JP2579567B2 - Protease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for producing L-asparagine C
It relates to a protease that selectively hydrolyzes only the terminal amide bond.

[0002]

2. Description of the Related Art A method of selectively recognizing a specific amino acid residue contained in a peptide chain and hydrolyzing only an N-terminal or C-terminal peptide bond thereof is known by analyzing the structure of a protein, modifying the protein, and the like. [Susumu Tsunazawa, Fumio Sakiyama, Seminar on Seikagaku Experiment (edited by the Biochemical Society of Japan), 2, 260-277 (198)
7)]. Conventionally, as means for this purpose, biochemical methods include hydrolysis of the C-terminal peptide bond of L-lysine, hydrolysis of the N-terminal peptide bond of L-lysine, and hydrolysis of the C-terminal peptide bond of L-arginine. Hydrolysis, hydrolysis of the C-terminal peptide bond of L-glutamic acid and L-aspartic acid, hydrolysis of the N-terminal peptide bond of L-aspartic acid, hydrolysis of the C-terminal peptide bond of L-proline, and Examples of the organic chemical method include cleavage of a peptide bond at the C-terminal side of L-methionine, and cleavage of N-terminal of L-cystine.
Cleavage of the terminal peptide bond and cleavage of the C-terminal peptide bond of L-tryptophan are known.

[0003]

In protein engineering, there is a demand for a method of specifically cleaving peptide bonds of various amino acids. However, there is no known amino acid-specific cleavage method other than the method described above. An object of the present invention is to provide a protease that selectively hydrolyzes only the amide bond at the C-terminal side of L-asparagine.

[0004]

SUMMARY OF THE INVENTION In general, the present invention relates to a protease which selectively hydrolyzes only the amide bond at the C-terminal side of L-asparagine, and has the following enzymatic properties: : Only the C-terminal amide bond of L-asparagine in the peptide chain is selectively hydrolyzed. (2) Substrate specificity: L-asparagine in a peptide chain consisting of neurotensin, β-endorphin, parathyroid hormone (1-34), bathoactive intestinal peptide, peptide T, bombesin, oxidized insulin B chain, and fisalamine Selectively hydrolyzes only the amide bond at the C-terminal side. (3) Optimum pH: 6.0 to 7.0 (4) pH stability: 4.5 to 6.5 (treatment at 37 ° C. for 6 hours in a buffer containing 1 mM DTT) (5) Optimum temperature : Around 45 ° C (6) Temperature stability Stable by heating at 55 ° C for 15 minutes in a buffer solution containing 1 mM DTT at pH 5.0. (7) It has a molecular weight of 26550 (gel filtration method).

In view of the fact that the amino acid at the C-terminal side of lectins and storage proteins present in plant seeds is L-asparagine, the present inventors have found that C-terminal amides of L-asparagine in peptide chains are present in plant seeds. When a protease that selectively hydrolyzes only the bond was searched for, a protease that hydrolyzes only the amide bond at the C-terminal side of L-asparagine in the peptide chain from the seeds of the beans ( Canavalia ensiformis ) was isolated and purified. Succeeded and completed the present invention. The present invention provides a method for producing the enzyme,
It has been completed by studying various properties of the enzyme.

Hereinafter, the present invention will be described in detail. (Production method of the present enzyme) The enzyme used in the present invention is obtained, for example, by solubilizing the present enzyme by homogenizing commercially available jack bean meal in a buffer solution of pH 4 to 6 in the presence of a suitable reducing agent. Insoluble matter is removed by means of centrifugation or the like, and the purification degree is increased by means of various purification methods such as ion exchange chromatography and gel filtration, and the specific inhibitor of this enzyme, paramercuribenzoic acid (PMB), is agarose-treated. The protein can be isolated as a single protein by performing affinity chromatography immobilized on the protein.

(Measurement method and unit for this enzyme) As a substrate, the peptide shown in SEQ ID NO: 1 in the sequence listing is used.
0.02 mM this substrate, 5 mM DTT, 2 mM EDT
A, 0.002 mM D as enzyme and internal standard
20 mM acetate buffer containing NP-L-Ser (pH 5.
After keeping the reaction solution comprising the mixture (0) at 37 ° C. for 10 to 20 minutes, the reaction was stopped by adding formic acid to a final concentration of 10%, and a part of the reaction solution was subjected to high-speed The enzyme activity can be measured by analyzing by liquid chromatography and comparing and quantifying the amount of the fragment having the sequence shown in SEQ ID NO: 2 in the generated sequence listing with DNP-L-Ser which is an internal standard substance. The unit of this enzyme activity was 1 unit of the amount of an enzyme that produced a fragment having the sequence shown in SEQ ID NO: 2 in 1 μmol of the sequence listing per minute.

(Substrate specificity of the enzyme) Neurotensin represented by SEQ ID NO: 3, β-endorphin represented by SEQ ID NO: 4, N-terminal poly-parathyroid hormone represented by SEQ ID NO: 5 Peptide (1-34), bathoactive intestinal peptide represented by SEQ ID NO: 6, peptide T represented by SEQ ID NO: 7, bombesin represented by SEQ ID NO: 8, oxidized insulin B represented by SEQ ID NO: 9 The ratio of the substrate (nmol) to the enzyme (mU) was 5 using the fisalamine represented by SEQ ID NO: 10 as a substrate.
As a result of performing the reaction at 37 ° C. for 15 hours at pH 5.0 at 0: 1, only the C-terminal amide bond of L-asparagine was hydrolyzed, and the other peptide bonds were not hydrolyzed at all. Tables 1 and 2 show the synthetic peptides used as substrates and the hydrolyzed amide bond sites.

[0009]

[Table 1] Table 1 ア ミ ド Base hydrolyzed amide bond ── ───────────────────────────────── ₅ ↓ ₆ Neurotensin Asn-Lys ₂₀ ↓ ₂₁ β-endorphin (human) Asn-Ala ₂₅ ↓ ₂₆ Asn-Ala ₁₀ ↓ ₁₁ Parathyroid hormone Asn-Leu (human, 1-34) ₁₆ ↓ ₁₇ Asn-Ser ₃₃ ↓ ₃₄ Asn-Phe ₉ ↓ ₁₀ Vaso active in Asn-Tyr testinal Peptide ₂₄ ↓ ₂₅ (human, pig) Asn-Ser ₂₈ ↓ Asn-NH ₂

[0010]

[Table 2] Table 2 ア ミ ド Base hydrolyzed amide bond ── ───────────────────────────────── ₆ ↓ ₇ Peptide T Asn-Tyr ₆ ↓ ₇ Bombesin Asn-Gln ₃ ↓ ₄ Insulin-oxidized B chain Asn-Gln ₅ ↓ ₆ Fisalamine Asn-Lys ───────────────────────────────────

[0011]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 500 g of commercially available jack bean meal (manufactured by Sigma) was added to 1
The mixture was homogenized with 20 mM acetate buffer (pH 5.0) containing mM DTT and 1 mM EDTA, and centrifuged to obtain a supernatant. After the supernatant was dialyzed in the above buffer, the precipitate formed again was removed by centrifugation, and SP-Toyopearl 65 was removed.
Ion exchange chromatography with 0M (manufactured by Tosoh Corporation) was performed. Adsorbed on SP-Toyopearl 650M column,
The active fraction eluted with 0.5M sodium chloride was reduced to 60%
After the precipitation with saturated ammonium sulfate, the buffer was dialyzed. continue,
Affinity chromatography was performed using an affinity column prepared by immobilizing PMB on agarose. The active fraction adsorbed on the column and eluted with 0.5 M sodium chloride was again dialyzed against the above buffer. Next, affinity chromatography using a leucyl argininal column (manufactured by Organo) was performed, and an active fraction not adsorbed to the column was collected. The active fraction was concentrated on an ultrafiltration membrane with a molecular weight cutoff of 10,000 and then concentrated with Toyopearl HW.
Gel filtration was performed using -55S (manufactured by Tosoh Corporation). The purified enzyme thus obtained was 2.6 mg, the activity was 180 mU, and was a single protein in C4-reverse phase chromatography.

Example 2 Using the purified enzyme obtained in Example 1, limited hydrolysis of commercially available soluble reduced lysozyme (manufactured by Seikagaku Corporation) was performed.
The reaction conditions were as follows: in 20 mM sodium acetate buffer (pH 5.0) containing 10 mM DTT and 1 mM EDTA, at a substrate concentration of 0.2 mM and an enzyme concentration of 4 mU / ml at 37 ° C.
The reaction was performed for 15 hours. After terminating the reaction by adding formic acid to a final concentration of 10%, the resulting peptide fragment was subjected to C4-reverse phase chromatography (Micro Bonder sphere, manufactured by Waters, 3.9 × 15).
0 mm), amino acid composition analysis and amino acid sequence analysis were performed on each fragment.

The amino acid sequence of soluble reduced lysozyme is shown in SEQ ID NO: 11 in the sequence listing, C4-reverse phase chromatography is shown in FIG. 1, the amino acid sequence of each fragment of peaks 1 to 16 is shown in the sequence listing, SEQ ID NO: 12
-18, peak No. 8 is SEQ ID Nos. 19 and 20, peak No. 9 is SEQ ID Nos. 21 and 22, peak Nos. 10-14
Is SEQ ID NO: 23-27, peak number 15 is SEQ ID NO: 2
8, 29 and 30, peak number 16 are shown as SEQ ID NO: 31, respectively. FIG. 1 is a graph showing the relationship between the time (minutes, horizontal axis) and the absorbance at 215 nm (vertical axis) after the soluble reduced lysozyme was digested with the present enzyme and fractionated by C4 reverse phase HPLC. . As a result, it was confirmed that the present enzyme cleaves only the C-terminal amide bond of L-asparagine contained in the soluble reduced lysozyme.

[0015]

As described in detail above, the present invention provides a protease which selectively hydrolyzes only the amide bond at the C-terminal side of L-asparagine in a peptide chain. Further, according to the present invention, L-asparagine and L-aspartic acid can be distinguished, which is also effective in confirming a product in genetic engineering. The protease of the present invention can be used as a very important means in protein structural analysis.

[Sequence list] SEQ ID NO: 1 Sequence length: 4 Sequence type: Number of amino acid chains: Single chain Topology: Linear Sequence type: Peptide Sequence characteristics: 1 2,4, -dinitrophenyl-L- proline 4 L-asparaginamide SEQ ID NO: 2 Sequence length: 4 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Sequence characteristics: 1 2,4, -dinitrophenyl-L-proline SEQ ID NO: 3 Sequence length: 13 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Sequence characteristics: 1 L-pyroglutamic acid SEQ ID NO: 4 Sequence length: 31 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Sequence: Tyr Gly Gly Phe Met Thr Ser Glu Lys Ser Gln Thr Pro Leu Val Thr 1 5 10 15 Leu Phe Lys Asn Ala Ile Ile Lys Asn Ala Tyr Lys Lys Gly Glu 20 25 30 SEQ ID NO: 5 Sequence length: 34 Sequence type: Amino acid Number of chains: 1-chain Topology: Linear fragment Type: N-terminal fragment Sequence type: Peptide Sequence: Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe SEQ ID NO: 6 Sequence length: 28 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Sequence characteristics: 28 L-asparaginamide Sequence: His Ser Asp Ala Val Phe Thr Asp Asn Tyr Thr Arg Leu Arg Lys Gln 1 5 10 15 Met Ala Val Lys Lys Tyr Leu Asn Ser Ile Leu Xaa 20 25 SEQ ID NO: : 7 Sequence length: 8 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide SEQ ID NO: 8 Sequence length: 14 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Sequence characteristics: 1 L-pyroglutamic acid 14 L-methioninamide Sequence: Xaa Gln Arg Leu Gly Asn Gln Trp Ala Val Gly His Leu Xaa 1 5 10 SEQ ID NO: 9 Sequence length: 24 Sequence type: Amino acid Number of chains: 1 chain Topology: Linear Sequence type: Peptide Sequence: Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe 20 SEQ ID NO: 10 Sequence length: 11 Sequence type: Amino acid Number of chains: Single strand Topology : Linear type of sequence: Peptide Sequence characteristics: 1 L-pyroglutamic acid 11 L-methioninamide SEQ ID NO: 11 Sequence length: 129 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Sequence characteristics: 6 S-3- (trimethylammonio) propylcysteine 30 S-3- (trimethylammonio) propylcysteine 64 S-3- (trimethylammonio) propylcysteine 76 S-3- (trimethylammonio) propylcysteine 80 S-3- (trimethylammonio) propylcysteine 94 S-3- (trimethylammonio) propylcysteine 115 S-3- (trimethylammonio) propylcysteine 127 S-3- (trimethylammonio) propylcysteine Sequence: Lys Val Phe Gly Arg Xaa Glu Leu Ala Ala Ala Met Lys Arg His Gly 1 5 10 15 Leu Asp Asn Tyr Arg Gly Tyr Ser Leu Gly Asn Trp Val Xaa Ala Ala 20 25 30 Lys Phe Glu Ser Asn Phe Asn Thr Gln Ala Thr Asn Arg Asn Thr Asp 35 40 45 Gly Ser Thr Asp Tyr Gly Ile Leu Gln Ile Asn Ser Arg Trp Trp Xaa 50 55 60 Asn Asp Gly Arg Thr Pro Gly Ser Arg Asn Leu Xaa Asn Ile Pro Xaa 65 70 75 80 Ser Ala Leu Leu Ser Ser Asp Ile Thr Ala Ser Val Asn Xaa Ala Lys 85 90 95 Lys Ile Val Ser Asp Gly Asn Gly Met Asn Ala Trp Val Ala Trp Arg 100 105 110 Asn Arg Xaa Lys Gly Thr Asp Val Gln Ala Trp Ile Arg Gly Xaa Arg 115 120 125 Leu SEQ ID NO: 12 Sequence length: 7 Sequence type: Number of amino acids Chain number: Single strand Topology: Linear Type Sequence type: Peptide Fragment type: Middle fragment (Egg white soluble reduced lysozyme) SEQ ID NO: 13 Sequence length: 10 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Fragment type: Middle fragment (egg white soluble reduced lysozyme) SEQ ID NO: 14 Sequence length: 8 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Fragment type: middle fragment (egg white soluble reduced lysozyme) SEQ ID NO: 15 Sequence length: 10 Sequence type: number of amino acid chains: 1-chain Topology: linear Sequence type: peptide Fragment type: middle fragment (egg white soluble reduced lysozyme) Sequence characteristics: 30 S -3- (trimethylammonio) propylcysteine SEQ ID NO: 16 Sequence length: 9 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Fragment type: Middle fragment (egg white soluble reduced lysozyme) SEQ ID NO: 17 Sequence length: 6 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Fragment type: middle fragment (egg white soluble reduced lysozyme) Sequence characteristics: 64 S -3- (trimethylammonio) propylcysteine SEQ ID NO: 18 Sequence length: 7 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: Middle fragment (egg white soluble reduced lysozyme) SEQ ID NO: 19 Sequence length: 17 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Fragment type: Middle fragment (egg white soluble reduced lysozyme) Sequence characteristics: 30 S -3- (trimethylammonio) propylcysteine Sequence: Trp Val Xaa Ala Ala Lys Phe Glu Ser Asn Phe Asn Thr Gln Ala Thr 28 32 37 42 Asn SEQ ID NO: 20 Sequence length: 21 Sequence type: Amino acid Number of chains: 1 Main chain Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment (egg white soluble reduced lysozyme) Sequence characteristics: 60 unknown Sequence: Thr Gln Ala Thr Asn Arg Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile 40 44 49 54 Leu Gln Ile Asn Xaa 59 SEQ ID NO: 21 Sequence length: 15 Sequence type: Amino acid Number of chains: 1-chain Topology: Linear Sequence type: Peptide Fragment type: Middle fragment (egg white soluble reduction) Zozyme) Sequence: Arg Asn Thr Asp Gly Ser Thr Asp Tyr Gly Ile Leu Gln Ile Asn 45 49 54 59 SEQ ID NO: 22 Sequence length: 18 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Peptide Fragment type: Intermediate fragment (egg white soluble reduced lysozyme) Sequence characteristics: 30 S-3- (trimethylammonio) propylcysteine 45 unknown Sequence: Trp Val Xaa Ala Ala Lys Phe Glu Ser Asn Phe Asn Thr Gln Ala Thr 28 32 37 42 Asn Xaa SEQ ID NO: 23 Sequence length: 19 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide Fragment type: N-terminal fragment (egg white soluble reduced lysozyme) Sequence characteristics: 6 S-3- (trimethylammonio) propylcysteine Sequence: Lys Val Phe Gly Arg Xaa Glu Leu Ala Ala Ala Met Lys Arg His Gly 1 5 10 15 Leu Asp Asn SEQ ID NO: 24 Sequence length: 10 Sequence length Type: number of amino acid chains: single-chain topo Gee: linear sequence type: peptide fragment type: intermediate portion fragment (egg white soluble reducing lysozyme) SEQ ID NO: 25 Sequence length: 16 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: C-terminal fragment (egg white soluble reduced lysozyme) Sequence characteristics: 115 S -3- (trimethylammonio) propylcysteine 127 S-3- (trimethylammonio) propylcysteine Sequence: Arg Xaa Lys Gly Thr Asp Val Gln Ala Trp Ile Arg Gly Xaa Arg Leu 114 118 123 128 SEQ ID NO: 26 Sequence length: 28 Sequence length: 28 Type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Fragment type: Middle fragment (egg white soluble reduced lysozyme) Sequence characteristics: 76 S-3- (trimethylammonio) propylcysteine 80 S-3- (trimethylammonio) propylcysteine Sequence: Asp Gly Arg Thr Pro Gly Ser Arg Asn Leu Xaa Asn Ile Pro Xaa Ser 66 70 75 80 SEQ ID NO: 27 Sequence length: 27 Sequence type: amino acid Number of chains: 1-chain Topology: linear Sequence type: peptide Fragment type: N-terminal fragment (egg white soluble reduced lysozyme) Sequence characteristics: 6 S -3- (trimethylammonio) propylcysteine Sequence: Lys Val Phe Gly Arg Xaa Glu Leu Ala Ala Ala Met Lys Arg His Gly 1 5 10 15 Leu Asp Asn Tyr Arg Gly Tyr Ser Leu Gly Asn 20 25 SEQ ID NO: 28 Sequence length : 21 Sequence type: amino acid Number of chains: 1 chain Topology: linear Sequence type: peptide Fragment type: Intermediate fragment (egg white soluble reduced lysozyme) Sequence characteristics: 30 S-3- (trimethylammonio) propylcysteine 48 unknown Sequence: Trp Val Xaa Ala Ala Lys Phe Glu Ser Asn Phe Asn Thr Gln Ala Thr 28 32 37 42 Asn Arg Asn Thr Xaa 47 SEQ ID NO: 29 Sequence length: 21 Sequence type: Amino acid Number of chains: 1 Chain Topology: linear Sequence type: pep De Fragment type: Middle fragment (egg white soluble reduced lysozyme) Sequence characteristics: 64 S-3- (trimethylammonio) propylcysteine 76 S-3- (trimethylammonio) propylcysteine 80 unknown Sequence: Ser Arg Trp Trp Xaa Asn Asp Gly Arg Thr Pro Gly Ser Arg Asn Leu 60 64 69 74 Xaa Asn Ile Pro Xaa 79 SEQ ID NO: 30 Sequence length: 16 Sequence type: Amino acid Number of chains: 1-chain Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment (egg white soluble reduced lysozyme) Sequence features: 115 S-3- (trimethylammonio) propylcysteine 127 S-3- (trimethylammonio) propylcysteine Sequence: Arg Xaa Lys Gly Thr Asp Val Gln Ala Trp Ile Arg Gly Xaa Arg Leu 114 118 123 128 SEQ ID NO: 31 Sequence length: 23 Sequence type: number of amino acid chains: single-chain Topology: linear Sequence type: peptide Fragment type: C-terminal fragment (egg white soluble reduced lysozyme) Features: 115 S-3- (trimethylammonio) propylcysteine 127 S-3- (trimethylammonio) propylcysteine Sequence: Ala Trp Val Ala Trp Arg Asn Arg Xaa Lys Gly Thr Asp Val Gln Ala 107 111 116 121 Trp Ile Arg Gly Xaa Arg Leu 126

[Brief description of the drawings]

FIG. 1. Digestion of soluble reduced lysozyme with this enzyme followed by C
FIG. 4 is a view showing chromatography fractionated by reverse phase HPLC.

Claims (1)

(57) [Claims] (1) Action: selectively hydrolyzes only the amide bond at the C-terminal side of L-asparagine in a peptide chain. (2) Substrate specificity: L-asparagine in a peptide chain consisting of neurotensin, β-endorphin, parathyroid hormone (1-34), bathoactive intestinal peptide, peptide T, bombesin, oxidized insulin B chain, and fisalamine Selectively hydrolyzes only the amide bond at the C-terminal side. (3) Optimum pH: 6.0 to 7.0 (4) pH stability: 4.5 to 6.5 (treatment at 37 ° C. for 6 hours in a buffer containing 1 mM DTT) (5) Optimum temperature : Around 45 ° C (6) Temperature stability Stable by heating at 55 ° C for 15 minutes in a buffer solution containing 1 mM DTT at pH 5.0. (7) A protease which selectively and specifically hydrolyzes only the C-terminal amide bond of L-asparagine, which has a molecular weight of 26550 (gel filtration method).