KR0180749B1 - Protease - Google Patents

Abstract

The present invention relates to a method for preparing a thermolysine-like neutral metallo-protease and a method for preparing the same, particularly a method for preparing benzyloxycarbonyl-alpha-L-aspartyl-L-phenylalanine methyl ester,

The modified protease according to the invention is a mutant of a thermostable neutral protease and is selected from the group consisting of 144th leucine residue, 150th aspartic acid residue, 187th glutamic acid residue and 227th asparagine residue. At least one amino acid residue of 1 is characterized by being substituted with an amino acid residue other than the above amino acid.

Description

Protease and its use

1 is a diagram used to prepare recombinant plasmid pUCT237 from known plasmid pMK4.

2 is a schematic used to prepare recombinant plasmid pUCTZ47 from plasmids pMK4 and pUCTZ37.

3 is a diagram used to prepare recombinant plasmid pUBTZ1 from known plasmids pUCTZ47 and pUB110.

4 is a schematic used to prepare plasmid pUBTZ2 from plasmid pUBTZ1.

5 is a diagram used to prepare recombinant plasmid pUCTZ55 from known plasmid pMK1.

6 is a diagram used to prepare M13TZSp-Bc, a recombinant M13 phage from plasmid pUCTZ55.

7 is a diagram used to prepare plasmid pUBTZ2 (mutants) obtained from plasmids pUBTZ2 and M13TZSp-Bc (mutants).

8 is a diagram used to prepare M13TZBa-Sp, a recombinant M13 phage from plasmid pUCTZ55.

Figure 9 shows the reaction mixture of PCR comprising mutagenesis primer (D150N) and the mutant PCR product obtained from plasmid pUBTZ2 (N227H) and the recombinant plasmid pUBTZ2 (mutant) from plasmid pUBTZ2 (L144S) (L144S-D150N-N227H or L144S). -D150H-N227H).

The present invention relates to a servolysine-like neutral metallo-protease and a process for preparing the same, particularly a method for preparing benzyloxycarbonyl-alpha-L-aspartyl-L-phenylalanine methyl ester.

Thermolysin is an enzyme that is used in a variety of fields and commercially useful, for example, in detergent compositions, food preparations and cosmetic preparations. The enzyme is also used in the synthesis of benzyloxycarbonyl-alpha-L-aspartyl-L-phenylalanine methyl ester (hereinafter abbreviated as Z-APM), a precursor of aspartame, an artificial sweetener.

Thermolysine has been extensively investigated since it was first discovered in Maeyang broth of Bacillus Thermoproteloyticus (Endo, S. (1962) J. Fermentation Tech., 40, 346-353).

Thus, for example, the amino sequence (Titani, K., et al., (1972) Nature New Biol., 238, 35-37) and the three-dimensional structure of the enzyme (Holmes, MA and Matthews, BW (1982) J. Mol. Biol. 160., (623-639), while the protease gene was cloned from Bacillus thermoproteolyticus (European Patent EP-A-0418625) and the amino acid sequence of the mature enzyme derived from the nucleotide sequence of this gene. The two parts differed from the primary structure indicated by Titani.

Thus, it was reported that the 37th amino acid residue (from the amino terminus) of the mature enzyme is asparagine rather than aspartic acid and the 119th amino acid residue is glutamine, not glutamic acid. This amino acid sequence is identical to that encoded by nprM, one of the protease genes cloned from Bacillus Stearothermophilus (Kubo, M., et al., Journal of Genreal Microbiology 134 (1988), p. 1887).

Thus the protease encoded by this nprM gene or gene from Bacillus thermoproteloyticus is referred to herein as the wild type thermolysine-like neutral metallo-protease.

Alterations in the stability and specific activity of thermolysine-like neutral metallo-proteases have been recorded (Kubo M., et al., (1992) Applid and Environmental Microbiology, 58, 3779-3783). This paper describes a variety of mutants that differ in one or more amino acid residues in the primary structure, particularly at 93, 110, 114, 115, 136, 137, 143, 151, 157, 193, 211, 217 and 221 positions. However, in this reference the activity is measured only by the casein digestion method. However, in this reference the activity is measured only by the casein digestion method. However, none of these mutants showed substantially improved activity related to Z-APM synthesis or digestion. It has also been found that casein digestion activity is not correlated with activity for Z-APM synthesis (as described in more detail in the Examples below), which is specific for Z-APM synthesis even if the specific activity for casein digestion is increased. It indicates that activity does not always increase.

Based on this observation, Z-APM such as relatively low activity of enzyme, condensation of product Z-APM with starting material L- or D, L-phenylalanine methyl ester (PM) and inactivation of enzyme during hydrolysis. Because of various problems due to long reaction times and / or inadequate pH in the enzymatic synthesis of N, it is necessary to develop improved enzymes having higher activity for the synthesis of Z-APM than wild type thermolysine.

The object of the present invention is to solve the above problem. It has been found that enzymes with excellent properties can be obtained when amino acids with unique positions at particular positions are selected from a variety of amino acids. From thermolysine-like neutral metallo-proteases having the (wild-type) amino acid sequence SEQ ID NO: 1 shown below, by replacing one or more amino acid residues at specific sites in the sequence having amino acid residues different from the original Protease was induced. In particular modified proteases according to the invention are obtained by substituting at least one of the following amino acid residues for another amino acid residue: 144 th (leucine), 150 th (aspartic acid), 187 th (glutamic acid) and 227 th (asparagine) Amino acid residues.

In particular, as shown above, at least one of the amino acid residues at positions 144, 150, 187, and 227 of the modified enzyme having an amino acid sequence has higher specific activity in digesting or synthesizing Z-APM than the wild type enzyme. Indicates. This solves the problem of how to enhance the activity of A-APM synthesis of thermolysine-like neutral metallo-proteases derived from Bacillus Stearothermophilus. In addition, it provides enzymes with higher activity at lower pH and thus less hydrolysis of Z-APM and PM during Z-APM synthesis.

It should be noted that enzymes obtained by substitution at two or more positions specified above are also included within the scope of the present invention. It should also be noted that enzymes obtained by substitution of at least one amino acid residue specified above and by further substitution of at least one of the other amino acid residues with other amino acids are also included within the scope of the present invention.

The proteases described in the specification according to the invention are derivatives having a modified sequence different from that of wild type thermolysine-like neutral metallo-proteases. In particular, this protease has increased activity against Z-APM synthesis and digestion. Typically this is determined by analyzing Z-APM synthesis and / or digestive activity and comparing the chemical activity with the activity of the wild type thermolysine-like neutral metallo-protease quantified in the same manner. This process is described further in the following examples.

Modified enzymes can be prepared by known methods by one of ordinary skill in the art.

A preferred method of making the modified protease of the present invention is by modifying the amino acid sequence to a predetermined position of at least one of positions 144, 150, 187 and 227 of the thermolysine-like neutral metallo-protease by recombinant DNA methods. The stability of the protease obtained can be measured by quantitative testes for the end use.

Various methods are known that are used to mutate cloned DNA. For example, the mutant nprM gene fragment is prepared using the M13 phage mutation generation method (see Vandeyar, M. et al., (1988) Gene, 65, 129).

Plasmids and phage DNA used as templates in this method can be derived from the known plasmid pMK1 (Kubo, M. and Imanaka, T. (1989) J. Bacteriol, 171, 4080-4082). Several restriction endonucleases are used to digest and clone nprM gene fragments into other plasmids or phage vectors.

Mutant primers should be complementary to the single-stranded template DNA containing the nprM gene except for the codon (s) encoding the substituted amino acid residue (s). Any desired amino acid substituent can be obtained by using mutagenic primers having different codon (s) for such substituted amino acid residue (s).

The nprM gene can also be synthesized using PCR techniques (polymerase chain reactions) using chemically synthesized primers (Higuchi, R. Krummel, B., and Saiki, R, K., (1988) Nucleic acids Res. 16. 7351-7367). May be mutated). This PCR method is particularly useful when the restriction enzyme site is near the mutation site. For example, since the cleavage site of the restriction enzyme Sph I is near the codon of aspartic acid at position 150 of the wild type thermolysine-like neutral metallo-protease, the mutant primer containing this SPH I site is It can be used to generate mutants in position.

Mutant primers are therefore used as detection primers. Oligonucleotides that can be used as reverse primers (antisenses) are complementary to the nprM gene, for example, from the Aat I cleavage position of the nprM gene.

Two methods are used to mutate at one or more positions. One method is to mutate all target positions simultaneously, while the other is to mutate one by one sequentially. Both methods actually provide plasmids that are mutated at one or more positions.

A general method for preparing recombinant thermolysine-like neutral metallo-proteases is described in Kubo, M. and Imanaka T., (1989) J. Bacteriol., 171, 4080-4082, which modify the thermostat. Insert the DNA encoding the lysine-like neutral metallo-protease into the expression vector, use the vector to transform host cells, incubate the transformant until the modified metallo-protease accumulates in the culture and And recovering the modified enzyme from the culture. However, the plasmid pMK1 used in this reference is more than 20 kb in size, and therefore it is difficult to substantially transform Escherichia coli with the plasmid. In addition, in Bacillus subtilis, plasmid pMK1 was also found to drop out significantly in the later stages of culture.

Therefore, in order to overcome such a problem, the inventors of the present invention prepared a shuttle vector capable of transforming the host Escherichia coli and Bacillus subtilis, and in these hosts, can express the nprM gene. Thus, two shuttle vectors containing the nprM gene were prepared as shown in FIGS. 1 to 4 (pUBTZ1 and puBTZ2).

When they are used to transform Escherichia coli strains such as HB101 and JM103, the nprM gene is expressed in these strains. In addition, transformation of Bacillus subtilis strains such as DB104, DB117 and MT-2 into these plasmids successfully expresses the nprM gene. In addition, no drop-out was observed in the later stages of culture.

Similar results and advantages of using these shuttle vectors are also obtained using modified thermolysine-like neutral metallo-proteases instead of wild-type genes. Modified thermolysine-like neutral metallo-proteases can be prepared in recombinant bacteria and secreted into the culture medium. Such proteases are recovered by ammonium sulfite precipitation and homogeneously purified by conventional methods such as hydrophobic interaction chromatography and / or gel filtration.

This modified protease is used for the synthesis of Z-APM, the precursor of aspartame, more effectively than the wild type thermolysine-like neutral metallo-protease. This is shown by comparing the activity of Z-APM digestion and Z-APM synthesis of these modified proteases with the activity of wild type enzymes, indicating that the activity of the modified protease is much higher than that of wild type enzymes. Measurements of these activities are described in the Examples.

In the present invention, positions 144, 150, 187, and 227 from the amino terminal amino acids of the thermolysine-like neutral metallo-protease enhance the activity of Z-APM digestion or synthesis, particularly when two or three of these positions are joined. I found it to be quite effective.

It should be noted that casein digestive activity is not related to Z-APM synthesis or activity on digestion. When comparing the activity of mutant enzymes for casein and Z-APM, it is clear that even if the activity for casein digestion is reduced, the activity for Z-APM crying and / or digestion can be greatly increased. The present invention will be described in more detail with reference to the following Examples, which are intended to illustrate the present invention, but the present invention is not limited thereto.

Example 1

[Synthesis of Mutant Thermolysine-Like Neutral Metallo-Protease Mutated to 187th (Example 1A) or 227th (Example 1B) Amino Acid Residues

1 μg of plasmid pMK1 containing the thermolysine-like neutral metallo-protease gene nprM derived from Bacillus Stearothermophilus MK 232 was mixed with 20 μl of Pst I in 20 μl of the reaction mixture (tris-HCl 50 mM, MgCl ₂ 10 mM, DTT 1 mM, pH 7.5). 5 units of BamH I were digested for 2 hours at 37 ° C. This sample was electrophoresed on a 1% agar gel to separate approximately 3.5 kb DNA fragments and purified using a Bio-101 Gene Clean DNA purification kit.

Meanwhile, 1 µg of plasmid pUC9 was treated in 20 µl of the reaction mixture described above for 5 hours at 37 ° C in 5 units of Pst I and BamH I, respectively.

The Pst I-BamH I fragment of the nprM gene was ligated to the Pst I-BamH I digest of pUC9 using a Takara Shuzo DNA ligation kit, and the ligation mixture was transferred to Escherichia coli JM 109 by conventional methods. The recombinant plasmid (pUCTZ55) containing the Pst I-BamH I fragment of the nprM gene was used for transformation (Figure 5).

1 μg of the recombinant plasmid pUCTZ55 was digested in 20 μl of the reaction mixture (TriS-HCl 50 mM, 10 mM MgCl ₂ , 100 mM NaCl, 1 mM DTT, pH 7.5) for 5 hours at 37 ° C. in 5 units of Sph I and Bcl I, respectively. This sample was subjected to 1% agar gel electrophoresis to separate approximately 550kbp DNA fragments and purified using a Bio-101 Gene Clean DNA Purifier. Meanwhile, 181 μg of phage vector M13mp was digested in 20 μl of the reaction mixture at 37 ° C. for 5 hours at 5 units of Sph I and BamH I.

The ShpI-BclI fragment of the nprM gene was ligated to the Sph I-BamH I digest of M13mp18 using a Takara Shuzo DNA Ligation Apparatus, and the ligation mixture was used to transform Escherichia coli JM109 in a conventional manner to the Sph I of the nprM gene. Recombinant phage (M137TZSp-Bc) containing -Bcl I fragment was obtained (FIG. 6).

Single chain DNA was prepared from M137TZSp-Bc by conventional methods and mutations were induced. Oligonucleotides used to induce mutations were prepared using an applied biosystem model 380B DNA synthesizer.

Mutant oligonucleotides used to replace the 187th residue (glutamic acid with glutamine, Example 1A) and the 227th residue (asparagine with histidine, Example 1B) are as follows.

Mutagenesis was performed using a USB T7-GEN in vitro mutation generator, followed by DNA sequencing to confirm mutations.

The double-stranded DNA of the mutated M13TZSp-Bc was prepared by a conventional method, and 1 μg of the double-stranded DNA was added to 20 μl of Sph I in a reaction mixture (50 mM Tris-HCl, 10 mM MgCl ₂ , 100 mM NaCl, 1 mM DDT at pH 7.5). 5 units of Aat I and 5 units of A2 were digested at 37 ° C. for 2 hours, and each sample was electrophoresed on 1% agar gel. About 550kbp DNA fragments were isolated from each M13TZSp-Bc digest and purified on a Bio-101 Gene Clean DNA Purifier.

At the same time, a vector fragment for the expression of Bacillus subtilis was prepared by the method described below. From plasmid pMK4 (Yamada et al., (1991) Gene, 99, 109-114), about 1.0 kb DNA fragment containing a portion of the nprM gene was digested with Bcl I, cloned to the BamH I site of plasmid pUC9 and plasmid pUCTZ37 Was prepared (FIG. 1). Plasmid pUCTZ37 is incomplete without the 5'-end of the nprM gene. Plasmid pUCTZ47 was prepared by digesting plasmid pUCTZ37 with restriction endonuclease HindIII and cloned about 1.2 kb HindIII fragment of pMK4 into larger pUCTZ37 fragment (Figure 2). Recombinant plasmid pUCTZ47 contains the entire sequence of nprM and its transcriptional promoter sequence.

To prepare a shuttle ejector between Escherichia coli and Bacillus subtilis, pUCTZ47 and pUB110 (Lacey et al., 1974) were digested with EcoR I and ligated with T4 polynucleotide kinase to prepare plasmid pUBTZ1 (see Figure 3). .

Finally, plasmid pUBTZ2 was prepared by deleting the DNA fragment between the restriction sites with endonuclease Sma I and PuvII from plasmid pUBTZ1 as shown in FIG. Plasmid pUBTZ2 has a single BamH I, Sph I and Aat I restriction position in the nprM gene. Substitution of the wild type gene with the mutant gene was carried out as follows.

Plasmid pUBTZ2 was digested with Sph I and Aat I and 7.6 kb fragments were isolated.

PUBTZ2 thus obtained was ligated to Shp I-Aat I fragments using Takara Shuzo DNA ligation politics to mutated Sph I-Aat I fragments (about 550bp) of the nprM gene. This ligation mixture was used to transform Escherichia coli JM103 in a conventional manner to prepare recombinant plasmids pUBTZ2 (E187Q) and pUBTZ2 (N227H) (see Figure 7).

Recombinant plasmid pUBTZ2 (mutant) was transformed with Bacillus subtilis strain MT-2 in a conventional manner. The cell suspension was spread on LB agar plates containing 1% casein and 5 μg / ml kanamycin and incubated overnight at 37 ° C. Finally, separation of the halo-forming colonies yielded the recombinant plasmid pUBTZ2 (mutant).

A single colony of recombinant Bacillus subtilis MT-2 / pUBTZ2 (mutant) was transferred to 5 ml LB medium containing kanamycin (5 μg / ml) and incubated overnight at 37 ° C. The culture was transferred to 500 ml of 2 L medium (2% bacto tryptone, 1% yeast extract, 0.5% NaCl) containing kanamycin (5 µg / ml) and incubated at 37 ° C for 20 hours. To remove the bacteria, the broth broth was centrifuged at 800 rpm for 30 minutes, ammonium sulfate was added to the supernatant to obtain 60% saturated solution, and the mixture was stirred at 4 ° C. overnight.

The precipitate was recovered by centrifugation and dissolved in 10 ml of Buffer A (20 mM Tris-HCl, 10 mM CaCl ₂ , pH 9.0). The solution was poured into 20 ml of butyl-Toyopearl and eluted with buffer A at a flow rate of 1.5 ml / min. The active part was bound and salted with 60% saturated ammonium sulfate. The precipitate was collected by centrifugation at 15,000 rpm for 30 minutes, and then dissolved in 5 ml of buffer B (20 mM of Tris-HCl, 10 mM CaCl ₂ , pH 7.5). The enzyme solution was further filtered on a gel-filtration column (TSK Gel G2000 SW 21.5 x 600 mm) and eluted with buffer B at a flow rate of 1 ml / min. The active eluate binds to produce purified enzyme.

Example 2

[Synthesis of Mutant Thermolysine-Like Neutral Metallo-Protease Mutated to 144th Amino Acid Residue by Submutation Generation]

Recombination comprising the BamH I-Sph I fragment of the nprM gene in the same manner as in Example 1 except that restriction enzymes (BamH I and Sph I instead of Pst I and BamH I) and M13 phage vectors (mp19 instead of mp18) were used Phage M13TZBa-Sp was prepared. The manufacturing process of the recombinant phage M13TZBa-Sp is shown in FIG.

Single-stranded DNA of M13TZBa-Sp was incubated in 20 ml of 0.25 M sodium acetate buffer containing IM NaNO ₂ (pH 4.3) for 4.5 hours at 21.5 ° C., and the DNA was recovered by ethanol precipitation.

The recovered DNA was PCR (Ph 8.8 Tris-HCl 67 mM, 16.6 mM ammonium sulfate, 6.7 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 0.2 mM dATP, 0.2 mM dTTIP, 0.2 mM dGTP, 0.1 mM dCTP, 1 μM M13 forward). Dissolved in the reaction mixture for universal primer, 1 μM M13 reverse primer) and 1 unit of Tth DNA polymorase was added. The solution was covered with 1 mineral oil. Denatured at 93 ° C. for 1 minute, annealed at 45 ° C. for 1 minute, and extension at 45 ° C. for 45 seconds was repeated 30 times. After the reaction, the aqueous layer was recovered, extracted with phenol, and ethanol-treated to recover amplified DNA by precipitation.

One half of the DNA was digested in 5 μl of BamH I and Sph I for 2 hours at 37 ° C. in 20 μl of the reaction mixture (Tris-HCl 50 mM, 10 mM, MgCl ₂ , 100 mM NaCl, 1 mM DTT, pH 7.5).

The reaction mixture was incubated at 70 ° C. for 5 minutes. BamH I-Sph I fragments of pUBTZ2 (7.4 kb) using the Takara Shuzo DNA ligation apparatus and BamH I-Sph I fragments of pUBTZ2 (7.4 kb) using the Takara Shuzo DNA ligation apparatus The fragment was ligated. This ligation mixture was transformed into E. Coli JM 103 by conventional methods to obtain JM103 / pUBTZ2 (mutants).

3 ml of alkaline solution (0.2N, NaoH, 0.2% SDS) was added to an agar plate containing transformant colonies to dissolve the colonies and recover 1 ml of the solution. 1 ml of 5 M potassium acetate was added to the solution and centrifuged for 20 minutes to remove the precipitate and left to stand on ice for 10 minutes. The supernatant was treated with phenol and the precipitate was obtained by treatment with ethanol.

The precipitate was dried after vacuum drying and used as a mutant library.

Bacillus subtilis strain MT-2 was transformed by mutant library in a conventional manner. The cell suspension was spread on LB agar plates containing 1% casein and 5 μg / ml kanamycin and incubated overnight at 37 ° C. to finally isolate 140 halo-forming colonies.

Single colonies were transferred to 3 ml of LB medium containing 5 μg / ml kanamycin and incubated overnight at 37 ° C. The culture was centrifuged at 10000 rpm for 5 minutes and 0.3 ml of saturated ammonium sulfate solution and 0.04 ml of butyl-Toyopearl 650S were added to 1.2 ml of the supernatant. After the mixture was stirred and left at room temperature for 5 minutes, the solution was centrifuged at 10,000 rpm for 1 minute to remove the supernatant, and the precipitate was washed with 8 mM Tris-HCl, 8 mM CaCl ₂ , pH 8.0, 20% saturated ammonium sulfate. ) Into a small pipette-type column. After washing with 0.6 ml wash solution, the enzyme was eluted with 0.2 ml of eluent (10 mM Tris-HCl, 10 mM CaCl ₂ , pH 8.0). The enzyme solution was desalted using gel filtration (Sepadex PD101) to obtain 0.5 mL of the finally purified enzyme solution.

Enzyme concentrations were determined by dye binding quantitation (Bradford, MM, (1976) Anal. Biochem., 72, 248-254), and Z-APM digestive activity was measured to screen for denatured enzymes in the manner described below. .

1 ml of substrate solution (10 mM of Tris-maleate, 10 mM CaCl ₂ , 10 mM Z-APM, pH 8.0) was mixed in a 0.05 ml enzyme solution and incubated at 37 ° C. for 20 minutes. After that, 0.5 ml of a stop solution (0.1 M acetic acid) was added, and 1 ml of ninhydrin solution was further added. After incubation at 100 ° C. for 15 minutes, 50% ethanol 2 ° C. was added and the absorbance was measured at 570 nm. Specific activity, defined as the change in absorbance per mg enzyme protein, is shown as a comparative value compared to that of the wild type enzyme.

Two colonies with high activity on Z-APM digestion were obtained.

It should be noted that these activity values are measured by the rough method. Plasmid DNA was extracted from these two clones and nucleotide sequences of these clones were determined. From the nucleotide sequence, we found that the two clones were identical and that the 38th glycine residue and the 144th leucine residue were substituted with glutamine and serine, respectively. This clone is referred to below as G-38Q-L144S.

1 μg of each of the plasmids pUBTZ2 (G38Q-L144S) and pUBTZ2 (wild type) was incubated for 2 hours at 37 ° C in 20 µl of a reaction mixture containing 5 units of HinIII (pH7.5, 50 mM, 10 mM MgCl ₂ , 1 mM DTT). did. These samples were electrophoresed on 1% agar gel to separate about 1.1 kb DNA fragments and about 7 kb DNA fragments, respectively, and the two fragments were purified using a Bio-101 gene clean DNA purification apparatus.

About 7 kb DNA fragments derived from pUBTZ2 (G38Q-L144S) and about 1.1 kb DNA fragments derived from pUBTZ2 (wild type) were screened using a Takara Shuzo DNA Ligation Apparatus, and each ligation mixture was subjected to E. Coli JM by conventional methods. 103 was used for transformation. Transformants were screened by rapid DNA isolation and DNA sequencing to isolate pUBTZ2 (L144S) clones.

Single colonies of transformants Bacillus subtilis MT-2 / pUBTZ2 (G38Q-L144S) and MT-2 / pUBTZ2 (L144S) were transferred to 5 ml of LB medium containing kanamycin (5 μg / ml) and incubated overnight at 37 ° C. It was. The culture was transferred to 500 ml of 2 L medium (2% bacto tryptone, 1% yeast extract, 0.5% NaCl) containing kanamycin (5 μg / ml) and incubated at 37 ° C. for 20 hours. To remove the bacteria, broth broth was centrifuged at 800 rpm for 30 minutes, and ammonium sulfate was added to the supernatant to obtain 60% saturated solution, and the mixture was stirred at 4 ° C. overnight. The precipitate was recovered by centrifugation and dissolved in 10 ml of Buffer A (20 mM Tris-HCl, 10 mM CaCl ₂ , pH 9.0). The solution was placed in 20 ml of butyl-toyopearl and buffer A was eluted at a flow rate of 1.5 ml / min. The active moiety is bound and salted with 60% saturated ammonium sulfate. The precipitate was collected by centrifugation at 15,000 rpm for 30 minutes and dissolved in 5 ml of Buffer B (20 mM Tris-HCl, 10 mM CaCl ₂ , pH 7.5). The enzyme solution was placed in a gel-filtration column (TSK gel G2000 SW 21.5 x 300 mm), and buffer B was eluted at a flow rate of 1 ml / min. The active moieties are combined to produce purified enzymes.

Example 3

[Synthesis of Mutant Thermolysine-Like Neutral Metallo-Protease Mutated to 150th Amino Acid Residue]

Oligonucleotides used for mutagenesis were described in Applied Biosystems Co. Synthesis was carried out using DNA synthesizer model 380B provided by LTD. The nucleotide sequence of the mutagenesis primer is as follows.

In addition, reverse primers were prepared having the following nucleotide sequences.

Tris-HCl 67 mM, 16.6 mM ammonium sulfate, 6.7 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 0.05 mM daTP, 0.05 mM dTTp, 0.05 mM dGTP, o.05 mM dCTP with 1 ng of plasmid pUBTZ2 for PCR , 1 μM mutagenesis primer, 1 μM reverse primer) were dissolved and 100 units of Tth DNA polymerase was added. The liquid level of this mixture was covered with 1 mineral oil. The process of denaturation at 90 ° C. for 1 minute, annealing at 45 ° C. for 1 minute, and extension at 72 ° C. for 45 seconds was repeated 30 times. After the reaction, the aqueous layer was collected, extracted with phenol, and ethanol-treated to recover the amplified DNA.

20 µl of the reaction mixture containing half the amount of DNA (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 100 mM NaCl, 1 mM DTT) was digested with 5 units of Sph I and Aat I at 37 ° C. for 2 hours. Incubated at 70 ° C. for 5 minutes. The denatured Sph I-Aat I fragment is ligated to Sph I-Aat I fragment (7.6 kb) of pUBTZ2 using a Takara Shuzo DNA ligation apparatus, and this ligation mixture is used to transform Escherichia coli JM103 by conventional methods. Transformant JM103 / pUBTZ2 (mutant) was formed. Substituted amino acids were identified by determining the nucleotide sequence of these plasmids.

A single colony of the transformant Bacillus subtilis MT-2 / pUBTZ2 (mutant) was transferred to 5 ml of LB medium containing kanamycin (5 μg / ml) and incubated overnight at 37 ° C. The culture solution was transferred to 500 ml of 2 L medium (2% bacto tryptone, 1% yeast extract, 0.5NaCl) containing kanamycin (5 µg / ml) and incubated at 37 ° C for 20 hours. The broth broth was centrifuged at 8000 rpm for 30 minutes to remove bacteria, ammonium sulfate was added to the supernatant to obtain 60% saturated solution and the mixture was stirred at 4 ° C. overnight.

The precipitate was recovered by centrifugation and dissolved in 10 ml of Buffer A (20 mM Tris-HCl, 10 mM CaCl ₂ , pH 9.0.) The solution was poured into 20 ml butyl-toyopearl and eluted with Buffer A at a flow rate of 1.5 ml / min. The active portion was bound and basified with 60% saturated ammonium sulfate, centrifuged at 15,000 rpm for 30 minutes to collect the precipitate and dissolved in 5 ml of Buffer B (tris-HCl 20 mM, 10 mM CaCl ₂ , pH 7.5). The solution was placed in a gel-filtration column (TSK Gel G2000 SW 21.5 x 600 mm) and eluted with Buffer B at a flow rate of 1 mL / min. The active moieties were combined to obtain purified enzyme.

Example 4

[Synthesis of Mutant Thermolysine-Like Neutral Metallo-Protease Mutated to Amino Acid Residue 144, 150, 227]

The three position mutants of thermolysine-like neutral metallo-protease were prepared as follows. Plasmid pUBTZ2 (N227H), representing a plasmid in which the codon of the 227th amino acid residue was mutated from asparagine to histidine, was used as a template for the polymerase chain reaction. Mutagenesis and reverse primers are the same as in Example 3.

Mutation Generating Primers:

Reverse primer:

1 ng of plasmid pUBTZ2 (N227H) was added to the reaction mixture for PCR (pH 8.8 Tris-HCl 67 mM, 16.6 mM ammonium sulfate, 6.7 mM MgCl ₂ , 10 mM 2-mercaptoethanol, 0.05 mM dATP, 0.05 mM dTTP, 0.05 mM dGTP, 0.05 100 μL of mM dCTP, 1 μM mutagenesis primer, and 1 μM reverse primer) were digested and 1 unit of Tth DNA polymerase was added. The liquid level of the solution was covered with 1 mineral oil. The procedure of denaturation at 93 ° C. for 1 minute, annealing at 45 ° C. for 1 minute, and extension at 72 ° C. for 45 seconds was repeated 30 times. After the reaction, the aqueous layer was recovered, extracted with phenol and precipitated with ethanol to recover amplified DNA (D150N-N227H (Example 4A) and D150H-N227H (Example 4B)).

20 μl of the reaction mixture containing half the amount of DNA (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ 100 mM NaCl, 1 mM DTT) was digested in 5 units of Sph I and Art I for 2 hours at 37 ° C. for 70 hours. Treatment was carried out at 5 ° C. The mutated Sph I-Aat I fragment was ligated to 7.6 kb Sph I-Aat I fragment of pUBTZ2 (L144S) using a Takara Shuzo DNA Ligation Apparatus, and the ligation mixture was transfected with E. Coli JM 103 by conventional methods. The transformant was used to obtain transformant JM 103 / pUBTZ2 (mutant). Substituted amino acids are identified by nucleotide sequence determination of these plasmids.

A single colony of transformant Bacillus subtilis MT-2 / pUBTZ2 (mutant) was incubated overnight at 37 ° C. in 5 ml of LB medium containing kanamycin (5 μg / ml). The culture was transferred to 500 ml of 2 L medium (2% bacto tryptone, 1% yeast extract, 0.5% NaCl) containing kanamycin (5 µg / ml) and incubated at 37 ° C for 20 hours. The broth broth was centrifuged at 8000 rpm for 30 minutes to remove bacteria, ammonium sulfate was added to the supernatant to give 60% saturated solution, and the mixture was stirred at 4 ° C. overnight.

The precipitate was recovered by centrifugation and dissolved in 10 ml of buffer (20 mM Tris-HCl, pH 10 CaM ₂ ), pH 9.0. The solution was placed in 20 mL butyl-toyopearl and eluted with the same buffer at a flow rate of 1.5 mL / min. The active moiety was combined and salted with 60% saturated ammonium sulfate. The precipitate was collected by centrifugation at 15,000 rpm for 30 minutes and dissolved in 5 ml of buffer (Tris-HCl 20 mM, 10 mM CaCl ₂ , pH 7.5). The enzyme solution was filtered through a gel-filtration column (TSK Cell G2000 SW 21.5 x 600 mm) and eluted with the same buffer at a flow rate of 1 ml / min. The active moieties were combined to obtain each purified enzyme [L144S-D150N-N227H (Example 4A), L144S-D150H-N227H (Example 5B)].

Two recombinant Bacillus subtilis strains expressing a modified thermolysine-like neutral metallo-protease according to the present invention, the 144th leucine residue is substituted with serine, the 150th aspartic acid residue is histidine, and the 227th asparagine residue is Bacillus subtilis MT-2 / pUBTZ2 (TZ-1) substituted with histidine, Bacillus subtilis MT with 1441 leucine residues serine, 150th aspartic acid residues asparagine, 227th, and asparagine residues histidine -2 / pUBTZ2 (TZ-2) are each a deposit number Nos. FERM BP-4112 and FERM BP-4113 have been deposited with Fermentation Research Research Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 1-3, Higashi 1-chome, Ibarakinken, Japan.

Figure 9 shows the scheme used to prepare recombinant plasmid pUBTZ2 (mutants) (L144S-D150N-N227H or L144S-D150H-N227H).

Example 5

Measurement of Casein Digestion and Z-APM Digestion]

Various modified enzymes were used to measure casein digestion and Z-APM digestion activity, which are as follows:

N227H, an enzyme containing histidine at the 227th amino acid residue (originally asparagine),

N227K, an enzyme containing lysine at the 227th amino acid residue (originally asparagine),

N227R, an enzyme containing arginine at the 227th amino acid residue (originally asparagine),

L144S, a marker containing serine at the 114th amino acid residue (original leucine),

E187Q, an enzyme containing glutamine at the 187th amino acid residue (original glutamic acid),

D150N, an enzyme containing asparagine at the 150th amino acid residue (originally aspartic acid),

D150H, an enzyme containing histidine at the 150th amino acid residue (originally aspartic acid),

L144S-D150N-N227H, an enzyme comprising serine at the 144th residue (original leucine), asparagine at the 150th residue (original aspartic acid), and hydridine at the 227th amino acid residue (original asparagine),

L144S-D150H-N227H, an enzyme comprising serine at the 144th residue (original leucine), histidine at the 150th residue (original aspartic acid), and thytidine at the 227th amino acid residue (original asparagine).

As a casein digestion method, 1 ml of each purified enzyme solution (prepared by dissolving the enzyme in a borate buffer of pH 8.0 and 10 mM of calcium sulfate) 1 ml of substrate solution (1% casein 1 / 15M, phosphate buffer of pH 7.2) Put in. After incubating the mixture for 10 minutes at 35 ° C., 2.0 ml of a stop solution (0.1 M TCA, 0.2 M sodium acetate, 0.3 M acetic acid) was added, and 1 ml of a poly solvent) was diluted 5 times with distilled water at 35 ° C.). It was added to 1 ml of the filtrate and incubated for 20 minutes at 35 degreeC. The absorbance of the mixture was measured at 660 nm. One unit (PU) of enzyme is derived from milk casein, a digestible amount capable of producing a polycolor (determined by an increase in absorbance at 660 nm), such as 1 μg of tyrosine per minute, at an initial stage during the reaction at 35 ° C., pH 7.2. Inactivity (PU / mg) was compared with wild type enzyme and the comparison is shown in Table 1.

Z-APM digestion was added to 0.5 ml of each purified enzyme solution (10 mM of Tris-maleic acid buffer, pH 8.0, 10 mM CaC ₂ , 10 mM Z-APM). After incubating the mixture at 37 ° C. for 20 minutes, 0.5 ml of a stop solution (0.1 M acetic acid) was added, and 1 ml of ninhydrin solution was further added.

After 15 minutes of treatment at 100 ° C, 2 ml of 50% ethanol was added and the absorbance was measured at 570 nm. Inactivation, defined as the change in absorbance per mg of enzyme, is shown as a comparison compared with that of wild-type urea and is shown in Table 1.

Example 6

[Activity for Z-APM Synthesis]

Various modified enzymes were used to determine Z-APM synthesis activity, these enzymes are as follows:

N227H, an enzyme containing histidine at the 227th amino acid residue (originally asparagine),

L144S, an enzyme containing serine at the 144th amino acid residue (original leucine),

150N, an enzyme containing asparagine at the 150th amino acid residue (originally aspartic acid),

D150H, an enzyme containing histidine at the 150th amino acid residue (originally aspartic acid),

L144S-D150N-N227H, an enzyme comprising serine at the 144th residue (original leucine), asparagine at the 150th residue (original aspartic acid), and histidine at the 227th amino acid residue (original asparagine),

L144S-D150H-N227H, an enzyme comprising serine at the 144th residue (original leucine), histidine at the 150th residue (original aspartic acid), and histidine at the 227th amino acid residue (original asparagine).

0.1 ml of each purified enzyme solution (1 mg / ml) was added to the substrate solution (0.1 M benzyloxycarbonyl-alpha-L-aspartic acid, 0.1 ml-phenylalanine methyl ester hydrochloride, 0.2 M Tris-maric acid buffer at pH 7.0). 1 ml, and the mixture was mixed and then subjected to an enzyme reaction at 37 ° C (water bath). After 10 minutes, 20 minutes and 30 minutes each 10 μl of the reaction mixture was taken as a sample and analyzed by HPLC on a reversed phase column. Using the standard sample of Z-APM as standard, the aroma of Z-APM produced by the enzymatic reaction was calculated, and the inactivation per mg of enzyme was shown as a comparison comparing with the wild type enzyme case and is shown in Table 2.

Example 7

[Influence of pH on Z-APM Synthesis of Mutant Enzyme]

The effect of pH on the synthesis of Z-APM was influenced by both mutant enzymes (L144S-D150H-N227H (TZ-1), L144S-D150H-N227H (TZ-2)) and wild type thermolysine-like neutral metallo-protease. Was measured. Except for pH (pH5.0 ~ 8.0) of Tris-maleic acid buffer solution was measured in the same manner as in Example 6, the results are shown in Table 3. Activity is shown as a comparison for wild type enzyme at pH 7.0. Z-APM synthesis activity was shown to be significantly affected by the pH of the reaction mixture.

Because lower hydrolysis of the methyl ester of the substrate occurs at lower pH, lower pH results in better Z-APM synthesis. The present invention exhibits high enzymatic activity at lower pH than wild type enzyme at its optimum pH. The activity of L144S-D150H-N227H (TZ-1) and L144S-D150H-N227H (TZ-2) at pH 6.0 is much greater than that of wild type enzyme at pH 7.0.

Although the present invention has been described with reference to specific embodiments, those skilled in the art can make various changes without departing from the spirit and scope of the present invention.

Claims (6)

In a modified protease of thermolysine-like neutral metallo-protease having an amino acid sequence of SEQ ID NO: 1, only one of the following methods for substituting amino acid residues is performed, i.e., the 144th leucine residue is substituted with a serine residue. Or the 150th aspartic acid residue is substituted with a sasparagine residue or a histidine residue, the 187th glutamic acid residue is replaced with a glutamine residue, or the 227th asparagine residue is replaced with a histidine residue or an arginine residue. Protease. The method of claim 1, wherein only one of the following two-digit substitution methods of the amino acid residues is performed, that is, the 144th leucine residue is substituted with serine and the 150th aspartic acid residue is substituted with an asparagine residue or histidine residue, or 144 A modified protease, wherein the second leucine residue is substituted with a serine residue and the 227th asparagine residue is substituted with hydridine. 2. The method of claim 1, wherein only one of the following two-digit substitutions of the two amino acid residues is performed, i.e., the 227th asparagine residue is replaced with a histidine residue and the 150th aspartic acid residue is substituted with an asparagine or histidine residue. A modified protease, characterized in that. 3. The method of claim 2, wherein only one of the following three-digit substitutions of amino acid residues is performed: the 144th leucine residue is substituted with a serine residue and the 150th aspartic acid residue is substituted with a histidine residue and the 227th asparagine The residues are each substituted with a histidine residue, or the 144th leucine residue is substituted with a serine residue, the 150th aspartic acid residue is replaced with an asparagine residue and the 227th asparagine residue is substituted with a histidine residue, respectively. Protease. Consisting of contacting the modified protease according to any one of claims 1 to 4 with a substrate solution containing benzyloxycarbonyl-alpha-L-aspartic acid and L- or D, L-phenylalanine methyl ester. A method for synthesizing benzyloxycarbonyl-alpha-L-aspartyl-L-phenylalanine methyl ester. Benzyl, characterized in that it comprises contacting the modified protease according to any one of claims 1 to 4 with a substrate solution containing benzyloxycamonyl-alpha-L-aspartyl-L-phenylalanine methyl ester. A method of digesting oxycarbonyl-alpha-L-aspartyl-L-phenylalanine methyl ester.