KR20170124401A - Enzyme Compositions for Detecting Cancer Biomarker Tyrosine - Google Patents

Abstract

The present invention relates to an enzymatic composition for detecting cancer biomarker tyrosine, which is intended to detect concentration of cancer biomarker tyrosine existing in urine, and consists of tyrosinase, dopachrome tautomerase (DCT), and tyrosinase related protein 1 (Tyrp1).

Description

[0001] Enzyme Compositions for Detecting Cancer Biomarker Tyrosine [

The present invention relates to an enzyme composition for detecting tyrosine, which is an oncological biomarker, and more particularly, to an enzyme composition for detecting tyrosine, tyrosine, tyrosine, and tyrosine for detecting the concentration of cancer biomarker tyrosine present in urine, Relates to an enzyme composition comprising a Dopachrome tautomerase (DCT) and tyrosinase-related protein 1 (Tyrp1).

Cancer markers are substances that occur in the malignant tumor cells themselves or are caused by reactions of normal tissues against cancer and exhibit abnormally high concentrations in the blood, urine or tissues, and the cancer marker concentration in the blood, urine or tissue You can diagnose, screen, and track progress and degeneration.

In general, the measurement or detection of the concentration of the cancer marker is carried out through the blood, but the blood has a problem that it is difficult for the general person to collect it.

Therefore, there has been developed a method of diagnosing cancer by detecting the concentration of cancer biomarker present in the urine using urine, which is not easy to obtain but is not a biomaterial such as blood or tissue.

On the other hand, it is known that the tyrosine can be used as the cancer biomarker because the concentration of tyrosine present in the urine is high in cancer patients as compared with the general public.

Korean Patent No. 10-0033547 relates to a mixed reagent for diagnosing cancer by detecting aromatic amine (Tyrosine) commonly present in urine of a cancer patient, wherein the mixed reagent is composed of mercury, nickel, nitric acid and distilled water .

However, the above-mentioned mixed reagent contains mercury and is currently prohibited because it has a great influence on the environment and human body.

Therefore, there is an urgent need to develop a composition for diagnosing cancer by eco-friendly detection of the concentration of cancer biomarker tyrosine (Tyrosine) present in the urine by using urine which is not easy to obtain but is not easy to obtain.

Korean Patent No. 10-0033547

As a result of intensive research to solve the above-mentioned problems in the enzyme composition for detecting cancer biomarker tyrosine of the present invention,

We have found that by using enzymes that are specialized for the conversion of L-tyrosine into Eumelanin, the concentration of tyrosine present in the urine can be detected environmentally and diagnosed as cancer.

Accordingly, an object of the present invention is to provide an enzyme composition capable of detecting the concentration of cancer biomarker tyrosine present in the urine environmentally.

Meanwhile, the present invention relates to a method for detecting tyrosine, tyrosinase, Dopachrome tautomerase (DCT) and tyrosinase-related protein 1 (Tyrp1). The present invention also provides an enzyme composition for detecting cancer biomarker tyrosine.

In one embodiment of the present invention, cancer is diagnosed by converting the cancer biomarker tyrosine (L-Tyrosine) present in the urine into melanin using the enzyme composition and measuring the concentration through the color of the melanin do.

In one embodiment of the present invention, the melanin is eumelanin.

In one embodiment of the present invention, the enzyme composition is characterized by comprising 40 units of tyrosinase in a weight ratio of 1: 0.5 to 2: 1 by weight of DCT to 0.5: 2 by weight of tyrosinase, and 0.5: 2 by weight of Tyrp1 .

In one embodiment of the present invention, the enzyme composition is characterized in that melanin is formed from the tyrosine to distinguish it from the color of urine which is a sample.

The enzyme composition for detection of cancer biomarkers according to the present invention contains enzymes that are characterized in converting L-tyrosine into Eumelanin, so that the concentration of tyrosine present in the urine is detected environmentally, Can be diagnosed.

Further, the enzyme composition according to the present invention can easily diagnose cancer without inconveniencing users to visit a specialized institution such as a hospital and undergo cancer diagnosis by using urine which can be easily collected.

Fig. 1 is a control palette for measuring the color of the produced eumelanin.
FIG. 2 is a photograph comparing the colors of the produced melanin.

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

The present invention relates to a pharmaceutical composition comprising tyrosinase, Dopachrome tautomerase (DCT) and tyrosinase-related protein 1 (Tyrp1) for detecting the concentration of the cancer biomarker tyrosine present in the urine And an enzyme composition for detecting cancer biomarker tyrosine.

In one embodiment of the present invention, an enzyme composition comprising the tyrosinase, Dopachrome tautomerase (DCT) and tyrosinase-related protein 1 (Tyrp1) Cancer can be diagnosed by converting the cancer biomarker tyrosine (L-Tyrosine) into melanin and measuring the concentration through the color of the melanin.

In one embodiment of the present invention, in converting the L-tyrosine into melanin using the enzyme composition, the melanin is characterized by being eumelanin.

Since the enzyme composition according to the present invention is for diagnosing cancer using urine, when the concentration is measured by means of Pheomelanin (yellowish brown color) or mixed melanin (brown melanin) in consideration of color of urine , It may be difficult to measure the concentration because it is similar to the color of the urine. Therefore, it is preferable that the enzyme composition according to the present invention measures the concentration through eumelanin showing black color.

The enzyme composition according to the present invention not only accelerates the pathway of eumelanin biosynthesis but also induces the color of the resulting eumelanin to a more black path by adding DCT and Tyrp1 enzyme to tyrosinase.

In one embodiment of the present invention, the tyrosine detection reaction is preferably performed under the conditions of a phosphate buffer solution or a Tris-HCl buffer solution at a final concentration of 50 to 100 mM, pH 7.0 to 8.5 Do.

Specifically, the phosphate buffer solution contained in the enzyme composition according to an embodiment of the present invention is 200 mM. When the total volume of 500 μl of the enzyme composition and 500 μl of the urine sample is 1000 μl, the phosphate buffer solution of the tyrosine detection reaction Concentration can be performed at 50 mM.

In one embodiment of the present invention, the concentration of tyrosine present in the urine can be measured through the color of the eumelanin produced by the reaction with the enzyme composition. When the color of the urine produced by the produced eumelanin is close to black, it can be seen that the concentration of tyrosine in the constant urine amount is high.

In one embodiment of the present invention, the enzyme composition is characterized by comprising 40 units of tyrosinase in a weight ratio of 1: 0.5 to 2: 1 by weight of DCT to 0.5: 2 by weight of tyrosinase, and 0.5: 2 by weight of Tyrp1 .

If the above content is not satisfied, it may be difficult to measure the concentration of tyrosine because the color of the produced melanin may appear to be a yellowish brown color and be mixed with the urine color.

Referring to Reaction Scheme 1 below, L-tyrosine is oxidized by tyrosinase to form 3,4-dihydroxyphenylalanine (DOPA), and further oxidized by tyrosinase to form DOPA quinone , And Leucodopachrome to form Dopachrome.

The dopachrome is a melanin precursor and is tautomerized by DCT to form 5,6-dihydroxyindole-2-carboxylic acid (DHICA).

The 5,6-dihydroxyindole-2-carboxylic acid is oxidized by Tyrp 1 to form indole-5,6-quinonecarboxylic acid and the indole-5,6-quinonecarboxylic acid is polymerized to form uamelanin .

 [Reaction Scheme 1]

The tyrosinase is a tyrosinase hydroxylase and a copper-containing enzyme having catalytic activity of dopa oxidase, which can be found in microorganisms and plant and organism tissues. In particular, the tyrosinase promotes the production of melanin and other pigments by oxidation of phenols such as tyrosine.

The Dopachrome tautomerase (DCT), also known as tyrosinase-related protein 2 (Tyrp 2), binds tyrosinase and Tyrp 1 and is involved in the conversion of L-tyrosine to melanin in melanocytes Specific melanin cell-specific enzyme.

The tyrosinase-related protein 1 (Tyrp 1) plays a role in stabilizing tyrosinase.

The enzyme composition according to the present invention is characterized in that the dopa chromium is converted to indole-5,6-quinone via 5,6-dihydroxyindole by tyrosinase (See Reaction Scheme 2 below), which is tautomerized by DCT and oxidized by Tyrp 1 to form melanin close to black to distinguish it from the color of the urine of the sample So that the measurement can be performed effectively.

[Reaction Scheme 2]

In one embodiment of the present invention, when measuring the concentration of tyrosine present in the urine through the color of the produced eumelanin, the color of the produced uromelanin is compared with the contrast color table of FIG. 1 to confirm the tyrosine concentration in the urine have. Through this, it is possible to diagnose the occurrence of cancer and metabolic diseases.

In one embodiment of the present invention, the enzyme composition comprises

Tyrosinase, DCT and Tyrp1 and can be prepared by adding phosphate buffer or Tris-HCl buffer solution and distilled water.

As described above, tyrosinase, DCT and Tyrp1 are characterized by comprising 40 units of tyrosinase in a weight ratio of 1: 0.5 to 2: 1 by weight of DCT and 0.5: 2 by weight of Tyrp1 relative to 1 part by weight of tyrosinase .

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are for illustrative purpose only and that the scope of the present invention is not limited to these embodiments.

Production Example 1: Preparation of each component of the enzyme composition

The tyrosinase enzyme was purchased from Sigma.

DCT and Tyrp1 were obtained from mouse blood by reference to the following steps and purified after protein expression in E. coli.

(i) RNA extraction from mouse blood

RiboEx RNA extraction solution kit (Jinol Biotechnology, Korea) was used in this embodiment. The process is as follows.

a. 300 μl of mouse blood and 600 μl of RiboEx RNA extraction solution (Jeolol Biotechnology, Korea) were mixed from the mouse blood, mixed strongly and left at room temperature for 5 ~ 10 minutes to dissolve the cell membrane.

b. 200 μl of chloroform was added and mixed vigorously. The mixture was centrifuged at 13,000 rpm at 4 ° C for 10 minutes using a centrifuge to separate the upper and lower layers.

c. After collecting only the upper layer, the same amount of the column-binding buffer was added and mixed well. Then, the solution was added to the column provided in the kit and spin-down to bind only RNA to the column.

d. The wash buffer provided in the kit was added to the column, and then the material other than RNA was removed by spin-down. The remaining wash buffer was removed by centrifugation for an additional 1 minute.

e. The elution buffer was added to recover the RNA bound to the column, and the RNA was recovered by spinning 5 minutes later.

(ii) preparing cDNA from the obtained RNA

 1 μg RNA, 5 units of reverse transcriptase (reverse transcriptase), oligo dT-50 pmol, 0.1 M DTT- 2 μl, 10x reaction buffer - 2 μl, water - about 20 μl; The mixture was prepared in the above composition and reacted at 45 ° C for 10 minutes and at 38 ° C for 3 hours or longer in a PCR instrument.

(iii) securing the gene (DCT, Tyrp1) from cDNA

a. In order to amplify the gene from the cDNA generated in the above step, the mixture was prepared with the following composition, and the gene amplification reaction (PCR) was performed under the following conditions.

Mixture composition: 2 μl of cDNA solution, 5 units of pfu DNA amplification enzyme, 2 μl of 10 mM dNTP, 4 μl of 10 × reaction buffer, 2 pmol of each primer pair, and water - about 40 μl.

Amplification reaction conditions: 95 ° C 4 min - 1 time; 95 ° C for 20 seconds, 60 ° C for 20 seconds, 72 ° C for 1 minute and 50 seconds - 35 times; 72 占 폚 10 min - 1 time; Continue at 4 ℃.

The information of the primer pair used in the above reaction is shown in Table 1 below.

division Base sequence Primer for DCT amplification Forward primer Atgggccttgtgggatggg (SEQ ID NO: 1) Reverse primer Ctaggcttcctccgtgtatctcttgc (SEQ ID NO: 2) Tyrp1 primer for amplification Forward primer Atgaaatcttacaacgtcctccccct (SEQ ID NO: 3) Reverse primer Tcagaccatggagtggttaggattcg (SEQ ID NO: 4)

The DCT and Tyrp1 enzymes were prepared by inserting the genes obtained by the above method into the pET28a vector and constructing a fusion protein in which 6x His was attached to the N-terminus and transformed into BL21 (DE3) Escherichia coli. The process is as follows.

(i) the step of culturing the transformed E. coli

a. NdeI of the pET28a vector, and DCT or Tyrp1 gene into the XhoI restriction enzyme site, thereby preparing a recombinant DNA which produces 6x His ~ protein.

b. E. coli transformed with BL21 (DE3) was prepared by heat shock transformation.

c. The E. coli strain was cultured in 3 L of Luria broth at 37 DEG C and 200 rpm at OD600 ~ 0.7, followed by treatment with 0.1M IPTG to induce protein production.

d. After further culturing at 30 DEG C for 4 hours, E. coli was precipitated by centrifugation.

   (ii) Escherichia coli disruption step

a. The precipitated E. coli was resuspended in 200 ml of 1x PBS and washed by centrifugation.

b. The precipitated E. coli was suspended in 40 ml of 0.5 M NaCl, 5 mM imidazole, 20 mM Tris-HCl, pH 7.9; Lt; / RTI >

c. Escherichia coli was disrupted in an ultrasonic cell disruptor under the condition of Energy 38% max, total cell breaking time 4 min (2 second sonic treatment / 4 sec pause). The bottle containing the E. coli sample remained ice-filled during the cell disruption process.

d. The shredded E. coli was centrifuged at 13,000 rpm, 30 minutes, and 4 ° C.

e. Only the supernatant was collected in a clean conical tube.

   (iii) Purification of his tag-fusion protein using Ni-NTA agarose resin

a. The water-soluble protein solution obtained in the above step was mixed with Ni-NTA agarose resin and shaken for 30 minutes at 4 ° C to induce binding of the his tag-fusion protein to the Ni-NTA agarose resin.

b. The protein solution and the resin mixture were put into the column, and the column cock was opened to extract the liquid.

c. 400 ml of washing buffer (0.5 M NaCl, 60 mM imidazole, 20 mM Tris-HCl, pH 7.9) was passed through to remove proteins other than impurities and fusion proteins.

d. 3 ml of elution buffer (0.25 M NaCl, 500 mM imidazole, 20 mM Tris-HCl, pH 7.9) was added and allowed to stand for 10 minutes.

e. d process was repeated twice.

(iii) Protein purification using FPLC-size exclusion method

a. The recovered proteins were loaded onto FPLC equipped with a superdex S200 (GE healthcare) column and the proteins were separated by size.

b. Among the obtained fraction samples, fractions corresponding to his tag fusion proteins were identified by polyacrylamide gel electrophoresis and finally the desired his tag fusion protein was obtained.

c. Abs 280 values were measured, and protein concentrations were converted from the obtained values, and they are shown in Table 2 below.

division Protein concentration (OD ₂₈₀ nm) 1 mg DCT / ml 1.65 1 mg Tyrp1 / ml 1.42

Example 1: Preparation of enzyme composition

40 [micro] l of tyrosinase enzyme (SEQ ID NO: 5) having an activity of 1000 unit / ml, 40 [micro] l of DCT (SEQ ID NO: 6) having activity of 1000 unit / ml prepared in Preparation Example 1, 40 μl of Tyrp1 (SEQ ID NO: 7) having 1000 units / ml, 250 μl of 200 mM Tris buffer pH 8, and 130 μl of distilled water were added to prepare an enzyme composition (stock solution).

Stock solution Volumn (500 [mu] L) 200 mM Tris buffer pH8 250 μl Tyrosinase (> = 1000 unit / ml) 40 μl DCT (> = 1000 unit / ml) 40 μl TYRP1 (> = 1000 unit / ml) 40 μl D.W 130 μl

Example 2: Urine sampling

Urine collection from normal and cancer patients was the same and was performed as follows.

The first urine from the urethra was not collected during the first urine collection after the morning wake, and 30 ml of the sample was collected from the urine.

Experimental Example 1: Measurement of color of melanin produced

In order to measure the concentration of tyrosine present in a certain amount, the same amount of 500 μl of the urine of each of the normal and cancer patients collected in Example 2 was reacted with the enzyme composition of Example 1 (total of 1000 μl each). Then, the color of the urine produced by each produced eumelanin is compared with the control color table of FIG. 1, and is shown in Table 4 below.

Color of urine by eumelanin Urine of a normal person Yellow to tan Urine of cancer patient black

As shown in Table 4, the urine color produced by the reaction of the normal human urine with the enzyme composition of the present invention was yellow to yellowish brown, while the urine of the cancer patient was reacted with the enzyme composition, The color of the urine was confirmed to be black.

This shows that tyrosine is present in large amounts in urine of cancer patients.

Experimental Example 2: Comparison of color of melanin produced

In order to examine that the enzyme composition containing DCT and Tyrp1 in addition to the enzyme composition containing only tyrosinase can cause the color of urine caused by the produced uromelain to appear more black, the following experiment was conducted .

(A) obtained by mixing 500 μl of normal human urine collected in Example 2 with 40 parts of tyrosinase, 20 units of DCT and 20 units of Tyrp1, and the mixture (A) obtained in Example 2 (B) in which 500 μl of urine of the patient was mixed with an enzyme composition containing only 40 units of tyrosinase, and 500 μl of urine of the cancer patient collected in Example 2 were mixed with 40 units of tyrosinase, 20 units of DCT and Tyrp1 (C) mixed with the enzyme composition of the present invention containing 20 units were reacted at 37 DEG C for 30 minutes, respectively.

2, the mixture (A) obtained by reacting the enzyme composition according to the present invention with the urine of a normal person shows yellow color, whereas the mixture (B) and (C) Respectively.

Especially, as a result of urine detection of a cancer patient, it was confirmed that the color of the mixture (C) containing DCT and Tyrp 1 is more black than the mixture (B) containing only tyrosinase.

Therefore, by adding the DCT and Tyrp1 enzyme to the tyrosinase, the enzyme composition according to the present invention not only accelerates the pathway of eumelanin biosynthesis, but also can induce the color of the resulting eumelanin to a more black-colored pathway Respectively.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. Do. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

<110> CubeBio <120> Enzyme Compositions for Detecting Cancer Biomarker Tyrosine <130> DP18030 <160> 7 <170> KoPatentin 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 1 atgggccttg tgggatggg 19 <210> 2 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 2 ctaggcttcc tccgtgtatc tcttgc 26 <210> 3 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 3 atgaaatctt acaacgtcct ccccct 26 <210> 4 <211> 26 <212> RNA <213> Artificial Sequence <220> <223> primer <400> 4 tcagaccatg gagtggttag gattcg 26 <210> 5 <211> 556 <212> PRT <213> Agaricus bisporus <400> 5 Met Ser Leu Ile Ala Thr Val Gly Pro Thr Gly Gly Val Lys Asn Arg   1 5 10 15 Leu Asn Ile Val Asp Phe Val Lys Asn Glu Lys Phe Phe Thr Leu Tyr              20 25 30 Val Arg Ser Leu Glu Leu Leu Gln Ala Lys Glu Gln His Asp Tyr Ser          35 40 45 Ser Phe Phe Gln Leu Ala Gly Ile His Gly Leu Pro Phe Thr Glu Trp      50 55 60 Ala Lys Glu Arg Pro Ser Met Asn Leu Tyr Lys Ala Gly Tyr Cys Thr  65 70 75 80 His Gly Gln Val Leu Phe Pro Thr Trp His Arg Thr Tyr Leu Ser Val                  85 90 95 Phe Glu Gln Ile Leu Gln Gly Ala Ala Ile Glu Val Ala Asn Lys Phe             100 105 110 Thr Ser Asn Gln Thr Asp Trp Ile Gln Ala Ala Gln Asp Leu Arg Gln         115 120 125 Pro Tyr Trp Asp Trp Gly Phe Glu Leu Met Pro Pro Asp Glu Val Ile     130 135 140 Lys Asn Glu Glu Val Asn Ile Thr Asn Tyr Asp Gly Lys Lys Ile Ser 145 150 155 160 Val Lys Asn Pro Ile Leu Arg Tyr His Phe His Pro Ile Asp Pro Ser                 165 170 175 Phe Lys Pro Tyr Gly Asp Phe Ala Thr Trp Arg Thr Thr Val Arg Asn             180 185 190 Pro Asp Arg Asn Arg Arg Glu Asp Ile Pro Gly Leu Ile Lys Lys Met         195 200 205 Arg Leu Glu Glu Gly Gln Ile Arg Glu Lys Thr Tyr Asn Met Leu Lys     210 215 220 Phe Asn Asp Ala Trp Glu Arg Phe Ser Asn His Gly Ile Ser Asp Asp 225 230 235 240 Gln His Ala Asn Ser Leu Glu Ser Val His Asp Asp Ile His Val Met                 245 250 255 Val Gly Tyr Gly Lys Ile Glu Gly His Met Asp His Pro Phe Phe Ala             260 265 270 Ala Phe Asp Pro Ile Phe Trp Leu His His Thr Asn Val Asp Arg Leu         275 280 285 Leu Ser Leu Trp Lys Ala Ile Asn Pro Asp Val Trp Val Thr Ser Gly     290 295 300 Arg Asn Arg Asp Gly Thr Met Gly Ile Ala Pro Asn Ala Gln Ile Asn 305 310 315 320 Asp Glu Thr Pro Leu Glu Pro Phe Tyr Gln Ser Glu Asp Lys Val Trp                 325 330 335 Thr Ser Ala Ser Leu Ala Asp Thr Ala Arg Leu Gly Tyr Ser Tyr Pro             340 345 350 Asp Phe Asp Lys Leu Val Gly Gly Thr Lys Glu Leu Ile Arg Asp Ala         355 360 365 Ile Asp Asp Leu Ile Asp Glu Arg Tyr Gly Ser Lys Pro Ser Ser Gly     370 375 380 Ala Arg Asn Thr Ala Phe Asp Leu Leu Ala Asp Phe Lys Gly Ile Thr 385 390 395 400 Lys Glu His Lys Glu Asp Leu Lys Met Tyr Asp Trp Thr Ile His Val                 405 410 415 Ala Phe Lys Lys Phe Glu Leu Lys Glu Ser Phe Ser Leu Leu Phe Tyr             420 425 430 Phe Ala Ser Asp Gly Gly Asp Tyr Asp Gln Glu Asn Cys Phe Val Gly         435 440 445 Ser Ile Asn Ala Phe Arg Gly Thr Thr Pro Glu Thr Cys Ala Asn Cys     450 455 460 Gln Asp Asn Glu Asn Leu Ile Gln Glu Gly Phe Ile His Leu Asn His 465 470 475 480 Tyr Leu Ala Arg Asp Leu Glu Ser Phe Glu Pro Gln Asp Val His Lys                 485 490 495 Phe Leu Lys Glu Lys Gly Leu Ser Tyr Lys Leu Tyr Ser Arg Glu Asp             500 505 510 Lys Ser Leu Thr Ser Leu Ser Val Lys Ile Glu Gly Arg Pro Leu His         515 520 525 Leu Pro Pro Gly Glu His Arg Pro Lys Tyr Asp His Thr Gln Asp Arg     530 535 540 Val Val Phe Asp Asp Val Ala Val His Val Ile Asn 545 550 555 <210> 6 <211> 517 <212> PRT <213> Mus musculus <400> 6 Met Gly Leu Val Gly Trp Gly Leu Leu Leu Gly Cys Leu Gly Cys Gly   1 5 10 15 Ile Leu Leu Arg Ala Arg Ala Gln Phe Pro Arg Val Cys Met Thr Leu              20 25 30 Asp Gly Val Leu Asn Lys Glu Cys Cys Pro Pro Leu Gly Pro Glu Ala          35 40 45 Thr Asn Ile Cys Gly Phe Leu Glu Gly Arg Gly Gln Cys Ala Glu Val      50 55 60 Gln Thr Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln  65 70 75 80 Asp Asp Arg Glu Gln Trp Pro Arg Lys Phe Phe Asn Arg Thr Cys Lys                  85 90 95 Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Gly Cys Lys Phe Gly             100 105 110 Trp Thr Gly Pro Asp Cys Asn Arg Lys Lys Pro Ala Ile Leu Arg Arg         115 120 125 Asn Ile His Ser Leu Thr Ala Gln Glu Arg Glu Gln Phe Leu Gly Ala     130 135 140 Leu Asp Leu Ala Lys Lys Ser Ile His Pro Asp Tyr Val Ile Thr Thr 145 150 155 160 Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro Gln Ile                 165 170 175 Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser             180 185 190 Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Lys Ala Ile Asp         195 200 205 Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr His Leu     210 215 220 Leu Trp Leu Glu Arg Glu Leu Gln Arg Leu Thr Gly Asn Glu Ser Phe 225 230 235 240 Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn Glu Cys Asp Val                 245 250 255 Cys Thr Asp Glu Leu Leu Gly Ala Ala Arg Gln Asp Asp Pro Thr Leu             260 265 270 Ile Ser Arg Asn Ser Arg Phe Ser Thr Trp Glu Ile Val Cys Asp Ser         275 280 285 Leu Asp Asp Tyr Asn Arg Arg Val Thr Leu Cys Asn Gly Thr Tyr Glu     290 295 300 Gly Leu Leu Arg Arg Asn Lys Val Gly Arg Asn Asn Glu Lys Leu Pro 305 310 315 320 Thr Leu Lys Asn Val Gln Asp Cys Leu Ser Leu Gln Lys Phe Asp Ser                 325 330 335 Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu             340 345 350 Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met Asn Leu         355 360 365 His Asn Leu Ala His Ser Phe Leu Asn Gly Thr Asn Ala Leu Pro His     370 375 380 Ser Ala Asn Asp Pro Val Phe Val Val Leu His Ser Phe Thr Asp 385 390 395 400 Ala Ile Phe Asp Glu Trp Leu Lys Arg Asn Asn Pro Ser Thr Asp Ala                 405 410 415 Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr Asn Met             420 425 430 Val Pro Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu Thr Ala         435 440 445 Glu Gln Leu Gly Tyr Asn Tyr Ala Val Asp Leu Ser Glu Glu Glu Ala     450 455 460 Pro Val Trp Ser Thr Thr Leu Ser Val Val Ile Gly Ile Leu Gly Ala 465 470 475 480 Phe Val Leu Leu Leu Gly Leu Leu Ala Phe Leu Gln Tyr Arg Arg Leu                 485 490 495 Arg Lys Gly Tyr Ala Pro Leu Met Glu Thr Gly Leu Ser Ser Lys Arg             500 505 510 Tyr Thr Glu Glu Ala         515 <210> 7 <211> 537 <212> PRT <213> Mus musculus <400> 7 Met Lys Ser Tyr Asn Val Leu Pro Leu Ala Tyr Ile Ser Leu Phe Leu   1 5 10 15 Met Leu Phe Tyr Gln Val Trp Ala Gln Phe Pro Arg Glu Cys Ala Asn              20 25 30 Ile Glu Ala Leu Arg Arg Gly Val Cys Cys Pro Asp Leu Leu Pro Ser          35 40 45 Ser Gly Pro Gly Thr Asp Pro Cys Gly Ser Ser Ser Gly Arg Gly Arg      50 55 60 Cys Val Ala Val Ile Ala Asp Ser Arg Pro His Ser Arg His Tyr Pro  65 70 75 80 His Asp Gly Lys Asp Asp Arg Glu Ala Trp Pro Leu Arg Phe Phe Asn                  85 90 95 Arg Thr Cys Gln Cys Asn Asp Asn Phe Ser Gly His Asn Cys Gly Thr             100 105 110 Cys Arg Pro Gly Trp Arg Gly Ala Ala Cys Asn Gln Lys Ile Leu Thr         115 120 125 Val Arg Arg Asn Leu Leu Asp Leu Ser Pro Glu Glu Lys Ser His Phe     130 135 140 Val Arg Ala Leu Asp Met Ala Lys Arg Thr Thr His Pro Gln Phe Val 145 150 155 160 Ile Ala Thr Arg Arg Leu Glu Asp Ile Leu Gly Pro Asp Gly Asn Thr                 165 170 175 Pro Gln Phe Glu Asn Ile Ser Val Tyr Asn Tyr Phe Val Trp Thr His             180 185 190 Tyr Tyr Ser Val Lys Lys Thr Phe Leu Gly Thr Gly Gln Glu Ser Phe         195 200 205 Gly Asp Val Asp Phe Ser His Glu Gly Pro Ala Phe Leu Thr Trp His     210 215 220 Arg Tyr His Leu Leu Gln Leu Glu Arg Asp Met Gln Glu Met Leu Gln 225 230 235 240 Glu Pro Ser Phe Ser Leu Pro Tyr Trp Asn Phe Ala Thr Gly Lys Asn                 245 250 255 Val Cys Asp Val Cys Thr Asp Asp Leu Met Gly Ser Arg Ser Asn Phe             260 265 270 Asp Ser Thr Leu Ile Ser Pro Asn Ser Val Phe Ser Gln Trp Arg Val         275 280 285 Val Cys Glu Ser Leu Glu Glu Tyr Asp Thr Leu Gly Thr Leu Cys Asn     290 295 300 Ser Thr Glu Gly Gly Pro Ile Arg Arg Asn Pro Ala Gly Asn Val Gly 305 310 315 320 Arg Pro Ala Val Gln Arg Leu Pro Glu Pro Gln Asp Val Thr Gln Cys                 325 330 335 Leu Glu Val Arg Phe Asp Thr Pro Pro Phe Tyr Ser Asn Ser Thr             340 345 350 Asp Ser Phe Arg Asn Thr Val Glu Gly Tyr Ser Ala Pro Thr Gly Lys         355 360 365 Tyr Asp Pro Ala Val Arg Ser Leu His Asn Leu Ala His Leu Phe Leu     370 375 380 Asn Gly Thr Gly Gly Gln Thr His Leu Ser Pro Asn Asp Pro Ile Phe 385 390 395 400 Val Leu Leu His Thr Phe Thr Asp Ala Val Phe Asp Glu Trp Leu Arg                 405 410 415 Arg Tyr Asn Ala Asp Ile Ser Thr Phe Pro Leu Glu Asn Ala Pro Ile             420 425 430 Gly His Asn Arg Gln Tyr Asn Met Val Pro Phe Trp Pro Pro Val Thr         435 440 445 Asn Thr Glu Met Phe Val Thr Ala Pro Asp Asn Leu Gly Tyr Ala Tyr     450 455 460 Glu Val Gln Trp Pro Gly Gln Glu Phe Thr Val Ser Glu Ile Ile Thr 465 470 475 480 Ile Ala Val Ala Ala Leu Leu Ala Val Ala Ile Phe Gly Val                 485 490 495 Ala Ser Cys Leu Ile Arg Ser Ser Ser Thr Lys Asn Glu Ala Asn Gln             500 505 510 Pro Leu Leu Thr Asp His Tyr Gln Arg Tyr Ala Glu Asp Tyr Glu Glu         515 520 525 Leu Pro Asn Pro Asn His Ser Met Val     530 535

Claims (6)

Cancer including tyrosinase, Dopachrome tautomerase (DCT) and tyrosinase-related protein 1 (Tyrp1) to detect the concentration of cancer biomarker tyrosine present in the urine, Enzyme composition for detecting biomarker tyrosine. The cancer composition according to claim 1, wherein the enzyme composition is used to convert cancer biomarker tyrosine (L-Tyrosine) present in the urine into melanin, and the cancer is diagnosed by measuring the concentration through the color of the melanin Enzyme composition for detecting biomarker tyrosine. 3. The enzyme composition according to claim 2, wherein the melanin is eumelanin. [Claim 3] The method according to claim 2, wherein the enzyme composition comprises 40 units of tyrosinase and 1 part by weight of tyrosinase, 0.5 to 2 parts by weight of DCT and 0.5 to 2 parts by weight of Tyrp1, Enzyme composition for detecting marker tyrosine. The method according to claim 2, wherein the tyrosine detection reaction through the color of the melanin is carried out in phosphate buffer or Tris-HCl buffer solution at a final concentration of 50 to 100 mM, pH 7.0 to 8.5 Wherein the enzyme is a protease. [Claim 3] The enzyme composition for detecting cancer biomarker tyrosine according to claim 2, wherein the enzyme composition forms melanin that shows black color from the tyrosine, thereby distinguishing it from urine color of a sample.

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