JP2004254647A - Method for producing l-penicillamine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing optically active L-penicillamine extremely important as a production intermediate for pharmaceuticals, agrochemicals and various industrial chemicals at a low cost. <P>SOLUTION: L-penicillamine is produced by treating DL-penicillanamide with microbial cells or treated cells capable of stereoselectively hydrolyzing L-penicillanamide. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００１０】

【００１１】

【００１２】
上記の菌学的性状から、２−１株はシュードモナス属と同定された。また、脂肪酸組成分析の結果、シュードモナス属のＲＮＡグループ１と同定された。さらに、１６ＳｒＤＮＡ遺伝子の部分塩基配列を解析した結果、シュードモナス アゼライカと１００％の相同率を示し、２−１株はシュードモナス アゼライカと同定された。
２−２株は、グラム染色陽性、カタラーゼ反応陽性、無芽胞、桿菌、ゼラチン加水分解試験項目で陽性、カゼインの加水分解性陽性、６％ＮａＣｌ存在下での生育性陽性を示したことから、ブレビバクテリウム属と同定された。
２−３株は、グラム染色陰性、オキシダーゼ反応陰性、ブドウ糖を発酵的に分解する桿菌など、腸内細菌様の性状を示し、リシンデカルボキシラーゼ、クエン酸の利用性、ウレアーゼ、アセトイン産生の試験項目で陽性、アルギニンジヒドロラーゼ、インドール産生の試験項目で陰性を示したことからクラブシエラ ニューモニアエと同定された。
【００１３】
本発明の微生物の培養は、通常資化し得る炭素源、窒素源、各微生物に必須の無機塩、栄養等を含有させた培地を用いて行われる。炭素源としては、ブドウ糖、乳糖、ショ糖、麦芽糖、デキストリン、でん粉、グリセリン、およびマンニトールなどが、また窒素源としては、肉エキス、酵母エキス、コーン・スティープ・リカー、ペプトン、尿素、およびアンモニアなどが用いられる。無機塩としては、ナトリウム、カリウム、カルシウム、およびマグネシウムなどを含む塩類、並びに鉄、マンガン、亜鉛、およびコバルトなどの金属塩類などが用いられる。その他に、アミノ酸、ペプチド、各種ビタミン類等も必要により用いられる。培養時のｐＨは、４〜１０の範囲であり、温度は２０〜５０℃である。培養は、１日〜１週間程度好気的に行われる。このようにして培養した微生物は、生菌体または該生菌体処理物、例えば培養液、分離菌体、菌体破砕物、さらには精製した酵素として反応に使用される。また、常法に従って菌体または酵素を固定化して使用することもできる。
【００１４】
ＤＬ−ペニシラミンアミドの生化学的不斉加水分解反応の条件は、ＤＬ−ペニシラミンアミド濃度０．０１〜２０ｗｔ％、ＤＬ−ペニシラミンアミドに対する微生物の使用量は、乾燥菌体として重量比０．０００１〜１０、反応温度１０〜６０℃、ｐＨ４〜１３の範囲である。
ＤＬ−ペニシラミンアミドの生化学的不斉加水分解反応で生成したＬ−ペニシラミンの、反応終了液からの単離は、例えば遠心分離あるいは濾過膜などの通常の固液分離手段により微生物菌体を除いた後、溶媒抽出やカラムクロマトグラフィー等の公知の方法を利用でき、特に制限はないが、イオン交換電気透析法や、イオン交換樹脂による吸脱着を利用した分離方法も有効である。
本発明の方法によって、ＤＬ−ペニシラアミドより、医薬品、農薬、および各種工業薬品の製造中間体として非常に重要な光学活性なＬ−ペニシラミンを安価に製造することができる。
【００１５】
【実施例】
本発明を実施例により更に具体的に説明するが、本発明はこれらの実施例に限定されるものではない。
実施例１
シュードモナス アゼライカ（Ｐｓｅｕｄｏｍｏｎａｓ ａｚｅｌａｉｃａ）２−１株（ＦＥＲＭ Ｐ−１９２１８）をＴＧＹ培地（トリプトン５ｇ、酵母エキス５ｇ、グルコース１ｇ、リン酸水素二カリウム１ｇ、蒸留水１Ｌ、ｐＨ７．０）２０ｍｌに接種し、２４時間、３０℃で振とう培養した。この培養液１．５ｍｌを次の組成を有する培地２５０ｍｌに接種し、２４時間、３０℃で振とう培養した。以下に、使用した培地組成、並びにビタミン混液および微量ミネラル混液の詳細を示す。
培地組成（ｐＨ７．０）
ソルビトール ５ｇ ＮＺアミン、タイプＡ ５ｇ
Ｋ_２ＨＰＯ_４ ２ｇ ＮａＣｌ １ｇ
ＭｎＣｌ_２・４Ｈ_２Ｏ ５０ｍｇ ビタミン混液 １０ｍｌ
微量ミネラル混液 １０ｍｌ 蒸留水 １Ｌ
ビタミン混液組成
塩酸チアミン ４ｍｇ リボフラビン ２ｍｇ
パントテン酸カルシウム ４ｍｇ ピリドキシン ４ｍｇ
ビオチン ２０μｇ ｐ−アミノ安息香酸 ２ｍｇ
ニコチン酸 ４ｍｇ 葉酸 ０．１ｍｇ
イノシトール ２０ｍｇ 蒸留水 １Ｌ
微量ミネラル混液組成
Ｔｉｔｒｐｌｅｘ ＩＶ ５００ｍｇ ＦｅＳＯ_４・７Ｈ_２Ｏ ２００ｍｇ
ＺｎＳＯ_４・７Ｈ_２Ｏ １０ｍｇ ＭｎＣｌ_２・４Ｈ_２Ｏ ３ｍｇ
Ｈ_３ＢＯ_４ ３０ｍｇ ＣｏＣｌ_２・６Ｈ_２Ｏ ２０ｍｇ
ＣｕＳＯ_４・２Ｈ_２Ｏ １ｍｇ ＮｉＣｌ_２・６Ｈ_２Ｏ ２ｍｇ
Ｎａ_２ＭｏＯ_４・２Ｈ_２Ｏ ３ｍｇ 蒸留水 １Ｌ
培養終了後、培養液１００ｇを８０００ｒｐｍで２０分間、遠心分離することにより、集菌し、５０ｍＭリン酸バッファー（ｐＨ８）を用いて調製した５０ｍＭのＤＬ−ペニシラミンアミド５ｍｌを加えて懸濁し、窒素気流下、３０℃で２４時間振とうした。遠心分離（１５０００ｒｐｍ、５分）後の上澄み液をＨＰＬＣにかけて生成したＬ−ペニシラミンを定量した結果、光学純度１００％のＬ−ペニシラミンが、ＤＬ−ペニシラミンアミド基準の収率４６％、Ｌ−ペニシラミンアミド基準の収率９２％で得られた。
【００１６】
実施例２
ブレビバクテリウム ｓｐ．（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ ｓｐ．）２−２株（ＦＥＲＭ Ｐ−１９２１９）を用いて、実施例１と同様に操作した結果、光学純度１００％のＬ−ペニシラミンが、ＤＬ−ペニシラミンアミド基準の収率４２％、Ｌ−ペニシラミンアミド基準の収率８４％で得られた。
【００１７】
実施例３
クレブシエラ ニューモニアエ（Ｋｌｅｂｓｉｅｌｌａ ｐｎｅｕｍｏｎｉａｅ）２−３株（自己寄託菌株）を用いて、実施例１と同様に操作した結果、光学純度１００％のＬ−ペニシラミンが、ＤＬ−ペニシラミンアミド基準の収率４１％、Ｌ−ペニシラミンアミド基準の収率８２％で得られた。
【００１８】
本発明の方法によれば、医薬品、農薬、および各種工業薬品の製造中間体として非常に重要な光学活性なＬ−ペニシラミンを反応が穏和かつ簡便で、経済的に有利な方法で製造することが可能となる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing L-penicillamine. More specifically, the present invention relates to a method for producing L-penicillamine by biochemically asymmetrically hydrolyzing DL-penicillamine amide.
L-penicillamine is an important substance as a production intermediate of pharmaceuticals, agricultural chemicals, and various industrial chemicals.
[0002]
[Prior art]
Conventionally, as a method for producing L-penicillamine, various methods have been known in which a racemic penicillamine derivative is optically resolved by a diastereomer salt method, and then L-penicillamine is derived. For example, a method of deriving racemic penicillamine into N-acetylpenicillamine and then dividing it using brucine or the like (for example, see Non-Patent Document 1), and a method of converting racemic penicillamine into 3-formyl-2,2 , 5,5-Tetramethyl-4-thiazolidinecarboxylic acid, followed by optical resolution (for example, see Non-Patent Document 2 and Patent Document 1). However, these optical resolutions by the diastereomer method are not economical because the resolving agent used is expensive.
On the other hand, a method of producing L-penicillamine by asymmetric hydrolysis of DL-penicillamine amide using an enzyme possessed by a microorganism has not yet been reported.
[0003]
[Non-patent document 1]
H. T. Clarke et al. , Eds, The Chemistry of Penicillin, Princeton Univ. Press, p466, 1949
[Non-patent document 2]
Biochem. Prepar. , 3, p116, 1953
[Patent Document 1]
Japanese Patent Publication No. 55-30711
[Problems to be solved by the invention]
An object of the present invention is to solve the above-mentioned problems in the prior art and to provide a method for inexpensively producing optically active L-penicillamine, which is very important as an intermediate for producing pharmaceuticals, agricultural chemicals, and various industrial chemicals. Is to do.
[0005]
[Means for Solving the Problems]
The present inventors have conducted intensive studies on a method for producing L-penicillamine at low cost, and as a result, have arrived at the present invention for producing L-penicillamine by asymmetric hydrolysis of DL-penicillamine amide biochemically.
[0006]
That is, the present invention is characterized in that L-penicillamine is produced by allowing DL-penicillamine amide to act on cells of a microorganism having an activity of stereoselectively hydrolyzing L-penicillamine amide or a treated product thereof. It relates to a method for producing L-penicillamine shown in (1) to (3).
(1) L-penicillamine is produced by reacting DL-penicillamine with a cell or a treated product of a microorganism having an activity of stereoselectively hydrolyzing L-penicillamine. A method for producing L-penicillamine.
(2) The method for producing L-penicillamine according to (1), wherein the microorganism having an activity of stereoselectively hydrolyzing L-penicillamine amide is a bacterium belonging to the genus Pseudomonas, Brevibacterium, and Klebsiella.
(3) A bacterium belonging to the genus Pseudomonas is Pseudomonas azelaica 2-1 (FERM P-19218), and a bacterium belonging to the genus Brevibacterium is Brevibacterium sp. (Brevobacterium sp.) 2-2 (FERM P-19219), and a bacterium belonging to the genus Klebsiella is Klebsiella pneumoniae 2-3 (self-deposited strain), any of (1) and (2). C. A method for producing L-penicillamine according to
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, details of the present invention will be described.
The microorganism used in the present invention may be any microorganism having an activity of stereoselectively hydrolyzing L-penicillamine amide, and is not particularly limited. Examples of such microorganisms include Pseudomonas, Brevibacterium, And bacteria belonging to the genus Klebsiella, specifically, Pseudomonas azelaica 2-1 strain, Brevibacterium sp. (Brevobacterium sp.) 2-2 strain and Klebsiella pneumoniae 2-3 strain, but are not limited thereto.
In addition, any of these strains, such as mutant strains derived from these microorganisms by artificial mutation means or recombinant strains derived by genetic engineering techniques such as cell fusion or gene recombination, has the above ability. If it has, it can be used in the present invention.
[0008]
Pseudomonas azelaica 2-1 strain, Brevibacterium sp. (Brevibacterium sp.) 2-2 strain and Klebsiella pneumoniae 2-3 strain are bacteria isolated from soil by the present inventors, and are registered with the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (AIST). , Pseudomonas azelaica 2-1 strain was deposited under the accession number FERM P-19218 (deposited on February 18, 2003) with Brevibacterium sp. (Brevobacterium sp.) 2-2 strain has been deposited under accession number FERM P-19219 (deposit date: February 18, 2003). Klebsiella pneumoniae 2-3 strain is stored as a self-deposited strain (February 18, 2003, the date of issue of the rejection certificate) because the bacterium belongs to the microorganism to be rejected at the center. (Hereinafter, each strain may be simply referred to as 2-1 strain, 2-2 strain, or 2-3 strain).
[0009]
Next, the mycological properties of the three strains of the present invention will be described (Tables 1, 2, and 3). In the table, + indicates that the relevant test item was positive,-indicates negative, and W indicates that the positive reaction was weak.

[0010]

[0011]

[0012]
From the above mycological properties, strain 2-1 was identified as Pseudomonas. In addition, as a result of fatty acid composition analysis, it was identified as RNA group 1 of the genus Pseudomonas. Furthermore, as a result of analyzing the partial nucleotide sequence of the 16S rDNA gene, the homology was 100% with Pseudomonas azelaica, and strain 2-1 was identified as Pseudomonas azelaica.
The 2-2 strain showed gram staining positive, catalase reaction positive, spore-free, bacillus, positive in gelatin hydrolysis test item, casein hydrolytic positive, growth positive in the presence of 6% NaCl, Brevibacterium was identified.
The 2-3 strain exhibits intestinal bacterial-like properties such as gram stain negative, oxidase negative, and bacterium that fermentatively degrades glucose, and test items for lysine decarboxylase, citric acid availability, urease, and acetoin production. Was positive for arginine dihydrolase and negative for indole production test items, and was identified as Crabsierra pneumoniae.
[0013]
The cultivation of the microorganism of the present invention is carried out using a medium containing a carbon source, a nitrogen source, an inorganic salt essential for each microorganism, nutrients, and the like, which can be usually utilized. Carbon sources include glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, and mannitol, and nitrogen sources include meat extract, yeast extract, corn steep liquor, peptone, urea, and ammonia. Is used. As the inorganic salt, salts including sodium, potassium, calcium, magnesium, and the like, and metal salts such as iron, manganese, zinc, and cobalt are used. In addition, amino acids, peptides, various vitamins, and the like are used as needed. The pH during the culturing is in the range of 4 to 10, and the temperature is 20 to 50C. Culture is performed aerobically for about one day to one week. The microorganism cultured in this manner is used in the reaction as a viable cell or a processed product of the viable cell, for example, a culture solution, a separated cell, a crushed cell, or a purified enzyme. In addition, cells or enzymes can be immobilized and used according to a conventional method.
[0014]
The conditions of the biochemical asymmetric hydrolysis reaction of DL-penicillamine amide are as follows: the concentration of DL-penicillamine amide is 0.01 to 20 wt%, and the amount of microorganism used for DL-penicillamine amide is 0.0001 to 0.001 wt. 10, the reaction temperature ranges from 10 to 60 ° C and the pH ranges from 4 to 13.
Isolation of L-penicillamine produced by the biochemical asymmetric hydrolysis reaction of DL-penicillamine amide from the reaction-terminated liquid is performed by removing microbial cells by ordinary solid-liquid separation means such as centrifugation or a filtration membrane. After that, known methods such as solvent extraction and column chromatography can be used, and there is no particular limitation. However, an ion exchange electrodialysis method and a separation method using adsorption and desorption with an ion exchange resin are also effective.
According to the method of the present invention, optically active L-penicillamine, which is very important as a production intermediate for pharmaceuticals, agricultural chemicals, and various industrial chemicals, can be produced at low cost from DL-penicillamide.
[0015]
【Example】
The present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Example 1
Pseudomonas azelaica strain 2-1 (FERM P-19218) was inoculated into 20 ml of TGY medium (5 g of tryptone, 5 g of yeast extract, 1 g of glucose, 1 g of dipotassium hydrogen phosphate, 1 L of distilled water, pH 7.0), The cells were cultured with shaking at 30 ° C. for 24 hours. 1.5 ml of this culture was inoculated into 250 ml of a medium having the following composition, and cultured with shaking at 30 ° C. for 24 hours. The details of the medium composition used and the vitamin mixture and the trace mineral mixture are shown below.
Medium composition (pH 7.0)
Sorbitol 5g NZ amine, type A 5g
K ₂ HPO ₄ 2 g NaCl 1 g
MnCl ₂ · _{4H 2} O 50mg vitamin mixture 10ml
Trace mineral mixture 10ml distilled water 1L
Vitamin mixture composition Thiamine hydrochloride 4mg Riboflavin 2mg
Calcium pantothenate 4mg Pyridoxine 4mg
Biotin 20 μg p-aminobenzoic acid 2 mg
Nicotinic acid 4mg Folic acid 0.1mg
Inositol 20mg distilled water 1L
Trace mineral mixture composition _{Titrplex IV 500mg FeSO 4 · 7H 2} O 200mg
_{ZnSO 4 · 7H 2 O 10mg MnCl} 2 · 4H 2 O 3mg
_{H 3 BO 4 30mg CoCl 2 ·} 6H 2 O 20mg
_{CuSO 4 · 2H 2 O 1mg NiCl} 2 · 6H 2 O 2mg
_{Na 2 MoO 4 · 2H 2 O} 3mg distilled water 1L
After completion of the culture, 100 g of the culture solution was centrifuged at 8000 rpm for 20 minutes to collect the bacteria, and suspended by adding 5 ml of 50 mM DL-penicillamine amide prepared using a 50 mM phosphate buffer (pH 8). The mixture was shaken at 30 ° C. for 24 hours. The supernatant obtained after centrifugation (15000 rpm, 5 minutes) was subjected to HPLC to determine the amount of L-penicillamine produced. Obtained with a standard yield of 92%.
[0016]
Example 2
Brevibacterium sp. (Brevibacterium sp.) 2-2 strain (FERM P-19219) was used and operated in the same manner as in Example 1. As a result, L-penicillamine having an optical purity of 100% was converted to a DL-penicillamine amide-based yield of 42%. The yield was 84% based on L-penicillamine amide.
[0017]
Example 3
Using Klebsiella pneumoniae 2-3 strain (self-deposited strain) in the same manner as in Example 1, L-penicillamine having an optical purity of 100% was converted into a 41% yield based on DL-penicillamine amide. The yield was 82% based on L-penicillamine amide.
[0018]
According to the method of the present invention, optically active L-penicillamine, which is very important as a production intermediate for pharmaceuticals, agricultural chemicals, and various industrial chemicals, can be produced by a mild and simple reaction, and an economically advantageous method. It becomes possible.

Claims (3)

L-penicillamine characterized in that L-penicillamine is produced by reacting DL-penicillamine with microbial cells or a processed product of a microorganism having an activity of stereoselectively hydrolyzing L-penicillamine. Manufacturing method. The method for producing L-penicillamine according to claim 1, wherein the microorganism having an activity of stereoselectively hydrolyzing L-penicillamine amide is a bacterium belonging to the genus Pseudomonas, the genus Brevibacterium, and the genus Klebsiella. A bacterium belonging to the genus Pseudomonas is Pseudomonas azelaica 2-1 (FERM P-19218), and a bacterium belonging to the genus Brevibacterium is Brevibacterium sp. (Brevacterium sp.) 2-2 strain (FERM P-19219), and the bacterium belonging to the genus Klebsiella is Klebsiella pneumoniae 2-3 strain (self-deposited strain). The method for producing L-penicillamine according to the above.