JPH0740935B2 - Enzymes involved in C-terminal amidation and methods for their production and use - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel enzyme involved in the C-terminal amidation of a C-terminal glycine adduct, its preparation and use, and the method for measuring the activity of the enzyme and its method. Relates to a method for searching for the enzyme.

[Prior Art] Peptides showing physiological activity only after the C-terminal is amidated, such as calcitonin, gastrin, secretin, vasoactive intestinal peptide (VIP), growth hormone releasing factor, and adrenocorticotropic hormone releasing factor, are It is known that it is produced from a glycine adduct by an enzymatic reaction in the body. Many of these physiologically active peptides are useful as pharmaceuticals, and calcitonin and secretin are currently marketed as pharmaceuticals.

Conventionally, these peptides have been obtained mainly by separation and purification from a living body, and the process is complicated and the living body of origin is also difficult to obtain. Therefore, the above commercially available peptides are very expensive.

Therefore, in recent years, attempts have been made to produce these physiologically active peptides using recombinant DNA technology.
However, the recombinant DNA technology using Escherichia coli, yeast, Bacillus subtilis, etc. as a host does not allow C-terminal amidation of the produced peptide, which is an obstacle in producing the peptide. Therefore, there is a demand for a technique capable of performing C-terminal amidation easily and inexpensively in vitro.

On the other hand, the enzyme involved in the above-mentioned in vivo enzymatic reaction, that is, the C-terminal amidation of the C-terminal glycine adduct of peptides is
Peptidase is called Jill glycine -α- amino dating monooxygenase (C-terminal amidating enzyme) (EC.1.14.17.3) (Bradbury et al., Nature, 298, 686,1982: Glembo
tski et al., J. Biol. Chem., 259 , 6385, 1984), are believed to catalyze the following reactions.

Attempts have been made to purify this enzyme for the purpose of elucidating the mechanism of amidation in vivo and C-terminal amidation of peptides produced by recombinant DNA technology in vitro. So far, as an example of purification in which the specific activity is 100 times higher than that of the starting material, bovine pituitary middle lobe (Murthy et al., J. Bi
ol.Chem., 261 , 1815, 1986), pig pituitary gland (Kizer et al., E.
ndocrinology. 118 , 2262, 1986: Bradbury et al., Eur. J. Bioch.
em., 169 , 579,1987), pig atrium (Kojima et al., J. Bioche)
m., 105 , 440,1989), Xenopus laevis skin (Mizuno)
Biochem.Biophys.Res.Commun., 137 , 984, 1986), rat thyroid tumor (Mehta et al. Arch. Biochem, Biophys., 26) .
1, 44,1988) those derived from have been reported.

All of these purified enzymes require an enzyme, copper ion, to express their activity, and by adding a reducing agent for ascorbic acid,
It is known to have the property of increasing activity. Recently, studies on the elucidation of the reaction mechanism using a purified enzyme have been conducted, and the existence of reaction intermediates has been suggested [Bradbury et al., Eur. J. Biochem., 169 , 579-584 (198
7), Ramer et al., J. Am. Chem. Soc., 110 , 8526-8532 (198
8), Young et al., J. Am. Chem. Soc., 111 , 1933-1934 (198).
9)].

However, at present, there are no examples showing the isolation of the intermediate and the relationship between the intermediate and the amidating enzyme.

[Problems to be Solved by the Invention]

As described above, the C-terminal amidating enzyme exhibits a very interesting effect in vivo, and a composition having a certain purity derived from a specific living organ is also known. However, it cannot be said that these compositions are sufficient for the production of C-terminal amidated products in vitro in terms of their purity and stability and the manufacturing cost. In order to solve these problems, it was considered necessary to collect the basic knowledge about the enzyme, that is, to elucidate the reaction mechanism for the C-terminal amidation reaction. The body was successfully isolated and its structure was determined. From this result, the C-terminal amidation reaction is not a one-step reaction as conventionally thought, but a two-step reaction via an intermediate (corresponding C-terminal α-hydroxylglycine adduct). all right. Therefore, as a result of intensive investigations on the search for an enzyme that catalyzes each reaction, a new substance having the desired enzyme activity was found, and the present invention was completed. That is, an object of the present invention is to provide a novel enzyme involved in C-terminal amidation, a method for preparing and using the same, a method for measuring the activity of the enzyme and a method for searching a substance having the enzyme activity using this method.

[Means for Solving the Problems] Therefore, according to the present invention, in order to solve the above-mentioned problems, the following formula (I) (In the above formula, A represents a residue other than an α-amino group or an imino group and an α-carboxyl group derived from a natural α-amino acid, and X represents an N atom via a hydrogen atom or a carbonyl group. (Representing the residue of an amino acid derivative that binds to), acting on a C-terminal glycine adduct represented by the following formula (II) C having a molecular weight of about 25,000 daltons, which forms a C-terminal α-hydroxylglycine adduct represented by the above formula (wherein A and X have the aforementioned meanings) and has a molecular weight of about 25,000 daltons by a molecular weight determination method using gel filtration An enzyme (hereinafter, sometimes referred to as “enzyme-I”) involved in the C-terminal amidation of a terminal glycine adduct is provided.

In addition, the hydrogen atom (H) in the parentheses in the formulas (I) and (II) and the following formula (III) does not have a hydrogen atom when A is derived from an α-amino acid having an α-imino group. Means

The C-terminal glycine adduct represented by the formula (I) in the present invention, that is, the substrate for the enzyme of the present invention, is generally
Of the above formula Is derived from natural or synthetic amino acid derivatives, particularly peptides or proteins, and its C-terminal amino acid residue [-
[Indicated by N (H) -A-CO-]] to glycine. As the C-terminal amic acid residue, natural α-amino acids, particularly protein-constituting amino acids, for example, aliphatic amino acids such as glycine and alanine; branched amino acids such as valine, leucine and isoleucine; hydroxy amino acids such as serine and threonine. Acidic amino acids such as aspartic acid and glutamic acid; amides such as asparagine and glutamine; basic amino acids such as lysine, hydroxylysine and arginine; sulfur containing amino acids such as cysteine, cystine and methionine; phenylalanine and tyrosine Aromatic amino acids; heterocyclic amino acids such as tryptophan and histidine; residues derived from imino acids such as proline and 4-hydroxyproline. In addition, the hydrogen atom bonded to the α-amino group or imino group of the amino acid residue or the residue of the residue of the amino acid derivative [represented by X-] is a single amino acid or the α-amino group. There is no limitation on the type of the constituent amino acid residues and the chain length of the peptide as long as the peptides can be peptide-bonded with each other, and phosphoric acid or sugar or other substituent is covalently bonded to the constituent amino acid residues. Or may form a complex with a lipid. Specific examples of the substituent include a group that substitutes for a guanidino group of an arginine residue corresponding to each amino acid residue, for example, an alkyl group such as a methyl group or an ethyl group or adenosine diphosphate ribose, Residue derived from citrulline or ornithine; group substituting ε-amino group of lysine residue, for example, glucosyl group, pyridoxyl group, biotinyl group, lipoyl group or acetyl group or phosphoric acid, compound having δ-hydroxyl group, δ A compound having a glycosyl group, a residue derived from glutaraldehyde or citraconic anhydride; a group substituting the imidazole group of a histidine residue, for example, a methyl group, a phosphate group or a residue derived from an iodine atom or flavin; proline A group that substitutes for a residue, such as a hydroxyl group, a dihydroxyl group or a glycosyl group. Oxy group; group substituting benze ring of phenylalanine residue, for example, hydroxyl group or glucosyloxy group; group substituting for hydroxyl group of tyrosine residue, for example, glycosyloxy group, sulfonic acid group, iodine atom, bromine atom or chlorine atom Or a compound having a hydroxyl group, a residue derived from bisether, adenine, uridine or RNA (ribonucleic acid); a group substituting the hydroxyl group of a serine residue, for example, a methyl group, a glycosyl group, a phosphopantetheine group,
Adenosine diphosphate ribosyl or phosphate group; group substituting for hydroxyl group of threonine residue, eg, glucosyl group, methyl group or phosphate group; group substituting for SH group of cysteine residue, eg, glycosyl group, cystinyl group ,
Dehydroalanyl group or a residue derived from a selenium atom or heme or flavin; a group substituting the carboxyl group of an aspartic acid or glutamic acid residue, for example, a methyl group, a phosphate group or a γ-carboxyl group;
A group which substitutes the amide group of an asparagine or glutamine residue, for example, a glycosyl group, a pyrrolidonyl group or an imino group can be mentioned.

The peptide in which glycine is peptide-bonded to the C-terminal residue as the substrate or a derivative thereof is either a naturally extracted peptide, a chemically synthesized peptide, or a recombinant DN.
It may be produced using A technology. Therefore, as the substrate of the present invention, the compound represented by the formula (I), peptides such as peptides having about 2 to 100 amino acid residues,
Phosphate peptides represented by casein, protein kinase, adenovirus EIA protein, RAS1 protein and their hydrolysates, lipoproteins represented by thromboplastin, α ₁ lipoprotein, lipoviterin and their hydrolysates, hemoglobin , Metalloproteins represented by myoglobin, hemocyanin, chlorophyll, phycocyanin, flavin, rhodopsin, and their hydrolysates, glycoproteins represented by collagen, laminin, interferon α, celloglycoid, avidin, etc., and other hydrolysates C-terminal carboxyl group is amidated and matured physiologically active peptides such as calcitonin, secretin, gastrin, vasoactive intestinal peptide (VIP), cholecystokinin, cerulein, pancreatic polypeptide, growth hormone. Down releasing factor, corticotropin releasing factor, C-terminal glycine adduct of peptides each to form a calcitonin gene-related peptide or (i.e., amide bond compound C-terminal carboxyl group and glycine) and the like. Of these, preferred substrates for confirming the enzyme activity of the enzyme composition of the present invention include:
For example, D-tyrosyl valyl glycine, D-tyrosyl tryptophanyl glycine, glycyl phenylalanyl glycine, phenylalanyl glycyl phenylalanyl glycine, D-tyrosyl leucyl asparaginyl glycine, arginyl phenylalanyl glycine, Arginyl alanyl Arginyl leucyl glycine, leucyl methionyl glycine, glycyl leucyl methionyl glycine,
Phenylalanylglycyl leucylmethionylglycine, asparaginylarginylphenylalanylglycine, tryptophanylasparaginylarginylphenylalanylglycine, alanylphenylalanylglycine, lysylalanylphenylalanylglycine, seryllydyl Alanyl phenylalanyl glycine, arginyl tyrosyl glycine, glycyl methionyl glycine, glycyl tyrosyl glycine, glycyl histidyl glycine,
Histidyl glycyl glycine, tryptophanyl glycyl glycine, glycyl cysteinyl glycine, etc. are mentioned (In addition, except glycine, what is not shown as D- shows L-form). On the other hand, as a preferable substrate for effective use of the enzyme composition (fifth aspect of the present invention described later), the C-terminal carboxyl group that is amidated to form a mature physiologically active peptide is used. Examples include peptides in which glycine is peptide-bonded.

The enzyme-I of the present invention acts on the above-mentioned substrate and has the following formula (II) [In the above formula, Has the meaning as defined for formula (I) above], and a C-terminal α-hydroxylglycine adduct represented by the formula can be produced.

The compound represented by the formula (II) is Hydrolyzed under conditions that do not adversely affect the corresponding C or by treatment with the second enzyme of the present invention described below.
It can be converted to a terminal amidated product.

Also, the Enzyme-I has a molecular weight of about 25,000 daltons by a molecular weight determination method using gel filtration. That is, this molecular weight is determined by the gel filtration method known per se [eg,
"Biochemistry Experiment Course 5, Enzyme Research Method, Volume 1, 283-298
P. ", Tokyo Kagaku Dojin (1975)]. Specifically, 50 mM Tris-HCl (pH 7.4) containing 100 mM potassium chloride was used as an equilibration and eluent, and Toyopearl HW- Gel filtration on 55S (Tosoh Corporation), β-amylase (MW200,000), alcohol dehydrogenase (MW150,000), BSA (MW66,000),
Carbonic anhydrase (MW29,000) and cytochrome C (MW15,400) were used as indicators.

The enzyme-I of the present invention is further specified by the following physicochemical properties. That is, (i) the optimum pH is about 5 to 7 and the stable pH is 4 to 9 (ii) the optimum temperature for action is in the range of 25 to 40 ° C., and (iii) metal ions and ascorbic acid are used as cofactors. To do.

The properties of (i) and (ii) above are obtained by using a commonly used buffer, specifically, Tris-hydrochloric acid, Meth-potassium hydroxide, Tess-sodium hydroxide, Hepes-potassium hydroxide buffer. It was measured. The enzyme composition of the present invention catalyzes the above reaction within the temperature range of 1 ° C to 55 ° C, but is inactivated at 56 ° C in about 10 minutes, and slightly deactivated at around 40 ° C.

Suitable metal ions are Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Fe ^{3+ and the} like, with Cu ²⁺ and Zn ²⁺ being particularly preferred.

In the C-terminal amidation, the enzyme of the present invention (Enzyme-I)
The following enzyme (enzyme-II) is mentioned as another type of enzyme that works together. That is, acting on the C-terminal α-hydroxylglycine adduct represented by the formula (II),
Formula (III) C-terminal glycine addition having a molecular weight of about 40,000 daltons, which produces a C-terminal amidated compound represented by the above formula (wherein A and X represent the above meanings) and glyoxylic acid, and has a molecular weight of about 40,000 daltons by a molecular weight determination method using gel filtration Enzymes involved in C-terminal amidation of the body (hereinafter sometimes referred to as "enzyme-II")
Will be provided. Used to identify this enzyme And the method of determining the molecular weight are the same as those used for the identification of enzyme-I. In addition, as a preferable substrate for confirming the enzyme activity of the enzyme-II, an α-hydroxylglycine compound corresponding to the substrate specifically listed for the enzyme-I can be mentioned. In addition, Enzyme-II is specified by having almost the same physicochemical properties as Enzyme-I. That is, (i) the optimum pH is about 5 to 7 and the stable pH is 4 to 9, (ii) the optimum temperature for action is in the range of 15 to 35 ° C, and (iii) the metal ion is used as a cofactor.

The properties of (i) and (ii) above are obtained by using a commonly used buffer, specifically, Tris-hydrochloric acid, Meth-potassium hydroxide, Tess-sodium hydroxide, Hepes-potassium hydroxide buffer. It was measured. The enzyme composition of the present invention catalyzes the above reaction within the temperature range of 1 ° C to 55 ° C, but is inactivated at 56 ° C in about 10 minutes, and slightly deactivated at around 40 ° C.

Suitable metal ions are Cu ²⁺ , Zn ²⁺ , Ni ²⁺ , Co ²⁺ , Fe ^{3+ and the} like, with Cu ²⁺ and Zn ²⁺ being particularly preferred.

Preparation of Enzyme The enzyme-I and enzyme-II of the present invention described above can be prepared from a raw material having the enzyme activity by a method of separating and purifying an enzyme known per se, but preferably in the present specification. It can be obtained by the following preparation method of the present invention disclosed. That is, substrate affinity chromatography using the enzyme activity-containing substance of enzyme-I or enzyme-II as a ligand with the C-terminal glycine adduct represented by the formula (I),
And a method for preparing Enzyme-I or Enzyme-II, which comprises treating with anion exchange chromatography.

The raw material used in this method may be any as long as it contains the enzyme of the present invention.
Those derived from an organism having a high content of enzyme-I or enzyme-II searched according to the search method of II are preferable. Generally, an organism having these enzymatic activities is a human,
Mammals such as cows, horses, pigs, sheep, rabbits, goats, rats and mice, birds such as chickens, porpoises and rock pigeons, reptiles such as turtles, pit bugs, rattlesnakes and cobras, newts, Xenopus laevis,
Amphibians such as bullfrogs and toads, fishes such as lampreys, eels, blue sharks, tortoises, sturgeons, herring, salmon, eels, troughs and Thailand, insects such as American cockroaches, silkworms, fruit flies and bees. Suitable extraction targets also include homogenates derived from organs such as brain, pituitary gland, stomach, heart and liver, and biological fluids including body fluids such as blood and lymph.

That is, a biological fluid containing the present enzyme as described above is converted into the following formula (I): (Wherein A and X have the above-mentioned meanings) Affinity chromatography of a substrate analog using a C-terminal glycine adduct as a ligand is used and, if necessary, is commonly used for purification of the enzyme (1) The enzyme of the present invention is used in combination with fractionation by precipitation, (2) heparin affinity chromatography, (3) molecular weight fractionation method by dialysis, gel filtration, etc., and / or (4) ion exchange chromatography. (Enzyme-I and Enzyme-
II) can be obtained.

Examples of the ligand include C represented by the above formula (I).
Although any terminal glycine adduct can be used, peptides including 2 to 6 amino acid residues including glycine specifically listed as preferable ones for confirming the enzyme activity can be mentioned. Of these, D-Tyr-T
rp-Gly, Phe-Gly-Phe-Gly and Gly-Phe-Gly are more preferable, but those that ligand Phe-Gly-Phe-Gly are strong against the enzyme of the present invention (also referred to as the present enzyme). It is particularly preferable because it has affinity.

These ligands are usually used by being bound to a water-insoluble carrier, but it is important that the carboxyl group of the C-terminal glycine residue of the peptide used as the ligand is in a free state for binding to the present enzyme. Yes, it is necessary to bind to the carrier via the amino group of the N-terminal amino acid residue. That is, the carrier may be anything as long as it can bind to the amino group of the peptide, and also an active group that reacts with an amino group may be chemically introduced into the carrier, or a commercially available carrier that has already been introduced may be used. May be used. The method of chemically introducing may be a commonly used method, for example,
As described in "Biochemistry Experimental Method Vol. 5, First Volume, pp. 257 to 281" by Kasai, Tokyo Kagaku Dojin (1975), for example, cyanogen bromide is used for agarose to introduce an imidocarboxyl group. Commercially available activated carriers include, for example, agarose-based, cellulose-based, hydrophilic polyvinyl-based, etc., when the substrate is used as an index, but any of these may be used. As an agarose-based carrier, CNBr-activated Sepharose 4B (manufactured by Pharmacia) using the CNBr method for binding to the amino group of the ligand, CH-Sepharose 4B using the carbodiimide method, ECH-Sepharose 4B (above,
(Pharmacia), Affi-Gel 10, Affi-Gel 15 (above, manufactured by Bauorad), Tresyl-activated Sepharose 4B using the tresyl chloride method (Pharmacia)
Etc. Examples of the cellulosic carrier include formyl cellulofine (manufactured by Chisso Corporation) using the formyl method. As the hydrophilic polyvinyl carrier, AF-carboxytoyopearl 650 using the carbodiimide method, AF-formyltoyopearl 650 using the formyl method, AF-tresiltoyopearl 650 using the tresyl chloride method,
AF-epoxy toyopearl 650 using epoxy activation method
(These are manufactured by Tosoh Corporation). The binding reaction with the ligand may be performed according to the instruction manual of each carrier.

Of these, the method for producing Affi-Gel 10 will be described.
The reaction between Affigel 10 and the peptide is carried out in a buffer such as 0.001 to 1 M, preferably 0.1 M mops-potassium hydroxide. The reaction conditions may be 0 to 20 ° C, 10 minutes to 24 hours, and pH 3 to 11 or so, but preferably 4 ° C, 4 to 24 hours, pH 5 to
Perform under the conditions of 9. The mixing ratio of Affigel 10 and the peptide used for binding is about 25 μm of peptide to 1 ml of Affigel.
Since the more the ol is added, the more the binding is performed, any amount may be used within the range, but from the viewpoint of binding efficiency, about 1 to 20 μmol is conveniently used. After the reaction, thoroughly wash with the buffer used during the reaction and add Tris-hydrochloric acid (pH 8.0) to a final concentration of 50 m.
M is added so that the unreacted active groups are blocked by a method such as shaking at 4 ° C. for 1 hour. The substrate analog affinity gel is prepared as described above.

Substrate affinity chromatography may be carried out batchwise or continuously packed in a column. The time for contacting the sample with the gel may be such that the present enzyme is sufficiently adsorbed, but it is usually 20 to 24 hours. The non-adsorbed components are thoroughly washed out with a buffer solution having the same composition as that used for equilibration of the gel and having a low ionic strength and a pH of 6.0 to 11.0, preferably 7.0 to 9.0, for example, 10 mM Hepes-potassium hydroxide (pH 7.0). . Of these, the fraction in which the enzyme activity is present is eluted. The eluate is
Any composition may be used as long as the enzyme can be efficiently obtained,
As a preferable one, a buffer solution having a pH of 7.0 to 9.0 containing 0.1 to 10 M sodium chloride together with about 1 to 40% acetonitrile, for example, 20 mM acetonitrile, 10 mM Hepes-sodium hydroxide containing 0.4 M sodium chloride (pH 7. 0). Further, elution may be carried out by applying a concentration gradient when packed in a column.

In some cases, before or after performing the substrate analog affinity chromatography [hereinafter represented by (5)] step, or both before and after the fractionation by precipitation described above [represented by (1) below]. ], Heparin affinity chromatography [hereinafter represented by (2)], molecular weight fractionation by dialysis, gel filtration, etc. [hereinafter represented by (3)] and / or ion exchange chromatography [hereinafter represented by (4)] ] You may implement a process. Thus, this enzyme (enzyme-I and enzyme-
II) can be separated from other contaminants, but it is effective to carry out the steps (3) and / or (4) for separating the enzyme-I and the enzyme-II. In general, it is preferable to carry out a total of 1 to 6 steps, and further carry out the above step (5) or (3) at the final stage. As a specific example of the combination of each step, only (5),
(1) → (5), (5) → (3), (2) → (5),
(1) → (3) → (5), (2) → (3) → (5),
(1) → (5) → (3), (2) → (5) → (3),
(2) → (1) → (5), (1) → (2) → (3) →
(5), (1) → (2) → (5) → (3), (1) →
(3) → (5) → (3), (1) → (2) → (1) →
(5), (1) → (2) → (1) → (3) → (5),
(2) → (1) → (5) → (3), (2) → (1) →
(3) → (5), (2) → (1) → (3) → (5) →
(3), (1) → (2) → (3) → (5) → (3),
(1) → (3) → (2) → (3) → (5), (1) →
(3) → (2) → (3) → (5) → (3), (4) →
(3) → (5), (5) → (3) → (5) → (3),
(1) → (5) → (3) → (5) → (3), (4) →
(5), (1) → (3) → (5) → (4) → (3),
(1) → (3) → (4) → (3) → (5), (1) →
(2) → (3) → (5) → (3) → (4), (1) →
(2) → (3) → (5) → (4) → (3) or (4) → (5) → (3). Of these, (1) → (2) → (3) → (5), (1) → (2)
→ (3) → (5) → (3), (1) → (3) → (2) →
(3) → (5) or (1) → (3) → (2) → (3)
→ (5) → (3), (1) → (2) → (3) → (5) →
It is more preferable to proceed in the order of (4) → (3).

Hereinafter, the steps (1) to (4) will also be described in detail. In addition, these are all 0 ℃ ~ 10 ℃, preferably 4 ℃
Done in.

Examples of the substance used for the fractionation by precipitation in (1) include salts such as ammonium sulfate, organic solvents such as ethanol and acetone, and polymers such as polyethylene glycol. The concentration to be added is not particularly limited, but it is preferable that the enzyme can be recovered in good yield and can be separated from other protein components. For example, when 30 to 50% saturated ammonium sulfate and 10 to 15% (w / v) polyethylene glycol 6000 were added, this enzyme came to the precipitation fraction, whereas most of the protein was present in the supernatant, so the efficiency was Well refined.
The addition is preferably carried out gradually with stirring with a stirrer. After completion of the addition, the mixture is allowed to stand for at least 1 hour or more and then centrifuged to collect a fraction containing the present enzyme. When the precipitated fraction is collected, it is dissolved in a suitable buffer. Buffer, pH 6.0-11.0, preferably pH 7.0-9.
The composition may be any as long as it is 0, and examples thereof include Tris-hydrochloric acid, Hepes-potassium hydroxide, and Tess-sodium hydroxide. The concentration is not limited as long as the buffer capacity can be maintained, but is preferably about 5 to 50 mM.

The active fraction obtained in (1) may then be subjected to (1) again or to any one of steps (2) to (5), but ammonium sulfate is added to the fraction in (1). When using (2) or (4) or (5) with a salt such as
It is necessary to reduce the salt concentration at which the enzyme can bind to the gel used in the next step by adding the appropriate buffer solution in the step (3). Further, when the precipitate is dissolved and left standing for 1 hour or more, or when dialysis is performed, an insoluble substance may be generated. Therefore, this is removed by, for example, centrifugation or filtration.

The heparin affinity chromatography (2) may be performed in a batch mode or in a continuous mode by packing a column with gel. Gels with heparin as a ligand are commercially available, heparin Zepharose CL-6B (manufactured by Pharmacia), Affigel heparin (manufactured by Bio-Rad), heparin agarose (manufactured by Sigma), AF-heparint yopal. There are 650 (manufactured by Tosoh Corporation) and others.

The biological extract is treated directly or after the fractionation treatment by precipitation shown in (1), and then contacted with a heparin affinity gel. The contact time may be such that the enzyme is sufficiently adsorbed, but it is usually 20 minutes to 12 hours. A component having no affinity for heparin, having a strong ionic strength to the extent that the enzyme is not eluted, and having a pH of 6.0 to 11.0, preferably 7.0 to 9.0, for example, 10 mM Hepes-potassium hydroxide (pH 7.0).
To remove enough. After that, the fraction containing this enzyme is eluted. As the eluate, one having a high recovery rate of the present enzyme activity is preferable, for example, one having a pH of 6.0 to 11.0 containing salts commonly used for enzyme purification such as 0.5M-2M sodium chloride, potassium chloride, and ammonium sulfate. preferable. When the column is packed, elution may be carried out in one step by a salt concentration gradient. For example, elute with 10 mM Hepes-potassium hydroxide buffer (pH 7.0) containing 0.3-2.0 M sodium chloride.

The active fraction obtained in the step (2) may be subjected to any of the steps (1) to (4) next, but the step (2) may be performed again.
When proceeding to (4) or (5), (3) is carried out first, or a large amount of a low ionic strength pH 6.0 to 11.0, preferably 7.0 to 9.0 buffer solution of 50 mM or less, for example 5 mM Hepes- It is necessary to add potassium hydroxide (pH 7.0) etc. to reduce the ionic strength so that the enzyme can be adsorbed on the gel used in (2), (4) or (5).

It is a step of removing low-molecular substances by dialysis, gel filtration, etc. of (3). In the case of dialysis, the membrane to be used may have a molecular weight cut-off such that the enzyme cannot pass through it. 00
0 to 10,000 is preferable. The dialysis method may be, for example, a commonly used method described in “Biochemistry Experiment Course, Vol. 5, First Volume, pp. 252 to 253” by Soda, Tokyo Kagaku Dojin (1975), and may be used for several hours to several days. , Low ionic strength, pH 6.0
~ 11.0, preferably pH 7.0-9.0 buffer, such as 10 mM Hepes-potassium hydroxide (pH 7.0), 10 mM Tris-hydrochloric acid (p
H7.5) etc. If an insoluble substance is deposited during dialysis, it is removed by, for example, centrifugation or filtration.

Any gel filtration may be used as long as it is generally used as a carrier for gel filtration. For example, Sephadex G-10, G-15, G-25, G-50, G-75, G-100, Sephacryl S-200, S-300 (all manufactured by Pharmacia) Toyopearl HW-40, HW-55 ( Tosoh Corporation), Biogel P-
2, P-4, P-6, P-10, P-30, P-60, P-100 (all manufactured by Bio-Rad Co.) and the like are preferable. The buffer solution to be used is
It is similar to the composition to be used in dialysis. However, if the ionic strength is too low, adsorption of the enzyme to the gel may occur, so the concentration should be 5 to 200 mM, preferably 10 mM.
~ 20mM. The gel filtration method may be carried out, for example, as described in “Biochemistry Experiment Course, Vol. 5, Vol. 1, pp. 283 to 298” by Soda, Tokyo Kagaku Dojin (1975). After adding a sample in an amount sufficient to achieve a sufficient separation capacity with respect to the bed volume of the gel filtration carrier, elution is carried out to collect a fraction having the present enzyme activity.

The active fraction obtained by the step (3) can be advanced to each step (1) to (5) without any special treatment.

Regarding the ion exchange chromatography, any commercially available carrier for ion exchange chromatography may be used. For example, Aminex, Dowex, Amberlite, S
P-Sephacryl M, Asahipak, DEAE-Toyopearl, DEAE-Seph
adex, CM-Sepharose, DEAE Bio-Gel A, CM-Cellulose, D
EAE-Cellulofine, Partisil SCY, Mono Q and Mono S are preferred. The buffer solution to be used and the method of use may be in accordance with the method described in the section of heparin affinity gel. The basic operation method may be according to the general method described in "New Basic Biochemistry Experimental Method 2, Extraction / Purification / Analysis I" (Maruzen, 1988).

The active fraction obtained in the step (4) may be subjected to any one of the steps (1) to (5) next, but the step (4) may be performed again or the step (2) or (5) may be performed. If you want to proceed,
Do (3) first, or use a large amount of low ionic strength of 50 mM or less.
pH 5.0 to 11.0, preferably 6.0 to 8.0 buffer, such as Hepes-sodium hydroxide (pH 7.0) and the like,
The gel used in (2), (4) or (5) needs to be reduced to an ionic strength at which this enzyme can be adsorbed. A crude product of the enzyme of the present invention can be obtained through the above purification steps. Such a crude product of the enzyme further comprises (3)
The enzyme preparation can be obtained by isolating into fractions having a peak at a molecular weight of about 25,000 and a fraction having a peak at a molecular weight of about 40,000 by the protein separation means using the gel filtration step.

Each of the above steps is carried out according to another method of measuring the activity of an enzyme according to the present invention, which will be described later, using the compound of the formula (I) or the formula (II) as a substrate, the enzyme-I and / or the enzyme. It is carried out by following the activity of -II and obtaining an active fraction.

Use of Enzyme Composition The use of the composition comprising the enzyme of the present invention provides the following fourth and fifth inventions, that is, C represented by the above formula (I).
A method for producing a C-terminal α-hydroxylglycine adduct represented by the formula (II), which comprises treating the terminal glycine adduct with a composition containing the enzyme-I, and the formula (II). A method for producing a C-terminal amidated compound represented by the above formula (III) can be provided by treating the adduct with a composition containing enzyme-II. Further, by combining these Enzyme-I and Enzyme-II, the compound of formula (I) can be converted to the compound of formula (III) in a single reaction composition. The use of Enzyme-II in the step of converting a compound of formula (I) to a compound of formula (III) means that in the presence of only the enzyme of type Enzyme-I, formula (II) to formula (III)
It will be apparent that this conversion is significant because the conversion can be achieved under milder enzymatic reaction conditions as compared to the need to be exposed to chemical hydrolysis conditions in the conversion to the compound. In particular, it is suitable for treating substrates that are unstable under alkaline conditions.

These production methods can be used as long as they contain the enzyme of the present invention regardless of their concentration and purity, but they are contaminated in consideration of isolation and purification of the product from the reaction mixture and the compound of formula (II). It is advantageous to use the enzyme inclusions according to the invention in which the protein has been significantly removed. If a biological extract having the present enzyme activity is used, it can be used as it is or simply as a concentrate thereof.

Compounds of formula (I) include all those mentioned above, but suitable for use in these processes are:
In particular, compounds of formula (III), such as arginine vasotocin (AVT), luteinizing hormone-releasing hormone (LH-RH),
Oxytocin, gastrin, gastrin secretagogue peptide (GGRP), calcitonin (CT), vasoactive intestinal peptide (VIP), thyrotropin-releasing hormone (TR)
H), melanophore stimulating hormone (MSH), MSH release inhibitory hormone (MIH), cholecystokinin octapeptide (CCK)
-8), substance P (SP), fat mobilizing hormone, pancreatic polypeptide (PP), growth hormone releasing factor, secretin, cerulein, mollusc cardiac excitatory neuropeptide,
Vasopressin, adrenocorticotropic hormone (ACTH), color-changing hormone, bombesin, photoadaptation hormone, motilin, apamin, aritesin, eledoisin, catsinin, granuriverin R, scotophobin, hylanbate cerulein, mast cell degranulation peptide, fisaremin, phylloserlein , Phylomezine, promeritin, bombinin, mastoparan, mastoparan-X, melittin-1,
Preference is given to their corresponding compounds of the formula (I) or formula (II), which can be converted into lanatensin, lanatensin-R and the like in the present production method.

The treatment can be carried out by adding ascorbic acid and catalase to the reaction solution in an ordinary buffer, especially in the reaction using enzyme-I. It is preferable to carry out the method in consideration of the conditions of the method for measuring the activity.

Method for measuring enzyme activity and method for searching for new enzyme using the same The enzyme-I and enzyme-II of the present invention described above can be traced by the following method for measuring activity, and this measuring method is Useful for rational implementation of enzyme preparation methods.

Needless to say, these measurement methods are not a one-step reaction in which the C-terminal amidation reaction has been considered in the past, but the reaction via an intermediate (C-terminal α-hydroxylglycine adduct)
It is based on the finding that it is a stepwise reaction.

First, the enzyme-I activity is (i) buffering a subject predicted to have that activity to pH 5-8, (ii) this buffer solution having the formula (I) shown above. Incubation with addition of C-terminal glycine adduct and L-ascorbic acid and catalase, and (iii) detection of reaction product represented by formula (III) by HPLC using acetonitrile-containing buffer (pH 6-10) It is measured by the method characterized by including the step of performing.

The activity of enzyme-II is (i) the step of buffering a subject predicted to have the activity to pH 4 to 8,
i) adding the C-terminal α-hydroxylglycine adduct represented by the formula (II) to the buffer solution and incubating, and (iii) detecting the resulting C-terminal amidated compound represented by the formula (III). The method is characterized by including a step.

These methods are provided as the sixth and seventh inventions of the present application, respectively.

The subject referred to in these inventions includes any fluid having those enzymatic activities, and in particular, biological fluids having those activities, that is, homogenized organism organs, as well as body fluids such as blood. And lymph, and treatment liquids such as purification treatments thereof. Further, a treatment liquid derived from microbial cells is also included in the biological fluid.

The buffer used for buffering these subjects is not particularly limited, and those commonly used can be used. For example Tris-
Examples thereof include hydrochloric acid and Hepes-potassium hydroxide. The concentration of the buffer in the buffer solution may be any concentration as long as the buffer action is achieved, but 20 to 200 mM is usually suitable.

Each buffer solution is the sixth invention (referred to as "former").
Is adjusted to about pH 6 to 8, preferably about pH 6.5, and for the seventh invention (referred to as "the latter"), adjusted to about pH 4 to 8, preferably about pH 6. The C-terminal glycine adduct added in the former to the buffer solution thus prepared is represented by the formula (I) listed as a substrate of the enzyme and a preferable substrate for confirming the activity of the above-mentioned enzyme-I. It is preferable to use those that are Generally, a suitable concentration of this compound is about 0.1 μM to 2 mM. Further, it is necessary to add L-ascorbic acid which is considered to function as a cofactor and catalase as an activator. In general,
The concentration of L-ascorbic acid is preferably 0.5 to 2 mM, and the concentration of catalase is suitably 40 to 100 μg / ml. In addition, a metal ion may be added to the buffer solution, but this addition is not particularly required for this activity measurement, and the addition may be performed because higher activity may be obtained as compared with the case of no addition. It is preferable. Zn ²⁺ , Cu ²⁺ , Ni ²⁺ , Co ²⁺ , Fe ^3+, etc. are suitable as the metal ions to be used.
²⁺ and Zn ²⁺ are preferred. The concentration of the metal ion in the buffer solution is 0 to 1000 μM, preferably 0 to 10 μM. Compounds that provide such metal ions include, but are not limited to, CuSO ₄ , CuCl ₂ , ZnCl ₂ , NiCl ₂ , CoCl ₂ , FeCl ₃
Etc.

As a concrete example of such a reaction composition, for example, the composition of Example 2 described later will be referred to. On the other hand, the reaction composition in the latter is prepared by using the corresponding compound of formula (II) instead of the compound of formula (I). In this case, cofactors such as ascorbic acid and catalase are not required.

In both measurement methods, the amount of test substance used can be variously changed without particular limitation, but is preferably a pm with respect to the amount of substrate (a nanomol (nmol)) present in the reaction system.
ol / hr or more, more preferably 10 × a pmol / hr or more, and most preferably 10 × a pmol / hr to a mol / hr [unit shows enzyme activity and can react at 37 ° C. for 1 hour. It is suitable to adjust so that the enzyme activity is expressed in picomoles (pmol).

Incubation is performed at 1 to 55 ° C., preferably 25 to 40 ° C. in the former case, particularly preferably 30 ° C. for 2 to 24 hours while shaking, and in the latter case, preferably 15 to 35 ° C., most preferably about 25 ° C. Let stand for 1 minute to 48 hours.

In the above steps, the compounds of the formula (II) and the compound of the formula (III) produced respectively are detected by their substrates and products, for example, in the former case, the compound of the formula (I) and the compound of the formula (II), In the latter case, any method can be used as long as it can separately measure the compound of formula (II) and the compound of formula (III). Generally, the separation and measurement can be carried out by separating and purifying by the following chromatography. Chromatography that can be used above includes ion exchange chromatography, reverse phase chromatography, gel filtration, affinity chromatography, high performance liquid chromatography (HPLC), thin layer chromatography (TLC) and the like. The substrate of formula (II) and the amidated product of formula (III) in the latter reaction system are C
The ends are a carboxyl group and an amide group, respectively, and have different charges. Ion exchange chromatography, reverse phase chromatography and the like using this property are preferable. Affinity chromatography using the antibody of the product is also effective. However, although the separation of the substrate represented by the formula (I) and the product represented by the formula (II) in the former reaction system is quite difficult by the usual chromatographic treatment, the present inventors tried acetonitrile-containing for the first time. High-performance liquid chromatography (HPLC) using a buffer solution (pH 6 to 10, preferably pH 9) as an eluent can advantageously perform separation and measurement. The eluate is preferably subjected to a linear concentration gradient of acetonitrile concentration. As the HPLC column, any commercially available column can be used as long as it is suitable for this purpose, and it is particularly advantageous to use Capsule Pack C18SG, 300Å (manufactured by Shiseido Co., Ltd.).

Thus, each separated substrate and product may be measured for any of their (optionally attached) chemical or physical labels. For this measurement, a known label and a known measurement method can be used, and it is generally convenient to use UV absorption derived from the amino acids forming the substrate peptide.

Since the above measuring methods are accurate and simple, by applying these to the above-mentioned biological fluid, it is possible to search for an enzyme having enzyme-I or enzyme-II activity, respectively. Such a search method is described in the eighth aspect of the present application.
And provided as a ninth invention.

The biological fluid to be searched is, of course, not limited to those predicted to have the above-mentioned enzyme activity, but also includes living cells, tissues and extracts of animals and plants. For example, the extract is "Experimental Biology Course 6, Cell Fractionation Method" (Maruzen, 1984), "Biochemistry Experiment Course 5, Enzyme Research Method (above)"
(Tokyo Kagaku Dojin, 1975), “Basic Biochemistry Experimental Method 1, How to Handle Biological Materials” (Maruzen 1974), and the like.

〔Example〕

Next, the present invention will be described in more detail with reference to examples. The present invention is not limited to this.

Example 1 Preparation of Substrate Analog Affinity Chromatography Gel 5 ml of Affi-Gel 10 was filled with 10 m of isopropanol.
l Weighed in Econo column (Bio-Rad).
After flushing out the isopropanol, it was washed with 50 ml of 10 mM sodium acetate buffer (pH 4.5) and then with 10 ml of 0.1 M mops-sodium hydroxide buffer (containing 80 mM calcium chloride, pH 7.5). After the gel was transferred to a 20 ml bottle, 40 mg (about 100 μmol) of phenylalanylglycyl, phenylalanylglycine (Phe-Gly-Phe-Gly, manufactured by Sigma) was dissolved in the above mops-sodium hydroxide buffer solution (10 ml). The mixture was mixed with and reacted by shaking at 4 ° C. for 18 hours. After that, 0.5 ml of 1 M Tris-hydrochloric acid buffer solution (pH 8.0) was added, and the mixture was reacted by shaking at 4 ° C. for 1 hour to inactivate unreacted active groups. The gel was washed with the Mops-sodium hydroxide buffer solution and then with ion-exchanged water, suspended in 0.02% NaN ₃ , packed in a column, and stored at 4 ° C. From the amount of peptide (Phe-Gly-Phe-Gly) used in the reaction and the amount of peptide in the recovered solution, it was calculated that about 10 µmol was bound per 1 ml of gel.

Example 2 Preparation of phenylalanylglycyl phenylalanylhydroxylglycine as a substrate Phenylalanylglycyl phenylalanylglycine (FGFG) (manufactured by Sigma) was weighed out in an amount of 3 mg to obtain 50 mM Hepes-
Potassium hydroxide buffer (pH 5.5), 3 mM ascorbic acid,
10 mM potassium iodide, 0.25 mg / ml catalase, 0.25 nM copper sulfate, 7.5% acetonitrile and international patent application JP89-005
200 μm of horse serum derived amidating enzyme composition as described in Example 2 of No. 21 to make a total volume of 10 ml, 30
The amidation reaction was carried out aerobically at 20 ° C for 20 hours. The reaction was stopped by the addition of 10% formic acid, and phenylalanylglycyl phenylalanylhydroxylglycine (FGFhyG) was fractionated using high performance liquid chromatography (HPLC). Capsule Pack C18SG, 300Å (made by Shiseido) was used as the HPLC column. The elution solvent used was 1 mM ammonium hydrogen carbonate (pH 9.0) and acetonitrile, and a linear concentration gradient was applied to increase acetonitrile from 0% to 40% in 30 minutes. The peptide was detected by absorption at 214 nm. The results are shown in Fig. 1. The phenylalanylglycyl phenylalanylglycine peak at 10.7 minutes almost disappeared due to the C-terminal amidation reaction. Along with that, peaks of the α-hydroxyglycine derivative at 9.9 minutes and the amidated product at 14.5 minutes were observed. The structures of these substances were confirmed by FAB-MS spectral analysis and NMR analysis. FIG. 2 shows the result of FAB-MS spectrum in glycerin solution. The parent peak represents a molecular weight of 442, and fragmentation confirmed 425 and 408 m / z fragments with one or two -OH groups present at the C-terminus, an α-hydroxylglycine adduct. Was showing that. The peak for 9.9 minutes was collected, promptly made into a 10% formic acid solution, and freeze-dried to prepare a substrate for the enzyme-II of the present invention. This substrate could be similarly prepared by using the enzyme-I-containing material of the present invention instead of the amidating enzyme composition. As described above, the α-hydroxylglycine derivative is stable under acidic conditions but unstable under alkaline conditions and decomposes into amidated products and glyoxylic acid regardless of the enzymatic reaction. Therefore,
The first C-terminal amidation reaction of this example was carried out under acidic conditions. At this time, if the reaction is carried out at pH 7.5 or higher, the α-hydroxylglycine derivative cannot be confirmed. A known C-terminal amidating enzyme is a C-terminal glycine adduct represented by the formula (I) and a C-terminal glycine represented by the formula (III).
It was thought that the conversion to the terminal amidation product and glyoxylic acid was due to the inclusion of the non-enzymatic conversion under this alkaline condition, and the catalytic reaction of the enzyme itself was represented by the formula (I)
Is a conversion reaction from the C-terminal glycine adduct represented by the formula (II) to the C-terminal α-hydroxyl glycine adduct represented by the formula (II). Therefore, the yield of the conventional amidation reaction by the C-terminal amidating enzyme under acidic conditions was generally low.

Example 3 Preparation of enzyme-I from horse serum (1) Polyethylene glycol 6000 (Wako Pure Chemical Industries) 25% aqueous solution (w / v) was added to 100 ml of commercially available horse serum (manufactured by Gibco).
Was gradually added with stirring to 100 ml, that is, a final concentration of 12.5%. In addition, all of the following operations were performed at 4 ° C. After standing for 12 hours, centrifuge (10,000 xg, 10
The resulting precipitate was dissolved in 120 ml of 10 mM Hepes-potassium hydroxide buffer (pH 7.0), and the mixture was allowed to stand for 2 hours, and then the resulting insoluble material was centrifuged again (10,000 × g,
It was removed by 10 minutes) to obtain a supernatant (127 ml) containing enzyme-I.

(2) The active fraction obtained in (1) above was packed with a column (1.6 × 15 cm) packed with heparin Sepharose CL-6B (Pharmacia) equilibrated with 10 mM Hepes-potassium hydroxide buffer (pH 7.0). ). After washing out non-adsorbed substances with 96 ml of the same buffer, 10 mM Hepes containing 0.5 M sodium chloride
Elution was performed with potassium hydroxide buffer (pH 7.0) (flow rate 3
0 ml (/ hr). The elution pattern is shown in FIG. Enzyme-I was eluted with 0.5 M sodium chloride-containing buffer [collection of fraction numbers No. 14 to 16 (100 ml)] (3) The above fractions were subjected to Sephadex G-25 Fine (Pharmacia) column chromatography. Graphography (5cmφ × 23c
Gel filtration was carried out using m). As the solvent, 10 mM Hepes-potassium hydroxide (pH 7.0) was used and eluted at a flow rate of 2 ml / min. The protein was detected by absorption at 280 nm, and 100 ml of protein-containing fractions were collected.

(4) Affigel 10-Phe-G prepared according to Example 1
Fill the column with 5 ml of ly-Phe-Gly gel (1.0 x 6.0 c
m), equilibrated with 10 mM Hepes-potassium hydroxide buffer (pH 7.0) containing 0.1 M sodium chloride. The sample (18.1 ml) obtained in the above (3) was applied to this column. In order to sufficiently adsorb the enzyme-I to the gel, the liquid passed through the column was circulated through the column many times (flow rate 20 ml / hr). 1
After 2 hours, the circulation was stopped and the non-adsorbed substance was washed out with 35 ml of the buffer used for equilibration.
Elution was performed with 8 mM Hepes-potassium hydroxide buffer (pH 7.0) containing% acetonitrile (flow rate 20 ml / hr). Enzyme
The activity of I was found only in the eluted fraction (10 ml).

(5) After subjecting the purified product obtained in (4) above to the treatment in (3) again, 10 mM Hepes-potassium hydroxide buffer (pH)
MonoQ column (Pharmacia 0.5 × 5) equilibrated with 7.0)
cm) and add a linear NaCl concentration gradient in the same buffer to a third
The protein was eluted by applying as shown in the figure. At this time, the flow rate was 0.5 ml / min.

Table 1 shows total protein, total enzyme activity, specific activity, yield and purification rate in each step of the purification performed in the above (1) to (5).

The activity was measured by carrying out a reaction according to the method for preparing FGFG from FGFhyG described in Example 2 for each purified product, and quantifying FGFhyG by the HPLC described therein. 1 U of enzyme activity was defined as the amount of 1 nmole of FGFhyG produced at 37 ° C. for 1 hour.

The amount of protein was measured by the modified Lowry method (Bensadou
n et al., Anal. Biochem., 70 , 265, 1976), and a standard curve was prepared with bovine serum albumin (fraction V, manufactured by Sigma).

As shown in Table 1, this enzyme could be purified about 500 times with a yield of 2%. When further purification is required, the above steps (3) to (5) may be repeated, or any one of these steps may be repeated.

Example 4 Preparation of Enzyme-II from Horse Serum Horse serum was treated in the same manner as in Example 3 except that each purification step was performed while following the activity of enzyme-II.

Table 2 shows the total protein, total enzyme activity, specific activity, yield and purification rate in each purification step (1) to (5).

The activity was measured by dissolving phenylalanylglycyl phenylalanylhydroxylglycine (FGFhyG) obtained in Example 1 at a concentration of 5 mM in 10 mM Hepes-potassium hydroxide (pH 6.5), and adding a sample at each step, 30 ℃ for 100μ
It was made to react with. After reacting for 1 hour, the reaction was stopped by adding 10% formic acid, and the reaction product was quantified by HPLC using the conditions of Example 2. At this time, a control reaction without addition of a sample was also performed, and it was confirmed that the non-enzymatic conversion hardly proceeded. The HPLC pattern of the reaction solution is shown in FIG. 4 (the reaction conditions in the figure are 37 ° C. and pH 6.9, and the reaction time is shown in the figure).
The activity was expressed in units (U). 1 U is 1 nmol of FGF-
NH ₂ was defined as the amount of enzyme produced at 30 ° C. for 1 hour.

The protein amount was measured in the same manner as in Example 3.

As shown in Table 2, this enzyme could be purified approximately 1800 times with a yield of 2%.

〔The invention's effect〕

The enzyme of the present invention can be used in the production of the C-terminal amidated product represented by the formula (III) from the C-terminal glycine adduct represented by the formula (I). Also, enzyme-I and enzyme-
When used in combination with II, the C-terminal α represented by the formula (II)
-Hydroxylglycine enables efficient production of the C-terminal amidated product of formula (III) under mild reaction conditions such that the adduct is not hydrolyzed. Furthermore, the preparation method of the present invention has the effect of efficiently preparing the enzyme composition of the present invention in a high yield.

Each enzyme activity measuring method of the present invention comprises an enzymatic activity for converting a C-terminal glycine adduct to a C-terminal α-hydroxylglycine adduct represented by the formula (II) and a C-terminal hydroxylglycine addition represented by the formula (II). The enzyme activity of converting the body to the C-terminal amidated product can be quantified quickly and accurately. In addition, the enzyme search method using this enables the search for an enzyme having the above-mentioned activity, which has hitherto been unknown.

[Brief description of drawings]

FIG. 1 is an HPLC pattern when FGFhyG was produced using FGFG as a substrate and the enzyme-I of the present invention. FIG. 2 shows the results of FAB-MS spectral analysis performed to confirm the molecular structure of the prepared FGFhyG. FIG. 3 is a chromatographic pattern showing the separation of Enzyme-I and Enzyme-II of the present invention by chromatography on a Mono Q column. A white square plot shows the enzyme-II activity, a black circle plot shows the enzyme-I activity, and a broken line shows a linear concentration gradient of sodium chloride. FIG. 4 is a time-dependent HPLC pattern when FGF-NH ₂ was produced using the enzyme-II of the present invention with FGFhyG as a substrate.

Front page continuation (72) Inventor Masahiro Tajima 1050 Shinba-cho, Kohoku-ku, Yokohama-shi, Kanagawa Inside Shiseido Laboratories, Inc. (72) Inventor Hiroshi Okamoto 2-15-3-205 Kakugoro, Aoba-ku, Sendai City, Miyagi Prefecture

Claims (5)

[Claims] 1. The following formula (I) (In the above formula, A represents a residue other than an α-amino group or an imino group and an α-carboxyl group derived from a natural α-amino acid, and X represents an N atom via a hydrogen atom or a carbonyl group. (Representing the residue of an amino acid derivative that binds to), acting on a C-terminal glycine adduct represented by the following formula (II) A C-terminal α-hydroxyglycine adduct represented by the above formula (wherein A and X represent the above meanings) and having a molecular weight of about 25,000 daltons by a molecular weight determination method using gel filtration. , Optimum pH is about 5-7 and stable pH
Is 4-9, and the optimum temperature for action is 25-40 ° C, C
An enzyme involved in the C-terminal amidation of a terminal glycine adduct. 2. The enzyme activity-containing material according to claim 1 is treated by substrate affinity chromatography using a C-terminal glycine adduct represented by the formula (I) as a ligand and anion exchange chromatography. Claim 1
A method for preparing an enzyme involved in the C-terminal amidation described. 3. A C-terminal α-hydroxyglycine adduct of formula (II), characterized in that the C-terminal glycine adduct of formula (I) is treated with the enzyme of claim 1. Production method. 4. (i) buffering a subject predicted to have the activity of the enzyme of claim 1 to pH 5 to 8, (ii) this buffer solution represented by the above formula (I) A step of adding and incubating a C-terminal glycine adduct, L-ascorbic acid and catalase, and (iii) an eluent containing the resulting C-terminal α-hydroxylglycine adduct represented by the formula (II) at pH 6 to 10 And a step of detecting by HPLC using the above method. 5. The method for searching for an enzyme according to claim 1, wherein the measuring method according to claim 4 is applied to a subject.