WO2018124623A1 - Novel recombinant tyrosinase - Google Patents

Novel recombinant tyrosinase Download PDF

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WO2018124623A1
WO2018124623A1 PCT/KR2017/015183 KR2017015183W WO2018124623A1 WO 2018124623 A1 WO2018124623 A1 WO 2018124623A1 KR 2017015183 W KR2017015183 W KR 2017015183W WO 2018124623 A1 WO2018124623 A1 WO 2018124623A1
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tyrosinase
seq
recombinant
present
cnk
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PCT/KR2017/015183
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Korean (ko)
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최유성
도현수
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충남대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/18—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with another compound as one donor, and incorporation of one atom of oxygen (1.14.18)
    • C12Y114/18001—Tyrosinase (1.14.18.1)

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  • the present invention relates to a novel recombinant tyrosinase prepared by newly redesigned genes based on tyrosinase present in marine polar ammonia oxidation-related bacteria.
  • Tyrosinase is classified similarly to laccase, which is involved in the breakdown of lignin, and hemocyanin, which is required for the respiration of invertebrates, and type-3 copper containing copper at the active site.
  • laccase which is involved in the breakdown of lignin, and hemocyanin, which is required for the respiration of invertebrates, and type-3 copper containing copper at the active site.
  • an enzyme monophenol monooxygenase, EC. 1.14.18.1
  • Tyrosinase is a catalyst that converts L-tyrosine (L-Tyr), which is an early stage of melanin biosynthesis, into L-3,4-dihydroxyphenylalanine (L-DOPA), and converts L-DOPA into dopaquinone.
  • L-Tyr L-tyrosine
  • L-DOPA L-3,4-dihydroxyphenylalanine
  • melanin protects cells from UV rays, free radicals and toxic heavy metals.
  • Tyrosinase-based melaninization reactions are involved in wound healing and primary immune responses in such a way that plants such as apples, potatoes and bananas are cut and cause browning of the cut surface when exposed to air. In animals, it affects the color of the skin, hair, and eyes, and may be involved in brown pigmentation of the skin or hardening of the wound.
  • tyrosinase can be used to convert various forms of monophenolic or diphenolic compounds into quinones using the reaction substrate.
  • tyrosinase not only catalyzes the oxidation reaction of phenolic compounds for melanination, but also purifies wastewater or contaminated water containing phenols, biosynthesis of antibiotics such as lincomycin, and medical materials for treating Parkinson's disease.
  • L-DOPA biosensor for measuring the amount of phenolic compounds, and cross-linking of proteins and sugars.
  • tyrosinase Although the gene sequence of tyrosinase associated with melanin biosynthesis is known in many organisms, relatively little information is known about the biochemical properties of purified tyrosinase. Usually, the genome of an organism that has a tyrosinase gene contains one or more tyrosinase genes, and each tyrosinase has different biochemical properties. For example, MelC2 and MelD2, which are representative tyrosinase produced from actinomycetes, are secreted out of cells and enzymes are active in various substrates, whereas MelD2 is expressed in cells and is highly substrate specific. In general, tyrosinase is a monomeric protein having a molecular weight of about 20 to 60 kDa, and has an optimum pH of about 7.5 and an optimum reaction temperature at 40 ° C.
  • Tyrosinase which is present in bacteria in recent years, depends on the need for additional proteins, called caddie proteins, for enzymes to be active, the domain composition of one motif that binds to oxygen, and two motifs that bind to copper. It is classified into different types.
  • tyrosinase which is currently used commercially, is produced by extracting A. bisporus tyrosinase from mushrooms sold by Sigma. Although not yet commercialized, various types of bacteria-derived tyrosinase are likewise extracted directly from strains carrying the gene or produced as recombinant proteins in foreign strains.
  • the present inventors have provided a novel recombinant tyrosinase derived from marine polar ammonia oxidation-related bacteria in a previous study, wherein the tyrosinase is a novel enzyme whose tyrosinase does not belong to the five existing tyrosinase classes.
  • the tyrosinase is a novel enzyme whose tyrosinase does not belong to the five existing tyrosinase classes.
  • mass production in Escherichia coli it showed high activity at weakly acidic pH and low temperature unlike conventional enzymes (Domestic Patent Application 10-2015-0053189).
  • the enzyme in order to produce a water-soluble protein, the enzyme must simultaneously express Chaperone (Chaperone) protein with tyrosinase, and stability is well maintained at 0 ° C., but is slightly low at room temperature (20 ° C.), such as L-tyrosine.
  • Chaperone Chaperone
  • tyrosinase tyrosinase
  • stability is well maintained at 0 ° C., but is slightly low at room temperature (20 ° C.), such as L-tyrosine.
  • L-tyrosine room temperature
  • the reaction of a substrate such as L-tyrosine present in a polymer such as mussel adhesive protein is not performed efficiently, and thus a separate protein for water-soluble protein expression is not required.
  • the inventors of the present invention while studying the tyrosinase that can overcome the existing limitations, the activity and stability when removing a certain length of the C-terminal amino acid sequence from the tyrosinase derived from marine polar ammonia oxidation-related bacteria In view of the novel industrial tyrosinase more excellent industrial utility in terms of the present invention was completed. It is therefore an object of the present invention to provide novel recombinant tyrosinase with improved activity and stability.
  • the present invention provides a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • the present invention also provides a composition for converting tyrosinase substrates comprising recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • the present invention also provides a composition for converting a monophenol or catechol substrate in a natural polymer or synthetic polymer, comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • the present invention also provides a polynucleotide encoding the recombinant tyrosinase.
  • the present invention also provides a vector comprising a polynucleotide encoding the recombinant tyrosinase.
  • the present invention also provides a transformant transformed with the vector.
  • the present invention comprises the steps of isolating and purifying recombinant tyrosinase from the transformant; It provides a method for producing a recombinant tyrosinase comprising.
  • the present invention comprises the steps of treating the tyrosinase substrate with a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3; It provides a tyrosinase substrate conversion method comprising a.
  • the present invention also provides a biological material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • the present invention provides a composition for producing a bioadhesive material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • the recombinant tyrosinase of the present invention can be mass-produced very easily from a transformant, and the reaction can proceed efficiently unlike conventional enzymes at a wide pH and room temperature and low temperature. Therefore, the recombinant tyrosinase of the present invention can be variously used for the production of functional medicines and biomaterials containing phenolic compounds, and using tyrosinase such as environmental purification process of phenolic compounds that are harmful to the environment. It can be usefully used in various industrial fields requiring a catalytic reaction.
  • FIG. 1 is a diagram showing the results of structural analysis of tyrosinase using the I-TASSER server.
  • A) of FIG. 1 shows the structure of total tyrosinase-CNK (1-415), and
  • (b) of FIG. 1 shows the predictive structure of mTyr-CNK (1-303) from which the C-terminus was removed.
  • Figure 3 shows the results of expression and purification analysis of tyrosinase-CNK, mTyr-CNK, cTyr-CNK (Sol; aqueous supernatant fraction; Ins: insoluble cell deris fraction; Elu: Ni-NTA affinity chromatography Purified tyrosinase used; Mk: protein molecular weight marker).
  • Figure 4 is a view showing the results of confirming the relative changes in the initial activity of mTyr-CNK with pH changes.
  • 5 is a view showing the results of confirming the relative initial activity change of mTyr-CNK with temperature changes.
  • Figure 6 is a diagram showing the thermal stability evaluation results of mTyr-CNK according to the culture time and temperature.
  • MAP mussel adhesive protein
  • the present invention provides a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • the recombinant tyrosinase of the present invention is a novel tyrosinase that exhibits new biochemical properties exhibiting high activity even at low temperatures to room temperature and a wide pH, and can be expressed as water-soluble without adding chaperone. In addition, it exhibits high activity in a wide pH range, and can perform enzymatic reactions while suppressing natural oxidation reactions occurring at weakly acidic, neutral and weakly basic pH conditions. It can maintain enzymatic activity and in particular catalyze the conversion of tyrosinase substrates present in the polymer.
  • the tyrosinase of the present invention consists of an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, 80% to 99%, preferably as long as it exhibits the same degree of activity and properties as tyrosinase of the present invention.
  • Tyrosinase having a homology of 85% to 99%, more preferably 90% to 99% can also be included in the tyrosinase of the present invention without limitation.
  • Tyrosinase of the present invention is derived from marine polar ammonia oxidation-related bacteria, preferably Candidatus nitrosofumilus corensis Nitrosopumilus Koreensis ) can be prepared by a method of recombining a portion of the tyrosinase sequence derived.
  • the tyrosinase of the present invention may be characterized in that the C-terminal extension is removed from the tyrosinase of SEQ ID NO: 1, which is part of the C-terminal extension of the tyrosinase sequence represented by SEQ ID NO: 304 to 415
  • the portion may have been removed, or 294 to 415, all of the C-terminal extension, may have been removed.
  • the enzyme activity may be different, and the recombinant tyrosinase of the present invention may include a core enzyme active domain site, and more preferably, a core enzyme active domain site and His294. And a Pro303 region.
  • the recombinant tyrosinase of the present invention may exhibit the highest activity at an optimum pH, such an optimum pH is pH 3.5 to pH 10, more preferably pH 4.5 to pH 9.5, more preferably pH 6 to 8 days Can be.
  • Such an optimal pH range is a broad active pH range similar to commercial mushroom tyrosinase.
  • the recombinant tyrosinase of the present invention may exhibit excellent activity even in more acidic conditions than conventional tyrosinase having an optimal pH, usually at about pH 7.5.
  • it has a high activity in a wide pH range, it is possible to perform the enzymatic reaction while suppressing the natural oxidation reaction occurring in weakly acidic, neutral and weakly basic pH conditions.
  • the recombinant tyrosinase of the present invention may be characterized as being active at low or normal temperatures, preferably at -20 to 35 ° C, more preferably at -10 to 30 ° C, even more preferably at 0 to 30 ° C. High activity.
  • the recombinant tyrosinase of the present invention is able to maintain about 80% of the maximum activity even at a temperature below 0 °C, there is an advantage that can be maintained at sub-zero storage temperature.
  • the recombinant tyrosinase of the present invention may exhibit optimal activity at 0 to 30 ° C., not 37 ° C., which is a general activity temperature. When it changes to 37 degreeC conditions, the activity can abruptly lose
  • the present invention provides a composition for converting tyrosinase substrate comprising recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • tyrosinase substrate may include without limitation various substrates in which enzymatic reactions can be caused by tyrosinase, monophenol, catechol, preferably tyrosine or DOPA (3,4-dihydroxyphenylalanine), More preferably L-tyrosine or L-DOPA.
  • tyrosinase substrate conversion means catalyzing and converting an enzymatic reaction of a substrate capable of reacting with tyrosinase. For example, tyrosinase tyrosine is converted to L-DOPA and tyrosinase substrate Converting L-DOPA to dopaquinone.
  • the recombinant tyrosinase of the present invention has a structure that facilitates the reaction between the active site and the substrate, and thus includes a natural polymer and a monophenol containing a monophenol or a catechol group. It is possible to effectively catalyze the reaction of monophenol or catechol of phenolic synthetic polymers containing catechol groups.
  • the natural polymer including the monophenol or catechol may be one or more selected from the group consisting of collagen, gelatin, tannin, lignin and soy protein, and the synthetic polymer is a phenolic resin such as Novolac. It may be a substrate included in.
  • a reaction comprising a substrate contained in the mussel adhesive protein, such as the L-tyrosine residue of the mussel adhesive protein.
  • a substrate contained in the mussel adhesive protein such as the L-tyrosine residue of the mussel adhesive protein.
  • mussel adhesive protein produced in Escherichia coli tyrosine residues are not modified. Therefore, it is necessary to convert tyrosine residues in vitro using a separate enzyme.
  • a substrate included in a polymer such as an mussel adhesive protein since the binding to the active site of the enzyme is not easy, there is a disadvantage that it is difficult to easily convert the substrate in vitro.
  • the recombinant tyrosinase of the present invention can react with the L-tyrosine of the mussel adhesive protein and convert it to L-DOPA with high efficiency, especially in comparison with the tyrosinase-CNK represented by SEQ ID NO: 1.
  • L-tyrosine can be converted to L-DOPA in yield.
  • the present invention is a natural polymer comprising a tyrosinase substrate conversion composition or recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3, characterized in that for converting tyrosinase substrate contained in the mussel adhesive protein
  • compositions for converting monophenol or catechol substrates in synthetic polymers are provided.
  • the present invention can be used for tyrosinase substrate conversion for use in a wide range of pH, low temperature and room temperature by using a recombinant tyrosinase represented by SEQ ID NO. .
  • the present invention provides a composition for converting tyrosinase substrate in an environment of pH 4.5 to pH 9.5 and a composition for converting tyrosinase substrate in an environment of -20 ° C to 35 ° C.
  • the present invention also provides a polynucleotide encoding a recombinant tyrosinase of SEQ ID NO: 2, which preferably includes without limitation the nucleotide sequence represented by SEQ ID NO: 4 or a homologous polynucleotide having a functional equivalent thereto Can be.
  • the present invention also provides a polynucleotide encoding a recombinant tyrosinase of SEQ ID NO: 3, which preferably includes without limitation the base sequence represented by SEQ ID NO: 5 or a homologous polynucleotide having a functional equivalent thereto Can be.
  • the homologous polynucleotide refers to a sequence that is similar or interchangeable between two or more polynucleotide sequences.
  • the homologous polynucleotide is at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably at least the polynucleotide sequence encoding the amino acid of SEQ ID NO: 2.
  • Any polynucleotide sequence that satisfies may also be included without limitation.
  • the present invention also provides a vector comprising a polynucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • vector is used to refer to DNA fragment (s), nucleic acid molecules that are delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells.
  • expression vector refers to a recombinant DNA molecule comprising a suitable nucleic acid sequence necessary to express a coding sequence of interest and a coding sequence operably linked in a particular host organism.
  • the expression vector may preferably comprise one or more selectable markers, such markers are typically nucleic acid sequences having properties that can be selected by chemical methods, to distinguish transformed cells from non-transformed cells. All genes present are this. Examples include, but are not limited to, antibiotic genes such as kanamycin, bleomycin, chloro ampinicol, ampicillin.
  • the present invention also provides a transformant transformed with the vector.
  • transformant refers to the introduction of a new target polynucleotide into a host cell
  • the host cell may include, but is not limited to, E. coli, yeast, animal, plant cell or insect cell.
  • the present invention provides a method for producing a recombinant tyrosinase comprising the step of isolating and purifying the prepared tyrosinase from the transformant.
  • the production method comprises the steps of cloning a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 into an expression vector to prepare a tyrosinase expression vector and transforming it into E. coli; Inducing expression of the recombinant tyrosinase protein in a transformed Escherichia coli via conventional methods; Dividing it into an aqueous and an insoluble fraction and separating and purifying using a column; It may include.
  • the recombinant tyrosinase of the present invention can be detected by overexpression by water solubility without forming an inclusion body in E. coli without a separate chaperone, mass production is possible.
  • the present invention comprises the steps of treating the tyrosinase substrate with a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3; It provides a tyrosinase substrate conversion method comprising a.
  • the tyrosinase substrate conversion method according to the present invention is preferably a tyrosinase substrate for recombinant tyrosinase at -20 to 35 ° C, more preferably at -10 to 30 ° C, even more preferably at 0 to 30 ° C.
  • the process may include the step of.
  • the present invention also provides a biological material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • Biomaterial of the present invention is a kind of medical material, and means a material that can replace some or all of its functions in place of tissues or organs of a human body for a certain period of time in a synthetic, natural or complex form thereof except for medicines. .
  • the present invention provides a composition for producing a bioadhesive material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  • the "bioadhesive material” is applied locally to replace a surgical suture, and can be easily and immediately bonded and sutured to a wound, to fill a total layer of defect tissue caused by skin burn or surgery, or It may include, without limitation, materials that can be used for regeneration in scar tissue and minimizing scarring.
  • biological tissue herein is not particularly limited and includes, for example, skin, nerves, brain, lungs, liver, kidneys, stomach, small intestine, rectum and bones and the like.
  • the recombinant tyrosinase of the present invention can efficiently convert L-DOPA from tyrosine, which plays an important role in the preparation of the bioadhesive material, it can be very useful for preparing the bioadhesive material.
  • tyrosinase-CNK recombinant tyrosinase with improved enzyme activity by improving the recombinant tyrosinase identified in Korean Patent Application No. 10-2015-0053189.
  • the structural homology model was analyzed using I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER), and the model structure was found in Discovery Studio software (Dassault Systems BIOVIA, Discovery Studio Modeling Environment, Release 2016, San Diego: Dassault Systems, 2017).
  • a novel recombinant enzyme from which the C-terminal extension is removed was prepared. As shown in FIG. 2, a recombinant tyrosinase was prepared in which a C-terminal extension portion consisting only of a sequence of 1-303 was removed except a portion (gray) corresponding to 304-415 of the entire tyrosinase sequence.
  • SEQ ID NO: 1 The sequence of total tyrosinase-CNK (1-415) is shown in SEQ ID NO: 1, and the new tyrosinase mTyr-CNK (1-303) recombined through genetic modification is in SEQ ID NO: 2, including the core sequence.
  • Recombinant novel tyrosinase cTyr-CNK (1-293) is shown in SEQ ID NO: 3.
  • Escherichia to Produce Selected Novel Tyrosinase coli DH5 ⁇ (Life Technologies, Carlsbad, Calif., USA) cells were used as host cells for recombinant vector preparation and E. coli BL21 (DE3) (Merck KGaA, Darmstadt, Germany) was used for recombinant tyrosinase expression.
  • the E. coli cells were cultured in LB medium containing 50 ⁇ g ampicillin mL -1 (Sigma-Aldrich, St. Louis, MO, USA).
  • tyrosinase-based tyrosinase derived from marine polar ammonia oxidation-related bacteria
  • its gene was synthesized by codon optimization and an expression vector including the same was prepared.
  • Tyrosinase expression vectors were prepared by adding restriction sites of Nde I and Xho I to both ends of the optimized DNA and cloning them into the expression vector pET23b (+) containing the promoter T7 for expression.
  • the expression vector includes a histidine tag (HHHHHH) consisting of six histidine sequences at the C-terminus and an ampicillin resistance gene to confirm gene introduction.
  • the prepared vector structure pmTyr-CNK was confirmed by direct sequencing.
  • the cTyr-CNK (1-293) gene was inserted into the pET23b + vector in a similar manner and the final vector was described as pcTyr-CNK.
  • E. coli was prepared to express the vector pmTyr-CNK and pcTyr-CNK prepared in order to effectively express the tyrosinase contained in the vector.
  • Vector pmTyr-CNK and pcTyr-CNK containing tyrosinase gene were introduced into E. coli BL21 (DE3) by thermal shock at 42 ° C. for 1 minute and 30 seconds, respectively. Since pmTyr-CNK and pcTyr-CNK were vectors resistant to ampicillin antibiotics, transformed Escherichia coli with the vector was selected by culturing in LB-agar medium containing ampicillin.
  • the transformant was incubated at 200 rpm in a conventional 37 ° C., 50 mL LB medium to which 50 ⁇ g / mL ampicillin was added, and when the absorbance (OD 600 ) of the culture medium reached 0.8 to 1.0, IPTG was an inducer. (isopropyl-ß-D-thiogalactopyranoside, 1 mM) was added to induce the expression of the recombinant tyrosinase protein. After incubation at 20 ° C. for an additional 20 hours after IPTG addition, the cultured cells were centrifuged at 15840 g for 10 minutes, then the supernatant was removed and the resulting pellets were stored at ⁇ 80 ° C.
  • lysis buffer 50 mM sodium phosphate buffer, pH 8.0, 10 mM imidazole, 300 mM NaCl
  • Lysate was centrifuged at 15,840 g and 4 ° C. for 10 minutes. The supernatant was collected and added to a Ni-nitrilotriacetic acid (Ni-NTA) affinity purification column (Qiagen, Germantown, MD, USA) and washed.
  • Ni-NTA Ni-nitrilotriacetic acid
  • mTyr-CNK has a very good improvement in total protein expression in cells compared to tyrosinase-CNK of SEQ ID NO: 1, resulting in some insoluble inclusion body, but tyrosinase-CNK of SEQ ID NO: Contrary to the requirement of chaperones called GroES and GroEL for functional expression, approximately 50% of the total protein was found to be water-soluble without chaperone.
  • mTyr-CNK was purified to a high purity of 95% or more, cTyr-CNK was also expressed and purified water-soluble under the same conditions.
  • Optimum pH was analyzed to analyze the properties of the recombinant tyrosinase protein prepared in Example 2.
  • L-DOPA was used as a reactant to analyze the formation of L-dopachrome at different pH. This assay was performed in 50 mM reaction buffer (glycine-HCl buffer at pH 1-3, sodium acetate buffer at pH 3.5). -5, sodium phosphate buffer at pH 5.5-7, tris-HCl buffer at pH 7.5-9, and glycine-NaOH buffer at pH 9.5-11), 0.01 mM CuSO 4 , 0.05 mM L-DOPA and 0.4 ⁇ M purified mTyr It was performed using -CNK.
  • mTyr-CNK showed the optimum activity at room temperature 20 °C to 25 °C, it was confirmed that even at 0 °C to maintain more than 80% of the maximum activity. That is, mTyr-CNK showed high activity even at low temperature conditions of 0 to 10 ° C. unlike conventional tyrosinase, and showed the highest activity at room temperature around 20 ° C. instead of 37 ° C., which is a general activity temperature. According to these results, it was confirmed that the recombinant mTyr-CNK is an enzyme having new properties that can exhibit high activity even at low temperature and room temperature, unlike the existing tyrosinase.
  • Kinetic parameters of mTyr-CNK activity were determined by L-dopachrome formation using L-tyrosine and L-DOPA analysis using spectrometric assay.
  • the assay was performed using 200 ⁇ L solution containing 50 mM Tris buffer (pH 6.0) and 0.01 mM CuSO 4 .
  • 0.4 ⁇ M purified enzyme and L-tyrosine and L-DOPA at 0.025-0.0625 mM concentration were used as substrates. Formation of L-dopachrome was confirmed by measuring absorbance at 475 nm, and the absorbance was converted using a molar extinction coefficient of 3600 M 1 cm. All measurements were repeated three times.
  • Thermal stability of mTyr-CNK was performed by checking how much activity of enzymes exposed to various temperatures can be maintained, 50 mM Tris buffer (pH 6.0), 0.01 mM CuSO 4 , 0.05 mM L-DOPA and A 200 ⁇ L solution containing 0.4 ⁇ M purified mTyr-CNK was used. Kinetic parameter measurements and thermal stability evaluation results are shown in Table 1 and FIG. 6, respectively.
  • the mTyr-CNK has a significantly higher V max mono / V max di (monophenolase / diphenolase activity) of 3.83, compared to 100 times lower monophenolase activity in many conventional tyrosinase activities than diphenolase activity. ratio, and the kcat / Km value of monophenolase reaction was 87%, which is slightly lower than diphenolase reaction.
  • the activity ratio of mTyr-CNK ( V max mono / V max di) is about 2 times higher than the known kinetic parameters (Table 2, pH6 and 25 ° C. conditions) of tyrosinase-CNK represented by SEQ ID NO: 1. Indicated.
  • mTyr-CNK protein was very stable at 30 °C or less, the half-life at 37 and 40 °C was confirmed that less than 25 minutes and 5 minutes, respectively.
  • Such instability of mTyr-CNK at temperatures above 30 ° C. can be used to selectively deactivate the enzyme after reaction in a process using the enzyme. This feature can be useful in cases where undesired autooxidation in catechol-related biocatalytic reactions should be minimized and the modification of the phenolic moiety of the polymer polymer should be enhanced.
  • mTyr-CNK can maintain the thermal stability well below 30 °C, it is expected that the activity can be well maintained even in the refrigeration (2 to 8 °C) and freezing (20 °C) conditions where the enzyme is usually stored.
  • the degradation of enzyme activity occurs rapidly at 37 ° C., which is an in vivo temperature, and the enzyme activity may quickly disappear when the enzyme reaction is performed at a temperature lower than the living temperature and the temperature is changed to an in vivo environment. It was expected that the applicability would be very high.
  • Mussel adhesive proteins are known as representative DOPA-tethered biomaterials, and DOPA is essential for fast and strong aquatic adhesion.
  • Recombinant mussel adhesive proteins have the advantage of being able to produce large amounts of protein, but less than 15% is an obstacle to low in vitro DOPA modification yields.
  • mTyr-CNK was applied to the in vitro DOPA modification of the recombinant mussel adhesive protein, the change in the DOPA content was confirmed using the amino acid composition analysis method.
  • fp-151 hereinafter referred to as 'MAP'
  • Lyophilized MAP powder was dissolved to a final concentration of 1 mg / mL using a solution containing 50 mM Tris buffer (pH 6.0) and 25 mM ascorbic acid.
  • Purified tyrosinase-CNK or mTyr-CNK of SEQ ID NO: 1 was added to a final concentration of 0.01 mg / mL.
  • the mixture was incubated for 3 hours with gentle stirring at room temperature, dialyzed with 10 mM acetic acid and stored at -80 ° C for further study. 25 ⁇ L of phenol was added to 500 ⁇ L of tyrosinase-treated MAP solution in 6 M HCl.
  • mTyr-CNK showed a significantly higher 53% strain yield compared to a strain rate of 15% or less reported in mushroom tyrosinase.
  • tyrosinase-CNK of SEQ ID NO: 1 used as a comparative example showed a modification yield of about 37%.
  • the above results show that mTyr-CNK shows a very high monophenolase / diphenolase activity ratio and can show increased enzymatic activity for L-tyrosine.
  • MTyr-CNK represented by SEQ ID NO: 2 of the present invention not only shows very good enzymatic activity, but also can maintain stability at a wide range of pH and temperature. Excellent excellence in maintaining a very stable state. This superiority is expected because the macromolecular phenolic substrate has relatively easy access to the active site of mTyr-CNK.
  • mTyr-CNK can be used for biosynthesis of pharmaceutical catechol intermediates and biosynthesis of phenolic phytochemicals, detoxification of tyrosinase-based biosensors for the detection and quantification of catechol-producing chemicals and phenolic and substituted phenolic compounds. It can be usefully used as monophenol monooxygenase for the same biomedical and industrial applications.

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Abstract

The present invention relates to a novel recombinant tyrosinase produced by newly redesigning a gene on the basis of a tyrosinase present in marine polar ammonia oxidation-related bacteria. Unlike conventionally known tyrosinases, the recombinant tyrosinase of the present invention can be very easily mass-produced from a transformant and, unlike conventional enzymes, can efficiently perform reactions over a wide pH range and at room temperature and low temperature. Therefore, the recombinant tyrosinase of the present invention can be variously utilized in the production of functional medicines and biomaterials, both of which contain phenolic compounds, and can be utilized in various industrial fields requiring catalytic reactions using tyrosinases, such as bioremediation processes of toxic phenolic compounds, which are harmful to the environment.

Description

신규 재조합 티로시나아제 New Recombinant Tyrosinase
본 발명은 해양 극지 암모니아 산화 관련 박테리아에 존재하는 티로시나아제를 바탕으로 새롭게 유전자를 재설계하여 제조한 신규 재조합 티로시나아제에 관한 것이다. The present invention relates to a novel recombinant tyrosinase prepared by newly redesigned genes based on tyrosinase present in marine polar ammonia oxidation-related bacteria.
티로시나아제는, 리그닌의 분해에 관여하는 락카아제(laccase) 및 무척추동물의 호흡에 필요한 헤모시아닌(hemocyanin) 등과 비슷하게 분류되며, 활성 부위(active site)에 구리를 함유하고 있는 type-3 구리 효소(monophenol monooxygenase, EC. 1.14.18.1)로 알려져 있다. 티로시나아제는 멜라닌 생합성의 초기단계인 L-tyrosine(L-Tyr)을 L-3,4-dihydroxyphenylalanine(L-DOPA)로 전환하고 L-DOPA를 도파퀴논(Dopaquinone)으로 변환하는 촉매로, 멜라닌의 생합성 초기 단계에 중요하게 관여하고 있다. 기본적으로 멜라닌은 세포를 자외선과 활성 산소 그리고 독성을 띄는 중금속으로부터 보호하는 역할을 한다. 티로시나아제에 기반한 멜라닌화 반응은 사과, 감자, 바나나와 같은 식물이 절단되어 공기 중에 노출되었을 때 절단면의 갈변 현상을 일으키는 등의 방식으로 상처 치료 및 1차 면역 반응에 관여한다. 동물의 경우, 피부, 털, 눈동자의 색깔에 영향을 주고 피부의 갈색 색소침착 또는 상처 부위의 경화에 관여하기도 한다. 또한, 티로시나아제는 다양한 형태의 모노페놀릭(monophenolic) 또는 다이페놀릭(diphenolic) 화합물을 반응 기질로 사용하여 퀴논(quinone)으로 전환하는데 사용할 수 있다. 따라서 티로시나아제는 멜라닌화를 위한 페놀릭 화합물의 산화 반응을 촉매할 뿐만 아니라, 페놀을 함유하는 폐수나 오염수의 정화, 린코마이신(lincomycin)과 같은 항생제의 생합성, 파킨슨 질환 치료제 등의 의료용 재료로 활용될 수 있는 L-DOPA의 생산, 페놀릭 화합물의 양을 측정하기 위한 바이오센서, 단백질 및 당의 가교(cross-linking) 등에 활용되고 있다.Tyrosinase is classified similarly to laccase, which is involved in the breakdown of lignin, and hemocyanin, which is required for the respiration of invertebrates, and type-3 copper containing copper at the active site. Known as an enzyme (monophenol monooxygenase, EC. 1.14.18.1). Tyrosinase is a catalyst that converts L-tyrosine (L-Tyr), which is an early stage of melanin biosynthesis, into L-3,4-dihydroxyphenylalanine (L-DOPA), and converts L-DOPA into dopaquinone. Is involved in the early stages of biosynthesis. Basically, melanin protects cells from UV rays, free radicals and toxic heavy metals. Tyrosinase-based melaninization reactions are involved in wound healing and primary immune responses in such a way that plants such as apples, potatoes and bananas are cut and cause browning of the cut surface when exposed to air. In animals, it affects the color of the skin, hair, and eyes, and may be involved in brown pigmentation of the skin or hardening of the wound. In addition, tyrosinase can be used to convert various forms of monophenolic or diphenolic compounds into quinones using the reaction substrate. Thus, tyrosinase not only catalyzes the oxidation reaction of phenolic compounds for melanination, but also purifies wastewater or contaminated water containing phenols, biosynthesis of antibiotics such as lincomycin, and medical materials for treating Parkinson's disease. L-DOPA, biosensor for measuring the amount of phenolic compounds, and cross-linking of proteins and sugars.
많은 생명체에서 멜라닌 생합성과 관련된 티로시나아제의 유전자 서열이 알려졌음에도 불구하고, 정제된 티로시나아제의 생화학적 특성에 대한 정보는 상대적으로 많이 알려져 있지 않다. 보통 티로시나아제 유전자를 갖고 있는 생명체의 유전체는 하나 이상의 티로시나아제 유전자를 갖고 있으며, 각각의 티로시나아제는 생화학적 특성이 다르다. 예를 들어 방선균으로부터 생산되는 대표적인 티로시나아제인 MelC2와 MelD2의 경우, MelC2는 세포 밖으로 분비되고 효소는 다양한 여러 기질에서 활성을 띄고 있는데 반하여, MelD2는 세포 안에서 발현되면서 그 기질특이성이 매우 높다. 대체적으로 티로시나아제는 모노머릭 단백질로 분자량이 약 20~60 kDa 정도되고, 약 7.5의 최적 pH와 40℃에서 반응 최적 온도를 갖고 있다.Although the gene sequence of tyrosinase associated with melanin biosynthesis is known in many organisms, relatively little information is known about the biochemical properties of purified tyrosinase. Usually, the genome of an organism that has a tyrosinase gene contains one or more tyrosinase genes, and each tyrosinase has different biochemical properties. For example, MelC2 and MelD2, which are representative tyrosinase produced from actinomycetes, are secreted out of cells and enzymes are active in various substrates, whereas MelD2 is expressed in cells and is highly substrate specific. In general, tyrosinase is a monomeric protein having a molecular weight of about 20 to 60 kDa, and has an optimum pH of about 7.5 and an optimum reaction temperature at 40 ° C.
최근 박테리아에 존재하는 티로시나아제는, 효소가 활성을 띄기 위해 caddie 단백질로 불리는 추가적인 단백질의 필요 여부와, 산소와 결합하는 하나의 모티프, 구리와 결합하는 두 개의 모티프 등의 도메인 구성에 따라, 5가지 유형으로 분류되고 있다.Tyrosinase, which is present in bacteria in recent years, depends on the need for additional proteins, called caddie proteins, for enzymes to be active, the domain composition of one motif that binds to oxygen, and two motifs that bind to copper. It is classified into different types.
한편, 현재 상업적으로 활용되고 있는 티로시나아제는 Sigma 회사에서 판매하고 있는 버섯 유래의 A. bisporus 티로시나아제로 추출에 의해서 생산되고 있다. 아직까지 상업화되지는 않았지만, 박테리아 유래 다양한 종류의 티로시나아제 또한 마찬가지로 해당 유전자를 갖고 있는 균주로부터 직접 추출하여 얻어내거나 외래 균주에서 재조합 단백질로 생산되고 있다. Meanwhile, tyrosinase, which is currently used commercially, is produced by extracting A. bisporus tyrosinase from mushrooms sold by Sigma. Although not yet commercialized, various types of bacteria-derived tyrosinase are likewise extracted directly from strains carrying the gene or produced as recombinant proteins in foreign strains.
이러한 측면에서 본 발명자들은 이전의 연구에서 해양 극지 암모니아 산화 관련 박테리아 유래의 신규 재조합 티로시나아제를 제공하였고, 상기 티로시나아제는 티로시나아제는 기존의 5가지의 티로시나아제 분류에 속하지 않는 신규 효소로서, 대장균에서 대량생산이 가능할 뿐만 아니라 기존 효소들과 달리 약산성 pH와 저온에서 높은 활성을 보여주었다 (국내특허출원 10-2015-0053189).In this respect, the present inventors have provided a novel recombinant tyrosinase derived from marine polar ammonia oxidation-related bacteria in a previous study, wherein the tyrosinase is a novel enzyme whose tyrosinase does not belong to the five existing tyrosinase classes. As well as mass production in Escherichia coli, it showed high activity at weakly acidic pH and low temperature unlike conventional enzymes (Domestic Patent Application 10-2015-0053189).
하지만, 상기 효소는 수용성 단백질로 생산하기 위하여 샤페론(Chaperone) 단백질을 티로시나아제와 동시 발현하여야 하며, 0℃ 에서는 안정성이 잘 유지되나 상온(20℃)에서도 안정성이 다소 낮고, L-티로신과 같은 상대적으로 작은 물질을 기질로 한 반응의 전환율은 높으나 홍합접착단백질과 같은 고분자에 존재하는 L-티로신과 같은 기질의 반응을 효율적으로 수행하지 못하여, 수용성 단백질 발현을 위한 별도의 단백질을 필요로 하지 않으면서 동시에 상온에서도 안정성이 뛰어나고 활용가능성이 높은 재조합 티로시나아제에 대한 필요성이 높은 상태이다.However, in order to produce a water-soluble protein, the enzyme must simultaneously express Chaperone (Chaperone) protein with tyrosinase, and stability is well maintained at 0 ° C., but is slightly low at room temperature (20 ° C.), such as L-tyrosine. Although the conversion rate of the reaction using a relatively small substance is high, the reaction of a substrate such as L-tyrosine present in a polymer such as mussel adhesive protein is not performed efficiently, and thus a separate protein for water-soluble protein expression is not required. At the same time, there is a high need for recombinant tyrosinase having high stability and high utility at room temperature.
본 발명자들은 기존의 한계점을 극복할 수 있는 티로시나아제에 대한 연구를 수행하던 중, 해양 극지 암모니아 산화 관련 박테리아 유래의 티로시나아제에서 일정 길이의 C-말단 아미노산 서열을 제거하였을 때, 활성과 안정성 측면에서 산업적 활용성이 더욱 뛰어난 신규 티로시나아제를 수득할 수 있음을 확인하고 본 발명을 완성하였다. 따라서 본 발명의 목적은 활성과 안정성이 개선된 신규한 재조합 티로시나아제를 제공하는 것이다. The inventors of the present invention, while studying the tyrosinase that can overcome the existing limitations, the activity and stability when removing a certain length of the C-terminal amino acid sequence from the tyrosinase derived from marine polar ammonia oxidation-related bacteria In view of the novel industrial tyrosinase more excellent industrial utility in terms of the present invention was completed. It is therefore an object of the present invention to provide novel recombinant tyrosinase with improved activity and stability.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 제공한다. In order to achieve the above object, the present invention provides a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 티로시나아제 기질 전환용 조성물을 제공한다. The present invention also provides a composition for converting tyrosinase substrates comprising recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는, 천연고분자 또는 합성 고분자 내 모노페놀 또는 카테콜 기질 전환용 조성물을 제공한다. The present invention also provides a composition for converting a monophenol or catechol substrate in a natural polymer or synthetic polymer, comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
또한 본 발명은 상기 재조합 티로시나아제를 암호화하는 폴리뉴클레오티드를 제공한다. The present invention also provides a polynucleotide encoding the recombinant tyrosinase.
또한 본 발명은 상기 재조합 티로시나아제를 암호화하는 폴리뉴클레오티드를 포함하는 벡터를 제공한다. The present invention also provides a vector comprising a polynucleotide encoding the recombinant tyrosinase.
또한 본 발명은 상기 벡터로 형질전환된 형질전환체를 제공한다. The present invention also provides a transformant transformed with the vector.
또한 본 발명은 상기 형질전환체로부터 재조합 티로시나아제를 분리 및 정제하는 단계; 를 포함하는 재조합 티로시나아제의 생산 방법을 제공한다. In another aspect, the present invention comprises the steps of isolating and purifying recombinant tyrosinase from the transformant; It provides a method for producing a recombinant tyrosinase comprising.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 티로시나아제 기질에 처리하는 단계; 를 포함하는 티로시나아제 기질 전환 방법을 제공한다. In another aspect, the present invention comprises the steps of treating the tyrosinase substrate with a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3; It provides a tyrosinase substrate conversion method comprising a.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 생체 소재를 제공한다. The present invention also provides a biological material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 생체 접착 소재 제조용 조성물을 제공한다. In another aspect, the present invention provides a composition for producing a bioadhesive material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
본 발명의 재조합 티로시나아제는 기존에 알려진 티로시나아제와 달리, 형질전환체로부터 매우 용이하게 대량생산이 가능하고, 넓은 pH 및 상온 및 저온에서도 기존 효소들과 달리 반응을 효율적으로 진행할 수 있다. 따라서 본 발명의 재조합 티로시나아제는 페놀성 화합물이 포함되어 있는 기능성 의약품 및 생체 재료의 생산에 다양하게 활용될 수 있으며, 환경에 유해한 독성을 띄는 페놀성 화합물의 환경 정화 공정 등 티로시나아제를 이용한 촉매 반응을 필요로 하는 다양한 산업 분야에서 유용하게 활용될 수 있다. Unlike tyrosinase known in the present invention, the recombinant tyrosinase of the present invention can be mass-produced very easily from a transformant, and the reaction can proceed efficiently unlike conventional enzymes at a wide pH and room temperature and low temperature. Therefore, the recombinant tyrosinase of the present invention can be variously used for the production of functional medicines and biomaterials containing phenolic compounds, and using tyrosinase such as environmental purification process of phenolic compounds that are harmful to the environment. It can be usefully used in various industrial fields requiring a catalytic reaction.
도 1은 I-TASSER server 를 이용한 티로시나아제의 구조분석 결과를 나타낸 도이다. 도 1의 (a)는 전체 티로시나아제-CNK (1-415) 의 구조를 나타내며, 도 1의 (b)는 C-말단이 제거된 mTyr-CNK (1-303) 의 예측 구조를 나타낸다. 1 is a diagram showing the results of structural analysis of tyrosinase using the I-TASSER server. (A) of FIG. 1 shows the structure of total tyrosinase-CNK (1-415), and (b) of FIG. 1 shows the predictive structure of mTyr-CNK (1-303) from which the C-terminus was removed.
도 2는 전체 티로시나아제 서열 중 제거된 C-말단 신장부의 서열 및 선택된 mTyr-CNK 의 서열을 나타낸다. 2 shows the sequence of the removed C-terminal extension and the sequence of selected mTyr-CNK in the total tyrosinase sequence.
도 3은 티로시나아제-CNK, mTyr-CNK, cTyr-CNK 의 발현 및 정제 분석 결과를 나타낸 도이다 (Sol; 수용성 상청액 분획; Ins: 불용성 세포 deris 분획; Elu: Ni-NTA 친화성 크로마토그래피를 이용한 정제된 티로시나아제; Mk: 단백질 분자량 마커). Figure 3 shows the results of expression and purification analysis of tyrosinase-CNK, mTyr-CNK, cTyr-CNK (Sol; aqueous supernatant fraction; Ins: insoluble cell deris fraction; Elu: Ni-NTA affinity chromatography Purified tyrosinase used; Mk: protein molecular weight marker).
도 4는 pH 변화에 따른 mTyr-CNK의 상대적인 초기 활성 변화를 확인한 결과를 나타낸 도이다. Figure 4 is a view showing the results of confirming the relative changes in the initial activity of mTyr-CNK with pH changes.
도 5는 온도 변화에 따른 mTyr-CNK의 상대적인 초기 활성 변화를 확인한 결과를 나타낸 도이다. 5 is a view showing the results of confirming the relative initial activity change of mTyr-CNK with temperature changes.
도 6은 배양 시간 및 온도에 따른 mTyr-CNK의 열안정성 평가 결과를 나타낸 도이다. Figure 6 is a diagram showing the thermal stability evaluation results of mTyr-CNK according to the culture time and temperature.
도 7은 in vitro DOPA 변형 수율 확인을 위한 MAP (mussel adhesive protein) 아미노산 조성물 분석결과를 나타낸 도이다.7 is a view showing the results of analysis of the mussel adhesive protein (MAP) amino acid composition for confirming the in vitro DOPA modification yield.
본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 제공한다. The present invention provides a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
본 발명의 재조합 티로시나아제는 기존의 티로시나아제와 달리 저온 내지 상온 및 넓은 pH 에서도 높은 활성을 나타내는 새로운 생화학적 물성을 나타내는 신규한 티로시나아제이며, 샤페론 추가없이도 수용성으로 발현될 수 있다. 또한 넓은 pH 영역에서 높은 활성을 나타내어, 약산성, 중성 및 약염기성 pH 조건에서 발생하는 자연산화 반응을 억제하면서 효소 반응을 수행할 수 있을 뿐만 아니라, 기존의 효소들과 달리 30℃ 이하의 저온에서도 높은 효소 활성을 유지할 수 있고 특히 고분자 내에 존재하는 티로시나아제 기질의 전환을 효과적으로 촉매할 수 있다. Unlike conventional tyrosinase, the recombinant tyrosinase of the present invention is a novel tyrosinase that exhibits new biochemical properties exhibiting high activity even at low temperatures to room temperature and a wide pH, and can be expressed as water-soluble without adding chaperone. In addition, it exhibits high activity in a wide pH range, and can perform enzymatic reactions while suppressing natural oxidation reactions occurring at weakly acidic, neutral and weakly basic pH conditions. It can maintain enzymatic activity and in particular catalyze the conversion of tyrosinase substrates present in the polymer.
이하, 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명의 티로시나아제는 서열번호 2 또는 서열번호 3으로 표시되는 아미노산 서열로 이루어지며, 본 발명의 티로시나아제와 동등한 정도의 활성 및 특성을 나타내는 한, 이와 80% 내지 99%, 바람직하게는 85 % 내지 99%, 더욱 바람직하게는 90% 내지 99%의 상동성을 갖는 티로시나아제도 제한없이 본 발명의 티로시나아제에 포함될 수 있다. The tyrosinase of the present invention consists of an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, 80% to 99%, preferably as long as it exhibits the same degree of activity and properties as tyrosinase of the present invention. Tyrosinase having a homology of 85% to 99%, more preferably 90% to 99% can also be included in the tyrosinase of the present invention without limitation.
본 발명의 티로시나아제는 해양 극지 암모니아 산화 관련 박테리아 유래, 바람직하게는 캔디다투스 니트로소푸밀루스 코렌시스(Candidatus Nitrosopumilus Koreensis) 유래 티로시나아제 서열의 일부를 재조합하는 방법으로 제조될 수 있다. Tyrosinase of the present invention is derived from marine polar ammonia oxidation-related bacteria, preferably Candidatus nitrosofumilus corensis Nitrosopumilus Koreensis ) can be prepared by a method of recombining a portion of the tyrosinase sequence derived.
특히 본 발명의 티로시나아제는 서열번호 1의 티로시나아제에서 C-말단 신장부가 제거된 것을 특징으로 할 수 있으며, 서열번호 1로 표시되는 티로시나아제 서열 중 C-말단 신장부 일부인 304 내지 415 부분이 제거된 것이거나, C-말단 신장부 전부인 294 내지 415 가 제거된 것일 수 있다. In particular, the tyrosinase of the present invention may be characterized in that the C-terminal extension is removed from the tyrosinase of SEQ ID NO: 1, which is part of the C-terminal extension of the tyrosinase sequence represented by SEQ ID NO: 304 to 415 The portion may have been removed, or 294 to 415, all of the C-terminal extension, may have been removed.
C-말단 신장부의 제거 부위에 따라 효소 활성은 상이하게 나타날 수 있으며, 본 발명의 재조합 티로시나아제의 경우 코어 효소 활성 도메인 부위를 포함하는 것일 수 있고, 더욱 바람직하게는 코어 효소 활성 도메인 부위 및 His294 및 Pro303 영역을 포함하는 것을 특징으로 할 수 있다. According to the removal site of the C-terminal kidney, the enzyme activity may be different, and the recombinant tyrosinase of the present invention may include a core enzyme active domain site, and more preferably, a core enzyme active domain site and His294. And a Pro303 region.
또한 본 발명의 재조합 티로시나아제는 최적 pH 에서 가장 높은 활성을 나타낼 수 있고, 이와 같은 최적 pH는 pH 3.5 내지 pH 10, 더욱 바람직하게는 pH 4.5 내지 pH 9.5, 더욱 바람직하게는 pH 6 내지 8일 수 있다. In addition, the recombinant tyrosinase of the present invention may exhibit the highest activity at an optimum pH, such an optimum pH is pH 3.5 to pH 10, more preferably pH 4.5 to pH 9.5, more preferably pH 6 to 8 days Can be.
상기와 같은 최적의 pH 범위는 상용의 버섯 티로시나아제와 유사한 정도의 넓은 활성 pH 범위이다. 이러한 pH 활성 범위에 의하여 본 발명의 재조합 티로시나아제는 통상 약 pH 7.5에서 최적 pH를 갖는 기존의 티로시나아제와 비교하여 보다 산성인 조건에서도 우수한 활성을 나타낼 수 있다. 또한 넓은 pH 영역에서 높은 활성을 띄고 있어, 약산성, 중성 및 약염기성 pH 조건에서 발생하는 자연산화 반응을 억제하면서 효소 반응을 수행할 수 있다. Such an optimal pH range is a broad active pH range similar to commercial mushroom tyrosinase. By such a range of pH activity, the recombinant tyrosinase of the present invention may exhibit excellent activity even in more acidic conditions than conventional tyrosinase having an optimal pH, usually at about pH 7.5. In addition, it has a high activity in a wide pH range, it is possible to perform the enzymatic reaction while suppressing the natural oxidation reaction occurring in weakly acidic, neutral and weakly basic pH conditions.
또한 본 발명의 재조합 티로시나아제는 저온 또는 상온에서 활성인 것을 특징으로 할 수 있으며, 바람직하게는 -20 내지 35℃, 더욱 바람직하게는 -10 내지 30℃, 더더욱 바람직하게는 0 내지 30℃ 에서 높은 활성을 나타낼 수 있다. In addition, the recombinant tyrosinase of the present invention may be characterized as being active at low or normal temperatures, preferably at -20 to 35 ° C, more preferably at -10 to 30 ° C, even more preferably at 0 to 30 ° C. High activity.
본 발명의 재조합 티로시나아제는 특히 0℃ 이하의 온도에서도 최대 활성의 약 80% 정도를 유지할 수 있으므로, 영하의 보관 온도에서도 활성을 유지할 수 있다는 장점이 있다. 또한 본 발명의 재조합 티로시나아제는 일반적인 활성 온도인 37℃가 아닌 0 내지 30℃ 에서 최적의 활성을 나타낼 수 있으며, 예컨대 생체 외 저온 및 상온 조건에서 활성을 나타내고, 생체 내 주입을 통해 생체 온도인 37℃ 조건으로 변화되는 경우, 그 활성을 급격하게 소실함으로써, 생체 내에서 원하지 않는 효소 촉매 반응이 발생하는 것을 용이하게 차단할 수 있다. In particular, the recombinant tyrosinase of the present invention is able to maintain about 80% of the maximum activity even at a temperature below 0 ℃, there is an advantage that can be maintained at sub-zero storage temperature. In addition, the recombinant tyrosinase of the present invention may exhibit optimal activity at 0 to 30 ° C., not 37 ° C., which is a general activity temperature. When it changes to 37 degreeC conditions, the activity can abruptly lose | disappear, and an unwanted enzyme catalysis can easily be prevented from occurring in a living body.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 티로시니아제 기질 전환용 조성물을 제공한다. In another aspect, the present invention provides a composition for converting tyrosinase substrate comprising recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
본 발명의 “티로시나아제 기질” 이란, 티로시나아제에 의해 효소 반응이 일어날 수 있는 다양한 기질을 제한없이 포함할 수 있으며, monophenol, catechol, 바람직하게는 티로신 또는 DOPA (3,4-dihydroxyphenylalanine), 더욱 바람직하게는 L-티로신 또는 L-DOPA 일 수 있다. The term "tyrosinase substrate" of the present invention may include without limitation various substrates in which enzymatic reactions can be caused by tyrosinase, monophenol, catechol, preferably tyrosine or DOPA (3,4-dihydroxyphenylalanine), More preferably L-tyrosine or L-DOPA.
본 발명의 “티로시나아제 기질 전환” 이란 티로시나아제와 반응할 수 있는 기질의 효소 반응을 촉매하여 전환시키는 것을 의미하며, 예컨대 티로시나아제 기질인 티로신을 L-DOPA로, 티로시나아제 기질인 L-DOPA를 도파 퀴논으로 전환시키는 것을 포함할 수 있다. The term “tyrosinase substrate conversion” of the present invention means catalyzing and converting an enzymatic reaction of a substrate capable of reacting with tyrosinase. For example, tyrosinase tyrosine is converted to L-DOPA and tyrosinase substrate Converting L-DOPA to dopaquinone.
특히 본 발명의 재조합 티로시나아제는 활성 부위와 기질의 반응이 용이한 구조를 가지고 있어, 모노페놀(monophenol) 또는 카테콜(catechol) 기(group) 을 포함하는 천연고분자 및 모노페놀(monophenol) 또는 카테콜(catechol) 기(group) 를 포함하는 페놀릭 합성고분자의 모노페놀(monophenol) 또는 카테콜(catechol)의 반응을 효과적으로 촉매할 수 있다. 상기 모노페놀 또는 카테콜을 포함하는 천연고분자는 콜라젠, 젤라틴, 탄닌, 리그닌 및 콩단백질로 이루어진 군에서 선택된 1종 이상일 수 있으며, 상기 합성 고분자는 노볼락(Novolac)과 같은 페놀레진(phenolic resin)에 포함된 기질일 수 있다. 본 발명의 바람직한 일 예에서는 홍합 접착 단백질 내에 포함된 기질, 예를 들어 홍합 접착 단백질의 L-티로신 잔기를 기질로 하는 반응을 제공한다. 대장균에서 생산된 홍합 접착 단백질의 경우 티로신 잔기들이 변형되어 있지 않아 별도의 효소 등을 이용하여 in vitro 상에서 티로신 잔기의 전환이 필요하다. 그러나 홍합 접착 단백질과 같은 고분자에 포함된 기질의 경우, 효소의 활성 부위와의 결합이 용이하지 않아, in vitro에서 기질의 전환이 용이하게 발생하기 어렵다는 단점이 있다. 그러나 본 발명의 재조합 티로시나아제는 홍합 접착 단백질의 L-티로신과 반응하여 이를 높은 효율로 L-DOPA로 전환시킬 수 있으며, 특히 서열번호 1로 표시되는 티로시나아제-CNK 와 비교하더라도 현저히 높은 변형 수율로 L-티로신을 L-DOPA로 전환시킬 수 있다. In particular, the recombinant tyrosinase of the present invention has a structure that facilitates the reaction between the active site and the substrate, and thus includes a natural polymer and a monophenol containing a monophenol or a catechol group. It is possible to effectively catalyze the reaction of monophenol or catechol of phenolic synthetic polymers containing catechol groups. The natural polymer including the monophenol or catechol may be one or more selected from the group consisting of collagen, gelatin, tannin, lignin and soy protein, and the synthetic polymer is a phenolic resin such as Novolac. It may be a substrate included in. In a preferred embodiment of the present invention provides a reaction comprising a substrate contained in the mussel adhesive protein, such as the L-tyrosine residue of the mussel adhesive protein. In the case of mussel adhesive protein produced in Escherichia coli, tyrosine residues are not modified. Therefore, it is necessary to convert tyrosine residues in vitro using a separate enzyme. However, in the case of a substrate included in a polymer such as an mussel adhesive protein, since the binding to the active site of the enzyme is not easy, there is a disadvantage that it is difficult to easily convert the substrate in vitro. However, the recombinant tyrosinase of the present invention can react with the L-tyrosine of the mussel adhesive protein and convert it to L-DOPA with high efficiency, especially in comparison with the tyrosinase-CNK represented by SEQ ID NO: 1. L-tyrosine can be converted to L-DOPA in yield.
따라서 본 발명은 홍합 접착 단백질 내 포함된 티로시나아제 기질 전환용인 것을 특징으로 하는, 티로시나아제 기질 전환용 조성물 또는 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는, 천연고분자 또는 합성 고분자 내 모노페놀 또는 카테콜 기질 전환용 조성물을 제공한다. Therefore, the present invention is a natural polymer comprising a tyrosinase substrate conversion composition or recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3, characterized in that for converting tyrosinase substrate contained in the mussel adhesive protein Provided are compositions for converting monophenol or catechol substrates in synthetic polymers.
또한 본 발명은 넓은 pH 범위와 저온 및 상온에서 활성을 나타내는 서열번호 2로 표시되는 재조합 티로시나아제를 이용함에 따라, 다양한 pH, 저온 및 상온 환경에 존재하는 티로시나아제 기질 전환용도로 이용할 수 있다. In addition, the present invention can be used for tyrosinase substrate conversion for use in a wide range of pH, low temperature and room temperature by using a recombinant tyrosinase represented by SEQ ID NO. .
따라서 본 발명은 pH 4.5 내지 pH 9.5 환경의 티로시나아제 기질 전환용 조성물 및 -20℃ 내지 35℃ 환경의 티로시나아제 기질 전환용 조성물을 제공한다. Accordingly, the present invention provides a composition for converting tyrosinase substrate in an environment of pH 4.5 to pH 9.5 and a composition for converting tyrosinase substrate in an environment of -20 ° C to 35 ° C.
또한 본 발명은 서열번호 2의 재조합 티로시나아제를 암호화하는 폴리뉴클레오 티드를 제공하며, 이는 바람직하게는 서열번호 4로 표시되는 염기서열 또는 이와 기능적 동등성이 있는 상동성 폴리뉴클레오티드를 제한없이 포함할 수 있다. The present invention also provides a polynucleotide encoding a recombinant tyrosinase of SEQ ID NO: 2, which preferably includes without limitation the nucleotide sequence represented by SEQ ID NO: 4 or a homologous polynucleotide having a functional equivalent thereto Can be.
또한 본 발명은 서열번호 3의 재조합 티로시나아제를 암호화하는 폴리뉴클레오 티드를 제공하며, 이는 바람직하게는 서열번호 5로 표시되는 염기서열 또는 이와 기능적 동등성이 있는 상동성 폴리뉴클레오티드를 제한없이 포함할 수 있다. The present invention also provides a polynucleotide encoding a recombinant tyrosinase of SEQ ID NO: 3, which preferably includes without limitation the base sequence represented by SEQ ID NO: 5 or a homologous polynucleotide having a functional equivalent thereto Can be.
상기 상동성 폴리뉴클레오티드란, 두 개 이상의 폴리뉴클레오티드 서열 사이에 유사한 또는 상호 교환 가능한 서열을 지칭한다. 바람직하게는, 상동성인 폴리뉴클레오티드는 서열번호 2의 아미노산을 코딩하는 폴리뉴클레오티드 서열에 적어도 70%, 바람직하게는 적어도 80%, 더 바람직하게는 적어도 90%, 더 바람직하게는 95%, 더 바람직하게는 97%, 더 바람직하게는 98%, 더더욱 바람직하게는 99%의 서열 동일성을 갖는 것을 말하며, 이와 같은 상동성 폴리뉴클레오티드는 서열번호 2의 아미노산을 코딩하는 폴리뉴클레오티드의 코돈 최적화 서열에 의해 상기 조건을 만족하게 되는 폴리뉴클레오티드 서열 역시 모두 제한없이 포함할 수 있다.The homologous polynucleotide refers to a sequence that is similar or interchangeable between two or more polynucleotide sequences. Preferably, the homologous polynucleotide is at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably at least the polynucleotide sequence encoding the amino acid of SEQ ID NO: 2. Refers to 97%, more preferably 98%, even more preferably 99% of sequence identity, wherein such homologous polynucleotides are conditioned by the codon optimization sequence of the polynucleotide encoding the amino acid of SEQ ID NO: 2. Any polynucleotide sequence that satisfies may also be included without limitation.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 아미노산 서열을 코딩하는 폴리뉴클레오티드 서열을 포함하는 벡터를 제공한다. The present invention also provides a vector comprising a polynucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.
용어 벡터는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고 숙주세포에서 독립적으로 재생산될 수 있다. 용어 발현 벡터는 목적한 코딩서열과 특정 숙주생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 발현벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 수 있으며, 이와 같은 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질전환된 세포를 비 형질전환 세포와 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 카나마이신, 블레오마이신, 클로로앰피니콜, 앰피실린과 같은 항생제 유전자가 있으나 이에 한정되는 것은 아니다. The term vector is used to refer to DNA fragment (s), nucleic acid molecules that are delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term expression vector refers to a recombinant DNA molecule comprising a suitable nucleic acid sequence necessary to express a coding sequence of interest and a coding sequence operably linked in a particular host organism. The expression vector may preferably comprise one or more selectable markers, such markers are typically nucleic acid sequences having properties that can be selected by chemical methods, to distinguish transformed cells from non-transformed cells. All genes present are this. Examples include, but are not limited to, antibiotic genes such as kanamycin, bleomycin, chloro ampinicol, ampicillin.
또한 본 발명은 상기 벡터로 형질전환된 형질전환체를 제공한다. The present invention also provides a transformant transformed with the vector.
용어 형질전환체는 숙주세포에 새로운 목적 폴리뉴클레오티드가 도입된 것을 말하며, 숙주세포는 대장균, 효모, 동물, 식물 세포 또는 곤충세포 등을 포함할 수 있으나 이에 제한되지 않는다. The term transformant refers to the introduction of a new target polynucleotide into a host cell, and the host cell may include, but is not limited to, E. coli, yeast, animal, plant cell or insect cell.
또한 본 발명은 상기 형질전환체로부터 제조합 티로시나아제를 분리 및 정제하는 단계;를 포함하는 재조합 티로시나아제의 생산 방법을 제공한다. In another aspect, the present invention provides a method for producing a recombinant tyrosinase comprising the step of isolating and purifying the prepared tyrosinase from the transformant.
보다 구체적으로, 상기 생산방법은 서열번호 2 또는 서열번호 3의 아미노산 서열을 코딩하는 폴리뉴클레오티드를 발현벡터에 클로닝하여 티로시나아제 발현벡터를 제조하고 이를 대장균에 형질전환시키는 단계; 형질전환된 대장균에서 통상의 방법을 통해 상기 재조합 티로시나아제 단백질의 발현을 유도하는 단계; 및 이를 수용성 분액과 불용성 분액으로 나누고 컬럼을 이용하여 분리 및 정제하는 단계; 를 포함할 수 있다. More specifically, the production method comprises the steps of cloning a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 into an expression vector to prepare a tyrosinase expression vector and transforming it into E. coli; Inducing expression of the recombinant tyrosinase protein in a transformed Escherichia coli via conventional methods; Dividing it into an aqueous and an insoluble fraction and separating and purifying using a column; It may include.
상기 생산방법에 따르면, 본 발명의 재조합 티로시나아제가 별도의 샤페론 없이도 대장균에서 inclusion body를 형성하지 않으면서 수용성으로 과량 발현되어 검출될 수 있으므로 대량 생산이 가능하다.According to the production method, since the recombinant tyrosinase of the present invention can be detected by overexpression by water solubility without forming an inclusion body in E. coli without a separate chaperone, mass production is possible.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 티로시나아제 기질에 처리하는 단계; 를 포함하는 티로시나아제 기질 전환 방법을 제공한다. In another aspect, the present invention comprises the steps of treating the tyrosinase substrate with a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3; It provides a tyrosinase substrate conversion method comprising a.
본 발명의 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제는 앞서 설명한 바와 같은 온도 및 pH 활성을 가지므로, 저온 및 상온, 예컨대 바람직하게는 -20 내지 35℃, 더욱 바람직하게는 -10 내지 30℃, 더더욱 바람직하게는 0 내지 30℃ 에서 기질을 효과적으로 전환시킬 수 있다. 따라서 본 발명에 따른 상기 티로시나아제 기질 전환 방법은 바람직하게는 -20 내지 35℃, 더욱 바람직하게는 -10 내지 30℃, 더더욱 바람직하게는 0 내지 30℃에서 재조합 티로시나아제를 티로시나아제 기질에 처리하는 단계를 포함할 수 있다.Since recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3 of the present invention has the temperature and pH activity as described above, it is low and normal temperature, such as preferably -20 to 35 ℃, more preferably -10 The substrate can be effectively converted at from 30 to 30 ° C, even more preferably from 0 to 30 ° C. Therefore, the tyrosinase substrate conversion method according to the present invention is preferably a tyrosinase substrate for recombinant tyrosinase at -20 to 35 ° C, more preferably at -10 to 30 ° C, even more preferably at 0 to 30 ° C. The process may include the step of.
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 생체 소재를 제공한다. The present invention also provides a biological material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
본 발명의 “생체소재” 란 의료용 재료의 일종으로, 의약품을 제외한 합성, 천연 또는 그들의 복합체 형태로 일정기간 인체의 조직 또는 기관을 대신하여 그 기능의 일부 또는 전부를 대체할 수 있는 소재를 의미한다. "Biomaterial" of the present invention is a kind of medical material, and means a material that can replace some or all of its functions in place of tissues or organs of a human body for a certain period of time in a synthetic, natural or complex form thereof except for medicines. .
또한 본 발명은 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 생체 접착 소재 제조용 조성물을 제공한다. In another aspect, the present invention provides a composition for producing a bioadhesive material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
본 발명에 있어 “생체 접착 소재” 란 생체에 국소적으로 적용되어 외과 수술용 봉합사를 대체해 손쉽고, 즉각적으로 상처에 접착 및 봉합할 수 있고, 피부 화상 또는 수술로 인해 발생한 전층 결함 조직을 메우거나, 결함 조직 내의 재생과 흉터 최소화를 위해 이용될 수 있는 소재를 제한없이 포함할 수 있다. 본 명세서에서 용어 “생체 조직”은 특별하게 제한되지 않으며, 예를 들어 피부, 신경, 뇌, 폐, 간, 신장, 위, 소장, 직장 및 뼈 등을 포함한다. In the present invention, the "bioadhesive material" is applied locally to replace a surgical suture, and can be easily and immediately bonded and sutured to a wound, to fill a total layer of defect tissue caused by skin burn or surgery, or It may include, without limitation, materials that can be used for regeneration in scar tissue and minimizing scarring. The term “biological tissue” herein is not particularly limited and includes, for example, skin, nerves, brain, lungs, liver, kidneys, stomach, small intestine, rectum and bones and the like.
본 발명의 재조합 티로시나아제는 생체 접착 소재의 제조에 있어 중요한 역할을 하는 L-DOPA 를 티로신으로부터 효율적으로 전환시킬 수 있으므로, 생체 접착 소재를 제조하는데 매우 유용하게 이용될 수 있다. Since the recombinant tyrosinase of the present invention can efficiently convert L-DOPA from tyrosine, which plays an important role in the preparation of the bioadhesive material, it can be very useful for preparing the bioadhesive material.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.
실시예Example 1. 재조합 티로시나아제의 선정 1. Selection of Recombinant Tyrosinase
국내특허출원 10-2015-0053189 에서 확인한 재조합 티로시나아제 (이하, 티로시나아제-CNK) 를 개선하여 효소 활성이 더욱 향상된 재조합 티로시나아제를 제조하기 위한 서열 분석 및 구조 분석을 수행하였다. 구조적 상동성 모델을 I-TASSER 서버 (http://zhanglab.ccmb.med.umich.edu/I-TASSER) 를 이용하여 분석하였으며, 모델 구조는 Discovery Studio software (Dassault Systems BIOVIA, Discovery Studio Modeling Environment, Release 2016, San Diego: Dassault Systems, 2017) 를 이용하여 분석하였다. 단백질 이온화도, residual pK 값, SAP (응집 스코어) 및 docking simulations (CDOCKER) 는 pH 6.0 에서 Discovery Studio modules 및 CHARMm force field 를 이용하여 확인하였다. PyMol 1.8.6.2 (Schrodinger, LLC, Cambridge, MA, USA) 을 이용하여 상기 결과를 시각화하였으며, 그 결과를 도 1에 나타내었다. Sequence analysis and structural analysis were performed to prepare recombinant tyrosinase with improved enzyme activity by improving the recombinant tyrosinase (hereinafter, tyrosinase-CNK) identified in Korean Patent Application No. 10-2015-0053189. The structural homology model was analyzed using I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER), and the model structure was found in Discovery Studio software (Dassault Systems BIOVIA, Discovery Studio Modeling Environment, Release 2016, San Diego: Dassault Systems, 2017). Protein ionization, residual pK value, SAP (aggregation score) and docking simulations (CDOCKER) were confirmed using Discovery Studio modules and CHARMm force field at pH 6.0. The results were visualized using PyMol 1.8.6.2 (Schrodinger, LLC, Cambridge, MA, USA) and the results are shown in FIG. 1.
도 1의 (a) 에 나타낸 바와 같이, I-TASSER server 를 이용한 상동성 모델에서 전체 티로시나아제-CNK 는 회색으로 표시된 C-말단 신장부 (304-415) 를 가지고 있는 것을 확인하였다. 또한 His294 및 Pro303 사이에 붉은 리본으로 표시된 헬리컬 영역을 가지고 있었다. SAP 분석에 의하면, 이는 소수성 패치의 dynamic 노출의 결과로 aggregation-prone 영역일 것으로 예측되었다. 이러한 결과를 통해 티로시나아제-CNK 의 일부 부위를 제거하면 남은 티로시나아제-CNK 부위의 기능적인 폴딩을 개선할 수 있을 것임을 예측하였다. 도 1의 (b) 에 나타낸 바와 같이, 전체 티로시나아제-CNK에서 C 말단 신장부를 제거하여 제조한 재조합 티로시나아제인 mTyr-CNK 는 CDOCKER docking 시뮬레이션을 이용한 L-tyrosinase 결합 복합체 구조 분석 결과 활성 부위의 진입 부분이 노출된 형태를 가질 수 있음을 예측하였다. As shown in (a) of FIG. 1, in the homology model using the I-TASSER server, it was confirmed that the whole tyrosinase-CNK had a C-terminal extension portion 304-415 shown in gray. It also had a helical region marked with a red ribbon between His294 and Pro303. According to SAP analysis, this is expected to be an aggregation-prone region as a result of dynamic exposure of hydrophobic patches. These results predicted that removing some sites of tyrosinase-CNK could improve the functional folding of the remaining tyrosinase-CNK sites. As shown in (b) of FIG. 1, mTyr-CNK, a recombinant tyrosinase prepared by removing the C-terminal extension from total tyrosinase-CNK, has an active site as a result of L-tyrosinase binding complex structure analysis using CDOCKER docking simulation. It was predicted that the entry portion of could have an exposed form.
이와 같은 C-말단 신장부의 제거를 통해 기존 티로시나아제-CNK 의 효소 활성을 더욱 개선할 수 있는지 여부를 확인하기 위해 C-말단 신장부를 제거한 신규한 재조합 효소를 제조하였다. 도 2에 나타낸 바와 같이, 전체 티로시나아제 서열 중 304-415 에 해당하는 부분 (회색)을 제외하고 1-303 의 서열로만 이루어진 C-말단 신장부가 제거된 재조합 티로시나아제를 제조하였다. 전체 티로시나아제-CNK (1-415) 의 서열은 서열번호 1에 나타내었으며, 유전자 변형을 통해 재조합된 신규한 티로시나아제 mTyr-CNK (1-303) 는 서열번호 2에, 코어 서열을 포함하는 재조합된 신규한 티로시나아제 cTyr-CNK (1-293)은 서열번호 3에 나타내었다. In order to confirm whether the removal of the C-terminal extension can further improve the enzymatic activity of existing tyrosinase-CNK, a novel recombinant enzyme from which the C-terminal extension is removed was prepared. As shown in FIG. 2, a recombinant tyrosinase was prepared in which a C-terminal extension portion consisting only of a sequence of 1-303 was removed except a portion (gray) corresponding to 304-415 of the entire tyrosinase sequence. The sequence of total tyrosinase-CNK (1-415) is shown in SEQ ID NO: 1, and the new tyrosinase mTyr-CNK (1-303) recombined through genetic modification is in SEQ ID NO: 2, including the core sequence. Recombinant novel tyrosinase cTyr-CNK (1-293) is shown in SEQ ID NO: 3.
실시예Example 2. 재조합 티로시나아제 발현 및 정제 2. Recombinant Tyrosinase Expression and Purification
선정된 신규한 티로시나아제를 제조하기 위하여 Escherichia coli DH5α (Life Technologies, Carlsbad, CA, USA) 세포를 재조합 벡터 제조를 위한 숙주세포로 사용하였으며 E. coli BL21 (DE3) (Merck KGaA, Darmstadt, Germany) 를 재조합 티로시나아제 발현을 위해 사용하였다. 상기 대장균 세포들은 50μg ampicillin mL-1 (Sigma-Aldrich, St. Louis, MO, USA) 이 포함된 LB 배지에서 배양하였다. 구체적으로 해양 극지 암모니아 산화 관련 박테리아 유래의 티로시나아제 기반 신규 재조합 티로시나아제를 제조하기 위하여 이의 유전자를 코돈 최적화하여 합성하고 이를 포함하는 발현 벡터를 제조하였다. 최적화된 DNA의 양 말단에는 NdeI, XhoI의 제한효소 사이트를 첨가하고 발현용 프로모터 T7 을 포함하는 발현벡터 pET23b(+)에 클로닝하여 티로시나아제 발현 벡터를 제조하였다. 발현 벡터에는 재조합 단백질의 정제를 위하여 C-말단에 6개의 히스티딘 서열로 이루어진 히스티딘 태그(histidine tag, HHHHHH)가 포함되도록 하였으며, 유전자 도입을 확인하기 위하여 앰피실린 저항성 유전자가 포함되도록 하였다. 제조된 벡터 구조 pmTyr-CNK 를 직접 서열 분석법을 통해 확인하였다. cTyr-CNK(1-293) 유전자를 유사한 방법으로 pET23b+ 벡터에 삽입하였으며, 최종 벡터는 pcTyr-CNK 로 기재하였다. Escherichia to Produce Selected Novel Tyrosinase coli DH5α (Life Technologies, Carlsbad, Calif., USA) cells were used as host cells for recombinant vector preparation and E. coli BL21 (DE3) (Merck KGaA, Darmstadt, Germany) was used for recombinant tyrosinase expression. The E. coli cells were cultured in LB medium containing 50 μg ampicillin mL -1 (Sigma-Aldrich, St. Louis, MO, USA). Specifically, in order to prepare a new recombinant tyrosinase-based tyrosinase derived from marine polar ammonia oxidation-related bacteria, its gene was synthesized by codon optimization and an expression vector including the same was prepared. Tyrosinase expression vectors were prepared by adding restriction sites of Nde I and Xho I to both ends of the optimized DNA and cloning them into the expression vector pET23b (+) containing the promoter T7 for expression. In order to purify the recombinant protein, the expression vector includes a histidine tag (HHHHHH) consisting of six histidine sequences at the C-terminus and an ampicillin resistance gene to confirm gene introduction. The prepared vector structure pmTyr-CNK was confirmed by direct sequencing. The cTyr-CNK (1-293) gene was inserted into the pET23b + vector in a similar manner and the final vector was described as pcTyr-CNK.
상기 벡터에 포함된 티로시나아제를 효과적으로 발현하기 위하여 제조한 벡터 pmTyr-CNK 과 pcTyr-CNK 을 각각 발현할 수 있는 대장균을 제조하였다. 티로시나아제 유전자를 포함하고 있는 벡터 pmTyr-CNK과 pcTyr-CNK 을 각각 42℃ 에서 1분 30초간 열충격을 가하여 대장균 BL21(DE3)에 도입하였다. pmTyr-CNK과 pcTyr-CNK 을 각각 앰피실린 항생제에서 저항 내성을 지니는 벡터이므로, 앰피실린이 첨가되어 있는 LB-한천 배지에서 배양하여 벡터가 도입된 형질전환 대장균을 선별하였다. E. coli was prepared to express the vector pmTyr-CNK and pcTyr-CNK prepared in order to effectively express the tyrosinase contained in the vector. Vector pmTyr-CNK and pcTyr-CNK containing tyrosinase gene were introduced into E. coli BL21 (DE3) by thermal shock at 42 ° C. for 1 minute and 30 seconds, respectively. Since pmTyr-CNK and pcTyr-CNK were vectors resistant to ampicillin antibiotics, transformed Escherichia coli with the vector was selected by culturing in LB-agar medium containing ampicillin.
상기 제조된 형질전환체를 50μg/mL 앰피실린이 첨가된 통상의 37℃, 50mL 의 LB 배지에서 200rpm으로 진탕 배양하여 배양액의 흡광도(OD600)가 0.8~1.0 정도에 도달했을 때 유도물질인 IPTG(isopropyl-ß-D-thiogalactopyranoside, 1 mM)를 첨가하여 상기 재조합 티로시나아제 단백질의 발현을 유도하였다. IPTG 첨가 후 추가로 20시간 동안 20℃에서 배양한 후, 배양된 세포를 15840g에서 10분간 원심분리한 다음 상등액을 제거하고 수득한 펠렛을 -80℃ 에서 보관하였다. 재조합 티로시나아제를 정제하기 위하여, 건중량 당 5ml lysis 완충액 (50 mM 인산나트륨 완충액(sodium phosphate buffer), pH 8.0, 10mM 이미다졸(imidazole), 300mM NaCl) 을 재부유시켰으며, 상기 세포들을 초음파 분쇄기를 이용하여 파쇄하였다 Lysate 를 10분 동안 15,840g 및 4℃ 조건에서 원심분리하였으며, 상청액을 수집하여 Ni-nitrilotriacetic acid (Ni-NTA) affinity purification column (Qiagen, Germantown, MD, USA) 에 가하고, 세척 완충액 (50 mM NaH2PO4, 300 mM NaCl 및 20 mM 이미다졸; pH 8.0) 및 용출 완충액 (50 mM NaH2PO4, 300 mM NaCl, 250 mM 이미다졸 및 0.02 mM CuSO4; pH 8.0) 를 이용하여 순수한 티로시나아제를 수득하였다. 발현 수준 및 단백질의 순도는 SDS-PAGE 를 이용하여 확인하였으며, 겔 분석은 이미지 분석 소프트웨어 (CLIQS; TotalLab Ltd., Newcastle upon Tyne, UK)를 이용하여 수행하였다. 상기 단백질의 농도는 BCA 어세이를 통해 확인하였다. 티로시나아제-CNK, mTyr-CNK 및 cTyr-CNK 단백질의 기능적 발현 및 정제 결과를 도 3에 나타내었다. The transformant was incubated at 200 rpm in a conventional 37 ° C., 50 mL LB medium to which 50 μg / mL ampicillin was added, and when the absorbance (OD 600 ) of the culture medium reached 0.8 to 1.0, IPTG was an inducer. (isopropyl-ß-D-thiogalactopyranoside, 1 mM) was added to induce the expression of the recombinant tyrosinase protein. After incubation at 20 ° C. for an additional 20 hours after IPTG addition, the cultured cells were centrifuged at 15840 g for 10 minutes, then the supernatant was removed and the resulting pellets were stored at −80 ° C. To purify recombinant tyrosinase, 5 ml lysis buffer (50 mM sodium phosphate buffer, pH 8.0, 10 mM imidazole, 300 mM NaCl) per dry weight was resuspended and the cells were resuspended. Lysate was centrifuged at 15,840 g and 4 ° C. for 10 minutes. The supernatant was collected and added to a Ni-nitrilotriacetic acid (Ni-NTA) affinity purification column (Qiagen, Germantown, MD, USA) and washed. Buffer (50 mM NaH 2 PO 4 , 300 mM NaCl and 20 mM imidazole; pH 8.0) and elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole and 0.02 mM CuSO 4 ; pH 8.0) To obtain pure tyrosinase. Expression levels and protein purity were confirmed using SDS-PAGE, and gel analysis was performed using image analysis software (CLIQS; TotalLab Ltd., Newcastle upon Tyne, UK). The concentration of the protein was confirmed through a BCA assay. The results of functional expression and purification of tyrosinase-CNK, mTyr-CNK and cTyr-CNK proteins are shown in FIG. 3.
도 3에 나타낸 바와 같이, 재조합 티로시나아제 단백질이 수용성으로 과량 발현되어 검출되었으며, 이를 통해 재조합 티로시나아제 단백질이 수용성 형태로 대량 생산될 수 있음을 확인하였다. 특히 mTyr-CNK 는 서열번호 1의 티로시나아제-CNK 와 비교하여 세포 내에서의 단백질 총 발현양이 매우 탁월하게 향상되어 일부 불용성의 inclusion body가 형성되었으나, 서열번호 1의 티로시나아제-CNK 는 기능적인 발현을 위해서 GroES 및 GroEL 라는 샤페론이 필요한 것과 달리 총 단백질의 약 50% 정도가 샤페론 없이도 수용성으로 발현됨을 확인하였다. mTyr-CNK는 95% 이상의 높은 순도로 정제되었으며, cTyr-CNK 역시 동일 조건에서 수용성으로 발현되고 정제되었다. As shown in FIG. 3, the recombinant tyrosinase protein was detected by overexpression with water solubility, thereby confirming that the recombinant tyrosinase protein could be mass produced in water-soluble form. In particular, mTyr-CNK has a very good improvement in total protein expression in cells compared to tyrosinase-CNK of SEQ ID NO: 1, resulting in some insoluble inclusion body, but tyrosinase-CNK of SEQ ID NO: Contrary to the requirement of chaperones called GroES and GroEL for functional expression, approximately 50% of the total protein was found to be water-soluble without chaperone. mTyr-CNK was purified to a high purity of 95% or more, cTyr-CNK was also expressed and purified water-soluble under the same conditions.
실시예Example 3. 재조합 티로시나아제 단백질 분석 3. Recombinant Tyrosinase Protein Analysis
3.1 최적 pH 분석3.1 Optimal pH Analysis
실시예 2에서 제조된 재조합 티로시나아제 단백질의 성질을 분석하기 위하여 최적 pH를 분석하였다. L-DOPA를 반응물로 이용하여 L-도파크롬의 형성을 각기 다른 pH 에서 측정하는 분석을 수행하였으며, 이와 같은 분석법은 50mM 반응 완충액 (glycine-HCl buffer at pH 1-3, sodium acetate buffer at pH 3.5-5, sodium phosphate buffer at pH 5.5-7, tris-HCl buffer at pH 7.5-9, 및 glycine-NaOH buffer at pH 9.5-11), 0.01 mM CuSO4, 0.05 mM L-DOPA 및 0.4μM 정제된 mTyr-CNK 를 이용하여 수행하였다. L-도파크롬의 형성은 472nm 에서 흡광도를 측정하는 방법으로 모니터링하였다. 상용의 버섯 티로시나아제 (Sigma-Aldrich) 를 비교군으로 이용하였으며 mTyr-CNK의 최적 pH 분석 결과를 도 4에 나타내었다. 모든 측정은 3회 반복하였다. Optimum pH was analyzed to analyze the properties of the recombinant tyrosinase protein prepared in Example 2. L-DOPA was used as a reactant to analyze the formation of L-dopachrome at different pH. This assay was performed in 50 mM reaction buffer (glycine-HCl buffer at pH 1-3, sodium acetate buffer at pH 3.5). -5, sodium phosphate buffer at pH 5.5-7, tris-HCl buffer at pH 7.5-9, and glycine-NaOH buffer at pH 9.5-11), 0.01 mM CuSO 4 , 0.05 mM L-DOPA and 0.4 μM purified mTyr It was performed using -CNK. Formation of L-dopachrome was monitored by measuring absorbance at 472 nm. Commercial mushroom tyrosinase (Sigma-Aldrich) was used as a comparison group and the optimum pH analysis of mTyr-CNK is shown in FIG. 4. All measurements were repeated three times.
도 4 에 나타낸 바와 같이, L-DOPA를 기질로 사용하였을 때 mTyr-CNK 의 상대적 초기 활성은 변화하였으며, 특히 pH 5 이상 pH 9 이하 범위에서 높은 활성을 나타낼 수 있음을 확인하였다. mTyr-CNK 는 상용의 버섯 티로시나아제와 유사하게 상대적으로 넓은 pH 영역에서 높은 활성을 나타내었으며, 기존에 알려진 티로시나아제가 약 pH 7.5에서 최적 pH를 갖는 것과 비교하여 보다 산성인 조건에서도 우수한 활성을 나타낼 수 있는 장점이 있음을 확인하였다. As shown in FIG. 4, when L-DOPA was used as a substrate, the relative initial activity of mTyr-CNK was changed, and it was confirmed that high activity could be exhibited especially in the range of pH 5 or more and pH 9 or less. mTyr-CNK showed high activity in a relatively wide pH range, similar to commercial mushroom tyrosinase, and superior activity even in more acidic conditions compared to the known tyrosinase having an optimal pH at about pH 7.5. It was confirmed that there is an advantage that can represent.
3.2 최적 온도 분석3.2 Optimal Temperature Analysis
pH 값을 6으로 고정하고 온도를 0 내지 60℃ 범위로 달리하며 mTyr-CNK의 최적의 온도를 확인하는 실험을 수행하였다. 온도에 따른 반응 분석은 50 mM Tris buffer (pH 6.0), 0.01 mM CuSO4, 0.05 mM L-DOPA 및 0.4 μM 정제된 mTyr-CNK 를 포함하는 200μL 부피의 반응액에서 수행하였다. 상용의 버섯 티로시나아제 (Sigma-Aldrich) 를 역시 비교군으로 이용하였다. 모든 측정은 3회 반복하였으며, 측정 결과를 도 5에 나타내었다. Experiments were performed to determine the optimal temperature of mTyr-CNK with the pH value fixed at 6 and with varying temperatures ranging from 0 to 60 ° C. Temperature-dependent reaction analysis was performed in a 200 μL volume of reaction solution containing 50 mM Tris buffer (pH 6.0), 0.01 mM CuSO 4 , 0.05 mM L-DOPA and 0.4 μM purified mTyr-CNK. Commercial mushroom tyrosinase (Sigma-Aldrich) was also used as a comparative group. All measurements were repeated three times, and the measurement results are shown in FIG. 5.
도 5에 나타낸 바와 같이, mTyr-CNK 는 실온인 20℃ 내지 25℃ 에서 최적의 활성을 나타내었으며, 심지어 0℃ 에서도 최대 활성의 80% 이상을 유지하는 것을 확인하였다. 즉 mTyr-CNK 는 통상의 티로시나아제와 달리 저온 조건인 0 내지 10℃ 에서도 높은 활성을 나타내었고, 일반적인 활성 온도인 37℃가 아닌 20℃ 부근의 상온에서 최고의 활성을 나타내었다. 이와 같은 결과에 따라 재조합 mTyr-CNK 가 기존의 티로시나아제와 달리 저온 및 상온에서도 높은 활성을 나타낼 수 있는 새로운 성질을 갖는 효소임을 확인하였다. As shown in Figure 5, mTyr-CNK showed the optimum activity at room temperature 20 ℃ to 25 ℃, it was confirmed that even at 0 ℃ to maintain more than 80% of the maximum activity. That is, mTyr-CNK showed high activity even at low temperature conditions of 0 to 10 ° C. unlike conventional tyrosinase, and showed the highest activity at room temperature around 20 ° C. instead of 37 ° C., which is a general activity temperature. According to these results, it was confirmed that the recombinant mTyr-CNK is an enzyme having new properties that can exhibit high activity even at low temperature and room temperature, unlike the existing tyrosinase.
3.3 3.3 mTyrmTyr -- CNKCNK 의  of 열안정성Thermal stability 및 kinetic parameter 분석  And kinetic parameter analysis
mTyr-CNK 활성의 kinetic parameter 를 L-티로신을 이용한 L-도파크롬 형성 측정방법 및 spectrometric assay 을 이용한 L-DOPA 분석을 통해 확인하였다. 분석은 50 mM Tris buffer (pH 6.0) 및 0.01 mM CuSO4 를 포함하는 200 μL 용액을 이용하여 수행하였다. Kinetic 분석을 위하여 0.4μM 정제된 효소와 기질인 0.025-0.0625 mM 농도의 L-티로신 및 L-DOPA 를 이용하였다. L-도파크롬의 형성은 475nm 에서의 흡광도 측정을 통해 확인하였으며, 3600 M1cm의 몰 흡광계수를 이용하여 흡광도를 변환하였다. 모든 측정은 3회 반복 수행하였다. mTyr-CNK 의 열안정성은 다양한 온도에 노출된 효소의 활성이 얼마나 유지될 수 있는지를 확인하는 방법을 통해 수행하였으며, 50 mM Tris buffer (pH 6.0), 0.01 mM CuSO4, 0.05 mM L-DOPA 및 0.4 μM 정제된 mTyr-CNK 를 포함하는 200 μL 용액을 이용하였다. Kinetic parameter 측정값과 열안정성 평가 결과를 각각 표 1 및 도 6 에 나타내었다. Kinetic parameters of mTyr-CNK activity were determined by L-dopachrome formation using L-tyrosine and L-DOPA analysis using spectrometric assay. The assay was performed using 200 μL solution containing 50 mM Tris buffer (pH 6.0) and 0.01 mM CuSO 4 . For kinetic analysis, 0.4 μM purified enzyme and L-tyrosine and L-DOPA at 0.025-0.0625 mM concentration were used as substrates. Formation of L-dopachrome was confirmed by measuring absorbance at 475 nm, and the absorbance was converted using a molar extinction coefficient of 3600 M 1 cm. All measurements were repeated three times. Thermal stability of mTyr-CNK was performed by checking how much activity of enzymes exposed to various temperatures can be maintained, 50 mM Tris buffer (pH 6.0), 0.01 mM CuSO 4 , 0.05 mM L-DOPA and A 200 μL solution containing 0.4 μM purified mTyr-CNK was used. Kinetic parameter measurements and thermal stability evaluation results are shown in Table 1 and FIG. 6, respectively.
Figure PCTKR2017015183-appb-T000001
Figure PCTKR2017015183-appb-T000001
표 1에 나타낸 바와 같이, 종래의 상당수 티로시나아제에서 monophenolase 활성이 diphenolase 활성에 비해 100 배 이상 낮은 것과 비교하여, mTyr-CNK는 3.83 이라는 현저하게 높은 Vmax mono/ Vmax di (monophenolase/diphenolase activity ratio) 를 나타내었으며, monophenolase 반응의 kcat /Km 값은 diphenolase 반응값보다 약간 낮은 87% 수준인 것으로 나타났다. mTyr-CNK의 활성 비 (Vmax mono/ Vmax di) 는 서열번호 1로 표시되는 티로시나아제-CNK 의 알려진 kinetic 파라미터 (표 2, pH6 및 25℃ 조건) 와 비교하여 약 2배 이상 높은 값을 나타내었다. As shown in Table 1, the mTyr-CNK has a significantly higher V max mono / V max di (monophenolase / diphenolase activity) of 3.83, compared to 100 times lower monophenolase activity in many conventional tyrosinase activities than diphenolase activity. ratio, and the kcat / Km value of monophenolase reaction was 87%, which is slightly lower than diphenolase reaction. The activity ratio of mTyr-CNK ( V max mono / V max di) is about 2 times higher than the known kinetic parameters (Table 2, pH6 and 25 ° C. conditions) of tyrosinase-CNK represented by SEQ ID NO: 1. Indicated.
L-티로신L-tyrosine kcat = 4.3 ± 1.08 (s-1) kcat = 4.3 ± 1.08 (s-1)
Km = 9.2 ± 2.4 (mM) Km = 9.2 ± 2.4 (mM)
kcat/Km = 0.47 (mM-1·s-1) kcat / Km = 0.47 (mM-1s-1)
L-DOPAL-DOPA kcat = 2.2 ± 0.47 (s-1) kcat = 2.2 ± 0.47 (s-1)
Km = 2.6 ± 0.56 (mM) Km = 2.6 ± 0.56 (mM)
kcat/Km = 0.85 mM-1·s-1) kcat / Km = 0.85 mM-1 s-1)
한편 His294 및 Pro303 사이의 헬리컬 영역이 제거된 cTyr-CNK의 kinetic 파라미터는 pH 6 및 25℃ 조건에서 표 3과 같이 나타났다. Meanwhile, kinetic parameters of cTyr-CNK from which the helical region between His294 and Pro303 were removed are shown in Table 3 at pH 6 and 25 ° C.
L-티로신L-tyrosine kcatkcat = 2.13 ± 0.79 (s-1) = 2.13 ± 0.79 (s-1)
Km = 1.60 ± 0.65 (Km = 1.60 ± 0.65 ( mMmM ),),
kcatkcat /Km = 1.37 ± 0.07 (/ Km = 1.37 ± 0.07 ( mMmM -1·s-1)-1 · s-1)
L-DOPAL-DOPA kcatkcat = 2.05 ± 0.10 (s-1) = 2.05 ± 0.10 (s-1)
Km = 0.56 ± 0.04 (Km = 0.56 ± 0.04 ( mMmM ))
kcatkcat /Km = 3.65 ± 0.09 (/ Km = 3.65 ± 0.09 ( mMmM -1·s-1)-1 · s-1)
이러한 결과는 mTyr-CNK와 cTyr-CNK 가 비록 동일한 코어 효소활성 도메인을 공유하지만 His294 및 Pro303 사이의 헬리컬 영역의 제거로 인하여 mTyr-CNK 의 효소활성이 변화됨을 보여주는 결과이다. 이러한 결과는 C-말단 도메인이 제거된 mTyr-CNK 은 코어 효소활성 도메인의 구조에는 직접적인 큰 영향을 받지 않으며, 오직 효소 활성 및 특이도만 변형되었음을 나타낸다. 한편 L-티로신에 대한 효소의 턴오버 수 (turnover number) 및 결합 친화도는 L-DOPA 에 대한 턴오버수 및 결합 친화도보다 높게 나타났다. These results show that although mTyr-CNK and cTyr-CNK share the same core enzymatic domain, the enzymatic activity of mTyr-CNK changes due to the removal of the helical region between His294 and Pro303. These results indicate that mTyr-CNK from which the C-terminal domain was removed was not directly affected by the structure of the core enzymatic activity domain, but only the enzyme activity and specificity were modified. Meanwhile, the turnover number and binding affinity of the enzyme for L-tyrosine were higher than the turnover number and binding affinity for L-DOPA.
또한 도 6 에 나타낸 바와 같이 mTyr-CNK 단백질은 30℃ 이하에서는 매우 안정하였으나, 37 및 40℃ 에서의 반감기는 각각 25분 및 5분 미만인 것을 확인하였다. 이와 같은 30℃ 를 초과하는 온도에서의 mTyr-CNK의 불안정성은 효소를 이용한 공정에서 반응 후 효소를 선택적으로 불활성화시키는데 이용될 수 있다. 이러한 특징은 카테콜관련 생촉매 반응에서 원하지 않는 자가산화반응이 최소화되어야 하고 폴리머 고분자의 페놀성 모이어티의 변형이 강화되어야 하는 경우 유용하게 사용될 수 있다. In addition, as shown in Figure 6 mTyr-CNK protein was very stable at 30 ℃ or less, the half-life at 37 and 40 ℃ was confirmed that less than 25 minutes and 5 minutes, respectively. Such instability of mTyr-CNK at temperatures above 30 ° C. can be used to selectively deactivate the enzyme after reaction in a process using the enzyme. This feature can be useful in cases where undesired autooxidation in catechol-related biocatalytic reactions should be minimized and the modification of the phenolic moiety of the polymer polymer should be enhanced.
한편 mTyr-CNK 는 30℃ 이하에서도 열안정성을 잘 유지할 수 있으므로, 통상적으로 효소가 보관되는 냉장 (2 내지 8℃) 및 냉동 (20℃) 조건에서도 활성을 잘 유지할 수 있을 것으로 예상되었다. 특히, 생체내의 온도인 37℃에서 효소의 활성 저하가 빠르게 일어나, 생체 온도보다 낮은 조건에서 효소반응을 수행하고 생체 내 환경으로 온도를 바꾸었을 때 효소의 활성이 빠르게 사라질 수 있으므로, 의료용 생체 소재에 적용 가능성이 매우 높을 것으로 기대되었다. On the other hand, mTyr-CNK can maintain the thermal stability well below 30 ℃, it is expected that the activity can be well maintained even in the refrigeration (2 to 8 ℃) and freezing (20 ℃) conditions where the enzyme is usually stored. In particular, the degradation of enzyme activity occurs rapidly at 37 ° C., which is an in vivo temperature, and the enzyme activity may quickly disappear when the enzyme reaction is performed at a temperature lower than the living temperature and the temperature is changed to an in vivo environment. It was expected that the applicability would be very high.
실시예 4. DOPA 변형활성 비교 Example 4 DOPA Modification Activity Comparison
홍합 접착 단백질은 대표적인 DOPA-tethered 생체재료로 알려져 있고, DOPA 는 빠르고 강한 수중 접착을 위해 필수적이다. 재조합 홍합 접착 단백질을 이용하는 것은 많은 양의 단백질을 생산할 수 있다는 점에서 장점이 있으나, 15% 이하라는 낮은 in vitro DOPA 변형 수율이 걸림돌로 작용한다. 본 발명의 mTyr-CNK 의 in vitro DOPA 변형 효율을 확인하기 위하여, mTyr-CNK를 재조합 홍합 접착 단백질의 in vitro DOPA 변형에 적용하였으며, 이에 따른 DOPA 함량의 변화를 아미노산 조성물 분석법을 이용하여 확인하였다. 모델 단백질로는 홍합 접착단백질 중 fp-151 (이하 'MAP') 를 이용하였다. Mussel adhesive proteins are known as representative DOPA-tethered biomaterials, and DOPA is essential for fast and strong aquatic adhesion. Recombinant mussel adhesive proteins have the advantage of being able to produce large amounts of protein, but less than 15% is an obstacle to low in vitro DOPA modification yields. In order to confirm the in vitro DOPA modification efficiency of mTyr-CNK of the present invention, mTyr-CNK was applied to the in vitro DOPA modification of the recombinant mussel adhesive protein, the change in the DOPA content was confirmed using the amino acid composition analysis method. As a model protein, fp-151 (hereinafter referred to as 'MAP') in mussel adhesive protein was used.
동결건조된 MAP 분말을 50 mM Tris buffer (pH 6.0) 및 25 mM 아스코르브산을 포함하는 용액을 이용하여 1mg/mL 의 최종 농도가 되도록 용해시켰다. 정제된 서열번호 1의 티로시나아제-CNK 또는 mTyr-CNK 를 최종 농도가 0.01mg/mL 이 되도록 추가하였다. 상기 혼합물을 실온에서 부드럽게 교반하면서 3시간 동안 배양하였으며, 10mM 아세트산으로 투석하고 추가 연구를 위하여 -80℃에 저장하였다. 25 μL의 페놀을 6 M HCl 에서 500 μL의 티로시나아제-처리된 MAP 용액에 추가하였다. 아르곤 가스를 이용하여 산소를 제거한 후 1시간 동안 156 ℃에서 시료를 배양하면서 산 가수분해를 수행하였다. 증류수 및 메탄올을 이용하여 단백질의 가수분해물을 세척하고 용매를 증발시켜다. 그 후 아미노산 조성물을 이온 교환 컬럼 및 닌하이드린-기반 검출 시스템 (Amino acid analyzer S4300; Sykam, Eresing, Germany) 을 이용하여 분석하였다. mTyr-CNK 및 티로시나아제-CNK 를 이용한 in vitro DOPA 변형 분석 결과를 도 7에 나타내었다. Lyophilized MAP powder was dissolved to a final concentration of 1 mg / mL using a solution containing 50 mM Tris buffer (pH 6.0) and 25 mM ascorbic acid. Purified tyrosinase-CNK or mTyr-CNK of SEQ ID NO: 1 was added to a final concentration of 0.01 mg / mL. The mixture was incubated for 3 hours with gentle stirring at room temperature, dialyzed with 10 mM acetic acid and stored at -80 ° C for further study. 25 μL of phenol was added to 500 μL of tyrosinase-treated MAP solution in 6 M HCl. After removing oxygen using argon gas, acid hydrolysis was performed while culturing the sample at 156 ° C. for 1 hour. Distilled water and methanol are used to wash the hydrolyzate of the protein and the solvent is evaporated. The amino acid composition was then analyzed using an ion exchange column and a ninhydrin-based detection system (Amino acid analyzer S4300; Sykam, Eresing, Germany). Results of in vitro DOPA modification analysis using mTyr-CNK and tyrosinase-CNK are shown in FIG. 7.
도 7에 나타낸 바와 같이, mTyr-CNK 는 버섯 티로시나아제에서 보고된 15% 이하의 변형율과 비교하여 현저하게 높은 53%의 변형 수율을 나타내었다. 한편 비교예로 사용된 서열번호 1의 티로시나아제-CNK는 37% 정도의 변형 수율을 나타내었다. 상기와 같은 결과는 mTyr-CNK가 매우 높은 monophenolase/diphenolase 활성 비율을 나타내며, L-티로신에 대하여 증가된 효소 활성을 나타낼 수 있음을 보여주는 결과이다. As shown in FIG. 7, mTyr-CNK showed a significantly higher 53% strain yield compared to a strain rate of 15% or less reported in mushroom tyrosinase. On the other hand, tyrosinase-CNK of SEQ ID NO: 1 used as a comparative example showed a modification yield of about 37%. The above results show that mTyr-CNK shows a very high monophenolase / diphenolase activity ratio and can show increased enzymatic activity for L-tyrosine.
본 발명의 서열번호 2로 표시되는 mTyr-CNK은 매우 우수한 효소 활성을 나타낼 뿐만 아니라 넓은 pH 및 온도에서 안정성을 유지할 수 있고, 특히 0℃ 에서 최대 활성의 80% 를 유지하고 30℃ 이하에서도 지속적으로 매우 안정한 상태를 유지하는 우수성을 나타낸다. 이러한 우수성은 거대 분자인 페놀성 기질이 상대적으로 mTyr-CNK 의 활성부위에 접근이 용이하기 때문인 것으로 예상된다. 따라서 mTyr-CNK는 약학적 카테콜 중간체의 생합성 및 페놀성 식물성 화학물질의 생합성, 카테콜 생성 화학물의 검출 및 정량을 위한 티로시나아제-기반 바이오 센서 및 페놀성 및 치환된 페놀성 화합물의 해독과 같은 바이오 의약 및 산업적 적용을 위한 모노페놀 모노옥시다아제(monophenol monooxygenase) 로 유용하게 사용될 수 있다. MTyr-CNK represented by SEQ ID NO: 2 of the present invention not only shows very good enzymatic activity, but also can maintain stability at a wide range of pH and temperature. Excellent excellence in maintaining a very stable state. This superiority is expected because the macromolecular phenolic substrate has relatively easy access to the active site of mTyr-CNK. Thus, mTyr-CNK can be used for biosynthesis of pharmaceutical catechol intermediates and biosynthesis of phenolic phytochemicals, detoxification of tyrosinase-based biosensors for the detection and quantification of catechol-producing chemicals and phenolic and substituted phenolic compounds. It can be usefully used as monophenol monooxygenase for the same biomedical and industrial applications.

Claims (17)

  1. 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제. Recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  2. 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제는 서열번호 1의 티로시나아제에서 C-말단 신장부가 제거된 것을 특징으로 하는, 재조합 티로시나아제.The recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3 is characterized in that the C-terminal extension is removed from the tyrosinase of SEQ ID NO: 1, recombinant tyrosinase.
  3. 제 1항에 있어서, 상기 재조합 티로시나아제는 pH 4.5 내지 pH 9.5 에서 활성인 것을 특징으로 하는, 재조합 티로시나아제. The recombinant tyrosinase according to claim 1, wherein the recombinant tyrosinase is active at pH 4.5 to pH 9.5.
  4. 제1항에 있어서, 상기 재조합 티로시나아제는 -20℃ 내지 35℃ 에서 활성을 나타내는 것을 특징으로 하는, 재조합 티로시나아제.The method of claim 1, wherein the recombinant tyrosinase is characterized in that the activity at -20 ℃ to 35 ℃, recombinant tyrosinase.
  5. 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 티로시나아제 기질 전환용 조성물. Tyrosinase substrate conversion composition comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  6. 제5항에 있어서, 상기 티로시나아제 기질은 티로신 또는 DOPA(3,4-dihydroxyphenylalanine) 인 것을 특징으로 하는, 티로시나아제 기질 전환용 조성물. The composition of claim 5, wherein the tyrosinase substrate is tyrosine or DOPA (3,4-dihydroxyphenylalanine).
  7. 제5항에 있어서, 상기 조성물은 홍합 접착 단백질 내 포함된 티로시나아제 기질 전환용인 것을 특징으로 하는, 티로시나아제 기질 전환용 조성물. The composition for converting tyrosinase substrate according to claim 5, wherein the composition is for converting tyrosinase substrate contained in mussel adhesive protein.
  8. 제5항에 있어서, 상기 조성물은 pH 4.5 내지 pH 9.5 환경의 티로시나아제 기질 전환용인 것을 특징으로 하는, 티로시나아제 기질 전환용 조성물. According to claim 5, The composition is characterized in that for converting tyrosinase substrate of pH 4.5 to pH 9.5 environment, composition for converting tyrosinase substrate.
  9. 제5항에 있어서, 상기 조성물은 -20℃ 내지 35℃ 환경의 티로시나아제 기질 전환용 인 것을 특징으로 하는, 티로시나아제 기질 전환용 조성물. The composition for tyrosinase substrate conversion according to claim 5, wherein the composition is for tyrosinase substrate conversion in an environment of -20 ° C to 35 ° C.
  10. 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는, 천연고분자 또는 합성 고분자 내 모노페놀 또는 카테콜 기질 전환용 조성물. A composition for converting a monophenol or catechol substrate in a natural polymer or a synthetic polymer, comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  11. 제1항의 재조합 티로시나아제를 암호화하는 폴리뉴클레오티드. A polynucleotide encoding the recombinant tyrosinase of claim 1.
  12. 제11항의 폴리뉴클레오티드를 포함하는 벡터. A vector comprising the polynucleotide of claim 11.
  13. 제12항의 벡터로 형질전환된, 형질전환체. A transformant transformed with the vector of claim 12.
  14. 제13항의 형질전환체로부터 재조합 티로시나아제를 분리 및 정제하는 단계; 를 포함하는 재조합 티로시나아제의 생산 방법. Isolating and purifying recombinant tyrosinase from the transformant of claim 13; Method for producing a recombinant tyrosinase comprising a.
  15. 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 티로시나아제 기질에 처리하는 단계; 를 포함하는 티로시나아제 기질 전환 방법. Treating the tyrosinase substrate with a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3; Tyrosinase substrate conversion method comprising a.
  16. 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 생체 소재.A biomaterial comprising recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
  17. 서열번호 2 또는 서열번호 3으로 표시되는 재조합 티로시나아제를 포함하는 생체 접착 소재 제조용 조성물. Composition for producing a bioadhesive material comprising a recombinant tyrosinase represented by SEQ ID NO: 2 or SEQ ID NO: 3.
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