JPWO2004014944A1 - Microbial polyamino acid or derivative thereof - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to production of a functional polyamino acid or a derivative thereof using a microorganism, and more specifically, a functional polyamino acid or a derivative thereof derived from a microorganism, particularly a fungus of the genus Epichloe, and a composition containing them. , A method for producing the polyamino acid or a derivative thereof, and a microorganism for producing the polyamino acid. The present invention also relates to a method for producing D-amino acid using a microorganism.

Description

  The present invention relates to the production of functional polyamino acids or derivatives thereof using microorganisms, and more particularly to the production of functional polyamino acids or derivatives thereof of microbial origin, compositions containing them, production of said polyamino acids or derivatives thereof. The present invention relates to a method and a microorganism producing the polyamino acid. The present invention also relates to a method for producing D-amino acid using a microorganism.

Polyamino acids have a structure in which multiple amino acids are linked by peptide bonds, and are basically biodegradable, and after exhibiting their intended functions in the living body or environment, they are easily decomposed and remain. It is attracting attention in various fields as a non-biodegradable functional substance.
Conventionally, polylysine which is a basic polyamino acid, white protein (protamine) (Bioindustry, Vol. 19, No. 3 (2002), pages 64-73), nisin which is one kind of bacteriocin (JP-A-5-146282, Chemistry and organisms Vol 38, No. 7, (2000) 439-446) and the like have antibacterial activity, and have been suggested to be useful for preventing spoilage of foods and the like. In addition, it is disclosed that low molecular weight ε-poly-L-lysine produced by Streptomyces strains can be used as a food additive (Patent Publication 2000-17159, 2000-69988). However, the antibacterial action of these polyamino acids is not always sufficient.
In addition, using phytokeratin, which is a polyamino acid (sulfur-containing peptide) having a high affinity for metals, a method of collecting heavy metals in the environment or measuring the concentration of heavy metals (JP-A-11-174054), A method of using a metal complex of a peptide as a medical contrast agent composition (JP-A-7-224050) is disclosed. However, for example, phytokeratin has a problem that it is easily altered because it contains cysteine, which is easily oxidized.
In addition, polylysine and polyarginine, which are basic polyamino acids, are allowed to form a complex with polynucleotides (DNA and RNA) that are acidic substances, and these are transported to animal culture cells for gene transfer or gene transfer in gene therapy. A method for use as a body is disclosed (for example, Japanese Patent Laid-Open No. 9-308484). Further, it has been reported that an arginine-rich peptide is transported into cells (J. Biol. Chemistry (2001) Volume 276, 5836-5840). In order to achieve this purpose better, it is desirable to provide a wider variety of polyamino acids and to effectively utilize their functions according to their respective properties. However, in the case of conventional homopolymers composed of a single amino acid, the relationship between amino acid composition and function may not be sufficiently elucidated, and it is necessary to develop a polyamino acid composed of multiple kinds of amino acids.
In addition, almost all proteins constituting organisms are made up of α-amino acids (hereinafter referred to as L-amino acids) whose absolute configuration belongs to the L series. On the other hand, when considered in a broader range of polymers of amino acids, D-amino acids, which are enantiomers of L-amino acids, exist in the peptidoglycan layer present in the bacterial cell wall, the viscous thread of Bacillus natto, and the like. In addition, in peptide antibiotics represented by polymyxin, D-amino acids are considered to play an important function as its constituent elements. For example, it is generally accepted that the inclusion of D-amino acids results in resistance to peptidases (peptidolytic enzymes) that are ubiquitously distributed in the living world. That is, a peptide containing a D-amino acid is considered to be biologically advantageous because it takes longer to decompose and lose its function than a peptide containing an L-amino acid.
D-amino acids can be produced by chemical synthesis, but there is a problem in that there is a high risk of environmental pollution due to reagents, solvents, wastes, etc. used in the reaction. Further, generally, a complicated technique is required and the manufacturing cost is high. In addition, the optical purity of the chemical product is stoichiometrically controlled, and the abundance ratio of the D-form and the L-form is usually 1:1 so that a division treatment is necessary. The optical resolution for separating the D-form and the L-form can be performed by chromatography or the like using the difference in affinity between the D-form and the L-form, but there are problems such as complicated operation and low product yield. Unsuitable for industrial scale mass production.
Therefore, optical resolution of amino acids using microorganisms has been proposed. For example, there is a method of adding a mixture of amino acids of D-form and L-form to a culture solution and recovering the remaining D-form by utilizing the property that the microorganism specifically consumes only the L-form. Kaihei 10-080297, JP-A-10-286098). In this case, a chemically synthesized mixture of D-form and L-form is required as a raw material.
There is also a method of producing D form exclusively from the stage of biosynthesis using a microorganism. For example, a chemically synthesized precursor substance such as N-substituted carbonyl-D,L-amino acid (JP-A 06-22789) or N-acylated amino acid (JP-A 11-113592) is used as a culture system. There is a method of introducing and recovering the reaction product D-form by an enzyme action specific to the D-form. Also in this case, a chemically synthesized precursor is required as a raw material.
Among various amino acids, histidine has an imidazole ring which is a dissociative functional group. Since its pKa value is 6.0, which is close to the physiological pH value, the dissociation/non-dissociation state change is involved in various chemical reactions in the living body including the enzymatic reaction. On the basis of the same principle, histidine or a peptide or derivative containing it is often physiologically active, and histidine has a great application value in medicines, cosmetics and the like. In particular, when D-histidine is introduced into raw materials such as pharmaceuticals, agricultural chemicals, cosmetics, food additives, etc., as described generally with respect to the above D-amino acids, it causes denaturation by enzymatic action, suppression of decomposition, and optical activity. It can be expected to have effects such as the addition of new functionality. Therefore, development of a method for efficiently producing D-histidine is strongly desired.
In order to meet the widespread demand for D-histidine, it is necessary to develop a production method that is safe and efficient and can be applied to industrial production. It is not always appropriate in terms of the effects of. Therefore, a production method by microbial fermentation is desirable. However, a method for producing D-histidine by microbial fermentation has not yet been known.
As described above, since polyamino acids can exhibit various functions and are extremely environmentally preferable substances, there is a strong demand for the development of novel polyamino acid derivatives having excellent functions. As mentioned above, most of the currently used polyamino acids are homopolymers composed of one kind of amino acid, but by combining two or more kinds of amino acids, more excellent properties (antibacterial activity, etc.) can be obtained. It is believed that a polymer having However, it is extremely difficult to select and provide a polyamino acid having a desired activity from among a myriad of combinations of amino acids and capable of being produced safely and efficiently.
Further, regarding the production method, the chemical synthesis of polyamino acid requires an expensive synthetic reagent and is high in cost, and it is difficult to dispose the polyamino acid because the reagents used include harmful chemicals. There are many problems. Furthermore, although it is possible to produce by a gene recombination method, it is not always an efficient method because of problems such as the antibacterial property of the product adversely affecting the host and the extraction method being complicated.
Furthermore, regarding D-amino acids such as D-histidine, a safe and efficient production method applicable to industrial production is also required, but such a method has not been known yet.
Therefore, in order to widely use polyamino acids and D-amino acids useful as functional polymers, it is essential to develop an efficient and safe production method.

［式中、Ｘは、下記の１）および２）：
１）アルギニン、リジン、オルニチン、シトルリン、ホモシトルリン、ホモアルギニン、カナバニンおよび２−アミノ−３−グアニジノプロピオン酸
２）ヒスチジン、システイン、ホモシステイン、トリプトファンおよびチロシンのいずれか一方の群から選択されるアミノ酸残基を表し、Ｙは他方の群から選択されるアミノ酸残基を表し、Ｒ_１は水素原子、糖残基、アシル基、ビオチニル基、チオール基、フェノール基、インドール基、または前記Ｙと同一のアミノ酸残基を表し、Ｒ_２は水酸基、糖残基、アシル基、ビオチニル基、チオール基、フェノール基、インドール基または前記Ｘと同一のアミノ酸残基を表し、ｎは２以上の整数を表す］
で示される、（１）に記載のポリアミノ酸またはその誘導体、
（３） Ｘがアルギニンまたはリジン残基であってＹがヒスチジンまたはシステイン残基である、（２）に記載のポリアミノ酸またはその誘導体、
（４） Ｘがアルギニン残基であってＹがヒスチジン残基である、（３）に記載のポリアミノ酸またはその誘導体、
（５） ｎが２〜５０の整数である、（２）〜（４）のいずれかに記載のポリアミノ酸またはその誘導体、
（６） Ｒ_１が水素原子でありＲ_２が水酸基である、（２）〜（５）のいずれかに記載のポリアミノ酸またはその誘導体、
（７） 銅、亜鉛、銀、ガドリニウム、マグネシウム、マンガン、コバルトおよびニッケルから選択される金属との錯体である、（１）〜（６）のいずれかに記載のポリアミノ酸またはその誘導体、
（８） Ｄ−アミノ酸残基を含む、（１）〜（７）のいずれかに記載のポリアミノ酸またはその誘導体、
（９） Ｄ−アミノ酸残基がＤ−ヒスチジン残基である、（８）に記載のポリアミノ酸またはその誘導体、
（１０） （１）〜（９）のいずれかに記載のポリアミノ酸またはその誘導体を有効成分として含有する組成物、
（１１） 抗菌性の組成物である、（１０）に記載の組成物、
（１２） 金属結合性の組成物である、（１０）に記載の組成物、
（１３） 酸性物質を吸着し、塩基性物質を排斥するための組成物である、（１０）に記載の組成物、
（１４） 物質を有効成分と化学結合した状態で、または混合した形で、細胞内へ輸送するための組成物である、（１０）に記載の組成物、
（１５） （１）〜（９）のいずれかに記載のポリアミノ酸を生産するエピクロエ（Ｅｐｉｃｈｌｏｅ）属の菌株、
（１６） エピクロエ キビエンシス（Ｅｐｉｃｈｌｏｅ ｋｉｂｉｅｎｓｉｓ）Ｅ１８株（ＦＥＲＭ Ｐ−１８９２３）またはそのポリアミノ酸生産能を有する変異体である、（１５）に記載の微生物、
（１７） （１５）または（１６）に記載の微生物を培養し、培地からポリアミノ酸を回収し、所望によりその遊離官能基を化学修飾し、および／または金属錯体を形成させるか、塩化することを特徴とする、（１）〜（９）のいずれかに記載のポリアミノ酸またはその誘導体の製造方法、
（１８） （８）または（９）に記載のポリアミノ酸の産生能を有する微生物を適当な培地で培養し、培地から分離した培養液、菌体または菌体破砕物からＤ−アミノ酸を回収するか、必要に応じてペプチド誘導体を加水分解した後、Ｄ−アミノ酸を回収することからなる、Ｄ−アミノ酸の製造方法、
（１９） 微生物がエピクロエ キビエンシス（Ｅｐｉｃｈｌｏｅ ｋｉｂｉｅｎｓｉｓ）Ｅ−１８株（ＦＥＲＭ Ｐ−１８９２３）またはそのＤ−アミノ酸生合成能を有する変異株である（１８）に記載の方法、および
（２０） （１８）または（１９）に記載の方法で製造されたＤ−アミノ酸、などに関する。An object of the present invention is to solve the above problems and provide a novel polyamino acid having excellent function and activity, and a derivative thereof.
Another object of the present invention is to provide a safe and easy method for producing such polyamino acids and their derivatives.
Another object of the present invention is to provide a novel method for producing a D-amino acid, especially D-histidine, which is excellent in safety and production efficiency, low in production cost, and applicable to mass production.
The present invention also aims to utilize polyamino acid derivatives, D-amino acids and the like.
The present inventors have conducted extensive studies in order to achieve the above-mentioned object, and as a result, two kinds of amino acids (at least one of which is a basic amino acid is It was also found that a polyamino acid consisting of (1) has a high antibacterial activity, and that some of the polyamino acids include D-histidine as an amino acid, and thus completed the present invention.
That is, the present invention is
(1) A polyamino acid consisting of 2 to 10 kinds of amino acids containing at least one kind of basic amino acid or a derivative thereof,
(2) General formula (I):

[Wherein, X is the following 1) and 2):
1) Arginine, lysine, ornithine, citrulline, homocitrulline, homoarginine, canavanine and 2-amino-3-guanidinopropionic acid 2) An amino acid selected from the group consisting of histidine, cysteine, homocysteine, tryptophan and tyrosine. Represents a residue, Y represents an amino acid residue selected from the other group, R ₁ represents a hydrogen atom, a sugar residue, an acyl group, a biotinyl group, a thiol group, a phenol group, an indole group, or the same as the above Y. R ₂ represents a hydroxyl group, a sugar residue, an acyl group, a biotinyl group, a thiol group, a phenol group, an indole group or the same amino acid residue as the above X, and n represents an integer of 2 or more. ]
And the polyamino acid or derivative thereof according to (1),
(3) The polyamino acid or derivative thereof according to (2), wherein X is an arginine or lysine residue and Y is a histidine or cysteine residue.
(4) The polyamino acid or a derivative thereof according to (3), wherein X is an arginine residue and Y is a histidine residue.
(5) The polyamino acid or derivative thereof according to any of (2) to (4), wherein n is an integer of 2 to 50.
(6) The polyamino acid or derivative thereof according to any of (2) to (5), wherein R ₁ is a hydrogen atom and R ₂ is a hydroxyl group.
(7) The polyamino acid or derivative thereof according to any one of (1) to (6), which is a complex with a metal selected from copper, zinc, silver, gadolinium, magnesium, manganese, cobalt and nickel.
(8) The polyamino acid or derivative thereof according to any one of (1) to (7), which comprises a D-amino acid residue.
(9) The polyamino acid or the derivative thereof according to (8), wherein the D-amino acid residue is a D-histidine residue.
(10) A composition containing the polyamino acid or derivative thereof according to any one of (1) to (9) as an active ingredient,
(11) The composition according to (10), which is an antibacterial composition,
(12) The composition according to (10), which is a metal-bonding composition,
(13) The composition according to (10), which is a composition for adsorbing an acidic substance and eliminating a basic substance,
(14) The composition according to (10), which is a composition for transporting a substance into cells in a state of being chemically bound to an active ingredient or in a mixed form.
(15) A strain of the genus Epichloe that produces the polyamino acid according to any one of (1) to (9),
(16) The microorganism according to (15), which is a strain having epichloe kibiensis E18 strain (FERM P-18923) or a polyamino acid-producing ability thereof,
(17) Culturing the microorganism according to (15) or (16), recovering the polyamino acid from the medium, chemically modifying the free functional group thereof, and/or forming a metal complex or salification. A method for producing the polyamino acid or derivative thereof according to any one of (1) to (9),
(18) The microorganism capable of producing the polyamino acid according to (8) or (9) is cultured in an appropriate medium, and the D-amino acid is recovered from the culture solution, the bacterial cells or the disrupted bacterial cells separated from the medium. Alternatively, a method for producing a D-amino acid, which comprises recovering the D-amino acid after hydrolyzing the peptide derivative if necessary,
(19) The method according to (18), wherein the microorganism is an Epichloe kibiensis E-18 strain (FERM P-18923) or a mutant strain thereof having a D-amino acid biosynthetic ability, and (20) (18) Alternatively, it relates to a D-amino acid produced by the method described in (19).

FIG. 1 is a chart showing the results of analyzing the polyamino acid produced by the E-18 strain purified in Examples 2(1) and (2) with an automatic Edman degradation device (Applied Biosystems, model 492). ..
FIG. 2 shows a MALDI-TOF Mass of a polyamino acid produced by the E-18 strain purified in Examples 2(1) and (2) using a time-of-flight mass spectrometer (Model Voyager DE manufactured by Perceptive). It is a figure which shows the result of the molecular weight measurement by the method.
FIG. 3 shows a molecular weight measurement of a mixture of synthetic polyarginyl histidine having 10 amino acid residues and zinc ion by a MALDI-TOF Mass method using a time-of-flight mass spectrometer (Model Voyager DE manufactured by Perceptive). It is a figure which shows the result of having used.
FIG. 4 is a graph showing the effect of various concentrations of zinc ions on the growth of Escherichia coli IFO3301 strain in modified synthetic liquid medium of Davis in the presence of polyarginyl histidine.
FIG. 5 shows that synthetic polyarginyl histidine having 10 amino acid residues is infiltrated on an agar plate containing the acidic dye PolyR-478 (A) or the basic dye methylene blue (B), and changes in the dye accompanying the diffusion are observed. It is a copy drawing of the photographed.
FIG. 6 shows the incorporation of the fluorescent dyes FAM (A, C) bound to the N-terminus of polyarginyl histidine having 10 amino acid residues or FAM alone (B, D) into the Candida boidinii MIP104 strain using a fluorescence microscope. It is a mimicking figure of the result observed under the microscope under (A, B) and normal light (C, D).
FIG. 7 is a chart showing the results of analyzing polyarginyl histidine (LR-DH) _n produced by the E-18 strain prepared in Example 2(2) by high performance liquid chromatography equipped with an optical resolution column. is there.
FIG. 8 is a chart showing the results of MALDI/TOF-MS analysis of various trypsin-treated polyarginyl histidines described in Example 15(2). Arrows indicate putative trypsin breakpoints.

［式中、Ｘは、下記の１）および２）：
１）アルギニン、リジン、オルニチン、シトルリン、ホモシトルリン、ホモアルギニン、カナバニンおよび２−アミノ−３−グアニジノプロピオン酸
２）ヒスチジン、システイン、ホモシステイン、トリプトファンおよびチロシンのいずれか一方の群から選択されるアミノ酸残基を表し、Ｙは他方の群から選択されるアミノ酸残基を表し、Ｒ_１は水素原子、糖残基、アシル基、ビオチニル基、チオール基、フェノール基、インドール基、または前記Ｙと同一のアミノ酸残基を表し、Ｒ_２は水酸基、糖残基、アシル基、ビオチニル基、、チオール基、フェノール基、インドール基または前記Ｘと同一のアミノ酸残基を表し、ｎは２以上の整数を表す］
で示されるポリアミノ酸およびその誘導体を挙げることができる。
上記式（Ｉ）において、Ｘがアルギニンまたはリジンであり、Ｙがヒスチジンまたはシステインであるか、Ｘがヒスチジンまたはシステインであり、Ｙがアルギニンまたはリジンであるポリアミノ酸がより好ましく、ＸおよびＹのいずれか一方がアルギニンであり、他方がヒスチジンであるポリアミノ酸が特に好ましい。さらに、ｎが２〜５０の整数であることが好ましく、Ｒ_１が水素原子でありＲ_２が水酸基であることがとりわけ好ましい。
また、いずれかのアミノ酸がＤ−アミノ酸であることが好ましく、特にヒスチジンがＤ−ヒスチジンであることが好ましい。
本発明のポリアミノ酸誘導体のペプチド鎖の長さは任意であり、目的に応じて選択されるが、通常、アミノ酸数は３以上である。好ましくは５〜１００の範囲、より好ましくは５〜５０、さらに好ましくは６〜２０、最も好ましくは６〜１２である。
＜ポリアミノ酸誘導体の製造＞
本発明のポリアミノ酸誘導体は、当該技術分野で既知の化学合成法、生化学的な方法、微生物発酵等により製造することができる。生化学的な方法は、ポリアミノ酸をコードするＤＮＡまたはＲＮＡの遺伝情報を鋳型として、生物に備わる転写、翻訳系の機能により、細胞内あるいは人工的反応液内で遺伝子工学的に製造することにより行うことができる。特に好ましい方法は通常の微生物発酵法である。本発明のポリアミノ酸誘導体は、遊離の形でも活性を有するが、抗菌活性が増強されたり、また安定性や水溶性の改善のために、適当な酸や塩基の塩や金属錯体にすることもできる。そのような塩や錯体は当該技術分野で既知の方法に従って製造することができる。
微生物発酵によるポリアミノ酸、Ｄ−アミノ酸の製造
微生物発酵によって本発明のポリアミノ酸またはＤ−アミノ酸を製造するためには、まず、目的のポリアミノ酸を産生する微生物を特定する必要がある。そのような微生物のスクリーニング法は当該技術分野で既知である［西川等（Ｎｉｓｈｉｋａｗａ，Ｍ．ｅｔ ａｌ．），Ａｐｐｌｉｅｄ Ａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ，（米国），ｖｏｌ６８，Ｎｏ．７，ｐ３３７５−３５８１］。例えば菌体外に塩基性ポリアミノ酸を分泌する、塩基性ポリアミノ酸生産性の微生物を選抜するには、微生物を含むサンプル（例えば土壌試料）を適当な組成の寒天平板培地上にて、個々の微生物集落に展開する。この寒天平板にあらかじめ酸性色素または塩基性色素を添加しておき、塩基性ポリアミノ酸の分泌を、酸性色素との結合、または塩基性色素に対する反発作用に基づいて検出する。
さらに、キレート形成能を有する優れた多座配位子であるポリアミノ酸誘導体を生産する微生物は、例えば、以下の方法でスクリーニングすることができる。菌体外にキレート形成性（金属配位性）のポリアミノ酸を分泌する微生物の選抜には、微生物を含む土壌試料を適当な組成の寒天平板培地上にて、個々の微生物集落に展開する。寒天平板にはあらかじめ微生物にとって有害な毒性金属、例えば銀、銅、亜鉛、ニッケル、コバルト、カドミウム、水銀、などを適当な濃度にて、添加しておく。キレート形成性ポリアミノ酸の分泌は、その金属への配位性に基づいて、該ポリアミノ酸が有害金属を捕捉、抱合する事により毒性が中和または緩和され、他のポリアミノ酸非生産微生物との間で増殖に差が認められることにより検出される。
塩基性ならびにキレート形成性を示すポリアミノ酸を生産する微生物は、上述の方法により、塩基性およびキレート形成性の二つの特性を、単独でまたは同時に試験することで選抜することができる。
本発明の好ましい態様では、本発明のポリアミノ酸誘導体を、エピクロエ属の菌株を用いて微生物学的に製造する。好ましい微生物はエピクロエ キビエンシス（Ｅｐｉｃｈｌｏｅ ｋｉｂｉｅｎｓｉｓ）Ｅ１８株（ＦＥＲＭ Ｐ−１８９２３）またはその変異体である。
エピクロエ キビエンシスＥ１８株（以下、「Ｅ１８株」と略称する）は、実施例に記載の方法により本発明者らが林地土壌中から、新たに分離した菌株であり、その菌学的性状は次のとおりである。
Ａ．培地上の生育状況
Ｅ１８株を、ポテトデキストロース培地（ＰＤＡ）、オートミール培地（ＯＡ）、および２％麦芽培地（ＭＥＡ）の各寒天平板上において、２５℃にて培養すると、菌糸は綿毛状で白色を呈する。生育はやや遅い。分生子の形成は少なく、分生子によるコロニー色調の変化は観察されない。可溶性色素の産生は認められない。
Ｂ．胞子の形成
光学顕微鏡下ではフィアロ型の分生子構造が確認される。分生子柄は多少直立し、気中菌糸より単生ないしは２から４個のフィアライドを輪生する。分生子は１細胞性で表面は平滑である。形は卵円形、涙形、楕円形、長円形など多様である。分生子は粘性を示し、塊状に集まる。培養８週間経過後においても三日月型の大型分生子や厚膜胞子の形成は観察されない。
Ｃ．生理学的性質
ポリアルギニルヒスチジンを生産する。
Ｄ．資化可能な炭素源
グリセロール、Ｄ−グルコース、Ｄ−ガラクトース、Ｄ−キシロースを利用しうる。
上記の菌学的性質を有する本菌株の分類学上の位置を、２８ｓリボソームＲＮＡ分子の塩基配列の種間比較に基づく分子生物学的手法と併せて検討した結果、エピクロエに属する新菌株である事が判明し、これをＥｐｉｃｈｌｏｅ ｋｉｂｉｅｎｓｉｓ Ｅ１８株と命名した。本菌株は、茨城県つくば市東１丁目１番地１ 中央第６、独立行政法人産業技術総合研究所 特許生物寄託センターに寄託されている（微生物の表示：Ｅｐｉｃｈｌｏｅ ｋｉｂｉｅｎｓｉｓ Ｅ１８、受領日：平成１４年７月５日；受託番号ＦＥＲＭ Ｐ−１８９２３）（国際寄託への変更日：平成１５年４月１１日；受託番号：ＦＥＲＭ ＢＰ−８３５８）。
Ｅ１８株を親株として、変異体誘導や組換え遺伝子技術などにより、ポリアルギニルヒスチジンの生産性を高めたり、アミノ酸の組成が変更されたポリアミノ酸を産生する、派生菌株を得ることができる。そのような変異株も本発明の範囲内である。派生菌株は、人為的に突然変異を誘発されたものや、スクリーニングで得られたもの等を包含する。
Ｅ１８株は塩基性ポリアミノ酸の示す抗菌作用に耐性であるので、任意の配列を持つ塩基性ポリアミノ酸、ペプチド、タンパク質の加工、製造に利用できる。例えば、▲１▼後述の実施例に記載のポリアルギニルヒスチジンの遺伝子を改変、加工してＥ１８株を形質転換する、あるいは▲２▼適当なポリアミノ酸をコードするＤＮＡを発現プロモーター下に、適当なベクター内で連結し、得られた発現ベクターでＥ１８株を形質転換する、等の方法が可能である。
本発明のポリアミノ酸を生産する微生物、例えば、エピクロエ キビエンシス Ｅ１８株などの培養は、微生物の性質に応じて適宜選択され、市販品から入手可能であるか、当業者既知の方法で調製することができる。液体状または固体状の適当な組成で構成される完全培地、合成培地、半合成培地を用いることができるが、操作の容易性等から液体培地が適する。培地は、一般的な成分として、炭素源、窒素源、無機塩及びその他の栄養物が含まれていれば、いかなるものでもよい。炭素源としてはグルコース、ガラクトース、フラクトース、グリセロール、スターチ等が挙げられ、その含有量は０．１〜１０％（ｗ／ｖ）が好ましい。窒素源としては、酵母エキス、ペプトン、カゼイン加水分解物、アミノ酸等の有機化合物や、硫酸アンモニウム、塩化アンモニウム、硝酸ナトリウムなどの無機アンモニウム塩等が挙げられ、その含有量は０．１〜５％（ｗ／ｖ）が好ましい。所望により、培地に他の栄養源（例えば無機塩類、例えば、リン酸イオン、カリウムイオン、ナトリウムイオン、マグネシウムイオン、亜鉛イオン、鉄イオン、マンガンイオン、ニッケルイオン、硫酸イオン等を与えるもの）、ビタミン類（例えばビタミンＢ_１）、抗生物質（例えばアンピシリン、テトラサイクリン、カナマイシン等））を加えてもよい。
培養は、好気的条件下で振盪培養、攪拌培養等により行うことができる。培養温度は約２５〜４０℃であり、培地のｐＨは２．０〜８．０、好ましくはｐＨ３．０〜８．０、より好ましくはｐＨ約５．０である。培養期間は、通常、１日〜１４日間であるが、それ以上の期間、培養を続けることができる。
Ｅ１８株を親株として誘導される上記の派生菌株（変異株）も同様に培養することができる。
生産されたポリアミノ酸が培養液中に分泌されている場合、培養物をろ過又は遠心分離して粗生成物を単離する。生成されたポリアミノ酸の精製は、回収した培養液上清から、天然又は合成のアミノ酸やタンパク質の精製、単離に用いられる当該技術分野で既知の方法（例えば、イオン交換樹脂処理法、活性炭吸着処理法、有機溶媒沈澱法、減圧濃縮法、凍結乾燥法、結晶化法等）を適宜組み合わせて実施することができる。生産されたポリアミノ酸が培養微生物のペリプラズム及び細胞質中に存在するときは、ろ過や遠心分離によって細胞を集め、それらの細胞壁及び／又は細胞膜を、たとえば超音波及び／又はリゾチーム処理によって破壊して、デブリス（細胞破砕物）を得る。このデブリスを適当な水溶液（例えば緩衝液）に溶解させ、上記の方法に準じて生成物を単離、精製することができる。
Ｄ−アミノ酸の製造
微生物により産生されるポリアミノ酸がＤ−アミノ酸を含んでいる場合、必要に応じて、化学的、酵素的な方法で加水分解し、Ｄ−アミノ酸を単離する。加水分解は、当該技術分野で既知の方法により行うことができ、例えば、塩酸、硫酸などの酸、水酸化ナトリウム、水酸化カリウムなどの塩基、アミノペプチダーゼ、カルボキシペプチダーゼ、エンドペプチダーゼなどのプロテアーゼを用いて実施することができる。
酸加水分解は、例えば６Ｎ塩酸により、約１００℃で約２０時間加熱すればよい。
加水分解産物からのＤ−アミノ酸モノマーの単離精製は上記した培養物からの単離、精製法に準じて行うことができ、例えば、クロマトグラフィー、分別晶析、酵素処理等が例示される。
Ｅ−１８株により産生されるポリアミノ酸の特性
上記の方法でＥ１８株等を培養すると、ポリアミノ酸として、ポリアルギニルヒスチジンが生成される。このポリアルギニルヒスチジンの物理化学的性質は以下のとおりである。
（ａ）６Ｎ塩酸溶液による加水分解処理により、アルギニンおよびヒスチジンのみが生成する。
（ｂ）ポリアルギニルヒスチジンおよびその加水分解物は坂口反応およびパウリ反応に陽性である。
（ｃ）モノマー間の結合様式はα位カルボキシル基とα位アミノ基間のペプチド結合である。
（ｄ）自動エドマン分解法により決定されるアミノ酸配列は、Ｎ末端をアルギニンとするアルギニンとヒスチジンの交互の繰り返しである。
（ｅ）ＭＡＬＤＩ−ＴＯＦ Ｍａｓｓ（マトリックス支援脱離イオン化−飛行時間測定）法による分子量測定では、分子量が約１，４８６である分子が主成分である。この他にも、約２９３の規則的な分子量の差をもった、異なる分子の混成物である。
（ｆ）生成したヒスチジンは薄層クロマトグラフィーにおいて、ヒスチジン標準品と同一のＲｆ値（０．１９）を示し、ニンヒドリン反応、パウリ反応に陽性である。
（ｇ）生成したヒスチジンの光学純度は、光学分割カラムによるクロマトグラフィー分析により、約８５％がＤ−体である。
従って、本発明は、本発明のポリアミノ酸誘導体を生産するエピクロエ（Ｅｐｉｃｈｌｏｅ）属の菌株を提供するものである。
好ましくは、エピクロエ キビエンシス（Ｅｐｉｃｈｌｏｅ ｋｉｂｉｅｎｓｉｓ）Ｅ１８株（ＦＥＲＭ Ｐ−１８９２３）またはそのポリアミノ酸誘導体生産能を有する変異体である。
さらに、本発明は、本発明の菌株を培地中で培養し、培養液からポリアミノ酸誘導体を回収することを特徴とする、ポリアミノ酸誘導体の製造法（以下、本発明製造法ともいう）を提供する。本発明菌株は、好ましくはエピクロエ キビエンシス（Ｅｐｉｃｈｌｏｅ ｋｉｂｉｅｎｓｉｓ）Ｅ１８株（ＦＥＲＭ Ｐ−１８９２３）であり、ポリアミノ酸誘導体はポリアルギニルヒスチジンである。
本発明はまたＤ−アミノ酸の微生物発酵による製造方法であって、Ｄ−アミノ酸を含むポリアミノ酸誘導体の産生能を有する微生物を適当な培地で培養し、培地から分離した培養液、菌体または菌体破砕物からＤ−アミノ酸を回収するか、必要に応じてペプチド誘導体を加水分解した後、Ｄ−アミノ酸を回収することからなる、Ｄ−アミノ酸の製造方法、およびそのようにして製造されたＤ−アミノ酸を提供するものである。
Ｅ−１８株の変異株は、Ｅ−１８株の細胞を変異誘発処理することによって得られる変異株やＥ−１８株の自然突然変異株を、ポリアルギニルヒスチジンを生産する性質などを指標としてスクリーニングすることによって得られる。変異誘発処理としては、紫外線照射やＮ−メチル−Ｎ−ニトロ−Ｎ’−ニトロソグアニジンなどの変異誘発物質による処理が挙げられる。ポリアルギニルヒスチジンを生産する性質などを指標とするスクリーニングは、例えば、後記実施例１に記載された方法によって行うことができる。
化学合成による製造および誘導体化
本発明のポリアミノ酸（例えばポリアルギニルヒスチジン）はペプチド合成法により化学合成によっても製造できる。化学合成に際しては、例えばポリアルギニルヒスチジンのアルギニン残基をホモアルギニン、リジン、オルニチン、シトルリンなど他の塩基性アミノ酸と置換し、および／または、ヒスチジンをシステイン、トリプトファン、チロシンなどと置換できる。
誘導体化に際しては、塩基性ポリアミノ酸中の塩基性官能基を有機または無機の酸との塩とすることができる。例えば、塩酸、硫酸、リン酸などの無機酸もしくは酢酸、プロピオン酸、フマル酸、リンゴ酸などの有機酸の塩の形で用いることができる。
また、当該技術分野で既知の方法で、適当な金属元素との錯体とすることができる。そのような金属原子の例として、マグネシウム、マンガン、コバルト、ニッケル、銅、亜鉛を挙げることができる。
さらに、ポリアミノ酸残基中の遊離した官能基には各種の化学修飾、例えばアシル化等を行うことが可能である。これらの製造方法および誘導体化の方法は当該技術分野で既知である。
ポリアルギニルヒスチジン中のアルギニン残基に側鎖として遊離するグアニジノ基をアルカリ加水分解することで、オルニチンに変更できる。アルギニンとオルニチンは、それぞれのグアニジノ基およびアミノ基の解離定数が異なるため、アルギニンとオルニチンの構成比を適宜調節して、用途目的に最適な電解性官能基の解離定数（ｐＫａ）を持ったポリアミノ酸を製造することができる。
ポリアミノ酸誘導体の用途
本発明のポリアミノ酸誘導体は、後述の実施例に示すように、高い抗菌活性を有しており（実施例３，４）、しかも既存のホモポリマーに比較して顕著に高い抗菌活性を有することが明らかになった（実施例８）。また、金属との結合性（実施例１０）、酸性物質の吸着活性（実施例１２）、生細胞への透過性（実施例１３）など、種々の機能を有する機能性分子であることが判明した。さらに、本発明のポリアミノ酸の抗菌活性は錯体形成により増強されることを示唆することが確認された（実施例１１）。従って、本発明のポリアミノ酸誘導体は生分解性の機能性ポリマーであり、広範な用途を有する。以下に示す用途には、ポリアミノ酸誘導体を単独で、または組成物としておよび／または他の成分と併用して使用することができる。ポリアミノ酸誘導体を含有する組成物は、当該技術分野で既知の方法で、適当な担体、賦形剤、希釈剤等と共に溶液、懸濁液、固形等の様々な形に調製することができる。本発明のポリアミノ酸は他の有効成分と併用することができ、そのような目的には予め一組成物中に共存させても良く、使用時に混合、併用してもよい。
本発明のポリアミノ酸誘導体の主な用途は、その抗菌活性、金属結合活性、酸性物質との結合または吸着活性等の各機能に関連する。例えば、抗菌素材、金属結合素材、酸性物質吸着作用に基づく塩基性物質の排斥用素材、または物質と化学結合した状態で、または混合した形で、該物質を細胞内へ輸送するための素材として使用しうる。ここで「素材」とは、それ自体が例えば抗菌性組成物として有用であるのみならず、各種製品の製造原料として機能することを意味する。以下にこれらを詳細に説明する。
１）抗菌性物質としての利用
本発明のポリアミノ酸誘導体は、グラム陰性細菌、グラム陽性細菌抗菌性、酵母菌、糸状真菌に対して増殖抑制効果を有する。この効果は金属錯体の形で特に優れている。
従って、本発明のポリアミノ酸誘導体は、抗菌性物質として食品や飼料の保存（食品添加物）、食品や飼料の日持ち性向上（食品添加物）、食品保存性を改善する食品容器の成分、冷蔵氷の食品保存性増強、発酵産業における微生物の制御、生簀や水槽の水質劣化防止、人、畜、魚類に投与する抗生物質（医薬品類）、無菌であることが求められる医療器具の保存剤、コンタクトレンズの保存剤、歯科補綴具の保存、抗菌効果を付した医療用繊維、化粧品や医薬部外品の品質保持、搾乳房の汚染管理、花卉の日持ち性向上、植物の根腐れ防止、植物種子の発芽管理、無菌栽培における微生物制御、有用微生物と不要微生物を分離するための選抜法、などに利用できる。
上記の抗菌活性に係る本発明のポリアミノ酸誘導体の使用形態は、特に限定されないが、本発明のポリアミノ酸誘導体の抗菌活性を最終目的物中に混合または練りこむ、表面に散布する、あるいは目的物の収容容器、包装材料等に含有させるか塗布する等の方法をとることができる。実施に際しては、当該技術分野で同様の目的で使用される方法に準じて行えばよい。そのような使用例は、例えば、特許１５３７２９７、特開平５−１４６２８２、特開平８−１７５９０１、特開平９−１２４４２２、特開平９−１０２８８、特開平１０−１０９９０６、特開平１０−２７９７９４、特開２０００−１８９１２９等に記載されており、当業者に既知である。
２）金属結合能を有する物質、または金属錯体としての利用
本発明のポリアミノ酸誘導体は金属錯体形成能を有することから、上記の抗菌性物質としての利用に加えて、有用金属の吸着、濃縮、金属定量用試薬として利用できる。さらには、重金属錯体として医療用造影剤にも利用可能である。さらには、放射性金属の吸着、濃縮、放射性金属の生体からの排出促進、生物に重金属耐性を付与するための細胞内含有物または細胞外分泌物（細胞内で生産後、細胞内部に蓄積されるか細胞外に分泌されることによる）、インク（顔料）の成分、金属含有医薬品の成分、ならびに家畜や魚類への金属投与用組成物または飼料の成分、生体必須金属の摂取源（健康食品）等に利用できる。
上記の金属結合活性に係る本発明のポリアミノ酸誘導体の使用形態は、特に限定されず、当該技術分野で同様の目的で使用される方法に準じて選択することができる。そのような使用例は、例えば、特開平７−２２４０５０、特許出願平１１−１７４０５４等に記載されており、当業者に既知である。
本発明のポリアミノ酸誘導体が錯体を形成している場合、金属イオンとしては、目的に応じて適宜選択される、特に限定されないが、例えば、食品、医薬、化粧品等の抗菌剤として用いる場合は、銅、亜鉛、銀、造影剤として用いる場合は常磁性のガドリニウム、マンガン等を挙げることができる。
３）静電的（イオン）反応性物質としての利用
本発明のポリアミノ酸誘導体は塩基性ポリマーであることから、静電的に酸性物質と結合し、塩基性物質とは反発する性質を有する。また、塩基性ポリアミノ酸は細胞内へ進入する性質を有することから、ポリアミノ酸に化学的に結合した物質やポリアミノ酸と親和した物質を細胞内へ輸送することができる。ここで、ポリアミノ酸と「親和した物質」とは、ポリアミノ酸水溶液中に溶解または懸濁された状態で、ポリアミノ酸が細胞膜の透過性の変化させた際に、細胞内へ進入しうる物質を指す。
例えば、ポリアルギニルヒスチジンと化学的に結合する物質（例えば、ペプチド、タンパク質、ＤＮＡ、ＲＮＡ、糖）は細胞内へ輸送され、細胞内でそのままか、あるいは分解、プロセッシングされて遊離され、所望の機能を発揮しうる。このように、酸性物質の吸着剤、酸性有害物質の除去剤、ドラッグ・ディリバリー法における担体、遺伝子治療に使用される核酸の運搬体、核酸の細胞への導入を促す担体、繊維の染色補助剤、などに利用できる。
好ましくは、本発明のポリアミノ酸誘導体はＲＮＡやＤＮＡなどの遺伝情報をコードするポリヌクレオチドのビークル（運搬媒体）として、利用される。そのようなビークルは、遺伝的、非遺伝的疾患の治療疾患の遺伝子治療や、発酵、農業、畜産、水産業等における有用な性質を持つ新たな細菌、動植物などを得るために、インビトロまたはインビボでの遺伝子導入に有用である。
上記の酸性物質との結合活性に係る本発明のポリアミノ酸誘導体の使用形態は、特に限定されず、当該技術分野で同様の目的で使用される方法に準じて選択することができる。そのような使用例は、例えば、特開平９−３０８４８４等に記載されており、当業者に既知である。
Ｄ−アミノ酸の用途
本発明方法により得られるＤ−アミノ酸は医薬、化粧品等の分野で、そのまま、あるいは中間体として極めて有用である。利用に際しては、Ｄ−アミノ酸単独で用いることも可能であるが、通常は、ペプチド結合により、ジペプチド、トリペプチド、ポリペプチド（残基数が４またはそれ以上）、タンパク質やアミノ酸構成が単純であるポリアミノ酸に導入する。さらに、Ｄ−アミノ酸がＤ−ヒスチジンである場合、化合物にイミダゾール環を導入する際の該環の供給源としても利用しうる。
Ｄ−アミノ酸が導入されたペプチド等は、例えば、Ｄ−アミノ酸が存在することに関連する特徴的な性質を具備し得る。
例えばＤ−アミノ酸がＤ−ヒスチジンである場合、本願発明に係るポリアルギニルヒスチジン以外にＤ−ヒスチジンの生物界での存在を示す報告例がないことは、Ｄ−アミノ酸またはそれを含有するペプチド誘導体が極めて稀な物質であることを示しており、これから、ペプチド分解酵素、アミノ酸酸化酵素、アミノ酸脱炭酸酵素、アミノ酸脱アミノ酵素など、Ｄ−ヒスチジンに作用する酵素の分布は少ないと予想される。従って、Ｄ−ヒスチジンを含有する化合物を生体、食品、環境などに導入した場合、Ｌ−ヒスチジンを含有する化合物と比較して、生物による酵素作用に対して感受性が低く（耐性が高く）、変性を受けにくいため、化合物としての寿命を長期化できると考えられる。この特性を活かして、医薬品、農薬品、化学品、化粧品、食品添加物等原料やその合成中間体などに利用できる。
また、生理活性物質にはＤ−アミノ酸を持つものがあり、Ｄ−アミノ酸の示す特異性が直接生理活性に関わる場合もある。従って、機能発現にＤ−系列のヒスチジンまたは誘導体が必要である化合物の製造において、本発明方法で得られるＤ−ヒスチジンは有用である。
以下に実施例を挙げて本発明をさらに詳細に説明するが、これら実施例は例示のみを目的としており、いかなる意味においても本発明を限定することを意図するものではない。
実施例１ ポリアミノ酸生産菌株の同定
ポリアルギニルヒスチジン生産菌の取得は、西川等（前掲）記載の方法に従い、下記のようにして行った。
（１）菌株の分離
固形培地（グリセロール；１０ｇ、硫酸アンモニウム；０．６６ｇ、りん酸二水素カリウム；０．６８ｇ、硫酸マグネシウム七水和物；０．２５ｇ、イーストエクストラクト；０．１ｇ、微量ミネラル要素、微量、の各成分を１Ｌの脱イオン水に溶解し、水酸化ナトリウム水溶液にてｐＨを７．０に調整し、粉末寒天１５ｇを加える）を滅菌（１２１℃、１５分間）した後、別に滅菌した色素、酸性色素ＰｏｌｙＲ−４７８（シグマ社製、終濃度０．０２％）を添加して、シャーレ中に固化する。１白金耳量の土壌試料を適当量の滅菌水にて分散し、一部を上記寒天平板培地上に塗布により植菌する。２８℃にて３〜１４日間培養し、コロニー（細胞集落）の周縁に形成される色素の吸着域の存在を特徴とする菌株を、塩基性ポリアミノ酸生産菌の候補として選抜する。
（２）菌学的性質
ポリアミノ酸生産菌の分離、特性化
上記（１）で選抜した菌株をポテトデキストロース培地（ＰＤＡ）、オートミール培地（ＯＡ）、および２％麦芽培地（ＭＥＡ）の各寒天平板に接種し、２８℃で培養し、菌学的性質を調べた。ポテトデキストロース培地（ＰＤＡ）、オートミール培地（ＯＡ）、および２％麦芽培地（ＭＥＡ）はいずれもディフコ／ベクトン−ディキンソン（Ｄｉｆｃｏ／Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ）社製である。
Ａ．培地上の生育状況
すべての寒天平板上において菌糸は綿毛状で白色を呈する。生育はやや遅く、ＰＤＡおよびＭＥＡの平板では培養開始後１週間で直径２０ｍｍに達し、ＯＡの平板では直径１５ｍｍに達する。分生子の形成は少なく、分生子によるコロニー色調の変化は観察されない。浸出液および可溶性色素の産生は認められない。
Ｂ．胞子の形成
光学顕微鏡下ではフィアロ型の無性生殖器官の形成が確認される。分生子柄は多少直立し、気中菌糸より単生ないしは２から４個のフィアライドを輪生する。また、二次的な分枝を示す分生子柄も若干観察される。フィアライドは細長い錐形で直線的、大きさは変化に富む。菌糸およびフィアライドの表面は平滑である。分生子は１細胞性で表面は平滑である。形は卵円形、涙形、楕円形、長円形など多様である。分生子は粘性を示し、分生子柄先端より塊状となる。培養８週間経過後の観察でも三日月型の大型分生子や厚膜胞子およびテレオモルフの形成は認められない。
Ｃ．生理学的性質
２１℃または２８℃にて良好に生育するが、３３℃では生育しない。ｐＨは２．５から８．８の間で生育する。後述のとおり、ポリアルギニルヒスチジンを生産する。
Ｄ．資化可能な炭素源
Ｄ−グルコース、Ｄ−ガラクトース、Ｄ−キシロース、Ｄ−ソルビトール、Ｄ−マンニトール、グリセロール、クエン酸、Ｌ−グルタミン酸を利用し得る。
（３）種の同定
上記（１）で選抜した菌株について、２８ｓリボソームＲＮＡ分子の塩基配列を決定した。この塩基配列はＤＮＡ Ｄａｔａ Ｂａｎｋ ｏｆ Ｊａｐａｎに、登録番号ＡＢ０８７３７３の下で登録されている。２８ｓリボソームＲＮＡ分子の種間比較に基づく分子生物学的手法により検討した結果、当該菌株はエピクロエに属する新菌株である事が判明した。これはＥｐｉｃｈｌｏｅ ｋｉｂｉｅｎｓｉｓ Ｅ１８株と命名され、独立行政法人産業技術総合研究所 特許生物寄託センターに受託番号ＦＥＲＭ ＢＰ−８３５８の下で寄託されている（受託日：平成１４年７月５日）。
実施例２ Ｅ１８株によるポリアミノ酸の生産と同定
（１）固体培養による生産
実施例１に記載の方法に従い、該実施例１に記載の固形培地（但し色素は含まない）でＥ１８株を培養した。１０日間培養した後、コロニー（細胞集落）周縁に分泌されるポリアミノ酸を寒天小片として回収した。寒天小片中に残留するポリアミノ酸以外の培地成分を、寒天小片を水に浸積することで除去した。次に寒天小片を１Ｎ塩酸溶液中で９０℃、１時間加熱することにより寒天成分を加水分解した。この溶液中に含まれるポリアミノ酸を陽イオン交換樹脂（アンバーライトＩＲＣ−５０、オルガノ社製）に吸着させ、０．２Ｎ酢酸を用いて洗浄し、０．１Ｎ塩酸にて溶出し、減圧乾燥することにより精製した。
（２）液体培養による生産
液体培地（Ｄ−ガラクトース、１０ｇ；硫酸アンモニウム、０．６６ｇ；りん酸二水素カリウム、０．６８ｇ；硫酸マグネシウム七水和物、０．２５ｇ；酵母エキス、０．２ｇ；微量ミネラル要素、微量、を１Ｌの脱イオン水に溶解し、ｐＨ値をＮａＯＨ水溶液にて７．０に調整する）の１００ｍＬ量を５００ｍＬ容バッフル付三角フラスコ入れ、滅菌（１２１℃、１５分間）する。植菌後、２８℃の恒温槽内で１００ｒｐｍの旋回振とうにより好気的に培養する。７日後、培養液の上清を、孔径０．４５ｍｍの合成高分子膜でろ過することにより回収し、この溶液中に含まれるポリアミノ酸を陽イオン交換樹脂（アンバーライトＩＲＣ−５０、オルガノ社製）に吸着させ、０．２Ｎ酢酸を用いて洗浄し、０．１Ｎ塩酸にて溶出し、減圧乾燥することにより精製した。
（３）モノマーの同定
１）上記（１）または（２）でＥ１８株の培養液から精製したポリアルギニルヒスチジンを、６Ｎ塩酸溶液中で１００℃、２０時間加熱することにより加水分解し、モノマーを調製した。モノマーを薄層クロマトグラフィー法［展開薄層板、シリカゲルまたはセルロースを塗布したもの；展開温度，２０℃；展開溶媒，ｎ−ブタノール：酢酸：ピリジン：水＝４：１：１：２の混液］にて展開し、ニンヒドリン試薬にて現像した。モノマー化されたアミノ酸は２種あり、それぞれ標準物質のアルギニンおよびヒスチジンと同一のＲｆ値（それぞれ、０．２２および０．１９）を示した。Ｒｆ値が０．２２であるモノマーは、グアニジノ基の検出法である坂口反応に陽性であった。また、Ｒｆ値が０．１９であるスポットはイミダゾール環の検出法であるパウリ反応に陽性であった。また加水分解処理していないポリアミノ酸は坂口反応およびパウリ反応に陽性であった。
以上の結果から、モノマーはアルギニンとヒスチジンであることが判明した。各側鎖上に存在するグアニジノ基とイミダゾール環はポリアミノ酸中に遊離した形で存在しており、アルギニンとヒスチジンは主鎖上のα位アミノ基とα位カルボキシル基間のペプチド結合により相互に連結されていることが判明した。
（４）ポリアミノ酸の配列決定
ポリアミノ酸の構成成分であるアルギニンとヒスチジンの配列を明らにするため、精製したポリアミノ酸を自動エドマン分解分析装置（アプライドバイオシステムズ社製、モデル４９２）により解析した。結果を図１に示す。
Ｎ末端のアミノ酸残基はアルギニンであり、第２残基はヒスチジンであった。これ以降は、奇数番目の残基がアルギニンであり、偶数番目の残基がヒスチジンであった。図１に示すように、第２残基はヒスチジンであり、第３残基はルギニンである。
以上から、ポリアミノ酸はアルギニンをＮ末端とし、アルギニンとヒスチジンが交互に連鎖した構造を持つことが判明した。
（５）分子量の測定
ポリアミノ酸の分子量を明かにするために、精製したポリアミノ酸をＭＡＬＤＩ／飛行時間型質量分析計（パーセプティブ社製、モデルＶｏｙａｇｅｒ ＤＥ）で測定した。図２に質量分析の結果を示す。検出された分子量はＲＨＲＨＲＨＲＨＲＨ（Ｒはアルギニン残基、Ｈはヒスチジン残基を表す、配列番号１）に由来する約１，４８６を中心とし、この他に約２９３の差をもった、より小さいか（ＲＨＲＨＲＨＲＨに由来する約１，１９３、配列番号２）、またはより大きい（ＲＨＲＨＲＨＲＨＲＨＲＨに由来する約１，７７９、配列番号３）シグナルが認められた。また、精製票品には、これら以外の不純物はほぼ含まれなかった。
以上から、Ｅ１８株が生産するポリアミノ酸は唯一の分子量をもつ単一分子ではなく、一定の範囲内に分子量分布をもつ分子の混成物であった。
実施例３ Ｅ１８株が生産するポリアルギニルヒスチジンの抗菌活性
抗菌性試験は以下の方法で行った。
実施例２の（２）で精製し、実施例２（３）〜（５）で同定したＥ１８株が生産するポリアルギニルヒスチジンを滅菌水にて溶解し、さらに一定の終濃度となるように適当な液体培地に添加した。液体培地は表１に示すとおり、感受性を評価される微生物（被検微生物）の増殖に適したものを選択した。各液体培地の１Ｌ中の組成は、１）Ｎ培地（ヌートリエント ブロス）：肉エキス，３ｇ；ペプトン，５ｇ，ｐＨ６．８、２）７０２培地：ポリペプトン，１０ｇ；酵母エキス，２ｇ；硫酸マグネシウム七水和物，１ｇ，ｐＨ７．０、３）ＹＰＤ培地：酵母エキス，１０ｇ；ペプトン，２０ｇ；Ｄ−グルコース，２０ｇ，ｐＨ６．５、４）ＰＤ培地：ジャガイモ浸出物，原料ジャガイモ２００ｇに相当する量；Ｄ−グルコース，２０ｇ，ｐＨ５．１、とした。ポリアルギニルヒスチジンを含む液体培地に被検微生物を接種し、それぞれの最適培養温度にて２０時間培養した。増殖の検定には、細胞の分散性が良好な微生物に対しては６００ｎｍの吸光度を測定し、不均一な細胞塊を形成する微生物に対しては目視にて行った。抗菌性試験の結果を表１に示す。表１に示すように、Ｅ１８株が生産したポリアルギニルヒスチジンはグラム陰性細菌、グラム陽性細菌、酵母菌、糸状真菌に対して、増殖を強く抑制した。

培地：Ｎ，ヌートリエント ブロス；ＹＰＤ，ｙｅａｓｔ ｅｘｔｒａｃｔ／ｐｅｐｔｏｎｅ／ｄｅｘｔｒｏｓｅ；ＰＤ，ポテト デキストロース培地；７０２，ＩＦＯ７０２培地．
＊増殖速度の低下が観察された。
相対濃度は、Ｅ１８の培養液から精製した抗菌物質を試験培地へ添加する際、その濃度が培養液中へ生産されたときの濃度を１とした場合の相対値（希釈倍率または濃縮倍率）を表す。ｘ１の濃度はおよそ４０μｇ／ｍＬと推定される。従って、相対濃度、ｘ１／１０、×１／２、ｘ１、ｘ５はそれぞれ、およそ４、２０，４０，２００μｇ／ｍｌに相当する。
実施例４ 化学合成したポリアルギニルヒスチジンの抗菌活性
アミノ酸残基数１０のポリアルギニルヒスチジンを化学合成法により作成し、試験に供した。化学合成は文献（Ａｔｈｅｒｔｏｎ，Ｅ ａｎｄ Ｓｈｅｐｐａｒｄ，Ｒ．Ｃ．，Ｓｏｌｉｄ ｐｈａｓｅ ｐｅｐｔｉｄｅ ｓｙｎｔｈｅｓｉｓ．Ａ ｐｒａｃｔｉｃａｌ ａｐｐｒｏａｃｈ，ＩＲＬ Ｐｒｅｓｓ，Ｏｘｆｏｒｄ（１９８９））に記載の方法に従って行った。生成物ポリアミノ酸のアミノ酸配列は、（Ｎ末端）（Ｌ−アルギニン）−（Ｌ−ヒスチジン）−（Ｌ−アルギニン）−（Ｌ−ヒスチジン）−（Ｌ−アルギニン）−（Ｌ−ヒスチジン）−（Ｌ−アルギニン）−（Ｌ−ヒスチジン）−（Ｌ−アルギニン）−（Ｌ−ヒスチジン）（Ｃ末端）である。このポリアルギニルヒスチジンを滅菌水に溶解、希釈し、一定の終濃度（１、１０、２０、５０、１００、１５０、２００、３００、４００、５００μｇ／ｍＬ）となるように実施例３に記載のものと同じ組成の液体培地に添加し、培養して抗菌活性を評価した。抗菌活性（増殖）の評価は上記の実施例３記載の方法に準じて行った。抗菌性試験の結果を表２に示す。

培地：Ｎ，ヌートリエント ブロス；ＹＰＤ，ｙｅａｓｔ ｅｘｔｒａｃｔ／ｐｅｐｔｏｎｅ／ｄｅｘｔｒｏｓｅ；ＰＤ，ポテト デキストロース培地；７０２，ＩＦＯ７０２培地．
化学合成ポリアルギニルヒスチジンはアミノ酸残基数１０のものを使用した。
＊増殖速度の低下が観察された。−は増殖を阻止せず。
表２に示すように、アミノ酸残基数１０のポリアルギニルヒスチジンはグラム陰性細菌およびグラム陽性細菌に対して抗菌性であり、酵母菌、糸状真菌に対して、増殖を強く抑制した。例外的に、Ｂａｃｉｌｌｕｓ ｃｅｒｅｕｓ ＩＦＯ３５１４株には無効であった。上記の表２に記載の結果は、合成ポリアミノ酸が、表１に記載のＥ１８株が産生するポリアミノ酸と同様の抗菌活性を有することを示している。これらの結果は、本発明のＥ１８株が所望のヘテロポリアミノ酸を産生することを証明するものでもある。なお、最小増殖阻止濃度の数値に若干の差があるが、これは天然のポリアミノ酸と化学合成ポリアミノ酸との純度等におけるに基づくと考えられる。
実施例５ アミノ酸残基の種類および数と抗菌性との関係
（１）ポリアルギニルヒスチジンのアミノ酸残基数と抗菌活性と関連を調べるために、アミノ酸残基数の異なるポリアルギニルヒスチジンを化学合成し、実施例３または４と同様の方法にて抗菌性試験を実施した。被検微生物には大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＩＦＯ３３０１株および枯草菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＩＦＯ３３３６株を使用した。抗菌性試験の結果を表３に示す。なお、表３に記載のポリアミノ酸のアミノ酸配列は配列番号４〜１３に記栽されている。

被検微生物：Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＩＦＯ３３３６およびＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＩＦＯ３３０１．
構造模式中のＲ、Ｈ、Ｋ、Ｇ、Ｃはそれぞれアルギニン、ヒスチジン、リジン、グリシン、システイン残基を表す。
培地，ヌートリエント ブロス。
−は増殖を阻止せず。
表３に示すように、アミノ酸残基数が６以上のポリアルギニルヒスチジン（配列番号５〜８）では抗菌性が認められた。最小増殖阻止濃度は残基数が８以上で飽和し、その値は１０μｇ／ｍＬであった。Ｎ末端はヒスチジンで、Ｃ末端はアルギニンである、ポリヒスチジルアルギニン（残基数１０、配列番号９）の最小増殖阻止濃度は１０μｇ／ｍＬであり、抗菌性について同残基数のＮ末端はアルギニンで、Ｃ末端はヒスチジンである、ポリアルギニルヒスチジンと変わりなかった。
実施例６ アミノ酸置換誘導体の抗菌性
ポリアルギニルヒスチジンのアミノ酸配列と抗菌活性と関連を調べるために、アミノ酸配列の異なるポリアミノ酸を化学合成し、実施例３または４と同様の方法にて抗菌性試験を実施した。被検微生物には大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）ＩＦＯ３３０１株および枯草菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）ＩＦＯ３３３６株を使用した。抗菌性試験の結果は前記表３に示されている。
表３に示すように、ポリアルギニルヒスチジンのアルギニンをリジンに置換した、残基数１０のポリアミノ酸（配列番号１０）は、最小増殖阻止濃度が１０μｇ／ｍＬであり、抗菌性について同残基数のポリアルギニルヒスチジンと変わりなかった。ポリアルギニルヒスチジンのヒスチジンをシステインに置換した、残基数１０のポリアミノ酸（配列番号１３）は、最小増殖阻止濃度が１０から２０μｇ／ｍＬであり、抗菌性について同残基数のポリアルギニルヒスチジンとほぼ変わりなかった。ポリアルギニルヒスチジンのヒスチジンをグリシンに置換した、残基数１０のポリアミノ酸（配列番号１１）は、最小増殖阻止濃度が１００μｇ／ｍＬであり、抗菌性について同残基数のポリアルギニルヒスチジンの１０分の１の効果しか示さなかった。ポリアルギニルヒスチジンのアルギニンをグリシンに置換した、残基数１０のポリアミノ酸（配列番号１２）は抗菌性を示さなかった。上記の結果から、相互に交換可能なアミノ酸の特性について以下の点が明らかである。即ち、アルギニンとリジンは、共に強い塩基性を有するアミノ酸であること、ヒスチジンとシステインはキレート形成能が高く、しかも、ペプチド結合によりポリアミノ酸を形成した状態でも優れたキレート形成能を維持していることが挙げられる。一方、ヒスチジンとグリシンの場合は、キレート形成能に関しては共通するが、ポリアミノ酸を形成した場合に、グリシンはキレート形成能の低下または喪失をきたす点で異なる。さらに、アルギニンとグリシンの場合は、塩基性において異なる。このように、抗菌性ポリアミノ酸を得るためには、塩基性アミノ酸が含まれること必要であり、さらにキレート形成能の優れたアミノ酸が存在することにより抗菌性が増強される事が分かった。
実施例７ アシル化誘導体の抗菌活性
極めて親水性であるポリアルギニルヒスチジンの極性変化が抗菌性に与える影響について調べるために、上記実施例に記載の残基数１０のポリアルギニルヒスチジン（ＲＨ_５）のＮ末端アルギニン残基のＮαアミノ基をミリスチル化し、残基数１０のミリスチル化ポリアルギニルヒスチジン（ｍｙｒｉｓｔｏｙｌ−ＲＨ_５）を化学合成した。これを用いて、実施例３または４と同様の方法で抗菌性試験を実施した。抗菌性試験の結果を表４に示す。

培地，ヌートリエント ブロス．
ＲＨ_５，ポリアルギニルヒスチジン（アミノ酸残基数は１０）；ｍｙｒｉｓｔｏｙｌ−ＲＨ_５，ポリアルギニルヒスチジン（アミノ酸残基数は１０，Ｎ末端アミノ基にミリスチル基を結合したもの）；εＰＬ，イプシロン−ポリ−Ｌ−リジン（アミノ酸残基数は２５から３６）。
＊増殖速度の低下が観察された。−は増殖を阻止せず。
表４に示すように、ｍｙｒｉｓｔｏｙｌ−ＲＨ_５は、アシル化されていない対応のポリアルギニルヒスチジン（ＲＨ_５）と比較して、より高い活性を有することが明らかになった。即ち、ｍｙｒｉｓｔｏｙｌ−ＲＨ_５はＲＨ_５が全く抗菌作用を示さないＢａｃｉｌｌｕｓ ｃｅｒｅｕｓ ＩＦＯ３５１４株に対して、最少増殖阻止濃度１０μｇ／ｍＬで増殖阻害作用を示した。また、非ミリスチル化ポリアミノ酸であるＲＨ_５が比較的弱い（最少増殖阻止濃度が５０μｇ／ｍＬ以上）増殖阻害作用を示すグラム陰性菌（Ｓａｌｍｏｎｅｌｌａ ｔｙｐｈｉｍｕｒｉｕｍ ＩＦＯ１２５２９）およびグラム陽性菌（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ ＩＦＯ１３２７６，Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ ＭＲＳＡ ＡＴＣＣ３３５９１）に対して高い増殖阻害作用（最少増殖阻止濃度１０μｇ／ｍＬ）を示した。このように、アシル化は抗菌活性の向上（抗菌スペクトルの拡大）に有効であることが証明された。
実施例８ ホモポリマーであるポリリジンとの抗菌活性における比較
既存のホモポリアミノ酸であるポリリジンは強い抗菌性を示す事が知られている。そこで、本発明のポリアルギニルヒスチジン（合成ＲＨ_５）の抗菌性との比較を行った。ポリリジンは和光純薬より入手したεポリ−Ｌ−リジンを使用した。抗菌活性の評価は実施例３，４に記載の方法と同様である。結果は上記の表４に示されている。この結果から明らかに、ポリリジンは試験したほぼすべての菌に対して強い抗菌性を示したが、Ｂ．ｃｅｒｅｕｓ ＩＦＯ３５１４には他の菌株に比べ１／５倍の効果しか示さなかった（５倍の抵抗性を示した）。一方、本発明の化学合成ポリアルギニルヒスチジン（残基数１０）は一部の菌株について十分な抗菌性を示さなかったが、ミリスチル化したポリアルギニルヒスチジンはすべての菌株の増殖を強く抑制し、ポリリジンに５倍の抵抗性を示したＢ．ｃｅｒｅｕｓ ＩＦＯ３５１４株に対しても同レベルで増殖を強く抑制した。
実施例９ 強アルカリ条件下での抗菌活性
既存品であるポリリジンの遊離アミノ基のｐＫａ値は８．９であり、ポリアルギニルヒスチジンの遊離グアニジノ基のｐＫａ値が１２．５であることから考えて、強アルカリ条件下では抗菌性はポリアルギニノヒスチジンの方がポリリジンより強いことがが予想される。そこで、好アルカリ細菌に対する抗菌性を調べた。培地には２０ｍＭのＣＡＰＳ／ＮａＯＨ緩衝液にてｐＨを１１．２に調製したヌートリエント ブロスを使用し、その他の試験方法は実施例３に準じて行った。結果を表５に示す。

培地，ヌートリエント ブロス（２０ｍＭ ＣＡＰＳ／ＮａＯＨ緩衝液）．
ＲＨ_５，ポリアルギニルヒスチジン（アミノ酸残基数は１０）；εＰＬ，イプシロン−ポリ−Ｌ−リジン（アミノ酸残基数は２５から３６）．
−は増殖を阻止せず。
表５に示すとおり、ｐＨ１１．２において、ポリアルギニルヒスチジンは抗菌性を示したのに対して、ポリリジンは抗菌性を示さなかった。よって、ポリアルギニルヒスチジンの抗菌性は、強アルカリ条件でも有効であることが示された。
実施例１０ 金属結合性試験
アミノ酸残基数１０のポリアルギニルヒスチジン（ＲＨ_５）を化学合成法により作成し、試験に供した。そのアミノ酸配列は、実施例２（５）におけるものと同質である。金属結合性は分子量の増加をもって評価した。ポリアルギニルヒスチジンを終濃度３０ｐｍｏｌ／μＬとなるように３０ｍＭトリス・塩酸緩衝液（ｐＨ７．５）で調製し、金属の塩化物塩または硫酸塩の水溶液と混和した。金属元素として、マグネシウム、マンガン、コバルト、ニッケル、銅、亜鉛を使用した。混和の比率は、モル比率で、ポリアルギニルヒスチジン：金属＝１：１０とした。分子量の測定は、ポリアルギニルヒスチジンと金属の混和物を測定器製造元が推奨する方法に従い、２、５−ジヒドロキシ安息香酸と混和し、乾燥後、飛行時間型質量分析計（パーセプティブ社製、モデルＶｏｙａｇｅｒ ＤＥ）を用い、ＭＡＬＤＩ−ＴＯＦ Ｍａｓｓ法で測定した。金属結合性試験の結果の一例を図３に示す。図３はＭＡＬＤＩ／ＴＯＦ−ＭＳ法によるポリアミノ酸−亜鉛複合体の検出結果を示し、Ａはポリアルギニルヒスチジン（残基数１０）のみ、Ｂはポリアルギニルヒスチジン（残基数１０）を硫酸銅（ポリアミノ酸の１０倍のモル数）と共存させた場合の結果を示す。
図３に示すように、ポリアルギニルヒスチジンと硫酸亜鉛を共存させた場合、硫酸亜鉛を共存させない場合にポリアルギニルヒスチジンが示すｍ／ｚ値１，４８６に加えて、亜鉛イオンの付加された分子が示すｍ／ｚ値１，５５０が観測された。他の金属についても、ポリアルギニルヒスチジンが単独で示すｍ／ｚ値に、おのおのの金属に固有な値の分だけ増したｍ／ｚ値が確認されたので、ポリアルギニルヒスチジンは混和したすべての種類の金属と金属の間で複合体を形成することが明らかとなった。
実施例１１ 抗菌性に対する金属の影響
実施例６に示したとおり、塩基性ポリアミノ酸の抗菌性の増強には、ヒスチジンやシステインなどのキレート形成性のアミノ酸残基が寄与している。実施例１０で示したとおり、ポリアルギニルヒスチジンは金属と錯体を形成する。そこで抗菌性に対する金属の依存性を調べるために、化学合成ポリアルギニルヒスチジン（アミノ酸残基数１０）を用いて抗菌性試験を行った。試験方法は、終濃度１０μｇ／ｍＬ（６．６μＭ）のポリアルギニルヒスチジンを含むＤａｖｉｓの改変合成液体培地中へ各種濃度の硫酸亜鉛水溶液を添加し、ここへ被検微生物としてＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉＩＦＯ３３０１株を接種、培養した。増殖の評価は２０時間倍養後の６００ｎｍの吸光度を測定して行った。Ｄａｖｉｓの改変合成液体培地１Ｌ中の組成は以下のとおりである：Ｄ−グルコース，１ｇ；リン酸水素二カリウム，７ｇ；リン酸二水素カリウム，２ｇ；クエン酸三ナトリウム二水和物，０．５ｇ；硫酸マグネシウム七水和物，１０ｍｇ；硫酸アンモニウム，１ｇ；カザミノ酸，１００ｍｇ；塩酸チアミン，２ｍｇ，ｐＨ７．０。結果を図４に示す。ポリアルギニルヒスチジン６．６μＭを含有する場合は斜線、ポリアルギニルヒスチジンを含有しない場合を白抜で示されている。
本試験で用いた条件（人工的な培地）においては、亜鉛イオンを全く含まないか、終濃度１．６５から３．３μＭの低濃度の範囲では、亜鉛イオンの有無に関わらず抗菌性は認められない。しかしながら、６．６μＭ以上の亜鉛が存在すると増殖は強く抑制された。対照実験として行ったポリアミノ酸を含まない場合の、同濃度の亜鉛の存在における増殖は全く抑制されないので、亜鉛の毒性の影響はない。同様の効果は、銅イオンでも観察された。
一方、前記の実施例３および４において、天然物に由来する培地を用いた場合には、特に亜鉛を添加しなくても抗菌作用が認められた。
これらの結果は、本実施例における実験条件には、何らかの理由でポリアルギニルヒスチジンの抗菌活性を抑制する阻害物質（因子）が含有されている可能性を示唆している。上記の結果はまた、亜鉛イオンが共存すると、そのような阻害物質の作用にもかかわらず、本発明のポリアミノ酸誘導体が抗菌活性を発揮しうることをも示唆している。そして、そのような阻害物質の存在下で十分な抗菌活性を得るためには、ポリアルギニルヒスチジンと等モル以上の亜鉛イオンを存在させることが有効であることを示している。
さらに、本発明のポリアルギニルヒスチジンを錯体とすると、単独では阻害物質の影響により抗菌活性の低下または喪失が懸念される場合でも、錯体の形なら、十分に抗菌活性を発揮することができることをも示している。
実施例１２ 酸性物質および塩基性物質との反応性
試験方法
アミノ酸残基数１０のポリアルギニルヒスチジン（ＲＨ_５）を化学合成法により作成し、試験に供した。そのアミノ酸配列は、実施例２（５）におけるものと同質である。酸性物質および塩基性物質との作用性の評価は、それぞれ酸性色素であるＰｏｌｙＲ−４７８および塩基性色素であるメチレンブルーを使用した。電荷をもった色素を含む寒天平板上に静置した直径６ｍｍのろ紙片に、化学合成したポリアルギニルヒスチジン１ｍｇを含む水溶液１０μＬを浸潤させ、拡散に伴う色素の変化を観察した。寒天、ＰｏｌｙＲ−４７８、塩基性色素メチレンブルーの濃度はそれぞれ１．５％、０．０２％、０．００２％であった。酸性物質および塩基性物質との作用性の評価試験の結果を図５に示す。
図５に示すように、ポリアルギニルヒスチジンは、ろ紙片から寒天平板中に拡散するのに伴って、酸性色素（ＰｏｌｙＲ−４７８）を吸着し濃縮する（Ａ）ことが観察された（矢印、色素が濃縮され、周辺より暗く見える）。一方、塩基性色素に関しては、これを排斥し、拡散の前線付近に濃縮する（Ｂ）ことが観察された（矢頭、色素が排斥され、周辺より明るく見える）。これらの結果から、ポリアルギニルヒスチジンは酸性物質を吸着し、逆に塩基性物質と反発する性質を有することが分かった。
実施例１３ ポリアルギニルヒスチジンの生細胞への透過性
ポリアルギニルヒスチジンが細胞に及ぼす影響を調べるために蛍光標識したポリアミノ酸の挙動を観察した。実施例４に準じて、アミノ酸残基数１０のポリアルギニルヒスチジンを化学合成法により作成し、さらにＮ末端の遊離アミノ基を蛍光色素であるＦＡＭ（ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ）によって化学標識した。Ｃａｎｄｉｄａ ｂｏｉｄｉｎｉｉ ＭＩＰ１０４株のけん濁液に終濃度１μｇ／μＬで添加し、５分間静置した後、けん濁液中の余剰の標識ポリアミノ酸を洗浄により除去したうえで、プレパラートを作成した。これを、同軸落射式蛍光顕微鏡にて通常光および蛍光励起光源下で観察、写真撮影した。対照実験として、蛍光色素ＦＡＭのみを単独で添加した試料も観察した。結果を図６に示す。図６におけるＡはＮ末端標識ポリアミノ酸（ＦＡＭ−ＲＨ５）を酵母Ｃａｎｄｉｄａ ｂｏｉｄｉｎｉｉ ＭＩＰ１０４株の細胞けん濁液に添加し、蛍光顕微鏡で観察した結果、Ｃは同じ視野を通常光で観察した結果、Ｂは対照実験ＦＡＭのみを同細胞けん濁液に添加し蛍光顕微鏡で観察した結果、Ｄは同じ視野を通常光で観察した結果を示す。
図６Ａに示すとおり、蛍光ラベルしたポリアルギニルヒスチジンは酵母生細胞内へ取りこまれた。一方、図６Ｂに示すとおり、蛍光色素だけでは細胞に取り込まれることはなく、ポリアミノ酸と結合した形においてのみ取り込みが観察された。従って、ポリアルギニルヒスチジンは、何ら特別の操作を経なくとも、酵母細胞の細胞壁および細胞膜を透過する能力を有することが確認された。
実施例１４ ヒスチジンの光学純度の検定
実施例２−（３）のポリアルギニルヒスチジンの加水分解物を、光学分割カラム（ダイセル工業製ＣＲＯＷＮＰＡＫ ＣＲ（＋）、移動相はｐＨ１．５の過塩素酸水溶液、カラム温度は４ｄ℃）を装着した高性能液体クロマトグラフィー装置（ＨＰＬＣ）にて分析した。検出は２００ｎｍの吸収を測定した。対照実験としては、Ｄ−ヒスチジン、Ｌ−ヒスチジン、Ｄ−アルギニン、Ｌ−アルギニンの４種のアミノ酸標準品、化学合成した２種のポリアミノ酸（Ｎ末端 Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ −Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ Ｃ末端およびＮ末端Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ Ｃ末端）を６Ｎ塩酸溶液中で１００℃、２０時間加熱することにより加水分解した標準品を、同じ条件でＨＰＬＣ分析した。
分析結果を図７に示す。図中、化学合成品、（ＬＲ−ＬＨ）_５、および（ＬＲ−ＤＨ）_５の構造はそれぞれ、［Ｎ末端 Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ Ｃ末端］および［Ｎ末端 Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ Ｃ末端］である。
図７に示すように、Ｅ−１８株が生産したポリアルギニルヒスチジンを構成するヒスチジンは、約８５％がＤ−体であった。また、アルギニンは１００％がＬ−体であった。化学合成品に含まれるヒスチジンは、加水分解操作に伴うラセミ化により、Ｌ−体からＤ−体への変換が約３．３％であり、Ｄ−体からＬ−体への変換が約０．９％であった。従って、加水分解操作に伴う人為的なラセミ化が、Ｅ−１８株のポリアルギニルヒスチジンに含有されるヒスチジンのＤ体とＬ体の存在比の数値に与える影響は小さいものであり、結論として、約８５％という光学純度の高いＤ−ヒスチジンを製造することができた。
実施例１５ Ｄ−ヒスチジンを含有するペプチドの機能性
（１）抗菌性試験
ポリアルギニルヒスチジンに含有されるヒスチジンの立体異性（Ｄ−体またはＬ−体）と抗菌活性との相関を見るために、化学合成法により、すべてのヒスチジン残基がＤ−体であるポリアルギニルヒスチジン（Ｎ末端 Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ Ｃ末端）と、すべてのヒスチジン残基がＬ−体であるポリアルギニルヒスチジン（Ｎ末端 Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ Ｃ末端）を調製し、上記実施例に記載のヌートリエントブロス（ＮＢ）またはポテトデキストロース培地の液体培地に培養開始時に添加した。植菌後、２８℃で振とう培養し、被検微生物の増殖を観察した。被検微生物には、緑膿菌（Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ、ＩＦＯ３４４５、財団法人発酵研究所より分譲）、サルモネラ菌（Ｓａｌｍｏｎｅｌｌａ ｔｙｐｈｉｍｕｒｉｕｍ、ＩＦＯ１２５２９、同）、カンジダ酵母（Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ、ＩＦＯ１３８５、同）、パン酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ、ＫＫ４、所属研究機関において保存）を使用した。
結果を表６に示す。Ｅ−１８株の生産したポリアルギニルヒスチジンの粗精製物は、８〜４０μｇ／ｍＬの最小増殖阻止濃度で、被検微生物の増殖を抑制した。ほぼ不純物の混入がないと考えられる化学合成品については、ヒスチジン残基がすべてＤ−体であるポリアルギニルヒスチジンの最小増殖阻止濃度は５〜１０μｇ／ｍＬであり、全アミノ酸がＬ−体であるポリアルギニルヒスチジンに比較して低い最小増殖阻止濃度を示した。これらの結果は、ポリアルギニルヒスチジン中のヒスチジン残基をＤ−体にすることにより、抗菌性が強められることを示している。

表６における化学合成品（_ＬＲ−_ＬＨ）_５および（_ＬＲ−_ＤＨ）_５の構造は、それぞれＮ末端［Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ］Ｃ末端、Ｎ末端［Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ］Ｃ末端である。ここで、ＲおよびＨは、それぞれアルギニンおよびヒスチジン残基を表す。
（２）ペプチド分解酵素に対する感受性試験
ポリアルギニルヒスチジンに含有されるヒスチジンの立体異性（Ｄ−体またはＬ−体）とペプチド分解酵素に対する感受性との相関を調べた。Ｄ−又はＬ−ヒスチジンを含有するポリアルギニルヒスチジンをペプチド分解酵素の一種であり、哺乳類消化管にも存在するトリプシンと反応させた後、分子量の変化を観察した。
具体的な試験方法は以下のとおりである。Ｅ−１８株が生産したか、又は化学合成した４種のポリアルギニルヒスチジン（▲１▼ Ｎ末端 Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ Ｃ末端；▲２▼ Ｎ末端 Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ Ｃ末端；▲３▼ Ｎ末端 Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ Ｃ末端；▲４▼ Ｎ末端 Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ Ｃ末端）のそれぞれを、約１００μｇづつ、２００μＬの１００ｍＭ炭酸水素アンモニウム水溶液（ｐＨ ７．８）に懸濁した担体固定型トリプシン（ピアース社製ＴＰＣＫ−Ｔｒｙｐｓｉｎ，ｉｍｍｏｂｉｌｉｚｅｄ）と混合し、強く攪拌しながら３０℃で６時間保温した。上清１μＬを９μＬの２，５−ジヒドロキシ安息香酸水溶液（１０ｍｇ／ｍＬ）と混合し、ＭＡＬＤＩ／ＴＯＦ−ＭＳ（マトリクス支援レーザー脱イオン化／飛行時間測定質量分析計、パーセプティブ社製、Ｖｏｙａｇｅｒ ＤＥ）にて分子量を測定した。
測定結果を図８に示す。図中、矢印は推定のトリプシンの切断点である。各チャートの被検物質は以下の通りである。（Ａ）Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ、（Ｂ）Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ、、（Ｃ）Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ、（Ｄ）Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ、（Ｅ）実施例２で調製した、Ｅ−１８株が生産するポリアルギニルヒスチジン。
トリプシンはペプチド鎖の内部に存在するアルギニンまたはリジン残基のカルボキシ末端側のペプチド結合を特異的に切断する活性を有する。一般に、この種の酵素は基質に対して立体選択性があり、切断点をはさむ２つのアミノ酸がともにＬ−体であることが必要とされている。そこで、化学合成した４種のポリアルギニルヒスチジンの、トリプシンによる分解物の分子量を分析することにより、立体選択性の確認を試みた。
その結果、（Ａ）に示すとおり、すべてのヒスチジン残基がＤ−体であるポリアルギニルヒスチジンはトリプシンによる分解を受けなかった。一方、（Ｂ）に示すとおり、すべてのヒスチジン残基がＬ−体であるポリアルギニルヒスチジンはトリプシンによる分解を受けて、ジペプチド（Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ、分子量約３１４）およびトリペプチド（Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ−Ｌ−ＡｒｇまたはＬ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｌ−Ｈｉｓ、分子量約４５１）を生じた。また、（Ｃ）に示すとおり、第８残基のヒスチジンのみがＬ−体であるポリアルギニルヒスチジンはトリプシンによる分解を受けて、トリペプチド（Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ、分子量約４５１）およびヘプタペプチド（Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ、分子量約１，０５６）を生じた。さらに、（Ｄ）に示すとおり、第６および第８残基のヒスチジンのみがＬ−体であるポリアルギニルヒスチジンはトリプシンによる分解を受けて、ジペプチド（Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ、分子量約３１４）、トリペプチド（Ｌ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ、分子量約４５１）、ペンタペプチド（Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ−Ｄ−Ｈｉｓ−Ｌ−Ａｒｇ、分子量約７６３）を生じた。以上を総合すると、トリプシンが基質として攻撃するのは、切断点をはさむ２つのアミノ酸、ここではアルギニンとヒスチジンが、ともにＬ−体である場合に限られることが明らかである。すなわち、Ｄ−ヒスチジンを含有するポリアルギニルヒスチジンは、Ｌ−体ヒスチジンを含有するポリアルギニルヒスチジンと比較して、トリプシンによる分解を受けにくいことが証明された。
（Ｅ）に示すとおり、Ｅ−１８株が生産したポリアルギニルヒスチジンは、上記（Ａ）の場合と同様、トリプシンによる分解を受けず、分子量はトリプシン処理する前の分子量と変化はなく、約１，４８７のままであった。この結果は、Ｅ−１８株が生産したポリアルギニルヒスチジンの、少なくとも第４、第６、第８残基がＤ−体であることを示している。従って、Ｅ−１８株により産生されるポリアルギニルヒスチジンは、Ｄ−ヒスチジンを含んでおり、Ｌ−体ヒスチジンを含むポリアルギニルヒスチジンと比較して、トリプシンによる分解を受けにくいことが分かった。  The polyamino acid of the present invention has a basic structure in which a plurality of amino acids are linked via peptide bonds, and includes peptides, polypeptides and proteins.
  The “polyamino acid or a derivative thereof” of the present invention means a polyamino acid in which amino acids are linked by a peptide bond, a chemically modified derivative of a free functional group thereof, or a metal complex or salt thereof. In the present specification, “polyamino acid or derivative thereof” is simply referred to as “polyamino acid derivative”. The polyamino acid derivative of the present invention contains at least one basic amino acid as a constituent unit of a peptide. Further, the polyamino acid derivative of the present invention includes a polyamino acid derivative in which at least one kind of amino acid is D-form.
  The “basic amino acid” used in the present invention is not particularly limited, provided that it is sufficiently basic to achieve the object of the present invention. For example, arginine, lysine, ornithine, citrulline, homocitrulline, homoarginine, canavanine and 2-amino-3-guanidinopropionic acid can be used. When the polyamino acid of the present invention is used as an antibacterial substance, an amino acid having strong basicity is more preferable, and arginine and lysine are particularly preferable.
  The polyamino acid derivative of the present invention has a basic structure in which a basic amino acid and appropriate other 1 to 9 kinds of amino acids are linked by a peptide bond.
  The amino acid to be contained in the polyamino acid derivative of the present invention together with the basic amino acid can be appropriately selected from known amino acids, but for the purpose of the present invention, an amino acid having a high chelating ability is preferable, and histidine, cysteine, homocysteine are preferred. , Tryptophan, tyrosine and the like can be used. Of these, histidine and cysteine are preferable. The amino acid may be either L-form or D-form.
  In the polyamino acid derivative of the present invention, an example of a polyamino acid consisting of two kinds of amino acids is represented by the formula (I):

[Wherein, X is the following 1) and 2):
1) Arginine, lysine, ornithine, citrulline, homocitrulline, homoarginine, canavanine and 2-amino-3-guanidinopropionic acid
2) An amino acid residue selected from the group of any one of histidine, cysteine, homocysteine, tryptophan and tyrosine, Y represents an amino acid residue selected from the other group, and R₁Represents a hydrogen atom, a sugar residue, an acyl group, a biotinyl group, a thiol group, a phenol group, an indole group, or the same amino acid residue as Y described above, and R_TwoRepresents a hydroxyl group, a sugar residue, an acyl group, a biotinyl group, a thiol group, a phenol group, an indole group or the same amino acid residue as the above X, and n represents an integer of 2 or more]
Examples thereof include polyamino acids and their derivatives.
  In the above formula (I), X is arginine or lysine, Y is histidine or cysteine, or a polyamino acid in which X is histidine or cysteine and Y is arginine or lysine is more preferable. Particularly preferred are polyamino acids, one of which is arginine and the other of which is histidine. Furthermore, n is preferably an integer of 2 to 50, and R is₁Is a hydrogen atom and R_TwoIs particularly preferably a hydroxyl group.
  Further, it is preferable that any one of the amino acids is a D-amino acid, and particularly histidine is preferably D-histidine.
  The length of the peptide chain of the polyamino acid derivative of the present invention is arbitrary and selected according to the purpose, but usually the number of amino acids is 3 or more. It is preferably in the range of 5 to 100, more preferably 5 to 50, still more preferably 6 to 20, and most preferably 6 to 12.
<Production of polyamino acid derivative>
  The polyamino acid derivative of the present invention can be produced by a chemical synthesis method, a biochemical method, microbial fermentation or the like known in the art. The biochemical method uses genetic information of DNA or RNA encoding a polyamino acid as a template, and is produced by genetic engineering in a cell or in an artificial reaction solution according to the functions of the transcription and translation systems of the organism. It can be carried out. A particularly preferred method is a conventional microbial fermentation method. The polyamino acid derivative of the present invention has activity even in a free form, but it may also be converted into an appropriate acid or base salt or metal complex for enhancing antibacterial activity and improving stability and water solubility. it can. Such salts and complexes can be prepared according to methods known in the art.
Production of polyamino acids and D-amino acids by microbial fermentation
  In order to produce the polyamino acid or D-amino acid of the present invention by microbial fermentation, it is first necessary to identify the microorganism that produces the target polyamino acid. Methods for screening such microorganisms are known in the art [Nishikawa, M. et al., Applied And Environmental Microbiology, (USA), vol68, No. 7, p3375-3581]. For example, in order to select a basic polyamino acid-producing microorganism that secretes a basic polyamino acid outside the cells, a sample containing the microorganism (for example, a soil sample) is placed on an agar plate medium of an appropriate composition and Expand to microbial communities. An acidic dye or a basic dye is added to this agar plate in advance, and the secretion of the basic polyamino acid is detected based on the binding with the acidic dye or the repulsive action to the basic dye.
  Furthermore, a microorganism producing a polyamino acid derivative which is an excellent polydentate ligand having a chelate-forming ability can be screened by, for example, the following method. In order to select a microorganism that secretes a chelate-forming (metal-coordinating) polyamino acid to the outside of a cell, a soil sample containing the microorganism is spread on an agar plate medium of an appropriate composition to form an individual microbial community. Toxic metals harmful to microorganisms such as silver, copper, zinc, nickel, cobalt, cadmium, and mercury are added to the agar plate in advance at an appropriate concentration. The chelate-forming polyamino acid is secreted based on its coordinating property to a metal, and the toxicity is neutralized or alleviated by the polyamino acid capturing and conjugating a harmful metal, and the polyamino acid does not react with other polyamino acid-producing microorganisms. It is detected by the difference in proliferation between the two.
  Microorganisms that produce basic and chelate-forming polyamino acids can be selected by testing the two properties, basic and chelating, alone or simultaneously by the method described above.
  In a preferred embodiment of the present invention, the polyamino acid derivative of the present invention is produced microbiologically using a strain of the genus Epichloe. Preferred microorganisms are Epichloe kibiensis strain E18 (FERM P-18923) or variants thereof.
  The E. coli strain E18 (hereinafter abbreviated as "E18 strain") is a strain newly isolated by the present inventors from the forest soil by the method described in Examples, and its mycological characteristics are as follows. It is as follows.
A. Growth status on medium
  When the E18 strain is cultivated at 25° C. on each agar plate of potato dextrose medium (PDA), oatmeal medium (OA), and 2% malt medium (MEA), mycelia appear fluffy and white. Growth is rather slow. The formation of conidia is small, and no change in the color tone of the colony due to conidia is observed. No soluble pigment production is observed.
B. Spore formation
  A fiaro-type conidial structure is confirmed under an optical microscope. The conidia peduncle is slightly erect and spins single or 2 to 4 phialides from aerial hyphae. Conidia are unicellular and have a smooth surface. There are various shapes such as oval, teardrop, oval, and oval. Conidia are viscous and cluster together. Formation of crescent-shaped large conidia and chlamydospores is not observed even after 8 weeks of culture.
C. Physiological properties
  Produces polyarginyl histidine.
D. Source of carbon that can be assimilated
  Glycerol, D-glucose, D-galactose, D-xylose can be used.
  The taxonomic position of this strain having the above-mentioned mycological properties was examined in combination with a molecular biology method based on interspecies comparison of the nucleotide sequences of 28s ribosomal RNA molecules, and as a result, it was a new strain belonging to Epichloe. It was found that this was named Epichloe kibiensis E18 strain. This strain has been deposited at the Japan Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology, Central No. 1, 1-1-1, Higashi Tsukuba, Ibaraki Prefecture (Labeling of microorganisms: Epichloe kibiensis E18, date of receipt: 2002-2002) 5th; deposit number FERM P-18923) (date of change to international deposit: April 11, 2003; deposit number: FERM BP-8358).
  By using the E18 strain as a parent strain, it is possible to obtain a derivative strain that enhances the productivity of polyarginyl histidine or produces a polyamino acid with a modified amino acid composition by mutation induction or recombinant gene technology. Such mutant strains are also within the scope of the present invention. Derivative strains include those artificially mutated, those obtained by screening, and the like.
  Since the E18 strain is resistant to the antibacterial action of basic polyamino acids, it can be used for processing and producing basic polyamino acids, peptides and proteins having arbitrary sequences. For example, (1) transform the E18 strain by modifying and processing the gene of polyarginyl histidine described in the examples below, or (2) appropriately insert a DNA encoding an appropriate polyamino acid under an expression promoter. The E18 strain is transformed with the resulting expression vector, and the like.
  Cultivation of the polyamino acid-producing microorganism of the present invention, for example, Epichloexeviensis E18 strain, is appropriately selected depending on the nature of the microorganism, is commercially available, or can be prepared by a method known to those skilled in the art. it can. A complete medium, a synthetic medium, or a semi-synthetic medium having an appropriate liquid or solid composition can be used, but a liquid medium is suitable in terms of easiness of operation and the like. The medium may be any medium as long as it contains a carbon source, a nitrogen source, an inorganic salt and other nutrients as general components. Examples of the carbon source include glucose, galactose, fructose, glycerol, starch and the like, and the content thereof is preferably 0.1 to 10% (w/v). Examples of the nitrogen source include yeast extract, peptone, casein hydrolyzate, organic compounds such as amino acids, ammonium sulfate, ammonium chloride, inorganic ammonium salts such as sodium nitrate, and the like, and the content thereof is 0.1 to 5% ( w/v) is preferred. If desired, other nutrient sources (for example, inorganic salts such as phosphate ion, potassium ion, sodium ion, magnesium ion, zinc ion, iron ion, manganese ion, nickel ion, sulfate ion, etc.) added to the medium, vitamins Kinds (eg vitamin B₁), antibiotics (eg ampicillin, tetracycline, kanamycin, etc.)) may be added.
  The culture can be performed under aerobic conditions by shaking culture, stirring culture, or the like. The culture temperature is about 25 to 40° C., and the pH of the medium is 2.0 to 8.0, preferably pH 3.0 to 8.0, more preferably pH about 5.0. The culture period is usually 1 to 14 days, but the culture can be continued for a longer period.
  The above-mentioned derivative strain (mutant strain) derived by using the E18 strain as a parent strain can be similarly cultured.
  If the polyamino acid produced is secreted into the culture medium, the culture is filtered or centrifuged to isolate the crude product. Purification of the produced polyamino acid is carried out by a method known in the art used for purification and isolation of natural or synthetic amino acid or protein from the collected culture supernatant (for example, ion exchange resin treatment method, activated carbon adsorption). A treatment method, an organic solvent precipitation method, a vacuum concentration method, a freeze-drying method, a crystallization method, etc.) can be appropriately combined and carried out. When the produced polyamino acid is present in the periplasm and cytoplasm of the cultured microorganism, cells are collected by filtration or centrifugation, and their cell wall and/or cell membrane is destroyed by, for example, ultrasonication and/or lysozyme treatment, Obtain debris (cell lysate). The debris can be dissolved in an appropriate aqueous solution (for example, a buffer solution), and the product can be isolated and purified according to the method described above.
Production of D-amino acids
  When the polyamino acid produced by the microorganism contains a D-amino acid, it is hydrolyzed by a chemical or enzymatic method as necessary to isolate the D-amino acid. Hydrolysis can be performed by a method known in the art, for example, using an acid such as hydrochloric acid or sulfuric acid, a base such as sodium hydroxide or potassium hydroxide, a protease such as aminopeptidase, carboxypeptidase, endopeptidase and the like. Can be implemented.
  The acid hydrolysis may be carried out, for example, by heating with 6N hydrochloric acid at about 100° C. for about 20 hours.
  Isolation and purification of the D-amino acid monomer from the hydrolyzate can be carried out according to the isolation and purification method from the culture described above, and examples thereof include chromatography, fractional crystallization, enzyme treatment and the like.
Properties of polyamino acids produced by E-18 strain
  When the E18 strain or the like is cultured by the above method, polyarginyl histidine is produced as a polyamino acid. The physicochemical properties of this polyarginyl histidine are as follows.
(A) Hydrolysis treatment with a 6N hydrochloric acid solution produces only arginine and histidine.
(B) Polyarginyl histidine and its hydrolyzate are positive for the Sakaguchi reaction and Pauli reaction.
(C) The bonding mode between the monomers is a peptide bond between the α-position carboxyl group and the α-position amino group.
(D) The amino acid sequence determined by the automated Edman degradation method is an alternating repeat of arginine and histidine with arginine at the N-terminus.
(E) In the molecular weight measurement by the MALDI-TOF Mass (matrix assisted desorption ionization-time of flight measurement) method, a molecule having a molecular weight of about 1,486 is the main component. In addition to this, it is a hybrid of different molecules with a regular molecular weight difference of about 293.
(F) The produced histidine showed the same Rf value (0.19) as the histidine standard product in thin layer chromatography, and was positive for the ninhydrin reaction and the Pauli reaction.
(G) The optical purity of the produced histidine is about 85% of the D-form by the chromatographic analysis by an optical resolution column.
  Therefore, the present invention provides a strain of the genus Epichloe that produces the polyamino acid derivative of the present invention.
  Preferably, it is Epichloe kibiensis E18 strain (FERM P-18923) or a mutant thereof having a polyamino acid derivative-producing ability.
  Furthermore, the present invention provides a method for producing a polyamino acid derivative, which comprises culturing the strain of the present invention in a medium and recovering the polyamino acid derivative from the culture solution (hereinafter, also referred to as the present production method). To do. The strain of the present invention is preferably Epichloe kibiensis E18 strain (FERM P-18923), and the polyamino acid derivative is polyarginyl histidine.
  The present invention is also a method for producing a D-amino acid by microbial fermentation, which comprises culturing a microorganism having an ability to produce a polyamino acid derivative containing a D-amino acid in an appropriate medium, and separating the culture solution, microbial cell or bacterium from the medium. A method for producing a D-amino acid, which comprises recovering the D-amino acid from a disrupted body, or hydrolyzing a peptide derivative as necessary, and the D-amino acid thus produced. -Provides amino acids.
  The mutant strain of the E-18 strain is a mutant strain obtained by subjecting the cells of the E-18 strain to a mutagenesis treatment or a natural mutant strain of the E-18 strain, using the property of producing polyarginyl histidine as an index. Obtained by screening. Examples of the mutagenesis treatment include ultraviolet irradiation and treatment with a mutagenesis substance such as N-methyl-N-nitro-N'-nitrosoguanidine. The screening using, for example, the property of producing polyarginyl histidine as an index can be performed by the method described in Example 1 below.
Manufacture and derivatization by chemical synthesis
  The polyamino acid (for example, polyarginyl histidine) of the present invention can also be produced by chemical synthesis by a peptide synthesis method. In chemical synthesis, for example, the arginine residue of polyarginyl histidine can be replaced with another basic amino acid such as homoarginine, lysine, ornithine, citrulline, and/or histidine can be replaced with cysteine, tryptophan, tyrosine, or the like.
  Upon derivatization, the basic functional group in the basic polyamino acid can be made into a salt with an organic or inorganic acid. For example, it can be used in the form of a salt of an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid or an organic acid such as acetic acid, propionic acid, fumaric acid or malic acid.
  Further, it can be made into a complex with an appropriate metal element by a method known in the art. Examples of such metal atoms include magnesium, manganese, cobalt, nickel, copper, zinc.
  Further, the released functional group in the polyamino acid residue can be subjected to various chemical modifications such as acylation. Methods for making these and methods for derivatization are known in the art.
  It can be converted to ornithine by alkaline hydrolysis of a guanidino group released as a side chain to an arginine residue in polyarginyl histidine. Since arginine and ornithine have different dissociation constants of guanidino group and amino group, respectively, the composition ratio of arginine and ornithine is appropriately adjusted to obtain a polyelectrolyte having a dissociation constant (pKa) of an electrolytic functional group most suitable for the purpose of use. Amino acids can be produced.
Uses of polyamino acid derivatives
  The polyamino acid derivative of the present invention has a high antibacterial activity, as shown in Examples described later (Examples 3 and 4), and has a remarkably high antibacterial activity as compared with existing homopolymers. Became clear (Example 8). In addition, it was found to be a functional molecule having various functions such as metal-binding property (Example 10), acidic substance adsorption activity (Example 12), and permeability to living cells (Example 13). did. Furthermore, it was confirmed that the antibacterial activity of the polyamino acid of the present invention was suggested to be enhanced by complex formation (Example 11). Therefore, the polyamino acid derivative of the present invention is a biodegradable functional polymer and has a wide range of applications. For the uses indicated below, the polyamino acid derivative can be used alone or as a composition and/or in combination with other components. The composition containing the polyamino acid derivative can be prepared in various forms such as a solution, suspension, and solid by a method known in the art, together with a suitable carrier, excipient, diluent and the like. The polyamino acid of the present invention can be used in combination with other active ingredients, and for such purpose, it may be allowed to coexist in one composition in advance, or may be mixed and used in combination at the time of use.
  The main uses of the polyamino acid derivative of the present invention relate to its functions such as antibacterial activity, metal binding activity, binding with acidic substances or adsorption activity. For example, as an antibacterial material, a metal binding material, a material for repulsing a basic substance based on an acidic substance adsorption action, or a material for transporting the substance into a cell in a state of being chemically bound to the substance or in a mixed form Can be used. Here, the "material" means that it is not only useful as an antibacterial composition itself, but also functions as a raw material for manufacturing various products. These will be described in detail below.
  1) Use as an antibacterial substance
  The polyamino acid derivative of the present invention has a growth inhibitory effect against Gram-negative bacteria, Gram-positive bacteria, antibacterial properties, yeasts, and filamentous fungi. This effect is particularly excellent in the form of metal complexes.
  Therefore, the polyamino acid derivative of the present invention is used as an antibacterial substance for preservation of foods and feeds (food additives), improvement of shelf life of foods and feeds (food additives), components of food containers for improving food preservation, refrigeration. Enhancement of food preservation of ice, control of microorganisms in the fermentation industry, prevention of water deterioration in cages and aquariums, antibiotics (pharmaceuticals) to be administered to humans, livestock and fish, preservatives for medical devices that are required to be sterile, Preservatives for contact lenses, preservation of dental prostheses, medical fibers with antibacterial effect, quality preservation of cosmetics and quasi-drugs, contamination control of breast milk, improvement of flower shelf life, prevention of root rot of plants, plants It can be used for seed germination control, microbial control in aseptic cultivation, selection method for separating useful microorganisms and unwanted microorganisms, and the like.
  The use form of the polyamino acid derivative of the present invention relating to the above-mentioned antibacterial activity is not particularly limited, but the antibacterial activity of the polyamino acid derivative of the present invention is mixed or kneaded into the final target product, sprayed on the surface, or the target product. It is possible to adopt a method such as containing or applying it in the container, packaging material, etc. In carrying out, it may be carried out according to the method used for the same purpose in the technical field. Examples of such use are, for example, Japanese Patent No. 1537297, Japanese Patent Laid-Open No. 5-146282, Japanese Patent Laid-Open No. 8-175901, Japanese Patent Laid-Open No. 9-124422, Japanese Patent Laid-Open No. 9-10288, Japanese Patent Laid-Open No. 10-109906, Japanese Patent Laid-Open No. 10-279794, and Japanese Patent Laid-Open No. 10-279794. 2000-189129 and the like and are known to those skilled in the art.
  2) Use as a metal-binding substance or metal complex
  Since the polyamino acid derivative of the present invention has the ability to form a metal complex, it can be used as a reagent for adsorbing, concentrating and quantifying a useful metal, in addition to being used as the above-mentioned antibacterial substance. Further, it can be used as a heavy metal complex for medical contrast agents. Furthermore, intracellular inclusions or extracellular secretions for adsorbing and concentrating radioactive metals, promoting the excretion of radioactive metals from living organisms, and conferring resistance to heavy metals on living organisms (whether they are accumulated inside cells after being produced inside cells) (Because it is secreted extracellularly), ink (pigment) components, metal-containing drug components, metal-administering compositions or feed components for livestock and fish, ingestion sources (health foods) of essential biological metals, etc. Available for
  The use form of the polyamino acid derivative of the present invention relating to the above metal binding activity is not particularly limited and can be selected according to the method used for the same purpose in the technical field. Examples of such use are described in, for example, Japanese Patent Application Laid-Open No. 7-224050, Patent Application No. 11-174054, etc., and are known to those skilled in the art.
  When the polyamino acid derivative of the present invention forms a complex, the metal ion is appropriately selected according to the purpose and is not particularly limited, but for example, when used as an antibacterial agent for foods, pharmaceuticals, cosmetics, etc., When used as a contrast agent, copper, zinc, silver, and paramagnetic gadolinium and manganese can be used.
  3) Use as an electrostatic (ion) reactive substance
  Since the polyamino acid derivative of the present invention is a basic polymer, it has a property of electrostatically binding to an acidic substance and repelling a basic substance. In addition, since the basic polyamino acid has a property of entering the cell, a substance chemically bound to the polyamino acid or a substance having an affinity for the polyamino acid can be transported into the cell. Here, the “affinity substance” with the polyamino acid means a substance that can enter the cell when the polyamino acid changes the permeability of the cell membrane in the state of being dissolved or suspended in the polyamino acid aqueous solution. Point to.
  For example, a substance that chemically binds to polyarginyl histidine (eg, peptide, protein, DNA, RNA, sugar) is transported into the cell and either left as it is in the cell or decomposed and processed to be released, It can exert its function. Thus, an adsorbent for acidic substances, a remover for acidic harmful substances, a carrier in the drug-delivery method, a carrier for nucleic acids used in gene therapy, a carrier for promoting the introduction of nucleic acids into cells, a dyeing aid for fibers. It can be used for.
  Preferably, the polyamino acid derivative of the present invention is used as a vehicle (carrying medium) for a polynucleotide encoding genetic information such as RNA and DNA. Such a vehicle can be used in vitro or in vivo for the treatment of genetic and non-genetic diseases, for gene therapy of diseases, and for obtaining new bacteria, plants and animals having useful properties in fermentation, agriculture, livestock, fisheries, etc. It is useful for gene transfer in.
  The form of use of the polyamino acid derivative of the present invention relating to the binding activity with the acidic substance is not particularly limited and can be selected according to the method used for the same purpose in the technical field. Examples of such use are described in, for example, JP-A-9-308484, and are known to those skilled in the art.
Uses of D-amino acids
  The D-amino acid obtained by the method of the present invention is extremely useful as it is or as an intermediate in the fields of medicine, cosmetics and the like. When used, the D-amino acid can be used alone, but usually it has a simple peptide, dipeptide, tripeptide, polypeptide (having 4 or more residues), protein or amino acid composition. Introduce into polyamino acid. Furthermore, when the D-amino acid is D-histidine, it can be used as a source of the imidazole ring when it is introduced into the compound.
  A peptide or the like into which a D-amino acid has been introduced may have a characteristic property related to the presence of the D-amino acid, for example.
  For example, when the D-amino acid is D-histidine, the fact that there is no report showing the existence of D-histidine in the biological world other than the polyarginylhistidine according to the present invention means that the D-amino acid or a peptide derivative containing the same. Is an extremely rare substance, and it is expected from this that the distribution of enzymes that act on D-histidine, such as peptide degrading enzyme, amino acid oxidase, amino acid decarboxylase, and amino acid deaminase, is small. Therefore, when a compound containing D-histidine is introduced into a living body, food, environment, etc., it is less sensitive (higher resistance) to the enzyme action by the organism and denatured as compared with a compound containing L-histidine. Since it is hard to receive, it is thought that the life as a compound can be extended. Utilizing this property, it can be used for raw materials such as pharmaceuticals, agricultural chemicals, chemicals, cosmetics, food additives, and their synthetic intermediates.
  Some physiologically active substances have D-amino acids, and the specificity of D-amino acids may directly relate to physiological activity. Therefore, the D-histidine obtained by the method of the present invention is useful in the production of a compound which requires a D-series histidine or derivative for functional expression.
  Hereinafter, the present invention will be described in more detail with reference to examples, but these examples are for the purpose of illustration only, and are not intended to limit the present invention in any sense.
Example 1 Identification of polyamino acid-producing strain
  The polyarginyl histidine-producing bacterium was obtained according to the method described in Nishikawa et al. (supra), as follows.
(1) Isolation of strains
  Solid medium (glycerol; 10 g, ammonium sulfate; 0.66 g, potassium dihydrogen phosphate; 0.68 g, magnesium sulfate heptahydrate; 0.25 g, yeast extract; 0.1 g, trace mineral elements, trace amount) Dissolve the components in 1 L of deionized water, adjust the pH to 7.0 with aqueous sodium hydroxide, and add 15 g of powder agar) and sterilize (121°C, 15 minutes), then sterilize separately The dye PolyR-478 (manufactured by Sigma, final concentration 0.02%) is added to solidify in a petri dish. One platinum loop of a soil sample is dispersed with an appropriate amount of sterilized water, and a part of the sample is inoculated on the agar plate medium by coating. After culturing at 28° C. for 3 to 14 days, a strain characterized by the presence of a dye adsorption region formed at the periphery of a colony (cell colony) is selected as a candidate for a basic polyamino acid-producing bacterium.
(2) Mycological properties
  Isolation and characterization of polyamino acid-producing bacteria
  The bacterial strain selected in (1) above was inoculated on each agar plate of potato dextrose medium (PDA), oatmeal medium (OA), and 2% malt medium (MEA), cultured at 28°C, and examined for mycological properties. It was Potato dextrose medium (PDA), oatmeal medium (OA), and 2% malt medium (MEA) are all manufactured by Difco/Becton Dickinson.
A. Growth status on medium
  Hyphae are fluffy and white on all agar plates. The growth is rather slow, and the PDA and MEA plates reach a diameter of 20 mm one week after the start of culture, and the OA plates reach a diameter of 15 mm. The formation of conidia is small, and no change in the color tone of colonies due to conidia is observed. No exudate or soluble pigment production is observed.
B. Spore formation
  Under a light microscope, formation of a fialo-type asexual reproductive organ is confirmed. The conidia peduncle is slightly erect and spins single or 2 to 4 phialides from aerial hyphae. Some conidia peduncles showing secondary branching are also observed. Phialides are elongated cones that are linear and vary in size. The surface of hyphae and phialides is smooth. Conidia are unicellular and have a smooth surface. There are various shapes such as oval, teardrop, oval, and oval. Conidia are viscous and become clumpy from the tip of the conidia peduncle. Observation after 8 weeks of culture does not reveal formation of crescent-shaped large conidia, chlamydospores, or teleomorphs.
C. Physiological properties
  It grows well at 21°C or 28°C, but not at 33°C. The pH grows between 2.5 and 8.8. As described below, polyarginyl histidine is produced.
D. Source of carbon that can be assimilated
  D-glucose, D-galactose, D-xylose, D-sorbitol, D-mannitol, glycerol, citric acid, L-glutamic acid can be used.
(3) Identification of species
  Regarding the strain selected in (1) above, the nucleotide sequence of 28s ribosomal RNA molecule was determined. This base sequence is registered in DNA Data Bank of Japan under the accession number AB087373. As a result of examination by a molecular biology method based on interspecies comparison of 28s ribosomal RNA molecules, the strain was found to be a new strain belonging to Epichloe. This is named Epichloe kibiensis E18 strain and has been deposited at the Patent Biological Depository Center, National Institute of Advanced Industrial Science and Technology under the deposit number FERM BP-8358 (deposit date: July 5, 2002).
Example 2 Production and identification of polyamino acid by E18 strain
(1) Production by solid culture
  According to the method described in Example 1, strain E18 was cultured in the solid medium described in Example 1 (however, the pigment was not included). After culturing for 10 days, the polyamino acid secreted in the periphery of the colony (cell colony) was recovered as agar pieces. Media components other than polyamino acids remaining in the agar pieces were removed by immersing the agar pieces in water. Next, the agar component was hydrolyzed by heating the agar piece in a 1N hydrochloric acid solution at 90° C. for 1 hour. The polyamino acid contained in this solution is adsorbed on a cation exchange resin (Amberlite IRC-50, manufactured by Organo), washed with 0.2N acetic acid, eluted with 0.1N hydrochloric acid, and dried under reduced pressure. It was purified by
(2) Production by liquid culture
  Liquid medium (D-galactose, 10 g; ammonium sulfate, 0.66 g; potassium dihydrogen phosphate, 0.68 g; magnesium sulfate heptahydrate, 0.25 g; yeast extract, 0.2 g; trace mineral elements, trace amount) Dissolve it in 1 L of deionized water and adjust the pH value to 7.0 with an aqueous NaOH solution) and put it in a 500 mL Erlenmeyer flask with a baffle to sterilize (121° C., 15 minutes). After inoculation, the cells are cultivated aerobically in a constant temperature bath at 28° C. with orbital shaking at 100 rpm. After 7 days, the supernatant of the culture solution was collected by filtration through a synthetic polymer membrane having a pore size of 0.45 mm, and the polyamino acid contained in this solution was cation exchange resin (Amberlite IRC-50, manufactured by Organo Corporation). ), washed with 0.2N acetic acid, eluted with 0.1N hydrochloric acid, and dried under reduced pressure for purification.
(3) Identification of monomer
  1) Polyarginyl histidine purified from the culture solution of E18 strain in the above (1) or (2) was hydrolyzed by heating in a 6N hydrochloric acid solution at 100° C. for 20 hours to prepare a monomer. Thin-layer chromatography method of monomers [developing thin-layer plate, silica gel or cellulose coated; developing temperature, 20° C.; developing solvent, n-butanol:acetic acid:pyridine:water=4:1:1:2 mixture] And developed with ninhydrin reagent. There were two kinds of monomerized amino acids, and they showed the same Rf values (0.22 and 0.19, respectively) as the standard substances arginine and histidine, respectively. The monomer having an Rf value of 0.22 was positive in the Sakaguchi reaction, which is a method for detecting a guanidino group. The spot with an Rf value of 0.19 was positive in the Pauli reaction, which is a method for detecting the imidazole ring. The polyamino acid that had not been hydrolyzed was positive for the Sakaguchi reaction and Pauli reaction.
  From the above results, it was revealed that the monomers were arginine and histidine. The guanidino group and imidazole ring present on each side chain are present in the polyamino acid in a free form, and arginine and histidine are mutually linked by the peptide bond between the α-amino group and the α-carboxyl group on the main chain. It turned out to be connected.
(4) Polyamino acid sequencing
  In order to clarify the sequences of arginine and histidine, which are components of polyamino acid, the purified polyamino acid was analyzed by an automatic Edman degradation analyzer (Model 492, manufactured by Applied Biosystems). The results are shown in Figure 1.
  The N-terminal amino acid residue was arginine and the second residue was histidine. From this point forward, the odd-numbered residue was arginine and the even-numbered residue was histidine. As shown in Figure 1, the second residue is histidine and the third residue is arginine.
  From the above, it was revealed that the polyamino acid has a structure in which arginine is the N-terminal and arginine and histidine are alternately linked.
(5) Measurement of molecular weight
  In order to clarify the molecular weight of the polyamino acid, the purified polyamino acid was measured by a MALDI/time-of-flight mass spectrometer (Model Voyager DE manufactured by Perceptive). FIG. 2 shows the result of mass spectrometry. The detected molecular weight is centered at about 1,486 derived from RHRHRHRHRH (R is an arginine residue, H is a histidine residue, and is SEQ ID NO: 1), and has a difference of about 293 in addition to this, is less than? A signal (about 1,193 from RHRHRHRH, SEQ ID NO:2) or a larger (about 1,779 from RHRHRHRHRHRH, SEQ ID NO:3) signal was observed. Further, the refined product contained almost no impurities other than these.
  From the above, the polyamino acid produced by strain E18 was not a single molecule having a unique molecular weight, but was a hybrid of molecules having a molecular weight distribution within a certain range.
Example 3 Antibacterial activity of polyarginyl histidine produced by E18 strain
  The antibacterial test was conducted by the following method.
  The polyarginyl histidine purified by Example 2 (2) and produced by the E18 strain identified in Examples 2 (3) to (5) was dissolved in sterilized water so that the final concentration became constant. Added to the appropriate liquid medium. As shown in Table 1, the liquid medium was selected to be suitable for the growth of the microorganism whose sensitivity is to be evaluated (test microorganism). The composition of 1 L of each liquid medium was as follows: 1) N medium (nutrient broth): meat extract, 3 g; peptone, 5 g, pH 6.8, 2) 702 medium: polypeptone, 10 g; yeast extract, 2 g; magnesium sulfate 7 Hydrate, 1 g, pH 7.0, 3) YPD medium: yeast extract, 10 g; peptone, 20 g; D-glucose, 20 g, pH 6.5, 4) PD medium: potato exudate, amount corresponding to 200 g of raw potato D-glucose, 20 g, pH 5.1 was used. A liquid culture medium containing polyarginyl histidine was inoculated with the test microorganism and cultured at each optimum culture temperature for 20 hours. For the assay of proliferation, the absorbance at 600 nm was measured for microorganisms having good cell dispersibility, and the microorganisms forming heterogeneous cell clusters were visually observed. The results of the antibacterial test are shown in Table 1. As shown in Table 1, the polyarginyl histidine produced by the E18 strain strongly suppressed the growth of Gram-negative bacteria, Gram-positive bacteria, yeasts and filamentous fungi.

Medium: N, nutrient broth; YPD, yeast extract/peptone/dextrose; PD, potato dextrose medium; 702, IFO702 medium.
* Decreased growth rate was observed.
The relative concentration is the relative value (dilution ratio or concentration ratio) when the concentration when the antibacterial substance purified from the culture medium of E18 is added to the test medium is 1 when the concentration is produced in the culture medium. Represent The x1 concentration is estimated to be approximately 40 μg/mL. Therefore, the relative concentrations, x1/10, x1/2, x1, and x5 correspond to approximately 4, 20, 40, and 200 μg/ml, respectively.
Example 4 Antibacterial activity of chemically synthesized polyarginyl histidine
  A polyarginyl histidine having 10 amino acid residues was prepared by a chemical synthesis method and subjected to a test. The chemical synthesis was performed according to the method described in the literature (Atherton, E and Sheppard, RC, Solid phase peptide synthesis. A practical approach, IRL Press, Oxford (1989)). The amino acid sequence of the product polyamino acid is (N-terminal)(L-arginine)-(L-histidine)-(L-arginine)-(L-histidine)-(L-arginine)-(L-histidine)-( L-arginine)-(L-histidine)-(L-arginine)-(L-histidine) (C-terminal). This polyarginyl histidine was dissolved and diluted in sterile water, and described in Example 3 so as to have a constant final concentration (1, 10, 20, 50, 100, 150, 200, 300, 400, 500 μg/mL). Antibacterial activity was evaluated by adding to a liquid medium having the same composition as the above and culturing. The antibacterial activity (proliferation) was evaluated according to the method described in Example 3 above. The results of the antibacterial test are shown in Table 2.

Medium: N, nutrient broth; YPD, yeast extract/peptone/dextrose; PD, potato dextrose medium; 702, IFO702 medium.
The chemically synthesized polyarginyl histidine used had 10 amino acid residues.
* Decreased growth rate was observed. -Does not prevent growth.
  As shown in Table 2, polyarginyl histidine having 10 amino acid residues was antibacterial against Gram-negative bacteria and Gram-positive bacteria, and strongly suppressed the growth against yeast and filamentous fungi. Exceptionally, it was ineffective against the Bacillus cereus IFO3514 strain. The results shown in Table 2 above show that the synthetic polyamino acids have the same antibacterial activity as the polyamino acids produced by the E18 strain shown in Table 1. These results also demonstrate that the E18 strain of the present invention produces the desired heteropolyamino acid. Although there is a slight difference in the numerical value of the minimum growth inhibitory concentration, it is considered that this is based on the purity and the like of the natural polyamino acid and the chemically synthesized polyamino acid.
Example 5 Relationship between type and number of amino acid residues and antibacterial property
(1) In order to investigate the relationship between the number of amino acid residues of polyarginyl histidine and the antibacterial activity, polyarginyl histidines having different numbers of amino acid residues were chemically synthesized, and the antibacterial activity was the same as in Example 3 or 4. The test was conducted. Escherichia coli IFO3301 strain and Bacillus subtilis IFO3336 strain were used as test microorganisms. Table 3 shows the results of the antibacterial test. The amino acid sequences of the polyamino acids shown in Table 3 are listed in SEQ ID NOs: 4 to 13.

Test microorganisms: Bacillus subtilis IFO3336 and Escherichia coli IFO3301.
R, H, K, G and C in the structural scheme represent arginine, histidine, lysine, glycine and cysteine residues, respectively.
Medium, Nutrient Broth.
-Does not prevent growth.
  As shown in Table 3, antibacterial properties were observed with polyarginyl histidine (SEQ ID NOs: 5 to 8) having 6 or more amino acid residues. The minimum growth inhibitory concentration was saturated when the number of residues was 8 or more, and the value was 10 μg/mL. N-terminal is histidine and C-terminal is arginine. The minimum inhibitory concentration of polyhistidylarginine (10 residues, SEQ ID NO: 9) is 10 μg/mL, and N-terminal with the same number of residues for antibacterial activity. Is arginine and histidine at the C-terminus is unchanged from polyarginyl histidine.
Example 6 Antibacterial properties of amino acid substitution derivatives
  In order to investigate the relationship between the amino acid sequence of polyarginyl histidine and the antibacterial activity, polyamino acids having different amino acid sequences were chemically synthesized, and an antibacterial test was conducted in the same manner as in Example 3 or 4. Escherichia coli IFO3301 strain and Bacillus subtilis IFO3336 strain were used as test microorganisms. The results of the antibacterial test are shown in Table 3 above.
  As shown in Table 3, a polyamino acid having 10 residues, in which arginine of polyarginyl histidine was substituted with lysine (SEQ ID NO: 10), had a minimum inhibitory concentration of 10 μg/mL and had the same residue for antibacterial activity. It was no different from the number of polyarginyl histidines. Polyarginyl histidine, in which histidine is replaced with cysteine, has a residue number of 10 and the polyamino acid (SEQ ID NO: 13) has a minimum inhibitory concentration of 10 to 20 μg/mL. It was almost the same as histidine. A polyamino acid (SEQ ID NO: 11) having a residue number of 10 in which histidine of polyarginyl histidine is replaced with glycine has a minimum inhibitory concentration of 100 μg/mL, and polyarginyl histidine having the same residue number as that of polyarginyl histidine has the same antibacterial activity. Only a tenth effect was shown. A polyamino acid with 10 residues (SEQ ID NO: 12) in which arginine of polyarginyl histidine was replaced with glycine did not show antibacterial properties. From the above results, the following points are clear regarding the properties of mutually interchangeable amino acids. That is, arginine and lysine are both amino acids having strong basicity, and histidine and cysteine have high chelate-forming ability, and further, excellent chelate-forming ability is maintained even in a state where polyamino acid is formed by peptide bond. It can be mentioned. On the other hand, histidine and glycine have common chelating ability, but differ in that glycine causes a decrease or loss of chelating ability when forming a polyamino acid. Furthermore, arginine and glycine differ in basicity. As described above, it was found that the basic amino acid needs to be contained in order to obtain the antibacterial polyamino acid, and the antibacterial property is enhanced by the presence of the amino acid having the excellent chelate forming ability.
Example 7 Antibacterial activity of acylated derivatives
  In order to investigate the effect of the polarity change of extremely hydrophilic polyarginyl histidine on the antibacterial activity, polyarginyl histidine (RH having 10 residues as described in the above examples)₅), N-amino group of N-terminal arginine residue is myristylated to give a myristylated polyarginyl histidine (myristyl-RH) having 10 residues.₅) Was chemically synthesized. Using this, an antibacterial test was carried out in the same manner as in Example 3 or 4. The results of the antibacterial test are shown in Table 4.

Medium, Nutrient Broth.
RH₅, Polyarginyl histidine (10 amino acid residues); myristoyl-RH₅, Polyarginyl histidine (the number of amino acid residues is 10, the myristyl group is bound to the N-terminal amino group); εPL, epsilon-poly-L-lysine (the number of amino acid residues is 25 to 36).
* Decreased growth rate was observed. -Does not prevent growth.
  As shown in Table 4, myristoyl-RH₅Is the corresponding non-acylated polyarginyl histidine (RH₅), it has become clear that it has a higher activity. That is, myristoyl-RH₅Is RH₅Showed an inhibitory effect on the growth of Bacillus cereus IFO3514 strain which showed no antibacterial effect at a minimum inhibitory concentration of 10 μg/mL. In addition, RH which is a non-myristylated polyamino acid₅Is relatively weak (minimum growth inhibitory concentration is 50 μg/mL or more) and has a growth inhibitory effect on Gram-negative bacteria (Salmonella typhimurium IFO12529) and Gram-positive bacteria (Staphylococcus aureus IFO13276, Staphylococcus aureus MRSA ATCC33 high growth inhibitory effect) The minimum growth inhibitory concentration was 10 μg/mL). Thus, it was proved that the acylation is effective in improving the antibacterial activity (extending the antibacterial spectrum).
Example 8 Comparison in antibacterial activity with homopolymer polylysine
  It is known that polylysine, which is an existing homopolyamino acid, exhibits a strong antibacterial property. Therefore, the polyarginyl histidine of the present invention (synthetic RH₅) Was compared with the antibacterial property. As polylysine, ε poly-L-lysine obtained from Wako Pure Chemical Industries, Ltd. was used. Evaluation of antibacterial activity is similar to the method described in Examples 3 and 4. The results are shown in Table 4 above. From this result, it is clear that polylysine showed strong antibacterial activity against almost all the tested bacteria. The cereus IFO3514 was only 1/5 times more effective than the other strains (5 times more resistant). On the other hand, although the chemically synthesized polyarginyl histidine (10 residues) of the present invention did not show sufficient antibacterial activity for some strains, myristylated polyarginyl histidine strongly suppressed the growth of all strains. , B., which showed a 5-fold resistance to polylysine. The cereus IFO3514 strain also strongly suppressed the growth at the same level.
Example 9 Antibacterial activity under strongly alkaline conditions
  Considering that the free amino group of polylysine, which is an existing product, has a pKa value of 8.9 and the free guanidino group of polyarginylhistidine has a pKa value of 12.5. Argininohistidine is expected to be stronger than polylysine. Therefore, the antibacterial activity against alkalophilic bacteria was investigated. Nutrient broth adjusted to pH 11.2 with a 20 mM CAPS/NaOH buffer was used as the medium, and the other test methods were performed according to Example 3. The results are shown in Table 5.

Medium, Nutrient Broth (20 mM CAPS/NaOH buffer).
RH₅, Polyarginyl histidine (the number of amino acid residues is 10); εPL, epsilon-poly-L-lysine (the number of amino acid residues is 25 to 36).
-Does not prevent growth.
  As shown in Table 5, at pH 11.2, polyarginyl histidine showed antibacterial properties, whereas polylysine did not. Therefore, it was shown that the antibacterial property of polyarginyl histidine is effective even under strong alkaline conditions.
Example 10 Metal bondability test
  Polyarginyl histidine (RH having 10 amino acid residues)₅) Was prepared by a chemical synthesis method and subjected to a test. The amino acid sequence is the same as that in Example 2(5). The metal binding property was evaluated by the increase in molecular weight. Polyarginyl histidine was prepared in 30 mM Tris-hydrochloric acid buffer (pH 7.5) so that the final concentration was 30 pmol/μL, and mixed with an aqueous solution of metal chloride or sulfate. Magnesium, manganese, cobalt, nickel, copper, and zinc were used as metal elements. The mixing ratio was a molar ratio of polyarginyl histidine:metal=1:10. The molecular weight was measured by mixing a mixture of polyarginyl histidine and a metal with 2,5-dihydroxybenzoic acid according to the method recommended by the measuring instrument manufacturer, and drying the mixture, followed by a time-of-flight mass spectrometer (model of Perceptive, model). Voyager DE) and was measured by the MALDI-TOF Mass method. An example of the result of the metal bondability test is shown in FIG. FIG. 3 shows the detection results of the polyamino acid-zinc complex by the MALDI/TOF-MS method, where A is polyarginyl histidine (10 residues) and B is polyarginyl histidine (10 residues) sulfated. The results when coexisting with copper (10 times the molar number of polyamino acid) are shown.
  As shown in FIG. 3, when polyarginyl histidine and zinc sulfate were allowed to coexist, zinc ions were added in addition to the m/z value of 1,486 shown by polyarginyl histidine when not coexisting with zinc sulfate. A m/z value of 1,550 indicated by the molecule was observed. For other metals, polyarginyl histidine was confirmed to have an m/z value increased by a value specific to each metal to the m/z value exhibited by polyarginyl histidine alone. It was revealed that a complex was formed between the two types of metals.
Example 11 Effect of metal on antibacterial property
  As shown in Example 6, chelate-forming amino acid residues such as histidine and cysteine contribute to the enhancement of antibacterial properties of basic polyamino acids. As shown in Example 10, polyarginyl histidine forms a complex with a metal. Therefore, in order to investigate the dependence of the metal on the antibacterial property, an antibacterial property test was performed using a chemically synthesized polyarginyl histidine (10 amino acid residues). The test method was to add various concentrations of aqueous zinc sulfate solution to a modified synthetic liquid medium of Davis containing polyarginyl histidine at a final concentration of 10 μg/mL (6.6 μM), and inoculate it with Escherichia coli IFO3301 strain as a test microorganism. , Cultured. The growth was evaluated by measuring the absorbance at 600 nm after doubling for 20 hours. The composition of 1 liter of modified synthetic liquid medium of Davis is as follows: D-glucose, 1 g; dipotassium hydrogen phosphate, 7 g; potassium dihydrogen phosphate, 2 g; trisodium citrate dihydrate, 0. 5 g; magnesium sulfate heptahydrate, 10 mg; ammonium sulfate, 1 g; casamino acid, 100 mg; thiamine hydrochloride, 2 mg, pH 7.0. The results are shown in Fig. 4. The case where 6.6 μM of polyarginyl histidine is contained is shown by hatching, and the case where polyarginyl histidine is not contained is shown by outline.
  Under the conditions (artificial medium) used in this test, no antibacterial property is observed regardless of the presence or absence of zinc ion in the range of low concentration of 1.65 to 3.3 μM, which does not contain zinc ion at all. I can't. However, the growth was strongly suppressed in the presence of 6.6 μM or more zinc. There is no effect of toxicity of zinc, since the growth in the presence of the same concentration of zinc in the absence of polyamino acid as a control experiment is not suppressed at all. Similar effects were observed with copper ions.
  On the other hand, in Examples 3 and 4 described above, when the medium derived from the natural product was used, the antibacterial action was observed without adding zinc.
  These results suggest that the experimental conditions in this example may contain an inhibitor (factor) that suppresses the antibacterial activity of polyarginyl histidine for some reason. The above results also suggest that, in the presence of zinc ions, the polyamino acid derivative of the present invention can exert antibacterial activity despite the action of such an inhibitor. And, in order to obtain a sufficient antibacterial activity in the presence of such an inhibitor, it has been shown that it is effective to allow zinc ions to be present in equimolar amounts or more to polyarginyl histidine.
  Furthermore, when the polyarginyl histidine of the present invention is used as a complex, even if it is feared that the antibacterial activity may be reduced or lost by the effect of the inhibitor alone, the complex form can sufficiently exhibit the antibacterial activity. Also shows.
Example 12 Reactivity with acidic substances and basic substances
Test method
  Polyarginyl histidine (RH having 10 amino acid residues)₅) Was prepared by a chemical synthesis method and subjected to a test. The amino acid sequence is the same as that in Example 2(5). For the evaluation of the action with an acidic substance and a basic substance, PolyR-478 which is an acidic dye and methylene blue which is a basic dye were used, respectively. 10 μL of an aqueous solution containing 1 mg of chemically synthesized polyarginyl histidine was infiltrated into a piece of filter paper having a diameter of 6 mm, which was left to stand on an agar plate containing a dye having a charge, and the change of the dye due to diffusion was observed. The concentrations of agar, PolyR-478 and basic dye methylene blue were 1.5%, 0.02% and 0.002%, respectively. The results of the evaluation test of the action with the acidic substance and the basic substance are shown in FIG.
  As shown in FIG. 5, it was observed that polyarginyl histidine adsorbs and concentrates the acidic dye (PolyR-478) (A) as it diffuses from the filter paper piece into the agar plate. The pigment is concentrated and looks darker than the surroundings). On the other hand, with regard to the basic dye, it was observed that this was rejected and concentrated in the vicinity of the diffusion front (B) (arrowhead, dye was rejected, and it appeared brighter than the surroundings). From these results, it was found that polyarginyl histidine has a property of adsorbing an acidic substance and, on the contrary, repelling a basic substance.
Example 13 Permeability of polyarginyl histidine into living cells
  In order to investigate the effect of polyarginyl histidine on cells, the behavior of fluorescently labeled polyamino acid was observed. In accordance with Example 4, polyarginyl histidine having 10 amino acid residues was prepared by a chemical synthesis method, and the N-terminal free amino group was chemically labeled with a fluorescent dye, FAM (carboxyfluorescein). A final concentration of 1 μg/μL was added to a suspension of Candida boidinii MIP104 strain at a final concentration of 1 μg/μL, the mixture was allowed to stand for 5 minutes, and then excess labeled polyamino acid in the suspension was removed by washing to prepare a preparation. This was observed and photographed with a coaxial epi-illumination fluorescence microscope under normal light and a fluorescence excitation light source. As a control experiment, a sample in which only the fluorescent dye FAM was added alone was also observed. The results are shown in Fig. 6. In FIG. 6, A is an N-terminal labeled polyamino acid (FAM-RH5) added to the cell suspension of the yeast Candida boidinii MIP104 strain, and observed with a fluorescence microscope. As a result, C shows the same field of view observed with normal light. Shows the result obtained by adding only FAM of the control experiment to the same cell suspension and observing with a fluorescence microscope, and D shows the result observing the same visual field with normal light.
  As shown in FIG. 6A, fluorescently labeled polyarginyl histidine was incorporated into live yeast cells. On the other hand, as shown in FIG. 6B, the fluorescent dye alone was not taken up by the cells, and the uptake was observed only in the form bound to the polyamino acid. Therefore, it was confirmed that polyarginyl histidine has the ability to penetrate the cell wall and cell membrane of yeast cells without any special manipulation.
Example 14 Assay of optical purity of histidine
  The hydrolyzate of polyarginyl histidine of Example 2-(3) was subjected to an optical resolution column (CROWNPAK CR(+) manufactured by Daicel Industries, Ltd., a mobile phase was an aqueous perchloric acid solution having a pH of 1.5, and a column temperature was 4 d° C.). It was analyzed by a high performance liquid chromatography device (HPLC) attached. For detection, absorption at 200 nm was measured. As a control experiment, four amino acid standards of D-histidine, L-histidine, D-arginine, and L-arginine, and two chemically synthesized polyamino acids (N-terminal L-Arg-D-His-L-Arg) were used. -D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His C-terminal and N-terminal L-Arg-L-His-L-Arg-L-His-L -Arg-L-His-L-Arg-L-His-L-Arg-L-His C-terminal) was hydrolyzed by heating the standard product in a 6N hydrochloric acid solution at 100°C for 20 hours under the same conditions. HPLC analysis was performed.
  The analysis result is shown in FIG. 7. In the figure, chemically synthesized product, (LR-LH)₅, And (LR-DH)₅[N-terminal L-Arg-L-His-L-Arg-L-His-L-Arg-L-His-L-Arg-L-His-L-Arg-L-His C-terminal] And [N-terminal L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His C-terminal].
  As shown in FIG. 7, about 85% of the histidines constituting the polyarginyl histidine produced by the E-18 strain were D-forms. In addition, 100% of arginine was L-form. Histidine contained in the chemically synthesized product was about 3.3% in conversion from L-form to D-form and about 0 in conversion from D-form to L-form due to racemization accompanying the hydrolysis operation. It was 0.9%. Therefore, the effect of artificial racemization accompanying the hydrolysis operation on the numerical value of the abundance ratio of the D-form and the L-form of histidine contained in the polyarginyl histidine of the E-18 strain is small. , D-histidine having a high optical purity of about 85% could be produced.
Example 15 Functionality of peptides containing D-histidine
(1) Antibacterial test
  In order to see the correlation between the stereoisomerism (D-form or L-form) of histidine contained in polyarginyl histidine and the antibacterial activity, polyarginine in which all histidine residues are D-form by a chemical synthesis method. Nylhistidine (N-terminal L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His C-terminal) and all The histidine residue of is an L-form polyarginyl histidine (N-terminal L-Arg-L-His-L-Arg-L-His-L-Arg-L-His-L-Arg-L-His-L -Arg-L-His C-terminal) was prepared and added to the liquid medium of Nutrient Broth (NB) or potato dextrose medium described in the above example at the start of the culture. After inoculation, the cells were cultured at 28° C. with shaking to observe the growth of test microorganisms. The microorganisms tested include Pseudomonas aeruginosa (IFO3445, from the Fermentation Research Institute), Salmonella bacterium (Salmonella typhimurium, IFO12529, the same), Candida albicans, Candida albicans, IFO1385, SIF1385, Sanda, IFO1385. , KK4, preserved in the research institution).
  The results are shown in Table 6. The crude purified product of polyarginyl histidine produced by the E-18 strain suppressed the growth of the test microorganisms at a minimum growth inhibitory concentration of 8 to 40 μg/mL. As for the chemically synthesized product that is considered to be almost free from impurities, the minimum growth inhibitory concentration of polyarginyl histidine in which all histidine residues are D-forms is 5 to 10 μg/mL, and all amino acids are L-forms. It showed a low minimum inhibitory concentration compared to some polyarginyl histidines. These results indicate that the antibacterial property is enhanced by making the histidine residue in polyarginyl histidine into the D-form.

  Chemically synthesized products in Table 6 (_LR-_LH)₅and(_LR-_DH)₅The structure of each is the N-terminal [L-Arg-L-His-L-Arg-L-His-L-Arg-L-His-L-Arg-L-His-L-Arg-L-His] C-terminal , N-terminal [L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His] C-terminal. Here, R and H represent arginine and histidine residues, respectively.
(2) Sensitivity test for peptide degrading enzymes
  The correlation between the stereoisomerism (D-form or L-form) of histidine contained in polyarginyl histidine and the sensitivity to the peptide-degrading enzyme was examined. After reacting polyarginyl histidine containing D- or L-histidine with trypsin which is a kind of peptide degrading enzyme and also exists in the digestive tract of mammals, a change in molecular weight was observed.
  The specific test method is as follows. Four kinds of polyarginyl histidine produced by the E-18 strain or chemically synthesized ((1) N-terminal L-Arg-D-His-L-Arg-D-His-L-Arg-D-His- L-Arg-D-His-L-Arg-D-His C-terminal; (2) N-terminal L-Arg-L-His-L-Arg-L-His-L-Arg-L-His-L-Arg -L-His-L-Arg-L-His C-terminal; <3> N-terminal L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-L- His-L-Arg-D-His C-terminal; (4) N-terminal L-Arg-D-His-L-Arg-D-His-L-Arg-L-His-L-Arg-L-His-L -Arg-D-His C-terminal) was mixed with about 100 μg each of them and carrier-fixed trypsin (TPCK-Trypsin, immobilized by Pierce) suspended in 200 μL of 100 mM ammonium hydrogencarbonate aqueous solution (pH 7.8). Then, the mixture was kept warm at 30° C. for 6 hours with vigorous stirring. 1 μL of the supernatant was mixed with 9 μL of an aqueous 2,5-dihydroxybenzoic acid solution (10 mg/mL), and the mixture was mixed with MALDI/TOF-MS (matrix-assisted laser deionization/time-of-flight mass spectrometer, Perceptive, Voyager DE). And the molecular weight was measured.
  The measurement result is shown in FIG. In the figure, the arrow indicates the putative trypsin breakpoint. The test substances in each chart are as follows. (A) L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg-D-His, (B) L-Arg- L-His-L-Arg-L-His-L-Arg-L-His-L-Arg-L-His-L-Arg-L-His, (C)L-Arg-D-His-L- Arg-D-His-L-Arg-D-His-L-Arg-L-His-L-Arg-D-His, (D)L-Arg-D-His-L-Arg-D-His-L -Arg-L-His-L-Arg-L-His-L-Arg-D-His, (E) Polyarginyl histidine produced by the E-18 strain prepared in Example 2.
  Trypsin has the activity of specifically cleaving the peptide bond at the carboxy-terminal side of the arginine or lysine residue present inside the peptide chain. In general, this type of enzyme has stereoselectivity for substrates, and it is required that the two amino acids sandwiching the cleavage point are both L-forms. Therefore, an attempt was made to confirm the stereoselectivity by analyzing the molecular weights of the decomposed products of four types of chemically synthesized polyarginyl histidines with trypsin.
  As a result, as shown in (A), polyarginyl histidine in which all histidine residues are D-forms was not decomposed by trypsin. On the other hand, as shown in (B), polyarginyl histidine in which all histidine residues are L-forms is decomposed by trypsin to give dipeptides (L-His-L-Arg, molecular weight about 314) and tripeptides (about 314). L-Arg-L-His-L-Arg or L-His-L-Arg-L-His, molecular weight about 451). In addition, as shown in (C), polyarginyl histidine in which only histidine at the eighth residue is in the L-form undergoes decomposition by trypsin to give tripeptide (L-His-L-Arg-D-His, molecular weight). About 451) and the heptapeptide (L-Arg-D-His-L-Arg-D-His-L-Arg-D-His-L-Arg, molecular weight about 1,056). Furthermore, as shown in (D), polyarginyl histidine, in which only histidines at the 6th and 8th residues are L-forms, was decomposed by trypsin to give a dipeptide (L-His-L-Arg, molecular weight about 314). ), tripeptide (L-His-L-Arg-D-His, molecular weight about 451), pentapeptide (L-Arg-D-His-L-Arg-D-His-L-Arg, molecular weight about 763). occured. Taken together, it is clear that trypsin attacks as a substrate only when two amino acids sandwiching the breakpoint, here arginine and histidine, are both L-forms. That is, it was proved that polyarginyl histidine containing D-histidine was less susceptible to decomposition by trypsin as compared with polyarginyl histidine containing L-histidine.
  As shown in (E), the polyarginyl histidine produced by the E-18 strain was not degraded by trypsin as in the case of (A) above, and the molecular weight did not change from the molecular weight before trypsin treatment, It remained 1,487. This result indicates that at least the 4th, 6th, and 8th residues of the polyarginyl histidine produced by the E-18 strain are D-forms. Therefore, it was found that the polyarginyl histidine produced by the E-18 strain contains D-histidine and is less susceptible to degradation by trypsin as compared with polyarginyl histidine containing L-histidine.

Industrial availability

The polyamino acid derivative of the present invention is a biodegradable functional polymer and exhibits excellent antibacterial activity, metal-binding activity, acid-material binding activity and repulsive action to basic substances. It has a wide range of uses as an adsorbent and a medium for transporting acidic substances. Moreover, since the polyamino acid derivative of the present invention can be easily produced by microbial fermentation and can be produced safely and efficiently, it can be produced and used without adversely affecting the environment.
When the polyamino acid derivative of the present invention contains a D-amino acid, the D-amino acid can be produced from a medium composition with extremely high efficiency and high optical purity. In particular, since Epichloe kibiensis E-18 strain (FERM P-18923) produces a polyamino acid containing D-histidine, it can be produced easily at a low production cost, and the chemicals and wastes that can affect the environment can be easily produced. It is possible to supply a large amount of D-histidine that is useful in the fields of medicine and cosmetics by reducing the amount as much as possible.

[Sequence list]

Claims (20)

A polyamino acid consisting of 2 to 10 kinds of amino acids containing at least one kind of basic amino acid or a derivative thereof. General formula (I):

[Wherein, X is the following 1) and 2):
1) Arginine, lysine, ornithine, citrulline, homocitrulline, homoarginine, canavanine and 2-amino-3-guanidinopropionic acid 2) An amino acid selected from the group consisting of histidine, cysteine, homocysteine, tryptophan and tyrosine. Represents a residue, Y represents an amino acid residue selected from the other group, R ₁ represents a hydrogen atom, a sugar residue, an acyl group, a biotinyl group, a thiol group, a phenol group, an indole group, or the same as the above Y. represents the amino acid residue, R ₂ is a hydroxyl group, sugar residue, acyl group, biotinyl group ,, a thiol group, a phenol group, an indole group, or the X the same amino acid residue, n represents an integer of 2 or more Represent]
The polyamino acid or derivative thereof according to claim 1, which is represented by: The polyamino acid or a derivative thereof according to claim 2, wherein X is an arginine or lysine residue and Y is a histidine or cysteine residue. The polyamino acid or a derivative thereof according to claim 3, wherein X is an arginine residue and Y is a histidine residue. The polyamino acid or derivative thereof according to any one of claims 2 to 4, wherein n is an integer of 2 to 50. The polyamino acid or a derivative thereof according to claim 2, wherein R ₁ is a hydrogen atom and R ₂ is a hydroxyl group. The polyamino acid or its derivative according to any one of claims 1 to 6, which is a complex with a metal selected from copper, zinc, silver, gadolinium, magnesium, manganese, cobalt and nickel. The polyamino acid or derivative thereof according to any one of claims 1 to 7, which comprises a D-amino acid residue. The polyamino acid or derivative thereof according to claim 8, wherein the D-amino acid residue is a D-histidine residue. A composition comprising the polyamino acid or derivative thereof according to claim 1 as an active ingredient. The composition according to claim 10, which is an antibacterial composition. The composition according to claim 10, which is a metal-binding composition. The composition according to claim 10, which is a composition for adsorbing an acidic substance and eliminating a basic substance. The composition according to claim 10, which is a composition for transporting a substance into a cell in a state of being chemically bound to an active ingredient or in a mixed form. A strain of the genus Epichloe that produces the polyamino acid according to any one of claims 1 to 9. 16. The microorganism according to claim 15, which is Epichloe kibiensis E18 strain (FERM P-18923) or a mutant thereof having a polyamino acid-producing ability. Culturing the microorganism according to claim 15 or 16, recovering the polyamino acid from the medium, optionally chemically modifying its free functional group, and/or forming a metal complex or salinizing. A method for producing the polyamino acid or the derivative thereof according to claim 1. The microorganism having the ability to produce the polyamino acid according to claim 8 or 9 is cultured in an appropriate medium, and the D-amino acid is recovered from the culture solution, the cells, or the disrupted cells separated from the medium, or if necessary. A method for producing a D-amino acid, which comprises recovering the D-amino acid after hydrolyzing the peptide derivative. 19. The method according to claim 18, wherein the microorganism is Epichloe kibiensis E-18 strain (FERM P-18923) or a mutant strain thereof having a D-amino acid biosynthetic ability. A D-amino acid produced by the method according to claim 18 or 19.

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