JP2009013256A - Novel production method of β-linked polyaspartic acid and β-linked polyaspartic acid produced by the method - Google Patents

Abstract

The present invention provides a method for producing β-bonded polyaspartic acid or a salt or ester thereof with high purity.
A method for synthesizing high-purity β-linked polyaspartic acid or a salt or ester thereof using aspartic acid or a salt or ester thereof as a substrate using polyaspartic acid hydrolase in an organic solvent.
[Selection figure] None

Description

  The present invention relates to a method for synthesizing high-purity β-linked polyaspartic acid or a salt or ester thereof using aspartic acid or a salt or ester thereof as a substrate using polyaspartic acid hydrolase in an organic solvent, and The present invention relates to a highly pure β-linked polyaspartic acid or a salt or ester thereof synthesized by the method.

  Polyaspartic acid (PAA) is a water-soluble polymer, and it has biodegradability and advantageous characteristics such as biocompatibility and bioabsorbability, so it can be used in various industries such as dispersants, surfactants, and pharmaceuticals. Used in the field.

  PAA can be chemically synthesized using thermal polymerization or a phosphoric acid catalyst. In this method, PAA is synthesized by thermal polymerization by hydrolyzing polysuccinimide obtained by polycondensation of aspartic acid.

  A method for enzymatic synthesis of PAA has also been found. In this method, PAA is synthesized by treating aspartic acid ester with an alkaline protease derived from actinomycetes (Patent Document 1).

  These methods can provide PAA easily and in large quantities, but the resulting polymer can be a mixture of D-form and L-form, and a mixture of α-position and β-position bonds. For example, PAA chemically synthesized by thermal polymerization (tPAA) contains α aspartic acid units and β aspartic acid units in a ratio of 3: 7 (FIG. 1), and these units are contained in PAA. It is known by analysis such as NMR that they are present at random, that the D-form and L-form aspartic acid units are present in equimolar amounts, and that they have branch units and irregular chain end groups. In addition, it has been found that poly L-aspartate synthesized using an alkaline protease contains about 90% α-position bond and about 10% β-position bond.

  In view of the fact that the structural unit contained in PAA acts on the PAA structure and can affect functions such as biodegradability, biocompatibility, and chelating ability (Non-patent Document 1), the synthesized PAA is uniform. It is preferable that a structural unit is included.

  However, in the conventional method, it was not possible to obtain only high-purity PAA composed of uniform structural units.

JP 2000-128898 A Nakato T. et al., "Relationship between Structure and Properties of Poly (aspartic acid) s", Macromolecules (1998), Vol. 31, pp.2107-2113

  PAA having only β-position bonds has advantages such as high resistance to degradation, and establishment of a method for obtaining PAA having only β-position bonds with high purity has been desired.

  The present invention relates to a method for synthesizing highly pure β-position PAA (β-PAA) or a salt or ester thereof using aspartic acid or a salt or ester thereof as a substrate using PAA hydrolase in an organic solvent. High-purity β-PAA or a salt or ester thereof synthesized by the method is provided.

  As a result of intensive studies to solve the above problems, the present inventors have isolated two types of PAA hydrolases (Pedobacter sp. KP-2) that recognize the β-position bond in PAA isolated by the present inventors. Β-PAA can be synthesized with high purity by allowing PAA to undergo polymerization using PAA hydrolase-1 derived from sucrose and polyaspartate hydrolase-1 derived from Sphingomonas sp. KT-1) As a result, the present invention has been completed.

That is, the present invention is as follows.
[1] A high-purity β-position bond that polymerizes aspartic acid or a salt or ester thereof using a polyaspartate hydrolase that recognizes between β-β peptide bonds in an organic solvent containing water. A method of synthesizing polyaspartic acid or a salt or ester thereof.
[2] The method of [1], wherein only β-linked polyaspartic acid or a salt or ester thereof is synthesized.
[3] The method according to [1] or [2], wherein the polyaspartate hydrolase that recognizes between β-β peptide bonds is polyaspartate hydrolase-1 derived from Pedobacter sp. KP-2.
[4] The method according to [1] or [2], wherein the polyaspartate hydrolase that recognizes between β-β peptide bonds is polyaspartate hydrolase-1 derived from Sphingomonas sp. KT-1.
[5] The method according to any one of [1] to [4], wherein the enzyme used is modified with an amphiphilic polymer substance.
[6] The method according to [5], wherein the amphiphilic polymer substance is polyethylene glycol.
[7] The method according to any one of [1] to [6], wherein the organic solvent is selected from the group consisting of acetonitrile, toluene, benzene, and THF.
[8] The method according to any one of [1] to [7], wherein the synthesis is performed in an organic solvent containing 0.1 to 10% of water.
[9] The method according to any one of [1] to [8], wherein the polymerization is performed within a temperature range of 4 to 60 ° C.
[10] High-purity β-linked polyaspartic acid, a salt or an ester thereof, produced by the method according to any one of [1] to [9].
[11] The β-position-bound polyaspartic acid according to [10], containing only β-position-bound polyaspartic acid, or a salt or ester thereof.
[12] The β-linked polyaspartic acid or a salt or ester thereof according to [10] or [11], which does not cause racemization.

  By the method of the present invention, it is possible to synthesize substantially β-linked polyaspartic acid or a salt or ester thereof using PAA hydrolase in an organic solvent. Racemization does not occur in the method of the present invention, and the resulting β-linked polyaspartic acid or a salt or ester thereof has high optical purity. The β-linked polyaspartic acid or a salt or ester thereof obtained by the method of the present invention is excellent in functionality and has high resistance to degradation while having biodegradability. Moreover, it is excellent in biocompatibility and is useful as a polymer material for pharmaceuticals, cosmetics and the like.

  β-PAA refers to a peptide bond using a β-position carboxyl group bonded to the β carbon of aspartic acid. That is, β-PAA is composed only of β-aspartic acid units (FIG. 2). According to the present invention, only this β-PAA can be synthesized.

  In the present invention, PAA can be synthesized using PAA hydrolase. PAA hydrolase is a hydrolase that can more selectively catalyze a polymerization reaction between a carboxyl group and an amino group between β-aspartic acid units, that is, a hydrolase that recognizes between β-β peptide bonds. Can be used, but preferably PAA hydrolase-1 derived from Pedobacter sp. KP-2 (FIG. 3A, SEQ ID NO: 1 (registration number AB290350) and SEQ ID NO: 2) and Sphingomonas sp. KT-1 PAA hydrolase-1 (FIG. 3B, SEQ ID NO: 3 (registration number AB112710) and SEQ ID NO: 4) is used. More preferably, PAA hydrolase-1 derived from Pedobacter sp. KP-2 is used.

PAA hydrolase-1 derived from Pedobacter sp. KP-2 and PAA hydrolase-1 derived from Sphingomonas sp. KT-1 were first isolated by the present inventors (Hiraishi T et al., Biomacromolecules 2003, 4, 80). The inventors of the present application have used Pedobacter sp. KP-2 and Sphingomonas sp. KT-1 as microbial cells that decompose α, β-poly (D, L-aspartic acid) (tPAA) obtained by thermal polymerization. And PAA hydrolase-1 was purified from each strain. From previous studies by the present inventors, it has been found that these PAA hydrolase-1s specifically degrade by recognizing amide bonds between β-aspartic acid units in PAA. FIGS. 4 (A) and (B) show the ¹³ C NMR spectra of tPAA and tPAA treated with PAA hydrolase-1 from Pedobacter sp. KP-2, respectively. Before the enzyme treatment, the ¹³ C resonance of the carbonyl carbon of the amide bond is caused between α aspartic acid units (α-α), between α aspartic acid units and β aspartic acid units (α- It is divided into three peaks of β and β-α) and amide bonds between β aspartic acid units (β-β) (FIG. 4A). Treatment with PAA hydrolase-1 derived from Pedobacter sp. KP-2 does not change the peak areas of α-α and α-β + β-α, but decreases the peak area of β-β amide bond (FIG. 4 ( B)). At the same time, a peak of an N-terminal β-aspartic acid unit is newly generated (arrow in FIG. 4 (B)), and from this result, PAA hydrolase-1 derived from Pedobacter sp. Thus, it can be seen that the PAA hydrolase-1 derived from Pedobacter sp. KP-2 and the PAA hydrolase-1 derived from Sphingomonas sp. KT-1 both have a molecular weight of 20000. It has been found that the above tPAA can be decomposed into oligomers having a molecular weight of about 1000. In addition, Pedobacter sp.KP-2 has the ability to decompose high molecular weight tPAA, while Sphingomonas sp. It is known that only 5000 tPAA or less can be decomposed.

  These enzymes can be recombinantly expressed and purified by general methods known in the art, as specifically described in the following examples.

  The purified PAA hydrolase can be immobilized and used. Immobilization is effective in that the enzyme is stabilized and can be used continuously and repeatedly. The immobilization of PAA hydrolase can be performed using a carrier binding method, a crosslinking method, or a comprehensive method. In the carrier binding method, PAA hydrolase is physically adsorbed and ion bound to a carrier (eg, a derivative of a polysaccharide such as cellulose, dextran, agarose, polyacrylamide gel, polystyrene resin, porous glass, metal oxide, etc.). And can be bound using a covalent bond or the like. In the cross-linking method, the enzyme is immobilized by cross-linking each other using a reagent having two or more functional groups. Cross-linking reagents include glutaraldehyde that forms Schiff bases, isocyanic acid derivatives that form peptide bonds, N, N'-ethylenemaleimide, bisdiazobenzines that perform diazo coupling, or N, N'-polymethylene bisiodo that alkylates Acetamide or the like can be used. In the inclusion method, a lattice type in which PAA hydrolase is incorporated in a fine lattice of a polymer gel and a microcapsule type in which PAA hydrolase is coated with a semipermeable polymer film are used. In the lattice type method, a polymer compound such as a synthetic polymer material such as polyacrylamide gel, polyvinyl alcohol, a photocurable resin, and a natural polymer material such as starch, konjac powder, gelatin, alginic acid, and carrageenan can be used. In the microcapsule type method, hexamethylenediamine, sebacoyl chloride, polystyrene, lecithin and the like can be used (Saburo Fukui, Ichiro Chibata, Shuichi Suzuki, “Enzyme Engineering”, published by Tokyo Chemical Dojin, 1981).

  In the present invention, PAA is synthesized by polymerizing a substrate using the reverse reaction of the hydrolysis reaction of PAA hydrolase in an organic solvent.

  PAA hydrolase is modified by binding an amphiphilic polymer. By making the enzyme amphiphilic, the enzyme is easily dissolved in the organic solvent, and the reaction efficiency is increased. Examples of the amphiphilic polymer substance to be bound to the enzyme include polyethylene glycol (PEG), monomethoxy-polyethylene glycol, poly (N-vinylpyrrolidone) polyethylene glycol, etc. Among them, PEG is preferable. The PEG is not limited, but has a molecular weight of 2000 to 10,000, preferably about 5000. An enzyme modified with PEG is sometimes called a PEGylase. Modification of an enzyme using an amphiphilic polymer substance can be performed by a general method in the art, such as an α-amino group at the N-terminal of the enzyme or an ε-amino group of lysine and an aldehyde. A covalent bond may be formed through a reactive group. One molecule of enzyme may be modified with a plurality of molecules of amphiphilic polymer material, but one molecule of enzyme is preferably modified with one molecule of amphiphilic polymer material. Further, after modifying the enzyme, an enzyme modified with one molecule of an amphiphilic polymer substance may be purified and used. Further, the modification rate of the enzyme with an amphiphilic polymer substance is about 60% or more, preferably about 70% or more. The enzyme modification rate can be determined, for example, by performing SDS-PAGE and measuring the band area.

  Examples of the organic solvent to be used include, but are not limited to, acetonitrile, toluene, THF, benzene, 1,1,1-trichloroethane, triglyceride, dimethyl sulfoxide (DMSO), ethanol, and methanol. Preferably, acetonitrile is used.

  Water is added to the reaction system to activate the enzyme. The amount of water added to the reaction system is 0.1 to 10% (v / v), preferably 1 to 10% (v / v), more preferably 4% (v / v)) of the whole reaction solution.

  The concentration of the enzyme added to the reaction system is preferably 1.5 to 6.1 mg / mL, more preferably 5.5 mg / mL.

  The reaction temperature is appropriately selected within the range of 4 to 60 ° C, preferably 20 to 60 ° C, more preferably 30 to 50 ° C, and particularly preferably 40 ° C. The reaction time is 5 hours to 30 days, preferably 24 hours to 15 days, and more preferably 48 hours.

  As the substrate to be used, those containing aspartic acid, a salt thereof, or an ester and a mixture thereof can be used. Aspartic acid may be L-form or D-form, but L-form is preferred. Preferably, L-aspartic acid diethyl hydrochloride is used. The substrate may be in the form of a monomer, dimer or polymer, or a mixture thereof. The concentration of the substrate contained in the reaction system is preferably 15 to 61 mg / mL, more preferably 55 mg / mL.

  The molar ratio of the enzyme to be added and the substrate is 1: 630 to 1: 2560, preferably 1: 2300.

The synthesis of PAA using the enzyme of the present invention may be performed by the following steps.
(1) PAA hydrolase is modified with an amphiphilic polymer such as PEG.
(2) The modified enzyme is dissolved in an organic solvent containing water. At this time, the first modified enzyme may be dissolved in a small amount of water, and an organic solvent may be added thereto.
(3) An enzyme dissolved in an organic solvent is mixed with a substrate and stirred to react.
(4) After the reaction, the enzyme and insoluble matter are removed, and a polymer is obtained by drying under reduced pressure.

  “High-purity β-linked polyaspartic acid” synthesized by the method of the present invention means that substantially only β-PAA is synthesized. That is, it means that 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more of the synthesized PAA is β-PAA. That is, the proportion of β bonds among peptide bonds in the synthesized PAA is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more. In the case where the ratio of β-PAA in the synthesized β-PAA is as described above, or the ratio of β bonds is as described above, in the present invention, “high-purity β-bonded polyaspartic acid”, “substantially It is referred to as “β-linked polyaspartic acid containing only β-PAA”, “β-linked polyaspartic acid containing only β-PAA”, and the like.

  Β-PAA synthesized by the method of the present invention is preferably heptamer or more, preferably octamer or more, preferably 9mer or more, preferably 10mer or more, preferably 11mer or more, preferably 12 Or more, preferably 13 or more, preferably 14 or more, preferably 15 or more, preferably 16 or more, preferably 17 or more, preferably 18 or more, preferably 19 or more. It is a body or more, preferably a 20-mer or more. The molecular weight of β-PAA synthesized by the method of the present invention is preferably 1047 or more, preferably 1190 or more, preferably 1333 or more, preferably 1476 or more, preferably 1619 or more, preferably 1762 or more, preferably 1905. Or more, preferably 2048 or more, preferably 2191 or more, preferably 2334 or more, preferably 2477 or more, preferably 2620 or more, preferably 2763 or more, preferably 2906 or more. Depending on the conditions, higher molecular weights, for example, tens of thousands or more can be synthesized.

  Furthermore, β-PAA synthesized by the method of the present invention does not cause racemization and has high optical purity.

  The synthesized β-PAA is obtained from the reaction solution by known methods used for protein purification, such as ammonium sulfate salting out, precipitation separation with an organic solvent (ethanol, methanol, acetone, etc.), ion exchange chromatography, isoelectric point chromatography. Chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrates or antibodies, chromatography such as reverse phase column chromatography, microfiltration, ultrafiltration, reverse osmosis filtration, etc. It is possible to purify using one or a combination of filtration processes and the like.

  The synthesized β-PAA can be measured for the degree of polymerization and the like by mass spectrometry such as NMR and MALDI TOF-MS.

  The present invention also includes β-PAA obtained by the synthesis method of the present invention.

  The obtained β-PAA can be suitably used as a material for pharmaceuticals, cosmetics and the like, for example.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Preparation of Pedobacter sp. KP-2 derived PAA hydrolase-1 (1) Purification of Pedobacter sp. KP-2 derived PAA hydrolase-1 Pedobacter sp. KP-2 collected from river water was 0.15. The cells were cultured at 25 ° C. for 24 hours in an inorganic medium (6 L) containing% (w / v) tPAA. After culturing, the cells were collected by centrifugation (5000 × g, 4 ° C., 15 minutes) and washed with 10 mM phosphate buffer (pH 7.0). The cells were suspended in 10 mM phosphate buffer (pH 7.0), and the suspension was sonicated on ice for 30 minutes at 20 kHz. The disrupted cells were centrifuged (150,000 × g, 4 ° C., 1 hour), and the supernatant was collected. All subsequent purification steps were performed at 0-4 ° C. The obtained supernatant was applied to an SP Sepharose HP column (25 mL, GE Health Bio-Science Corp.) previously equilibrated with 10 mM phosphate buffer (pH 7.0). The column was washed with 3 volumes of 10 mM phosphate buffer (pH 7.0). Subsequently, the enzyme was eluted at 3 mL / min using 90 mL of a 0-500 mM NaCl linear gradient and fractions were collected every 2 minutes. The collected fraction was dialyzed against 10 mM sodium phosphate buffer (pH 6.0) and pre-equilibrated with 10 mM phosphate buffer (pH 7.0) Mono S column (1 mL, GE Health Bio-Science Corp.) and the column was washed with 3 volumes of 10 mM phosphate buffer (pH 7.0). Subsequently, the enzyme was eluted at 1 mL / min using a 20 mL linear gradient of 0-500 mM NaCl, and fractions were collected every 2 minutes.

(2) Gene cloning of PAA hydrolase-1 from Pedobacter sp. KP-2
In order to determine the N-terminal amino acid sequence of Pedobacter sp. KP-2 derived PAA hydrolase-1, the enzyme was separated by SDS-PAGE according to a method known in the art, and then transferred to an Immobilon-P transfer membrane. Transferred and subjected to Applied Biosystems 473A protein sequencer.

As a result, the sequence of the N-terminal amino acid of mature PAA hydrolase-1 derived from Pedobacter sp. KP-2 was found to be XEGVGEFIYQDYKPLDNKPI (X means an undefined amino acid residue, SEQ ID NO: 5). In order to amplify the downstream region of the sequence inside the determined sequence (EGVGEFIYQDYKP) (SEQ ID NO: 6), the following degenerate primers used for LA PCR in vitro cloning were designed:
Primer S1 (5'-GARGGIGTIGGIGARTTYATHTA-3 ') (SEQ ID NO: 7)
Primer S2 (5′-TTIATITAYCARGAYYAYAARCC-3 ′) (SEQ ID NO: 8).

  LA PCR in vitro cloning was performed using a kit (TAKARA BIO INC.) According to the manufacturer's instructions. The genomic DNA of KP-2 strain was completely digested with EcoRI. This DNA fragment was ligated to a cassette oligonucleotide and used as a PCR template. After LA PCR, the obtained PCR product was ligated to pGEM-T Easy (Promega), nucleotide sequence was determined using CEQ2000XL DNA sequencer (Beckman Coulter Inc.), and GENETYX program (Software Development Co.) was It was confirmed that the obtained PCR product was a partial sequence and did not contain a stop codon.

In order to obtain the full-length PAA hydrolase-1 gene, LA PCR in vitro cloning was again performed using two pairs of primers different from the above primers. These primers were designed based on the partial sequence of PAA hydrolase-1. The following primers for downstream of the partial sequence:
S3 (5'-AATAAGCCCATAAAAGTAAGATATTATAATCC-3 ') (SEQ ID NO: 9)
S4 (5′-GGGAAAAATGACGCCCAAGTATTGTTCATTATGC-3 ′) (SEQ ID NO: 10)
And the following primers upstream of the partial sequence:
S5 (5′-GCATAATGAACAATACTTGGGCGTCATTTTTCCC-3 ′) (SEQ ID NO: 11)
S6 (5′-GATTATAATATCTTACTTTTATGGGCTTATTG-3 ′) (SEQ ID NO: 12)
Was made. The obtained PCR product was ligated to pGEM-T Easy, the nucleotide sequence was determined and analyzed by a database such as BLAST, and the target PAA hydrolase-1 gene was confirmed.

(3) Construction of Pedobacter sp. KP-2 derived PAA hydrolase-1 expression plasmid
The PAA hydrolase-1 gene has the following synthetic nucleotide primers:
S7 (5′-CAACCCACATATGGACGAGGGCGTAGGCGAG-3 ′) (SEQ ID NO: 13)
S8 (5′-GAGGGATCCGATTTATTTATCTTCGAACAGC-3 ′) (SEQ ID NO: 14)
And it amplified using the template DNA derived from Pedobacter sp. KP-2. Nde I and BamH I restriction enzyme sites were added to the S7 and S8 primers, respectively. The PCR product was digested with NdeI and BamHI and introduced into pET-15b (+), which was also pretreated with NdeI and BamHI. The obtained plasmid (hereinafter referred to as pPAA1KP2) was introduced into E. coli BL21 (DE3) cells by a method known in the art and plated on an LB plate containing 100 μg / mL ampicillin.

(4) Expression and purification of recombinant PAA hydrolase-1 from Pedobacter sp. KP-2
Recombinant E. coli BL21 (DE3) cells carrying pPAA1KP2 were cultured overnight at 37 ° C. in 1.75 mL LB medium supplemented with ampicillin (100 μg / mL). Of these, 200 μl was transferred to 20 mL of fresh LB medium containing 100 μg / mL ampicillin and cultured at 37 ° C. overnight. Of this, 10 mL was transferred to 1 L of a new LB medium containing 100 μg / mL ampicillin and cultured at 37 ° C. When the cell density reached OD = 0.5-1.0, isopropyl-β-D-thiogalactopyranoside (IPTG) (at a final concentration of 0.05 mM) was added to the medium. After culturing at 20 ° C. for 20 hours, the cells were collected by centrifugation (5000 × g, 4 ° C., 10 minutes). The collected cells were suspended in 10 mM buffer A (50 mM NaH ₂ PO ₄ , 300 mM NaCl and 10 mM imidazole) (pH 8.0) and crushed using a French press (4 times at 14000 psi). . The suspension was centrifuged (18800 × g, 4 ° C., 30 minutes), and the supernatant was filtered using a 0.45 μm cellulose acetate filter.

The following steps were performed at 0-4 ° C. The obtained soluble fraction was applied to a Ni-NTA Superflow column (10 mL, QIAGEN) previously equilibrated with buffer A. The column was washed with 10 volumes of buffer A. The enzyme was eluted stepwise with 4-60% strength buffer B (50 mM NaH ₂ PO ₄ , 300 mM NaCl and 250 mM imidazole) (pH 8.0) and 5 mL fractions were collected. The enzyme fraction was concentrated and applied to a Superdex 200 pg column (120 mL, GE Health Bio-Sciences Corp.) equilibrated with 10 mM phosphate buffer (pH 7.0) containing 0.1 M NaCl. The eluted enzyme fraction was collected, concentrated and dialyzed against 10 mM phosphate buffer (pH 7.0).

Example 2 Modification of Recombinant PAA Hydrolase-1 Derived from Pedobacter sp. KP-2 with Activated PEG Dialyze the purified protein against 20 mM borate buffer (pH 9.0) (Slide-A-Lyzer Dialysis, After 10000 MWCO, PIERCE), the concentration was measured and modified with activated PEG (mPEG-SMB-5000; MW 5763 Da, Nektar).

Both activated PEG and protein were adjusted to 2.5 mL with 20 mM borate buffer so that the final concentration was 6 × 10 ⁻⁴ M. After stirring at 4 ° C. for 24 hours, dialysis (Slide-A-Lyzer Dialysis, 10000 MWCO, PIERCE) was performed against MilliQ water to remove unreacted PEG. The result of the PEG modification rate is shown in FIG. 5 (FIG. 5). The PEG modification ratio was measured by SDS-PAGE band area using ATTO Densito graph 4.1. From this result, it was found that 70% of the total PAA hydrolase-1 was modified with PEG. Next, in order to investigate the influence of PEG modification on enzyme activity, the activity test of PEG modified enzyme and unmodified enzyme was performed. The activity test was conducted by a turbidity measuring method. The substrate tPAA was charged in a 10 mM sodium phosphate buffer (pH 7.0) to a final concentration of 0.15% (w / v), and reacted with the enzyme solution at 30 ° C. After the reaction, sodium chloride was sequentially added to a final concentration of 50 mM and ethanol was added to a final concentration of 50% (v / v), followed by stirring. After stirring, the turbidity of the cloudy solution of PAA produced by the addition of ethanol was measured. The turbidity measurement was performed by measuring absorbance at a wavelength of 595 nm using an absorptiometer (Microplate Reader Model 550, BioRad). The result of the activity test is shown in FIG. 6 (FIG. 6). PEG-PAA hydrolase-1 was found to retain the same activity as unmodified PAA hydrolase-1.

Example 3 Synthesis and Purification of β-PAA
(1) Dehydrochlorination of substrate
500 mg of L-aspartic acid diethyl salt was dissolved in 100 mL of MilliQ water, neutralized with NaOH, and then extracted with ethyl acetate. After evaporating the solvent, drying under reduced pressure was carried out overnight to obtain 227 mg of diethyl L-aspartate as a yellow oil. This was used as a substrate.

(2) Synthesis and purification of β-PAA
After adding 55 mg of the substrate to 1 mL of acetonitrile mixed solution containing 40 μL of MilliQ water, 5.5 mg of PEG-PAA hydrolase-1 was added and mixed well to be uniform. The reaction solution was stirred at 40 ° C. for 48 hours under a nitrogen atmosphere. After the reaction, anhydrous sodium sulfate (1.5 g) was added for dehydration, and insoluble matters were removed by filtration. Next, the obtained filtrate was mixed with 5 mL CHCl ₃ / TFA (100: 3, v / v), the insoluble enzyme was filtered through a 0.1 μm filter, the solvent was removed, and then drying under reduced pressure was performed overnight. As a liquid substance, 35.5 mg of product was obtained.

Example 4 Analysis of product by MALDI-TOF MS The product obtained in Example 3 was subjected to MALDI-TOF MS. MALDI TOF-MS (JNM-AL 400 (JEOL)) The matrix used for the measurement was 2,5-dihydroxybenzoic acid (DHB) in acetonitrile: 1% TFA (trifluoroacetic acid, 99%, ALDRICH) (1: 2, v / v) What was dissolved in the mixed solution to 10 mg / mL was used. The measurement conditions were Reflector mode, Positive, and the number of integrations was 150. Calibration was performed using DHB.

  FIG. 7 shows the results of MS spectrum. The monomer has a complex peak around 300 to 800, but the product has decreased low molecular weight complex peaks and many peaks appearing on the polymer side. From the peak top value seen in the product, the molecular weight of the synthesized polymer was expressed as m / z = 143n + 46 + 23. As described above, from the result of MS spectrum, a polymer exceeding the 16-mer with the 11-mer peak was obtained.

Example 5 Analysis of the product by NMR
^{For 1} H-NMR measurement, 99.9% dimethyl sulfoxide-d ₆ (Wako) containing 0.05 v / v% TMS was used as a solvent. Integration was performed 96 times.
FIG. 8 shows the result of ¹ H-NMR. From the top, spectra of PAA synthesized with PEG-PAA degrading enzyme-1, PAA synthesized with Bioprase, and diethyl aspartate (monomer) are shown. Peaks derived from methine hydrogen contained in the α unit and β unit are observed at 4.56 ppm and 4.30 ppm, respectively. In PAA synthesized using protease (Bioprase), a peak derived from α unit was observed around 4.56 ppm and a peak derived from β unit was observed near 4.30 ppm, and the peak area ratio was 10: 7.5. . On the other hand, in the PAA synthesized using the PEG-modified PAA hydrolase-1 of the present invention, only the peak derived from the β unit was confirmed.

  From this result, it became clear that only β-PAA can be synthesized by using the method according to the present invention.

  Since the present invention makes it possible to synthesize highly pure β-PAA or a salt or ester thereof using aspartic acid or a salt or ester thereof as a substrate using PAA hydrolase in an organic solvent. It is possible to produce a large amount of homogeneous uniform PAA at a relatively low temperature.

It is a figure which shows the structure of tPAA ((alpha), (beta) -poly (D, L-aspartic acid)). It is a figure which shows the structure of (beta) position coupling | bonding poly aspartic acid ((beta) -PAA). It is a figure which shows the nucleotide sequence (sequence number 1) and amino acid sequence (sequence number 2) of Pedobacter sp. KP-2 origin PAA hydrolase-1. It is a figure which shows the nucleotide sequence (sequence number 3) and amino acid sequence (sequence number 4) of Sphingomonas sp. KT-1 origin PAA hydrolase-1. (A) It is a figure which shows the ¹³ C NMR spectrum of tPAA. (B) ¹³ C NMR spectrum of tPAA treated with PAA hydrolase-1 derived from Pedobacter sp. KP-2. It is a figure which shows the result of PEG modification. (A) shows SDS-PAGE of PEG-modified PAA-degrading enzyme-1. From left, marker, 5 mg / mL unmodified PAA-degrading enzyme-1, 5 mg / mL PEG-modified PAA-degrading enzyme-1, 1 mg / mL unmodified PAA-degrading enzyme-1, 1 mg / mL PEG-modified PAA-degrading enzyme -1, 0.5 mg / mL unmodified PAA-degrading enzyme-1, 0.5 mg / mL PEG-modified PAA-degrading enzyme-1 is shown. (B) shows the PEG modification rate of 1 mg / mL unmodified PAA-degrading enzyme-1 and 1 mg / mL PEG-PAA-degrading enzyme-1. It is a figure which shows the result of the activity test of unmodified PAA degrading enzyme-1 and PEG modification PAA degrading enzyme-1. It is a figure which shows the MS spectrum of the synthetic product obtained by the method of this invention. It is a diagram showing the ¹ H-NMR spectrum of (C) monomer; synthesized PAA by using the (B) protease (Bioprase); PAA was synthesized using (A) PEG-modified PAA enzymes -1.

Claims (12)

  High-purity β-linked polyaspartic acid polymerized using aspartic acid or a salt or ester thereof as a substrate using polyaspartic acid hydrolase that recognizes between β-β peptide bonds in an organic solvent containing water Or a method of synthesizing a salt or ester thereof.   The process according to claim 1, wherein only β-linked polyaspartic acid or a salt or ester thereof is synthesized.   The method according to claim 1 or 2, wherein the polyaspartate hydrolase that recognizes between β-β peptide bonds is polyaspartate hydrolase-1 derived from Pedobacter sp. KP-2.   The method according to claim 1 or 2, wherein the polyaspartate hydrolase that recognizes between β-β peptide bonds is polyaspartate hydrolase-1 derived from Sphingomonas sp. KT-1.   The method according to claim 1, wherein the enzyme used is modified with an amphiphilic polymer substance.   6. The method according to claim 5, wherein the amphiphilic polymer substance is polyethylene glycol.   The method according to any one of claims 1 to 6, wherein the organic solvent is selected from the group consisting of acetonitrile, toluene, benzene, and THF.   The method according to any one of claims 1 to 7, wherein the synthesis is carried out in an organic solvent containing 0.1 to 10% water.   The method according to any one of claims 1 to 8, wherein the polymerization is carried out within a temperature range of 4 to 60 ° C.   A high-purity β-linked polyaspartic acid, a salt or an ester thereof, produced by the method according to claim 1.   The β-position binding polyaspartic acid or a salt or ester thereof according to claim 10, comprising only β-position polyaspartic acid.   The β-bonded polyaspartic acid or a salt or ester thereof according to claim 10 or 11, wherein no racemization has occurred.

Images