JP3110087B2 - Method for producing thermostable β-tyrosinase by genetic recombination - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing thermostable .beta.-tyrosinase by a genetic recombination technique, and a gene, an expression vector and a host microorganism useful for the method.

[0002]

2. Description of the Related Art Conventionally, β-tyrosinase has been used as vitamin B ₆
With the involvement of pyridoxal-5'-phosphate (PLP), which is a type of L-tyrosine, pyruvic acid, phenol,
It was discovered as an enzyme that catalyzes an α, β elimination reaction that decomposes to ammonia, and then a β-substitution reaction that synthesizes L-tyrosine or L-DOPA from phenol or pyrocatechol with L-serine, or pyruvate, ammonia, phenol or It is known as an enzyme that catalyzes the reverse reaction of the α, β elimination reaction for synthesizing L-tyrosine or L-dopa from pyrocatechol. For example, Escherichia intermedia , Proteus.
Retogeri (Proteus rettgeri), are found from a microorganism around the enterobacteria are such mesophile such Erwinia herbicola (Erwinia herbicola).

[0003] However, β-produced by such a normal-temperature bacterium.
Tyrosinase has a technical difficulty in that it has low heat resistance and is significantly restricted in terms of reaction temperature, thermal stability, and the like when used. Therefore, the present inventors have conducted intensive studies to create a β-tyrosinase excellent in heat resistance that overcomes such technical difficulties, and as a result, it was found that the natural medium alone and the L-tyrosine-containing synthetic medium alone could be used alone. Although it does not grow, Bacillus sp. S strain ( Bacillus sp stra
in S; co-produced by S. Biotechnol. No. 809), a thermostable β-tyrosinase-producing bacillus Cymbiobacterium having a unique growth characteristic which is completely unknown and has not been described in any known literature. Thermophilum ( Symbiobacter
ium thermophilum ; No. 810 of Microtechnical Research Laboratories) ( mixed form with Bacillus sp. S strain) was successfully separated and collected from a soil sample, and the new bacillus was about 80 ° C.
(PH 8.0) has an optimum temperature of action, and is approximately 80 ° C. (pH
8.0, a retention time of 20 minutes), producing a heat-resistant β-tyrosinase which is a novel enzyme completely different from β-tyrosinase produced by the above-mentioned normal temperature bacteria in that it exhibits thermostability with little heat inactivation. (Japanese Patent Laid-Open No. 62-2857)
No. 85).

[0004]

However, the heat resistance β
The tyrosinase-producing bacterium Symbiobacterium thermophilum cannot grow alone and can only grow in symbiosis with the Bacillus sp. S strain; for its cultivation, the symbiotic relationship with the Bacillus sp. Since the culture must be carried out while maintaining the mixture, the culture system is complicated and large-scale cultivation is difficult. In addition, heat-resistant β-
There are problems that the amount of tyrosinase protein is small and that the purification of the enzyme is difficult.

Accordingly, the present invention is to solve the above-mentioned various problems by a genetic engineering technique.

[0006]

Means for Solving the Problems Accordingly, the present inventors have:
Using a genetic engineering technique for the purpose of simplifying the culture system, improving the productivity per cell, and simplifying the enzyme purification, isolating the thermostable β-tyrosinase structural gene from the above Symbiobacterium thermophilum, As a result of inserting the structural gene into an appropriate expression vector and transforming a host microorganism using it to produce a large amount of thermostable β-tyrosinase, this study showed that thermophilic symbiotic bacteria Chromosomal DNA of Symbiobacterium thermophilum
DN containing a more thermostable β-tyrosinase structural gene
The A fragment can be cloned, and this DNA fragment is inserted into an appropriate expression vector, and Escherichia coli is transformed with the DNA fragment, whereby a large amount of thermostable β-tyrosinase was successfully expressed. It has been confirmed that β-tyrosinase has the same properties as the enzyme produced by Symbiobacterium thermophilum used as a starting material for gene acquisition, and the present invention has been completed.

Accordingly, the present invention provides the following properties: (1) action: L
-Generates pyruvic acid, phenol and ammonia from tyrosine; (2) Substrate specificity: decomposes L-tyrosine; (3) Range of optimal pH and stable pH: optimal pH at a reaction temperature of 70 ° C is about 7 to 7.5, and the stable pH when left at 25 ° C. for 2 days is 6 to 11; (4) Optimal temperature and heat resistance: The optimal temperature at pH 8.0 is about 80. 80 ° C when kept at pH 8.0 for 20 minutes.
(5) molecular weight of about 4 as determined by SDS gel electrophoresis
8,000; the molecular weight estimated from the amino acid sequence deduced from the genetic sequence is about 52,300;
A host transformed with an expression vector into which a gene encoding the enzyme obtained from a microorganism belonging to the genus Symbiobacterium and having the gene encoding the enzyme is cultivated. Provided is a method for producing a thermostable β-tyrosinase, comprising collecting an enzyme.

[0008] The present invention further provides a DNA encoding a thermostable β-tyrosinase protein having the amino acid sequence shown in SEQ ID NO: 1. The present invention further relates to the aforementioned D.
An expression vector comprising NA is provided. The invention further provides
Also provided is a host transformed with the expression vector.

[0009]

DETAILED DESCRIPTION OF THE INVENTION The DNA fragment containing the thermostable β-tyrosinase structural gene of the present invention is prepared by preparing a gene library from the chromosomal DNA of the aforementioned Symbiobacterium thermophilum, and using an appropriate probe, for example, the above-mentioned JP Determination of the primary amino acid sequence of heat-stable β-tyrosinase purified and isolated according to the method described in JP-A-62-285785, reveals that there is high homology at the amino acid level. The chromosomal DNA library can be obtained by screening the above chromosomal DNA library using the gene of the enzyme as a probe and cloning a DNA fragment containing the thermostable β-tyrosinase of the present invention.

[0010] The nucleotide sequence of the structural gene portion of the DNA fragment thus cloned can be determined by a method known per se, for example, the Zanger method using M13 phage. The structural gene of tyrosinase consists of 1377 bases, and by analyzing the amino acid residues encoded by it, the primary structure of a heat-resistant β-tyrosinase protein with a molecular weight of 52,269 consisting of 458 amino acid residues could be elucidated. (SEQ ID NO: 1).

However, as is well known, for many amino acids, the gene DNA encoding them is used.
There are a plurality of base sequences. The base sequence is not uniquely determined and there are many possibilities. In the case of the gene encoding the amino acid sequence of the thermostable β-tyrosinase protein of Symbiobacterium thermophilum disclosed by the present inventors, the DNA base sequence is not limited to the DNA base sequence of the natural gene. However, the gene DNA of the present invention is not limited to the natural DNA base sequence, but may be any other DNA encoding the amino acid sequence of the heat-resistant β-tyrosinase protein disclosed by the present invention. It also includes the base sequence.

According to the gene recombination technique, a specific site of a basic DNA is not changed or the basic characteristic of the enzyme activity of the heat-resistant β-tyrosinase encoded by the DNA is changed. Mutations can be artificially induced to improve. Also provided by the present invention, a gene DNA having a natural base sequence or a gene DNA having a base sequence different from the natural one can be similarly artificially inserted, deleted, and substituted to obtain a natural gene. The characteristics can be equivalent or improved, and the present invention includes such a mutant gene.

The DNA fragment containing the thermostable β-tyrosinase of the present invention can be obtained by preparing an expression vector suitable for a host in which the thermostable β-tyrosinase is expressed in the microbial cells, for example, pUC18 (lacking the lac promoter of Escherichia coli). (Takara Shuzo) or the like, it is possible to construct an expression plasmid that produces heat-resistant β-tyrosinase in the host microorganism cells, for example, in Escherichia coli.

The expression plasmid having the heat-resistant β-tyrosinase gene of the present invention can be transformed into a heat-resistant β-tyrosinase gene by introducing the plasmid into an appropriate host microorganism such as Escherichia coli.
-It is possible to create a transformed microorganism that produces a tyrosinase protein. The transformed microorganism thus created is cultured in an appropriate medium and under appropriate conditions to obtain a heat-resistant β-
It is possible to produce tyrosinase in large quantities. Isolation of the heat-stable β-tyrosinase protein from the culture after culturing can be performed, for example, by disrupting the cells with ultrasonic waves, heat-treating and centrifuging to heat-denature and remove proteins derived from Escherichia coli. A cell extract containing β-tyrosinase protein with high purity can be obtained with high efficiency.

In the present invention, a host of Escherichia coli is used.
Not only vector systems, but also host-vector systems such as Bacillus subtilis, yeast, Pseudomonas fungi, and actinomycetes can be used, and heat-resistant β-
Mass production of tyrosinase can be performed. next,
The present invention will be described more specifically with reference to examples. Embodiment 1 FIG . Clonin of thermostable β-tyrosinase gene
Grayed (1) Chromosomal DNA thermostable β- tyrosinase producing bacterium
Since the thermostable β-tyrosinase-producing bacterium Symbiobacterium thermophilum does not grow alone, it can grow only in coexistence with Bacillus sp. S strain (Microtechnological Research Institute No. 809), The cells were isolated by the method described in JP-A-61-282076.

That is, a Trp-PEP liquid medium (0.2% L
-Tryptophan (115 ° C, 15 minutes), 0.5% polypeptone, 0.1% yeast extract, 0.3% K ₂ HPO ₄ , 0.1% KH ₂ PO ₄ ,
_{0.05% MgSO 4 · 7H 2 O} , 0.05% pyridoxal-5'
A mixed culture of the producing bacterium and Bacillus sp. S strain, which had been statically cultured at 60 ° C. for 20 hours with phosphoric acid (121 ° C., 20 minutes), was mixed with a CPE solution (100 mM sodium phosphate buffer (pH 7.0)). ), 3
% Sodium citrate, 0.02% disodium ethylenediaminetetraacetate, each sterilized separately at 121 ° C for 20 minutes)
Suspended, added egg white lysozyme (Seikagaku Corporation, crystallized 6 times) to a concentration of 0.3 mg / ml, allowed to act at 35 ° C for 15 minutes,
Bacillus sp. S strain was selectively lysed to obtain cells of Symbiobacterium thermophilum.

[0017] In accordance with isolation and purification to conventional methods of chromosomal DNA containing the gene of thermostable β- tyrosinase from the producing bacterium cells prepared, Biochim.Biophys.Acta, 72. 619-629 (19
63), according to the phenol method. (2) Primary amino acid sequence of the thermostable β-tyrosinase
The thermostable β-tyrosinase was separated and purified by a conventional method from an ultrasonically crushed extract of a mixed culture of the producing bacteria Symbiobacterium thermophilum and Bacillus sp. S strain. That is, Tyr-PEP liquid medium (0.2% L-tyrosine, 0.5% polypeptone, 0.1% yeast extract, 0.3%
_{% K 2 HPO 4, 0.1%} KH 2 PO 4, 0.05% MgSO 4 · 7H 2 O, 0.05
% Pyridoxal-5'-phosphate (sterilized for 20 minutes at 121 ° C) and 0.05% L-tryptophan (sterilized separately for 15 minutes at 115 ° C) at 60 ° C for 30 hours. After sonicating the obtained cells, various column chromatography was performed by the method described in JP-A-62-285785, and the amino-terminal amino acid sequence of the purified thermostable β-tyrosinase was subjected to the Edman degradation method. Was analyzed using an Applied Biosystems protein sequencer 470A, which could not be determined because the amino terminus was blocked.

Therefore, the purified thermostable β-tyrosinase is chemically decomposed with CNBr according to a conventional method, and the decomposed products are purified by high performance liquid column chromatography (HPLC method). The primary sequence was determined. Peptide No. 1 (Met) -X-Glu-Glu-Trp-Ile-Leu-Gln-Lys-
Ala-Z peptide No.2 (Met) -Val-Tyr-Glu-Pro-Pro-Thr-Leu-Ar
g-Phe-Phe (X, Z; undecided) (3) Cloning of thermostable β-tyrosinase gene Step 1: Preparation of DNA probe According to the amino acid primary structure of thermostable β-tyrosinase protein determined in (2) above, The heat-resistant tryptophanase structural gene DNA of this bacterium, which was found to have high homology at the primary structure level of amino acids, was treated with the restriction enzyme Sma I, and then separated by electrophoresis. Sma I containing the second half of the tryptophanase structural gene.
A 0.55 kb DNA fragment was recovered.

Next, the recovered DNA fragment was labeled with ³² P as follows. 20 ng of the above DNA fragment was ligated with 50 μCi of α- ³² P dCTP (Amersham International Co Ltd, PB1
0205) and a multi-prime DNA labeling kit (Amersham
After labeling with phosphoric acid using International Co Ltd, the labeled DNA was separated by gel filtration using Sephadex G-50 (Pharmacia) and used as a ³² P-labeled DNA probe.

The structural gene for thermostable tryptophanase was obtained as follows. First, JP-A-61-282076
No. 5,019,098, the amino-terminal amino acid sequence of a thermostable tryptophanase protein produced by Symbiobacterium thermophilum purified by the method described in Japanese Patent Application Publication No.
It was determined using a sequencer 470A. A synthetic mixed oligonucleotide having a gene sequence predicted from the amino acid sequence was prepared, and its 5'-end was labeled with T4 polynucleotide kinase and [γ- ³² P] ATP.

Using this as a probe, a restriction enzyme for chromosomal DNA of Symbiobacterium thermophilum is used.
The Bam HI digest was subjected to Southern hybridization followed by colony hybridization according to a conventional method to obtain plasmids having 5.5 kb and 1.8 kb inserted Bam HI DNA fragments, respectively. The nucleotide sequence in the vicinity of the portion hybridized with the probe prepared above in the inserted gene fragment of these plasmids was determined according to the method of Zanger et al., And the entire gene structure of the thermostable tryptophanase produced by Symbiobacterium thermophilum was determined. Were determined. The presence of the portion encoding the amino terminal amino acid sequence determined above was confirmed from the determined base sequence, and it was confirmed that the thermostable tryptophanase gene was obtained. It was also confirmed that Escherichia coli transformed with the plasmid into which the full-length gene had been inserted produced a thermostable tryptophanase having the same enzyme activity. Step 2: Obtaining a heat-resistant β-tyrosinase gene DNA fragment by Southern hybridization The chromosomal DNA of the heat-resistant β-tyrosinase-producing bacterium obtained in the preceding section (1) was digested with a restriction enzyme Sac I (Takara Shuzo). After fractionation by 1% agarose gel electrophoresis, the cells were transferred and fixed to a nylon membrane Hybond N + (Amersham International Co Ltd) by alkali blotting.

The ³² P-labeled DNA prepared in step 1 above
Southern hybridization using probes
(J. Mol. Biol. 98 , 503-517 (1975)).
The size of the DNA fragment containing the thermostable β-tyrosinase gene in the SacI fragment of A was specified to be 3.8 kb, and the size was 3.8 kb.
The Sac I DNA fragment was placed in a 1% agarose gel (Anal. Bioch.
em. 101, 339 (1980)). Step 3: Chromosome D of thermostable β-tyrosinase producing bacterium
Preparation of NA Library Plasmid pUC was screened by a conventional method for screening for a thermostable β-tyrosinase gene.
A chromosomal DNA library of a thermostable β-tyrosinase-producing bacterium was prepared using 18 as a vector.

That is, 1 μg of the plasmid pUC18 (Takara Shuzo) was digested with the restriction enzyme Sac I, and the 5′-terminal phosphate of the vector DNA was removed using Escherichia coli alkaline phosphatase (Takara Shuzo). (1: 1, TE buffer saturated) was added and mixed, and the supernatant was subjected to 1% agarose gel electrophoresis, and the vector DNA was recovered from the gel.

The chromosomal DNA Sac obtained in step 2 above
After ligation of the I fragment and the above vector DNA fragment using T4 DNA ligase (Takara Shuzo), Escherichia coli JM105 strain (Amersham) was used to ligate the fragment by the standard method.
l, 119 , 1072 (1974). The E. coli was spread on an LB agar plate medium containing ampicillin at a final concentration of 50 μg / ml, and cultured at 37 ° C. overnight. Step 4: Cloning of thermostable β-tyrosinase gene by colony hybridization The colonies on the plate were replicated on a nitrocellulose filter, and the filter was used for ampicillin (50 μg).
/ Ml) and cultured at 37 ° C for about 10 hours. The filter was passed through solution A (0.5N NaOH) and solution B
1.0M Tris (pH8.0), Solution C (0.5M Tris (pH7.5)
+ 1.5M NaCl) for 5 minutes each, washed with solution D (2xSSC; 30mM sodium citrate + 0.3M NaCl (pH 7.0)) for 5 minutes, air-dried, and baked at 80 ° C for 2 hours under reduced pressure. Was.

The filter is subjected to colony hybridization using the ³² P-labeled synthetic DNA probe prepared in the above step 1, a colony giving a positive signal is picked up from the master plate, and the same colony hybridization is performed again. The positive clone E. coli JM105 / pTYAl was cloned. Embodiment 2 FIG . Structural analysis of thermostable β-tyrosinase gene Plasmid pTYAl was obtained from the isolated positive clone Escherichia coli JM105 / pTYAl according to a conventional method. The plasmid pTYAl was replaced with the restriction enzymes Bam HI, Pst I, Eco RV, Xho I, Kpn
Cleavage with I, Bgl II, and Sal I, and analysis by 1% agarose electrophoresis, the inserted DNA fragment Sac I 3.
An 8 kb restriction enzyme map was prepared (see FIG. 1). further,
Analysis of the restriction enzyme-cleaved DNA fragment by Southern hybridization using the ³² P-labeled synthetic DNA probe in Step 1 described above revealed that the DNA sequence hybridizing with the thermostable tryptophanase gene had an inserted Sac.
And determining the position of the IDNA fragment in (Bam HI- Bam HI
Between 1.0 kb sites). Next, Escherichia coli JM105 transformed with this pTYAl produced heat-resistant β-tyrosinase protein, and the heat-resistant β-tyrosinase activity was detected. Therefore, the thermostable β-tyrosinase gene is used for this 3.
It was found to be present in the 8 kb Sac I DNA fragment.

[0026] After the area around the Bam HI- Bam HI 1.0kb DNA fragment which hybridized with the probe prepared in Step 2 above (2) was digested with various restriction enzymes,
The obtained DNA fragments were inserted into the multiple cloning site of the M13mp18 or M13mp19 phage vector plasmid. Escherichia coli JM105 was transformed with each of these obtained phage plasmids, cultured at 37 ° C., and single-stranded DN
A was obtained.

Using this single-stranded DNA as a template, the Sequenase Vers
ion2.0 Kits (United States Biochemical Corporatio
n) using the method of Zanger et al. Proc. Natl. Acad. Sc
The entire nucleotide sequence of the β-tyrosinase gene was determined according to i. USA, 74 , 5463-5467 (1977). The elucidated nucleotide sequence and the amino acid sequence corresponding to the nucleotide sequence of the thermostable β-tyrosinase gene are shown in SEQ ID NO: 1.

The heat-stable β-tyrosinase gene identified here has an ORF region of 1,377 bases beginning with an initiation codon ATG and ending with an end codon TAG, and encodes a protein having a molecular weight of 52,269 consisting of 458 amino acid residues. Was. Embodiment 3 FIG . Escherichia coli with thermostable β-tyrosinase gene
Transformant JM 105 / pTYAl, which is expressed in large amounts, was inoculated in an LB liquid medium containing ampicillin, cultured at 37 ° C. with shaking overnight, and then inoculated to a fresh LB liquid medium containing ampicillin at a concentration of 1%.
The cells were shake-cultured at 37 ° C. for 24 hours while inducing genes in the presence of mM IPTG. The collected cells were buffered with 50 mM potassium phosphate + 1 mM 2-mercaptoethanol + 250 μM
PMSF + 100 μM pyridoxal-5'-phosphate (pH 8.
0) and sonication, followed by Yamada et al. (Kyoto Univ.)
Enzymatic reaction was carried out according to Agric. Biol. Chem., 38 (11), 2065-2072, (1974).

That is, L-tyrosine 2.5 mM, pyridoxal-5'-phosphate 0.1 mM, K ₂ HPO ₄ -KH ₂ PO ₄ (pH 8.0) 2
Using 4.0 ml of a reaction solution containing 50 mM and the enzyme,
After reacting for 1 minute, the reaction was stopped by adding 1.0 ml of a 30% aqueous solution of trichloroacetic acid. Then, pyruvate generated in the reaction solution was converted to Friedemann-Haugen [Friedemann-Haugen, J. Bio.
l. Chem., 147 , 415 (1943)], and the enzyme activity was determined.

The enzyme activity per medium was higher in the transformant JM105 / pTYAl than in the thermostable β-tyrosinase-producing bacterium Symbiobacterium thermophilum.
In order to further improve the productivity, an experiment was conducted to properly arrange the E. coli lac promoter and the start codon ATG of the thermostable β-tyrosinase gene in the expression vector pUC18, that is, the 5′-terminal untranslated region of the β-tyrosinase gene was deleted. An experiment to remove it was performed.

First, pTYA1 was digested with a restriction enzyme Eco RI,
After both ends of the linear DNA were digested with BAL31 nuclease at about 300 bases each, this was subjected to 1% agarose electrophoresis, and a DNA fragment was recovered from the gel. The reaction with BAL31 nuclease was performed under the following conditions. 5 μl of restriction enzyme H buffer (Takara Shuzo) is added to 40 μl of 2 μg of plasmid pTYA1.
1,4 U of restriction enzyme EcoRI was added and reacted at 37 ° C. for 1 hour. Next, a 5-fold concentrated buffer for BAL31 nuclease (100 mM Tris.HCl (pH 8.0), 3,000 mM NaCl,
10 μl of 60 mM CaCl ₂ , 60 mM MgCl ₂ , 5 mM EDTA) and BAL 31
After adding 0.6 U of nuclease (Takara Shuzo) and reacting at 37 ° C. for 1 minute or 2 minutes, 200 mM EDTA (pH 8.0) was added.
Was added to stop the reaction. 1% of this reaction solution
Agarose gel electrophoresis was performed, and the linear DNA cleaved from the gel was recovered.

This DNA fragment was digested with the restriction enzyme XhoI and subjected to 1% agarose electrophoresis. Then, a DNA fragment in which the upstream region of the thermostable β-tyrosinase gene had been cut was recovered from the gel. The obtained DNA fragment was used for the expression vector pUC18.
Was inserted between SmaI and SalI at the multiple cloning site of E. coli, and used to transform Escherichia coli JM105 strain. In this transformant, E. coli clone JM105 / pTYA2 having high heat-resistant β-tyrosinase activity was cloned.

The Escherichia coli JM 105 / pTYA2 was deposited on July 29, 1991 with the Institute of Microbiological Engineering, National Institute of Industrial Science as Microtechnical Research No. 3495 (FERM BP-3495 ) . The transformant JM105 / pTYA2 was cultured in the same manner as described above, and the obtained cells were disrupted by sonication.
(Laemmli) et al., Nature, 227, 680-685.
According to (1970), the whole cell protein was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the cells were stained with Coomassie Brilliant Blue R-250 and analyzed with a densitometer. As a result, it was found that the ratio of the heat-resistant β-tyrosinase protein to the total amount of the transformant protein was about 30%.

The heat-resistant β produced by Symbiobacterium thermophilum (Jikokenjo No. 810) was used.
The amount of tyrosinase is less than 1% of the total protein; Embodiment 4 FIG . Thermostable β-tyrosinase produced in Escherichia coli
The purification methods transformant JM 105 / pTYA2, after cultivation in the same manner as in Example 3, cells were collected and suspended in buffer and sonicated.
Since the obtained heat-resistant β-tyrosinase has higher heat resistance than β-tyrosinase protein derived from room temperature bacteria, heat treatment at 70 ° C. causes heat denaturation of Escherichia coli cell proteins other than the heat-resistant β-tyrosinase. In order to cause coagulation and precipitation, heat-resistant β-
Purification of the tyrosinase protein can be performed.

That is, the above transformant JM 105 / pTYA2
Was maintained at 70 ° C. for 1 hour, cooled on ice, and cooled and centrifuged (15,000 g, 10 minutes, 4 ° C.) to remove the precipitate. The resulting supernatant was subjected to SDS-polyacrylamide gel electrophoresis and analyzed with a densitometer. The ratio of heat-stable β-tyrosinase in the total protein was concentrated from about 30% before the treatment to substantially pure. did it.

Therefore, by using the above preparation method,
It was revealed that a heat-resistant β-tyrosinase protein can be easily and efficiently purified from a transformant Escherichia coli cell protein. As described above, the thermostable β-tyrosinase produced by Escherichia coli JM105 / pTYA2 was purified by various types of chromatography, and the optimal pH, pH stability, and optimal action were determined by the method described in JP-A-62-285785. The appropriate temperature and temperature stability were studied.

Next, the properties of the obtained enzyme will be described. (1) Action With the involvement of pyridoxal phosphate, L-tyrosine produces pyruvate, phenol and ammonia. (2) Substrate specificity Decomposes tyrosine. (3) Optimum pH and stable pH 50 mM potassium phosphate buffer (pH 5.9 to 7.9) and 50 mM
Glycine-NaOH buffer solution (pH 6.4 to 9.0) and the pH of the enzyme of the present invention under each pH condition (pH at 70 ° C.).
-Tyrosinase activity was measured. The optimum pH is determined to be around 7 at 70 ° C.

Various 50 mM buffers containing 10 mM KCl and 10 μM pyridoxal 5'-phosphate [citrate-
Na ₂ HPO ₄ buffer (pH 4.5 to 6.5); K ₂ HPO ₄ -KH ₂ PO ₄ buffer (pH 6.0 to 8.0); Tris-HCl buffer (pH 8.5 to 10.
5); glycine-NaOH buffer solution (pH 11.0 to 11.5)] under each pH condition (pH at 25 ° C).
A stable pH range of 6 to 11 (25%) was determined by measuring the residual β-tyrosinase activity after treating the enzyme of the present invention at 2 ° C. for 2 days.
° C, 2 days). (4) Range of optimal temperature of action 50 mM adjusted to pH 8.0 at each measurement temperature
Β-tyrosinase activity was determined by allowing an enzyme reaction to take place in a K ₂ HPO ₄ -KH ₂ PO ₄ buffer solution (pH 8.0) for 10 minutes. From these results, the optimum action temperature is determined to be about 80 ° C. (pH = 8.0). (5) Deactivation conditions depending on pH, temperature, etc. Thermal deactivation is not performed until about 80 ° C (pH = 8.0, retention time 20 minutes).

At about 85 ° C. (pH = 8.0, retention time 20 minutes), about 75
% Residual activity. (6) The molecular weight by SDS gel electrophoresis is about 48,000, and the molecular weight calculated from the amino acid sequence deduced from the base sequence is about 52,300. The titer of the thermostable β-tyrosinase of the present invention is measured as follows. L- Tyrosine 2.5 mM, pyridoxal phosphate _{(PLP) 0.1mM, K 2 HPO} 4 -KH 2 PO 4 (pH8.0) 50
After the reaction at 70 ° C. for 10 minutes using 4.0 ml of a reaction solution containing mM and enzyme, the reaction is stopped by adding 1.0 ml of a 30% aqueous solution of trichloroacetic acid (TCA). Pyruvic acid produced in the reaction system was converted to Friedemann-Haugen (Friedemann-Haugen, J. Biol. Ch.
em., 147 , 415 (1943)]. The activity was determined by measuring the number of μmoles of pyruvic acid generated per minute by 1 U (μmol
es / min).

[0040]

[Sequence List] SEQ ID NO: 1 Sequence length: 2471 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Symbiobacterium thermophilum ) direct origin: plasmid pTYA1 sequence GGATCCGGCC TCGGCGGAGG GGTTGCGCCA GGCGGGCGCA GAGGACGCCC 50 GCGCGCTGGT GGCGCTGACC AGCGACGAAC GCTCCAACCT CGCCGCGGCT 100 CGCCTGGGCC GCGGGGAGTT CGGCATCCCG CGCGCCATCC TCTTCGCAAC 150 CAGCCCGGAC GGGCTGGCGG AAGCCCGGCA GGAAGGACTG GAGGCGGTGA 200 ATCCGGACCT GGCGCCGGCC CTGCTGGTGG AGAGCCTGCT CTCCAGCCCG 250 GCCGCGACCG AGCTCACGGG CGGGACCAGG ACGTGCGATC CAGGACTTCC 300 TGCTGGCCGG CGGACCCCTG GCGGGCACCG CCCTGCGGGA TGCGCCGCTG 350 CCCGCCGGGG TGCTGGTGGT GTCGGTGAAG CGGGGCGCCG AGAAGATCGT 400 ACCCCACGGT CACACCGTGC TGCAGCCTTT CCTTTCCCCT GTTCAGCCGC 450 GCTGTTTTAT TCAGGTAAAG ACAGCTTGTT CTGGCAGTTC GTGCCCTC 500 ATACTGGTCG TGGTGACGACGAGACGACGAGAGACGACGAGAGACGAGGAGAGACGACGAGCAGGAGCGAGAGACGCGGAGACGACGAGAGAGACGCGGAGACGACGACGACGACGACGAGACGACGAGAGACGCGGAGACGACGAGAGCGAGACGACGAGAGACGCAGA A CCG TAC AAG ATC AAG GCG 595 Met Gln Arg Pro Trp Ala Glu Pro Tyr Lys Ile Lys Ala 5 10 GTG GAA CCC ATC CGC ATG ACG ACC CGG GAG TAC CGG GAA CAG GCG 640 Val Glu Pro Ile Arg Met Thr Thr Arg Glu Tyr Arg Glu Gln Ala 15 20 25 ATC CGG GAG GCC GGC TAC AAC ACC TTC CTG CTG CGC AGC GAG GAC 685 Ile Arg Glu Ala Gly Tyr Asn Thr Phe Leu Leu Arg Ser Glu Asp 30 35 40 GTG TAC ATC GAC CTG CTG ACG GAC TCG GGA ACC AAC GCG ATG TCG 730 Val Tyr Ile Asp Leu Leu Thr Asp Ser Gly Thr Asn Ala Met Ser 45 50 55 GAC CGG CAG TGG GGC GCC CTG ATG ATG GGC GAT GAG GCC TAC GCC 775 Asp Arg Gln Trp Gly Ala Leu Met Met Gly Asp Glu Ala Tyr Ala 60 65 70 GGC GCA CGC TCC TTC TTC CGC CTG GAG GAG GCG GTG CGC GAG ATC 820 Gly Ala Arg Ser Phe Phe Arg Leu Glu Glu Ala Val Arg Glu Ile 75 80 85 TAC GGC TTC AAG TAC GTG GTG GTG CCG ACC CAT CAG GGG CGC GGT GCC 865 Tyr Gly Phe Lys Tyr Val Val Pro Thr His Gln Gly Arg Gly Ala 90 95 100 GAG CAC CTG ATC TCC CGC ATC CTC ATC AAG CCC GGC GAC TAC ATC 910 Glu His Leu Ile Ser Arg Ile Leu Ile Lys Pro Gly As p Tyr Ile 105 110 115 CCC GGC AAC ATG TAC TTC ACG ACG ACG CGC ACC CAC CAG GAG CTG 955 Pro Gly Asn Met Tyr Phe Thr Thr Thr Thr Arg Thr His Gln Glu Leu 120 125 130 CAG GGG GGC ACG TTC GTC GAC GTG ATC ATC GAC GAG GCC CAC GAT 1000 Gln Gly Gly Thr Phe Val Asp Val Ile Ile Asp Glu Ala His Asp 135 140 145 CCC CAG GCC AGT CAC CCG TTC AAG GGG AAC GTG GAC ATC GCC AAG 1045 Pro Gln Ala Ser His Pro Phe Lys Gly Asn Val Asp Ile Ala Lys 150 155 160 TTC GAG GCG CTC ATC GAC CGG GTC GGC CGC GAC AAG ATC CCG TAC 1090 Phe Glu Ala Leu Ile Asp Arg Val Gly Arg Asp Lys Ile Pro Tyr 165 170 175 ATC AAC GTG GCG CTT ACG GTC AAC ATG GCC GGT GGT CAG CCC GTC 1135 Ile Asn Val Ala Leu Thr Val Asn Met Ala Gly Gly Gln Pro Val 180 185 190 TCC ATG GCC AAC CTG CGT GAG GTG CGG AAG GTT TGC GAC CGG CAC 1180 Ser Met Ala Asn Leu Arg Glu Val Arg Lys Val Cys Asp Arg His 195 200 205 GGC ATC CGG ATG TGG AGC GAC GCG ACC CGC GCC GTG GAG AAC GCC 1225 Gly Ile Arg Met Trp Ser Asp Ala Thr Arg Ala Val Glu Asn Ala 210 215 220 TAC TTC ATC AAG GAG CGG GAG GAG GGC TAC CAG GAC AAG CCG GTC 1270 Tyr Phe Ile Lys Glu Arg Glu Glu Gly Tyr Gln Asp Lys Pro Val 225 230 235 CGG GAG ATC CTG AAG GAG ATG ATG TCC TAC TTC GAC GGC TGC ACC 1315 Arg Glu Ile Les Lys Glu Met Met Ser Tyr Phe Asp Gly Cys Thr 240 245 250 ATG TCG GGA AAG AAG GAC TGC CTG GTC AAC ATC GGC GGC TTC CTG 1360 Met Ser Gly Lys Lys Asp Cys Leu Val Asn Ile Gly Gly Phe Leu 255 260 265 GCC ATG AAC GAG GAG TGG ATC CTC CAG AAG GCC CGG GAG CAG GTG 1405 Ala Met Asn Glu Glu Trp Ile Leu Gln Lys Ala Arg Glu Gln Val 270 275 280 GTC ATC TTC GAG GGC ATG CCC ACG TAC GGC GGC CTG GCC GGG CGC 1450 Val Ile Phe Glu Gly Met Pro Thr Tyr Gly Gly Leu Ala Gly Arg 285 290 295 GAC ATG GAG GCG ATC GCT CAG GGC ATC TAC GAG ATG GTG GAC GAC 1495 Asp Met Glu Ala Ile Ala Gln Gly Ile Tyr Glu Met Val Asp Asp 300 305 310 GAC TAC ATC GCC CAC CGG ATC CAT CAG GTG CGC TAC CTG GGC GAG 1540 Asp Tyr Ile Ala His Arg Ile His Gln Val Arg Tyr Leu Gly Glu 315 320 325 CAG CTG CTG GAG GCG GGC ATC CCC ATC GTG CAG CCC ATC GGC GGC 1 585 Gln Leu Leu Glu Ala Gly Ile Pro Ile Val Gln Pro Ile Gly Gly 330 335 340 CAC GCC GTC TTC CTG GAC GCC CGG GCC TTC CTG CCT CAC ATC CCG 1630 His Ala Val Phe Leu Asp Ala Arg Ala Phe Leu Pro His Ile Pro 345 350 355 CAG GAC CAG TTC CCT GCC CAG GCG CTG GCG GCG GCG CTC TAC GTC 1675 Gln Asp Gln Phe Pro Ala Gln Ala Leu Ala Ala Ala Ala Leu Tyr Val 360 365 370 CAC TCC GGG GTG CGC GCG ATG GAG CGG GGC ATC GTC TCC GCC GGC 1720 His Ser Gly Val Arg Ala Met Glu Arg Gly Ile Val Ser Ala Gly 375 380 385 CGC AAC CCC CAG ACC GGT GAG CAC AAC TAT CCC AAG CTG GAG CTG 1765 Arg Asn Pro Gln Thr Gly Glu His Asn Tyr Pro Lys Leu Glu Leu 390 395 400 GTG CGC CTC ACG ATC CCG CGG CGG GTC TAC ACC GAC CGG CAC ATG 1810 Val Arg Leu Thr Ile Pro Arg Arg Val Tyr Thr Asp Arg His Met 405 410 415 GAC GTG GTG GCG TAC TCG GTG AAG CAC CTG TGG AAG GAG CGC GAC 1855 Asp Val Val Ala Tyr Ser Val Lys His Leu Trp Lys Glu Arg Asp 420 425 430 ACC ATC CGC GGG CTG CGC ATG GTC TAC GAG CCG CCC ACC CTG CGG 1900 Thr Ile Arg Gly Leu Arg Met Val Tyr G lu Pro Pro Thr Leu Arg 435 440 445 TTC TTC ACG GCC CGC TTC GAG CCG ATC AGC TAG CAGATCCGCA 1943 Phe Phe Thr Ala Arg Phe Glu Pro Ile Ser ^*** 450 455 ACCCCGCCCG GAGCCGGCGT GAGCACCGGCCCCCGCCGCCCCCCCGCGCGCCCCCCCGCGCGCCCCCCCGCGCGCCCCCC AACCGGCGGC GTGCACATAC GCTGGACCTG AACTCGTATG 2093 CGCGAAAGGA GAGGAGAAGG ATGCAGCAGC CCGGCATGGT GACCTACAGC 2143 CCGCAGGCGC CGGTTCAGAC CCAGCCGCAG GCAGCCCTGG CTGCGCCGGT 2193 CCCCTTCGCC GCCGGACCAT TCGGCAGCGC GCCGCCCTTC GCCCAGACCG 2243 GCCTGCCCGT GCAGGGGCAG CAGCCCAACC CCGCCCAGGT GCTGACCCAG 2293 CAGCTGGTGC AGACGGCCCG GTCCCTGGAG CAGCTCTTCC CCGGTTACCA 2343 GGCTCTGCTC TCGCTCCTGC TGGAGGCCAG CAGTAACGCC CCCTCGCCCG 2393 CCCTGAACGA GGCGGTGCGC AGCGTCGAGG AGGGGCTCTA CTACCACGGG 2443 GCCGCGCTGG GAGCGATCCG GAGGATCC 2471

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/88 C12R 1:19) (C12N 15/09 ZNA C12R 1:01) (72) Inventor Makoto Nishiyama Shinjuku-ku, Tokyo 2-16-11 Nishi Ochiai Maple House 1F (72) Inventor Sueharu Horinouchi 1-316 Ecchujima 1-chome, Koto-ku, Tokyo (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15 / 00-15/90 C12N 1/00-1/38 C12N 9/00-9/99 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) MEDLINE (STN) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A method for producing a thermostable β-tyrosinase having the amino acid sequence shown in SEQ ID NO: 1, comprising culturing a host cell transformed with an expression vector having a gene encoding the enzyme, and culturing the host cell. A method for producing a thermostable β-tyrosinase, comprising collecting the enzyme from a substance. 2. The method of claim 1, wherein said host is E. coli. 3. A DNA encoding a thermostable β-tyrosinase protein having the amino acid sequence shown in SEQ ID NO: 1.
A. 4. An expression vector containing the DNA according to claim 3. A host transformed with the expression vector according to claim 4. 6. An Escherichia coli host according to claim 5.