JPH03216187A - Enzyme participating in c-terminal amidation, production thereof and use thereof - Google Patents

Abstract

NEW MATERIAL:An enzyme having about 25000 dalton molecular weight, participating in C-terminal amidation of a C-terminal glycine adduct and capable of acting on a C-terminal glycine adduct of formula I (A is natural alpha-amino acid-derived alpha-amino group or imino group or residue except alpha-carboxyl group; X is H or amino acid derivative residue bonding to N atom through carbonyl group) and capable of producing a C-terminal alpha-hydroxylglycine adduct of formula II. Optimum pH is about 5-7 and optimum reaction temperature is 25-40 deg.C. Substrates are compounds in which glycine is bonded to a C-terminal amino acid residue [-N(H)-A-CO-]. USE:Formation of a C-terminal alpha-hydroxylglycine adduct as an intermediate in C-terminal amidation. PREPARATION:An enzymatic activity-containing material is treated with substrate-affinity chromatography in which the ligand is a C-terminal glycine adduct of formula I and anion-exchange chromatography for preparation of the objective enzyme.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention provides a novel enzyme involved in the C-terminal amidation of a C-terminal glycine adduct, a method for preparing and using the same, and a method for measuring the activity of the enzyme and a method thereof. The present invention relates to a method for searching for the enzyme using.

[Conventional technology]

Peptides that exhibit physiological activity only when their C-terminus is amidated, such as calcitonin, gastrin, secretin vasoactive intestinal peptide (VIP), growth hormone-releasing factor, and corticotropin-releasing factor, are converted to glycine by an enzymatic reaction in vivo. It is known that it is produced from adducts. Many of these physiologically active peptides are useful as pharmaceuticals, and calcitonin, secretin, and the like are currently commercially available as pharmaceuticals.

Conventionally, these bebutides have been obtained mainly by separation and purification from living organisms, which is a complicated process and also makes it difficult to obtain the original living organisms. Therefore, the above-mentioned peptides currently available on the market are extremely expensive.

Therefore, in recent years, attempts have been made to produce these physiologically active peptides using recombinant DNA technology. However, recombinant DNA technology using Escherichia coli, yeast, Bacillus subtilis, etc. as hosts does not allow C-terminal amidation of the produced peptides, which is an obstacle in producing the above-mentioned peptides. Therefore, there is a need for a technology that allows C-terminal amidation to be easily and inexpensively performed in vitro.

On the other hand, the enzymes involved in the above-mentioned in-vivo enzymatic reaction, that is, the C-terminal amidation of C-terminal glycine adducts of peptides, are
Beptidylglycine-α-amidating monooxygenase (C-terminal amidating enzyme) (EC.1.14.1
7.3) (Bradbury et al., Nat.
ure, 298, 686. 1982: G.I.E.M.
Botski et al., J. Biol. Chem. , 259
, 6385. (1984), is thought to catalyze the following reactions.

Elucidation of in vivo amidation mechanism and recombinant DNA
Attempts have been made to purify this enzyme for the purpose of in vitro C-terminal amidation of bebutide produced by this technique. Up to now, examples of purification in which the specific activity has been increased more than 100 times compared to the starting material include bovine pituitary middle lobe (Murth).
y et al., J. Biol. Chem. , 2Ejiang, 1815
．． 1986), porcine pituitary gland (Kizer et al., En
docrinology, month 8. 2262, 19
86: Bradbury et al., Eur. J. Bio
chem. , 169, 579. 1987), porcine atrium (Xojima et al., J. Biochem., 1
05, 440. 1989), African frog body skin (Mizuno et al., Biochem. Biophys
．． Res. Comllun. , 137, 984.
1986), rat thyroid tumor (Mehta et al., Arc
h. Biochem, Biophys. +261+
44, 1988) has been reported.

It is known that all of these purified enzymes require oxygen and copper ions to express their activity, and that their activity increases by the addition of an ascorbic oxygen reducing agent. Recently, research has been progressing to elucidate the reaction mechanism using purified enzymes, and the existence of reaction intermediates has been suggested (Bradbury et al., Eur. J. Bioch.
em. , 169, 579-584 (I987), R
awer et al., J. Am. Chem. Soc. ,Month. 0
．． 8526-8532 (I988), Young
et al., J. Eight meters. Chem. Soc. , 111
．． 1933-1934 (I989)).

However, at present, there is no example in which the isolation of the intermediate or the relationship between the intermediate and the amidation enzyme has been clarified.

[Problem to be solved by the invention]

As mentioned above, C-terminal amidation enzymes exhibit very interesting effects in vivo, and compositions with a certain degree of purity derived from specific biological organs are also known. However, these compositions cannot be said to be sufficient in terms of purity, stability, and manufacturing cost for use in the production of C-terminal amidated products in vitro. In order to solve these problems, we believed that it was necessary to collect basic knowledge about the enzyme, that is, to elucidate the reaction mechanism that performs the C-terminal amidation reaction, and when we attempted to isolate the intermediate product, we found that We succeeded in isolating the body and determining its structure. From this result, the C-terminal amidation reaction is different from the one previously thought.
It was found that the reaction was not a stepwise reaction, but a two-step reaction via an intermediate (corresponding C-terminal α-hydroxylglycine adduct). Therefore, as a result of intensive investigation into the search for enzymes that catalyze each reaction, we discovered a new substance that has the desired enzyme activity, and completed the present invention. That is, an object of the present invention is to provide a novel enzyme group 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 for substances having the enzyme activity using this method. .

[Means to solve the problem]

Therefore, according to the present invention, in order to solve the above-mentioned problems, the following formula (I) (wherein A is a group other than the α-amino group or imino group derived from a natural α-amino acid and the X represents a residue of an amino acid derivative bonded to an N atom via a hydrogen atom or a carbonyl group), and the following formula (II) In the above formula, A and X represent the above meanings)
By producing a C-terminal α-hydroxylglycine adduct shown as
An enzyme involved in the C-terminal amidation of a C-terminal glycine adduct with a molecular weight of 5,000 daltons (hereinafter referred to as “enzyme-
■") is provided.

In addition, formulas (I) and (II) and the following formula (I[I
) means that if A is derived from an α-amino acid having an α-imino group, it does not have a hydrogen atom. In the present invention, the C-terminal glycine adduct represented by formula (I), that is, (H) as a substrate for the enzyme of the present invention, is generally a C-terminal glycine adduct of the formula (I) in which part of the Amino acid derivatives, in particular derived from peptides or proteins, whose C-terminal amino acid residue (-N(
Examples include compounds in which glycine is bonded to peptide H) - A-CO-. C-terminal amino acid residues include natural α-amino acids, especially proteinogenic amino acids, such as aliphatic amino acids such as glycine and alanine; fractional 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, glutamine; basic amino acids such as lysine, hydroxylysine, arginine;
Residues derived from sulfur-containing amino acids such as cysteine, cystine, and methionine; aromatic amino acids such as phenylalanine and tyrosine; heterocyclic amino acids such as tributophane and histidine; and imino acids such as proline and 4-hydroxyproline. Can be mentioned. In addition, the hydrogen atom or residue of an amino acid derivative [represented by As long as the peptides can bind to peptides, there are no restrictions on the types of amino acid residues that constitute them or the chain length of the peptide, and even if the amino acid residues have phosphates, sugars, or other substituents covalently bonded to them. It may also form a complex with lipids. Specific examples of the substituents include groups to be substituted for the guanidino group of the arginine residue, such as alkyl groups such as methyl or ethyl groups, ribose adenosine diphosphate, and citrulline, corresponding to each amino acid residue. or a residue derived from ornithine; a group substituting the ε-amino group of a lysine residue, such as a glucosyl group, pyridoxyl group, biotinyl group, lipoyl group or acetyl group, or phosphoric acid, δ
Compounds having a monohydroxyl group, compounds having a δ-glycosyl group, glutaraldehyde, << is a residue derived from citraconic anhydride; a group substituting the imidazole group of a histidine residue, such as a methyl group, a phosphoric acid group, or an iodine atom or a residue derived from flavin; a group substituting on a proline residue, such as a hydroxyl group, dihydroxyl group, or a glycosyloxy group; a group substituting on a benzene ring of a phenylalanine residue, such as a hydroxyl group or a glycosyloxy group; Groups to substitute for hydroxyl groups, such as glycosyloxy groups, sulfonic acid groups, iodine atoms, bromine atoms, compounds having chlorine atoms or hydroxyl groups, bisethers,
Residues derived from adenine, uridine or RNA (ribonucleic acid); groups substituting the hydroxyl group of serine residues, such as methyl groups, glycosyl groups, phosphobantetheine groups, adenosine diphosphate ribosyl or phosphate groups; threonine residues A group substituting the hydroxyl group of, for example, a glycosyl group,
Methyl group << is a phosphate group; a group substituting the SH group of a cysteine residue, such as a glycosyl group, a cystinyl group, a dehydroalanyl group or a selenium atom, or a residue derived from heme or flavin; an aspartic acid or glutamic acid residue A group substituting the carboxyl group of the group, e.g.
Methyl group, phosphoric acid group or T-carboxyl group; groups substituting the amide group of asparagine or glutamine residues, such as glycosyl group, pyrrolidonyl group or imino group.

Bebutide or its derivatives in which glycine is peptide-bound to the C-terminal residue serving as the substrate may be extracted from nature, produced by chemical synthesis, or produced using recombinant DNA technology. Therefore, the substrate of the present invention, the compound represented by formula (I), includes peptides, for example, peptides having about 2 to 100 amino acid residues, casein, protein kinase, adenovirus EIA protein, RAS 1 protein, etc. phosphopeptides and their hydrolysates, lipoproteins and their hydrolysates represented by thromboplastin, α1 riboprotein, ribovitellin, etc., hemoglobin, myoglobin, hemocyanin, chlorophyll, phycocyanin, flavin, rhodobusin, etc. metalloproteins and their hydrolysates; glycoproteins and their hydrolysates represented by collagen, laminin, interferon α, seroglycoids, avidin, etc.; and other physiologically active mature forms in which the C-terminal hydroxyl group is amidated. Peptides forming peptides such as calcitonin, secretin, gastrin, vasoactive intestinal peptide (VIP), cholecystokinin, caerulein, pancreatic polypeptide, growth hormone-releasing factor, corticotropin-releasing factor, calcitonin gene-related peptides, etc. Each C-terminal glycine adduct (ie, an amide linkage compound of a C-terminal hydroxyl group and glycine). Among these, preferred substrates for confirming the enzyme activity of the enzyme composition of the present invention include, for example, D-tyrosylvalylglycine, D-tyrosyltryptophanylglycine, glycylphenylalanylglycine, and phenylalanylglycine. Leucyl phenylalanylglycine, D-tyrosylleucylasparaginylglycine, Arginylphenylalanylglycine, Arginylalanylarginylleucylglycine, Leucylmethionylglycine, Glycylleucilmethionylglycine, Phenylalani Luglycylleusylmethionylglycine, Asparaginylarginylphenylalanylglycine, Tributophanylasparaginylarginylphenylalanylglycine, Alanylphenylalanylglycine, Lysylalanylphenylalanylglycine, Seryl Lysyl Examples include alanylphenylalanylglycine, arginylthylglycine, glycylmethionylglycine, glycylthylglycine, glycylhistidylglycine, histidylglycylglycine, tributophanylglycylglycine, and glycylcysteinylglycine ( In addition, except for glycine, D
1 and those not specifically indicated indicate L1 type). On the other hand, as a preferable substrate for the effective use of the present enzyme composition (the fifth aspect of the present invention described below), the C-terminal carboxyl group is amidated to form a mature physiologically active peptide. Examples include peptides in which glycine is linked to a peptide.

The enzyme-■ of the present invention acts on the substrate to form the following formula (n) (H) [In the above formula, a specific example of the X-N-A-CO moiety is the formula (I)
C-terminal α-hydroxylglycine adducts having the meanings as defined above can be produced.

It can be converted into the corresponding C-terminal amidation product by hydrolysis under conditions that do not adversely affect the amino acid, or by treatment with the second enzyme of the present invention described below.

Further, the enzyme=1 has a molecular weight of about 25,000 Daltons according to a molecular weight determination method using gel filtration. That is, this molecular weight can be calculated using gel filtration methods known per se [for example, "Biochemistry Experimental Course 5, Enzyme Research Methods, Volume 1, 283-
298゜', Tokyo Kagaku Dojin (I975). Specifically, 50 mM Tris-HCl (pH 7.4) containing 100 mM potassium chloride was used as the equilibration and eluent, and Toyovar I-55S
(manufactured by Tosoh Corporation), and β-amylase (
M. W. 200,000), alcohol dehydrogenase (M.W. 150,000), BSA (M.W.
66,000), carbonic anhydrase (M.
W. 29. OOO) and cytochrome C (M.L1
5,400) was determined as an index.

Enzyme-■ 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 about 4 to 9, (ii) the optimum temperature for action is in the range of 25 to 40°C, and (i)
ii) Metal ions and ascorbic acid as cofactors.

The properties (i) and (ii) above were measured using commonly used buffer solutions, specifically, Tris-hydrochloric acid, mes-potassium hydroxide, Tes-sodium hydroxide, and Hebes-potassium hydroxide buffers. be. The enzyme composition of the present invention catalyzes the above reaction within a temperature range of 1°C to 55°C, but is deactivated in about 10 minutes at 56°C, and some deactivation is observed even around 40°C. It will be done.

As metal ions, Cu”, Zn”, Ni”
．． Co"Fe3+ etc. are suitable, especially Cu"
"rZn" is preferred.

The present invention further provides the following another type of enzyme. That is, by acting on the C-terminal α-hydroxylglycine adduct represented by the above formula (n), the following formula (III) (H) X-NA-CONHz (III)
(wherein A and An enzyme involved in C-terminal amidation of glycine adducts (hereinafter sometimes referred to as "enzyme 1") is provided.

(H) The significance of the χ-N-A-Co moiety and the method for determining the molecular weight used to identify this enzyme are the same as those used to identify enzyme-■. Preferable substrates for confirming the enzymatic activity of Enzyme 1.1 include α-hydroxyglycinate corresponding to the substrates specifically listed for Enzyme 1.1. In addition, Enzyme 1 is specified by having other physical and chemical properties that are almost the same as Enzyme 1.

That is, (i) the optimum pH is about 5 to 6 and the stable pu is in the range of 4 to 9, (i:) the optimum temperature for action is in the range of 15 to 35°C, and (mountain) metal ions are used as cofactors. do.

The properties (i) and (ii) above were measured using commonly used buffer solutions, specifically, Tris-HCl, Meth potassium hydroxide, Tes-Sodium hydroxide, and Hepes Potassium hydroxide buffers. be. The enzyme composition of the present invention catalyzes the above reaction within a temperature range of 1°C to 55°C, but is inactivated in about 10 minutes at 56°C, and catalyzes the reaction at 40°C.
Some deactivation can also be seen in the vicinity.

As metal ions, Cu”, Zn”, Ni”
, Co"Fe"", etc. are suitable, especially Cu",
Zn'' is preferred.

Enzyme-1 and Enzyme-1 of the present invention explained above can be prepared from the raw materials having the enzyme activity by a known enzyme separation and purification method, but preferably, according to the present invention. It can be obtained by the following preparation method of the present invention disclosed in the book. That is, substrate affinity chromatography using the enzyme-■ or enzyme-activated substance of the enzyme-■ as a ligand is the C-terminal glycine adduct represented by the formula (I);
A method for preparing enzyme-1 or enzyme-1, which is characterized by treatment with anion exchange chromatography and anion exchange chromatography, can be used.

Any raw material used in this method can be used as long as it contains the enzyme of the present invention;
It is preferable to use a substance derived from an organism having a high content of enzyme-■ or enzyme-■, which is searched according to the search method of item (1). In general, organisms that have these enzyme activities include humans,
Chewing mammals such as cows, horses, pigs, sheep, rabbits, goats, rats and mice, chickens, birds such as jungle fowl and rock pigeons, reptiles such as stone turtles, pit vipers, rattlesnakes and cobras, newts, African clawed frogs,
Included are amphibians such as bullfrogs and toads, fish such as lampreys, hagfish, oil sharks, rays, sturgeon, herring, salmon, eels, tiger puffers and sea bream, insects such as American cockroaches, silkworms, fruit flies and honey bees. Suitable extraction targets also include biological fluids, including homogenates derived from organs such as the brain, pituitary gland, stomach, heart and liver, and body fluids such as blood and lymph.

That is, a biological fluid having the present enzyme as described above is mixed with a substrate analogue having a C-terminal glycine adduct represented by the following formula (I) (wherein A and X have the above-mentioned meanings) as a ligand. Using body affinity chromatography, if necessary, molecular weight fractionation methods commonly used for enzyme purification include (I) fractionation by precipitation, (2) heparin affinity chromatography, (3) dialysis, gel filtration, etc.; / or (4) the enzyme of the present invention (enzyme-■ and enzyme-
■) can be obtained.

The above-mentioned ligand may be C represented by the above-mentioned formula (I).
Although any terminal glycine adduct can be used, peptides consisting of 2 to 6 amino acid residues including glycine, which are specifically listed as preferable for confirming the enzyme activity, can be used. Among these, D-Tyr
-Trp-Gly, Phe-GlyPhe-Gly and Gly-Phe-Gly are more preferred, but those having Phe-Gly-Phe-Gly as a ligand are resistant to the enzyme of the present invention (also referred to as the present enzyme). It is particularly preferred since it has affinity.

These ligands are usually used by binding to a water-insoluble carrier, but it is important for the carboxyl group of the C-terminal glycine residue of the peptide used as a ligand to be in a free state for binding to this enzyme. It is necessary to bind to the carrier via the amino group of the N-terminal amino acid residue. In other words, any carrier can be used as long as it can bind to the amino group of the peptide, and even if an active group that reacts with the amino group is chemically introduced into the carrier, it may also be a commercially available carrier that has already been introduced. may be used. The chemical introduction method may be a commonly used method, for example,
As described in "Biochemistry Experimental Methods, Volume 5, Volume 1, pp. 257-281" by Kasai, Tokyo Kagaku Dojin (I975), for example, cyanogen bromide is used to introduce an imidocarboxyl group into agarose.

Commercially available activated carriers include, for example, agarose-based, cellulose-based, hydrophilic polyvinyl-based, etc. based on the base material, and any of these may be used.

Examples of agarose-based carriers include CNBr-activated Sepharose 4B (manufactured by Pharmacia), which uses the CNBr method for binding to the amino group of the ligand, CH-Sepharose 4B, which uses the carbodiimide method, and ECH-Sepharose 4B.
(manufactured by Pharmacia), Affigel 10, Affigel 15 (manufactured by Bioland), Tresyl-activated Sepharose 4B using the tresyl chloride method (manufactured by Pharmacia), and the like. Examples of cellulose-based carriers include Formylcell Kuchifine (manufactured by Chisso Corporation), which uses the formyl method. Examples of hydrophilic polyvinyl carriers include AF-Carpoxy Toyopearl 650 using the carbodiimide method, AF-Formil Toyopearl 650 using the formyl method, and AF-Carpoxy Toyopearl 650 using the tresyl chloride method.
1-Resil Toyopearl 650, AF-Eboxy Toyopearl 650 using epoxy activation method (manufactured by Tosoh Corporation)
etc. The binding reaction with the ligand may be carried out according to the instruction manual for each carrier.

Among these, the manufacturing method for Affigel 10 will be described. The reaction between Affigel 10 and peptide is 0.001 to I
M is preferably 0. It is carried out in a buffer such as 1 M Motsupu potassium hydroxide. The reaction conditions may be 0 to 20''C, 10 minutes to 24 hours, and pH 3 to 11, but preferably 4°C, 4 to 24 hours, and pH 5 to 9.

The mixing ratio of Affi-Gel 10 and the peptide used for binding may be any value within this range, since the more peptides are added to Affi-Gel IFR1, the more they will bind, but from the viewpoint of binding efficiency, it is 1 to 20 μmol.
Degrees are conveniently used. After the reaction, after thoroughly washing with the buffer used during the reaction, add Tris-HCl (pH 8.0) to a final concentration of 50 mM, and shake at 4''C for 1 hour to remove unreacted active groups. Block the substrate analog affinity gel.

Substrate affinity chromatography may be performed either batchwise or continuously by filling a column. The time period for which the sample and gel are brought into contact may be as long as the enzyme is sufficiently adsorbed, but is usually 20 minutes to 24 hours. pH 6.0-1 at low ionic strength with the same composition used to equilibrate the gel.
1.0 preferably 7.0 to 9.0 buffer, e.g. 10
Thoroughly wash out non-adsorbed components with mM Hepes potassium hydroxide (pH 7.0). Of these, both fractions containing the enzyme activity are eluted.

The eluate may have any composition as long as the present enzyme can be obtained efficiently, but it is preferably an eluate containing 0.1 to IOM sodium chloride with about 1 to 40% acetonitrile and a pH of 7.0 to 9. Buffers between .

Furthermore, when the column is packed, elution may be performed by applying a concentration gradient.

In addition, in some cases, the above-mentioned fractionation by precipitation [hereinafter represented as (I)] may be carried out before or after, or both before and after carrying out the step of substrate analog affinity chromatography [hereinafter represented as (5)]. ], hebarin affinity chromatography [hereinafter referred to as (2)], molecular weight fractionation by dialysis, gel filtration, etc. [hereinafter referred to as (3)], and/or ion exchange chromatography [hereinafter referred to as (4)] ] step may be carried out. In this way, this enzyme (enzyme-■ and enzyme-
■) can be separated from other contaminants, but the separation of enzyme-■ and enzyme-H requires (3) and/or (
It is effective to implement step 4). Generally, it is preferable to carry out a total of 1 to 6 of these steps, and further carry out the above step (5) or (3) as the final stage. As specific examples of combinations of each step, only (5), (I
)→(5). (5) → (3), (2) → (5), (I)
→(3)→(5),(2)→(3)→(5). (I) →
(5)→(3). (2) → (5) → (3), (2) → (
I) → (5), (I) → (2) → (3) → (5), (I
) → (2) → (5) → (3), (I) → (3) → (5)
→(3),(I)→(2)→(I)→(5),(I)→
(2) → (I) → (3) → (5). (2) → (I) → (
5) → (3L (2) → (I) → (3) → (5). (2)
→(I)→(3)→(5)→(3),(I)→(2)→
(3)→(5)→(3). (I) → (3) → (2) → (
3) → (5), (I) → (3) → (2) → (3) → (5
) → (3), (4) → (3) → (5). (5) → (3)
→(5)→(3),(I)→(5)→(3)→(5)→
(3), (4) → (5), (I) → (3) → (5) → (
4)→(3). (I) → (3) → (4) → (3) → (5
). (I) → (2) → (3) → (5) → (3) → (4)
, (I) → (2) → (3) → (5) → (4) → (3),
or (4)→(5)→(3), etc. Among these, (I)→(2)→(3)→(5). (I)
→(2)→(3)→(5)→(3), (I)→(3)→
(2) → (3) → (5) or (I) → (3) → (2)
→(3)→(5)→(3). (I)→(2)→(3)→
It is more preferable to proceed with the steps in the order of (5)→(4)→(3).

Hereinafter, the steps (I) to (4) will also be explained in detail. In addition, all of these are carried out at O''C-10''C, preferably 4°C.

Substances used for fractionation by precipitation (I) include salts such as ammonium sulfate, organic solvents such as ethanol and acetone, and polymers such as polyethylene glycol. There are no particular restrictions on the concentration of the enzyme added, but conditions are preferred that allow the enzyme to be recovered in good yield and to be separated from other protein components. For example, when adding 30 to 50% saturated ammonium sulfate and 10 to 15% (w/v) polyethylene glycol 6000, the enzyme is present in the precipitate fraction, whereas most of the protein is present in the supernatant fraction, so it is not efficient. Well refined. Note that the addition is preferably carried out gradually while stirring with a stirrer. After the addition is completed, the mixture is allowed to stand for at least 1 hour, and then the fraction containing the enzyme is collected by centrifugation. When the precipitate fraction is collected, it is dissolved in an appropriate buffer.

Buffer, pH 6.0 to I1.0, preferably pH 7.0
~9. Any composition may be used as long as it is 0, and examples thereof include Tris-hydrochloric acid, Hepes-potassium hydroxide, Tes-Sodium hydroxide, and the like. The concentration is not particularly limited as long as the buffering capacity can be maintained, but it is preferably about 5 to 50%.

The active fraction obtained in (I) may then be subjected to (I) again or to any one of steps (2) to (5), but ammonium sulfate is added to the fraction (I). When proceeding to (2), (4) or (5) using salts such as (
It is necessary to reduce the salt concentration to a level that allows the enzyme to bind to the gel used in step 3) or the next step by adding an appropriate buffer. Further, when a precipitate is dissolved and left to stand for more than one hour, or when dialysis is performed, insoluble substances may be generated, and these are removed by, for example, centrifugation or filtration.

Heparin affinity chromatography (2) may be carried out either batchwise or continuously by filling a column with gel. Commercially available gels with heparin as a ligand include Hebarin Sepharose CL-6B (manufactured by Pharmacia), Affigel Heparin (manufactured by Bioland), Heparin Agarose (manufactured by Sigma), and AF-Hebarin Toyovar. 650 (manufactured by Tosoh Corporation), etc.

The biological extract is brought into contact with a heparin affinity gel either directly or after being subjected to the fractionation treatment by precipitation shown in (I). The contact time may be as long as the enzyme is sufficiently adsorbed, but it is usually 20 minutes to 12 hours. Components that have no affinity for heparin are contained in an ionic strength low enough that the present enzyme is not eluted, and 9H is 6.0 to 11.0, preferably 7.
0 to 9.0 buffer solution, for example, 10 mM Hepes potassium hydroxide (pH 7.0). Thereafter, the fraction containing this enzyme is eluted. The eluate should preferably have a high recovery rate of the enzyme activity, for example, 0.5M-
pH 6.0 containing salts commonly used for enzyme purification such as 2M sodium chloride, potassium chloride, ammonium sulfate, etc.
-11.0 is preferred. When the column is packed, elution may be performed by a salt concentration gradient or by one-step elution. For example, 0. 3-2. 0M
Elute with 10a+M Hepes-potassium hydroxide buffer (pH 7.0) containing sodium chloride.

The active fraction obtained in step (2) may be subjected to any of steps (I) to (4) next, but it is also possible to perform (2) again,
When proceeding to (4) or (5), either perform (3) first, or use a large amount of pns with a low ionic strength of 50 mM or less. o~
11.0, preferably 7.0 to 9.0, such as 5sM Hepes potassium hydroxide (pH 7.0), and add this enzyme to the gel used in (2), (4) or (5). It is necessary to lower the ionic strength to a level that allows it to be adsorbed.

In the step (3) of removing low-molecular substances by dialysis, gel filtration, etc., in the case of dialysis, the membrane used only needs to have a molecular weight cut-off that does not allow the enzyme to pass through.
000 to 10,000 is preferred. The dialysis method is described in, for example, “Biochemistry Experimental Course Vol. 5, Volume 1, pp. 252-253.”
Commonly used ones such as those described in Tokyo Kagaku Doujin (I975) written by Soda may be used, and the pH is low for several hours to several days, with a low ionic strength, and preferably at a pH of 6.0 to 11.0.
7.0 to 9.0 buffer solution, such as 10mM Hepes-potassium hydroxide (pH 7.0), 10mM Tris-HCl (pH 7.5), etc. Also, during dialysis,
If insoluble substances precipitate, they are removed by, for example, centrifugation, filtration, or the like.

Regarding gel filtration, any carrier that is generally used as a carrier for gel filtration may be used.

For example, Se7 Atex G-10, G-15. G-25,
G-50. G-75, G-100, Se7y'
) Lil s-200, S-300 (manufactured by Pharmacia) Toyobar HW-40, f{L55 (manufactured by Tosoh), Hyogel P-2. P-4, P-6, P
-10, P-30, P-60, P-100 (manufactured by Bio-Rad), etc. are preferred. Note that the buffer solution to be used has the same composition as that to be used during dialysis. However, if the ionic strength is too low, adsorption of this enzyme to the gel may occur, so the concentration should be adjusted to 5-200+nM.
Preferably it is 10-20mM. The gel filtration method is
For example, “Biochemistry Experiment Course Volume 5, Volume 1, pp. 283-298
It can be carried out as described in "Tokyo Kagaku Dojin (I975)" written by Soda. After adding an amount of sample that provides sufficient separation power to the head volume of the gel filtration carrier, elution is performed to determine the activity of the enzyme. Collect the fraction in which .

The active fraction obtained in step (3) can be proceeded to each step of (I) to (5) without special treatment.

For ion exchange chromatography, any commercially available carrier for ion exchange chromatography may be used. For example, Aminex, [low
ex, Amberlite, SP-Sephacr
yl M+8 sahipak, DEAE-Toyo
pearl, DEAE-Sephadex. CM-
Sepharose. DEAE, Bio-Gel
A+ CM-Cellulose, DEAE-Cell
ulofine, Partisil SCY, Mo
No Q, Mono S, etc. are preferred. Note that the buffer solution to be used and the method for use may be in accordance with the method described in the section on heparin affinity gel. In addition, the basic operating method is described in "New Basic Biochemical Experimental Methods 2, Extraction, Purification, and Analysis I.
The general method described in J (Maruzen, 1988) etc. may be followed.

The active fraction obtained in step (4) may be subjected to any of steps (I) to (5) next, but it is also possible to perform (4) again, or to perform (2) or (5). If you proceed, (3)
or add a large amount of a low ionic strength buffer of pH 5.0 to 11.0, preferably 6.0 to 8.0, such as Hepes sodium hydroxide (pH 7.0).
), etc., to lower the ionic strength to a level that allows the enzyme to be adsorbed to the gel used in (2), (4), or (5). Through the above purification steps, a crude enzyme product of the present invention can be obtained. The crude enzyme product is
Furthermore, by the protein separation means using the gel filtration step (3), a protein with a molecular weight of about 25,000 and a molecular weight of about 40
Both fractions, each having a peak at ,000, can be isolated and used as the enzyme preparation.

Each of the above steps is performed according to the method for measuring enzyme activity, which is another aspect of the present invention, which will be described later.
This is carried out by using the compound of n) as a substrate to monitor the activity of Enzyme-■ and/or Enzyme-H to obtain an active fraction.

By using the composition containing the enzyme of the present invention, the following fourth and fifth present inventions, that is, C represented by the formula (I)
A method for producing a C-terminal α-hydroxylglycine adduct represented by the formula (I[), which comprises treating the terminal glycine adduct with a composition containing the enzyme -■, and a method for producing a C-terminal α-hydroxylglycine adduct represented by the formula (I[); It is possible to provide a method for producing a C-terminally amidated product represented by the formula (III), which comprises treating the adduct obtained by the present invention with a composition containing the enzyme -2. Furthermore, by using these enzymes -1 and -2 in combination, the compound of formula (I) can be converted to the compound of formula (I[) in a single reaction composition. From formula (I) to formula (Ilr
) The use of enzyme -■ in the step of conversion to the compound of formula (III) means that in the presence of only the enzyme -■ type of enzyme, the chemical hydrolysis conditions are lower during the conversion of formula (It) to the compound of formula (III). It will be appreciated that the conversion can be achieved under milder enzymatic reaction conditions compared to the need for exposure to .

It is particularly suitable for treating substrates that are unstable under alkaline conditions.

These production methods can be used without worrying about the concentration or purity of the enzyme as long as it contains the enzyme of the present invention, but considering the isolation and purification of the product from the reaction mixture and the compound of formula (JT), contamination may occur. It is advantageous to use enzyme-containing compositions according to the invention which are largely free of proteins. Note that if a biological extract having the present enzyme activity is used, it can be used as it is or simply as a concentrate.

Compounds of formula (I) include all those mentioned above, but those suitable for use in these preparation methods include:
In particular compounds of formula (I), such as arginine vasotocin (
AVT), luteinizing hormone-releasing hormone (LH-R
H), oxytocin, gastrin, gastrin secretagogue peptide (GGRP), calcitonin (CT), vasoactive intestinal peptide (VIP), thyroid-stimulating hormone-releasing hormone (TRH), melanophore-stimulating hormone (
MSH), MSH release inhibitory hormone (MI}l)
, cholecystokinin octapeptide (CCK-8)
, substance P (SP), fat mobilization hormone, pancreatic polypeptide (PP), growth hormone releasing factor, secretin, caerulein, soft motor cardiac excitatory peptide,
vasopressin, adrenocorticotropic hormone (ACTH),
Color-changing hormone, bombesin, photopic hormone, motilin, avamin, alitesin, eredoisin, katushinin,
Granuliberin R1 scotophobin, hylambate cerulein, mast cell degranulation peptide, physalemin, phyllocerulein, phyllomezucin, promelitin, pombinin, mastoparan, mastonoglan-X, melittin-
1. Their corresponding formulas (I) that can be converted into lanatensin and lanatensin-R etc. by this production method
Alternatively, a compound represented by formula (II) is preferred.

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

Enzyme-1 and Enzyme-1 of the present invention described above can be tracked by the following activity measuring method, and this measuring method is useful for rationally carrying out the method for preparing the present enzyme described above.

Of course, these measurement methods require that the C-terminal amidation reaction is not a one-step reaction as previously thought, but an intermediate (
This is based on the knowledge that it is a two-step reaction via a C-terminal α-hydroxylglycine adduct).

First, the activity of the enzyme-■ is determined by (i) buffering a test substance predicted to have the activity to pH 5 to 8, (ii) adding to this buffer solution the formula (I) shown above. Adding and incubating the C-terminal glycine adduct and L-ascorbic acid and catalase, and (in) adding the reaction product of formula (II[) using an acetonitrile-containing buffer (pH 6 to 10). It is measured by a method characterized by including a step of detecting with HPLC.

In addition, the activity of enzyme-■ can be determined by (i) buffering the test substance predicted to have the activity to pH 4 to 8;
ii) A step of adding a C-terminal α-hydroxylglycine adduct represented by formula (ff) to this buffer solution and incubating it, and (ij) producing a compound of formula (II).
It is measured by a method characterized by including the step of detecting the C-terminal amidation product shown in I).

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 that has these enzymatic activities, and in particular biological fluids that have these activities, i.e., homogenized organisms' organs, as well as body fluids, such as blood. and lymph fluid, as well as treatment solutions such as those for purification treatment. Biological fluids also include processing liquids derived from microbial cells.

The buffer used for buffering these test substances is not particularly limited, and commonly used buffers can be used.

Examples include tris-hydrochloric acid and hepes-potassium hydroxide. The concentration of the buffer in the buffer solution may be any concentration as long as the buffering effect is achieved, but it is usually between 20 and 200 a
+M is appropriate.

Each buffer solution is the sixth invention (referred to as the "former").
For the seventh invention (referred to as "the latter"), the pH is adjusted to 6 to 8, preferably around 16.5.
Adjust the pH to 4 to 8, preferably around 6. The C-terminal glycine adduct added to the buffer solution prepared in this way is a substrate of the enzyme, and the aforementioned enzyme -
It is preferable to use those represented by formula (I) listed as preferred substrates for confirming the activity of . The concentration of this compound is 0. Approximately 1 m to 2 mM is appropriate. Furthermore, it is possible that it functions as a cofactor - ascorbic acid, requiring the addition of catalase as an activator. Generally, the concentration of L-ascorbic acid is
0.5-2mM is preferable, and the concentration of catalase is 4
0 to 100x/d is appropriate. Additionally, metal ions may be added to the buffer solution, but this addition is not particularly required for this activity measurement, and adding metal ions may result in higher activity than without addition. It is preferable.

The metal ions used are zn2+, CuZ*.
Hil+Co", Fe", etc. are suitable, especially Cu
and Zn2+ are preferred. The concentration of the metal ion in the buffer solution is suitably between 0 and 1000, preferably between 0 and 10. Compounds that provide such metal ions include, but are not limited to, CIISO41 CuCf, , Z
nClz. Ni(/!2, CoCfz, FeCf3
etc. can be mentioned.

As a specific example of such a reaction composition, the composition of Example 2 described below may be referred to, for example.

On the other hand, the reaction composition in the latter case is prepared using the corresponding compound of formula (II) in place of the compound of formula (I). In this case, cofactors such as ascorbic acid, catalase, etc. are not required.

In both measurement methods, the amount of the test substance used can be varied without particular limitation, but the amount of substrate present in the reaction system (
a nanomol (nmol)), preferably at least apn+ol/hr, more preferably 10Xa
pmof /hr or more, most preferably lQXa
pmoj! /hr-a mol/hr (The unit indicates enzyme activity, and is expressed in the amount of substrate that can react in 1 hour at 37°C (for example, expressed as bicomole (pmof)).It is appropriate to adjust the enzyme activity to contain the following. be.

Incubation is carried out for 2 to 24 hours with shaking at 1 to 55°C, particularly preferably at 25 to 40°C, particularly preferably around 30°C for the former, and preferably at 15 to 35°C for the latter. Most preferably, it is left standing at around 25°C for 1 minute to 48 hours.

In the above steps, the detection of the compound of formula (II) and the compound of formula (II[) produced respectively is performed by detecting their substrates and products, for example, in the former case, the compound of formula (I) and the compound of formula (ff).
In the latter case, the compound of formula (II) and the compound of formula (II[)
Any method that can separate and measure the compounds can be used. Generally, separation measurements can be performed by separating and purifying by chromatography as described below. Chromatography that can be used for the above includes ion exchange chromatography, reversed phase chromatography, gel filtration,
Affinity chromatography, high performance liquid chromatography (HPLC), thin layer chromatography (T
LC), etc. In the latter reaction system, the formula (I
The substrate represented by I) and the amidated product represented by formula (I[[) have a carboxyl group and an amide group at the C-terminus, respectively, and have different charges. Ion-exchange chromatography, reversed-phase chromatography, etc. that utilize this property are suitable. Affinity stomatography using product antibodies is also effective. However, in the former reaction system, it is quite difficult to separate the substrate represented by formula (I) and the product represented by formula (II) by ordinary chromatography, but the present inventors attempted for the first time to separate the substrate represented by formula (I) and the product represented by formula (II). Separation and measurement can be advantageously carried out by high performance liquid chromatography (HPLC) using a buffer solution (pH 6 to 10, preferably po 9) as an eluent. The elution is preferably carried out by applying a linear concentration gradient of acetonitrile concentration. }As the IPLcO column, any type of commercially available column can be used as long as it is suitable for this purpose, but in particular, Capsule Pack C18
It is advantageous to use 3G, 300 (manufactured by Shiseido).

Thus, each separated substrate and product may be assayed for any of their (optionally conjugated) chemical or physical labels.

For this measurement, known labels and known measurement methods can be used.
Generally, U derived from the amino acids that make up the substrate peptide
It may be advantageous to utilize V absorption.

Since the above measurement methods are accurate and simple, by applying them to the above-mentioned biological fluids, it is possible to search for enzymes having the activity of Enzyme-1 or Enzyme-H, respectively. Such search methods are described in Section 8 of this application, respectively.
And it is provided as a ninth invention.

Biological fluids to be searched include not only those predicted to have the above-mentioned enzyme activity, but also living cells, tissues, and extracts of other animals and plants. For example, the extract can be used in "Experimental Biology Course 6, Cell Fractionation" (
Maruzen, 1984), “Biochemistry Experiment Course 5, Enzyme Research Methods (
1)" (Tokyo Kagaku Doujin, 1975), r Basic Biochemical Experiment Methods 1, How to Handle Biological Materials" (Maruzen 1974),
It may be prepared according to the general extraction method described in .

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples. Note that the present invention is not limited to this.

5 d of Affigel 10 was weighed out into a 101 volume Econocolumn (manufactured by BioRad) filled with isopropanol. After pouring out the isopropanol, 501IIl
of 1On+M sodium acetate buffer (pH 4.5), then 10-M of sodium acetate buffer (pH 4.5). 1 M Motsupu Sodium Hydroxide Buffer (contains 80 mM calcium chloride, pH 7.5)
Washed with. After transferring the gel to a 20d bottle, 40
(approximately 100 μmof) of phenylalanylglycine (Phe-Gly-Phe-G)
Ly, manufactured by Sigma Corporation) was mixed with the above-mentioned Motsupusu sodium hydroxide buffer solution 10-1, and reacted with shaking at 4°C for 18 hours. After that, 0. 5d LM} Lys-HCl buffer (pH 8.0) was added and the mixture was shaken at 4°C for 1 hour to inactivate unreacted active groups. After washing the gel with the above-mentioned MopSu sodium hydroxide buffer and then with ion-exchanged water, 0.02% NaN. Suspend in and fill in column, 4
Stored at °C. In addition, the peptide (Phe
-Gly-Phe-Gly) and the amount of peptide in the recovered solution, it was calculated that about 10 μmof was bound per gel.

Phenylalanylglycylphenylalanylglycine (
FGFG) (manufactured by Sigma) Measure 3■, 50ff
1M hebes-potassium hydroxide buffer (pH 5.5)
, 3mM ascorbic acid, 10mM potassium iodide,
0.25■/ml! catalase, 0.25 nM copper sulfate,
7.5% acetonitrile and international patent application JP89-0
200 IJ1 of the amidation enzyme composition derived from horse serum as described in Example 2 of Specification No. 0521 was added to make a total amount of 1
The amidation reaction was carried out aerobically at 30°C for 20 hours. The reaction was stopped by adding 10% formic acid, and phenylalanylglycylphenylalanylhydrocylglycine (FGFhyG) was fractionated using high performance liquid chromatography (HPLC). The HPLC column used was Capsule Pack C18SG, 300 (manufactured by Shiseido). The elution solvent was 1mM ammonium bicarbonate (p
H9.0) and acetonitrile, a linear concentration gradient was applied increasing the acetonitrile from 0% to 40% in 30 minutes. Veptides were detected by absorption at 214 nm. The results are shown in Figure 1. The peak of phenylalanylglycine at 10.7 minutes almost disappeared due to the C-terminal amidation reaction. Along with this, 9.9 minutes of α-hydroxyglycine derivative and 14
．． A peak of the amidated product at 5 minutes was observed. The structures of these substances were confirmed by FAB-MS spectrum analysis and NMR analysis. Figure 2 shows FAB in glycerin solution.
-The results of MS spectra are shown. The parent peak has a molecular weight of 4
42, and as a result of fragmentation, fragments of 425 and 408 m/z in which one or two 10H groups present at the C-terminus were missing were confirmed, indicating that they are α-hydroxylglycine adducts. was.

The peak at 9.9 minutes was fractionated, immediately made into a 10% formic acid solution, and lyophilized to prepare a substrate for the enzyme (1) of the present invention. This substrate could be similarly prepared using the enzyme-I-containing composition of the present invention instead of the amidating enzyme composition. As mentioned above, α-hydroxylglycine derivatives are stable under acidic conditions, but unstable under alkaline conditions, and decompose into amidates and glyoxylic acid irrespective of oxygen reactions. Therefore, the C-terminal amidation reaction performed at the beginning of this example was performed under acidic conditions.

At this time, if the reaction is carried out at a pH of 7.5 or higher, the α-hydroxylglycine derivative cannot be confirmed. It was thought that the known C-terminal amidation enzymes convert the C-terminal glycine adduct represented by the above formula (I) into the C-terminal amidation product represented by the formula (I[[) and glyoxylic acid.
This is because the non-enzymatic conversion under alkaline conditions was included, and the catalytic reaction of the enzyme itself is the C
From the terminal glycine adduct to the C-terminal α represented by formula (II)
This is a conversion reaction to a hydroxylglycine adduct. Therefore, the yield of amidation reactions under acidic conditions using conventional C-terminal amidation enzymes has generally been low.

(I) 100 d of commercially available horse serum (manufactured by Gibco) was added with a 25% aqueous solution (w/v) of polyethylene glycol 6000 (Wako Pure Chemical Industries).
) was gradually added with stirring to a final concentration of 12.5%. The following operations were all performed at 4°C. After standing for 12 hours, centrifugation (I0
, OOOx g, 10 minutes), and the resulting precipitate was
R1 10mM Hepes potassium hydroxide buffer (pH 7)
．． After further standing for 2 hours, the generated insoluble substances were centrifuged again (I0,000 x g, 10 minutes).
The supernatant (I27d) containing Enzyme I was obtained.

(2) The active fraction obtained in (I) above was transferred to a column (1.6 x 15C1 m ).

After washing out non-adsorbed substances with the same buffer solution 96d, 0.5M
Elution was performed with 10n+M Hepes-potassium hydroxide buffer (pH 7.0) containing sodium chloride (flow rate 30IR).
Figure 3 shows the elution pattern. 0.5M
Enzyme I was eluted with a buffer containing sodium chloride [Fraction No. Collect 14-l6 (I0
0d)) (3) The above fraction was treated with Sephadex G-
Gel filtration was performed using 25 Fine (manufactured by Pharmacia) column chromatography (5 cmφ x 23 cm).

The solvent was 10n+M Hepes potassium hydroxide (pH 7
．． 0) at a flow rate of 2 d/a+in. Protein was detected by absorbance at 280 nm, and 100 fractions containing protein were collected.

(4) Affigel 10Phe produced according to Example 1
- Fill the column with Gly-Phe-Gly gel 5d (
I. OX6.3CI1), 10mM hebes-potassium hydroxide buffer containing 0.1M sodium chloride (pH 7.
0). The sample (I8.1d) obtained in (3) above was applied to this column. In order to sufficiently adsorb enzyme-■ onto the gel, the liquid that had passed through the column was circulated through the column many times (flow rate 20III1/hr).

After 12 hours, the circulation was stopped, unadsorbed substances were washed out with the buffer used for equilibration of 35-alpha, and 0. Elution was performed with 8 mM Hepes potassium hydroxide buffer (pH 7.0) containing 4 M sodium chloride, 20% acetonitrile (
flow rate 201 d/hr). The activity of enzyme-■ is measured in the elution fraction (I
0d).

(5) The purified product obtained in (4) above was treated again in (3) above, and then equilibrated with 10 mM Hepes potassium hydroxide buffer (pH 7.0) using a Mono ram (0. 5 x 5 cm) and injected with NaC in the same buffer.
The protein was eluted by applying a linear concentration of acidic acid of 1 as shown in Fig. 3. At this time, the flow rate was 0.5i1/IIIin.

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

This may be due to the influence of the white matter degrading enzyme present in the serum of my body for the next few days, but the activity is relatively low because the substrate and product were partially degraded.

The activity measurement method is F (; FG to Fl;
A reaction similar to the method for preparing FhyG was carried out for each purified product, and FGFhyfl was quantified by HPLC as described therein. Enzyme activity IU is lnmate
The amount of F(;FhyG produced in 1 hour at 37° C.) was taken as the amount.

The amount of fluorescent light was measured using Lowry's improved method (Bens
adown et al., Anal. Biochem. , 70,
265. (1976), and a standard curve was prepared using bovine serum albumin (fraction Ⅰ, manufactured by Sigma).

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

Example 1: Horse blood Horse serum was treated in the same manner as in Example 3, except that each of the purification steps of -1 and 1 was carried out while monitoring the activity of enzyme 1.

Table 2 shows the total protein, total enzyme activity, specific activity, yield, and purification rate for each of the purification steps (I) to (5).

Margin below 2 Horse serum 7.500 2 100" 0.28 This may be due to the influence of proteolytic enzymes present in the serum, but the activity is relatively low because the substrate and product were partially degraded. .

Activity measurements were performed using 10mM hebesu-potassium hydroxide (pH 6).
．． 5) Phenylalanylglycylphenylalanylhydroxylglycine (FGFhyG) obtained in Example 1
was dissolved to a concentration of 51, and samples at each stage were added to make a total of 10
The reaction was carried out at 30°C. After 1 hour reaction 1
The reaction was stopped by adding 0% formic acid, and the reaction product was quantified by HPLC using the conditions of Example 2. At this time, a control reaction in which no sample was added was also performed, and it was confirmed that non-enzymatic conversion had hardly progressed. HP of reaction solution
The LC pattern is shown in Figure 4 (the reaction conditions in the figure are 37
The reaction time was shown in the figure at °C pH 6.9) The activity was expressed in units (U). IU is 1 nmole of FG
F-NHZ was defined as the amount of enzyme produced in 1 hour at 30°C.

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

As shown in Table 2, this enzyme has a yield of 2% and approximately 180
It was able to be purified 0 times.

〔Effect of the invention〕

The enzyme of the present invention can be used in the production of a C-terminal amidated product represented by formula (II) from a C-terminal glycine adduct represented by formula (I). Furthermore, by using enzyme-■ and enzyme-■ in combination, the C-terminal α-hydroxylglycine represented by formula (II) can be efficiently converted to formula (I[ It becomes possible to produce the C-terminal amidated product shown in I).

Furthermore, the preparation method of the present invention has the effect that the enzyme composition of the present invention can be prepared efficiently and with high yield.

Each of the enzyme activity measurement methods of the present invention includes enzyme activity for converting a C-terminal glycine adduct to a C-terminal α-hydroxyl glycine adduct represented by formula (n) and
It is possible to quickly and accurately quantify the enzyme activity that converts the C-terminal hydroxylglycine adduct to the C-terminal amidated product. Furthermore, the enzyme search method using this method makes it possible to search for enzymes having the above-mentioned activities that were previously unknown.

[Brief explanation of drawings]

FIG. 1 shows an HPLC pattern when pcphyr is produced using enzyme I of the present invention using FGFG as a substrate. FIG. 2 shows the results of FAB-MS spectrum analysis performed to confirm the molecular structure of the prepared FGFhyG. FIG. 3 is a chromatographic pattern showing the separation of Enzyme-■ and Enzyme-H of the present invention by Mono Q column chromatography. The white square plot shows the activity of enzyme-■, the black circle plot shows the activity of enzyme-■, and the broken line shows the linear concentration gradient of salt. FIG. 4 shows the enzyme IH of the present invention using pcphyc as a substrate.
HP over time when producing FGF-NHZ using
This is an LC pattern.

Claims (1)

[Claims] 1. The following formula (I) ▲ Numerical formulas, chemical formulas, tables, etc. ▼ (I) (In the above formula, A is an α-amino group or an imino group derived from a natural α-amino acid; (representing a residue other than an α-carboxyl group, and X represents a residue of an amino acid derivative bonded to an N atom via a hydrogen atom or a carbonyl group), The following formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(II) (In the above formula, A and X represent the above meanings) A C-terminal α-hydroxylglycine adduct is produced, and , approximately 25,0 by molecular weight determination using gel filtration.
An enzyme involved in the C-terminal amidation of a C-terminal glycine adduct with a molecular weight of 00 Daltons. 2. Acting on the C-terminal α-hydroxylglycine adduct shown by the above formula (II), the following formula (III) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (III) (In the above formula, A and X are C-terminal amidation of a C-terminal glycine adduct having a molecular weight of about 40,000 Daltons as determined by a molecular weight determination method using gel filtration, which is produced by a C-terminal amidation product represented by the formula (with the above meaning) and glyoxylic acid. Enzymes involved in. 3. The enzymatic activity-containing material according to claim 1 or 2 is treated with substrate affinity chromatography using the C-terminal glycine adduct represented by formula (I) as a ligand and anion exchange chromatography. A method for preparing an enzyme involved in C-terminal amidation according to claim 1 or 2. 4. A method for producing a C-terminal α-hydroxylglycine adduct represented by the formula (II), which comprises treating the C-terminal glycine adduct represented by the formula (I) with the enzyme according to claim 1. 5. A method for producing a C-terminal amidated product represented by the formula (III), which comprises treating the C-terminal α-hydroxylglycine adduct represented by the formula (II) with the enzyme according to claim 2. 6. (i) Buffering a test substance predicted to have the activity of the enzyme according to claim 1 to pH 5 to 8; (ii) adding C-terminal glycine represented by formula (I) to this buffer solution; (iii) using an acetonitrile-containing eluate at pH 6 to 10 to remove the resulting C-terminal α-hydroxylglycine adduct represented by formula (II); The method for measuring enzyme activity, which comprises the step of detecting by HPLC. 7. (i) buffering the subject predicted to have the enzyme activity according to claim 2 to pH 4 to 8; (ii)
A step of adding and incubating the C-terminal α-hydroxyglycine adduct represented by the formula (II) to this buffer solution, and (i) a step of detecting the generated C-terminal amidation represented by the formula (III), The method for measuring enzyme activity, which also comprises: 8. The method for searching for an enzyme according to claim 1, which comprises applying the measuring method according to claim 6 to a subject. 9. The method for searching for an enzyme according to claim 2, characterized in that the measuring method according to claim 7 is applied to a subject.