A, C151R, F188G, T213R and V239I were selected as target mutants.
(Kcal / mol)
Example 4-3: Q150A , C151A , C151R , F188G, T213R , V239I Mutant Naphthalene activity
As in Example 4-1, the 150th glutamine residue, which is a residue showing low binding energy at the time of mutation in the substrate binding site, was substituted with alanine, the 151st residue cysteine was replaced with alanine and arginine, and the 188th residue phenylalanine Was substituted with glycine, threonine as the 213th residue was substituted with arginine, and valine as the 239th residue was replaced with isoleucine. The mutants were prepared using the methods of Examples 2 and 3. As shown in Table 4, it was found that the T213R mutant had the highest activity against the naphthalene substrate.
(nmoles / min / mg)
<110> Konkuk University Industrial Cooperation Corp <120> A mutant Soluble Methane monooxygenase derived from Methylosinus trichosporium OB3b with improved activity <130> HY151110 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atggcgatca gtctcgctac gaaa 24 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ccccgatccg ctcgccaaat tctga 25 <210> 3 <211> 526 <212> PRT <213> Artificial Sequence <220> <223> mutant <400> 3 Met Ala Ile Ser Leu Ala Thr Lys Ala Ala Thr Asp Ala Leu Lys Val 1 5 10 15 Asn Arg Ala Pro Val Gly Val Glu Pro Gln Glu Val His Lys Trp Leu 20 25 30 Gln Ser Phe Asn Trp Asp Phe Lys Glu Asn Arg Thr Lys Tyr Pro Thr 35 40 45 Lys Tyr His Met Ala Asn Glu Thr Lys Glu Gln Phe Lys Val Ile Ala 50 55 60 Lys Glu Tyr Ala Arg Met Glu Ala Ala Lys Asp Glu Arg Gln Phe Gly 65 70 75 80 Thr Leu Leu Asp Gly Leu Thr Arg Leu Gly Ala Gly Asn Lys Val His 85 90 95 Pro Arg Trp Gly Glu Thr Met Lys Val Ile Ser Asn Phe Leu Glu Val 100 105 110 Gly Glu Tyr Asn Ala Ile Ala Ala Ser Ala Met Leu Trp Asp Ser Ala 115 120 125 Thr Ala Glu Gln Lys Asn Gly Tyr Leu Ala Gln Val Leu Asp Glu 130 135 140 Ile Arg His Thr His Gln Cys Ala Phe Ile Asn His Tyr Tyr Ser Lys 145 150 155 160 His Tyr His Asp Pro Ala Gly His Asn Asp Ala Arg Arg Thr Arg Ala 165 170 175 Ile Gly Pro Leu Trp Lys Gly Met Lys Arg Val Phe Ala Asp Gly Phe 180 185 190 Ile Ser Gly Asp Ala Val Glu Cys Ser Val Asn Leu Gln Leu Val Gly 195 200 205 Glu Ala Cys Phe Arg Asn Pro Leu Ile Val Ala Val Thr Glu Trp Ala 210 215 220 Ser Ala Asn Gly Asp Glu Ile Thr Pro Thr Val Phe Leu Ser Val Glu 225 230 235 240 Thr Asp Glu Leu Arg His Met Ala Asn Gly Tyr Gln Thr Val Val Ser 245 250 255 Ile Ala Asn Asp Pro Ala Ser Ala Lys Phe Leu Asn Thr Asp Leu Asn 260 265 270 Asn Ala Phe Trp Thr Gln Gln Lys Tyr Phe Thr Pro Val Leu Gly Tyr 275 280 285 Leu Phe Glu Tyr Gly Ser Lys Phe Lys Val Glu Pro Trp Val Lys Thr 290 295 300 Trp Asn Arg Trp Val Tyr Glu Asp Trp Gly Gly Ile Trp Ile Gly Arg 305 310 315 320 Leu Gly Lys Tyr Gly Val Glu Ser Pro Ala Ser Leu Arg Asp Ala Lys 325 330 335 Arg Asp Ala Tyr Trp Ala His His Asp Leu Ala Leu Ala Ala Tyr Ala 340 345 350 Met Trp Pro Leu Gly Phe Ala Arg Leu Ala Leu Pro Asp Glu Glu Asp 355 360 365 Gln Ala Trp Phe Glu Ala Asn Tyr Pro Gly Trp Ala Asp His Tyr Gly 370 375 380 Lys Ile Phe Asn Glu Trp Lys Lys Leu Gly Tyr Glu Asp Pro Lys Ser 385 390 395 400 Gly Phe Ile Pro Tyr Gln Trp Leu Leu Ala Asn Gly His Asp Val Tyr 405 410 415 Ile Asp Arg Val Ser Gln Val Pro Phe Ile Pro Ser Leu Ala Lys Gly 420 425 430 Thr Gly Ser Leu Arg Val His Glu Phe Asn Gly Lys Lys His Ser Leu 435 440 445 Thr Asp Trp Gly Glu Arg Gln Trp Leu Ile Glu Pro Glu Arg Tyr 450 455 460 Glu Cys His Asn Val Phe Glu Gln Tyr Glu Gly Arg Glu Leu Ser Glu 465 470 475 480 Val Ile Ala Glu Gly His Gly Val Arg Ser Asp Gly Lys Thr Leu Ile 485 490 495 Ala Gln Pro His Thr Arg Gly Asp Asn Leu Trp Thr Leu Glu Asp Ile 500 505 510 Lys Arg Ala Gly Cys Val Phe Pro Asp Pro Leu Ala Lys Phe 515 520 525 <210> 4 <211> 1578 <212> DNA <213> Artificial Sequence <220> <223> mutant <400> 4 atggcgatca gtctcgctac gaaagcggcg accgatgctc tgaaggtcaa ccgcgctccg 60 gtcggcgtgg agcctcagga ggtccacaaa tggctgcaga gcttcaactg ggacttcaaa 120 gagaccgga cgaagtatcc gaccaaatat cacatggcga atgagaccaa ggagcagttc 180 aaggtcatcg ccaaggaata cgcccgcatg gaggcggcca aggacgagcg ccagttcggc 240 actcttctcg acggcctcac ccgcctcggc gccggcaaca aggtccatcc ccgctggggc 300 gagacgatga aggtgatctc gaacttcctc gaggtcggcg aatataacgc gatcgccgct 360 tcggccatgc tttgggacag cgccaccgcc gccgagcaga agaacggcta tctcgcgcag 420 gtgctcgacg agattcgtca cacgcatcaa tgcgccttca tcaatcacta ttactccaag 480 cattatcacg atccggccgg ccacaatgac gcccgtcgca cgcgcgcgat cggtccgctg 540 tggaagggca tgaagcgcgt cttcgccgac ggcttcatct ccggcgacgc cgtggagtgc 600 tcggtcaatc tgcagctggt cggcgaagcc tgcttccgca atccgctcat cgtcgccgtc 660 accgaatggg cttcggccaa tggcgacgag atcacgccga ccgtgttcct ctcggtggag 720 accgacgagc tgcgtcatat ggcgaacggc taccagaccg tggtgtcgat cgccaatgat 780 ccggcttcgg cgaagttcct caacaccgat ctgaacaacg ccttctggac gcagcagaaa 840 tatttcacgc ccgtcctcgg ctatctgttc gagtatggct ccaagttcaa ggtcgagccc 900 tgggtgaaga cctggaaccg ctgggtctac gaggattggg gtggaatctg gatcggccgt 960 ctcggcaaat atggcgtcga gagcccggcg tcgctgcgcg acgccaagcg cgacgcttac 1020 tgggcgcatc acgatctggc gctcgccgcc tatgcgatgt ggccgctcgg cttcgcgcgt 1080 ctcgctcttc ccgacgagga agatcaggcg tggttcgagg cgaattatcc gggctgggcc 1140 gatcactacg gcaagatctt caacgagtgg aagaagctcg gctatgaaga tcccaagagc 1200 ggcttcatcc cctatcagtg gctcctcgcg aacggtcacg acgtctatat cgaccgtgtc 1260 tcgcaggttc cgttcattcc gtcgctggcc aagggcacgg gctcgctgcg cgtgcacgag 1320 ttcaacggca agaagcattc gctgacggat gattggggtg agcgccagtg gctgatcgag 1380 ccggagcgct acgagtgcca caacgtcttc gagcagtatg agggacgcga attgtccgag 1440 gtgatcgccg agggccatgg cgttcgctcc gacggcaaga cgctgatcgc tcagccgcac 1500 acgcgcggcg acaatctgtg gacgctcgag gacatcaagc gggccggctg cgtgttcccc 1560 gatccgctcg ccaaattc 1578
Claims (10)
Wherein the mutant enzyme is composed of the amino acid sequence of SEQ ID NO: 3. 3. The mutant methionine oxidase (sMMO) according to claim 1,
Wherein the gene comprises the nucleotide sequence of SEQ ID NO: 4.
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