JP3512237B2 - Thermostable methionine aminopeptidase and its gene - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel thermostable methionine aminopeptidase useful in the field of protein engineering, a gene encoding the enzyme, and a method for producing the enzyme by genetic engineering.

[0002]

2. Description of the Related Art It is known that in protein biosynthesis in cells, the amino terminus thereof starts from methionine (formylmethionine in prokaryotes) corresponding to the initiation codon. However, this methionine may be removed by subsequent processing,
The methionine is usually absent in many secreted mature proteins. On the other hand, using genetic recombination technology,
It has been found that when the protein is produced in vivo, the methionine remains. It has been pointed out that the precursor protein in which methionine is added to the amino terminus has an increased antigenicity as compared with the mature form, as seen in, for example, recombinant human growth hormone. Therefore, in order to use the precursor protein produced by using the gene recombination technique as a drug, it is necessary to selectively remove the amino-terminal methionine to obtain a mature protein. The most preferable method for this is to use methionine aminopeptidase. Examples of methionine aminopeptidases that can be used in this method include Escherichia coli (Japanese Patent Laid-Open No. 62-115281, and Journal of Bacteriology, No. 1).
69 (2), 751-757 (1987)], Salmonella typhimuriu.
m) [Tokuyohei 3-502400, and European Journal of Biochemistry (European
Journal of Biochemistry), Volume 180, Nos. 23-3
2 (1989)], yeast (Japanese Patent Publication No. 4-500155), and Bacillus subtilis (Japanese Patent Laid-Open No. 3-1993).
No. 285684), a methionine aminopeptidase has been proposed. These methionine aminopeptidases have been isolated as proteins, and their genes have been revealed. On the other hand, methionine aminopeptidase, which is used in the process of protein engineering, heats the target protein, reversibly unwinds its three-dimensional structure, and acts in such a state that the excess amino-terminal methionine is exposed, resulting in high heat resistance. Stability is required. Known four types of methionine aminopeptidases are difficult to use under the above-mentioned conditions, and E. coli, Salmonella,
In the amino acid sequence of methionine aminopeptidase derived from yeast and Bacillus subtilis, since all cysteine residues are contained in plural, when highly expressed by genetic engineering,
There is a high possibility that a disulfide bond will be erroneously formed, and there is a risk in the production method that there is a high risk of accumulating a large amount of inactive substance. Therefore, in order to allow methionine aminopeptidase to act in a state in which the target protein is heated, the three-dimensional structure is reversibly unwound, and excess amino-terminal methionine is exposed, the amino-terminal methionine is removed under these conditions. It is desired to develop a methionine aminopeptidase that has a high thermostability that does not lose the removing activity and has a more reliable high-expression system by genetic engineering.

[0003]

In order to solve these problems, the present invention provides a novel thermostable methionine aminopeptidase, a gene encoding the enzyme, and a thermostable methionine aminopeptidase gene using the enzyme gene. An engineering manufacturing method is provided.

[0004]

If outlined present invention According to an aspect of the first invention of the present invention, it can have been processed at 75 ° C. 60 minutes 9
Thermostable methionine that retains at least 5 % residual activity
It is a minopeptidase and has the following physicochemical properties: (1) Optimum temperature: 70 to 95 ° C (2) Optimum pH: 7.0 to 8.5 (3) Molecular weight: SDS polyacrylamide gel electrophoresis
According to the present invention, it has a thermostable methionine aminopeptidase of 37,000 . The second invention is
Relates Single <br/> isolated gene refractory methionine aminopeptidase of the first aspect of the invention, heat resistance methionine aminopeptidase d'<br/> over peptidase comprising an amino acid sequence represented by SEQ ID NO: 1 And an isolated gene consisting of the nucleotide sequence represented by SEQ ID NO: 2 in the sequence listing. A third invention of the present invention relates to an industrial production method, wherein a transformant introduced with a recombinant plasmid containing the gene of the second invention is cultured, and a thermostable methionine aminopeptidase is added from the culture. Characterized by collecting.

The present inventors found a methionine aminopeptidase of Pyrococcus furiosus DSM3638 as a methionine aminopeptidase excellent in heat resistance, and further attempted to screen the gene of the enzyme. As a method for cloning a gene of an enzyme of interest, there is, for example, an expression cloning method, for example, Pyrococcus bosei
pullulanase gene from ccus woesei) (International Publication 92
No. / 02614) was obtained by this method. Generally, a plasmid vector is used in the expression cloning method, but in this case, a restriction enzyme that cuts the target gene into a relatively small DNA fragment that can be inserted into the plasmid vector without being cut midway is used. Must be used and not necessarily applicable to cloning all enzyme genes. Furthermore, the expression of enzyme activity has to be examined for a large number of clones, and the operation is complicated. The present inventors have used a cosmid vector capable of retaining a larger DNA fragment (35 to 50 kb) instead of the plasmid vector, and used Pyrococcus furiosus DNA.
An attempt was made to isolate a methionine aminopeptidase gene by constructing a cosmid library prepared from the genome and searching for a cosmid clone expressing methionine aminopeptidase activity in the library. By using the cosmid vector, the risk of internal cleavage of the enzyme gene is reduced and the number of transformants to be screened can be reduced. On the other hand, since the cosmid vector does not have a high copy number in the host as the plasmid vector, the expression level of the enzyme is low and the activity may not be detected. The present inventors focused on the fact that the enzyme of interest has high thermostability, and individually cultivated the transformants in the cosmid library, and obtained a lysate containing only thermostable proteins from the obtained bacterial cells. The steps of preparing were combined. This group of lysates is named a cosmid protein library, and by using the cosmid protein library for the detection of enzyme activity, the detection sensitivity is higher than that of the method using colonies of transformants, and the protein derived from the host, etc. It is possible to eliminate adverse effects such as background and inhibition of enzyme activity.

The present inventors searched the cosmid protein library derived from Pyrococcus furiosus and obtained several cosmid clones showing methionine aminopeptidase activity. The present inventors have succeeded in isolating a heat-resistant methionine aminopeptidase gene from various DNA engineering-containing DNA fragments contained in these clones using various genetic engineering techniques. The nucleotide sequence of the enzyme gene was determined. Furthermore, we succeeded in producing thermostable methionine aminopeptidase by genetic engineering using the enzyme gene, and clarified various enzymatic properties of the enzyme. Furthermore, the present inventors have completed the present invention by discovering that thermostable methionine aminopeptidase can be collected from the culture by culturing Pyrococcus furiosus cells. The expression cloning method using this cosmid protein library is not necessarily applicable to all thermostable enzymes, and its success depends on the gene of interest. For example, the present inventors have used this method to produce α-glucosidase derived from Pyrococcus furiosus [Journal of Bacteriology, Volume 172, 3654-36].
Page 60 (1990)] also tried to isolate the gene encoding the α-glucosidase, but the isolation of the gene has not been achieved.

The present invention will be described in more detail below. The microorganism used in the present invention is not particularly limited as long as it is a microorganism that produces a thermostable methionine aminopeptidase gene. For example, a strain belonging to the genus Pyrococcus which is a hyperthermophile can be used. For example, a gene of interest can be screened and obtained from a cosmid library of genomes such as Pyrococcus furiosus and Pyrococcus bousey. Pyrococcus furiosus is Pyrococcus furiosus DSM 3638 and Pyrococcus bousey is Pyrococcus bousey D
SM 3773 can be used and both strains are Deutsche Sammlung von Mi (Deutsche Sammlung von Mi).
This strain is available from Kroorganismen und Zellkulturen GmbH). For example, a Pseudococcus furiosus genome cosmid library is obtained by partially degrading Pyrococcus furiosus DSM 3638 genomic DNA using an appropriate restriction enzyme, for example, Sau3AI (Takara Shuzo), and size fractionating to 35 to 50 kb. Each DN
After ligating the A fragment with an appropriate cosmid vector, for example, a triple helix cosmid vector (manufactured by Stratagene), the in vitro packaging method was used to package the genomic DNA fragment of Pyrococcus furiosus into lambda phage particles, and the resulting phage solution was prepared. Suitable for use in E. coli, eg E. coli DH5αMCR
(Manufactured by BRL) can be transformed and prepared. Next, cosmid DNA is prepared from several colonies of the transformant, and insertion of a genomic DNA fragment of 35 to 50 kb is confirmed. The number of colonies to be cultured is usually 300 to 700.
It is good with the individual. After culturing each colony, the cultured bacterial cells were collected, heat-treated (100 ° C, 10 minutes), sonicated, and reheated (100 ° C, 10 minutes) to obtain a lysate obtained as a cosmid protein library. The presence or absence of methionine aminopeptidase activity in the synthetic peptide Met
-Pro-Ala-Ala-Gly (SEQ ID NO: 3 in the sequence listing) can be used as a substrate for screening. This makes it possible to obtain some cosmid clones containing a thermostable methionine aminopeptidase gene that expresses a thermostable methionine aminopeptidase that is stable to the above heat treatment. Furthermore, the cosmid prepared from the cosmid clone thus obtained can be fragmented with an appropriate restriction enzyme to prepare a recombinant plasmid into which each fragment has been introduced. Then, by examining the activity of the thermostable methionine aminopeptidase produced by the transformant obtained by introducing the plasmid into an appropriate microorganism, a recombinant plasmid containing the desired thermostable methionine aminopeptidase gene can be obtained. You can That is,
Cosmid D prepared from one of the above cosmid clones
DNA obtained by digesting NA with XhoI (Takara Shuzo)
The fragment is used as a plasmid vector pUC118 (Takara Shuzo).
A recombinant plasmid introduced into the SalI site of can be obtained. Next, this recombinant plasmid was transformed into Escherichia coli JM.
Introduced into 109 (manufactured by Takara Shuzo Co., Ltd.) and measuring the thermostable methionine aminopeptidase activity of the obtained colonies by the method used for screening the cosmid protein library, a plasmid is prepared from the colonies in which the activity is recognized.

As shown in the examples, one of the recombinant plasmids is designated pMAP1. Furthermore, this recombinant plasmid was cleaved with several kinds of restriction enzymes,
The resulting DNA fragments of various lengths are inserted into a suitable vector. By removing the thermostable methionine aminopeptidase-free DNA fragment from the above plasmid pMAP1 by measuring the thermostable methionine aminopeptidase activity of the transformant obtained by introducing the obtained recombinant plasmid into Escherichia coli JM109. You can
That is, a DNA fragment obtained by digesting the above-mentioned plasmid pMAP1 with BlnI (manufactured by Takara Shuzo) is introduced into the XbaI site of the plasmid vector pUC118 to prepare a recombinant plasmid. A thermostable methionine aminopeptidase activity of the transformant obtained by introducing the obtained recombinant plasmid into Escherichia coli JM109 is measured to prepare a plasmid from a colony in which the activity is recognized. The recombinant plasmid is designated as pMAP2T.

Furthermore, a DNA fragment containing no thermostable methionine aminopeptidase gene can be removed from the above plasmid pMAP2T as follows. That is,
About 3.5 kb Not obtained by digesting the plasmid pMAP2T with NotI (Takara Shuzo) and BlnI
The I-BlnI fragment was used as a plasmid vector pUCN19.
[PUC19 (Takara Shuzo) was digested with HincII, No
tI linker (manufactured by Takara Shuzo Co., Ltd.)]
After being introduced into the I-XbaI site, it is introduced into Escherichia coli JM109. The thermostable methionine aminopeptidase activity of the obtained colony is measured, and a plasmid is prepared from the colony showing the activity. The plasmid is named pMAP2P. FIG. 1 shows a restriction map of the plasmid pMAP2P. In the figure, the bold solid line is the plasmid vector p.
The inserted DNA fragment into UCN19 is shown.

Furthermore, a DNA fragment containing no thermostable methionine aminopeptidase gene can be removed from the above plasmid pMAP2P as follows. That is,
About 1.3 kb Eco obtained by digesting the plasmid pMAP2P with EcoRI (Takara Shuzo) and XhoI
The RI-XhoI fragment was used as a plasmid vector pUC18.
(Takara Shuzo) EcoRI-SalI site, and then E. coli JM109. The thermostable methionine aminopeptidase activity of the obtained colony is measured, and a plasmid is prepared from the colony showing the activity. This plasmid is designated as plasmid pMAP8. Figure 2
A restriction map of the plasmid pMAP8 is shown in FIG. In the figure,
The thick solid line is the DNA inserted into the plasmid vector pUC18.
A fragment is shown. E. coli JM109 transformed with the plasmid pMAP8 is Escherichia coli JM109 / pM
It is named and displayed as AP8 and has been deposited as FERM P-14344 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

A part of the nucleotide sequence of a DNA fragment of about 1.3 kb inserted in the plasmid pMAP8 is shown in SEQ ID NO: 2 in the sequence listing. That is, SEQ ID NO: 2 in the sequence listing is a base sequence of an example of the thermostable methionine aminopeptidase gene obtained by the present invention. Further, the amino acid sequence of the gene product deduced from the base sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1 in the sequence listing. That is, SEQ ID NO: 1 in the sequence listing is an amino acid sequence of an example of an enzyme protein produced by using the thermostable methionine aminopeptidase gene obtained by the present invention.

Escherichia coli JM109 / pMAP
8 was cultured at 37 ° C. under normal culture conditions, for example, 2 × TY medium containing 100 μg / ml of ampicillin (tryptone 16 g / liter, yeast extract 10 g / liter, NaCl 5 g / liter, pH 7.2). It is possible to express thermostable methionine aminopeptidase in cultured cells. After the completion of the culture, the cultured bacterial cells were collected, and the obtained supernatant of the bacterial cells after ultrasonic treatment was used as a crude enzyme solution. The enzyme solution was heat-treated at 100 ° C. for 10 minutes to denature the contaminant proteins, The thermostable methionine aminopeptidase can be purified by using a combination of methods usually used for purification of enzymes such as dialysis treatment and ion exchange chromatography. For example, Escherichia
After culturing Escherichia coli JM109 / pMAP8, the cultured bacterial cells were collected, and the centrifuged supernatant after ultrasonic treatment of the obtained bacterial cells was used as a crude enzyme solution, and the enzyme solution was contaminated by heat treatment at 100 ° C for 10 minutes. After denaturing the protein, centrifugation and dialysis were performed, followed by DEAE-Sepharose CL-6B.
Apply to an ion exchanger (Pharmacia) to pass the active fraction. The obtained active fraction is further applied to CM-Sepharose CL-6B ion exchanger (Pharmacia) to elute the active fraction. By such treatment, the enzymatic and physicochemical properties of the thermostable methionine aminopeptidase as the enzyme preparation (1) were measured.

The thermostable methionine aminopeptidase obtained by expressing the thermostable methionine aminopeptidase gene of the present invention is a strain belonging to the genus Pyrococcus, for example, Pyrococcus furiosus DSM 363.
Alternatively, it can be obtained by culturing 8 or Pyrococcus bousei DSM 3773 in an appropriate growth medium and purifying the cells and the culture solution. For culturing Pyrococcus cells, a method generally used for culturing hyperthermophilic bacteria can be used, and the nutrient source added to the medium may be any that can be utilized by the strain to be used. As the carbon source, for example, starch or the like can be used, as the nitrogen source, for example, tryptone, peptone, or the like can be used, and as the other nutrient source, for example, yeast extract or the like can be used. During the culturing, a metal salt such as a magnesium salt, a sodium salt or an iron salt may be added as a trace element. Also, for example, it is advantageous to use artificial seawater for the preparation of the medium. Further, the medium is preferably a transparent medium containing no solid sulfur, and the growth of the bacterial cells can be easily monitored by measuring the optical density by using the medium. The culture can be performed by static culture or agitation culture, and for example, applied and environmental microbiology (Applied and En
vironmental Microbiology), Volume 55, 2086-
Dialysis culture methods may be used as described on page 2088 (1992). Generally, the culture temperature is preferably around 95 ° C., and heat-resistant methionine aminopeptidase accumulates significantly in the culture in about 16 hours. It is natural that the culture conditions are set so as to maximize the amount of heat-resistant methionine aminopeptidase produced depending on the cells used and the composition of the medium.

In collecting the thermostable methionine aminopeptidase, for example, cells may be collected from the culture solution by centrifugation, filtration, etc., and then the cells may be crushed.
Examples of the method for disrupting bacterial cells include ultrasonic disruption, bead disruption, and lytic enzyme treatment, and these methods can be used to extract heat-resistant methionine aminopeptidase from bacterial cells. The enzyme extract may be prepared as a crude enzyme solution by a method having the highest extraction effect depending on the cells used. In isolating the thermostable methionine aminopeptidase from the thus obtained crude enzyme solution, a method generally used for enzyme purification can be used. For example, salting out with ammonium sulfate, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography and the like can be used in combination. For example, a crude enzyme solution prepared from cultured cells of Pyrococcus furiosus DSM 3638 is applied to a DEAE-Sepharose CL-6B ion exchanger to allow the active fraction to pass through. The obtained active fraction is HT
A P-cartridge column (manufactured by Bio-Rad) is applied to elute the active fraction. By such a treatment, a heat-resistant methionine aminopeptidase crude enzyme solution can be obtained.

That is, tryptone 1%, yeast extract 0.5%, soluble starch 1%, Jamarin S. Solid (manufactured by Jamarin Laboratory) 3.5%, Jamarin S. Liquid (manufactured by Jamarin Laboratory) 0. 5
%, MgSO ₄ 0.003%, NaCl 0.001
_{%, FeSO 4 · 7H 2 O} 0.0001%, CoSO
_{4 0.0001%, CaCl 2 · 7H} 2 O 0.000
_{1%, ZnSO 4 0.0001%,} CuSO 4 · 5H
₂ O 0.1 ppm, KAl (SO ₄ ) ₂ 0.1 pp
_{m, H 3 BO 3 0.1ppm,} Na 2 MoO 4 · 2H
_{2 O 0.1ppm, NiCl 2 · 6H} 2 O 0.25
2 liters of a medium having a composition of ppm was placed in a 2 liter medium bottle, sterilized at 120 ° C. for 20 minutes, nitrogen gas was blown to remove dissolved oxygen, and the strain was inoculated into the medium to obtain 95 ° C., 16 The culture was allowed to stand for a time. After culturing, the bacterial cells were collected by centrifugation. The collected cells were disrupted by ultrasonication and centrifuged, and the resulting supernatant was dialyzed, and the crude enzyme solution was added to DEAE-Sepharose CL-
Apply to a 6B ion exchanger and pass through the active fraction. The obtained active fraction is applied to an HTP-cartridge column to elute the active fraction. By such treatment, the resulting crude enzyme solution was used as an enzyme preparation (2) to measure the enzymatic and physicochemical properties of thermostable methionine aminopeptidase activity.

The enzymatic and physicochemical properties of the enzyme preparations (1) and (2) are shown below. (1) Action The enzyme of the present invention produces an amino-terminal methionine of a protein or peptide when methionine is present at the amino-terminal of the protein or peptide.

(2) Method for measuring enzyme activity The enzyme activity is measured by the peptide Met-Pro-Ala-A.
It can be assayed using la-Gly (SEQ ID NO: 3) as a substrate. That is, an enzyme preparation whose enzyme activity is to be measured is appropriately diluted, and 0.625 m is added to 5 μl of the enzyme solution.
20 mM PIPES sodium buffer containing M CoCl ₂ [pH 7.2 (pH at 75 ° C.)] 40
μl was added, and further 8 mM Met-Pro-Ala-A
5 μl of la-Gly (SEQ ID NO: 3) was added, and the mixture was reacted at 75 ° C. for 5 minutes and then cooled with ice, and 0.1 M EDTA (p
The reaction is stopped by adding 10 μl of H7.5). Thereafter, the released methionine was washed with 50 μl of the coloring reagent.
l [L-amino acid oxidase (Sigma) 18μ
g, o-dianisidine (manufactured by Sigma) 9 μg, and peroxidase (Peroxidase, manufactured by Sigma) 2.5 μg in 0.1 M sodium phosphate buffer (pH 7.5)] were added and reacted at 37 ° C. for 10 minutes. , 30% acetic acid 10μ
The reaction is stopped by adding 1 and quantified by measuring the absorbance at 450 nm. Alternatively, as another method, Met-Pro-Ala-Ala-Gly
After reaction with (SEQ ID NO: 3), amino acid analysis is performed to quantify. One unit of enzyme is the amount of enzyme that produces 1 μmol of methionine per minute at 75 ° C. The enzyme preparations (1) and (2) had a measured pH of 7.2, 7
Met-Pro-Ala-Ala-Gly at 5 ° C
(SEQ ID NO: 3) had a degrading activity. In the screening of the cosmid protein library, the solution whose enzyme activity is to be measured is appropriately diluted, and 20 μl of the sample solution is diluted with 2 mM Met-Pro-Ala-Ala.
-0.2 mM CoCl ₂ containing Gly (SEQ ID NO: 3)
0.1M sodium phosphate buffer (pH 7.
5) Add 100 μl and react at 95 ° C. for 30 minutes.
Then, after cooling with ice to stop the reaction, 100 .mu.l of a coloring reagent [36 .mu.g of L-amino acid oxidase, 18 .mu.g of o-dianisidine, 5 .mu.g of peroxidase containing 0.
1M sodium phosphate buffer (pH 7.5)] is added and reacted at 37 ° C. for 30 minutes, and then the absorbance at 450 nm is measured to quantify the released methionine. In addition, aminopeptidase is also Met-Pro-Ala-A.
As a blank, the synthetic peptide Leu-Pro-Ala-Ala reacts with la-Gly (SEQ ID NO: 3).
-Gly (SEQ ID NO: 4) substrate was reacted in the same manner,
No color was developed with Leu-Pro-Ala-Ala-Gly (SEQ ID NO: 4), and Met-Pro-Ala-Ala-
Those which reacted only with Gly (SEQ ID NO: 3) are selected.
Furthermore, the reaction product is separated by an HPLC column, and it is confirmed by amino acid analysis that only methionine is released.

(3) For the optimum temperature measurement, an enzyme preparation of 2 mm unit was used and the reaction was carried out at various temperatures. The enzyme preparation (1) was active in the range of 20 ° C to 100 ° C at pH 7.2 measured as shown in Fig. 3, and its optimum temperature was 70 to 95 ° C. FIG. 3 is a diagram showing the optimum temperatures of the enzyme preparations (1) and (2), in which the vertical axis represents the relative activity rate (%) and the horizontal axis represents the reaction temperature (° C.).
Indicates. In the figure, the white circles show the results of the enzyme preparation (1), and the black circles show the results of the enzyme preparation (2).

(4) For the optimum pH measurement, an enzyme preparation of 2.5 mm unit is used, and the buffer solution used for the activity measurement is 20 m at pH 4.1 to 5.1.
M sodium acetate buffer, pH 5.8-7.2, 20 mM pipet sodium buffer, pH 8.0-9.
5 mM 20 mM sodium borate buffer, pH
In 9.9 to 10.6, 20 mM disodium hydrogen phosphate-sodium hydroxide buffer was prepared and used (each pH is at 75 ° C.,
All with 0.625 mM CoCl ₂ ). The enzyme preparations (1) and (2) had a pH of 7.0 to 7.0 as shown in FIG.
The maximum activity was shown around 8.5. Figure 4 shows the enzyme preparation (1)
It is a figure which shows the optimal pH of and (2), and a vertical axis | shaft shows a relative activity rate (%) and a horizontal axis shows pH. In the figure, the white circles show the results of the enzyme preparation (1), and the black circles show the results of the enzyme preparation (2).

(5) Temperature stability A 17.5 mM pipette sodium buffer solution (pH 7.50) containing 75 mM units of enzyme and 0.5 mM CoCl ₂ .
After 2) was treated at 75 ° C. for various times, a part of it was used to measure the residual enzyme activity. As shown in FIG. 5, the enzyme preparations (1) and (2) retained 95% or more activity even after treatment at 75 ° C. for 1 hour. FIG. 5 is a diagram showing the temperature stability of the enzyme preparations (1) and (2) at 75 ° C., the vertical axis shows the residual activity rate (%), and the horizontal axis shows the heat treatment time. In the figure, the white circles show the results of the enzyme preparation (1), and the black circles show the results of the enzyme preparation (2).

(6) pH stability A buffer solution containing 16 milliunits of enzyme and containing 0.5 mM CoCl ₂ at each pH was treated at 75 ° C. for 1 hour, and then a part of the remaining enzyme activity was used. Was measured. 20 mM sodium acetate buffer at pH 4.1 to 5.1, 20 mM pipet sodium buffer at pH 5.8 to 7.2, 20 mM sodium borate buffer at pH 8.0 to 9.5. pH 9.9-10.
In 6, the measurement was performed using 20 mM disodium hydrogen phosphate-sodium hydroxide buffer. Figure 6
As shown in, between pH 4.1 and 10.6, 75 ℃,
After the treatment for 1 hour, the activity was maintained at 85% or more. FIG. 6 is a diagram showing the pH stability of the enzyme preparations (1) and (2) obtained by the present invention, the vertical axis shows the residual activity rate (%), and the horizontal axis shows the pH. In the figure, the white circles show the results of the enzyme preparation (1), and the black circles show the results of the enzyme preparation (2).

(7) Molecular weight The enzyme preparation (1) obtained by the present invention shows about 37,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

(8) Effects of various reagents Using enzyme preparations (1) and (2) in units of 2 mm,
The enzyme activity was measured in the presence of various reagents. No activity was seen when EDTA was added at a final concentration of 0.2 mM. Moreover, when CoCl ₂ with a final concentration of 0.2 mM to 5 mM was added, an increase in enzyme activity was observed, and MnCl
_An increase was also seen with the addition of ₂ .

As described in detail above, the present invention provides a thermostable methionine aminopeptidase and a gene encoding the enzyme, and a method for producing a thermostable methionine aminopeptidase by genetic engineering using the enzyme gene. To be done. The enzyme is a novel enzyme having a high degree of thermostability and is useful in protein engineering treatment under high temperature. Further, by using the gene isolated according to the present invention, a thermostable methionine aminopeptidase capable of hybridizing with the gene and an enzyme gene similar to the enzyme can be easily cloned.

[0025]

EXAMPLES Examples of the present invention will be shown below, but the present invention is not limited to the scope of the following examples. In the examples,% means% by weight.

Example 1 (Preparation of Pyrococcus furiosus genomic DNA) Pyrococcus furiosus DSM3638 was cultured as follows. Tryptone 1%, yeast extract 0.5
%, Soluble starch 1%, Jamarin S.Solid 3.
5%, Jamarin S. Liquid 0.5%, MgSO ₄
0.003%, 0.001% of NaCl, FeSO ₄ ·
7H ₂ O 0.0001%, CoSO ₄ 0.0001
%, CaCl ₂ .7H ₂ O 0.0001%, ZnSO
_{4 0.0001%, CuSO 4 · 5H} 2 O 0.1p
pm, KAl (SO ₄ ) ₂ 0.1 ppm, H ₃ BO _{3
0.1ppm, Na 2 MoO 4 · 2H} 2 O 0.1p
pm, NiCl ₂ · 6H ₂ O 0.25ppm composition medium into a 2 liter medium bottle, 120
After sterilization at 20 ° C. for 20 minutes, nitrogen gas was blown into the mixture to remove dissolved oxygen, and the strain was inoculated into the mixture, followed by static culture at 95 ° C. for 16 hours. After culturing, the bacterial cells were collected by centrifugation. Next, the collected cells were added with 0.05M containing 25% sucrose.
It was suspended in 4 ml of Tris-HCl (pH 8.0), and 0.8 ml of lysozyme [5 mg / ml, 0.2
5M Tris-HCl (pH 8.0)], 2 ml of 0.2
After adding M EDTA and incubating at 20 ℃ for 1 hour, 2
4 ml SET solution [150 mM NaCl, 1 mM
EDTA, 20 mM Tris-HCl (pH 8.0)]
5% SDS (4 ml) and proteinase K (1
0 μg / ml) (400 μl) was added, and the mixture was reacted at 37 ° C. for 1 hour. After completion of the reaction, phenol-chloroform extraction,
Subsequently, ethanol precipitation was performed to obtain about 3.2 mg of genome D.
NA was prepared.

(Preparation of Cosmid Protein Library) 400 μg of Pyrococcus furiosus DSM3638 genomic DNA was partially digested with Sau3AI and size-fractionated to 35 to 50 kb by density gradient ultracentrifugation.
Next, add 1 μg of the triple-helix cosmid vector to B
After digestion with amHI, dephosphorylation was performed using alkaline phosphatase (Takara Shuzo Co., Ltd.), and the above-mentioned fractionated 35-35 was used.
Ligation was performed by mixing with 140 μg of 50 kb DNA, and a fragment of Pyrococcus furiosus genomic DNA was packaged in lambda phage particles by the in vitro packaging method using Geiger Pack Gold (manufactured by Stratagene). Escherichia coli DH5α MCR using a part of the obtained phage solution
Was transformed into a library. After selecting several of the obtained colonies to prepare cosmids and confirming that there was an insert fragment of an appropriate size, we again selected about 500 colonies from the above library and individually selected them. The transformant was treated with 150 ml of LB medium containing 100 μg / ml of ampicillin (10 g of tryptone).
/ Liter, yeast extract 5 g / liter, NaCl 5
Cultured in g / l, pH 7.2). The culture was centrifuged, and the recovered bacterial cells were treated with 20 mM Tris-HCl (p
H8.0) was suspended in 1 ml and heat-treated at 100 ° C. for 10 minutes. Then, ultrasonic treatment is performed, and then 100 times again.
Heat treatment was performed at 10 ° C. for 10 minutes. The lysate obtained as the supernatant after centrifugation was used as a cosmid protein library.

(Selection of Cosmid Containing Thermostable Methionine Aminopeptidase Gene) The methionine aminopeptidase activity was determined by the peptide Met-Pro-Ala-Ala.
-Gly (SEQ ID NO: 3) degradation activity was measured by quantifying the methionine produced. That is, 20 μl of each lysate was individually taken from the above cosmid protein library, and 2 mM Met-Pro-
0.2 m containing Ala-Ala-Gly (SEQ ID NO: 3)
100 μl of 0.1 M sodium phosphate buffer (pH 7.5) containing M CoCl ₂ was added, and the mixture was reacted at 95 ° C. for 30 minutes. Then, after cooling with ice to stop the reaction, 1
00 μl of coloring reagent [L-amino acid oxidase 36 μ
g, o-dianisidine 18 μg, peroxidase 5 μ
0.1 M sodium phosphate buffer (pH 7.
5)] was added and reacted at 37 ° C. for 30 minutes, and then 45
The absorbance at 0 nm was measured to quantify the released methionine. In addition, aminopeptidase and the like can also be used for Met-Pr
As a blank for reacting with o-Ala-Ala-Gly (SEQ ID NO: 3), Leu-Pro-Ala-Ala was used as a blank.
-Gly (SEQ ID NO: 4) substrate was reacted in the same manner,
No color was developed with Leu-Pro-Ala-Ala-Gly (SEQ ID NO: 4), and Met-Pro-Ala-Ala-
Those that reacted only with Gly (SEQ ID NO: 3) were selected.
By the above operation, 6 cosmid clones having methionine aminopeptidase activity were obtained from the cosmid protein library.

(Preparation of plasmid pMAP1 containing thermostable methionine aminopeptidase gene) Cosmid DNA was prepared from one of the six cosmid clones having methionine aminopeptidase activity, digested with XhoI, and ligated to the SalI site of plasmid vector pUC118. did. After introducing this recombinant plasmid into E. coli JM109, it was spread on an LB plate containing 100 μg / ml of ampicillin, and several of the emerged colonies were individually cultured in 5 ml of LB medium containing 100 μg / ml of ampicillin. went. The bacterial cells recovered by centrifugation of the culture were treated with 50 mM Tris-HCl.
The cells were suspended in 50 μl (pH 7.5), heat-treated at 100 ° C. for 10 minutes, and then disrupted by sonication. Further, heat treatment was further performed at 100 ° C. for 10 minutes, and centrifugation was performed to obtain a lysate. The methionine aminopeptidase activity of this lysate was measured. That is, 0.4 μM Met-Pro was added to 1 μl of the lysate.
-Ala-Ala-Gly and 0.2mM CoCl ₂
50 mM pipet sodium buffer (pH 7.
2) 50 μl was added and reacted at 95 ° C. for 30 minutes. After cooling with ice, a coloring reagent [18 μg of L-amino acid oxidase,
9 μg of o-dianisidine, 2.5 μg of peroxidase
0.1 M sodium phosphate buffer (pH 7.
5)] 50 μl was added and reacted at 37 ° C. for 30 minutes, and then the absorbance at 450 nm was measured to quantify the released methionine. A plasmid was prepared from a colony having methionine aminopeptidase activity, and this was named plasmid pMAP1.

(Preparation of plasmid pMAP2T containing thermostable methionine aminopeptidase gene) DN obtained by digesting the above plasmid pMAP1 with BlnI
The A fragment was inserted into the XbaI site of pUC118. This recombinant plasmid was introduced into Escherichia coli JM109, and the resulting colonies were examined for thermostable methionine aminopeptidase activity by the method described above. A plasmid was prepared from a colony in which thermostable methionine aminopeptidase activity was observed, and this was named pMAP2T.

(Preparation of Plasmid pMAP2P Containing Thermostable Methionine Aminopeptidase Gene) About 3.5 kb NotI-BlnI DNA fragment obtained by digesting the above plasmid pMAP2T with NotI and BlnI
UCN19 (pUC19 was cut with HincII, Not
(I linker was introduced) NotI-XbaI site was introduced. This recombinant plasmid was transformed into E. coli JM109
The heat-resistant methionine aminopeptidase activity was examined for the colonies that had been introduced into and appeared by the method described above. A plasmid was prepared from a colony in which thermostable methionine aminopeptidase activity was observed, and this was named pMAP2P. FIG. 1 shows a restriction map of the plasmid pMAP2P.

(Preparation of plasmid pMAP8 containing thermostable methionine aminopeptidase gene) The above plasmid pMAP2P was digested with EcoRI and XhoI, and then subjected to agarose gel electrophoresis, and a DNA fragment of about 1.3 kb was recovered from the agarose gel. This DNA fragment was introduced into the plasmid vector pUC18 at the EcoRI-SalI site. E. coli J
The colonies that had been introduced into M109 and appeared were examined for thermostable methionine aminopeptidase activity by the method described above. A plasmid was prepared from a colony in which thermostable methionine aminopeptidase activity was observed, and this was used as pMAP.
It was named 8. FIG. 2 shows a restriction enzyme map of the plasmid pMAP8. Escherichia coli JM109 transformed with the plasmid pMAP8 is Escherichia coli JM109 / pM.
It is named and displayed as AP8 and has been deposited as FERM P-14344 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

(Determination of nucleotide sequence of thermostable methionine aminopeptidase gene) A DNA fragment of about 1.3 kb containing the thermostable methionine aminopeptidase gene inserted into the above-mentioned plasmid pMAP8 was fragmented into various sizes with various restriction enzymes. After subcloning the fragment into a plasmid vector, the nucleotide sequence of each fragment was determined.
The nucleotide sequence was determined by the dideoxy method using BcaBEST dideoxy sequencing kit (Takara Shuzo). The plasmid pMAP8 is shown in SEQ ID NO: 2 in the sequence listing.
2 shows the nucleotide sequence of a DNA fragment containing the thermostable methionine aminopeptidase gene inserted in the DNA fragment. The amino acid sequence deduced from SEQ ID NO: 2 in the sequence listing is shown in SEQ ID NO: 1 in the sequence listing.

Escherichia coli JM109 / pMAP
8 is a normal culture condition, for example, 2 × TY medium containing 100 μg / ml of ampicillin (tryptone 16 g / liter, yeast extract 10 g / liter, NaCl 5 g / liter, pH 7.2), and cultured at 37 ° C. After the completion, the cultured bacterial cells were collected, and the obtained bacterial cells were subjected to ultrasonic treatment, and the centrifuged supernatant was used as a crude enzyme solution. The enzyme solution was heat-treated at 100 ° C. for 10 minutes and then denatured to contaminating proteins and centrifuged. After treatment and dialysis treatment, DEAE-Sepharose CL-6B
It was applied to an ion exchanger and the active fraction was passed through. The obtained active fraction was further applied to CM-Sepharose CL-6B ion exchanger to elute the active fraction. The eluted active fraction was used as an enzyme preparation (1) of thermostable methionine aminopeptidase, and its enzymatic and physicochemical properties were measured.

Also, Pyrococcus furiosus DSM
3638 tryptone 1%, yeast extract 0.5%, soluble starch 1%, Jamarin S.solid 3.5%,
Jamarin S. Liquid 0.5%, MgSO ₄ 0.0
0.3%, 0.001% of NaCl, FeSO ₄ · 7H ₂
O 0.0001%, CoSO ₄ 0.0001%, Ca
Cl ₂ .7H ₂ O 0.0001%, ZnSO ₄ 0.
_{0001%, CuSO 4 · 5H 2} O 0.1ppm, K
Al (SO ₄ ) ₂ 0.1ppm, H ₃ BO ₃ 0.1p
_{pm, Na 2 MoO 4 · 2H} 2 O 0.1ppm, Ni
2 liters of medium having a composition of 0.25 ppm Cl ₂ .6H ₂ O was placed in a 2 liter medium bottle, and 120
After sterilization at 20 ° C. for 20 minutes, nitrogen gas was blown into the mixture to remove dissolved oxygen, and the strain was inoculated into the mixture, followed by static culture at 95 ° C. for 16 hours. After culturing, the bacterial cells were collected by centrifugation. The harvested cells were sonicated to disrupt the cells, centrifuged, and the resulting supernatant was dialyzed.
EAE-Sepharose CL-6B ion exchanger,
The active fraction was passed through, and the obtained active fraction was applied to an HTP-cartridge column to elute the active fraction. By this treatment, the resulting crude enzyme solution was used as an enzyme preparation (2) to measure the enzymatic and physicochemical properties of thermostable methionine aminopeptidase activity.

The enzymatic and physicochemical properties of the enzyme preparations (1) and (2) are shown below. (1) Action The enzyme of the present invention produced an amino-terminal methionine of a protein or peptide when methionine was present at the amino-terminal of the protein or peptide.

(2) Method for measuring enzyme activity The enzyme activity was measured by the peptide Met-Pro-Ala-A.
The assay was performed using la-Gly (SEQ ID NO: 3) as a substrate. The enzyme preparation whose enzyme activity is to be measured is appropriately diluted, and 5 μl of the enzyme solution is diluted with 0.625 mM CoCl 2.
20 mM pipese sodium buffer containing ₂ [pH 7.
2 (pH at 75 ° C.)] 40 μl, and further 8 m
After adding 5 μl of M Met-Pro-Ala-Ala-Gly (SEQ ID NO: 3) and reacting at 75 ° C. for 5 minutes,
After ice-cooling, 10 μl of 0.1 M EDTA (pH 7.5)
The reaction was stopped by the addition. Thereafter, the released methionine was added with 50 μl of a coloring reagent [18 μg of L-amino acid oxidase, 9 μg of o-dianisidine, and a 0.1 M sodium phosphate buffer (pH 7.5) containing 2.5 μg of peroxidase] at 37 ° C. for 10 minutes. The reaction is carried out for a minute, the reaction is stopped by adding 10 μl of 30% acetic acid, and the absorbance is measured at 450 nm for quantification. Alternatively, as another method, Met-Pro-
After reaction with Ala-Ala-Gly (SEQ ID NO: 3), quantification was performed by performing amino acid analysis. One unit of enzyme was the amount of enzyme that produced 1 μmol of methionine per minute at 75 ° C. The enzyme preparations (1) and (2) are
Met-Pro at measured pH 7.2, 75 ° C
It had an -Ala-Ala-Gly (SEQ ID NO: 3) degrading activity.

(3) For the optimum temperature measurement, an enzyme preparation of 2 mm unit was used and the reaction was carried out at various temperatures. The enzyme preparation (1) was active in the range of 20 ° C to 100 ° C at pH 7.2 measured as shown in Fig. 3, and its optimum temperature was 70 to 95 ° C. FIG. 3 is a diagram showing the optimum temperatures of the enzyme preparations (1) and (2), in which the vertical axis represents the relative activity rate (%) and the horizontal axis represents the reaction temperature (° C.).
Indicates. In the figure, the white circles show the results of the enzyme preparation (1), and the black circles show the results of the enzyme preparation (2).

(4) 2.5 mm unit enzyme preparation was used for optimum pH measurement, and the buffer solution used for activity measurement was 20 m at pH 4.1-5.1.
M sodium acetate buffer, pH 5.8-7.2, 20 mM pipet sodium buffer, pH 8.0-9.
5 mM 20 mM sodium borate buffer, pH
In 9.9 to 10.6, 20 mM disodium hydrogen phosphate-sodium hydroxide buffer was prepared and used (each pH is at 75 ° C.,
All with 0.625 mM CoCl ₂ ). The enzyme preparations (1) and (2) had a pH of 7.0 to 7.0 as shown in FIG.
The maximum activity was shown around 8.5. Figure 4 shows the enzyme preparation (1)
It is a figure which shows the optimal pH of and (2), and a vertical axis | shaft shows a relative activity rate (%) and a horizontal axis shows pH. In the figure, the white circles show the results of the enzyme preparation (1), and the black circles show the results of the enzyme preparation (2).

(5) Temperature stability Each of 17.5 mM buffer solutions containing 75 milliunits of enzyme and 0.5 mM CoCl ₂ was treated at 75 ° C. for various times, and some of the buffer solutions remained. Enzyme activity was measured. As shown in FIG. 5, the enzyme preparations (1) and (2) were 7
Even after treatment at 5 ° C. for 1 hour, 95% or more of the activity was retained. Figure 5 shows enzyme preparations (1) and (2) at 75 ° C.
FIG. 3 is a graph showing the temperature stability in, where the vertical axis represents the residual activity rate (%) and the horizontal axis represents the heat treatment time. In the figure, the white circles show the results of the enzyme preparation (1), and the black circles show the results of the enzyme preparation (2).

(6) pH stability A buffer solution containing 16 milliunits of enzyme and containing 0.5 mM CoCl ₂ at each pH was treated at 75 ° C. for 1 hour, and then a part of the remaining enzyme activity was used. Was measured. 20 mM sodium acetate buffer at pH 4.1 to 5.1, 20 mM pipet sodium buffer at pH 5.8 to 7.2, 20 mM sodium borate buffer at pH 8.0 to 9.5. pH 9.9-10.
In 6, the measurement was performed using 20 mM disodium hydrogen phosphate-sodium hydroxide buffer. Figure 6
As shown in, between pH 4.1 and 10.6, 75 ℃,
After the treatment for 1 hour, the activity was maintained at 85% or more. FIG. 6 is a diagram showing the pH stability of the enzyme preparations (1) and (2) obtained by the present invention, the vertical axis shows the residual activity rate (%), and the horizontal axis shows the pH. In the figure, the white circles show the results of the enzyme preparation (1), and the black circles show the results of the enzyme preparation (2).

(7) Molecular weight The enzyme preparation (1) obtained by the present invention shows about 37,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

(8) Effect of various reagents Using enzyme preparations (1) and (2) in units of 2 mm,
The enzyme activity was measured in the presence of various reagents. No activity was seen when EDTA was added at a final concentration of 0.2 mM. Moreover, when CoCl ₂ with a final concentration of 0.2 mM to 5 mM was added, an increase in enzyme activity was observed, and MnCl
_An increase was also seen with the addition of ₂ .

[0044]

INDUSTRIAL APPLICABILITY According to the present invention, methionine aminopeptidase having extremely high thermostability and a gene encoding the enzyme were obtained. Using this gene, thermostable methionine aminopeptidase can be supplied in high purity in a large amount.

[0045]

[Sequence list]

SEQ ID NO: 1 Array length: 295 Sequence type: Amino acid Number of chains: Single chain Topology: linear Sequence type: Peptide Array:      Met Asp Thr Glu Lys Leu Met Lys Ala Gly Glu Ile Ala Lys Lys        1 5 10 15      Val Arg Glu Lys Ala Ile Lys Leu Ala Arg Pro Gly Met Leu Leu                       20 25 30      Leu Glu Leu Ala Glu Ser Ile Glu Lys Met Ile Met Glu Leu Gly                       35 40 45      Gly Lys Pro Ala Phe Pro Val Asn Leu Ser Ile Asn Glu Ile Ala                       50 55 60      Ala His Tyr Thr Pro Tyr Lys Gly Asp Thr Thr Val Leu Lys Glu                       65 70 75      Gly Asp Tyr Leu Lys Ile Asp Val Gly Val His Ile Asp Gly Phe                       80 85 90      Ile Ala Asp Thr Ala Val Thr Val Arg Val Gly Met Glu Glu Asp                       95 100 105      Glu Leu Met Glu Ala Ala Lys Glu Ala Leu Asn Ala Ala Ile Ser                      110 115 120      Val Ala Arg Ala Gly Val Glu Ile Lys Glu Leu Gly Lys Ala Ile                      125 130 135      Glu Asn Glu Ile Arg Lys Arg Gly Phe Lys Pro Ile Val Asn Leu                      140 145 150      Ser Gly His Lys Ile Glu Arg Tyr Lys Leu His Ala Gly Ile Ser                      155 160 165      Ile Pro Asn Ile Tyr Arg Pro His Asp Asn Tyr Val Leu Lys Glu                      170 175 180      Gly Asp Val Phe Ala Ile Glu Pro Phe Ala Thr Ile Gly Ala Gly                      185 190 195      Gln Val Ile Glu Val Pro Pro Thr Leu Ile Tyr Met Tyr Val Arg                      200 205 210      Asp Val Pro Val Arg Val Ala Gln Ala Arg Phe Leu Leu Ala Lys                      215 220 225      Ile Lys Arg Glu Tyr Gly Thr Leu Pro Phe Ala Tyr Arg Trp Leu                      230 235 240      Gln Asn Asp Met Pro Glu Gly Gln Leu Lys Leu Ala Leu Lys Thr                      245 250 255      Leu Glu Lys Ala Gly Ala Ile Tyr Gly Tyr Pro Val Leu Lys Glu                      260 265 270      Ile Arg Asn Gly Ile Val Ala Gln Phe Glu His Thr Ile Ile Val                      275 280 285      Glu Lys Asp Ser Val Ile Val Thr Thr Glu                      290 295

SEQ ID NO: 2 Array length: 885 Sequence type: Nucleic acid Number of chains: double-stranded Topology: linear Sequence Type: Genomic DNA Antisense: NO Array: ATGGATACTG AAAAACTTAT GAAAGCCGGA GAAATAGCAA AAAAAGTAAG AGAGAAAGCT 60 ATTAAACTTG CTAGACCTGG GATGTTGTTG TTAGAACTTG CAGAGTCTAT AGAAAAGATG 120 ATAATGGAAC TTGGGGGTAA ACCTGCTTTC CCAGTAAATT TATCAATTAA TGAAATTGCA 180 GCTCACTATA CTCCTTACAA GGGAGATACT ACTGTTCTGA AAGAGGGGGA TTATCTAAAG 240 ATCGACGTGG GGGTTCACAT AGATGGATTT ATAGCAGATA CTGCAGTTAC AGTTAGAGTA 300 GGGATGGAAG AAGATGAGCT TATGGAGGCT GCCAAGGAAG CGTTAAACGC CGCAATTTCT 360 GTAGCTAGGG CGGGAGTGGA GATAAAGGAA CTAGGAAAGG CAATAGAAAA TGAAATTAGG 420 AAGAGAGGAT TCAAACCAAT AGTTAATCTA AGTGGGCACA AGATAGAAAG ATACAAGCTT 480 CATGCAGGGA TTAGCATTCC GAACATTTAT AGACCGCATG ATAACTATGT TTTAAAGGAA 540 GGAGATGTTT TCGCAATTGA GCCTTTCGCT ACTATAGGTG CTGGTCAAGT AATTGAGGTT 600 CCCCCAACCT TAATCTACAT GTACGTTAGA GATGTTCCAG TTAGAGTGGC CCAAGCTAGG 660 TTCCTTTTGG CTAAGATAAA AAGGGAATAT GGAACCCTAC CCTTTGCCTA TAGGTGGCTT 720 CAGAATGACA TGCCAGAAGG ACAGCTTAAG TTGGCCCTAA AAACCCTCGA AAAGGCTGGA 780 GCTATATATG GCTATCCAGT GCTTAAAGAA ATTAGAAATG GCATTGTGGC ACAATTTGAG 840 CACACAATCA TTGTTGAAAA GGATTCTGTG ATAGTGACGA CAGAA 885

SEQ ID NO: 3 Sequence length: 5 Sequence type: Number of amino acid chains: Single-chain topology: Linear sequence type: Peptide

SEQ ID NO: 4 Sequence length: 5 Sequence type: Number of amino acid chains: Single-chain topology: Linear sequence type: Peptide

[Brief description of drawings]

FIG. 1 is a diagram showing a restriction enzyme map of plasmid pMAP2P.

FIG. 2 is a diagram showing a restriction enzyme map of plasmid pMAP8.

FIG. 3 is a graph showing the optimum temperature of an enzyme preparation of thermostable methionine aminopeptidase of the present invention.

FIG. 4 is a graph showing the optimum pH of an enzyme preparation of thermostable methionine aminopeptidase of the present invention.

FIG. 5 is a graph showing the temperature stability of an enzyme preparation of thermostable methionine aminopeptidase of the present invention.

FIG. 6 is a graph showing pH stability of an enzyme preparation of thermostable methionine aminopeptidase of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Susumu Tsunazawa 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Takara Shuzo Co., Ltd. (72) Inventor Ikuno Kato 3-chome Seta, Otsu City, Shiga Prefecture No. 4 No. 1 in the Central Research Laboratory of Takazake Co., Ltd. (56) Reference JP-A-3-285684 (JP, A) JP-A-62-115281 (JP, A) JP-A-6-62869 (JP, A) (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 9/50-9/64 C12N 15/09-15/90 ZNA BIOSIS / MEDLINE / WPIDS (STN) GenBank / EMBL / DDBJ / GeneSeq SwissProt / PIR / GeneS eq

Claims (5)

(57) [Claims] 1. A 75 ° C. in holding the treated and can more than 95% residual activity 60 minutes heat methionine aminopeptidase
A heat-resistant methionine aminopeptidase, which is a peptidase and has the following physicochemical properties . (1) Optimum temperature: 70 to 95 ° C (2) Optimum pH: 7.0 to 8.5 (3) Molecular weight: SDS polyacrylamide gel electrophoresis
By 37,000 2. Amino acid represented by SEQ ID NO: 1 in Sequence Listing
Thermostable methionine aminopeptidase consisting of sequences. 3. A thermostable methionine aminopeptidase comprising an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing
To over de isolated thermostable methionine aminopeptidase gene. 4. A base sequence represented by SEQ ID NO: 2 in the sequence listing.
4. The isolated thermostable methionine aminopeptidase gene according to claim 3 , which comprises 5. A transformant introduced with the recombinant plasmid containing the thermostable methionine aminopeptidase gene according to claim 3 is cultured, and the thermostable methionine aminopeptidase is collected from the culture. A method for producing a thermostable methionine aminopeptidase.