JPH0556779A - Production of thermostable beta-tyrosinase by gene recombination - Google Patents

Abstract

PURPOSE:To provide a new DNA useful for production of a thermostable beta- tyrosinase. CONSTITUTION:A DNA coding a thermostable beta-tyrosinase protein and having an amino acid sequence of the formula. A gene library obtained from a chromosome DNA of Symbiobacterium thermophilium is screened by using a gene of a thermostable tryptophanase as a probe and DNA fragments containing a thermostable beta-tyrosinase are cloned, thus obtaining the objective substance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing thermostable β-tyrosinase by a gene recombination technique, and a gene, an expression vector and a host microorganism useful for the production method.

[0002]

2. Description of the Related Art Conventionally, β-tyrosinase is vitamin B ₆
Of L-tyrosine with the participation of pyridoxal-5'-phosphate (PLP), which is one of the
It was discovered as an enzyme that catalyzes α, β elimination reactions that decompose into ammonia, and then β-substitution reaction that synthesizes L-tyrosine or L-DOPA from phenol or pyrocatechol and L-serine, as well as pyruvic acid, ammonia, phenol or It is known as an enzyme that catalyzes the reverse reaction of α, β elimination reaction for synthesizing L-tyrosine or L-dopa from pyrocatechol, and for example, Escherichia intermedia , Proteus
It is found from microorganisms such as Proteus rettgeri , Erwinia herbicola, etc., mainly in the group of enterobacteria which are room temperature bacteria.

However, β-produced by such room temperature bacteria
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 inventors of the present invention have conducted diligent research to create β-tyrosinase excellent in thermostability that overcomes such technical difficulties, and as a result, in both natural medium and L-tyrosine-containing synthetic medium, It does not grow, but Bacillus sp. S strain ( Bacillus sp.
in S; Microtechnology Research Article No. 809), which is a thermostable β-tyrosinase-producing bacillus symbiobacterium that grows in symbiosis with a completely unknown and has not been described in any known literature. Thermophilum ( Symbiobacteriu
m thermophilum ; Mikori Kenjo No. 810) is Bacillus
We succeeded in separating and collecting the S. S strain mixed form) with a soil sample, and the novel bacillus was about 80 ° C. (pH 8.
0) has the optimum temperature for action, and exhibits thermostability with almost no heat inactivation up to about 80 ° C. (pH 8.0, holding time 20 minutes). Found that a completely different novel enzyme, thermostable β-tyrosinase, was produced (JP-A-62-285785).

[0004]

However, this heat resistance β
-The tyrosinase-producing bacterium Symbiobacterium thermophilum does not grow alone and can grow only in symbiosis with the Bacillus sp. S strain. For its culturing, a symbiotic relationship with the Bacillus sp. S strain is required. Since it is necessary to carry out mixed culture while maintaining it, the culture system is complicated and large-scale culture is difficult, and the heat-resistant β-
There are problems that the amount of tyrosinase protein is small and that the enzyme is difficult to purify.

Therefore, the present invention is intended to solve the above-mentioned various problems by genetic engineering techniques.

[0006]

Therefore, the present inventors have
Utilizing the technology of genetic engineering for the purpose of simplifying the culture system, improving productivity per bacterium, and simplifying enzyme purification, the thermostable β-tyrosinase structural gene was isolated from the Symbiobacterium thermophilum, Inserting the structural gene into an appropriate expression vector, using it to transform a host microorganism, repeated research aiming at large-scale production of thermostable β-tyrosinase. Chromosomal DNA of Symbiobacterium thermophilum
DN containing more thermostable β-tyrosinase structural gene
The A fragment can be cloned, and this DNA fragment was incorporated into an appropriate expression vector, and E. coli was transformed with the expression vector, whereby a large amount of thermostable β-tyrosinase was successfully expressed. It was 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 was completed.

Therefore, the present invention has the following properties: (1) Action: L is involved in the involvement of pyridoxal phosphate.
-Pyruvate, phenol and ammonia are produced from tyrosine; (2) Substrate specificity: L-tyrosine is decomposed; (3) Action optimum pH and stable pH range: optimum pH at reaction temperature 70 ° C is about 7 to 7.5, and stable pH is 6 to 11 when left at 25 ° C for 2 days; (4) Optimum action temperature and heat resistance: The optimum action temperature at pH 8.0 is about 80. 80 ℃ when kept at pH 8.0 for 20 minutes
It does not heat inactivate up to ℃; and (5) The molecular weight measured by SDS gel electrophoresis is about 4
8,000; and the molecular weight predicted from the amino acid sequence deduced from the genetic sequence is about 52,300;
A host transformed with an expression vector having 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 thermostable β-tyrosinase, which comprises collecting the enzyme.

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 provides the above D
An expression vector containing NA is provided. The invention further comprises
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 Symbiobacterium thermophilum described above, and using a suitable probe such as that described in the above-mentioned Japanese Patent The thermostable tryptophan of this bacterium was found to have high homology at the amino acid level by determining the amino acid primary sequence of thermostable β-tyrosinase purified and isolated by the method described in Japanese Patent Publication No. 62-285785. It can be obtained by screening the above-mentioned chromosomal DNA library using the gene for nase as a probe and cloning the DNA fragment containing the thermostable β-tyrosinase of the present invention.

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, and as a result, the thermostable β-according to the present invention. The structural gene of tyrosinase consists of 1377 bases, and by analyzing the amino acid residues encoded by it, we were able to elucidate the primary structure of the thermostable β-tyrosinase protein with a molecular weight of 52,269 consisting of 458 amino acid residues. (SEQ ID NO: 1).

However, as is well known, many DNAs are gene DNAs that code for them.
There are multiple base sequences. The base sequence is not uniquely determined, and there are many possibilities. Also in the case of the gene encoding the amino acid sequence of the thermostable β-tyrosinase protein of Symbiobacterium thermophilum revealed by the present inventors, its 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, and other DNA encoding the amino acid sequence of the thermostable β-tyrosinase protein revealed by the present invention. It also includes the base sequence.

[0012] According to the gene recombination technique, the basic characteristic of the enzymatic activity of thermostable β-tyrosinase encoded by the DNA is not changed at a specific site of the basic DNA, or its characteristic is changed. Artificial mutations can be made to improve. Regarding the gene DNA having a natural base sequence or the gene DNA having a base sequence different from the natural one, which is provided by the present invention, it can be replaced with a natural gene by artificially inserting, deleting, or substituting. The characteristics may be the same or improved, and the present invention also includes such a mutant gene.

The thermostable β-tyrosinase-containing DNA fragment of the present invention is a suitable expression vector depending on the host in which the thermostable β-tyrosinase is expressed in the microbial cells, such as pUC18 (which holds the lac promoter of E. coli. An expression plasmid for producing thermostable β-tyrosinase in the host microbial cells, for example, Escherichia coli, can be constructed by connecting to Takara Shuzo or the like.

The expression plasmid carrying the thermostable β-tyrosinase gene of the present invention is introduced into a suitable host microorganism such as Escherichia coli, etc.
-It is possible to create transformed microorganisms that produce tyrosinase protein. The transformed microorganism thus created can be heat-resistant β-
It is possible to produce tyrosinase in large quantities. Isolation of the heat-resistant β-tyrosinase protein from the culture after culturing can be carried out, for example, by crushing the cells with ultrasonic waves and then heat-treating and centrifuging to heat-denaturate and remove the protein derived from E. coli. A bacterial cell extract containing the β-tyrosinase protein in high purity can be obtained with high efficiency.

In the present invention, the host of E. coli is
Not only vector systems, but also host-vector systems such as Bacillus subtilis, yeast, Pseudomonas, actinomycetes, etc. can be used, and the heat-resistant β-
Mass production of tyrosinase can be performed. next,
The present invention will be described in more detail by way of examples. Example 1 . Clonin of thermostable β-tyrosinase gene
Grayed (1) Chromosomal DNA thermostable β- tyrosinase producing bacterium
The thermostable β-tyrosinase-producing bacterium Symbiobacterium thermophilum does not grow alone, but can grow only in symbiosis with the Bacillus sp. S strain (Mikokenjojo No. 809). The cells were isolated by the method described in JP-A-61-282076.

That is, 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'
CPE solution (100 mM sodium phosphate buffer (pH 7.0) was added to the mixed culture of the production bacterium and Bacillus sp. S strain that had been statically cultivated in phosphoric acid (121 ° C, 20 minutes) at 60 ° C for 20 hours. ) 3
% Sodium citrate, 0.02% ethylenediaminetetraacetic acid disodium, each sterilized at 121 ° C for 20 minutes),
Suspend, add egg white lysozyme (Seikagaku Corporation, crystallize 6 times) to 0.3 mg / ml, act at 35 ° C for 15 minutes,
The Bacillus sp. S strain was selectively lysed to obtain symbiobacterium thermophilum cells.

Isolation and purification of the chromosomal DNA containing the gene of thermostable β-tyrosinase from the prepared bacterial cells of the producing strain is carried out in accordance with a conventional method. Biochim. Biophys. Acta, 72. 619-629 (19
It was carried out by the phenol method described in 63). (2) Primary amino acid sequence of the thermostable β-tyrosinase
The thermostable β-tyrosinase was isolated and purified from an ultrasonically disrupted extract of a mixed culture of the producing bacterium Symbiobacterium thermophilum and Bacillus sp. S strain by a conventional method. 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 at 121 ° C. for 20 minutes) and 0.05% L-tryptophan (115 ° C., sterilized separately for 15 minutes) were incubated at 60 ° C. for 30 hours. After ultrasonic disruption of the obtained bacterial cells, various column chromatography was carried out by the method described in JP-A-62-285785, and the amino terminal amino acid sequence of the purified thermostable β-tyrosinase was analyzed by Edman degradation method. Was analyzed by a protein sequencer 470A manufactured by Applied Biosystems, but it could not be determined because the amino terminal was blocked.

Then, the purified thermostable β-tyrosinase was chemically decomposed by CNBr according to a conventional method, and the decomposed products were purified by high performance liquid column chromatography (HPLC method) to obtain two peptides. 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 By the amino acid primary structure of thermostable β-tyrosinase protein determined in (2) above, The structural gene DNA of the thermostable tryptophanase 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 latter half of the tryptophanase structural gene
A 0.55 kb DNA fragment was recovered.

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

The thermostable tryptophanase structural gene was obtained as follows. First of all, JP 61-282076
The amino-terminal amino acid sequence of the thermostable tryptophanase protein produced by Symbiobacterium thermophilum purified by the method described in Japanese Patent Publication No.
Determined using 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
The Bam HI degradation product was subjected to Southern hybridization and colony hybridization according to a conventional method to obtain plasmids having inserted Bam HI DNA fragments of 5.5 kb and 1.8 kb, respectively. The nucleotide sequences near the portion of the inserted gene fragments of these plasmids that hybridize with the probe prepared above were determined according to the method of Zanger et al., And the entire gene structure of thermostable tryptophanase produced by Symbiobacterium thermophilum was determined. Were determined. From the determined nucleotide sequence, the presence of a portion encoding the amino-terminal amino acid sequence determined above was confirmed, and it was confirmed that a thermostable tryptophanase gene was obtained. It was also confirmed that Escherichia coli transformed with the plasmid having the full-length gene inserted produced a thermostable tryptophanase having the same enzymatic activity. Step 2: Acquisition of thermostable β-tyrosinase gene DNA fragment by Southern hybridization After cleaving the chromosomal DNA of the thermostable β-tyrosinase-producing bacterium obtained in the previous section (1) with restriction enzyme Sac I (Takara Shuzo), After fractionation was performed by 1% agarose gel electrophoresis, it was transferred and fixed on a nylon membrane Hybond N + (Amersham International Co Ltd) by an alkali blotting method.

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

That is, 1 μg of the plasmid pUC18 (Takara Shuzo) was cleaved with the restriction enzyme Sac I to remove the 5′-terminal phosphate of vector DNA using Escherichia coli alkaline phosphatase (Takara Shuzo), and then an equal amount of phenol: chloroform was added. (1: 1, TE buffer saturated) was added and mixed, and the supernatant was subjected to 1% agarose gel electrophoresis to recover the vector DNA from the gel.

Chromosomal DNA Sac obtained in step 2 above
The I fragment and the above vector DNA fragment were ligated with T4 DNA ligase (Takara Shuzo), and then E. coli JM 105 strain (Amersham) was used in a conventional method J. Bacterio.
L., 119 , 1072 (1974). The Escherichia coli was spread on an LB agar plate medium containing a final concentration of 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. Step 4: Cloning of thermostable β-tyrosinase gene by colony hybridization Replicate the colonies on the plate to a nitrocellulose filter, and filter the ampicillin (50 μg
/ ML) and transferred to an LB plate medium and cultured at 37 ° C. for about 10 hours. Liquid A (0.5N NaOH), liquid B
1.0M Tris (pH8.0), C solution (0.5M Tris (pH7.5)
Soak in + 1.5M NaCl for 5 minutes each, wash with liquid D (2xSSC; 30mM sodium citrate + 0.3M NaCl (pH 7.0)) for 5 minutes, air-dry, and bake at 80 ° C for 2 hours under reduced pressure. It was

Colony hybridization of the filter was carried out using the ³² P-labeled synthetic DNA probe prepared in the above step 1, colonies giving a positive signal were picked up from the master plate, and the same colony hybridization was carried out again. The positive clone E. coli JM 105 / pTYAl was cloned. Example 2 . Structural analysis of thermostable β-tyrosinase gene Plasmid pTYAl was obtained from the isolated positive clone Escherichia coli JM 105 / pTYAl according to a conventional method. The plasmid pTYAl was digested with restriction enzymes Bam HI, Pst I, Eco RV, Xho I, Kpn
The insert DNA fragment Sac I 3. was digested with I, Bgl II and Sal I and analyzed by 1% agarose electrophoresis.
An 8 kb restriction map was prepared (see Fig. 1). further,
Analysis of the above-mentioned restriction enzyme-cut DNA fragment by the Southern hybridization method using the ³² P-labeled synthetic DNA probe in the above step 1 revealed that the inserted Sac of the DNA sequence that hybridizes with the thermostable tryptophanase gene.
And determining the position of the IDNA fragment in (Bam HI- Bam HI
(Between 1.0 kb sites). Next, Escherichia coli JM 105 transformed with this pTYAl produced a thermostable β-tyrosinase protein, and the thermostable β-tyrosinase activity was detected. Therefore, the thermostable β-tyrosinase gene is
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 multi-cloning site of M13mp18 or M13mp19 phage vector plasmid. Escherichia coli JM 105 was transformed with each of the obtained phage plasmids, cultured at 37 ° C., and single-stranded DNA of the phage was isolated from the culture supernatant by a conventional method.
Got A.

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

The thermostable β-tyrosinase gene revealed here has a 1,377-base ORF region which starts at the start codon ATG and ends at the end codon TAG, and encodes a protein having a molecular weight of 52,269 consisting of 458 amino acid residues. Was. Example 3 . Escherichia coli cells of thermostable β-tyrosinase gene
The large amount of transformant JM 105 / pTYAl was planted in LB liquid medium containing ampicillin, shaken overnight at 37 ° C., and then inoculated into fresh LB liquid medium containing ampicillin at a final concentration of 1%.
The cells were shake-cultured at 37 ° C. for 24 hours while inducing the gene in the presence of mM IPTG. The collected cells are buffered with 50 mM potassium phosphate + 1 mM 2-mercaptoethanol + 250 μM
PMSF + 100 μM pyridoxal-5'-phosphate (pH 8.
0) and ultrasonic crushing, then Yamada et al. (Kyoto Univ.)
The 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
00 Using reaction mixture containing 50 mM and enzyme, 4.0 ml at 70 ℃
After reacting for a minute, the reaction was stopped by adding 1.0 ml of a 30% trichloroacetic acid aqueous solution. Then, the pyruvic acid produced in the reaction solution was converted to Friedemann-Haugen, J. Bio.
l. Chem., 147 , 415 (1943)] method to determine the enzyme activity.

The enzyme activity per medium was higher in the transformant JM 105 / pTYAl than in the thermostable β-tyrosinase-producing bacterium Symbiobacterium thermophilum.
In order to further improve productivity, an experiment in which the E. coli lac promoter of the expression vector pUC18 and the initiation codon ATG of the thermostable β-tyrosinase gene were appropriately arranged, that is, the 5'-untranslated region of the β-tyrosinase gene was An experiment was conducted to remove it.

First, pTYAl was cleaved with the restriction enzyme Eco RI,
Both ends of the linear DNA were digested with BAL 31 nuclease at about 300 bases each, and then subjected to 1% agarose gel electrophoresis to recover a DNA fragment from the gel. The reaction with BAL 31 nuclease was performed under the following conditions. 2 μg of plasmid
Restriction enzyme H buffer solution (Takara Shuzo) 5 μl, 4 in 40 μl of pTYAl
V RI restriction enzyme Eco RI was added, and the mixture was reacted at 37 ° C for 1 hour. Next, 5 times concentrated buffer for BAL 31 nuclease (100 mM T
ris HCl (pH 8.0), 3,000 mM NaCl, 60 mM CaCl ₂ , 60 mM
After adding 10 μl of MgCl ₂ , 5 mM EDTA) and 0.6 V of BAL 31 nuclease (Takara Shuzo) and reacting at 37 ° C. for 1 minute or 2 minutes, the reaction was stopped by adding 50 μl of 200 mM EDTA (pH 8.0). .. This reaction solution was subjected to 1% agarose gel electrophoresis,
The truncated linear DNA was recovered from the gel.

This DNA fragment was cleaved with a restriction enzyme Xho I and subjected to 1% agarose electrophoresis, and then a DNA fragment obtained by truncating the upstream region of the thermostable β-tyrosinase gene was recovered from the gel. The obtained DNA fragment was used as an expression vector pUC 18
E. coli JM 105 strain was transformed with Sma I- Sal I of the multi-cloning site of Escherichia coli. Among these transformants, Escherichia coli clone JM 105 / pTYA2 having high thermostable β-tyrosinase activity was cloned.

This Escherichia coli JM 105 / pTYA2 was deposited at the Institute of Microbial Engineering, Institute of Industrial Science, on July 29, 1991 as Micro Engineering Research Article No. 3495 (FERM BP-3495. Transformant JM Cultivate 105 / pTYA2 in the same manner as above, sonicate the resulting cells, and
li) et al., Nature, 227, 680-685, (1970)
The whole cell protein was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, Coomassie Brilliant Blue R-250
When stained with densitometer and analyzed with a densitometer, it was found that the ratio of thermostable β-tyrosinase protein to the total protein of the transformant was about 30%.

The heat-resistant β produced by Symbiobacterium thermophilum (Kikuyoji No. 810)
The amount of tyrosinase is less than 1% of the total protein. Example 4 . Thermostable β-tyrosinase produced in E. coli
Purification method of the transformant The transformant JM 105 / pTYA2 was cultured in the same manner as in Example 3, the cells were collected, suspended in a buffer solution, and ultrasonically disrupted.
Since the obtained thermostable β-tyrosinase has higher thermostability than β-tyrosinase protein derived from normal temperature bacteria, heat treatment at 70 ° C causes heat denaturation of Escherichia coli cell proteins other than the thermostable β-tyrosinase. In order to cause aggregation / precipitation, it is possible to efficiently remove the heat-resistant β-
Purification of the tyrosinase protein can be performed.

That is, the above transformant JM 105 / pTYA2
The ultrasonic cell disruption extract of (1) was kept at 70 ° C for 1 hour, then cooled with ice and subjected to cooling centrifugation (15,000 g, 10 minutes, 4 ° C) to remove the precipitate. When the obtained supernatant was subjected to SDS-polyacrylamide gel electrophoresis and analyzed with a densitometer, the percentage of thermostable β-tyrosinase in the total protein was concentrated from about 30% before treatment to substantially pure. did it.

Therefore, by using the above preparation method,
It was revealed that the thermostable β-tyrosinase protein can be easily and highly purified from the transformant Escherichia coli cell protein. As described above, the thermostable β-tyrosinase produced by Escherichia coli JM 105 / pTYA2 was purified by various chromatographies, and the optimal pH, pH stability, and optimal action were obtained by the method described in JP-A-62-285785. The optimum temperature and temperature stability were investigated.

Next, the properties of the obtained enzyme will be described. (1) Action Pyruvic acid, phenol and ammonia are produced from L-tyrosine under the participation of pyridoxal phosphate. (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
Of the enzyme of the present invention under the conditions of each pH (pH at 70 ° C.) using each of the glycine-NaOH buffers (pH 6.4 to 9.0).
-Tyrosinase activity was measured. The optimum pH is determined to be around 7 at 70 ° C.

50 mM various 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 11.0-11.5)] under each pH (pH at 25 ° C.) conditions.
After measuring the residual β-tyrosinase activity after treating the enzyme of the present invention for 2 days at ℃, the stable pH range was 6-11 (25
C, 2 days). (4) Optimum temperature range of action 50mM adjusted to pH 8.0 at each measurement temperature
The β-tyrosinase activity was determined by performing an enzymatic reaction for 10 minutes in the K ₂ HPO ₄ -KH ₂ PO ₄ buffer solution (pH 8.0). From this result, the optimum action temperature is determined to be about 80 ° C (pH = 8.0). (5) Conditions for deactivation due to pH, temperature, etc. Heat deactivation does not occur up to about 80 ° C (pH = 8.0, holding time 20 minutes).

Approximately 75 at approx. 85 ° C. (pH = 8.0, holding time 20 minutes)
% Residual activity is shown. (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 thermostable β-tyrosinase of the present invention is measured as follows. L-tyrosine 2.5 mM, pyridoxal phosphate (PLP) 0.1 mM, K ₂ HPO ₄ —KH ₂ PO ₄ (pH 8.0) 50
After using 4.0 ml of a reaction solution containing mM and enzyme for 10 minutes at 70 ° C., 1.0 ml of 30% trichloroacetic acid (TCA) aqueous solution is added to stop the reaction. The pyruvic acid produced in the reaction system was converted to Friedemann-Haugen, J. Biol. Ch.
em., 147 , 415 (1943)] method. The activity is 1 U (μ mol
es / min).

[0040]

[Sequence listing] SEQ ID NO: 1 Sequence length: 2471 Sequence type: Nucleic acid Number of strands: Double strand 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 CTGGCAGGTC GAGCGACCGA CCGCGATCGA CCGCGATCGA CCGATCGACGA 500GACGATCGA 500GA 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 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 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 Leu 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 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 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 GAGCCGGGGT GCGCACCCGCCGCCGCGCGCGCAGCCG1993CGTCGACGCC19931993CGCCGACGCC 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

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[Procedure amendment]

[Submission date] August 30, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0003

[Correction method] Change

[Correction content]

However, β-produced by such room temperature bacteria
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 inventors of the present invention have conducted diligent research to create β-tyrosinase excellent in thermostability that overcomes such technical difficulties, and as a result, in both natural medium and L-tyrosine-containing synthetic medium, It does not grow, but Bacillus sp. S strain ( Bacillus sp.
in S; Microtechnical Research Institute No. 809), which is a thermostable β-tyrosinase-producing bacillus Symbiobacterium, which has a unique growth characteristic that is completely unknown and has not been described in any known literature. Thermophilum ( Symbiobacter
ium thermophilum ; Mikori Kenjoyori No. 810) ( mixed form with Bacillus sp. S strain) was successfully separated and collected with a soil sample, and the novel bacillus was about 80 ° C.
It has an optimum temperature of action at (pH 8.0) and about 80 ℃ (pH
It produces a thermostable β-tyrosinase, which is a novel enzyme completely different from the β-tyrosinase produced by the room temperature bacterium in that it exhibits thermostability with almost no heat inactivation up to 8.0 and a holding time of 20 minutes). It has been found that (Japanese Patent Laid-Open No. 62-2857).
No. 85).

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0031

[Correction method] Change

[Correction content]

First, pTYA1 was cleaved with the restriction enzyme Eco RI,
About 300 bases of each end of the linear DNA was digested with BAL 31 nuclease, and this was electrophoresed on 1% agarose gel to recover a DNA fragment from the gel. The reaction with BAL 31 nuclease was performed under the following conditions. 40 μl of 2 μg plasmid pTYA1 was added with 5 μ of restriction enzyme H buffer (Takara Shuzo).
1,4 U of restriction enzyme Eco RI was added, and the mixture was reacted at 37 ° C for 1 hour. Next, a 5-fold concentrated buffer solution for BAL 31 nuclease (100 mM Tris.HCl (pH 8.0), 3,000 mM NaCl,
60 mM CaCl ₂ , 60 mM MgCl ₂ , 5 mM EDTA) 10 μl, 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)
The reaction was stopped by adding 50 μl of 1% of this reaction solution
Agarose gel electrophoresis was performed to recover the truncated linear DNA from the gel.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0033

[Correction method] Change

[Correction content]

This Escherichia coli JM 105 / pTYA2 was deposited on July 29, 1991 at the Institute of Microbial Engineering, Institute of Industrial Technology, as Micro Engineering Research Article No. 3495 (FERM BP-3495 ) . The transformant JM 105 / pTYA2 was cultured in the same manner as above, and the obtained bacterial cells were ultrasonically disrupted.
(Laemmli) et al. Nature, 227, 680-685.
According to (1970), whole cell protein was subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, staining with Coomassie Brilliant Blue R-250 and analysis with a densitometer revealed that the ratio of thermostable β-tyrosinase protein to the total transformant protein was about 30%.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12N 1/21 C12R 1:19) (C12N 15/60 C12R 1:01) (72) Inventor Makoto Nishiyama 2-16-11 Nishiochiai, Shinjuku-ku, Tokyo Maple House 1st floor (72) Inventor Suenji Horinouchi 1-3-16, Etchujima, Koto-ku, Tokyo 403

Claims (6)

[Claims] 1. The following properties: (1) Action: L is involved with the participation of pyridoxal phosphate.
-Pyruvate, phenol and ammonia are produced from tyrosine; (2) Substrate specificity: L-tyrosine is decomposed; (3) Action optimum pH and stable pH range: optimum pH at reaction temperature 70 ° C is about 7 to 7.5, and stable pH is 6 to 11 when left at 25 ° C for 2 days; (4) Optimum action temperature and heat resistance: The optimum action temperature at pH 8.0 is about 80. 80 ℃ when kept at pH 8.0 for 20 minutes
It does not heat inactivate up to ℃; and (5) The molecular weight measured by SDS gel electrophoresis is about 4
8,000; and the molecular weight predicted from the amino acid sequence deduced from the gene sequence is about 52,300;
A host transformed with an expression vector having a gene encoding the enzyme obtained from a microorganism belonging to the genus Symbiobacterium and having the gene encoding the enzyme is cultivated, A method for producing thermostable β-tyrosinase, which comprises collecting the enzyme. 2. The method of claim 1, wherein the host is E. coli. 3. A DN 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. 5. A host transformed with the expression vector according to claim 4. 6. The E. coli host according to claim 5.