JP2008029270A - Mutant type protease and method for synthesizing peptide using the protease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protease having an excellent peptide synthetic activity without substantially requiring periodic cleaning of a reaction vessel. <P>SOLUTION: The mutant type protease has a specific amino acid sequence in which the 114th amino acid residue in the amino acid sequence is substituted with phenylalanine. Furthermore, in the mutant type protease, one or several amino acids other than the 114th amino acid residue are deleted, substituted or added. A method for synthesizing the mutant type protease comprises a step of bringing the mutant type protease into contact with a substrate solution containing one or more kinds selected from an amino acid or a derivative thereof. The method for synthesizing benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester comprises a step of bringing the mutant type protease into contact with the substrate solution containing benzyloxycarbonyl-α-L-aspartic acid and L-phenylalanine methyl ester. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a mutant protease. The present invention also relates to a peptide synthesis method using the protease. In particular, the present invention relates to a method for synthesizing benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester (hereinafter also referred to as “Z-Asp-PheOMe”), which is an aspartame precursor.

  Thermolysin is known as an industrially useful protease. Thermolysin is used in the fields of detergents, food production, cosmetics, and the like, and is particularly used for the synthesis of aspartame precursor Z-Asp-PheOMe, which is a kind of artificial sweetener (see Patent Document 1).

  Thermolysin is an excellent enzyme, but since there are four calcium ions per molecule, the presence of calcium ions in the reaction system is important in order to carry out the enzyme reaction stably (see Non-Patent Document 1). ).

  Therefore, normally, when performing a peptide synthesis reaction using thermolysin, an excessive amount of calcium ions is added to the reaction solution (see Non-Patent Document 2). In particular, when aspartame precursors are produced continuously using thermolysin, the calcium ions of thermolysin are removed by the chelating action of N-benzyloxycarbonylaspartic acid (Z-Asp) as the substrate, so the reaction solution It is necessary to continue to add calcium ions to (see Non-Patent Documents 3 and 4).

  On the other hand, it has been suggested that calcium ions form a sparingly soluble salt with Z-Asp and the like, causing scaling of the equipment (see Non-Patent Document 5). Therefore, when the reaction is performed using thermolysin, it is necessary to periodically wash the reaction vessel.

  On the other hand, as an enzyme useful for peptide synthesis, PST-01 protease produced by the microorganism Pseudomonas aeruginosa PST-01 strain is known (see Non-Patent Document 6).

  The PST-01 protease gene has already been cloned and the nucleotide sequence has been clarified (see Non-Patent Document 7).

  PST-01 protease contains one calcium ion per molecule, and the calcium ion does not easily leave, so it is not particularly necessary to add calcium ion. Therefore, it is substantially unnecessary to clean the reaction vessel as in the case of using thermolysin.

However, the reaction rate of the Z-Asp-PheOMe synthesis reaction using PST-01 protease is slower than that using thermolysin.
Japanese Patent Laid-Open No. 6-181761 J. Feder, LR Garrett, and BS Wildi, Role of calcium in thermolysin, Biochemistry 10 (24), 4552-4556 (1971) JS Fruton, Proteinases as catalysts of peptide bond synthesis, Transactions of the New York Academy of Sciences 41, 49-56 (1983) K. Nakanishi and R. Matsuno, Recent developments in the synthetic synthesis of peptides, Advances in Biotechnological Processes 10, 173-202 (1988) Kazuhiro Nakanishi, Synthesis of oligopeptides using water-organic solvent bilayer system, Bioscience and Industry, 47 (8), 19-26 (1989)) : Takeshi Nakai, supervised by Takehisa Ohashi, Chiral Technology, SMC Publishing, January 31, 1998, pages 65-67 H. Ogino, M. Yamada, F. Watanabe, H. Ichinose, M. Yasuda, and H. Ishikawa, Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems, Journal of Bioscience and Bioengineering, 88 (5), 513-518 (1999) H. Ogino, J. Yokoo, F. Watanabe, and H. Ishikawa, Cloning and sequencing of a gene of organic solvent-stable protease secreted from Pseudomonas aeruginosa PST-01 and its expression in Escherichia coli, Biochemical Engineering Journal, 5 (3 ), 191-200 (2000)

  The main object of the present invention is to provide a novel protease having excellent peptide synthesis activity and substantially eliminating the need for periodic washing of a reaction vessel, and a peptide synthesis method using the protease. .

  As a result of repeated studies with the main purpose of solving the above-mentioned problems, the present invention has found that a mutant of PST-01 protease has excellent peptide synthesis activity, and has been completed through further studies. It was.

  That is, the present invention relates to the following mutant protease, a peptide synthesis method using the protease, and a Z-Asp-PheOMe synthesis method.

  Item 1: A mutant protease having the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein tyrosine, the 114th amino acid residue in the amino acid sequence, is substituted with phenylalanine.

  In other words, a mutant PST-01 protease having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing.

  Item 2: The mutant protease according to Item 1, wherein one or several amino acids other than position 114 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing are further deleted, substituted, or added.

  In other words, a mutant PST-01 protease in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing.

  Preferably, the mutant protease according to Item 1, wherein one or more amino acids selected from the 132nd, 142nd, 148th and 197th positions in the amino acid sequence of SEQ ID NO: 1 in the sequence listing are further substituted.

  Item 3: A method for synthesizing a peptide, comprising the step of bringing the mutant protease according to Item 1 or 2 into contact with a substrate solution containing one or more selected from amino acids and derivatives thereof.

Item 4: A method for synthesizing benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester,
Item 3. A method comprising the step of contacting the mutant protease according to Item 1 or 2 with a substrate solution containing benzyloxycarbonyl-α-L-aspartic acid and L-phenylalanine methyl ester.

  Hereinafter, the present invention will be described in detail.

1. Mutant PST-01 Protease The protease of the present invention has an amino acid sequence in which the 114th tyrosine is substituted with phenylalanine in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing of the PST-01 protease.

  In other words, the mutant PST-01 protease of the present invention has the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.

Production method The protease of the present invention is a PST-01 protease gene encoding the amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing, wherein the portion encoding the 114th tyrosine residue in the PST-01 protease amino acid sequence encodes phenylalanine It can be produced using a gene substituted with a base sequence.

  Examples of the PST-01 protease gene include a gene having the base sequence set forth in SEQ ID NO: 3 in the Sequence Listing. For example, PST-01 protease gene derived from Pseudomonas aeruginosa PST-01 strain obtained according to the method disclosed in Non-Patent Document 7 (Biochemical Engineering Journal, 5 (2000) 191-200) is exemplified. be able to.

In addition, the PST-01 protease gene has the nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing according to a conventional method such as the phosphite triester method (Nature, 310, 105 (1984)) or by a DNA synthesizer. It can also be obtained by a method such as synthesizing all or a part.

  In addition, it uses proteases produced from microorganisms with similar properties to Pseudomonas aeruginosa PST-01 strains, and genes encoding proteases in which one or several amino acids have been deleted, substituted, or added in PST-01 protease It is also possible to obtain it.

In the PST-01 protease gene, the portion encoding the 114th tyrosine residue is replaced with a sequence encoding phenylalanine. For example, the method of Kunkel (TA Kunkel, Proc. Natl. Acad. Sci. USA, 82, 488 (1985)), double primer method (P. Carter, H. Bedouelle, G. Winter, Nucleic Acid Res., 13 , 4431 (1985)), a method using thionucleotide (JW Taylor, J. Ott, F. Eckstern, Nucleic Acid Res., 13 , 8764 (1985)), cassette mutagenesis method (JA Wells, et al., Gene, 34 , 315 (1985)), and the like. It is also possible to use a commercially available gene mutation kit.

  An example of a specific replacement method is that a plasmid containing the PST-01 protease gene is used as a template, a pair of mutation primers containing the base to be replaced and a DNA polymerase are used to repeat the extension reaction, and further a restriction enzyme is used. By digesting the template, a plasmid containing the site-specific mutated gene can be obtained, and the PST-01 protease gene into which the target mutation has been introduced can be obtained.

  A mutation primer for substituting phenylalanine for tyrosine, the 114th amino acid from the N-terminal of PST-01 protease, can be appropriately designed according to a conventional method. For example, a primer having the base sequence described in SEQ ID NO: 5 and SEQ ID NO: Primers having the base sequence described in 6 can be used.

  The mutant PST-01 protease is constructed by constructing an expression vector using the PST-01 protease gene into which the mutation has been introduced as described above, and transforming an appropriate host using the vector. It can obtain by collect | recovering the protease produced by.

  The type of expression vector is not particularly limited as long as it can express such a mutated gene. For example, phages, plasmids, cosmids and the like can be used. Preferred vectors include plasmids derived from E. coli and Bacillus subtilis.

  In addition, in order to efficiently express the mutant protease gene, the expression vector may appropriately include a promoter, a selection marker, a replication origin, and the like.

  The construction method of the expression vector can be appropriately performed according to a known method.

  The host to be transformed with the expression vector can be appropriately determined according to the type of the selected expression vector. For example, in the case of a vector derived from E. coli, E. coli can be used as a host. Moreover, if it is a vector derived from Bacillus subtilis, Bacillus subtilis can be used as a host.

  Further, transformation can be appropriately performed according to a known method according to the selected expression vector or host.

  By culturing the obtained transformant in a medium and collecting an enzyme having protease activity from the culture, a mutant protease can be obtained.

  The culture conditions such as the medium and the culture recovery method can be appropriately set according to the type of the host.

  In addition, the culture temperature and the culture time are also determined according to the type of the host. When Escherichia coli is used, the culture temperature is usually 10 to 40 ° C, preferably about 20 to 37 ° C, and the culture time is 1 to 30 hours, preferably about 2 to 12 hours.

The method for recovering the enzyme from the culture obtained by the above culture can be performed according to a conventional method.

  For example, when the host secretes protease outside the cells, the culture is centrifuged to obtain a culture supernatant, and the obtained culture supernatant can be used as a crude enzyme solution.

  Moreover, when it is not secreted outside a microbial cell, a microbial cell is crushed by ultrasonic crushing etc., Centrifugation can be performed and a crude enzyme solution can be obtained.

  A method for isolating and purifying the mutant protease from the crude enzyme solution can also be performed according to a conventional method.

  Specifically, salting out using ammonium sulfate, electrophoresis, affinity chromatography, dialysis, hydrophobic chromatography, or a combination thereof can be used.

  Thus, a mutant PST-01 protease can be obtained by constructing a mutant PST-01 protease gene.

  In addition, for example, in the amino acid sequence of SEQ ID NO: 1, a sequence in which the 114th amino acid is substituted with phenylalanine is synthesized by peptide synthesis using a peptide synthesizer or the like, or by using a cell-free protein synthesis method. Can also be obtained.

Sequence The mutant protease of the present invention has an amino acid sequence in which the amino acid corresponding to the 114th tyrosine is phenylalanine in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, that is, the amino acid sequence represented by SEQ ID NO: 2. .

  In the mutant protease of the present invention, one or several amino acids other than the 114th tyrosine in the amino acid sequence represented by SEQ ID NO. It may be substituted or added.

In other words, the mutant protease of the present invention is
(1) an amino acid sequence in which the amino acid corresponding to the 114th tyrosine is substituted with phenylalanine in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or
(2) In the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, the amino acid corresponding to the 114th tyrosine is substituted with phenylalanine, and one or several amino acids other than the 114th are deleted or substituted Or a protease having an amino acid sequence that has been mutated by addition.

  Examples of the mutation site other than the 114th site include one or more sites selected from the 132nd, 142nd, 148th and 197th sites.

  Specifically, examples of mutations other than the 114th include substitution of the 132nd amino acid with phenylalanine and substitution of the 148th amino acid with asparagine, histidine or tryptophan. These mutations can improve peptide synthesis activity, enzyme stability, and the like.

  Examples also include substitution of the 142nd amino acid with serine and substitution of the 197th amino acid with glycine, alanine, valine, phenylalanine or tyrosine. These mutations can further improve enzyme activity.

Properties The mutant protease of the present invention is excellent in peptide synthesis activity and ester synthesis activity, and can be used as a catalyst for peptide synthesis reaction and ester synthesis reaction.

  In particular, the mutant protease of the present invention has a Z-Asp-PheOMe synthesis activity equivalent to that of thermolysin, and exhibits a reaction rate equivalent to that of thermolysin.

  In addition, the reaction using thermolysin requires the addition of calcium ions, and it is also necessary to periodically wash the reaction vessel. However, when the mutant PST-01 protease of the present invention is used, the reaction system uses calcium ions. Is not necessarily required, and periodic cleaning of the reaction vessel is substantially unnecessary (see FIG. 1).

  In addition, the mutant PST-01 protease of the present invention is excellent in stability in the presence of an organic solvent, and can be used for peptide synthesis reaction and ester synthesis reaction using an organic solvent.

  For example, it is stable in the presence of an organic solvent such as dimethyl sulfoxide, 1,5-pentadiol, ethylene glycol, 1,4-butanediol, glycerol, methanol, ethanol, 2-propanol, dimethylformamide and the like.

  Further, when the protease of the present invention is placed in the presence of a chelate such as ethylenediaminetetraacetic acid, the zinc ion at the active center is removed and becomes an apoenzyme, so that the enzyme activity can be lost or reduced. In addition, the enzyme activity can be restored by adding zinc ions, cobalt ions, or the like to the apoenzyme. Therefore, in the reaction using the protease of the present invention, the progress or termination of the reaction can be controlled by adding a chelating agent or a metal ion. In addition, for example, when the substrate has a chelating action, the enzyme activity can be improved by adding zinc ions or cobalt ions.

2. Peptide Synthesis Method The present invention further provides a peptide synthesis method using the above mutant PST-01 protease.

  The peptide synthesis reaction includes a step of bringing the mutant PST-01 protease into contact with a substrate solution containing one or more selected from amino acids, peptides, or derivatives thereof.

  As the amino acid, those appropriately selected according to the target peptide can be used. For example, the amino acid is selected from the group consisting of alanine, lysine, arginine, threonine, glycine, glutamic acid, phenylalanine, asparagine, aspartic acid, leucine, and tyrosine. One or more can be selected.

  Examples of the peptide include a peptide formed by combining a plurality of the above amino acids.

  Examples of amino acid or peptide derivatives include those obtained by modifying the carboxyl group or amino group of the amino acid or peptide with a protecting group.

  Examples of the protecting group for the carboxyl group include methyl ester, ethyl ester, amino, tert-butyl ester, hydrazino, anilino, diphenylmethyl ester and the like.

  Examples of amino-protecting groups include acetyl, t-butyloxycarbonyl, benzoyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl and the like.

  In particular, the mutant PST-01 protease in the present invention is suitable for the synthesis of aspartame precursors using benzyloxycarbonyl-α-L-aspartic acid and L-phenylalanine methyl ester as substrates.

  The concentration of the mutant PST-01 protease in the reaction solution can be appropriately set depending on the type of substrate and the like, but is usually about 1 μg / mL to 1 g / mL, preferably about 10 μg / mL to 10 mg / mL.

  The concentration of the substrate in the reaction solution can be appropriately set depending on the type of the substrate, but is usually about 1 μM to 1 M, preferably about 1 mM to 1 M.

  Although the kind of solvent can be suitably set, for example, water, an organic solvent, a mixed solvent of water and an organic solvent, or the like can be given.

  The ratio of water to the organic solvent is usually about water: organic solvent = 100: 0 to 0: 100, preferably about 100: 0 to 30:70 in a volume ratio.

  Examples of the organic solvent include dimethyl sulfoxide, 1,5-pentadiol, ethylene glycol, 1,4-butanediol, glycerol, methanol, ethanol, 2-propanol, dimethylformamide and the like.

  In particular, a mixed solvent of one or more organic solvents selected from dimethyl sulfoxide, 1,5-pentadiol, ethylene glycol, 1,4-butanediol, glycerol, methanol, and ethanol, with water, yield and reaction rate. It is preferably used in that it is excellent in.

  The reaction temperature can be appropriately set, but is usually about -20 ° C to 60 ° C, preferably about 25 ° C to 40 ° C. The reaction time can be appropriately set according to the amount of enzyme and the like, but is usually about 1 minute to 1 week, preferably about 1 hour to 2 days.

  Other components can be appropriately added to the reaction system as necessary.

  For example, when the substrate has a strong chelating action, zinc ions, cobalt ions, calcium ions, and the like can be added in small amounts for the purpose of improving enzyme activity and productivity.

  Moreover, a suitable salt and a buffer component can also be mix | blended suitably.

  After the reaction, a method for isolating and purifying the target peptide can be performed according to a known method. For example, crystallization, chromatography, membrane separation, extraction or a combination thereof can be mentioned.

  The peptide synthesis method using the mutant PST-01 protease of the present invention exhibits excellent equilibrium yield and initial reaction rate.

  Furthermore, in the peptide synthesis reaction of the present invention, addition of calcium is not particularly necessary, and periodic cleaning of the reaction vessel is substantially unnecessary (see FIG. 1).

  Furthermore, in the peptide synthesis method of the present invention, an organic solvent can be used as a reaction solvent, and the yield of the peptide can be further improved. In addition, contamination with bacteria and growth of bacteria in the reaction solution can be achieved. It can also be reduced.

3. Method for Synthesizing Z-Asp-PheOMe The present invention further provides a method for synthesizing Z-Asp-PheOMe using the mutant PST-01 protease.

  Z-Asp-PheOMe is synthesized by contacting the mutant PST-01 protease with a substrate solution containing L-phenylalanine methyl ester (L-PheOMe) and N-benzyloxycarbonylaspartic acid (Z-Asp). Process.

  The concentration of the mutant PST-01 protease in the reaction solution is usually about 1 μg / mL to 1 g / mL, preferably about 10 μg / mL to 10 mg / mL.

  The concentration of L-phenylalanine methyl ester (L-PheOMe) in the substrate solution is about 10 mM to 1 M, preferably about 50 mM to 0.7 M.

  The concentration of N-benzyloxycarbonylaspartic acid (Z-Asp) in the substrate solution is about 1 mM to 1 M, preferably about 1 mM to 60 mM.

  The use ratio of L-PheOMe to Z-Asp is about L-PheOMe: Z-Asp = 1: 1 to 1000: 1, preferably about 1: 1 to 100: 1.

  Although the kind of solvent can be suitably set, for example, water, an organic solvent, a mixed solvent of water and an organic solvent, or the like can be given.

  The ratio of water to the organic solvent is usually about water: organic solvent = 100: 0 to 0: 100, preferably about 100: 0 to 30:70 in a volume ratio.

  Examples of the organic solvent include dimethyl sulfoxide, 1,5-pentadiol, ethylene glycol, 1,4-butanediol, glycerol, methanol, ethanol, 2-propanol, dimethylformamide and the like.

  In particular, a mixed solvent of one or more organic solvents selected from dimethyl sulfoxide, 1,5-pentadiol, ethylene glycol, 1,4-butanediol, glycerol, methanol, and ethanol, with water, yield and reaction rate. It is preferably used in that it is excellent in.

  Among them, it is preferable to use a mixed solvent of water and dimethyl sulfoxide (DMSO), and in particular, by volume ratio, water: DMSO = about 80:20 to 30:70, especially about 70:30 to 50:50. A certain mixed solvent is preferable in that a high reaction rate and a high yield can be obtained.

  The reaction temperature can be appropriately set, but is usually about -20 ° C to 60 ° C, preferably about 25 ° C to 40 ° C. The reaction time can be appropriately set according to the amount of enzyme and the like, but is usually about 1 minute to 1 week, preferably about 1 hour to 2 days.

  Other components can be appropriately added to the reaction system as necessary.

  For example, when the substrate has a strong chelating action, a small amount of zinc ion, cobalt ion, or calcium ion can be added for the purpose of improving enzyme activity or productivity.

  Moreover, a suitable salt and a buffer component can also be mix | blended suitably.

  After the reaction, the method for isolating and purifying Z-Asp-PheOMe can be performed according to a known method. For example, crystallization, chromatography, extraction, or a combination thereof can be mentioned.

  In the Z-Asp-PheOMe synthesis method using the mutant PST-01 protease of the present invention, the same equilibrium yield and initial reaction rate as when thermolysin is used can be obtained.

  Furthermore, in the reaction using thermolysin, it is necessary to add calcium, and it is also necessary to periodically wash the reaction vessel. However, in the Z-Asp-PheOMe synthesis reaction of the present invention, addition of calcium is not particularly necessary. Periodic cleaning of the reaction vessel is also substantially unnecessary (see FIG. 1).

  Furthermore, in the Z-Asp-PheOMe synthesis method of the present invention, an organic solvent can be used as a reaction solvent, and the yield of Z-Asp-PheOMe can be further improved. It is also possible to reduce the growth of various germs.

  The present invention provides a mutant PST-01 protease in which the 114th tyrosine of PST-01 protease is substituted with phenylalanine.

  The mutant PST-01 protease of the present invention has excellent peptide synthesis activity, particularly Z-Asp-PheOMe synthesis activity, and has an equilibrium yield and initial reaction rate equivalent to those of thermolysin.

  In addition, the reaction using thermolysin requires the addition of calcium ions, and the reaction vessel must be periodically washed. In the reaction using the mutant PST-01 protease of the present invention, Addition is not particularly necessary, and regular cleaning of the reaction vessel is also substantially unnecessary.

  In addition, the mutant PST-01 protease of the present invention is excellent in stability in the presence of an organic solvent, and can be used for peptide synthesis reaction and ester synthesis reaction using an organic solvent to obtain a high synthesis yield. Can do.

  The present invention also provides a peptide synthesis method using a mutant PST-01 protease and a Z-Asp-PheOMe synthesis method. The synthesis method of the present invention exhibits an excellent reaction rate and equilibrium yield. In particular, in the Z-Asp-PheOMe synthesis method, the same equilibrium yield and initial reaction rate as when thermolysin is used can be obtained. Furthermore, in the synthesis method of the present invention, addition of calcium is not always necessary, and periodic cleaning of the reaction vessel is substantially unnecessary. In addition, the synthesis method of the present invention can use an organic solvent, and can further improve the yield. Moreover, contamination of germs and growth of germs in the reaction solution can be reduced.

  Thus, the present invention provides a novel protease useful for peptide synthesis and provides an efficient peptide synthesis method.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, a comparative example, etc., this invention is not limited to these Examples.

1. Construction of plasmid pPC1 Plasmid pPC1 was transformed into H. Ogino, J. Yokoo, F. Watanabe, and H. Ishikawa, Cloning and sequencing of a gene of organic solvent-stable protease secreted from Pseudomonas aeruginosa PST-01 and its expression in Escherichia coli, It was prepared according to the method described in Biochemical Engineering Journal, 5 (3), 191-200 (2000).

  Specifically, the chromosomal DNA of Peudomonas aeruginosa PST-01 was partially digested with Sau3A I and inserted into the BamH I site of pUC19, and then Escherichia coli JM109 was transformed. PPC1 was obtained by extracting a plasmid from a transformant imparted with protease activity. PPC1 is deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center under the deposit number FERM P-20964 (the deposit date is July 18, 2006).

  The analysis result of the entire base sequence of pPC1 is shown in SEQ ID NO: 4 in the sequence listing.

2. Introduction of site-specific mutation
Use pPC1 as a template, incubate at 95 ° C for 1 minute using a pair of mutant primers containing the base to be substituted and DNA polymerase, then denaturate at 95 ° C for 50 seconds, anneal at 60 ° C for 50 seconds, and at 68 ° C. The extension reaction for 5.2 minutes was repeated 18 times. Furthermore, after incubation at 68 ° C. for 7 minutes, the template was digested with Dpn I to construct a plasmid into which a site-specific mutation was introduced.

  When tyrosine, the 114th amino acid from the N-terminal of PST-01 protease, is substituted with phenylalanine, the mutation primer is a base sequence represented by SEQ ID NO: 5 in the sequence listing, that is, 5'-GCA GCG TGG AGA ACG CCT tCT GGG ACG GCA CGG CG-3 ′ and the nucleotide sequence represented by SEQ ID NO: 6 in the sequence listing, that is, 5′-CGC CGT GCC GTC CCA GaA GGC GTT CTC CAC GCT GC-3 ′ were used. (Lower case letters indicate mutation sites).

  When isoleucine which is the 190th amino acid is substituted with valine, the mutation primer is a base sequence represented by SEQ ID NO: 7 in the sequence listing, that is, 5'-CCT GAT CGG CTA CGA CgT CAA GAA GGG CAG CGG -3 ′ and the nucleotide sequence represented by SEQ ID NO: 8 in the sequence listing, that is, 5′-CCG CTG CCC TTC TTG AcG TCG TAG CCG ATC AGG-3 ′ were used (lower case letters indicate mutation sites).

  PST-01-Y114F protease in which tyrosine, the 114th amino acid of PST-01 protease, is substituted with phenylalanine, and PST-01-I190V, in which isoleucine, the 190th amino acid is replaced with valine, by mutagenesis using pPC1 as a template A protease and a gene for PST-01-Y114F-I190V protease in which the 114th and 190th amino acids were substituted with phenylalanine and valine, respectively, were constructed.

3. Preparation of crude enzyme solution
LB medium containing 50 mg / L ampicillin of Escherichia coli JM109 transformed with a plasmid containing a mutant PST-01 protease gene prepared by mutagenesis using pPC1 containing pST-01 protease gene or pPC1 as a template ( 1.0% (w / v) Tryptone, 0.5% (w / v) Yeast extract, 1.0% (w / v) NaCl, pH 7.0) was used for shaking culture at 37 ° C. and 150 rpm for 16 hours. The culture broth was centrifuged at 9,840 × g for 5 minutes at 4 ° C., and the precipitated cells were collected. The cells were suspended in 200 ml of 10 mM Tris-HCl buffer solution (pH 8.0) and then disrupted by irradiating with ultrasonic waves while cooling with ice. Further, the mixture was centrifuged at 19,400 × g for 20 minutes at 4 ° C., and the centrifuged supernatant was recovered as a crude enzyme solution.

4). Purification of enzyme Ammonium sulfate (ammonium sulfate) was added to the crude enzyme solution to a saturation of 40%. To the supernatant obtained by centrifugation at 11,900 × g for 10 minutes at 4 ° C., further ammonium sulfate was added to 75% saturation, and the precipitate obtained by centrifugation under the same conditions was recovered. The precipitate was dissolved in 10 mM Tris-HCl buffer solution (pH 8.0) containing 1 M ammonium sulfate and adsorbed on a Butyl Toyopearl 650M column equilibrated with 10 mM Tris-HCl buffer solution (pH 8.0) containing 1 M ammonium sulfate. It was. The adsorbed enzyme uses 10 mM Tris-HCl buffer solution (pH 8.0) containing 1 M ammonium sulfate and 10 mM Tris-HCl buffer solution (pH 8.0) containing no ammonium sulfate. And eluted. Fractions having protease activity were collected. Again, chromatography was performed using a Butyl Toyopearl 650M column in the same manner, and a fraction having protease activity was recovered to obtain a purified enzyme. The purified enzyme was concentrated by adding ammonium sulfate to 75% saturation and precipitation.

5. Enzymatic hydrolysis activity
Pre-heat 5 ml of 50 mM sodium tetraborate-HCl buffer solution (pH 8.5) containing 0.6% (w / v) casein (Hammarsten1 Grade manufactured by ICN Pharmaceuticals, Inc.) at 30 ° C for 5 minutes, and then enzyme solution 100 μl was added, mixed well, and reacted at 30 ° C. 10 minutes later, add 1 ml of TCA solution (5.44% (w / v) trichloroacetic acid, 6.6% (w / v) acetic acid and 5.46% (w / v) sodium acetate aqueous solution) and leave at 4 ° C for 30 minutes or more Then, it was filtered with a filter paper (Toyo Filter Paper No. 5C). The concentration of the degraded protein was measured by measuring the absorbance of the filtrate at a wavelength of 280 nm. As a blank for the reaction, a reaction was immediately stopped with a TCA solution after the enzyme solution was added. As a standard, tyrosine was used, and the amount of protein that decomposes in 1 minute was defined as 1 unit of enzyme activity that solubilized casein corresponding to the absorbance of 1 μmol of tyrosine.

6. Preparation of substrate L-phenylalanine methyl ester hydrochloride manufactured by Tokyo Chemical Industry Co., Ltd. was dissolved in water, neutralized by adding equimolar sodium carbonate, and L-phenylalanine methyl ester was extracted with chloroform. Chloroform was removed under reduced pressure to obtain L-phenylalanine methyl ester (L-PheOMe).

  N-benzyloxycarbonylaspartic acid (Z-Asp) was purchased from Kanto Chemical.

7. Aspartame precursor synthesis reaction and analysis conditions
30 mM Z-Asp, 500 mM L-PheOMe and 30 U / mL (about 1.5 mg / mL) enzyme were prepared, and the volume ratio of water and dimethyl sulfoxide (DMSO) was adjusted to 50:50. A 50 mM Tris-HCl buffer solution (pH 7.0) was reacted by incubating at 30 ° C.

  After starting the reaction, collect the reaction solution regularly, add 50 mM sodium phosphate buffer solution (pH 2.5) containing 9 times the amount of 60% (v / v) acetonitrile, and cool to -20 ° C. And using a Cosmosil 5C18-AR-II manufactured by Nacalai Tesque, with a mobile phase of 50 mM sodium phosphate buffer solution (pH 2.3) containing 33% (v / v) acetonitrile at a flow rate of 0.8 ml / min The amount of substrate and product was analyzed by analyzing with a liquid chromatograph.

8. Synthesis results of aspartame precursor (Z-Asp-PheOMe) using various enzymes
Synthesis of Z-Asp-PheOMe in the presence of 50% DMSO using PST-01 protease wild-type and various mutant enzymes and thermolysin (Thermolysin from Bacillus thermoproteolyticus rokko (Protease Type X) manufactured by Sigma-Aldrich Japan KK) Went.

  The amount of Z-Asp-PheOMe produced was measured with a high performance liquid chromatograph (LC-10A manufactured by Shimadzu Corporation).

  The reaction solution up to about 3 hours from the start of the reaction was sampled periodically, and the initial reaction rate was determined by measuring the amount of product contained in the sample.

  In addition, 6 days and 7 days after the start of the reaction, the amount of product contained in the reaction solution was measured, and it was confirmed that there was no difference, and the equilibrium yield was obtained.

  The initial reaction rate and the equilibrium yield are shown in FIG.

  The equilibrium yields ranged from about 71 to 84%, and the equilibrium yields of wild type PST-01 protease, PST-01-Y114F protease, PST-01-Y114F-I190V protease, and thermolysin were almost the same.

  On the other hand, the initial reaction rate of wild-type PST-01 protease and PST-01-I190V protease was less than half that of thermolysin, but PST-01-Y114F protease and PST-01-Y114F-I190V protease were the same as thermolysin. The initial reaction rate of was shown.

1 is a drawing comparing the outline of a reaction process using thermolysin and a reaction process using a mutant PST-01 protease of the present invention. The initial reaction rate and equilibrium yield of the Z-Asp-PheOMe synthesis reaction in the presence of 50% DMSO using the wild-type PST-01 protease, various mutant enzymes, and thermolysin were performed in the examples. It is drawing which shows the result of a rate.

Claims (4)

A variant of a protease having the amino acid sequence of SEQ ID NO: 1 in the sequence listing, wherein tyrosine which is the 114th amino acid residue in the amino acid sequence is substituted with phenylalanine. Furthermore, 1 or several amino acids other than the 114th in the amino acid sequence of sequence number 1 of a sequence table are deleted, substituted, or added. A method for synthesizing a peptide, comprising the step of bringing the mutant protease according to claim 1 or 2 into contact with a substrate solution containing one or more selected from amino acids, peptides or derivatives thereof. A method for synthesizing benzyloxycarbonyl-α-L-aspartyl-L-phenylalanine methyl ester,
A method comprising a step of contacting the mutant protease according to claim 1 or 2 with a substrate solution containing benzyloxycarbonyl-α-L-aspartic acid and L-phenylalanine methyl ester.

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