JPH09220090A - New thermolysin-like protease and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme improved in stability, having a thermolysin-like protease activity and useful for highly effectively synthesizing the precursor of an artificial sweetener, aspartame, etc., by changing amino acids at the specific site of thermolysin. SOLUTION: The volume of a pore expressed by an electron density and surrounded by at least the 144-Leu, 149-Thr, 240-Ala, 270-Ala and 288-Ala in thermolysin having an amino acid sequence of the formula is changed by replacing one or more of the amino acid residues with one or more of amino acid residues having larger hydrophobicity and bulkiness than those of the above amino acid residues or by giving an influence to the amino acid residues surrounding the pore of the electron density to obtain the new thermolysin-like protease in which the pore is partially filled to reduce its volume. The new thermolysin-like protease is improved in stability and useful for the efficient production of a precursor for synthesizing an artificial sweetener, α-L-aspartyl-L- phenylalanine methyl ester (aspartame), etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to N in an aqueous medium.
-Benzyloxycarbonyl aspartic acid (hereinafter Z
-Asp is abbreviated, and this Z-Asp is ZL-Asp.
Or Z-Asp racemate) and phenylalanine methyl ester (hereinafter abbreviated as PM, where PM means L-PM or PM racemate) using a thermolysin-like protease for enzymatic reaction. Benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester (hereinafter referred to as Z-
Abbreviation for APM).

The present invention also relates to novel highly stabilized thermolysin-like proteases.

[0003] Z-APM has a sweetness of about 200 times that of sugar, and is an important artificial sweetener having excellent palatability such as no remaining bitterness, α-L-aspartyl-L-phenylalanine methyl ester ( Hereinafter, it is abbreviated as APM).

[0004]

PRIOR ART A thermolysin-like protease is used for the coupling of N-benzyloxycarbonylaspartic acid and phenylalanine methyl ester in an aqueous medium, and benzyloxycarbonyl-
A method for synthesizing α-L-aspartyl-L-phenylalanine methyl ester by an enzymatic reaction is described in JP-A-8-1.
96292. In this process, Z-Asp and L-PM are used in almost equimolar amounts, the pH of the reaction system is in the range of 4.5 to 6.0, a high concentration of salt is required, and the amount of thermolysin in the reaction is increased. Although the activity is quite high, it is known that the activity of the enzyme at the end of the reaction is significantly lower than that at the start of the reaction, and this decrease leads to an increase in the amount of the enzyme used and an increase in cost.

Now, in order to further understand the present invention,
Information on thermolysin and its stability is given below.

[0006] Thermolysin is a metalloprotease first discovered in the culture solution of Bacillus thermoproteo-lyticus (End.
O, S. (1962) J. Fermentation Tech., 40 , 346-353).
Its amino acid sequence (Titani, K., et al., (1972) Nature N
ew Biol., 238 , 35-37; EP-A-0418625; Miki, Y. (199
4), J. Mol. Biol. 160 , 623-639) and three-dimensional structure (Vrien
d, G. (1993), J. Computer- Aided Molecular Design, 7
, 367-396; Holmes, MA and Mattews, BW, (198
2), J. Mol. Biol. 160 , 623-639) and catalytic mechanism (Kester,
R., K., and Mattews BW, (1982) J. Biol. Chem. 252 ,
7704-7710) is described.

According to these descriptions, it is pointed out that the glutamic acid residue at position 143 and histidine at position 231 are important for the expression of protease activity in thermolysin as well as the zinc ion contained in the molecule of thermolyline. Was done. Hereinafter, these 143rd and 231st amino acid residues are abbreviated as catalytic residues for protease reaction.

A protease having an amino acid sequence similar to that of thermolysin and having a catalytic activity similar to that of thermolysin is described in the literature (eg, Imanaka, et. Al., (1986) Nat.
ure, 324 , 695-697). Studies on introducing mutations at specific positions in the amino acid sequence of thermolysin have also been reported (Kubo, M., et al , (1992) Appl. Environ.
Microbiol., 58, 3779-3787) and patent application (EP-A-
0.616.033). In that patent application, mutagenesis at positions 144, 150, 187 and 227 was shown to improve enzyme activity. Hereinafter, a protease having an activity value 0.2 to 10 times higher than the activity value of wild-type thermolysin for synthesizing Z-APM is abbreviated as "thermolysin-like protease".

As described above, the term "thermolysin-like protease" as used herein refers to a protease whose amino acid sequence matches the amino acid sequence shown in Sequence Listing 1, or Sequence Listing 1.
In the amino acid sequence shown in, one or more amino acid residues are replaced with other amino acid residues, or one or more amino acid residues at one or more positions. Or a metalloprotease having an amino acid sequence in which one or more amino acid residues are omitted.

[0010] Wild-type thermolysin and thermolysin into which a mutation is introduced in this way are very stable in a buffer solution near neutrality, and have very high activity at such a pH. The stability of thermolysin and "thermolysin-like proteases" is diminished in weak acids at pH's in the range of about 4.5-6; thermolysin is known to be completely inactivated below pH 4.

Further, the stability of thermolysin is determined by the fact that benzyloxycarbonyl-α-aspartic acid (hereinafter Z
-Abbreviated as Asp. ) Is known to change depending on the concentration (Japanese Patent Application Laid-Open No. 8-131170), in which thermolysin is stabilized by the presence of Z-Asp in a molar ratio of 30 times or more, and this effect is Z. -It is stated that it may be advantageous for coupling reactions for the synthesis of APM. However, in this patent, the stability of thermolysin during the synthesis of Z-APM over a long period of time and the Z- at very low active catalyst concentrations were observed.
The APM coupling formation reaction is not described at all.

Further, according to the recent study by the present inventors, p
In the range of H = 4.5 to 6, the stability of the enzyme is not sufficient in the coupling reaction, and the coupling reaction is efficiently performed even at a Z-Asp concentration of 0.3 mol / liter or more. It became clear that it was not possible.

On the other hand, the literature (Nakanishi et al (1990) App
L. Microbiol. Biotechnol. 32 , 633-636).
On the contrary, the presence of Asp has been shown to adversely affect the stability of thermolysin.

On the other hand, contrary to thermolysin, pH = 7
The Z-APM is unstable in the vicinity and more stable in weak acids with pH = 4.5-6.

The instability of Z-APM near pH = 7 and the instability of thermolysin near pH = 4.5-6 are major obstacles to the efficient synthesis of Z-APM using thermolysin. Has become. When Z-APM is synthesized by the coupling reaction of Z-Asp and PM using thermolysin at around pH = 7, the produced Z-APM and PM as the starting material are decomposed, and thermolysin is stable. , Z-APM recovery rate decreases. Further, when this reaction is carried out at around pH = 4.5 to 6, the activity of thermolysin tends to decrease, and the raw material and the product are stable, but Z-AP
Efficient synthesis of M is difficult.

Therefore, if the stability of thermolysin around pH = 4.5 to 6 is improved, Z-APM can be treated at this pH.
It can be efficiently synthesized in the area.

As a prior art document (Japanese Patent Laid-Open No. 6-18176)
In 1), the 144th leucine residue, the 150th aspartic acid residue, the 187th glutamic acid residue and 22
By substituting at least one amino acid residue of the 7th asparagine residue with another amino acid residue, ZA
It is disclosed that the enzymatic activity for PM degradation and Z-APM synthesis is improved. Here, Z-Asp / P
The molar ratio of M is about 0.5, and Z-AP is around pH = 7.
M was decomposed or synthesized. However, the stability of this protease, in particular Z-Asp /
The stability when used for the synthesis of Z-APM at a PM molar ratio of 0.8 to 1.7 has not been investigated at all.

As another prior art document, the above-mentioned Japanese Patent Laid-Open No. 8-13
1170 discloses the stabilization of metalloproteases by the addition of N-protected amino acids. here,
Z-Asp / PM molar ratio is about 0.5, pH = 6
Z-APM was decomposed or synthesized in the vicinity. However, even in this document, the stability of this protease,
In particular, the stability when used for the synthesis of Z-APM at pH = 6 or less and a Z-Asp / PM molar ratio of 0.8 to 1.7 has not been studied at all.

[0019]

Accordingly, there is a need for improved processes that reduce the loss of enzyme activity. In addition, more flexible reaction conditions are expected in terms of substrate molar ratio and pH range.

The object of the present invention is to solve the above-mentioned problems and improve the stability of conventional thermolysin-like proteases, particularly in the pH range of about 4.5 to 6.0, and to improve the stability. Was used in the coupling reaction of Z-Asp with L-PM to synthesize benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester, and to improve the efficiency of this synthetic method. It is to provide a way to improve.

[0021]

[Means for Solving the Problems]
nd, G. and Eijsink, V. (1993), J. Computer-Aided Mol
ecular Design, 7, 367-396; Holmes, MA and Mattew
s, BW, (1982), J. Mol. Biol. 160, 623-639), the detailed analysis of the three-dimensional structure of thermolysin revealed that there was a void in the center of the enzyme molecule, and that this void should be filled. It has been found that the stability of thermolysin-like protease can be improved if the above is achieved. Furthermore, as a result of detailed examination, in the thermolysin-like protease, a leucine residue at the 144th position, a threonine residue at the 149th position, an alanine residue at the 240th position, 270
The volume of the vacancy at the electron density surrounded by the alanine residue at position 288, the alanine residue at position 288, and one or more amino acid residues of those residues are more hydrophobic than the amino acid residue. Or by substituting with a bulky amino acid residue, or by affecting the amino acid residue surrounding the hole of electron density (performing amino acid substitution in the surrounding region), It is partially filled, and it is possible to reduce the volume of its pores, and such thermolysin-like protease is more stable than other proteases at low pH and high Z-Asp concentration. It was found that the properties are excellent, and the present invention has been completed.

That is, according to the present invention, ZA is used in an aqueous medium.
In a method for synthesizing Z-APM by an enzymatic reaction using a thermolysin-like protease in a coupling reaction between sp and PM, at least the 144th leucine residue and the 149th leucine residue present in the thermolysin having the amino acid sequence of Sequence Listing 1 Of the hole density at the electron density surrounded by the threonine residue, the 240th alanine residue, the 270th alanine residue, and the 288th alanine residue of one or more of those residues. Amino acid residues are substituted by amino acid residues that are more hydrophobic and bulkier than that amino acid residue, or affect the amino acid residues that surround the electron density vacancy ( By performing amino acid substitution), at least a part of the vacancy is filled, and the volume of the vacancy is reduced. Z-APM synthesizing method according to the enzyme reaction, characterized by the use of lysine-like proteases, in particular, the amino acid residue to be substituted, histidine, isoleucine, leucine, phenylalanine, threonine,
An amino acid residue selected from the group of tyrosine and valine is preferable, and it is particularly preferable to use a novel thermolysin-like protease characterized in that the leucine residue at position 144 is replaced with a phenylalanine residue or a tyrosine residue. preferable.

In Table 1 below, the bulk size of amino acid residues (unit: cm ³ / mol, reference (Zamyatnin, AA (198
4) Ann. Rev. Biophys. Bioeng., 13, 145-165) and hydrophobicity (π value, reference (Fauchere, JL
and Pliska, V. (1983) Eur. J. Med Chem., 18, 369-37
The value from (5)) is shown.

[0024]

[Table 1]

As is clear from this table, an amino acid residue having a larger bulk than the original amino acid residue may be selected and substituted. For example, for the leucine residue at the 144th position, a tyrosine residue is used. And a phenylalanine residue are suitable, and a phenylalanine residue is preferable because it has not only a large bulkiness but also a large hydrophobicity. For the 149th threonine residue, isoleucine, histidine and valine are also suitable in addition to the tyrosine residue and phenylalanine residue. In particular, other than histidine, it is preferable because not only is it bulky but also hydrophobic.

The conditions for synthesizing the Z-APM are as follows:
PH of the aqueous medium = 4.3-8.0, Z-Asp /
JP-A-6-181761, characterized in that the molar ratio of PM is in the range of 0.8 to 1.7 (usually 1: 2) and the concentration of Z-Asp charged is 0.2 mol / kg or more. The present invention also relates to a method used for synthesizing Z-APM in a novel reaction system in which the conventional thermolysin-like protease cannot be used.

As a method for substituting an amino acid residue, a method of mutagenesis of M13 phage described in JP-A-6-181761 (Vandeyar M., et al., (1988) Gene,
65 , 129). This method was first carried out on the plasmid pU.
Taking out the Sph I- Bam HI fragment from CTZ55 ,
M13mp1 digested with restriction enzymes Sph I and Bam HI
Recombinant phage M13
Make TZSp-Bc. This recombinant phage M
Using 13TZBa-Sp as a template, a primer for mutagenesis and a reverse primer were used to amplify between both primers by PCR reaction, and the resulting mutagenized DNA fragment was used as a restriction enzyme S.
Digested with ph I and Bam HI, and plasmid pUBTZ
In this method, the mutant is obtained by replacing the Sph I- Bam HI fragment of No. 2 with E. coli and transforming it.

Recombinant phage M13TZBa-Sp
Is used as a template and a forward mutagenesis primer and a M13 reverse mutagenesis primer are used to amplify between both primers by PCR reaction (PCR1). Apart from this, M13TZ
Using Ba-Sp as a template, a reverse mutagenesis primer and M
Using 13 forward mutation-introducing primers, amplification between both primers is performed by PCR reaction (PCR2). After removing the primers in the reaction solution, both mutated DNA fragments and the M13 forward mutagenesis primer and the M13 reverse mutagenesis primer are used to extend both DNA fragments by PCR reaction (PCR3). The resulting mutagenized DN
The A fragment is digested with restriction enzymes Sph I and Bam HI, replaced with the Sph I- Bam HI fragment of the plasmid pUBTZ2, and transformed into Escherichia coli to obtain a mutant. This construction method is shown in FIG.

The present invention will be specifically described with reference to the following examples and comparative examples, but the present invention is not limited to these examples.

[0030]

【Example】

Example 1 A method for producing the above-mentioned protease using the gene npr M of protease having the same amino acid sequence as thermolysin described above will be described. In the following, npr M
The protease encoded by the gene is called a wild-type enzyme. That is, a method for substituting the 144th leucine residue of the wild-type enzyme with a phenylalanine residue or a tyrosine residue will be described.

1 μ of plasmid pMK1 containing npr M
g in 20 microliters of a reaction solution (50 mM Tris-hydrochloric acid buffer pH 7.5, 10 mM magnesium chloride, 100 mM sodium chloride, 1 mM DTT) with 3 units each of 5 units of Bam HI and Pst I.
It was decomposed at 7 ° C. for 2 hours. A DNA fragment of about 3.7 kb was separated from this sample by 1% agarose gel electrophoresis and purified using Gene Clean DNA Purification Kit of Bio 101.

On the other hand, 1 μ of the plasmid vector pUC9
20 microliters of reaction solution containing 50 g (50 mM Tris-HCl buffer pH 7.5, 10 mM magnesium chloride, 100 mM sodium chloride, 1 mM DT
In T), 5 units each of Pst I- Bam HI were reacted at 37 ° C. for 2 hours.

NPr of pMK1 thus obtained
Pst I- Bam HI fragment of M gene and Ps of pUC9
The digested product of t I- Bam HI was reacted with a DNA ligation kit manufactured by Takara Shuzo, and transformed into Escherichia coli JM109 according to a conventional method, and Pst I of npr M gene was transformed.
-Recombinant plasmid with Bam HI fragment (pUCT
Z55) was obtained (FIG. 1).

Furthermore, 1 μg of the plasmid pUCTZ55
20 microliter of reaction solution (50 mM Tris-
Hydrochloric acid buffer solution pH 7.5, 10 mM magnesium chloride, 100 mM sodium chloride, 1 mM DTT), 5 units each of Sph I and Bam HI were added, and the mixture was decomposed at 37 ° C. for 2 hours. A DNA fragment of about 730 kb was separated from this sample by 1% agarose gel electrophoresis and purified using Gene Clean DNA Purification Kit of Bio 101.

On the other hand, 1 μg of the phage vector M13mp19 was added to 20 microliters of the reaction solution (50 mM).
Tris-hydrochloric acid buffer pH 7.5, 10 mM magnesium chloride, 100 mM sodium chloride, 1 mM DT
In T), 5 units each of Sph I and Bam HI were added and the mixture was decomposed at 37 ° C. for 2 hours.

Of the npr M gene thus obtained
Sph I and Bam HI fragments and M13mp19 Sp
A ligation reaction was carried out with the degradation product of h I and Bam HI using a DNA ligation kit manufactured by Takara Shuzo. The reaction mixture was transformed into E. coli JM109 in a conventional manner to obtain a recombinant phage (M13TZBa-Sp) containing Sph I- Bam HI fragment of the npr M gene (Fig. 2).

The resulting recombinant phage (M13TZB
Single-stranded DNA was prepared from a-Sp) by a usual method and used for the introduction of amino acid substitution.

The oligonucleotide for introducing amino acid substitution used for the substitution of the leucine residue at the 144th position with phenylalanine was represented by the following SEQ ID: No. 2 is shown.

[0039] In addition, the oligonucleotide for introducing amino acid substitution used for the substitution with tyrosine was represented by the following SEQ ID: No. 3
It was shown to.

[0040] These oligonucleotides were prepared using a 380B type DNA synthesizer manufactured by Applied Biosystems.
Hereinafter, these are referred to as reverse synthetic primers.

PCR was performed on 0.5 μg of the prepared single-stranded DNA.
Reaction solution (67 mM Tris (pH 8.8), 16.6
mM ammonium sulfate, 6.7 mM magnesium chloride, 10 mM 2-mercaptoethanol, 0.2 mM
dATP, 0.2 mM dGTP, 0.2 mM dT
TP, 0.2 mM dCTP, 1 μM forward M13
Universal primer, 1 μM reverse synthetic primer), 1 unit of Tth DNA polymerase was added, and a drop of mineral oil was overlaid to denature 9
The gene amplification reaction was repeated 30 cycles at 3 ° C. for 1 minute, annealing at 45 ° C. for 1 minute, extension at 72 ° C. for 45 seconds. After completion of the reaction, the aqueous layer was recovered, phenol extraction and ethanol precipitation were performed according to a conventional method, and the amplified DNA was recovered. 20 microliters of a reaction solution containing 50% of the DNA thus obtained (50 mM Tris-hydrochloric acid buffer pH 7.5, 10 mM magnesium chloride,
Ba to 100 mM sodium chloride 1 mM DTT)
m HI, reacted for 2 hours at 37 ° C. by adding Sph I each 5 units. The thus-mutated npr M
An approximately 730 bp fragment of the gene was obtained.

On the other hand, the shuttle vector already constructed by Miyake et al., That is, both Escherichia coli and Bacillus subtilis can be transformed, and npr M can be used in both hosts.
A shuttle vector pUBTZ2 (Japanese Patent Laid-Open No. 184363/1993) capable of expressing a gene was constructed with Bam H
A 7.4 kb fragment treated with I and Sph I was obtained. This 7.4 kb fragment was reacted with the mutated 730 bp fragment using a DNA ligation kit manufactured by Takara Shuzo Co., and transformed into Escherichia coli JM109 according to a conventional method, and the mutated recombinant plasmid pUBTZ2 (L144
F) and pUBTZ2 (L144Y) were obtained (FIG. 3).

On the agar medium on which the transformant E. coli was grown, an alkaline solution (0.2 N sodium hydroxide, 0.2
% SDS) (3 ml) was added to lyse the cells to recover 1 ml. 1 ml of 5 M potassium acetate was added to the recovered solution, the mixture was allowed to stand in ice for 10 minutes, and then centrifuged at 8000 rpm for 20 minutes to remove the precipitate. The obtained supernatant was treated with phenol and precipitated with ethanol according to a conventional method, dried under reduced pressure, and then washed with water.
It was dissolved in 2 ml of TE (10 mM Tris-HCl buffer (pH 7.5), 1 mM EDTA) to obtain a plasmid. The thus-prepared plasmid was transformed into Bacillus subtilis MT-2 strain according to a conventional method and applied to an agar medium containing 1% casein and 20 μg / ml kanamycin to select 140 halo-forming colonies.

20 μg / each of these colonies
Culturing was carried out at 37 ° C. in 3 ml of LB medium containing ml of kanamycin overnight, and the plasmid pUBTZ2 was extracted from the culture solution according to a conventional method. Bam HI, Sph
Treatment with I was performed to recover a 730 bp DNA fragment.
The nucleotide sequence of the recovered DNA fragment was analyzed by a DNA sequencer 373A manufactured by Applied Biosystems (currently Perkin Elmer), and the gene npr M (L14) in which the 144th amino acid residue was a phenylalanine residue was analyzed.
4F) or a gene that is a tyrosine residue, npr M (L1
44Y) were selected.

The target enzymes L144F and L144Y were obtained from the strain having the enzyme gene thus obtained as follows. That is, Bacillus subtilis MT-2 strain / pUBTZ2 (L144F) or Bacillus subtilis M having the gene of the present invention
T-2 strain / pUBTZ2 (L144Y) was inoculated into 5 ml of LB medium containing kanamycin at a concentration of 5 μg / ml, and cultured at 37 ° C. for 8 hours. The culture was inoculated into a 2L medium containing twice the components of the LB medium except for salt, and the strain was cultured at 37 ° C for 20 hours. The amount of the culture seed at the time of culturing was such that the absorbance of the culture solution at 660 nm had a value of 0.02. After culturing, the culture solution was centrifuged at 8000 rpm for 20 minutes to obtain a supernatant of the culture solution, and then ammonium sulfate was added to the supernatant to 60% saturation. The resulting solution was 16 at 4 ° C.
After standing for a time, the enzyme was completely precipitated. The enzyme-containing precipitate was collected by centrifugation at 8,000 rpm for 20 minutes. Buffer A in which the precipitate was dissolved (10 mM Tris-hydrochloric acid, pH 9, 10 mM calcium chloride,
100 ml of 0.3 M ammonium sulphate) is passed through a column of 20 ml of Butyl Toyopearl to adsorb the enzyme, and buffer B (10 mM Tris-HCl, pH 9, 10 mM calcium chloride) is eluted separately for the enzyme and the impurities. For this, a flow rate of 1.5 ml / min was applied. The eluent containing the enzyme was collected, salted out with 60% saturated ammonium sulfate, and the precipitate was left to stand overnight at 4 ° C. for aging. This precipitation is 10,000 rpm
Centrifugation at 10 minutes and the precipitate thus obtained was buffered with C (10 mM Tris-HCl, pH 7.0,
Dissolve in 3 ml of 10 mM calcium chloride, 100 mM sodium chloride, and gel filtration column (TS
K Gel G2000 SW21.5 × 600mm)
And L144F and L144Y purified enzymes were obtained using the buffer and as eluent.

Example 2 (Evaluation of Stability of Novel Protease) Purified L144
F or L144Y is dissolved in a 0.1 M sodium acetate buffer (pH 5.0) containing 0.8 M Z-Asp and 10 mM calcium chloride, and remains in the solution after being kept at 40 ° C. for 16 hours. The amount of enzyme was quantified by high performance liquid chromatography. TSK gel Phenyl-5P
WRP (4.6 mm ID × 7.5 cm) was used as a column for high performance liquid chromatography, and the elution was
Performed at a flow rate of 1.2 ml / min using 10 mM potassium acetate containing acetonitrile as the eluent. Enzyme detection was performed by light absorption at 280 nm. The enzyme residual ratio after culturing the solution is shown in FIG. Both L144Y and L144F enzymes showed about 2-fold higher survival rate than the wild-type enzyme having the same sequence as the thermolysin-like amino acid sequence, demonstrating that both enzymes of the present invention have high stability. It was

Comparative Example 1 A thermolysin-like protease in which the 144th leucine residue was substituted with an amino acid residue other than a phenylalanine residue and a tyrosine residue was prepared and compared with Example 2 to study their stability. . These proteases were produced by the same procedure as in Example 1 except that the random primer described by the base sequence shown below was used, and the stability of the protease was examined by the same procedure as in Example 2.

[0048] A protease in which the leucine residue at the 144th position was replaced with an amino acid residue other than the phenylalanine residue and the tyrosine residue was identified by determining the nucleotide sequence of the plasmid.

The results of their stability are shown in FIG. L144T (leucine residue is replaced by threonine residue) and L144H (leucine residue is replaced by histidine residue) are both more stable than the wild-type enzyme, but L144F or It was revealed to be lower than the stability of L144Y. L144S was below the stability of the wild-type enzyme.

Example 3 Double mutant protease having high activity for Z-APM degradation and Z-APM synthesis (the aspartic acid residue at position 150 was replaced with a tryptophan residue, and the asparagine residue at position 227 was replaced with D15 substituted with a histidine residue
0W-N227H) was used as a starting material for the production of highly stable enzymes.

Transformant Bacillus subtilis MT-2 / pUBTZ2
A plasmid was extracted from (D150W-N227H) according to a conventional method, and 20 μl of a reaction solution (50 mM Tris-hydrochloric acid buffer pH 7.
5, 10 mM magnesium chloride, 100 mM sodium chloride, 1 mM DTT) was digested with 5 units each of Aat I and Sph I at 37 ° C. for 2 hours, and further 7
The reaction was carried out at 0 ° C for 5 minutes. This sample was electrophoresed on a 1% agarose gel to give D150 containing double mutations.
A DNA fragment of about 530 bp of W-N227H was obtained.

On the other hand, 21 μg of the plasmid pUBTZ containing the mutation of L144F prepared in Example 1 was digested with each of 5 units of Aat I and Sph I at 37 ° C. for 2 hours and further reacted at 70 ° C. for 5 minutes. Let This sample was electrophoresed on a 1% agarose gel to obtain a DNA fragment of about 7.6 kp containing the L144F mutation.

The DNA fragment of about 7.6 kb thus obtained from the plasmid pUBTZ2 (L144F) and the DNA fragment of about 530 bp thus obtained from the plasmid pUBTZ2 (D150W-N227H) were ligated by Takara Shuzo Co., Ltd. The reaction mixture was transformed into Escherichia coli JM103 according to a conventional method, and the transformant JM103 / pU having the mutation introduced thereinto was ligated.
BTZ2 was obtained. The replaced amino acid residue was confirmed by identification of the nucleotide sequence of the plasmid. FIG. 6 shows the recombinant plasmid pUBTZ2 (L144F-D150W
-N227H) is constructed.

Example 1 using this plasmid DNA
Transformant Bacillus subtilis MT-2 / pUBTZ2 (L
144F-D150W-N227H) was prepared, and transformant Bacillus subtilis MT-2 / pUBTZ2 (L144Y-D1) was prepared.
50W-N227H) was also created.

Two mutant enzymes L144F-D150W-N227H and L14 from this transformant Bacillus subtilis
4Y-D150W-N227H was purified by buffer D (10 mM Tris-HCl pH8.
Purification was carried out according to the method described in Example 1, except that 5, 0.5 M guanidine hydrochloride, 10 mM calcium chloride) was used.

When the stability of the enzyme prepared as described above was evaluated in the same manner as in Example 2, L144F-D15 was evaluated.
0W-N227H and L144Y-D150W-N22
Both 7H are wild type enzyme or original D150W-N227
The stability was higher than that of H. The stability evaluation result is shown in FIG. 7.

Example 4 L144F-D150W-N prepared by the method of Example 3
The actual Z-APM synthesis was carried out using 227H.
L-PM (189 g; 712.6 mmol) and ZA
A suspension was prepared by dissolving sp (113 g: 633.3 mmol) in water. The pH of the solution was adjusted to 5.3 with a 22% sodium hydroxide solution and 155 g sodium chloride was added. The prepared solution was transparent. At the same time, an enzyme solution was prepared by adding 2.5 g of enzyme and 3.36 g of CaCl ₂ to water.

Both solutions were mixed to start the enzyme coupling reaction. The solution after this mixing had the composition shown in Table 2 below.

[0059]

[Table 2]

The enzymatic reaction has a diameter of approximately 13 cm and is 40
It was carried out in a glass reaction vessel kept at 0 ° C. In this reactor, 0.5 cm from the bottom and 2 c from the liquid surface.
A variable speed stirrer with 11 cm long blades was installed at a depth of m. The stirring speed is 1 for 50 minutes after starting the reaction.
The speed was adjusted to 150 rpm and thereafter 300 rpm for 1 minute. The pH of the reaction mixture was continuously measured during the reaction, and when the pH reached 5.3 or more, it was adjusted to 5.3 by adding 16% hydrochloric acid. After a certain period of time, a sample was taken out of the reaction vessel, and Z
The concentrations of Asp, L-PM and residual enzyme were analyzed.

Z-Asp and L-PM are transferred to G2000SW.
Quantitation was performed at 25 ° C at a flow rate of 1 ml / min by an HPLC system using an XL (TSK-GEL) column. The eluent used was a 70/30 acetonitrile / water mixture containing 0.15 M acetate buffer, pH = 5.6. Detection of eluted components was performed at 254 nm. The concentration of the remaining enzyme is also Phenyl-5PW.
Quantitation was performed at 25 ° C. at a flow rate of 1.2 ml / min by an HPLC system using an RP (TSK-GEL) column. In this analysis, eluent A (5/95 acetonitrile / water mixture containing calcium acetate buffer pH = 6) and eluent B (60/40 containing calcium acetate buffer pH = 6) were used. A gradient eluent consisting of acetonitrile / water mixture) was used. The following gradient was used: 0-4 minutes, A / B = 90/10, 4-1
A / B was changed from 90/10 to 40/60 in 0 minutes and 6 minutes. The eluted enzyme was detected by absorption at 280 nm.

Table 3 shows the relationship between the conversion rate of Z-Asp to Z-APM and the enzyme residual rate.

[0063]

[Table 3]

Comparative Examples 2-3 Wild type enzyme and D150W-N22 obtained in Example 3
The same operation as in Example 4 was carried out using 7H and a solution having a slightly different composition shown in Table 4 below.

[0065]

[Table 4]

The results obtained are shown in Table 5 below.

[0067]

[Table 5]

As is clear from this table, the triple mutant enzyme (L144F-D150W-N227H) of the present invention is
Conventional wild type enzyme, double mutant enzyme (D150W-N2
It has been revealed that the stability is superior to that of (27H).

[0069]

INDUSTRIAL APPLICABILITY The novel thermolysin-like protease of the present invention is a wild-type thermolysin or other mutated thermolysin-like protease (for example, the aspartic acid residue at position 150 is replaced with a tryptophan residue, and the asparagine residue at position 227 is used). Is more stable than the thermolysin-like protease in which the histidine residue is replaced with
It is stable even in a solution around = 5. In addition, this thermolysin-like protease is used as the starting materials Z-Asp and L-
A new Z that cannot be used with conventional thermolysin-like proteases, such as a PM molar ratio of 0.8-1.7 (usually 2) and a reaction solution pH of 4.3-3.8 (usually 7).
-APM synthesis system can also be used. Due to such effects, excellent effects are exhibited in that the reaction can be continued for a long time and the amount of the recovered enzyme after the reaction is improved.

The sequence listing 1 in the present invention is shown as follows.

[Sequence Listing] SEQ ID NO: 1 Sequence length: 316 Sequence type: Amino acid Topology-: Linear Sequence type: Protein Origin: Bacillus sequence Ile Thr Gly Thr Ser Thr Val Gly Val Gly Arg Gly Val Leu Gly 1 5 10 15 Asp Gln Lys Asn Ile Asn Thr Thr Tyr Ser Thr Tyr Tyr Tyr Leu 20 25 30 Gln Asp Asn Thr Arg Gly Asn Gly Ile Phe Thr Tyr Asp Ala Lys 35 40 45 Tyr Arg Thr Thr Leu Pro Gly Ser Leu Trp Ala Asp Ala Asp Asn 50 55 60 Gln Phe Phe Ala Ser Tyr Asp Ala Pro Ala Val Asp Ala His Tyr 65 70 75 Tyr Ala Gly Val Thr Tyr Asp Tyr Tyr Lys Asn Val His Asn Arg 80 85 90 Leu Ser Tyr Asp Gly Asn Asn Ala Ala Ile Arg Ser Ser Val His 95 100 105 Tyr Ser Gln Gly Tyr Asn Asn Ala Phe Trp Asn Gly Ser Gln Met 110 115 120 Val Tyr Gly Asp Gly Asp Gly Gln Thr Phe Ile Pro Leu Ser Gly 125 130 135 Gly Ile Asp Val Val Ala His Glu Leu Thr His Ala Val Thr Asp 140 145 150 Tyr Thr Ala Gly Leu Ile Tyr Gln Asn Glu Ser Gly Ala Ile Asn 155 160 165 Glu Ala Ile Ser Asp Ile Phe G ly Thr Leu Val Glu Phe Tyr Ala 170 175 180 Asn Lys Asn Pro Asp Trp Glu Ile Gly Glu Asp Val Tyr Thr Pro 185 190 195 Gly Ile Ser Gly Asp Ser Leu Arg Ser Met Ser Asp Pro Ala Lys 200 205 210 Tyr Gly Asp Pro Asp His Tyr Ser Lys Arg Tyr Thr Gly Thr Gln 215 220 225 Asp Asn Gly Gly Val His Ile Asn Ser Gly Ile Ile Asn Lys Ala 230 235 240 Ala Tyr Leu Ile Ser Gln Gly Gly Thr His Tyr Gly Val Ser Val 245 250 255 Val Gly Ile Gly Arg Asp Lys Leu Gly Lys Ile Phe Tyr Arg Ala 260 265 270 Leu Thr Gln Tyr Leu Thr Pro Thr Ser Asn Phe Ser Gln Leu Arg 275 280 285 Ala Ala Ala Val Gln Ser Ala Thr Asp Leu Tyr Gly Ser Thr Ser 290 295 300 Gln Glu Val Ala Ser Val Lys Gln Ala Phe Asp Ala Val Gly Val 305 310 315 Lys

[Brief description of drawings]

FIG. 1: Plasmid pMK1 to plasmid pUCTZ
It is a process drawing which builds 55.

FIG. 2 is a process diagram for constructing M13 phage M13TZBa-Sp from plasmid pUCTZ55.

FIG. 3: Plasmid pU using M13TZBa-Sp
It is a process drawing which introduce | transduces a mutation into the nprM gene cloned on BTZ2 .

FIG. 4 is a diagram showing a mutation introduction method by the PCR method.

FIG. 5 is a diagram showing the results of examining the stability of a thermolysin-like protease in which the leucine residue at position 144 was replaced with another amino acid residue.

[Explanation of symbols]

 S: Serine Q: Glutamine C: Cysteine W: Tryptophan E: Glutamic acid A: Alanine L: Leucine N: Asparagine H: Histidine T: Threonine F: Phenylalanine Y: Tyrosine

FIG. 6 shows a plasmid pUBTZ2 (L144F) in which the nprM gene in which the leucine residue at the 144th position was replaced with a phenylalanine residue was cloned, and a plasmid pUBTZ2 (D150W in which the double mutant enzyme was cloned.
-N227H) to the plasmid (L144F-D150
FIG. 6 is a process drawing for constructing W-N227H).

FIG. 7 is a diagram showing the results of measuring the stability of thermolysin-like protease.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shunichi Josho 1-9-2 Minamidai, Sagamihara-shi, Kanagawa (72) Inventor Kimiko Endo 1733-10 Kanemori, Machida-shi, Tokyo (72) Inventor Akiyoshi Wada Akasaka, Tokyo 8-11-4

Claims (13)

[Claims] 1. Benzyloxycarbonyl-by enzymatic reaction using a thermolysin-like protease for coupling with N-benzyloxycarbonylaspartic acid and phenylalanine methyl ester in an aqueous medium.
In the method for synthesizing α-L-aspartyl-L-phenylalanine methyl ester, there is at least a leucine residue at the 144th position, a leucine residue at the 149th position, a threonine residue at the 149th position, which is present in thermolysin having the amino acid sequence of Sequence Listing 1.
0th alanine residue, 270th alanine residue, 2
The volume of the hole at the electron density surrounded by the 88th alanine residue is defined as the amino acid residue in which one or more amino acid residues of these residues are more hydrophobic and bulkier than the amino acid residue. Substituted by a group, or
A novel feature of the electron density is that at least part of the vacancy is filled by affecting the amino acid residues surrounding the vacancy, and the volume of the vacancy is reduced. A method for synthesizing benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester by an enzymatic reaction characterized by using a thermolysin-like protease. 2. The synthetic method according to claim 1, wherein the volume of the holes at the electron density is 144th, 149th,
The 240th, 270th and 288th amino acid residues have a larger molar volume than that residue and are replaced by an amino acid residue selected from the group of histidine, isoleucine, leucine, phenylalanine, threonine, tyrosine and valine. A method for synthesizing benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester by an enzymatic reaction characterized in that the volume of the pores is reduced. 3. The synthetic method according to claim 2, wherein 14
Benzyloxycarbonyl-α-L-aspartyl-by enzymatic reaction characterized in that the fourth leucine residue is substituted with a phenylalanine residue or a tyrosine residue
A method for synthesizing L-phenylalanine methyl ester. 4. The synthetic method according to claim 1, wherein at least one of the 150th aspartic acid residue, the 187th glutamic acid residue and the 227th asparagine residue is replaced with another amino acid. The method for synthesizing benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester by an enzymatic reaction characterized in that 5. The synthetic method according to any one of claims 1 to 4, wherein the enzyme coupling reaction is carried out at a pH of 4.3 to 8.
A method for synthesizing benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester by an enzymatic reaction characterized by being in the range of 0. 6. The synthetic method according to claim 5, wherein N-
Benzyloxycarbonyl-L-aspartic acid and L
-Benzyloxycarbonyl-α-L-aspartyl-L by enzymatic reaction characterized by using phenylalanine methyl ester as a substrate for enzyme coupling
-A method for synthesizing phenylalanine methyl ester. 7. The synthetic method according to claim 6, wherein L-
Benzyloxycarbonyl-α-by enzymatic reaction, characterized in that the molar ratio of N-benzyloxycarbonyl-L-aspartic acid to aspartyl-L-phenylalanine methyl ester is in the range of 0.8 to 1.7.
A method for synthesizing L-aspartyl-L-phenylalanine methyl ester. 8. The method according to any one of claims 1 to 7, wherein the concentration of N-benzyloxycarbonyl-L-aspartic acid is 0.2 mol / kg or more. Benzyloxycarbonyl-α
A method for synthesizing -L-aspartyl-L-phenylalanine methyl ester. 9. A leucine residue at the 144th position, a threonine residue at the 149th position, an alanine residue at the 240th position, an alanine residue at the 270th position, an alanine residue at the 270th position, which is present in thermolysin having the amino acid sequence of Sequence Listing 1. The volume of vacancies at the electron density surrounded by the alanine residues of the amino acid residue is such that one or more amino acid residues of those residues are more hydrophobic and bulkier than the amino acid residue. By substituting, or by affecting the amino acid residues surrounding the vacancy in electron density, filling at least part of the vacancy, reducing the volume of the vacancy. A novel thermolysin-like protease characterized. 10. The novel thermolysin-like protease according to claim 9, wherein the vacancy volume at the electron density is 144, 149, 240, 270 and 288 amino acid residues are the residues thereof. It has a larger molar volume than Histidine, isoleucine, leucine, phenylalanine, threonine, tyrosine and valine by replacing it with an amino acid residue, thereby reducing the volume of its pores. A novel thermolysin-like protease characterized. 11. The novel thermolysin-like protease according to claim 9, wherein the 144th leucine residue is replaced with a tyrosine residue. 12. The novel thermolysin-like protease according to claim 9, wherein at least one of the 150th aspartic acid residue, the 187th glutamic acid residue and the 227th asparagine residue is replaced with another amino acid. A novel thermolysin-like protease characterized by being present. 13. Benzyloxycarbonyl-.alpha.-L by enzymatic reaction substantially as described in the description and examples.
-A method for the synthesis of aspartyl-L-phenylalanine methyl ester and a highly stabilized thermolysin-like protease.