KR101936975B1 - A mutant of Methylosinus trichosporium OB-3b sMMO hydroxylase improved in activity and use of the same - Google Patents

Abstract

The present invention utilizes quantum mechanics and molecular mechanics techniques and partial mutation techniques to increase the stability and enzymatic activity of sMMO hydroxylase from methylosynthritol trisporosporium OB-3b. More particularly, the present invention relates to a method for screening for a residue affecting enzyme stability through screening based on quantum mechanics and molecular mechanics, a sMMO hydroxylase whose enzyme activity is improved by a mutation of the detected residue, a nucleic acid molecule encoding the enzyme, A vector containing the vector, a transformant containing the vector, a mutant of the sMMO hydroxylase, and a method for producing the improved sMMO hydroxylase.

Description

[0001] The present invention relates to a mutant soluble methane single-element digestive enzyme hydroxylase derived from methylosinus tricosporium having improved activity and a use thereof.

The present invention relates to a mutant sMMO hydroxylase derived from methylosinus tricosporium OB-3b having improved activity and its use.

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, methane oxidizing bacteria oxidize methane to methanol and methanol to formaldehyde again. The formaldehyde is then oxidized to formic acid, which ultimately converts it to less toxic carbon dioxide. Although the methanotrophic bacteria does not utilize an organic compound having a carbon-carbon bond as a growth material, it can also oxidize many alkanes and aromatics by the action of MMO enzymes. On the other hand, a lot of researches for recycling the methane generated in the waste disposal site are being carried out, and studies for converting methane to methanol using methane oxidizing bacteria are underway. However, the enzyme has disadvantages of instability and low enzyme activity.

[Prior Patent Literature]

 Korean Patent Publication No. 1020110006964

DISCLOSURE OF THE INVENTION An object of the present invention is to solve the above-mentioned problems and to provide a process for the production of sMMO-hydroxysuccinimide .

A second object of the present invention is to provide a recombinant expression vector containing the improved sMMO hydroxylase gene.

A third object of the present invention is to provide all the transformation strains comprising the recombinant methylorthosynthesis tricosporium transformed with the improved gene.

A fourth object of the present invention is to provide a recombinant sMMO hydroxylase using recombinant methylorthosynthesis tricosporium transformed with an improved enzyme.

A fifth object of the present invention is to propose residues which affect the activity of sMMO hydrolytic enzymes using the enzyme.

In order to achieve the above object, the present invention provides a mutant sMMO selected from the group consisting of a mutant in which the 97th amino acid proline of the sMMO hydroxylase of SEQ ID NO: 1 is substituted with leucine and a 191th amino acid glycine is replaced by isoleucine methane monooxygenase) hydroxylase.

In one embodiment, the enzyme when methyl Taunus tricot sports Solarium OB-3b (Methylosinus trichosporium OB- 3b ), but sMMO hydroxylase produced by chemical synthesis or genetic engineering methods is also covered by 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 or 5, but it is preferable that the mutant enzyme has an amino acid sequence of SEQ ID NO: 3 or 5, Mutants are also included within the scope of the present invention.

The present invention also provides a gene encoding the enzyme of the present invention.

The present invention also provides a recombinant vector comprising the gene of the present invention.

The present invention also provides a composition for degrading aromatic hydrocarbons and aromatic compounds comprising the mutant enzyme of the present invention as an active ingredient.

The present invention also provides a composition for decomposing aromatic hydrocarbons and aromatic compounds containing the gene of the present invention as an active ingredient.

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.

Hereinafter, the present invention will be described.

The present invention provides an underlying technology for identifying the determinants of activity of hydroxylase by suggesting several residues that play an important role in enzyme activity using the sMMO hydroxylase of the present invention.

The present invention also provides sMMO hydroxylase having high activity using the mutant sMMO hydroxylase of the present invention.

Hereinafter, the present invention will be described in detail.

SEQ ID NOS: 4 and 6 denote the nucleotide sequences of the mutated sMMO hydroxylase gene of the present invention, and SEQ ID NOS: 3 and 5 denote the amino acid sequences encoded by the genes. As described above, as long as the polypeptide having the amino acid sequence has the sMMO hydroxylase activity, there may be mutations such as deletion, substitution, and addition to several amino acids. In addition, the gene of the present invention includes, in addition to those having a base sequence encoding the amino acid sequence represented by SEQ ID NOS: 4 and 6, a prodrug that encodes the same polypeptide that differs only in the axial codon. Here, mutations 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.

In the present invention, the gene of sMMO-hydroxylase was cloned from methylosynthostricosporium OB-3b in order to secure a highly active enzyme and to suggest some residues which play an important role in the activity of the enzyme. The recombinant strains inserted with the electric gene showed several residues which play an important role in the high activity, and it was confirmed that it is possible to provide an underlying technology for the identification of highly active determinants.

The metabolism rate of P97L mutant was 4.60 ± 0.1 when toluene was used as a substrate for mutants in which the residues that play an important role in the activity were substituted with the sMMO hydroxylase derived from methylcystinosetricosporium OB-3b obtained in the present invention nmol min ^-1 mg ^{- 1} and the metabolic rotation rates of P97L and G191I variants were 0.37 ± 0.4 and 0.35 ± 0.3 nmol min ^-1 mg ^{- 1} , respectively, when biphenyl was used as a substrate.

In the present invention, sMMO hydroxylase mutant exhibiting high activity could be used to increase the metabolic rotation rate to the substrate. This will be useful for the production of methanol from methane by overcoming the low enzymatic activity problems of the existing sMMO hydroxides.

Methanol, which is a greenhouse gas generated by sMMO hydroxycarboxylase, has activity to produce methanol, which is a useful substance, from the substrate, but it has disadvantages such as instability of the enzyme and low enzyme activity.

Therefore the present invention to develop a metabolic enzyme speed improved by mutation of residues that play an important role in the activity of Janus tricot sports Solarium OB-3b (Methylosinus trichosporium OB -3b ) sMMO hydroxide enzyme derived during a methyl. Also, mutants of the sMMO hydroxylase and improved sMMO hydroxylase can be prepared.

Figure 1 shows the amino acid sequence of wild-type sMMO hydroxylase.
Figure 2 shows the nucleotide sequence of wild-type sMMO hydroxylase.
Fig. 3 shows the amino acid sequence of the sMMO hydroxylase P97L mutant.
Figure 4 shows the nucleotide sequence of the sMMO hydroxylase P97L mutant.
Fig. 5 shows the amino acid sequence of the mutant of sMMO hydroxylase G191I.
FIG. 6 shows the nucleotide sequence of the mutant of sMMO hydroxylase G191I.

Hereinafter, the present invention will be described in more detail with reference to the following non-limiting examples, but the present invention is not limited by the following examples.

Example  One: sMMO  Gene of hydroxylase Cloning

The mutations were amplified using primers described in detail in Table 1 using four primer overlap-extension PCR methods (Smith TJ, et al. Appl. Environ. Microbiol, 68, 5265-5273, 2002; Ho SN, et al. Gene, 77, 51-59, 1989). The mutated PCR products were cloned into the remainder of the sMMO operon in pT2ML using Bam HI and Nde I. The PCR-derived portion of all replicons was confirmed to be free of unwanted mutations by dye termination sequencing.

Primer Oligonucleotide sequence 5 '→ 3' P3 ATT CGA GCT CAA ACG TTC GAA C P4 GGG CTC TCG ACG CCA TAT TTG P97L TCC ATC TCC GCT GGG GCG AGA G191I TTC GCC GAC ATC TTC ATC TCC GG

Table 1 is a table of oligonucleotide sequences used for mutation.

Example  2: sMMO  Identification of the activity of hydroxylase

A 5 ml aliquot of the bacterial suspension was prepared by stirring the substrate (15 μl toluene 5 μl liquid or solid substrate biphenyl 15 mg) with 5 mM sodium formate for 24 hours at 200 rpm to provide a reduced amount of sMMO Lt; / RTI > The hydroxylated product was extracted with 1.25 ml diethyl ether and evaporated to a final volume of 50 [mu] l, vanillin (20 nmoles) was used as the internal standard. Samples (1 μl) were analyzed by GC-MS using a 5890 GC (Hewlett Packard) coupled with a Trio-1 mass spectrometer. GC was fitted with a Hewlett Packard HP-5 column with a (5% phenyl) methylpolysiloxane coating (0.32 mm × 50 m, coating thickness 0.25 μm) and operated as a carrier gas (nitrogen) flow rate of 1.5 ml min -1. Separation without split was performed and the column temperature was increased from 80 ° C to 126 ° C by 10 ° C per minute and then increased from 126 ° C to 129 ° C by 0.1 ° C per minute and finally increased from 129 ° C to 250 ° C by 10 ° C per minute.

Example  3: with high activity sMMO  Hydroxylase Mutant  making

Example  3-1: play an important role in activity Residue  Confirm

Quantum mechanics / molecular mechanics of Discovery Studio 3.1 and Meterials Studio 6.0 (Accelrys Inc. San Diego, Calif., USA) to explore residues that play an important role in high activity using sMMO hydroxylase. Method. The binding energy (Kcal / mol) of the mutants in which the residues of the substrate binding site of the enzyme were replaced with alanine was compared with the binding energy of the wild enzyme. The binding energy is known to be stronger at lower values. As shown in Table 2, the P97L and G191I mutants had lower binding affinities than wild-type enzymes, and P97L and G191I residues were selected as the target residues for mutagenesis.

enzyme A + B (enzyme substrate complex) A
(temperament) B
(enzyme) BE _QM
(Hartree) Bonding energy
(Kcal / mol) WT -12815.40 -569.62 -12245.7189 -0.062 -39.0 P97L -12628.70 -569.62 -12059.0203 -0.066 -41.9 L191I -12608.94 -569.62 -12039.2535 -0.068 -42.8

Example  3-2: P97L , G191I Mutant  Metabolic rotation speed

As in Example 3-1, residues at the 97th position and 191th position were substituted with Leu and Ile, respectively, in the mutants in the substrate binding site. As shown in Tables 3 and 4, when the toluene was used as a substrate, the mutant P97L was 2.9 times more active than the pure enzyme. When biphenyl was used as a substrate, the activity of P97L mutant and G191I mutant increased 6 times and 4.8 times as compared with pure enzyme, respectively.

Enzyme Benzyl alcohol o-crseol m-crseol p-crseol turnover rate
(nmol min ^-1 mg ^-1 ) WT 63.1 ± 2.6 ND ND 33.9 ± 2.6 1.60 ± 0.3 P97L 67.1 ± 1.7 ND ND 32.9 ± 1.7 4.60 ± 0.1 G191I 80.0 ± 0.3 ND ND 20.0 ± 0.3 1.2 ± 0.3

Enzyme 2-hydroxy biphenyl 3-hydroxy biphenyl 4-hydroxy biphenyl turnover rate
(nmol min ^-1 mg ^-1 ) WT 6.1 ± 1.5 ND 93.9 ± 1.5 0.062 ± 0.01 P97L 0.94 0.22 1.17 0.30 97.9 ± 0.51 0.37 ± 0.1 G191I 0.00 ± 1.29 7.77 ± 1.29 92.2 ± 1.29 0.30 ± 0.1

Tables 3 and 4 show the activity of pure sMMO hydroxylase and mutants on mono-aromatic (toluene) and di-armoatic (biphenyl) substrates, respectively.

<110> Konkuk University Industrial Cooperation Corp <120> A mutant of Methylosinus trichosporium OB-3b sMMO hydroxylase          improved in activity and use of the same <130> HY141015 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 526 <212> PRT <213> Methylosinus trichosporium <400> 1 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 Thr 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 Ser 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> 2 <211> 1581 <212> DNA <213> Methylosinus trichosporium <400> 2 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 cacgcatcag tgcgccttca tcaatcacta ttactccaag 480 cattatcatg atccggccgg tcacaacgac gcccgtcgca cccgtgcgat cggccccttg 540 tggaagggca tgaagcgcgt cttcgccgac ggcttcatct ccggcgacgc cgtggaatgc 600 tcggtcaatc tgcagctggt cggcgaagcc tgcttcacca atccgctgat cgtcgccgtc 660 accgaatggg cctcggccaa tggcgacgag atcacgccca ccgtcttcct ctcggtggag 720 accgacgagc tgcgtcatat ggcgaacggc taccagaccg tggtgtcgat cgccaatgat 780 ccggcctcgg cgaagttcct caacaccgat ctcaacaacg ccttctggac gcagcagaaa 840 tatttcacgc ccgtcctcgg ctatctgttc gagtacggct ccaagttcaa ggtcgagccg 900 tgggtgaaga cctggaaccg ctgggtctac gaggattggg gtggaatctg gatcggccgt 960 ctcggcaagt atggcgtcga gagcccggct tcgctgcgcg acgccaagcg cgacgcctat 1020 tgggcgcatc acgatctggc gctcgccgcc tatgcgatgt ggccgctcgg cttcgcgcgt 1080 ctcgctcttc cggacgagga ggaccaggcg tggttcgagg cgaattatcc gggctgggcc 1140 gatcactacg gcaagatctt caacgagtgg aagaagctcg gctatgagga tcccaagagc 1200 ggattcatcc cctacaagtg gctcctcgag aacggtcacg acgtctacat cgaccgcgtc 1260 tcgcaggttc cgttcattcc gtcgctggcc aagggctcgg gctcgctccg cgttcacgag 1320 ttcaacggca agaagcattc gctgacggat gattggggtg agcgccagtg gctgatcgag 1380 ccggagcgct acgagtgcca caatgtcttc gagcagtacg agggacgcga attgtccgag 1440 gtgatcgccg agggccatgg cgttcgctcc gatggcaaga cgctgatcgc tcagcctcac 1500 acgcgcggcg acaatctctg gacgctcgag gacatcaagc gcgcgggctg cgtgttcccc 1560 gatccgctcg ccaagttctg a 1581 <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 Leu 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 Thr 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 Ser 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> 1581 <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 aggtccatct ccgctggggc 300 gagacgatga aggtgatctc gaacttcctc gaggtcggcg aatataacgc gatcgccgct 360 tcggccatgc tttgggacag cgccaccgcc gccgagcaga agaacggcta tctcgcgcag 420 gtgctcgacg agattcgtca cacgcatcag tgcgccttca tcaatcacta ttactccaag 480 cattatcatg atccggccgg tcacaacgac gcccgtcgca cccgtgcgat cggccccttg 540 tggaagggca tgaagcgcgt cttcgccgac ggcttcatct ccggcgacgc cgtggaatgc 600 tcggtcaatc tgcagctggt cggcgaagcc tgcttcacca atccgctgat cgtcgccgtc 660 accgaatggg cctcggccaa tggcgacgag atcacgccca ccgtcttcct ctcggtggag 720 accgacgagc tgcgtcatat ggcgaacggc taccagaccg tggtgtcgat cgccaatgat 780 ccggcctcgg cgaagttcct caacaccgat ctcaacaacg ccttctggac gcagcagaaa 840 tatttcacgc ccgtcctcgg ctatctgttc gagtacggct ccaagttcaa ggtcgagccg 900 tgggtgaaga cctggaaccg ctgggtctac gaggattggg gtggaatctg gatcggccgt 960 ctcggcaagt atggcgtcga gagcccggct tcgctgcgcg acgccaagcg cgacgcctat 1020 tgggcgcatc acgatctggc gctcgccgcc tatgcgatgt ggccgctcgg cttcgcgcgt 1080 ctcgctcttc cggacgagga ggaccaggcg tggttcgagg cgaattatcc gggctgggcc 1140 gatcactacg gcaagatctt caacgagtgg aagaagctcg gctatgagga tcccaagagc 1200 ggattcatcc cctacaagtg gctcctcgag aacggtcacg acgtctacat cgaccgcgtc 1260 tcgcaggttc cgttcattcc gtcgctggcc aagggctcgg gctcgctccg cgttcacgag 1320 ttcaacggca agaagcattc gctgacggat gattggggtg agcgccagtg gctgatcgag 1380 ccggagcgct acgagtgcca caatgtcttc gagcagtacg agggacgcga attgtccgag 1440 gtgatcgccg agggccatgg cgttcgctcc gatggcaaga cgctgatcgc tcagcctcac 1500 acgcgcggcg acaatctctg gacgctcgag gacatcaagc gcgcgggctg cgtgttcccc 1560 gatccgctcg ccaagttctg a 1581 <210> 5 <211> 526 <212> PRT <213> Artificial Sequence <220> <223> Mutant <400> 5 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 Ile 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 Thr 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 Ser 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> 6 <211> 1581 <212> DNA <213> Artificial Sequence <220> <223> Mutant <400> 6 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 cacgcatcag tgcgccttca tcaatcacta ttactccaag 480 cattatcatg atccggccgg tcacaacgac gcccgtcgca cccgtgcgat cggccccttg 540 tggaagggca tgaagcgcgt cttcgccgac atcttcatct ccggcgacgc cgtggaatgc 600 tcggtcaatc tgcagctggt cggcgaagcc tgcttcacca atccgctgat cgtcgccgtc 660 accgaatggg cctcggccaa tggcgacgag atcacgccca ccgtcttcct ctcggtggag 720 accgacgagc tgcgtcatat ggcgaacggc taccagaccg tggtgtcgat cgccaatgat 780 ccggcctcgg cgaagttcct caacaccgat ctcaacaacg ccttctggac gcagcagaaa 840 tatttcacgc ccgtcctcgg ctatctgttc gagtacggct ccaagttcaa ggtcgagccg 900 tgggtgaaga cctggaaccg ctgggtctac gaggattggg gtggaatctg gatcggccgt 960 ctcggcaagt atggcgtcga gagcccggct tcgctgcgcg acgccaagcg cgacgcctat 1020 tgggcgcatc acgatctggc gctcgccgcc tatgcgatgt ggccgctcgg cttcgcgcgt 1080 ctcgctcttc cggacgagga ggaccaggcg tggttcgagg cgaattatcc gggctgggcc 1140 gatcactacg gcaagatctt caacgagtgg aagaagctcg gctatgagga tcccaagagc 1200 ggattcatcc cctacaagtg gctcctcgag aacggtcacg acgtctacat cgaccgcgtc 1260 tcgcaggttc cgttcattcc gtcgctggcc aagggctcgg gctcgctccg cgttcacgag 1320 ttcaacggca agaagcattc gctgacggat gattggggtg agcgccagtg gctgatcgag 1380 ccggagcgct acgagtgcca caatgtcttc gagcagtacg agggacgcga attgtccgag 1440 gtgatcgccg agggccatgg cgttcgctcc gatggcaaga cgctgatcgc tcagcctcac 1500 acgcgcggcg acaatctctg gacgctcgag gacatcaagc gcgcgggctg cgtgttcccc 1560 gatccgctcg ccaagttctg a 1581

Claims (8)

A mutant sMMO hydroxylase characterized in that the 97th amino acid proline of sMMO hydroxylase of SEQ ID NO: 1 is a mutant substituted with leucine. 2. The mutant sMMO hydroxylase according to claim 1, wherein said enzyme is sMMO-hydroxylase derived from methyl- rhosinostricosporium OB-3b. 2. The mutant sMMO hydroxylase according to claim 1, wherein the mutant enzyme comprises the amino acid sequence of SEQ ID NO: 3. A gene encoding the enzyme of claim 1. 5. The gene according to claim 4, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 4. A recombinant vector comprising the gene of claim 4. A composition for decomposing a chain-like hydrocarbon or an aromatic compound comprising the enzyme of claim 1 as an active ingredient. A composition for degrading a chain-like hydrocarbon or an aromatic compound comprising the gene of claim 4 as an active ingredient.

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