JP3330670B2 - Alkene monooxygenase, gene encoding the same, transformed microorganism and alkene epoxidation method - Google Patents

Alkene monooxygenase, gene encoding the same, transformed microorganism and alkene epoxidation method

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Publication number
JP3330670B2
JP3330670B2 JP10517193A JP10517193A JP3330670B2 JP 3330670 B2 JP3330670 B2 JP 3330670B2 JP 10517193 A JP10517193 A JP 10517193A JP 10517193 A JP10517193 A JP 10517193A JP 3330670 B2 JP3330670 B2 JP 3330670B2
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Japan
Prior art keywords
ala
leu
glu
val
arg
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JP10517193A
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Japanese (ja)
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JPH06292571A (en
Inventor
尚史 佐伯
敬三 古橋
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Eneos Corp
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Japan Energy Corp
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Priority to JP10517193A priority Critical patent/JP3330670B2/en
Publication of JPH06292571A publication Critical patent/JPH06292571A/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

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  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【０００１】[0001]

【産業上の利用分野】本発明は、アルケンのエポキシ化
に関与する酵素である新規なモノオキシゲナーゼ及びレ
ダクダーゼに関する。また、本発明は、これらの複合酵
素系である新規なアルケンモノオキシゲナーゼの遺伝
子、この遺伝子を用いて形質転換した微生物及びこの形
質転換微生物を用いてアルケンをエポキシ化する方法に
関する。The present invention relates to novel monooxygenases and reductases which are enzymes involved in alkene epoxidation. The present invention also relates to a novel alkene monooxygenase gene which is a complex enzyme system, a microorganism transformed with the gene, and a method for epoxidizing an alkene using the transformed microorganism.

【０００２】[0002]

【従来の技術】エポキシ化合物、特にその光学活性体
は、医薬や農薬さらには強誘電性液晶化合物等の機能性
化合物合成原料として有用なものである。この光学活性
なエポキシ化合物は、ノカルディア・コラリーナ（Noca
rdia corallina) 等のエポキシ化能を有している微生物
を用いて、各種アルケンを酸化することにより得ること
ができる。しかしながら、この微生物のエポキシ化合物
の生産性は未だ十分とは言えず、また自然からのスクリ
ーニングや突然変異処理等では生産性の向上に限界があ
り、遺伝子組変え技術を用いて新たな改良菌を創生する
ことが望まれていた。2. Description of the Related Art Epoxy compounds, especially optically active compounds thereof, are useful as raw materials for synthesizing functional compounds such as pharmaceuticals and agricultural chemicals and ferroelectric liquid crystal compounds. This optically active epoxy compound is available from Nocardia Coralina ( Noca
It can be obtained by oxidizing various alkenes using a microorganism having epoxidation ability such as rdia corallina ). However, the productivity of the epoxy compound of this microorganism is not yet sufficient, and there is a limit to the improvement of the productivity by natural screening or mutation treatment. It was desired to be created.

【０００３】[0003]

【発明が解決しようとする課題】本発明者は上記現状に
鑑み、遺伝子組換えに必要なエポキシ化酵素の遺伝子に
ついて鋭意探索した結果、アルケンをエポキシ化する酵
素は、アルケンに酸素を添加する酵素である２つのサブ
ユニットからなるモノオキシゲナーゼと補酵素NADHから
このモノオキシゲナーゼに電子を伝達する還元酵素であ
るレダクダーゼを含む複合酵素系からなること（この複
合酵素基をアルケンモノオキシゲナーゼという）及びこ
の酵素の遺伝子を見出し、さらにこの酵素のアミノ酸配
列を決定した。本発明はかかる知見に基づきなされたも
ので、本発明の目的は遺伝子組換え技術に有用な遺伝
子、さらにはこの遺伝子を用いて形質転換した微生物並
びにこの形質転換微生物を用いるアルケンのエポキシ化
方法、さらには新規なモノオキシゲナーゼ、レダクダー
ゼまたはこれらの複合酵素系である新規なアルケンモノ
オキシダーゼを提供することにある。In view of the above-mentioned situation, the present inventors have conducted intensive searches for genes of epoxidation enzymes necessary for gene recombination. As a result, the enzyme that epoxidizes alkene is an enzyme that adds oxygen to alkene. A complex enzyme system containing a monooxygenase consisting of two subunits and a reductase that transfers electrons from the coenzyme NADH to this monooxygenase (this complex enzyme group is called an alkene monooxygenase) and this enzyme And the amino acid sequence of this enzyme was determined. The present invention has been made based on such findings, and an object of the present invention is a gene useful for gene recombination technology, a microorganism transformed with the gene, and an alkene epoxidation method using the transformed microorganism. It is still another object of the present invention to provide a novel alkene monooxidase which is a novel monooxygenase, reductase or a complex enzyme thereof.

【０００４】[0004]

【課題を解決するための手段】本発明者等はノカルディ
ア・コラリーナから、アルケンのエポキシ化に関与して
いるアルケンモノオキシゲナーゼ（以下、AMO という)
遺伝子の全鎖長に相当するDNA を単離し、そのDNA を本
来エポキシ化能を有しない大腸菌に導入し、遺伝子が導
入された大腸菌がアルケンのエポキシ化能を付与された
ことを証明することによってこの遺伝子の機能を確認し
た。さらにこの遺伝子の塩基配列を決定し、本発明を完
成するに到った。Means for Solving the Problems The present inventors have obtained from Nocardia coralina an alkene monooxygenase (hereinafter referred to as AMO) involved in the epoxidation of alkenes.
By isolating the DNA corresponding to the entire length of the gene, introducing the DNA into E. coli that does not originally have epoxidation ability, and proving that the gene-introduced E. coli has conferred the alkene epoxidation ability. The function of this gene was confirmed. Furthermore, the nucleotide sequence of this gene was determined, and the present invention was completed.

【０００５】すなわち、本発明は、次のとおりである。 （１）次の式１及び式２のアミノ酸配列を含み、アルケ
ンに酸素を添加する能力を有するモノオキシゲナーゼ。That is, the present invention is as follows. (1) A monooxygenase comprising the amino acid sequences of the following formulas 1 and 2, and having the ability to add oxygen to alkenes.

【０００６】[0006]

【式１】 5 10 15 Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu Glu Gly His 20 25 30 Arg Thr Phe Thr Trp Phe Thr Pro Ala Arg Arg Lys Pro Thr Glu 35 40 45 Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro Asp Glu Trp 50 55 60 Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp Gly Arg Ala Pro 65 70 75 Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu Trp Ser Ala 80 85 90 Tyr Arg Asp Pro His Gln Leu Trp Gln Arg Pro Tyr Val Ser Thr 95 100 105 Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val Pro Val Leu 110 115 120 Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser Gln Lys Ile 125 130 135 Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu Tyr Gly Leu 140 145 150 Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met Ser Asp Thr 155 160 165 Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg Met Arg Leu 170 175 180 Leu Gln Asp Ile Val His His Leu Asp His Leu Gln Glu Ser Pro 185 190 195 Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met Ser Asp Ser 200 205 210 Thr Leu Val Pro Ile Arg Glu Val Ile Glu Arg Ile Ala Ala Ser 215 220 225 Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu Val Phe Glu 230 235 240 Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe Ser Arg Arg 245 250 255 Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val Leu Ala Ser 260 265 270 Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val Gln Ala Leu 275 280 285 Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp Gln Asn Gln 290 295 300 Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro Arg Cys Lys 305 310 315 Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp Cys Gly Ile 320 325 330 Asp Ala Lys Glu Ser Ala Asn Ala Leu Ser Arg Ala Leu Ala Asn 335 340 Gln Arg Ala Ala Val Glu Gly Ala Gly Ile Thr Ala [Equation 1] 5 10 15 Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu Glu Gly His 20 25 30 Arg Thr Phe Thr Trp Phe Thr Pro Ala Arg Arg Lys Pro Thr Glu 35 40 45 Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro Asp Glu Trp 50 55 60 Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp Gly Arg Ala Pro 65 70 75 Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu Trp Ser Ala 80 85 90 Tyr Arg Asp Pro His Gln Leu Trp Gln Arg Pro Tyr Val Ser Thr 95 100 105 Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val Pro Val Leu 110 115 120 Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser Gln Lys Ile 125 130 135 Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu Tyr Gly Leu 140 145 150 Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met Ser Asp Thr 155 160 165 Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg Met Arg Leu 170 175 180 Leu Gln Asp Ile Val His His Leu Asp His Leu Gln Glu Ser Pro 185 190 195 Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met Ser Asp Ser 200 205 210 Thr Leu Val Pro Ile Arg Glu Val Ile Glu Arg Ile Ala Ala Se r 215 220 225 Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu Val Phe Glu 230 235 240 Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe Ser Arg Arg 245 250 255 Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val Leu Ala Ser 260 265 270 Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val Gln Ala Leu 275 280 285 Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp Gln Asn Gln 290 295 300 Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro Arg Cys Lys 305 310 315 Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp Cys Gly Ile 320 325 330 Asp Ala Lys Glu Ser Ala Asn Ala Leu Ser Arg Ala Leu Ala Asn 335 340 Gln Arg Ala Ala Val Glu Gly Ala Gly Ile Thr Ala

【０００７】[0007]

【式２】 5 10 15 Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys Ser Tyr 20 25 30 Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe Glu Thr 35 40 45 Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg Val Leu 50 55 60 Ile Arg Asp Tyr Met Lys Met Glu Ala Glu Lys Asp Asp Arg Thr 65 70 75 His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala Thr Arg 80 85 90 Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val Pro Gln 95 100 105 Leu Thr Asn Ala Glu Tyr Gln Ala Val Ala Gly Cys Gly Met Ile 110 115 120 Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr Ala Ala 125 130 135 Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met Thr Leu 140 145 150 Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly Phe Asp 155 160 165 Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu Val Ser 170 175 180 Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu Asp Val 185 190 195 Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr Asn Ile 200 205 210 Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly Asp Asn 215 220 225 Ala Met Ala Ser Val Phe Leu Ser Ile Gln Ser Asp Glu Ala Arg 230 235 240 His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu Glu Asn 245 250 255 Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg His Phe 260 265 270 Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp Cys Ser 275 280 285 Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala Gln Trp 290 295 300 Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile Asp Arg 305 310 315 Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly Ala Ala 320 325 330 Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln Val Leu 335 340 345 Ser Ala Val Trp Pro Leu Asn Phe Trp Arg Ser Asp Ala Met Gly 350 355 360 Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly Trp Ser 365 370 375 Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu Ala Asp 380 385 390 Pro Ala Gly Gly Arg Ile Met Leu Gln Glu Leu Pro Gly Leu Pro 395 400 405 Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro Arg Leu 410 415 420 Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gln Lys Ile 425 430 435 Ala Leu Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn Trp Pro 440 445 450 Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr His Gly 455 460 465 Trp Asp Leu Ala Asp Val Ile Val Asp Leu Gly Tyr Ile Arg Pro 470 475 480 Asp Gly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met Glu Arg 485 490 495 Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Glu Val Lys 500 Asp Pro Leu Gln Glu Ala (Equation 2) 5 10 15 Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys Ser Tyr 20 25 30 Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe Glu Thr 35 40 45 Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg Val Leu 50 55 60 Ile Arg Asp Tyr Met Lys Met Glu Ala Glu Lys Asp Asp Arg Thr 65 70 75 His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala Thr Arg 80 85 90 Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val Pro Gln 95 100 105 Leu Thr Asn Ala Glu Tyr Gln Ala Val Ala Gly Cys Gly Met Ile 110 115 120 Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr Ala Ala 125 130 135 Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met Thr Leu 140 145 150 Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly Phe Asp 155 160 165 Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu Val Ser 170 175 180 Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu Asp Val 185 190 195 Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr Asn Ile 200 205 210 Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly Asp Asn 215 220 225 Ala Met Ala Ser Val Phe Leu Ser Ile Gln Ser Asp Glu Ala Arg 230 235 240 His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu Glu Asn 245 250 255 Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg His Phe 260 265 270 Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp Cys Ser 275 280 285 Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala Gln Trp 290 295 300 Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile Asp Arg 305 310 315 Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly Ala Ala 320 325 330 Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln Val Leu 335 340 345 Ser Ala Val Trp Pro Leu Asn Phe Trp Arg Ser Asp Ala Met Gly 350 355 360 Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly Trp Ser 365 370 375 Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu Ala Asp 380 385 390 Pro Ala Gly Gly Arg Ile Met Leu Gln Glu Leu Pro Gly Leu Pro 395 400 405 Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro Arg Leu 410 415 420 Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gl n Lys Ile 425 430 435 Ala Leu Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn Trp Pro 440 445 450 Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr His Gly 455 460 465 465 Trp Asp Leu Ala Asp Val Ile Val Asp Leu Gly Tyr Ile Arg Pro 470 475 480 Asp Gly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met Glu Arg 485 490 495 Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Glu Val Lys 500 Asp Pro Leu Gln Glu Ala

【０００８】（２）上記モノオキシゲナーゼのアミノ酸
配列をコードする塩基配列を含むアルケンモノオキシゲ
ナーゼ遺伝子。 （３）次の式３のアミノ酸配列を含み、NADH補酵素から
モノオキシゲナーゼに電子を伝達する能力を有するレダ
クダーゼ。[0008] (2) alkene monooxygenase gene comprising a nucleotide sequence encoding the amino acid sequence of the monooxygenase. (3) an amino acid sequence of the following formula 3, Redakudaze having the ability to transfer electrons to the monooxygenase from NADH coenzyme.

【０００９】[0009]

【式３】 5 10 15 Met Thr Thr Ile Asn Val Gln Pro Phe Ser His Glu Tyr Ser Cys 20 25 30 Glu Asp Gly Glu Ser Leu Leu Asp Gly Ala Leu Arg Asn Ser Leu 35 40 45 Leu Leu Lys Tyr Gly Cys Lys His Gly Gly Cys Gly Thr Cys Lys 50 55 60 Val Arg Leu Leu Asp Gly Asp Val Glu Glu Pro Gly Ser Ser Phe 65 70 75 Ala Leu Thr Pro Glu Asp Arg Glu Asn Asp Val Ile Leu Ala Cys 80 85 90 Ala Ser Val Pro Leu Glu Pro Cys Thr Ile Asp Val Glu Pro Ser 95 100 105 Gly Leu Thr Glu Glu Glu Phe Phe Ser Gly Asp Thr Ser Arg Glu 110 115 120 Phe Gln Thr Val Val Gly Gly Val Glu Phe Leu Thr Ala Asp Ile 125 130 135 Ala Arg Val Arg Leu Arg Leu Glu Pro Gly Glu Glu Ile Ala Phe 140 145 150 Thr Ala Gly Gln Phe Val Asn Val Glu Val Pro Gly Thr Gly Leu 155 160 165 Leu Arg Thr Phe Ser Leu Ala Asn Ala Pro Asp Asp Pro Ser Val 170 175 180 Val Glu Leu Ile Cys Lys Leu Tyr Pro Asp Gly Leu Phe Ser Arg 185 190 195 Phe Leu Arg Asp Glu Ala Ala Pro Gly Thr Pro Val Arg Val Phe 200 205 210 Gly Pro Tyr Gly Gln Leu Lys Ile Arg Leu Ser His Arg Pro Ile 215 220 225 Leu Met Ile Ala Gly Gly Ser Gly Leu Ala Pro Leu Leu Ser Met 230 235 240 Leu Arg Asp Leu Ala Ala Lys Lys Cys Asp Arg Pro Val Ser Met 245 250 255 Phe Phe Gly Ala Arg Ser Val Asp Asp Leu Tyr Leu Ile Glu Glu 260 265 270 Ile Arg Glu Ile Gly Glu Ser Leu Ala Asp Phe Glu Phe Ile Pro 275 280 285 Val Leu Ser Glu Ser Ser Pro Ala Asp Trp His Gly Glu Thr Gly 290 295 300 Met Val Thr Asp Ala Leu Leu Arg Trp Arg Ala Glu Leu Ala His 305 310 315 Asp Val Tyr Leu Cys Gly Pro Pro Pro Met Ile Asp Ala Ala Val 320 325 330 Pro Leu Leu Val Glu Arg Gly Val Arg Pro Arg Asn Ile Tyr Tyr 335 340 Asp Ala Phe Thr Pro Ala Ala Gln Val Val Val Val (Formula 3) 5 10 15 Met Thr Thr Ile Asn Val Gln Pro Phe Ser His Glu Tyr Ser Cys 20 25 30 Glu Asp Gly Glu Ser Leu Leu Asp Gly Ala Leu Arg Asn Ser Leu 35 40 45 Leu Leu Lys Tyr Gly Cys Lys His Gly Gly Cys Gly Thr Cys Lys 50 55 60 Val Arg Leu Leu Asp Gly Asp Val Glu Glu Pro Gly Ser Ser Phe 65 70 75 Ala Leu Thr Pro Glu Asp Arg Glu Asn Asp Val Ile Leu Ala Cys 80 85 90 Ala Ser Val Pro Leu Glu Pro Cys Thr Ile Asp Val Glu Pro Ser 95 100 105 Gly Leu Thr Glu Glu Glu Phe Phe Ser Gly Asp Thr Ser Arg Glu 110 115 120 Phe Gln Thr Val Val Gly Gly Val Glu Phe Leu Thr Ala Asp Ile 125 130 135 Ala Arg Val Arg Leu Arg Leu Glu Pro Gly Glu Glu Ile Ala Phe 140 145 150 Thr Ala Gly Gln Phe Val Asn Val Glu Val Pro Gly Thr Gly Leu 155 160 165 Leu Arg Thr Phe Ser Leu Ala Asn Ala Pro Asp Asp Pro Ser Val 170 175 180 Val Glu Leu Ile Cys Lys Leu Tyr Pro Asp Gly Leu Phe Ser Arg 185 190 195 Phe Leu Arg Asp Glu Ala Ala Pro Gly Thr Pro Val Arg Val Phe 200 205 210 Gly Pro Tyr Gly Gln Leu Lys Ile Arg Leu Ser His Arg Pro Ile 215 220 225 Leu Met Ile Ala Gly Gly Ser Gly Leu Ala Pro Leu Leu Ser Met 230 235 240 Leu Arg Asp Leu Ala Ala Lys Lys Cys Asp Arg Pro Val Ser Met 245 250 255 Phe Phe Gly Ala Arg Ser Val Asp Asp Leu Tyr Leu Ile Glu Glu 260 265 270 Ile Arg Glu Ile Gly Glu Ser Leu Ala Asp Phe Glu Phe Ile Pro 275 280 285 Val Leu Ser Glu Ser Ser Pro Ala Asp Trp His Gly Glu Thr Gly 290 295 300 Met Val Thr Asp Ala Leu Leu Arg Trp Arg Ala Glu Leu Ala His 305 310 315 Asp Val Tyr Leu Cys Gly Pro Pro Pro Met Ile Asp Ala Ala Val 320 325 330 Pro Leu Leu Val Glu Arg Gly Val Arg Pro Arg Asn Ile Tyr Tyr 335 340 Asp Ala Phe Thr Pro Ala Ala Gln Val Val Val Val

【００１０】（４）上記レダクダーゼのアミノ酸配列を
コードする塩基配列を含むアルケンモノオキシゲナーゼ
遺伝子。 （５）次の式４の塩基配列を含有するアルケンモノオキ
シゲナーゼ遺伝子。[0010] (4) alkene monooxygenase gene comprising a nucleotide sequence encoding the amino acid sequence of the Redakudaze. (5) alkene monooxygenase genes containing nucleotide sequence of the following formula 4.

【００１１】[0011]

【式４】 GGA TCC CCA GGT CCC GAG CCA CCG CGG CGA TCG ACT TCC CCG TCT 45 CAT GCA CGA TCC GCA CAG CCC CCT CAC GGA ACT CCC GGT CGT ACT 90 TTT CCG CTT CTC TGA CAT CCC TAC CCC TTA TAG CTG ATG CCT CCA 135 CGG TCT CGG GGA ATG CCA ATA TGA CTT GGT CAG ATC GTA GGC GGC 180 AAG AAC CAT GGC TTC CTT GGC GGA CTT CAC AGG GGC CTC TCC TCG 225 AGG GCT CGT TCG GGC ATG GGT AGC ACC GTC GAG CGA ACC CGA GGA 270 GAG GCC CCG ATC CGG GGA GGC GAT CGG GAT GAA CGG GGA CGT GTC 315 GAG AAT CGA TCA GAC TTG GTG GGC AGA TTC GTG TCC GCC GGT GGG 360 CAG AAC CCG ATG GCC ATC CAC GGG CAC TTT CGT GGC CAT CAG TGG 405 GCG GTT TCG TGA CCC CCT ATG GGC AGT TTT CCA TGG CCG CCG ACA 450 CAC AGG CCC GCA CCG ATG ACG AGG TAC TGC ACG ACC CGG CCG ACC 495 GCG CGG TGG TTT GCT GCA ACA AAT GAC AAC TTG ATC AAC GCA TCG 540 ATC ACG ACC GGC GCC ACT AAC GCC GCC GCG GCA AGC ATC GAC ATC 585 AAC ACC ATC GCG ACC GTC CGC CCT TCA CAT CGA CGA CAC CTC GGT 630 CGA TGT GTG CAT ACC GCA TGC ATC CGG CAA GAA CCC CGC GGA CTC 675 CGC AGC CGG TGC GGC TAG CCG ATT TCA CCC AAC CTG GGA CGT TGG 720 CTA CGG AAC GTC TCG TGG ATG ACG GCG CCT GCA TGC TGC ATA GAC 765 CCG TCC GTG GCC GCA ATG TCC TGG TGA GGC ATG CGA GCG GCT TCG 810 ATT CCT CTC GGC GGG GCC AAG ACC GGC GGG CCG CGC CTG TCT AAC 855 ATC GCG GCA CAA GGA AAT CGC AAC CGG ACC GAA ACG ATG GAA GGC 900 GTA GCG ATG ACG ACA GAG GCG ACG GTG GCC CGA CCG GTG GAG CTC 945 (amoA) Met Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu GAA GGT CAC CGG ACA TTC ACC TGG TTC ACG CCC GCC AGG CGA AAG 990 Glu Gly His Arg Thr Phe Thr Trp Phe Thr Pro Ala Arg Arg Lys CCG ACG GAG TAC GAG CTC TAC ACC GTG GGT CAA CAG TCC ACT CCG 1035 Pro Thr Glu Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro GAC GAG TGG CTG CAT GTG GAC TGG CCG CTG CGC TTC GAC GAC GGC 1080 Asp Glu Trp Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp Gly CGC GCC CCG TGG GAG GAG GAG TCG AGT GCG GTA CGG ACC TCG GAG 1125 Arg Ala Pro Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu TGG TCG GCT TAC CGC GAC CCA CAC CAA CTG TGG CAG CGT CCC TAC 1170 Trp Ser Ala Tyr Arg Asp Pro His Gln Leu Trp Gln Arg Pro Tyr GTC AGC ACG TGC AAC CAG GAC CAG CAG GCC CTC GCG CGG CTG GTC 1215 Val Ser Thr Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val CCC GTC CTG ACC ATG GGG TCG GCG GCG ATC ACG CCC ATC TGG TCG 1260 Pro Val Leu Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser CAG AAG ATC CTC GCC AGG TCC TAC GCC GCC TGG CCA TTC GTC GAG 1305 Gln Lys Ile Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu TAC GGG CTC TTC CTG AGC CTG GCC TAC GCC GTG CGC CAG GCC ATG 1350 Tyr Gly Leu Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met TCC GAC ACG GTC CAG TTC AGC GTG GTG TTC CAG GCC GTG GAC CGC 1395 Ser Asp Thr Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg ATG CGG CTG CTC CAG GAC ATC GTC CAC CAC CTG GAC CAC CTG CAG 1440 Met Arg Leu Leu Gln Asp Ile Val His His Leu Asp His Leu Gln GAG TCG CCG GAA TTC AGC GAC GCC GGG GCC CGC GAG GCC TGG ATG 1485 Glu Ser Pro Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met TCC GAC TCC ACC CTG GTC CCG ATC CGG GAA GTG ATC GAG CGC ATC 1530 Ser Asp Ser Thr Leu Val Pro Ile Arg Glu Val Ile Glu Arg Ile GCC GCC AGC CAG GAC TGG GTG GAG ATC CTG GTC GCC GGC ACG CTC 1575 Ala Ala Ser Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu GTC TTC GAG CCT CTG GTC GGC CAC CTG GCG AAG GCC GAG TTG TTC 1620 Val Phe Glu Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe AGC CGC CGT GCG CCA ATG TTC GGG GAC GGG ACC ACG CCG GCG GTG 1665 Ser Arg Arg Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val CTG GCG TCG GCC CTG CTG GAC AGC GGC AGG CAC CTC GAA TCG GTC 1710 Leu Ala Ser Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val CAG GCG CTC GTC CGC CTC GTC TGC CAA GAC CCC GTC CAT GGC GAC 1755 Gln Ala Leu Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp CAG AAC CAG GCG ACT GTG CGG CGG TGG ATC GAG GAA TGG CAG CCG 1800 Gln Asn Gln Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro CGG TGC AAG GCG GCG GCC CAG TCC TTC CTG CCG ACG TTC TCC GAC 1845 Arg Cys Lys Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp TGC GGC ATC GAC GCC AAG GAA AGC GCC AAC GCG CTG TCC CGG GCG 1890 Cys Gly Ile Asp Ala Lys Glu Ser Ala Asn Ala Leu Ser Arg Ala CTG GCG AAC CAG CGG GCC GCC GTC GAG GGC GCC GGC ATC ACG GCA 1935 Leu Ala Asn Gln Arg Ala Ala Val Glu Gly Ala Gly Ile Thr Ala (amoB) M TG ACA GAC GTT AAG GAG ACC ACT GTG ACC AGC ACC CCC TCG GCC 1979 * et Thr Asp Val Lys Glu Thr Thr Val Thr Ser Thr Pro Ser Ala GCC GTG CCG GGA ACC AAG AAC CGC CGG GTT GGT ATC TCG CTG ATC 2024 Ala Val Pro Gly Thr Lys Asn Arg Arg Val Gly Ile Ser Leu Ile AGC AGC AGC GAC ACC GAG GCA GCT GTC GAG CAC ATC GCG GAG ACC 2069 Ser Ser Ser Asp Thr Glu Ala Ala Val Glu His Ile Ala Glu Thr CAG CCG GAC GCG AAG ATC GAC TTT CGG GAC TGC TTC TAC AAG ATC 2114 Gln Pro Asp Ala Lys Ile Asp Phe Arg Asp Cys Phe Tyr Lys Ile GAG CGT GAC GGG CAG CTC AGT TTC GAC ATG GCA GAG CTC AGT GAG 2159 Glu Arg Asp Gly Gln Leu Ser Phe Asp Met Ala Glu Leu Ser Glu ATC GCC GGT CGC GAC ATC GAC ACC GAC ATC TTC CTG GTG AAC ATG 2204 Ile Ala Gly Arg Asp Ile Asp Thr Asp Ile Phe Leu Val Asn Met AGC ACC TAC TAC GGC CGG ATC GTC GTC AGT GAC GGC CGG GTC GAC 2249 Ser Thr Tyr Tyr Gly Arg Ile Val Val Ser Asp Gly Arg Val Asp ATC TAC GCC GAA ATC CAG CCG GCC CGC TTC AAG GAC TGA GAG GAA 2294 Ile Tyr Ala Glu Ile Gln Pro Ala Arg Phe Lys Asp * ACA CC ATG GCA TCG AAC CCC ACC CAG CTC CAC GAG AAG TCG AAG 2338 (amoC) Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys TCC TAC GAC TGG GAC TTC ACC TCC GTC GAG CGG CGC CCC AAG TTC 2383 Ser Tyr Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe GAG ACG AAG TAC AAG ATG CCC AAG AAG GGC AAG GAC CCG TTC CGC 2428 Glu Thr Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg GTC CTG ATC CGT GAC TAC ATG AAG ATG GAA GCG GAG AAG GAC GAC 2473 Val Leu Ile Arg Asp Tyr Met Lys Met Glu Ala Glu Lys Asp Asp CGG ACC CAT GGC TTC CTC GAC GGC GCC GTG CGG ACG CGT GAG GCC 2518 Arg Thr His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala ACC AGG ATT GAG CCG CGG TTC GCT GAG GCC ATG AAG ATC ATG GTG 2563 Thr Arg Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val CCG CAG CTG ACC AAC GCC GAG TAC CAG GCG GTG GCG GGC TGC GGA 2608 Pro Gln Leu Thr Asn Ala Glu Tyr Gln Ala Val Ala Gly Cys Gly ATG ATC ATC TCG GCC GTC GAG AAC CAG GAG CTC CGT CAG GGC TAC 2653 Met Ile Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr GCC GCT CAG ATG CTC GAT GAG GTG CGG CAC GCG CAG CTC GAG ATG 2698 Ala Ala Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met ACG CTA CGC AAC TAC TAC GCG AAG CAC TGG TGC GAT CCC TCC GGC 2743 Thr Leu Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly TTC GAC ATC GGT CAG CGC GGC CTG TAC CAG CAC CCC GCG GGG CTG 2788 Phe Asp Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu GTG TCC ATC GGC GAG TTC CAG CAC TTC AAT ACT GGT GAC CCG CTT 2833 Val Ser Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu GAC GTC ATC ATC GAT CTC AAC ATC GTG GCC GAG ACG GCG TTC ACG 2878 Asp Val Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr AAC ATC CTG CTG GTG GCC ACT CCA CAG GTC GCC GTG GCC AAC GGG 2923 Asn Ile Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly GAC AAC GCG ATG GCC AGC GTG TTC CTC TCG ATC CAG TCG GAC GAG 2968 Asp Asn Ala Met Ala Ser Val Phe Leu Ser Ile Gln Ser Asp Glu GCC AGG CAC ATG GCC AAC GGG TAC GGC TCG GTC ATG GCG CTG CTG 3013 Ala Arg His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu GAG AAC GAG GAC AAC CTC CCG CTG CTC AAC CAG TCT CTC GAT CGG 3058 Glu Asn Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg CAC TTC TGG CGT GCC CAC AAG GCC TTG GAC AAC GCG GTC GGA TGG 3103 His Phe Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp TGT TCG GAG TAT GGC GCC CGC AAG CGG CCA TGG AGC TAC AAG GCC 3148 Cys Ser Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala CAG TGG GAG GAA TGG GTC GTC GAC GAC TTC GTG GGC GGC TAC ATC 3193 Gln Trp Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile GAC CGA CTC AGC GAG TTC GGC GTT CAG GCT CCG GCC TGC CTT GGC 3238 Asp Arg Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly GCG GCC GCC GAC GAG GTC AAG TGG TCG CAC CAC ACG CTC GGT CAG 3283 Ala Ala Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln GTG CTG TCG GCG GTG TGG CCG CTG AAC TTC TGG CGC TCG GAC GCC 3328 Val Leu Ser Ala Val Trp Pro Leu Asn Phe Trp Arg Ser Asp Ala ATG GGA CCG GCG GAC TTC GAG TGG TTC GAG AAC CAC TAC CCG GGC 3373 Met Gly Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly TGG AGC GCG GCC TAC CAG GGT TAC TGG GAG GGC TAC AAG GCG CTC 3418 Trp Ser Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu GCC GAC CCA GCA GGC GGA CGC ATC ATG CTC CAG GAG CTG CCG GGT 3463 Ala Asp Pro Ala Gly Gly Arg Ile Met Leu Gln Glu Leu Pro Gly CTG CCG CCG ATG TGT CAG GTG TGC CAG GTG CCG TGC GTG ATG CCG 3508 Leu Pro Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro CGG CTG GAT ATG AAC GCC GCG CGG ATC ATC GAG TTC GAG GGG CAG 3553 Arg Leu Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gln AAA ATC GCG CTG TGC AGC GAA CCC TGC CAG CGG ATC TTC ACC AAC 3598 Lys Ile Ala Leu Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn TGG CCG GAG GCG TAC CGC CAC CGC AAG CAA TAC TGG GCC CGC TAC 3643 Trp Pro Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr CAC GGA TGG GAC CTG GCG GAC GTC ATC GTT GAT CTC GGC TAC ATC 3688 His Gly Trp Asp Leu Ala Asp Val Ile Val Asp Leu Gly Tyr Ile CGC CCG GAC GGC AAG ACC CTC ATC GGC CAG CCG CTG CTC GAG ATG 3733 Arg Pro Asp Gly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met GAG CGG CTG TGG ACC ATC GAC GAC ATC CGG GCC CTT CAG TAC GAA 3778 Glu Arg Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Glu GTC AAG GAC CCG TTG CAG GAG GCG TG ATG ACG ACG ATC AAT GTG 3822 Val Lys Asp Pro Leu Gln Glu Ala * * (amoD) Met Thr Thr Ile Asn Val CAG CCC TTC TCA CAC GAG TAC TCG TGC GAG GAC GGC GAG AGC CTC 3867 Gln Pro Phe Ser His Glu Tyr Ser Cys Glu Asp Gly Glu Ser Leu CTC GAC GGC GCC CTG CGC AAC AGC CTG CTC CTG AAG TAC GGG TGC 3912 Leu Asp Gly Ala Leu Arg Asn Ser Leu Leu Leu Lys Tyr Gly Cys AAG CAC GGG GGC TGC GGG ACC TGC AAG GTC CGG CTG CTC GAC GGC 3957 Lys His Gly Gly Cys Gly Thr Cys Lys Val Arg Leu Leu Asp Gly GAC GTA GAG GAA CCC GGG TCG TCG TTC GCG CTG ACG CCG GAG GAC 4002 Asp Val Glu Glu Pro Gly Ser Ser Phe Ala Leu Thr Pro Glu Asp CGC GAG AAC GAC GTG ATC CTC GCG TGC GCC AGC GTG CCG CTG GAA 4047 Arg Glu Asn Asp Val Ile Leu Ala Cys Ala Ser Val Pro Leu Glu CCG TGC ACC ATC GAC GTC GAG CCG AGC GGC CTC ACG GAG GAG GAG 4092 Pro Cys Thr Ile Asp Val Glu Pro Ser Gly Leu Thr Glu Glu Glu TTC TTC TCG GGC GAC ACC TCG CGC GAG TTC CAG ACG GTC GTG GGC 4137 Phe Phe Ser Gly Asp Thr Ser Arg Glu Phe Gln Thr Val Val Gly GGT GTC GAG TTT CTC ACG GCG GAC ATC GCC CGG GTC CGG CTC CGG 4182 Gly Val Glu Phe Leu Thr Ala Asp Ile Ala Arg Val Arg Leu Arg CTA GAG CCG GGC GAG GAG ATC GCC TTC ACC GCC GGT CAG TTC GTC 4227 Leu Glu Pro Gly Glu Glu Ile Ala Phe Thr Ala Gly Gln Phe Val AAC GTC GAG GTG CCG GGC ACG GGT CTG CTG CGG ACC TTC TCG CTG 4272 Asn Val Glu Val Pro Gly Thr Gly Leu Leu Arg Thr Phe Ser Leu GCA AAC GCC CCT GAC GAC CCG TCA GTG GTG GAG CTG ATC TGC AAG 4317 Ala Asn Ala Pro Asp Asp Pro Ser Val Val Glu Leu Ile Cys Lys CTC TAC CCG GAT GGC CTC TTC TCC CGC TTC CTG AGG GAC GAG GCT 4362 Leu Tyr Pro Asp Gly Leu Phe Ser Arg Phe Leu Arg Asp Glu Ala GCC CCG GGC ACG CCG GTC CGG GTG TTC GGG CCG TAT GGT CAG CTC 4407 Ala Pro Gly Thr Pro Val Arg Val Phe Gly Pro Tyr Gly Gln Leu AAG ATC CGC TTG TCC CAC CGG CCG ATC CTG ATG ATC GCC GGT GGG 4452 Lys Ile Arg Leu Ser His Arg Pro Ile Leu Met Ile Ala Gly Gly TCC GGT CTC GCC CCG CTG CTC TCG ATG CTG CGA GAC TTG GCC GCC 4497 Ser Gly Leu Ala Pro Leu Leu Ser Met Leu Arg Asp Leu Ala Ala AAG AAG TGC GAC CGG CCG GTC TCG ATG TTC TTC GGC GCA CGC AGC 4542 Lys Lys Cys Asp Arg Pro Val Ser Met Phe Phe Gly Ala Arg Ser GTC GAC GAC CTG TAC CTC ATC GAG GAG ATC CGC GAG ATC GGC GAG 4587 Val Asp Asp Leu Tyr Leu Ile Glu Glu Ile Arg Glu Ile Gly Glu TCG CTA GCC GAT TTC GAG TTC ATC CCC GTG CTC TCG GAG TCG TCG 4632 Ser Leu Ala Asp Phe Glu Phe Ile Pro Val Leu Ser Glu Ser Ser CCA GCC GAC TGG CAC GGC GAG ACG GGC ATG GTC ACC GAC GCC TTG 4677 Pro Ala Asp Trp His Gly Glu Thr Gly Met Val Thr Asp Ala Leu CTG CGG TGG CGC GCC GAA CTG GCG CAT GAC GTC TAC CTG TGC GGG 4722 Leu Arg Trp Arg Ala Glu Leu Ala His Asp Val Tyr Leu Cys Gly CCG CCA CCC ATG ATC GAC GCC GCT GTG CCG CTG CTC GTC GAG CGG 4767 Pro Pro Pro Met Ile Asp Ala Ala Val Pro Leu Leu Val Glu Arg GGC GTG CGC CCA CGC AAC ATC TAC TAC GAC GCA TTC ACC CCA GCT 4812 Gly Val Arg Pro Arg Asn Ile Tyr Tyr Asp Ala Phe Thr Pro Ala GCT CAG GTA GTC GTC GTC TGA TGG TGC ATA TCC GAT TGG GCG GCC 4857 Ala Gln Val Val Val Val * GGT ACC GGC GGG GTT AGG GCA GGG TAA TCG GCC GCG ATA GAG GCA 4902 GCC AGC AGA GAT GAT GCG CGT GCA AAG CGA AGC TTC GAC TCA GTT 4947 GAC ACC TTC GGC AAT GTG CTG TGC GAG CAC GAT AAC GGT GTG TTA 4992 CGG GTG AGG CTC AAT CGC ACC GGT GCC CTG AGC GCA ATC AAT TCA 5037 GCT ATG GCT GAT CAA TTG CGG GAA GCG TGG GTG TGG GTG CGC AGC 5082 CAA ACC GGA ATT CAT TCA ATC GTC ATC TCG GCT TCG CGG CCG GAG 5127 TCG TTT TTG CAT CGG TTT TGA TCC GGC CGA CCC GCC GGA GCC GAT 5172 GTT CGA TCG CCC CAT CAG CCC GAA GGA ATG CGG CGT CGA TCA ACG 5217 CGT CAT AGT CGC GGT GAA CGG CAT CGC GTG CCG CAT CCT GAG CGC 5262 CGG CGA ACC GAC ACT GCG TAA GCC GGT GAC GGC GGA ACA GGC GAT 5307 GGA GTC GGG CTT GGT TCG GGA GGT CGT ACC GCT TGC ATC GGC TGC 5352 GTT GTC GAG CGA ATA GCT CAG GCG GCA GGA CAT CGT ATA GGG ATT 5397 CAG GTG GGT AGT TGA TCT TCC GCG TGT CGA TGT GTG ACG GGC CGG 5442 ATC TGG CCT ACG AAC AAC ATC AGC AAG CGT GAC CTT CGG CCC ACA 5487 ACA TCG TGC CGT CCG CGA CCG GTG CCG ACC GTG ATC TCG GCG AAG 5532 TCC TGC CGG AGT TCG CGG GGC GGT TCC ACA GCG TTG GGG GTC CCT 5577 CGG CTA CAC CTC GGC GCT GTT CGT CAG CTC CGA CAT CAT CGG TGA 5622 CCC ACA CTC CTG CGC GGT GTT CGG CAA CAT GGT GCA CGA CGG CCA 5667 GGT GAA CCT TCG GCT GGT ACG ACA ACG AAT GGG CGA TGC CAA TCG 5712 GTT GCT CGA CCT CGT CGG GGT CAT GGC TGG CGT TCG CTG ACT AGG 5757 CGG CCT GTT CGT GCC GGC CGC AAC ATC CAG CCC GTG AGG AGT TGC 5802 GTA CCG GTC GGC GAG AAA CTC CAC GAC CCG GGC AAC CTC GTC GGG 5847 CTC ACC GAG TCG GCC GAC TGG GAT CTT CGC GAT GAC CCG ATC GAG 5892 CGC CTC CTT GGG CAC CGC AGC AAC TAT CTC GGT GGA GAT GTA GCC 5937 AGG CGG AAC GGA ATT GAC GGT GAT GCC CTT GCG GGC GGT CTC CTG 5982 GGC CAG GGT CTG TGT CCT TAT CTC TTG TCG ACC GAA CAC GGT GTG 6027 CGG TCG AGT TGG TCG GTT TGA TCA GGC GGG AAC GCG GAA TAG GAG 6072 GGC TTC GCC CTG ACC GCC GCC GCC GCA CAG GCC GGC CGC CCC GAC 6117 GCC GCC GCC GCG GCG GGC CAG CTC GAG CAC GAG ATG CAG CGC GAT 6162 CCG GGC GCC GGA GGC GCC GAC CGG GTG CCC CAT GGC GAT CGC GCC 6207 GCC GTT GAC GTT GAC GTT GTC GGG GCC GAT CTG CAG GTC GCG CAT 6252 GGA TTG AAT AGC GAC GGC CGC GAA TGC CTC GTT GAT CTC GTA CAG 6297 GTC CAC ATC CAT GGC GGA GAG TCC TTC GCG GCG CAG CGC CCT GGC 6342 GAT CGC GTT GGA GGG TTG CGA CAG CAG CGA GGG ATC C 6379 〔式中 (amoA) 、 (amoB) 、 (amoC) 、 (amoD) は、DN
A の塩基配列に対応するアミノ酸配列を示し、また＊は
翻訳の終止コドンを示す〕[Equation 4] GGA TCC CCA GGT CCC GAG CCA CCG CGG CGA TCG ACT TCC CCG TCT 45 CAT GCA CGA TCC GCA CAG CCC CCT CAC GGA ACT CCC GGT CGT ACT 90 TTT CCG CTT CTC TGA CAT CCC TAC CCC TTA TAG CTG ATG CCT CCA 135 CGG TCT CGG GGA ATG CCA ATA TGA CTT GGT CAG ATC GTA GGC GGC 180 AAG AAC CAT GGC TTC CTT GGC GGA CTT CAC AGG GGC CTC TCC TCG 225 AGG GCT CGT TCG GGC ATG GGT AGC ACC GTC GAG CGA ACC CGA GGA GAG GCC CCG ATC CGG GGA GGC GAT CGG GAT GAA CGG GGA CGT GTC 315 GAG AAT CGA TCA GAC TTG GTG GGC AGA TTC GTG TCC GCC GGT GGG 360 CAG AAC CCG ATG GCC ATC CAC GGG CAC TTT CGT GGC CAT CAG TGG 405 GCG TCG TGA CCC CCT ATG GGC AGT TTT CCA TGG CCG CCG ACA 450 CAC AGG CCC GCA CCG ATG ACG AGG TAC TGC ACG ACC CGG CCG ACC 495 GCG CGG TGG TTT GCT GCA ACA AAT GAC AAC TTG ATC AAC GCA TCG 540 ATC ACG ACC GCC GCC ACT AAC GCC GCC GCG GCA AGC ATC GAC ATC 585 AAC ACC ATC GCG ACC GTC CGC CCT TCA CAT CGA CGA CAC CTC GGT 630 CGA TGT GTG CAT ACC GCA TGC ATC CGG CAA GAA CCC CGC GGA CTC 675 CGC AGC CGG TGC GGC CCG A TT TCA CCC AAC CTG GGA CGT TGG 720 CTA CGG AAC GTC TCG TGG ATG ACG GCG CCT GCA TGC TGC ATA GAC 765 CCG TCC GTG GCC GCA ATG TCC TGG TGA GGC ATG CGA GCG GCT TCG 810 ATT CCT CTC GGC GGG GGC AAGACC GGG CCG CGC CTG TCT AAC 855 ATC GCG GCA CAA GGA AAT CGC AAC CGG ACC GAA ACG ATG GAA GGC 900 GTA GCG ATG ACG ACA GAG GCG ACG GTG GCC CGA CCG GTG GAG CTC 945 (amoA) Met Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu GAA GGT CAC CGG ACA TTC ACC TGG TTC ACG CCC GCC AGG CGA AAG 990 Glu Gly His Arg Thr Phe Thr Trp Phe Thr Pro Ala Arg Arg Lys CCG ACG GAG TAC GAG CTC TAC ACC GTG GGT CAA CAG TCC ACT CCG 1035 Pro Thr Glu Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro GAC GAG TGG CTG CAT GTG GAC TGG CCG CTG CGC TTC GAC GAC GGC 1080 Asp Glu Trp Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp Gly CGC GCC CCG TGG GAG GAG GAG TCG AGT GCG GTA CGG ACC TCG GAG 1125 Arg Ala Pro Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu TGG TCG GCT TAC CGC GAC CCA CAC CAA CTG TGG CAG CGT CCC TAC 1170 Trp Ser Ala Tyr Arg As p Pro His Gln Leu Trp Gln Arg Pro Tyr GTC AGC ACG TGC AAC CAG GAC CAG CAG GCC CTC GCG CGG CTG GTC 1215 Val Ser Thr Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val CCC GTC CTG ACC ATG GGG TCG GCG GCG ATC ACG CCC ATC TGG TCG 1260 Pro Val Leu Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser CAG AAG ATC CTC GCC AGG TCC TAC GCC GCC TGG CCA TTC GTC GAG 1305 Gln Lys Ile Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu TAC GGG CTC TTC CTG AGC CTG GCC TAC GCC GTG CGC CAG GCC ATG 1350 Tyr Gly Leu Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met TCC GAC ACG GTC CAG TTC AGC GTG GTG TTC CAG GCC GTG GAC C395 Ser Asp Thr Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg ATG CGG CTG CTC CAG GAC ATC GTC CAC CAC CTG GAC CAC CTG CAG 1440 Met Arg Leu Leu Leu Gln Asp Ile Val His His Leu Asp His Leu Gln GAG TCG CCG GAA TTC AGC GAC GCC GGG GCC CGC GAG GCC TGG ATG 1485 Glu Ser Pro Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met TCC GAC TCC ACC CTG GTC CCG ATC CGG GAA GTG ATC GAG CGC ATC 1530 Ser Asp Ser Thr Leu Va l Pro Ile Arg Glu Val Ile Glu Arg Ile GCC GCC AGC CAG GAC TGG GTG GAG ATC CTG GTC GCC GGC ACG CTC 1575 Ala Ala Ser Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu GTC TTC GAG CCT CTG GTC GGC CAC CTG GCG AAG GCC GAG TTG TTC 1620 Val Phe Glu Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe AGC CGC CGT GCG CCA ATG TTC GGG GAC GGG ACC ACG CCG GCG GTG 1665 Ser Arg Arg Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val CTG GCG TCG GCC CTG CTG GAC AGC GGC AGG CAC CTC GAA TCG GTC 1710 Leu Ala Ser Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val CAG GCG CTC GTC CGC CTC GTC TGC CAA GAC CCC GTC CAT GGC GAC 1755 Gln Ala Leu Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp CAG AAC CAG GCG ACT GTG CGG CGG TGG ATC GAG GAA TGG CAG CCG 1800 Gln Asn Gln Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro CGG TGC AAG GCG GCG GCC CAG TCC TTC CTG CCG ACG TTC TCC GAC 1845 Arg Cys Lys Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp TGC GGC ATC GAC GCC AAG GAA AGC GCC AAC GCG CTG TCC CGG GCG 1890 Cys Gly Ile Asp Ala Ly s Glu Ser Ala Asn Ala Leu Ser Arg Ala CTG GCG AAC CAG CGG GCC GCC GTC GAG GGC GCC GGC ATC ACG GCA 1935 Leu Ala Asn Gln Arg Ala Ala Val Glu Gly Ala Gly Ile Thr Ala (amoB) M TG ACA GAC GTT AAG GAG ACC ACT GTG ACC AGC ACC CCC TCG GCC 1979 * et Thr Asp Val Lys Glu Thr Thr Val Thr Ser Thr Pro Ser Ala GCC GTG CCG GGA ACC AAG AAC CGC CGG GTT GGT ATC TCG CTG ATC 2024 Ala Val Pro Gly Thr Lys Asn Arg Arg Val Gly Ile Ser Leu Ile AGC AGC AGC GAC ACC GAG GCA GCT GTC GAG CAC ATC GCG GAG ACC 2069 Ser Ser Ser Asp Thr Glu Ala Ala Val Glu His Ile Ala Glu Thr CAG CCG GAC GCG AAG ATC GAC TTT CGG GAC TGC TTC TAC AAG ATC 2114 Gln Pro Asp Ala Lys Ile Asp Phe Arg Asp Cys Phe Tyr Lys Ile GAG CGT GAC GGG CAG CTC AGT TTC GAC ATG GCA GAG CTC AGT GAG 2159 Glu Arg Asp Gly Gln Leu Ser Phe Asp Met Ala Glu Leu Ser Glu ATC GCC GGT CGC GAC ATC GAC ACC GAC ATC TTC CTG GTG AAC ATG 2204 Ile Ala Gly Arg Asp Ile Asp Thr Asp Ile Phe Leu Val Asn Met AGC ACC TAC TAC GGC CGG ATC GTC GTC AGT GAC GGC CGG GTC GAC 2249 Ser Thr Tyr T yr Gly Arg Ile Val Val Ser Asp Gly Arg Val Asp ATC TAC GCC GAA ATC CAG CCG GCC CGC TTC AAG GAC TGA GAG GAA 2294 Ile Tyr Ala Glu Ile Gln Pro Ala Arg Phe Lys Asp * ACA CC ATG GCA TCG AAC CCC ACC CAG CTC CAC GAG AAG TCG AAG 2338 (amoC) Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys TCC TAC GAC TGG GAC TTC ACC TCC GTC GAG CGG CGC CCC AAG TTC 2383 Ser Tyr Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe GAG ACG AAG TAC AAG ATG CCC AAG AAG GGC AAG GAC CCG TTC CGC 2428 Glu Thr Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg GTC CTG ATC CGT GAC TAC ATG AAG ATG GAA GCG GAG AAG GAC GAC 2473 Val Leu Ile Arg Asp Tyr Met Lys Met Glu Ala Glu Lys Asp Asp CGG ACC CAT GGC TTC CTC GAC GGC GCC GTG CGG ACG CGT GAG GCC 2518 Arg Thr His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala ACC AGG ATT GAG CCG CGG TTC GCT GAG GCC ATG AAG ATC ATG GTG 2563 Thr Arg Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val CCG CAG CTG ACC AAC GCC GAG TAC CAG GCG GTG GCG GGC TGC GGA 2608 Pro Gln Leu Thr Asn Ala G lu Tyr Gln Ala Val Ala Gly Cys Gly ATG ATC ATC TCG GCC GTC GAG AAC CAG GAG CTC CGT CAG GGC TAC 2653 Met Ile Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr GCC GCT CAG ATG CTC GAT GAG GTG CGG CAC GCG CAG CTC GAG ATG 2698 Ala Ala Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met ACG CTA CGC AAC TAC TAC GCG AAG CAC TGG TGC GAT CCC TCC GGC 2743 Thr Leu Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly TTC GAC ATC GGT CAG CGC GGC CTG TAC CAG CAC CCC GCG GGG CTG 2788 Phe Asp Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu GTG TCC ATC GGC GAG TTC CAG CAC TTC AAT ACT GGT GAC CCG CTT 2833 Val Ser Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu GAC GTC ATC ATC GAT CTC AAC ATC GTG GCC GAG ACG GCG TTC ACG 2878 Asp Val Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr AAC ATC CTG CTG GTG GCC ACT CCA CAG GTC GCC GTG GCC AAC GGG 2923 Asn Ile Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly GAC AAC GCG ATG GCC AGC GTG TTC CTC TCG ATC CAG TCG GAC GAG 2968 Asp Asn Ala Met Ala Ser V al Phe Leu Ser Ile Gln Ser Asp Glu GCC AGG CAC ATG GCC AAC GGG TAC GGC TCG GTC ATG GCG CTG CTG 3013 Ala Arg His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu GAG AAC GAG GAC AAC CTC CCG CTG CTC AAC CAG TCT CTC GAT CGG 3058 Glu Asn Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg CAC TTC TGG CGT GCC CAC AAG GCC TTG GAC AAC GCG GTC GGA TGG 3103 His Phe Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp TGT TCG GAG TAT GGC GCC CGC AAG CGG CCA TGG AGC TAC AAG GCC 3148 Cys Ser Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala CAG TGG GAG GAA TGG GTC GTC GAC GAC TTC GTG GGC GGC TAC ATC 3193 Gln Trp Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile GAC CGA CTC AGC GAG TTC GGC GTT CAG GCT CCG GCC TGC CTT GGC 3238 Asp Arg Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly GCG GCC GCC GAC GAG GTC AAG TGG TCG CAC CAC ACG CTC GGT CAG 3283 Ala Ala Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln GTG CTG TCG GCG GTG TGG CCG CTG AAC TTC TGG CGC TCG GAC GCC 3328 Val Leu Ser Ala Val Trp P ro Leu Asn Phe Trp Arg Ser Asp Ala ATG GGA CCG GCG GAC TTC GAG TGG TTC GAG AAC CAC TAC CCG GGC 3373 Met Gly Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly TGG AGC GCG GCC TAC CAG GGT TAC TGG GAG GGC TAC AAG GCG CTC 3418 Trp Ser Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu GCC GAC CCA GCA GGC GGA CGC ATC ATG CTC CAG GAG CTG CCG GGT 3463 Ala Asp Pro Ala Gly Gly Arg Ile Met Leu Gln Le Pro Gly CTG CCG CCG ATG TGT CAG GTG TGC CAG GTG CCG TGC GTG ATG CCG 3508 Leu Pro Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro CGG CTG GAT ATG AAC GCC GCG CGG ATC ATC GAG TTC GAG GGG CAG 3553 Arg Leu Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gln AAA ATC GCG CTG TGC AGC GAA CCC TGC CAG CGG ATC TTC ACC AAC 3598 Lys Ile Ala Leu Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn TGG CCG GAG GCG TAC CGC CAC CGC AAG CAA TAC TGG GCC CGC TAC 3643 Trp Pro Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr CAC GGA TGG GAC CTG GCG GAC GTC ATC GTT GAT CTC GGC TAC ATC 3688 His Gly Trp Asp Leu Ala A sp Val Ile Val Asp Leu Gly Tyr Ile CGC CCG GAC GGC AAG ACC CTC ATC GGC CAG CCG CTG CTC GAG ATG 3733 Arg Pro Asp Gly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met GAG CGG CTG TGG ACC ATC GAC GAC ATC CGG GCC CTT CAG TAC GAA 3778 Glu Arg Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Glu GTC AAG GAC CCG TTG CAG GAG GCG TG ATG ACG ACG ATC AAT GTG 3822 Val Lys Asp Pro Leu Gln Glu Ala * * (amoD) Met Thr Thr Ile Asn Val CAG CCC TTC TCA CAC GAG TAC TCG TGC GAG GAC GGC GAG AGC CTC 3867 Gln Pro Phe Ser His Glu Tyr Ser Cys Glu Asp Gly Glu Ser Leu CTC GAC GGC GCC CTG CGC AAC AGC CTG CTC CTG AAG TAC GGG TGC 3912 Leu Asp Gly Ala Leu Arg Asn Ser Leu Leu Leu Lys Tyr Gly Cys AAG CAC GGG GGC TGC GGG ACC TGC AAG GTC CGG CTG CTC GAC GGC 3957 Lys His Gly Gly Cys Gly Thr Cys Lys Val Arg Leu Leu Asp Gly GAC GTA GAG GAA CCC GGG TCG TCG TTC GCG CTG ACG CCG GAG GAC 4002 Asp Val Glu Glu Pro Gly Ser Ser Phe Ala Leu Thr Pro Glu Asp CGC GAG AAC GAC GTG ATC CTC GCG TGC GCC AGC GTG CCG CTG GAA 4047 Arg Glu Asn Asp Val Ile Leu Ala Cys Ala Ser Val Pro Leu Glu CCG TGC ACC ATC GAC GTC GAG CCG AGC GGC CTC ACG GAG GAG GAG 4092 Pro Cys Thr Ile Asp Val Glu Pro Ser Gly Leu Thr Glu Glu Glu TTC TTC TCG GGC GAC ACC TCG CGC GAG TTC CAG ACG GTC GTG GGC 4137 Phe Phe Ser Gly Asp Thr Ser Arg Glu Phe Gln Thr Val Val Gly GGT GTC GAG TTT CTC ACG GCG GAC ATC GCC CGG GTC CGG CTC CGG 4182 Gly Val Glu Phe Leu Thr Ala Asp Ile Ala Arg Val Arg Leu Arg CTA GAG CCG GGC GAG GAG ATC GCC TTC ACC GCC GGT CAG TTC GTC 4227 Leu Glu Pro Gly Glu Glu Ile Ala Phe Thr Ala Gly Gln Phe Val AAC GTC GAG GTG CCG GGC ACG GGT CTG CTG CGG ACC TTC TCG CTG 4272 Asn Val Glu Val Pro Gly Thr Gly Leu Leu Arg Thr Phe Ser Leu GCA AAC GCC CCT GAC GAC CCG TCA GTG GTG GAG CTG ATC TGC AAG 4317 Ala Asn Ala Pro Asp Asp Pro Ser Val Val Glu Leu Ile Cys Lys CTC TAC CCG GAT GGC CTC TTC TCC CGC TTC CTG AGG GAC GAG GCT 4362 Leu Tyr Pro Asp Gly Leu Phe Ser Arg Phe Leu Arg Asp Glu Ala GCC CCG GGC ACG CCG GTC CGG GTG TTC GGG CCG TAT GGT CAG CTC 4407 Ala Pro Gly Thr Pro Val Arg Val Phe Gly Pro Tyr Gly Gln Leu AAG ATC CGC TTG TCC CAC CGG CCG ATC CTG ATG ATC GCC GGT GGG 4452 Lys Ile Arg Leu Ser His Arg Pro Ile Leu Met Ile Ala Gly Gly TCC GGT CTC GCC CCG CTG CTC TCG ATG CTG CGA GAC TTG GCC GCC 4497 Ser Gly Leu Ala Pro Leu Leu Ser Met Leu Arg Asp Leu Ala Ala AAG AAG TGC GAC CGG CCG GTC TCG ATG TTC TTC GGC GCA CGC AGC 4542 Lys Lys Cys Asp Arg Pro Val Ser Met Phe Phe Gly Ala Arg Ser GTC GAC GAC CTG TAC CTC ATC GAG GAG ATC CGC GAG ATC GGC GAG 4587 Val Asp Asp Leu Tyr Leu Ile Glu Glu Ile Arg Glu Ile Gly Glu TCG CTA GCC GAT TTC GAG TTC ATC CCC GTG CTC TCG GAG TCG TCG 46 Ser Leu Ala Asp Phe Glu Phe Ile Pro Val Leu Ser Glu Ser Ser CCA GCC GAC TGG CAC GGC GAG ACG GGC ATG GTC ACC GAC GCC TTG 4677 Pro Ala Asp Trp His Gly Glu Thr Gly Met Val Thr Asp Ala Leu CTG CGG TGG CGC GCC GAA CTG GCG CAT GAC GTC TAC CTG TGC GGG 4722 Leu Arg Trp Arg Ala Glu Leu Ala His Asp Val Tyr Leu Cys Gly CCG CCA CCC ATG ATC GAC GCC GCT GTG CCG CTG CTC GTC GAG CGG 4767 Pro Pro Pro Met Ile Asp Ala Ala Val Pro Leu Leu Val Glu Arg GGC GTG CGC CCA CGC AAC ATC TAC TAC GAC GCA TTC ACC CCA GCT 4812 Gly Val Arg Pro Arg Asn Ile Tyr Tyr Asp Ala Phe Thr Pro Ala GCT CAG GTA GTC GTC GTC TGA TGG TGC ATA TCC GAT TGG GCG GCC 4857 Ala Gln Val Val Val Val * GGT ACC GGC GGG GTT AGG GCA GGG TAA TCG GCC GCG ATA GAG GCA 4902 GCC AGC AGA GAT GAT GCG CGT GCA AAG CGA AGC TTC GAC TCA GTT 4947 GAC ACC TTC GGC AAT GTG CTG TGC GAG CAC GAT AAC GGT GTG TTA 4992 CGG GTG AGG CTC AAT CGC ACC GGT GCC CTG AGC GCA ATC AAT TCA 5037 GCT ATG GCT GAT CAA TTG CGG GAA GCG TGG GTG TGG GTG CGC AGC 5082 CAA ACC GGA ATT ATC GTC ATC TCG GCT TCG CGG CCG GAG 5127 TCG TTT TTG CAT CGG TTT TGA TCC GGC CGA CCC GCC GGA GCC GAT 5172 GTT CGA TCG CCC CAT CAG CCC GAA GGA ATG CGG CGT CGA TCA ACG 5217 CGT CAT AGT CGC GGT GAGG CGC GTG CCG CAT CCT GAG CGC 5262 CGG CGA ACC GAC ACT GCG TAA GCC GGT GAC GGC GGA ACA GGC GAT 5307 GGA GTC GGG CTT GGT TCG GGA GGT CGT ACC GCT TGC ATC GGC TGC 5352 GTT GTC GAG CGA ATA GCT CAG GCG GCA GGA CAT CGT ATA GGG ATT 5397 CAG GTG GGT AGT TGA TCT TCC GCG TGT CGA TGT GTG ACG GGC CGG 5442 ATC TGG CCT ACG AAC AAC ATC ATC AGC AAG CGT GAC CTT CGG CCC ACA 5487 ACA TCG TGC CGT CCG CCGA CCTGCG ACC GTG ATC TCG GCG AAG 5532 TCC TGC CGG AGT TCG CGG GGC GGT TCC ACA GCG TTG GGG GTC CCT 5577 CGG CTA CAC CTC GGC GCT GTT CGT CAG CTC CGA CAT CAT CGG TGA 5622 CCC ACA CTC CTG CGC GGT GTT GGG CAA GCA CGA CGG CCA 5667 GGT GAA CCT TCG GCT GGT ACG ACA ACG AAT GGG CGA TGC CAA TCG 5712 GTT GCT CGA CCT CGT CGG GGT CAT GGC TGG CGT TCG CTG ACT AGG 5757 CGG CCT GTT CGT GCC GGC CGC AAC ATC CAG CTG AGT TGC 5802 GTA CCG GTC GGC GAG AAA CTC CAC GAC CCG GGC AAC CTC GTC GGG 5847 CTC ACC GAG TCG GCC GAC TGG GAT CTT CGC GAT GAC CCG ATC GAG 5892 CGC CTC CTT GGG CAC CGC AGC AAC TAT CTC GGT GGA GTA GTA 5937 AGG CGG AAC GGA ATT GAC GGT GAT GCC CTT GCG GGC GGT CTC CTG 5982 GGC CAG GGT CTG TGT CCT TAT CTC TTG TCG ACC GAA CAC GGT GTG 6027 CGG TCG AGT TGG TCG GTT TGA TCA GGC GGG AAC GCG GAA T AG GAG 6072 GGC TTC GCC CTG ACC GCC GCC GCC GCA CAG GCC GGC CGC CCC GAC 6117 GCC GCC GCC GCG GCG GGC CAG CTC GAG CAC GAG ATG CAG CGC GAT 6162 CCG GGC GCC GGA GGC GCC GAC CGG GTG CCC CAT GGC GCC 6207 GCC GTT GAC GTT GAC GTT GTC GGG GCC GAT CTG CAG GTC GCG CAT 6252 GGA TTG AAT AGC GAC GGC CGC GAA TGC CTC GTT GAT CTC GTA CAG 6297 GTC CAC ATC CAT GGC GGA GAG TCC TTC GCG GCG CAG CGC CCT GGC CGC GTT GGA GGG TTG CGA CAG CAG CGA GGG ATC C 6379 (where (amoA), (amoB), (amoC) and (amoD) are DN
The amino acid sequence corresponding to the nucleotide sequence of A is shown, and * indicates the translation stop codon.]

【００１２】（６）上記（５）のアルケンモノオキシゲ
ナーゼ遺伝子で形質転換された微生物。 （７）上記（６）の形質転換微生物を用いてアルケンを
エポキシ化するアルケンのエポキシ化方法。(6) A microorganism transformed with the alkene monooxygenase gene of (5). (7) An alkene epoxidation method for epoxidizing an alkene using the transformed microorganism of (6).

【００１３】以下に本発明について詳細に説明する。本
発明のAMO 遺伝子を含むDNA の供給源としては、ノカル
ディア属菌、ロドコッカス属菌等のアルケン資化性菌が
使用される。具体的にはプロピレン資化性菌として分離
されたノカルディア・コラリーナB-276 株が挙げられ
る。この微生物は工業技術院微生物工業研究所にFERM-P
-4094 として寄託されている。Hereinafter, the present invention will be described in detail. As a source of the DNA containing the AMO gene of the present invention, alkene-utilizing bacteria such as Nocardia and Rhodococcus are used. Specific examples include Nocardia coralina B-276 strain isolated as a propylene assimilating bacterium. This microorganism was sent to FERM-P
Deposited as -4094.

【００１４】本菌株からのDNA の分離は、ストレプトミ
セス属からのDNA を分離する方法を用いて行なうことが
できる〔Genetic manipulation of Streptomyces. A la
boratory manual, John Innes Foundation, Norwich, E
ngland(1985)〕。次に、このDNA からAMO 遺伝子を単離
するため、大腸菌によるクローニングを行う。クローニ
ングのためのプローブにはAMO 遺伝子の一構成成分であ
るモノオキシゲナーゼの２つのサブユニットのN-末端ア
ミノ酸配列から推定した遺伝子の塩基配列を持つ合成DN
A （式５参照）を使い、サザン・ハイブリダイゼーショ
ン法によってモノオキシゲナーゼ遺伝子を含むDNA の断
片集団を特定する。その後、大腸菌を使ったコロニー・
ハイブリダイゼーションにより合成DNA がハイブリダイ
ズする陽性コロニーを検出する。Isolation of DNA from the present strain can be carried out by a method for isolating DNA from Streptomyces [Genetic manipulation of Streptomyces.
boratory manual, John Innes Foundation, Norwich, E
ngland (1985)]. Next, cloning with Escherichia coli is performed to isolate the AMO gene from this DNA. As a probe for cloning, a synthetic DN having the nucleotide sequence of the gene deduced from the N-terminal amino acid sequence of the two subunits of monooxygenase which is a component of the AMO gene
Using A (see formula 5), a population of DNA fragments containing the monooxygenase gene is identified by Southern hybridization. After that, colonies using E. coli
Positive colonies to which the synthesized DNA hybridizes are detected by hybridization.

【００１５】[0015]

【式５】 1 2 3 4 5 6 7 8 9 10 11 12 subunit 1 Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu ACI ACI GAA GCI ACI GTI GCI CGI CCI GTI GA G A 1 2 3 4 5 6 7 8 9 10 11 12 subunit 2 Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys AAT CCI ACI CAI CTI CAT GAI AA C T C(Equation 5) 1 2 3 4 5 6 7 8 9 10 11 12 subunit 1 Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu ACI ACI GAA GCI ACI GTI GCI CGI CCI GTI GA GA 1 2 3 4 5 6 7 8 9 10 11 12 subunit 2 Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys AAT CCI ACI CAI CTI CAT GAI AA CTC

【００１６】次に、陽性菌株から、例えば〔Genetic ma
nipulation of Streptomyces. A laboratory manual, J
ohn Innes Foundation, Norwich, England(1985)〕に記
載の方法によってプラスミドを抽出、精製し、各種制限
酵素による分析を行い、制限酵素地図を作成する。次に
サザン・ハイブリダイゼーション法により合成DNA プロ
ーブがハイブリダイズする断片を決定する。次にその断
片の塩基配列を決定し、その前後の塩基配列も決定する
と共に取得した遺伝子を例えばプラスミドベクターpUC1
8 のような大腸菌用ベクターに結合し、大腸菌に導入
し、導入された大腸菌がアルケンのエポキシ化能を発現
することを証明することによって、遺伝子の機能を確認
する。上記の操作によって導入された遺伝子について決
定されたDNA 塩基配列およびこのDNA 塩基配列から推定
されるアミノ酸配列を式４に示す。さらに、本発明はこ
のようにして得られた大腸菌等の微生物を用いてアルケ
ンをエポキシ化する。このエポキシ化には、従来の微生
物によるアルケンのエポキシ化に用いられた方法がその
まま使用される。次に、実施例を挙げて本発明を更に具
体的に説明するが、本発明は実施例によって限定される
ものでない。Next, for example, [Genetic ma
nipulation of Streptomyces.A laboratory manual, J
The plasmid is extracted and purified by the method described in Ohn Innes Foundation, Norwich, England (1985)], analyzed with various restriction enzymes, and a restriction enzyme map is prepared. Next, a fragment to which the synthetic DNA probe hybridizes is determined by the Southern hybridization method. Next, the nucleotide sequence of the fragment was determined, the nucleotide sequences before and after the fragment were determined, and the obtained gene was, for example, plasmid vector pUC1.
The gene function is confirmed by ligating to a vector for Escherichia coli such as 8 and introducing into Escherichia coli, and proving that the introduced Escherichia coli expresses alkene epoxidation ability. Formula 4 shows the DNA base sequence determined for the gene introduced by the above operation and the amino acid sequence deduced from this DNA base sequence. Further, in the present invention, the alkene is epoxidized by using a microorganism such as Escherichia coli thus obtained. For this epoxidation, the method used for the conventional epoxidation of alkenes by microorganisms is used as it is. Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the examples.

【００１７】[0017]

【実施例】【Example】

１．ノカルディア・コラリーナからのDNA の単離 ノカルディア・コラリーナ(Nocordia corallina)B-276
(FERM P-4094)菌株を100ml の１％グリシン含有NBG 培
地〔 Nutrient Broth No.2(OXOID社製)28g、グルコース
10ｇ：１リットルイオン交換水(pH7.5) 〕にて30℃で21
時間振とう培養後ペニシリンG(5mg/ml) 溶液を 0.1％添
加してさらに３時間振とう培養をし菌体を回収した。こ
の菌体から〔Genetic manipulation of Streptomyces.
A laboratory manual, John Innes Foundation, Norwic
h, England(1985)〕に記載の方法に従ってDNA の調製を
行い約 0.5μg/μl のDNA 溶液２mlを得た。1. Isolation of DNA from Nocardia coralina Nocordia corallina B-276
(FERM P-4094) 100 g of a 1% glycine-containing NBG medium [Nutrient Broth No. 2 (manufactured by OXOID) 28 g, glucose
10g: 1 liter ion-exchanged water (pH 7.5)] at 30 ° C
After shaking culture for 0.1 hour, 0.1% of a penicillin G (5 mg / ml) solution was added, and shaking culture was further performed for 3 hours to collect cells. From this cell (Genetic manipulation of Streptomyces.
A laboratory manual, John Innes Foundation, Norwic
h, England (1985)] to prepare 2 ml of a DNA solution of about 0.5 μg / μl.

【００１８】２．アルケンモノオキシゲナーゼの構成酵
素であるモノオキシゲナーゼ遺伝子のN-末端部分の配列
を持つと考えられる合成DNA プローブの作成 ノカルディア・コラリーナ B-276株を NBG培地(Nutrien
t Broth No.2 25g、グルコース10ｇ：１リットルイオン
交換水）で30℃、72時間培養した後、集菌し、洗浄して
緩衝液(10mM リン酸カリウム(pH7.5) 、1mM エチレンジ
アミン四酢酸ナトリウム、0.5mM ジチオスレイトール)
に懸濁し、超音波で細胞を破砕、遠心分離、フィルター
濾過を行った。これをフェニルセファロース、DEAEトヨ
パールにかけ、活性分画を酵素液として得た。これを濃
縮したのちSDS-PAGE 200V 、30分によって分子量約3900
0 のサブユニット (以下サブユニット１という）と分子
量約59000 のサブユニット (以下サブユニット２とい
う）に展開し、それをポリビニリデンジフルオライド(P
VDF)膜 (バイオラッド社製) にブロッティングし、それ
ぞれのサブユニットを切り出した。これを各々自動アミ
ノ酸シークエンサー (アプライド バイオシステム社
製) を使ってＮ末端アミノ酸配列を調べた。これをもと
にＮ端アミノ配列部分のDNA 配列を推定し、前記推定DN
A 配列部分を自動DNA 合成機 (ベックマン社製) を用い
て合成した。決定したモノオキシゲナーゼN-末端アミノ
酸配列及びそれに対応させて作成した合成DNA 配列を式
５に示す。2. Preparation of a synthetic DNA probe that is considered to have the sequence of the N-terminal part of the monooxygenase gene that is a constituent enzyme of alkene monooxygenase Nocardia coralina strain B-276 was transferred to NBG medium (Nutrien
t Broth No.2 25 g, glucose 10 g: Cultured at 30 ° C. for 72 hours in 1 liter ion-exchanged water, and then collected, washed and buffered (10 mM potassium phosphate (pH 7.5), 1 mM ethylenediaminetetraacetic acid). (Sodium, 0.5 mM dithiothreitol)
, And the cells were disrupted by ultrasonic waves, centrifuged, and filtered. This was applied to phenyl sepharose and DEAE Toyopearl to obtain an active fraction as an enzyme solution. After concentrating this, SDS-PAGE 200V, molecular weight about 3900 by 30 minutes
0 subunit (hereinafter referred to as subunit 1) and a subunit having a molecular weight of about 59000 (hereinafter referred to as subunit 2), which are then combined with polyvinylidene difluoride (P
(VDF) membrane (Bio-Rad) and each subunit was cut out. The N-terminal amino acid sequence of each of them was examined using an automatic amino acid sequencer (manufactured by Applied Biosystems). Based on this, the DNA sequence of the N-terminal amino acid sequence was estimated,
The A sequence was synthesized using an automatic DNA synthesizer (Beckman). The determined monooxygenase N-terminal amino acid sequence and the corresponding synthetic DNA sequence are shown in Formula 5.

【００１９】 ３．N 端アミノ酸配列部分のDNA の位置確認 前記１の方法によってノカルディア・コラリーナより調
製した上記 DNA溶液２μl に各種の制限酵素を加え37
℃、２時間反応させた。各種反応液をアガロースゲル電
気泳動に流し、ナイロンフィルター（アマシャム社製）
にDNA を吸着させた。次に、合成DNA 10pmol、〔γ−32
Ｐ〕ATP(370MBq/ml)５μl 、10倍濃度リン酸化緩衝液１
μl 、T4ポリヌクレオチドキナーゼ（宝酒造社製）１μ
l および10mMトリス/1mM EDTA(pH8.0)で全量を10μl と
し、37℃で30分間反応させた後、セファデックスG-50
（ファルマシア社製）カラムを通して精製した合成DNA
を用い65℃で一晩前記ナイロンフィルターに吸着させた
DNA とハイブリダイゼーションを行った。65℃で30分洗
浄後、オートラジオグラムを行い、制限酵素BamHI 分解
物において２種類の合成DNA プローブがいづれも約 6.4
kbの位置にハイブリダイズすることを確認した。[0019] 3. Confirmation of N-terminal amino acid sequence DNA position Various restriction enzymes were added to 2 μl of the above DNA solution prepared from Nocardia coralina by the method described in 1 above.
The reaction was carried out at 2 ° C. for 2 hours. Run various reaction solutions on agarose gel electrophoresis and use nylon filter (Amersham).
DNA was adsorbed to the DNA. Next, 10 pmol of synthetic DNA, [γ- 32
P] ATP (370MBq / ml) 5μl, 10 times concentration phosphorylation buffer 1
μl, T4 polynucleotide kinase (Takara Shuzo) 1μ
l and 10 mM Tris / 1 mM EDTA (pH 8.0) to make a total volume of 10 μl, and after reacting at 37 ° C for 30 minutes, Sephadex G-50
(Pharmacia) Synthetic DNA purified through column
Was adsorbed to the nylon filter at 65 ° C. overnight.
Hybridization with DNA was performed. After washing at 65 ° C for 30 minutes, an autoradiogram was performed to confirm that both of the two types of synthetic DNA probes were about 6.4
It was confirmed that it hybridized to the position of kb.

【００２０】 ４．DNA の約6.4kb BamHI 分解断片のベクターへの挿入 DNA 50μl を制限酵素BamHI(東洋紡社製) で分解し 0.7
％アガロースゲル電気泳動を行い、〔Anal. Biochem.,
101 巻, 339 〜341 頁(1980)〕記載の方法により6.4kb
前後の断片を回収し、20μl の10mMトリス/1mM EDTA(pH
8.0)に溶解した。一方、プラスミドベクターpUC18(約
0.5μg/μl) 2μl に制限酵素BamHI(東洋紡社製）２μl
を加え、37℃、２時間反応後大腸菌由来のアルカリフ
ォスファターゼ（東洋紡社製)1.0μl を加え混合し、65
℃で30分間脱リン酸化反応を行った。更にフェノール処
理後エタノール沈澱を行い、沈澱物を10mMトリス/1mM E
DTA緩衝液(pH8.0) 10μl に溶解してリガーゼ反応に供
した。即ちリガーゼ反応は先に調製した約6.4kb BamHI
分解断片５μl 、BamHI 分解後脱リン酸化したpUC18溶
液５μl 、反応液（宝酒造社製 DNA Ligation kit A
液）80μl 、T4リガーゼ(宝酒造社製DNA Ligation kit
B 液）10μl を混合し、16℃で30分反応させた。[0020] 4. Insertion of about 6.4 kb BamHI digestion fragment of DNA into vector 50 μl of DNA was digested with restriction enzyme BamHI (manufactured by Toyobo) and 0.7
% Agarose gel electrophoresis, [Anal. Biochem.,
101, 339-341 (1980))
The front and rear fragments were collected, and 20 μl of 10 mM Tris / 1 mM EDTA (pH
8.0). On the other hand, the plasmid vector pUC18 (about
0.5μg / μl) 2μl to 2μl restriction enzyme BamHI (Toyobo)
After reaction at 37 ° C for 2 hours, 1.0 µl of Escherichia coli-derived alkaline phosphatase (manufactured by Toyobo Co., Ltd.) was added and mixed.
A dephosphorylation reaction was performed at 30 ° C. for 30 minutes. Further, after phenol treatment, ethanol precipitation was performed, and the precipitate was treated with 10 mM Tris / 1 mM E.
It was dissolved in 10 μl of DTA buffer (pH 8.0) and subjected to a ligase reaction. That is, the ligase reaction was performed using the previously prepared 6.4 kb BamHI
5 μl of the digested fragment, 5 μl of pUC18 solution dephosphorylated after BamHI digestion, and the reaction solution (Takara Shuzo DNA Ligation kit A
Solution) 80 µl, T4 ligase (Takara Shuzo DNA Ligation kit)
(Solution B) was mixed and reacted at 16 ° C for 30 minutes.

【００２１】５．大腸菌の形質転換 上記リガーゼ反応を行った液をHanahan の方法(J. Mol.
Biol., 166, 557, 1983) で調製した大腸菌のコンピテ
ントセルJM109 に加えて形質転換を行い、アンピシリン
50μg/ml, IPTG(Isopropyl- β-D-thiogalactopyranosi
de)2mM, X-gal(5-Bromo-4-chloro-3-indolyl-β-D-gala
ctoside) 2.5 ×10-3％を含むL-ブロス寒天培地にま
き、37℃で一晩培養した。5. Transformation of Escherichia coli The solution subjected to the ligase reaction was subjected to Hanahan's method (J. Mol.
Biol., 166, 557, 1983) and transformed with E. coli competent cell JM109.
50μg / ml, IPTG (Isopropyl-β-D-thiogalactopyranosi
de) 2mM, X-gal (5-Bromo-4-chloro-3-indolyl-β-D-gala
ctoside) The cells were spread on an L-broth agar medium containing 2.5 × 10 −3 % and cultured at 37 ° C. overnight.

【００２２】上記で得られた形質転換体のうち白いコロ
ニー、すなわち、組換えプラスミドを持つものだけを新
たなL-ブロス寒天培地上及び別のL-ブロス寒天培地上の
ナイロンフィルターに植菌し、合成DNA プローブを用
い、コロニーハイブリダイゼーション法により陽性株を
取得した。Of the transformants obtained above, only white colonies, that is, those having the recombinant plasmid, were inoculated on nylon filters on a new L-broth agar medium and another L-broth agar medium. A positive strain was obtained by a colony hybridization method using a synthetic DNA probe.

【００２３】６．アルケンモノオキシゲナーゼ遺伝子の
クローン化の確認 上記陽性株から〔Genetic manipulation of Streptomyc
es. A laboratory manual, John Innes Foundation,
Norwich, England(1985)〕記載のアルカリ法によってプ
ラスミドを分離し、各種制限酵素で切断後 0.7％アガロ
ースゲル電気泳動による解析を行うと共にサザン・ハイ
ブリダイゼーションを行った結果、 6.4kbの挿入断片が
含まれており、モノオキシゲナーゼのサブユニット１の
N-末端アミノ酸に対応する合成DNA プローブは制限酵素
BamHI-EcoRI 1.5kb 断片に、サブユニット２のN-末端ア
ミノ酸に対応する合成DNA プローブは制限酵素EcoRI-Kp
nI断片である3.5kb 断片にハイブリダイズすることがわ
かった。制限酵素BamHI-EcoRI 断片である1.5kb 断片を
ジデオキシシークエンス法により塩基配列を決定したと
ころサブユニット１のN-末端アミノ酸配列をコードする
読み取り枠が、制限酵素BamHI 切断部位から910bp の位
置にあり、Pst I 切断部位に向かってコードされてい
た。そこで更に6.4kb のBamHI 断片の全塩基配列を同様
にして決定した。その結果、クラスターを形成している
４つのオープンリーディングフレームが存在した（以下
転写順にamoA, amoB, amoC, amoDとする) 。このうちam
oAはモノオキシゲナーゼサブユニット１のN-末端アミノ
酸配列をコードしamoCはサブユニット２のN-末端アミノ
酸配列をコードし、amoDはレダクターゼのアミノ酸配列
をコードしていることを確認した。このDNA 配列を式４
に示す。6. Confirmation of cloning of alkene monooxygenase gene From the above positive strain, [Genetic manipulation of Streptomyc
es.A laboratory manual, John Innes Foundation,
The plasmid was isolated by the alkaline method described in Norwich, England (1985), digested with various restriction enzymes, analyzed by 0.7% agarose gel electrophoresis, and subjected to Southern hybridization. Subunit 1 of monooxygenase
Synthetic DNA probe corresponding to N-terminal amino acid is a restriction enzyme
A synthetic DNA probe corresponding to the N-terminal amino acid of subunit 2 was added to the restriction enzyme EcoRI-Kp
It was found to hybridize to the 3.5 kb fragment which is the nI fragment. The nucleotide sequence of the 1.5 kb fragment, a restriction enzyme BamHI-EcoRI fragment, was determined by the dideoxy sequencing method. It was coded towards the Pst I cleavage site. Therefore, the entire base sequence of the 6.4 kb BamHI fragment was determined in the same manner. As a result, there were four open reading frames forming a cluster (hereinafter referred to as amoA, amoB, amoC, and amoD in the order of transcription). Am
It was confirmed that oA encoded the N-terminal amino acid sequence of monooxygenase subunit 1, amoC encoded the N-terminal amino acid sequence of subunit 2, and amoD encoded the amino acid sequence of reductase. This DNA sequence is expressed by Formula 4
Shown in

【００２４】７．大腸菌形質転換体によるアルケンのエ
ポキシ化能の確認 ノカルディア・コラリーナからクローン化したamo A B
C D の上流には大腸菌リボソームの認識部位、いわゆる
SD配列が存在していた。そこでモノオキシゲナーゼ遺伝
子が発現ベクターpUC18 のプロモーター下流になるよう
につなげたプラスミドを用いて形質転換した大腸菌（前
記５で調製したもの）を後述の８の方法でエポキシ化能
を調べたが活性は検出されなかった。そこで、このDNA
(6.4kb のBamHI 断片) を制限酵素Bal 31 (東洋紡社製)
で処理して両端の一部を欠失させ、発現ベクターpUC18
のプロモーターの直下にamo A B C D がつながるよう
にしたベクターpDBB-1を大腸菌E.coli JM109株に挿入し
て形質転換し、エポキシ化能を後述の８の方法で確認し
た。[7] Confirmation of alkene epoxidation ability by E. coli transformant amo AB cloned from Nocardia coralina
Upstream of the CD, the E. coli ribosome recognition site,
SD sequence was present. Therefore, Escherichia coli (prepared in 5) transformed with a plasmid in which the monooxygenase gene was connected to the downstream of the promoter of the expression vector pUC18 was examined for epoxidation ability by the method of 8 described below, but the activity was detected. Was not done. So this DNA
(6.4 kb BamHI fragment) with restriction enzyme Bal 31 (Toyobo)
To delete a part of both ends, the expression vector pUC18
Escherichia coli E. coli JM109 strain was transformed by inserting the vector pDBB-1 in which amo ABCD was connected immediately below the promoter, and the epoxidation ability was confirmed by the method described in 8 below.

【００２５】本発明で得られた大腸菌E.coli JM109(pDB
B-1)を工業技術院微生物工業研究所に寄託した（受託番
号FERM-BP 4250号) 。The Escherichia coli E. coli JM109 (pDB
B-1) was deposited with the Research Institute of Microbial Industry of the Agency of Industrial Science and Technology (Accession No. FERM-BP 4250).

【００２６】８．アルケンのエポキシ化方法 上記７で形質転換した大腸菌E.coli JM109 (pDBB-1)(FE
RM BP-4250) を50μg/mlのアンピシリン含有LB培地1.5m
l に接種し、30℃で一晩培養し、この培養物を、再度同
様の培地1.5ml に 150μl 接種し、30℃で２時間培養し
た。これに、終濃度が１mMとなるようにIPTGを添加し、
さらに30℃で５時間培養した。集菌後、この菌体を反応
培地(K2HPO4 1.74g/l 、MgSO4 ・7H2O 1.5g/l 、FeSO4
・7H2O 50mg/l 、pH8.0)２mlに懸濁し、さらに10％のグ
ルコース溶液0.1ml を添加した。これをＬ字型試験官に
入れ、ゴム栓で密封した。これにトリフルオロプロペン
４mlを圧入し、30℃て16時間反応させた。反応終了後、
反応生成物をガスクロマトグラフィーにより測定した結
果、トリフルオロプロペンオキシドの生成が確認され
た。8. Alkene Epoxidation Method E. coli JM109 (pDBB-1) (FE
RM BP-4250) in 50 μg / ml ampicillin-containing LB medium 1.5 m
The resulting culture was inoculated again at 150 ° C. into 1.5 ml of the same medium, and cultured at 30 ° C. for 2 hours. To this, IPTG was added to a final concentration of 1 mM,
The cells were further cultured at 30 ° C. for 5 hours. After harvesting, the cell reaction medium (K 2 HPO 4 1.74g / l , MgSO 4 · 7H 2 O 1.5g / l, FeSO 4
(7H 2 O 50 mg / l, pH 8.0) was suspended in 2 ml, and 0.1 ml of a 10% glucose solution was further added. This was placed in an L-shaped tester and sealed with a rubber stopper. To this, 4 ml of trifluoropropene was injected and reacted at 30 ° C. for 16 hours. After the reaction,
As a result of measuring the reaction product by gas chromatography, formation of trifluoropropene oxide was confirmed.

【００２７】[0027]

【発明の効果】本発明の新規なDNA 断片は各種のアルケ
ンをエポキシ化することのできるアルケンモノオキシゲ
ナーゼ(AMO) をコードする遺伝子を含むDNA 断片であっ
て、該DNA 断片を遺伝子操作により微生物に導入するこ
とによりエポキシ化能を付与したりあるいはその能力を
高めることが可能となる。Industrial Applicability The novel DNA fragment of the present invention is a DNA fragment containing a gene encoding an alkene monooxygenase (AMO) capable of epoxidizing various alkenes. By introducing it, it becomes possible to impart epoxidation ability or to enhance the ability.

【００２８】[0028]

【配列表】[Sequence list]

配列番号：1 配列の長さ：342 配列の型 ：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 5 10 15 Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu Glu Gly His 20 25 30 Arg Thr Phe Thr Trp Phe Thr Pro Ala Arg Arg Lys Pro Thr Glu 35 40 45 Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro Asp Glu Trp 50 55 60 Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp Gly Arg Ala Pro 65 70 75 Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu Trp Ser Ala 80 85 90 Tyr Arg Asp Pro His Gln Leu Trp Gln Arg Pro Tyr Val Ser Thr 95 100 105 Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val Pro Val Leu 110 115 120 Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser Gln Lys Ile 125 130 135 Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu Tyr Gly Leu 140 145 150 Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met Ser Asp Thr 155 160 165 Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg Met Arg Leu 170 175 180 Leu Gln Asp Ile Val His His Leu Asp His Leu Gln Glu Ser Pro 185 190 195 Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met Ser Asp Ser 200 205 210 Thr Leu Val Pro Ile Arg Glu Val Ile Glu Arg Ile Ala Ala Ser 215 220 225 Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu Val Phe Glu 230 235 240 Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe Ser Arg Arg 245 250 255 Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val Leu Ala Ser 260 265 270 Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val Gln Ala Leu 275 280 285 Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp Gln Asn Gln 290 295 300 Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro Arg Cys Lys 305 310 315 Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp Cys Gly Ile 320 325 330 Asp Ala Lys Glu Ser Ala Asn Ala Leu Ser Arg Ala Leu Ala Asn 335 340 Gln Arg Ala Ala Val Glu Gly Ala Gly Ile Thr Ala  SEQ ID NO: 1 Sequence length: 342 Sequence type: Amino acid Topology: Linear Sequence type: Peptide 5 10 15 Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu Glu Gly His 20 25 30 Arg Thr Phe Thr Trp Phe Thr Pro Ala Arg Arg Lys Pro Thr Glu 35 40 45 Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro Asp Glu Trp 50 55 60 Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp Gly Arg Ala Pro 65 70 75 Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu Trp Ser Ala 80 85 90 Tyr Arg Asp Pro His Gln Leu Trp Gln Arg Pro Tyr Val Ser Thr 95 100 105 Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val Pro Val Leu 110 115 120 Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser Gln Lys Ile 125 130 135 Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu Tyr Gly Leu 140 145 150 Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met Ser Asp Thr 155 160 165 Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg Met Arg Leu 170 175 180 Leu Gln Asp Ile Val His His Leu Asp His Leu Gln Glu Ser Pro 185 190 195 Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met Ser Asp Ser 200 205 210 Thr Leu Val Pro Ile Arg Glu Val Ile Glu Arg Ile Ala Ala Ser 215 220 225 Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu Val Phe Glu 230 235 240 Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe Ser Arg Arg 245 250 255 Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val Leu Ala Ser 260 265 270 Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val Gln Ala Leu 275 280 285 Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp Gln Asn Gln 290 295 300 Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro Arg Cys Lys 305 310 315 Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp Cys Gly Ile 320 325 330 Asp Ala Lys Glu Ser Ala Asn Ala Leu Ser Arg Ala Leu Ala Asn 335 340 Gln Arg Ala Ala Val Glu Gly Ala Gly Ile Thr Ala

【００２９】配列番号：2 配列の長さ：501 配列の型 ：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 5 10 15 Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys Ser Tyr 20 25 30 Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe Glu Thr 35 40 45 Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg Val Leu 50 55 60 Ile Arg Asp Tyr Met Lys Met Glu Ala Glu Lys Asp Asp Arg Thr 65 70 75 His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala Thr Arg 80 85 90 Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val Pro Gln 95 100 105 Leu Thr Asn Ala Glu Tyr Gln Ala Val Ala Gly Cys Gly Met Ile 110 115 120 Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr Ala Ala 125 130 135 Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met Thr Leu 140 145 150 Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly Phe Asp 155 160 165 Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu Val Ser 170 175 180 Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu Asp Val 185 190 195 Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr Asn Ile 200 205 210 Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly Asp Asn 215 220 225 Ala Met Ala Ser Val Phe Leu Ser Ile Gln Ser Asp Glu Ala Arg 230 235 240 His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu Glu Asn 245 250 255 Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg His Phe 260 265 270 Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp Cys Ser 275 280 285 Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala Gln Trp 290 295 300 Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile Asp Arg 305 310 315 Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly Ala Ala 320 325 330 Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln Val Leu 335 340 345 Ser Ala Val Trp Pro Leu Asn Phe Trp Arg Ser Asp Ala Met Gly 350 355 360 Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly Trp Ser 365 370 375 Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu Ala Asp 380 385 390 Pro Ala Gly Gly Arg Ile Met Leu Gln Glu Leu Pro Gly Leu Pro 395 400 405 Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro Arg Leu 410 415 420 Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gln Lys Ile 425 430 435 Ala Leu Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn Trp Pro 440 445 450 Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr His Gly 455 460 465 Trp Asp Leu Ala Asp Val Ile Val Asp Leu Gly Tyr Ile Arg Pro 470 475 480 Asp Gly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met Glu Arg 485 490 495 Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Glu Val Lys 500 Asp Pro Leu Gln Glu Ala SEQ ID NO: 2 Sequence length: 501 Sequence type: amino acid Topology: linear Sequence type: peptide 5 10 15 Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys Ser Tyr 20 25 30 Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe Glu Thr 35 40 45 Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg Val Leu 50 55 60 Ile Arg Asp Tyr Met Lys Met Glu Ala Glu Lys Asp Asp Arg Thr 65 70 75 His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala Thr Arg 80 85 90 Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val Pro Gln 95 100 105 Leu Thr Asn Ala Glu Tyr Gln Ala Val Ala Gly Cys Gly Met Ile 110 115 120 Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr Ala Ala 125 130 135 Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met Thr Leu 140 145 150 Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly Phe Asp 155 160 165 Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu Val Ser 170 175 180 Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu Asp Val 185 190 195 Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr Asn Ile 200 205 210 Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly Asp Asn 215 220 225 Ala Met Ala Ser Val Phe Leu Ser Ile Gln Ser Asp Glu Ala Arg 230 235 240 His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu Glu Asn 245 250 255 Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg His Phe 260 265 270 Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp Cys Ser 275 280 285 Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala Gln Trp 290 295 300 Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile Asp Arg 305 310 315 Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly Ala Ala 320 325 330 Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln Val Leu 335 340 345 Ser Ala Val Trp Pro Leu Asn Phe Trp Arg Ser Asp Ala Met Gly 350 355 360 Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly Trp Ser 365 370 375 Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu Ala Asp 380 385 390 Pro Ala Gly Gly Arg Ile Met Leu Gln Glu Leu Pro Gly Leu Pro 395 400 405 Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro Arg Leu 410 415 420 Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gln Lys Ile 425 430 435 Ala Leu Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn Trp Pro 440 445 450 Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr His Gly 455 460 465 Trp Asp Leu Ala Asp Val Ile Val Asp Leu Gly Tyr Ile Arg Pro 470 475 480 Asp Gly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met Glu Arg 485 490 495 Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Glu Val Lys 500 Asp Pro Leu Gln Glu Ala

【００３０】配列番号：3 配列の長さ：342 配列の型 ：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 5 10 15 Met Thr Thr Ile Asn Val Gln Pro Phe Ser His Glu Tyr Ser Cys 20 25 30 Glu Asp Gly Glu Ser Leu Leu Asp Gly Ala Leu Arg Asn Ser Leu 35 40 45 Leu Leu Lys Tyr Gly Cys Lys His Gly Gly Cys Gly Thr Cys Lys 50 55 60 Val Arg Leu Leu Asp Gly Asp Val Glu Glu Pro Gly Ser Ser Phe 65 70 75 Ala Leu Thr Pro Glu Asp Arg Glu Asn Asp Val Ile Leu Ala Cys 80 85 90 Ala Ser Val Pro Leu Glu Pro Cys Thr Ile Asp Val Glu Pro Ser 95 100 105 Gly Leu Thr Glu Glu Glu Phe Phe Ser Gly Asp Thr Ser Arg Glu 110 115 120 Phe Gln Thr Val Val Gly Gly Val Glu Phe Leu Thr Ala Asp Ile 125 130 135 Ala Arg Val Arg Leu Arg Leu Glu Pro Gly Glu Glu Ile Ala Phe 140 145 150 Thr Ala Gly Gln Phe Val Asn Val Glu Val Pro Gly Thr Gly Leu 155 160 165 Leu Arg Thr Phe Ser Leu Ala Asn Ala Pro Asp Asp Pro Ser Val 170 175 180 Val Glu Leu Ile Cys Lys Leu Tyr Pro Asp Gly Leu Phe Ser Arg 185 190 195 Phe Leu Arg Asp Glu Ala Ala Pro Gly Thr Pro Val Arg Val Phe 200 205 210 Gly Pro Tyr Gly Gln Leu Lys Ile Arg Leu Ser His Arg Pro Ile 215 220 225 Leu Met Ile Ala Gly Gly Ser Gly Leu Ala Pro Leu Leu Ser Met 230 235 240 Leu Arg Asp Leu Ala Ala Lys Lys Cys Asp Arg Pro Val Ser Met 245 250 255 Phe Phe Gly Ala Arg Ser Val Asp Asp Leu Tyr Leu Ile Glu Glu 260 265 270 Ile Arg Glu Ile Gly Glu Ser Leu Ala Asp Phe Glu Phe Ile Pro 275 280 285 Val Leu Ser Glu Ser Ser Pro Ala Asp Trp His Gly Glu Thr Gly 290 295 300 Met Val Thr Asp Ala Leu Leu Arg Trp Arg Ala Glu Leu Ala His 305 310 315 Asp Val Tyr Leu Cys Gly Pro Pro Pro Met Ile Asp Ala Ala Val 320 325 330 Pro Leu Leu Val Glu Arg Gly Val Arg Pro Arg Asn Ile Tyr Tyr 335 340 Asp Ala Phe Thr Pro Ala Ala Gln Val Val Val Val SEQ ID NO: 3 Sequence length: 342 Sequence type: amino acid Topology: linear Sequence type: peptide 5 10 15 Met Thr Thr Ile Asn Val Gln Pro Phe Ser His Glu Tyr Ser Cys 20 25 30 Glu Asp Gly Glu Ser Leu Leu Asp Gly Ala Leu Arg Asn Ser Leu 35 40 45 Leu Leu Lys Tyr Gly Cys Lys His Gly Gly Cys Gly Thr Cys Lys 50 55 60 Val Arg Leu Leu Asp Gly Asp Val Glu Glu Pro Gly Ser Ser Phe 65 70 75 Ala Leu Thr Pro Glu Asp Arg Glu Asn Asp Val Ile Leu Ala Cys 80 85 90 Ala Ser Val Pro Leu Glu Pro Cys Thr Ile Asp Val Glu Pro Ser 95 100 105 Gly Leu Thr Glu Glu Glu Phe Phe Ser Gly Asp Thr Ser Arg Glu 110 115 120 Phe Gln Thr Val Val Gly Gly Val Glu Phe Leu Thr Ala Asp Ile 125 130 135 Ala Arg Val Arg Leu Arg Leu Glu Pro Gly Glu Glu Ile Ala Phe 140 145 150 Thr Ala Gly Gln Phe Val Asn Val Glu Val Pro Gly Thr Gly Leu 155 160 165 Leu Arg Thr Phe Ser Leu Ala Asn Ala Pro Asp Asp Pro Ser Val 170 175 180 Val Glu Leu Ile Cys Lys Leu Tyr Pro Asp Gly Leu Phe Ser Arg 185 190 195 Phe Leu Arg Asp Glu Ala Ala Pro Gly Thr Pro Val Arg Val Phe 200 205 210 Gly Pro Tyr Gly Gln Leu Lys Ile Arg Leu Ser His Arg Pro Ile 215 220 225 Leu Met Ile Ala Gly Gly Ser Gly Leu Ala Pro Leu Leu Ser Met 230 235 240 Leu Arg Asp Leu Ala Ala Lys Lys Cys Asp Arg Pro Val Ser Met 245 250 255 Phe Phe Gly Ala Arg Ser Val Asp Asp Leu Tyr Leu Ile Glu Glu 260 265 270 270 Ile Arg Glu Ile Gly Glu Ser Leu Ala Asp Phe Glu Phe Ile Pro 275 280 285 Val Leu Ser Glu Ser Ser Pro Ala Asp Trp His Gly Glu Thr Gly 290 295 300 Met Val Thr Asp Ala Leu Leu Arg Trp Arg Ala Glu Leu Ala His 305 310 315 Asp Val Tyr Leu Cys Gly Pro Pro Pro Met Ile Asp Ala Ala Val 320 325 330 Pro Leu Leu Val Glu Arg Gly Val Arg Pro Arg Asn Ile Tyr Tyr 335 340 Asp Ala Phe Thr Pro Ala Ala Gln Val Val Val Val

【００３１】配列番号：４ 配列の長さ：６３７９ 配列の型 ：核酸 鎖の数 ：一本鎖 トポロジー：直鎖状 配列の種類：ペプチド 起 源 ：ノカルディア・コラリーナ B-276(FERM P
-4094)907 −1935 amo A1935 −2285 amo B 2300−3802 amo C 3805−4830 amo D GGA TCC CCA GGT CCC GAG CCA CCG CGG CGA TCG ACT TCC CCG TCT 45 CAT GCA CGA TCC GCA CAG CCC CCT CAC GGA ACT CCC GGT CGT ACT 90 TTT CCG CTT CTC TGA CAT CCC TAC CCC TTA TAG CTG ATG CCT CCA 135 CGG TCT CGG GGA ATG CCA ATA TGA CTT GGT CAG ATC GTA GGC GGC 180 AAG AAC CAT GGC TTC CTT GGC GGA CTT CAC AGG GGC CTC TCC TCG 225 AGG GCT CGT TCG GGC ATG GGT AGC ACC GTC GAG CGA ACC CGA GGA 270 GAG GCC CCG ATC CGG GGA GGC GAT CGG GAT GAA CGG GGA CGT GTC 315 GAG AAT CGA TCA GAC TTG GTG GGC AGA TTC GTG TCC GCC GGT GGG 360 CAG AAC CCG ATG GCC ATC CAC GGG CAC TTT CGT GGC CAT CAG TGG 405 GCG GTT TCG TGA CCC CCT ATG GGC AGT TTT CCA TGG CCG CCG ACA 450 CAC AGG CCC GCA CCG ATG ACG AGG TAC TGC ACG ACC CGG CCG ACC 495 GCG CGG TGG TTT GCT GCA ACA AAT GAC AAC TTG ATC AAC GCA TCG 540 ATC ACG ACC GGC GCC ACT AAC GCC GCC GCG GCA AGC ATC GAC ATC 585 AAC ACC ATC GCG ACC GTC CGC CCT TCA CAT CGA CGA CAC CTC GGT 630 CGA TGT GTG CAT ACC GCA TGC ATC CGG CAA GAA CCC CGC GGA CTC 675 CGC AGC CGG TGC GGC TAG CCG ATT TCA CCC AAC CTG GGA CGT TGG 720 CTA CGG AAC GTC TCG TGG ATG ACG GCG CCT GCA TGC TGC ATA GAC 765 CCG TCC GTG GCC GCA ATG TCC TGG TGA GGC ATG CGA GCG GCT TCG 810 ATT CCT CTC GGC GGG GCC AAG ACC GGC GGG CCG CGC CTG TCT AAC 855 ATC GCG GCA CAA GGA AAT CGC AAC CGG ACC GAA ACG ATG GAA GGC 900 GTA GCG ATG ACG ACA GAG GCG ACG GTG GCC CGA CCG GTG GAG CTC 945 (amoA) Met Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu GAA GGT CAC CGG ACA TTC ACC TGG TTC ACG CCC GCC AGG CGA AAG 990 Glu Gly His Arg Thr Phe Thr Trp Phe Thr Pro Ala Arg Arg Lys CCG ACG GAG TAC GAG CTC TAC ACC GTG GGT CAA CAG TCC ACT CCG 1035 Pro Thr Glu Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro GAC GAG TGG CTG CAT GTG GAC TGG CCG CTG CGC TTC GAC GAC GGC 1080 Asp Glu Trp Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp Gly CGC GCC CCG TGG GAG GAG GAG TCG AGT GCG GTA CGG ACC TCG GAG 1125 Arg Ala Pro Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu TGG TCG GCT TAC CGC GAC CCA CAC CAA CTG TGG CAG CGT CCC TAC 1170 Trp Ser Ala Tyr Arg Asp Pro His Gln Leu Trp Gln Arg Pro Tyr GTC AGC ACG TGC AAC CAG GAC CAG CAG GCC CTC GCG CGG CTG GTC 1215 Val Ser Thr Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val CCC GTC CTG ACC ATG GGG TCG GCG GCG ATC ACG CCC ATC TGG TCG 1260 Pro Val Leu Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser CAG AAG ATC CTC GCC AGG TCC TAC GCC GCC TGG CCA TTC GTC GAG 1305 Gln Lys Ile Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu TAC GGG CTC TTC CTG AGC CTG GCC TAC GCC GTG CGC CAG GCC ATG 1350 Tyr Gly Leu Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met TCC GAC ACG GTC CAG TTC AGC GTG GTG TTC CAG GCC GTG GAC CGC 1395 Ser Asp Thr Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg ATG CGG CTG CTC CAG GAC ATC GTC CAC CAC CTG GAC CAC CTG CAG 1440 Met Arg Leu Leu Gln Asp Ile Val His His Leu Asp His Leu Gln GAG TCG CCG GAA TTC AGC GAC GCC GGG GCC CGC GAG GCC TGG ATG 1485 Glu Ser Pro Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met TCC GAC TCC ACC CTG GTC CCG ATC CGG GAA GTG ATC GAG CGC ATC 1530 Ser Asp Ser Thr Leu Val Pro Ile Arg Glu Val Ile Glu Arg Ile GCC GCC AGC CAG GAC TGG GTG GAG ATC CTG GTC GCC GGC ACG CTC 1575 Ala Ala Ser Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu GTC TTC GAG CCT CTG GTC GGC CAC CTG GCG AAG GCC GAG TTG TTC 1620 Val Phe Glu Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe AGC CGC CGT GCG CCA ATG TTC GGG GAC GGG ACC ACG CCG GCG GTG 1665 Ser Arg Arg Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val CTG GCG TCG GCC CTG CTG GAC AGC GGC AGG CAC CTC GAA TCG GTC 1710 Leu Ala Ser Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val CAG GCG CTC GTC CGC CTC GTC TGC CAA GAC CCC GTC CAT GGC GAC 1755 Gln Ala Leu Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp CAG AAC CAG GCG ACT GTG CGG CGG TGG ATC GAG GAA TGG CAG CCG 1800 Gln Asn Gln Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro CGG TGC AAG GCG GCG GCC CAG TCC TTC CTG CCG ACG TTC TCC GAC 1845 Arg Cys Lys Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp TGC GGC ATC GAC GCC AAG GAA AGC GCC AAC GCG CTG TCC CGG GCG 1890 Cys Gly Ile Asp Ala Lys Glu Ser Ala Asn Ala Leu Ser Arg Ala CTG GCG AAC CAG CGG GCC GCC GTC GAG GGC GCC GGC ATC ACG GCA 1935 Leu Ala Asn Gln Arg Ala Ala Val Glu Gly Ala Gly Ile Thr Ala (amoB) M TG ACA GAC GTT AAG GAG ACC ACT GTG ACC AGC ACC CCC TCG GCC 1979 * et Thr Asp Val Lys Glu Thr Thr Val Thr Ser Thr Pro Ser Ala GCC GTG CCG GGA ACC AAG AAC CGC CGG GTT GGT ATC TCG CTG ATC 2024 Ala Val Pro Gly Thr Lys Asn Arg Arg Val Gly Ile Ser Leu Ile AGC AGC AGC GAC ACC GAG GCA GCT GTC GAG CAC ATC GCG GAG ACC 2069 Ser Ser Ser Asp Thr Glu Ala Ala Val Glu His Ile Ala Glu Thr CAG CCG GAC GCG AAG ATC GAC TTT CGG GAC TGC TTC TAC AAG ATC 2114 Gln Pro Asp Ala Lys Ile Asp Phe Arg Asp Cys Phe Tyr Lys Ile GAG CGT GAC GGG CAG CTC AGT TTC GAC ATG GCA GAG CTC AGT GAG 2159 Glu Arg Asp Gly Gln Leu Ser Phe Asp Met Ala Glu Leu Ser Glu ATC GCC GGT CGC GAC ATC GAC ACC GAC ATC TTC CTG GTG AAC ATG 2204 Ile Ala Gly Arg Asp Ile Asp Thr Asp Ile Phe Leu Val Asn Met AGC ACC TAC TAC GGC CGG ATC GTC GTC AGT GAC GGC CGG GTC GAC 2249 Ser Thr Tyr Tyr Gly Arg Ile Val Val Ser Asp Gly Arg Val Asp ATC TAC GCC GAA ATC CAG CCG GCC CGC TTC AAG GAC TGA GAG GAA 2294 Ile Tyr Ala Glu Ile Gln Pro Ala Arg Phe Lys Asp * ACA CC ATG GCA TCG AAC CCC ACC CAG CTC CAC GAG AAG TCG AAG 2338 (amoC) Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys TCC TAC GAC TGG GAC TTC ACC TCC GTC GAG CGG CGC CCC AAG TTC 2383 Ser Tyr Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe GAG ACG AAG TAC AAG ATG CCC AAG AAG GGC AAG GAC CCG TTC CGC 2428 Glu Thr Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg GTC CTG ATC CGT GAC TAC ATG AAG ATG GAA GCG GAG AAG GAC GAC 2473 Val Leu Ile Arg Asp Tyr Met Lys Met Glu Ala Glu Lys Asp Asp CGG ACC CAT GGC TTC CTC GAC GGC GCC GTG CGG ACG CGT GAG GCC 2518 Arg Thr His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala ACC AGG ATT GAG CCG CGG TTC GCT GAG GCC ATG AAG ATC ATG GTG 2563 Thr Arg Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val CCG CAG CTG ACC AAC GCC GAG TAC CAG GCG GTG GCG GGC TGC GGA 2608 Pro Gln Leu Thr Asn Ala Glu Tyr Gln Ala Val Ala Gly Cys Gly ATG ATC ATC TCG GCC GTC GAG AAC CAG GAG CTC CGT CAG GGC TAC 2653 Met Ile Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr GCC GCT CAG ATG CTC GAT GAG GTG CGG CAC GCG CAG CTC GAG ATG 2698 Ala Ala Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met ACG CTA CGC AAC TAC TAC GCG AAG CAC TGG TGC GAT CCC TCC GGC 2743 Thr Leu Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly TTC GAC ATC GGT CAG CGC GGC CTG TAC CAG CAC CCC GCG GGG CTG 2788 Phe Asp Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu GTG TCC ATC GGC GAG TTC CAG CAC TTC AAT ACT GGT GAC CCG CTT 2833 Val Ser Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu GAC GTC ATC ATC GAT CTC AAC ATC GTG GCC GAG ACG GCG TTC ACG 2878 Asp Val Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr AAC ATC CTG CTG GTG GCC ACT CCA CAG GTC GCC GTG GCC AAC GGG 2923 Asn Ile Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly GAC AAC GCG ATG GCC AGC GTG TTC CTC TCG ATC CAG TCG GAC GAG 2968 Asp Asn Ala Met Ala Ser Val Phe Leu Ser Ile Gln Ser Asp Glu GCC AGG CAC ATG GCC AAC GGG TAC GGC TCG GTC ATG GCG CTG CTG 3013 Ala Arg His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu GAG AAC GAG GAC AAC CTC CCG CTG CTC AAC CAG TCT CTC GAT CGG 3058 Glu Asn Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg CAC TTC TGG CGT GCC CAC AAG GCC TTG GAC AAC GCG GTC GGA TGG 3103 His Phe Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp TGT TCG GAG TAT GGC GCC CGC AAG CGG CCA TGG AGC TAC AAG GCC 3148 Cys Ser Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala CAG TGG GAG GAA TGG GTC GTC GAC GAC TTC GTG GGC GGC TAC ATC 3193 Gln Trp Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile GAC CGA CTC AGC GAG TTC GGC GTT CAG GCT CCG GCC TGC CTT GGC 3238 Asp Arg Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly GCG GCC GCC GAC GAG GTC AAG TGG TCG CAC CAC ACG CTC GGT CAG 3283 Ala Ala Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln GTG CTG TCG GCG GTG TGG CCG CTG AAC TTC TGG CGC TCG GAC GCC 3328 Val Leu Ser Ala Val Trp Pro Leu Asn Phe Trp Arg Ser Asp Ala ATG GGA CCG GCG GAC TTC GAG TGG TTC GAG AAC CAC TAC CCG GGC 3373 Met Gly Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly TGG AGC GCG GCC TAC CAG GGT TAC TGG GAG GGC TAC AAG GCG CTC 3418 Trp Ser Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu GCC GAC CCA GCA GGC GGA CGC ATC ATG CTC CAG GAG CTG CCG GGT 3463 Ala Asp Pro Ala Gly Gly Arg Ile Met Leu Gln Glu Leu Pro Gly CTG CCG CCG ATG TGT CAG GTG TGC CAG GTG CCG TGC GTG ATG CCG 3508 Leu Pro Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro CGG CTG GAT ATG AAC GCC GCG CGG ATC ATC GAG TTC GAG GGG CAG 3553 Arg Leu Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gln AAA ATC GCG CTG TGC AGC GAA CCC TGC CAG CGG ATC TTC ACC AAC 3598 Lys Ile Ala Leu Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn TGG CCG GAG GCG TAC CGC CAC CGC AAG CAA TAC TGG GCC CGC TAC 3643 Trp Pro Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr CAC GGA TGG GAC CTG GCG GAC GTC ATC GTT GAT CTC GGC TAC ATC 3688 His Gly Trp Asp Leu Ala Asp Val Ile Val Asp Leu Gly Tyr Ile CGC CCG GAC GGC AAG ACC CTC ATC GGC CAG CCG CTG CTC GAG ATG 3733 Arg Pro Asp Gly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met GAG CGG CTG TGG ACC ATC GAC GAC ATC CGG GCC CTT CAG TAC GAA 3778 Glu Arg Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Glu GTC AAG GAC CCG TTG CAG GAG GCG TG ATG ACG ACG ATC AAT GTG 3822 Val Lys Asp Pro Leu Gln Glu Ala * * (amoD) Met Thr Thr Ile Asn Val CAG CCC TTC TCA CAC GAG TAC TCG TGC GAG GAC GGC GAG AGC CTC 3867 Gln Pro Phe Ser His Glu Tyr Ser Cys Glu Asp Gly Glu Ser Leu CTC GAC GGC GCC CTG CGC AAC AGC CTG CTC CTG AAG TAC GGG TGC 3912 Leu Asp Gly Ala Leu Arg Asn Ser Leu Leu Leu Lys Tyr Gly Cys AAG CAC GGG GGC TGC GGG ACC TGC AAG GTC CGG CTG CTC GAC GGC 3957 Lys His Gly Gly Cys Gly Thr Cys Lys Val Arg Leu Leu Asp Gly GAC GTA GAG GAA CCC GGG TCG TCG TTC GCG CTG ACG CCG GAG GAC 4002 Asp Val Glu Glu Pro Gly Ser Ser Phe Ala Leu Thr Pro Glu Asp CGC GAG AAC GAC GTG ATC CTC GCG TGC GCC AGC GTG CCG CTG GAA 4047 Arg Glu Asn Asp Val Ile Leu Ala Cys Ala Ser Val Pro Leu Glu CCG TGC ACC ATC GAC GTC GAG CCG AGC GGC CTC ACG GAG GAG GAG 4092 Pro Cys Thr Ile Asp Val Glu Pro Ser Gly Leu Thr Glu Glu Glu TTC TTC TCG GGC GAC ACC TCG CGC GAG TTC CAG ACG GTC GTG GGC 4137 Phe Phe Ser Gly Asp Thr Ser Arg Glu Phe Gln Thr Val Val Gly GGT GTC GAG TTT CTC ACG GCG GAC ATC GCC CGG GTC CGG CTC CGG 4182 Gly Val Glu Phe Leu Thr Ala Asp Ile Ala Arg Val Arg Leu Arg CTA GAG CCG GGC GAG GAG ATC GCC TTC ACC GCC GGT CAG TTC GTC 4227 Leu Glu Pro Gly Glu Glu Ile Ala Phe Thr Ala Gly Gln Phe Val AAC GTC GAG GTG CCG GGC ACG GGT CTG CTG CGG ACC TTC TCG CTG 4272 Asn Val Glu Val Pro Gly Thr Gly Leu Leu Arg Thr Phe Ser Leu GCA AAC GCC CCT GAC GAC CCG TCA GTG GTG GAG CTG ATC TGC AAG 4317 Ala Asn Ala Pro Asp Asp Pro Ser Val Val Glu Leu Ile Cys Lys CTC TAC CCG GAT GGC CTC TTC TCC CGC TTC CTG AGG GAC GAG GCT 4362 Leu Tyr Pro Asp Gly Leu Phe Ser Arg Phe Leu Arg Asp Glu Ala GCC CCG GGC ACG CCG GTC CGG GTG TTC GGG CCG TAT GGT CAG CTC 4407 Ala Pro Gly Thr Pro Val Arg Val Phe Gly Pro Tyr Gly Gln Leu AAG ATC CGC TTG TCC CAC CGG CCG ATC CTG ATG ATC GCC GGT GGG 4452 Lys Ile Arg Leu Ser His Arg Pro Ile Leu Met Ile Ala Gly Gly TCC GGT CTC GCC CCG CTG CTC TCG ATG CTG CGA GAC TTG GCC GCC 4497 Ser Gly Leu Ala Pro Leu Leu Ser Met Leu Arg Asp Leu Ala Ala AAG AAG TGC GAC CGG CCG GTC TCG ATG TTC TTC GGC GCA CGC AGC 4542 Lys Lys Cys Asp Arg Pro Val Ser Met Phe Phe Gly Ala Arg Ser GTC GAC GAC CTG TAC CTC ATC GAG GAG ATC CGC GAG ATC GGC GAG 4587 Val Asp Asp Leu Tyr Leu Ile Glu Glu Ile Arg Glu Ile Gly Glu TCG CTA GCC GAT TTC GAG TTC ATC CCC GTG CTC TCG GAG TCG TCG 4632 Ser Leu Ala Asp Phe Glu Phe Ile Pro Val Leu Ser Glu Ser Ser CCA GCC GAC TGG CAC GGC GAG ACG GGC ATG GTC ACC GAC GCC TTG 4677 Pro Ala Asp Trp His Gly Glu Thr Gly Met Val Thr Asp Ala Leu CTG CGG TGG CGC GCC GAA CTG GCG CAT GAC GTC TAC CTG TGC GGG 4722 Leu Arg Trp Arg Ala Glu Leu Ala His Asp Val Tyr Leu Cys Gly CCG CCA CCC ATG ATC GAC GCC GCT GTG CCG CTG CTC GTC GAG CGG 4767 Pro Pro Pro Met Ile Asp Ala Ala Val Pro Leu Leu Val Glu Arg GGC GTG CGC CCA CGC AAC ATC TAC TAC GAC GCA TTC ACC CCA GCT 4812 Gly Val Arg Pro Arg Asn Ile Tyr Tyr Asp Ala Phe Thr Pro Ala GCT CAG GTA GTC GTC GTC TGA TGG TGC ATA TCC GAT TGG GCG GCC 4857 Ala Gln Val Val Val Val * GGT ACC GGC GGG GTT AGG GCA GGG TAA TCG GCC GCG ATA GAG GCA 4902 GCC AGC AGA GAT GAT GCG CGT GCA AAG CGA AGC TTC GAC TCA GTT 4947 GAC ACC TTC GGC AAT GTG CTG TGC GAG CAC GAT AAC GGT GTG TTA 4992 CGG GTG AGG CTC AAT CGC ACC GGT GCC CTG AGC GCA ATC AAT TCA 5037 GCT ATG GCT GAT CAA TTG CGG GAA GCG TGG GTG TGG GTG CGC AGC 5082 CAA ACC GGA ATT CAT TCA ATC GTC ATC TCG GCT TCG CGG CCG GAG 5127 TCG TTT TTG CAT CGG TTT TGA TCC GGC CGA CCC GCC GGA GCC GAT 5172 GTT CGA TCG CCC CAT CAG CCC GAA GGA ATG CGG CGT CGA TCA ACG 5217 CGT CAT AGT CGC GGT GAA CGG CAT CGC GTG CCG CAT CCT GAG CGC 5262 CGG CGA ACC GAC ACT GCG TAA GCC GGT GAC GGC GGA ACA GGC GAT 5307 GGA GTC GGG CTT GGT TCG GGA GGT CGT ACC GCT TGC ATC GGC TGC 5352 GTT GTC GAG CGA ATA GCT CAG GCG GCA GGA CAT CGT ATA GGG ATT 5397 CAG GTG GGT AGT TGA TCT TCC GCG TGT CGA TGT GTG ACG GGC CGG 5442 ATC TGG CCT ACG AAC AAC ATC AGC AAG CGT GAC CTT CGG CCC ACA 5487 ACA TCG TGC CGT CCG CGA CCG GTG CCG ACC GTG ATC TCG GCG AAG 5532 TCC TGC CGG AGT TCG CGG GGC GGT TCC ACA GCG TTG GGG GTC CCT 5577 CGG CTA CAC CTC GGC GCT GTT CGT CAG CTC CGA CAT CAT CGG TGA 5622 CCC ACA CTC CTG CGC GGT GTT CGG CAA CAT GGT GCA CGA CGG CCA 5667 GGT GAA CCT TCG GCT GGT ACG ACA ACG AAT GGG CGA TGC CAA TCG 5712 GTT GCT CGA CCT CGT CGG GGT CAT GGC TGG CGT TCG CTG ACT AGG 5757 CGG CCT GTT CGT GCC GGC CGC AAC ATC CAG CCC GTG AGG AGT TGC 5802 GTA CCG GTC GGC GAG AAA CTC CAC GAC CCG GGC AAC CTC GTC GGG 5847 CTC ACC GAG TCG GCC GAC TGG GAT CTT CGC GAT GAC CCG ATC GAG 5892 CGC CTC CTT GGG CAC CGC AGC AAC TAT CTC GGT GGA GAT GTA GCC 5937 AGG CGG AAC GGA ATT GAC GGT GAT GCC CTT GCG GGC GGT CTC CTG 5982 GGC CAG GGT CTG TGT CCT TAT CTC TTG TCG ACC GAA CAC GGT GTG 6027 CGG TCG AGT TGG TCG GTT TGA TCA GGC GGG AAC GCG GAA TAG GAG 6072 GGC TTC GCC CTG ACC GCC GCC GCC GCA CAG GCC GGC CGC CCC GAC 6117 GCC GCC GCC GCG GCG GGC CAG CTC GAG CAC GAG ATG CAG CGC GAT 6162 CCG GGC GCC GGA GGC GCC GAC CGG GTG CCC CAT GGC GAT CGC GCC 6207 GCC GTT GAC GTT GAC GTT GTC GGG GCC GAT CTG CAG GTC GCG CAT 6252 GGA TTG AAT AGC GAC GGC CGC GAA TGC CTC GTT GAT CTC GTA CAG 6297 GTC CAC ATC CAT GGC GGA GAG TCC TTC GCG GCG CAG CGC CCT GGC 6342 GAT CGC GTT GGA GGG TTG CGA CAG CAG CGA GGG ATC C 6379Sequence number: 4 Sequence length: 6379 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Peptide Origin: Nocardia coralina B-276 (FERM P
-4094) 907 −1935 amo A 1935 −2285 amo B 2300−3802 amo C 3805−4830 amo D GGA TCC CCA GGT CCC GAG CCA CCG CGG CGA TCG ACT TCC CCG TCT 45 CAT GCA CGA TCC GCA CAG CCC CCT CAC GGA ACT CCC GGT CGT ACT 90 TTT CCG CTT CTC TGA CAT CCC TAC CCC TTA TAG CTG ATG CCT CCA 135 CGG TCT CGG GGA ATG CCA ATA TGA CTT GGT CAG ATC GTA GGC GGC 180 AAG AAC CAT GGC TTC CTT GGC GGA CTT CAC AGG GTC C TCC TCG 225 AGG GCT CGT TCG GGC ATG GGT AGC ACC GTC GAG CGA ACC CGA GGA 270 GAG GCC CCG ATC CGG GGA GGC GAT CGG GAT GAA CGG GGA CGT GTC 315 GAG AAT CGA TCA GAC TTG GTG GGC AGA TTC GTG TCC GCC GGT 360 CAG AAC CCG ATG GCC ATC CAC GGG CAC TTT CGT GGC CAT CAG TGG 405 GCG GTT TCG TGA CCC CCT ATG GGC AGT TTT CCA TGG CCG CCG ACA 450 CAC AGG CCC GCA CCG ATG ACG AGG TAC TGC ACG ACC CGG CCG ACC CGCG CGG TGG TTT GCT GCA ACA AAT GAC AAC TTG ATC AAC GCA TCG 540 ATC ACG ACC GGC GCC ACT AAC GCC GCC GCG GCA AGC ATC GAC ATC 585 AAC ACC ATC GCG ACC GTC CGC CCT TCA CAT CGA CGA CAC CTC GGT TG CGTGT CAT ACC GCA TG C ATC CGG CAA GAA CCC CGC GGA CTC 675 CGC AGC CGG TGC GGC TAG CCG ATT TCA CCC AAC CTG GGA CGT TGG 720 CTA CGG AAC GTC TCG TGG ATG ACG GCG CCT GCA TGC TGC ATA GAC 765 CCG TCC GTG GCC GCA ATG TCC TGCGG TGA GGC ATG CGA GCG GCT TCG 810 ATT CCT CTC GGC GGG GCC AAG ACC GGC GGG CCG CGC CTG TCT AAC 855 ATC GCG GCA CAA GGA AAT CGC AAC CGG ACC GAA ACG ATG GAA GGC 900 GTA GCG ATG ACG ACA GAG GCG GCG GTG GCG CGA CCG GTG GAG CTC 945 (amoA) Met Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu GAA GGT CAC CGG ACA TTC ACC TGG TTC ACG CCC GCC AGG CGA AAG 990 Glu Gly His Arg Thr Phe Thr Trp Phe Thr Pro Ala Arg Arg Lys CCG ACG GAG TAC GAG CTC TAC ACC GTG GGT CAA CAG TCC ACT CCG 1035 Pro Thr Glu Tyr Glu Leu Tyr Thr Val Gly Gln Gln Ser Thr Pro GAC GAG TGG CTG CAT GTG GAC TGG CCG CTG CGC TTC GAC GAC GGC 1080 Asp Glu Trp Leu His Val Asp Trp Pro Leu Arg Phe Asp Asp Gly CGC GCC CCG TGG GAG GAG GAG TCG AGT GCG GTA CGG ACC TCG GAG 1125 Arg Ala Pro Trp Glu Glu Glu Ser Ser Ala Val Arg Thr Ser Glu TGG TCG GCT TAC CGC GAC CCA CAC CAA CTG TGG CAG CGT CCC TAC 1170 Trp Ser Ala Tyr Arg Asp Pro His Gln Leu Trp Gln Arg Pro Tyr GTC AGC ACG TGC AGC CAG GAC CAG CAG GCC CTC GCG CGG CTG GTC 1215 Val Ser Thr Cys Asn Gln Asp Gln Gln Ala Leu Ala Arg Leu Val CCC GTC CTG ACC ATG GGG TCG GCG GCG ATC ACG CCC ATC TGG TCG 1260 Pro Val Leu Thr Met Gly Ser Ala Ala Ile Thr Pro Ile Trp Ser CAG AAG ATC CTC GCC AGG TCC TAC GCC GCC TGG CCA TTC GTC GAG 1305 Gln Lys Ile Leu Ala Arg Ser Tyr Ala Ala Trp Pro Phe Val Glu TAC GGG CTC TTC CTG AGC CTG GCC TAC GCC GTG CGC CAG GCC ATG 1350 Tyr Gly Leu Phe Leu Ser Leu Ala Tyr Ala Val Arg Gln Ala Met TCC GAC ACG GTC CAG TTC AGC GTG GTG TTC CAG GCC GTG GAC CGC 1395 Ser Asp Thr Val Gln Phe Ser Val Val Phe Gln Ala Val Asp Arg ATG CGG CTG CTC CAG GAC ATC GTC CAC CAC CTG GAC CAC CTG CAG 1440 Met Arg Leu Leu Gln Asp Ile Val His His Leu Asp His Leu Gln GAG TCG CCG GAA TTC AGC GAC GCC GGG GCC CGC GAG GCC TGG ATG 1485 Glu Ser Pro Glu Phe Ser Asp Ala Gly Ala Arg Glu Ala Trp Met TCC GAC TCC ACC CTG GTC CCG ATC CGG GAA GTG ATC GAG CGC ATC 1530 Ser Asp Ser Thr Leu Val Pro Ile Arg Glu Val Ile Glu Arg Ile GCC GCC AGC CAG GAC TGG GTG GAG ATC CTG GTC GCC GGC ACG CTC 1575 Ala Ala Ser Gln Asp Trp Val Glu Ile Leu Val Ala Gly Thr Leu GTC TTC GAG CCT CTG GTC GGC CAC CTG GCG AAG GCC GAG TTG TTC 1620 Val Phe Glu Pro Leu Val Gly His Leu Ala Lys Ala Glu Leu Phe AGC CGC CGT GCG CCA ATG TTC GGG GAC GGG ACC ACG CCG GCG GTG 1665 Ser Arg Arg Ala Pro Met Phe Gly Asp Gly Thr Thr Pro Ala Val CTG GCG TCG GCC CTG CTG GAC AGC GGC AGG CAC CTC GAA TCG GTC 1710 Leu Ala Ser Ala Leu Leu Asp Ser Gly Arg His Leu Glu Ser Val CAG GCG CTC GTC CGC CTC GTC TGC CAA GAC CCC GTC CAT GGC GAC 1755 Gln Ala Leu Val Arg Leu Val Cys Gln Asp Pro Val His Gly Asp CAG AAC CAG GCG ACT GTG CGG CGG TGG ATC GAG GAA TGG CAG CCG 1800 Gln Asn Gln Ala Thr Val Arg Arg Trp Ile Glu Glu Trp Gln Pro CGG TGC AAG GCG GCG GCC CAG TCC TTC CTG CCG ACG TTC TCC GAC 1845 Arg Cys Lys Ala Ala Ala Ala Gln Ser Phe Leu Pro Thr Phe Ser Asp TGC GGC ATC GAC GCC AAG GAA AGC GCC AAC GCG CTG TCC CGG GCG 1890 Cys Gly Ile Asp Ala Lys Glu Ser Ala Asn Ala Leu Ser Arg Ala CTG GCG AAC CAG CGG GCC GCC GTC GAG GGC GCC GGC ATC ACG GCA 1935 Leu Ala Asn Gln Arg Ala Val Glu Gly Ala Gly Ile Thr Ala (amoB) M TG ACA GAC GTT AAG GAG ACC ACT GTG ACC AGC ACC CCC TCG GCC 1979 * et Thr Asp Val Lys Glu Thr Thr Val Thr Ser Thr Pro Ser Ala GCC GTG CCG GGA ACC AAG AAC CGC CGG GTT GGT ATC TCG CTG ATC 2024 Ala Val Pro Gly Thr Lys Asn Arg Arg Val Gly Ile Ser Leu Ile AGC AGC AGC GAC ACC GAG GCA GCT GTC GAG CAC ATC GCG GAG ACC 2069 Ser Ser Ser Asp Thr Glu Ala Ala Val Glu His Ile Ala Glu Thr CAG CCG GAC GCG AAG ATC GAC TTT CGG GAC TGC TTC TAC AAG ATC 2114 Gln Pro Asp Ala Lys Ile Asp Phe Arg Asp Cys Phe Tyr Lys Ile GAG CGT GAC GGG CAG CTC AGT TTC GAC ATG GCA GAG CTC AGT GAG 2159 Glu Arg Asp Gly Gln Leu Ser Phe Asp Met Ala Glu Leu Ser Glu ATC GCC GGT CGC GAC ATC GAC ACC GAC ATC TTC CTG GTG AAC ATG 2204 Ile Ala Gly Arg Asp Ile Asp Thr Asp Ile Phe Leu Val Asn Met AGC ACC TAC TAC GGC CGG ATC GTC GTC AGT GAC GGC CGG GTC GAC 2249 Ser Thr Tyr Tyr Gly Arg Ile Val Val Ser Asp Gly Arg Val Asp ATC TAC GCC GAA ATC CAG CCG GCC CGC TTC AAG GAC TGA GAG GAA 2294 Ile Tyr Ala Glu Ile Gln Pro Ala Arg Phe Lys Asp * ACA CC ATG GCA TCG AAC CCC ACC CAG CTC CAC GAG AAG TCG AAG 2338 (amoC) Met Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys TCC TAC GAC TGG GAC TTC ACC TCC GTC GAG CGG CGC CCC AAG TTC 2383 Ser Tyr Asp Trp Asp Phe Thr Ser Val Glu Arg Arg Pro Lys Phe GAG ACG AAG TAC AAG ATG CCC AAG AAG GGC AAG GAC CCG TTC CGC 2428 Glu Thr Lys Tyr Lys Met Pro Lys Lys Gly Lys Asp Pro Phe Arg GTC CTG ATC CGT GAC TAC ATG AAG ATG GAA GCG GAG AAG GAC GAC 2473 Val Leu Ile Arg Asp Tyr Met Lys Met Glu Ala Glu Lys Asp Asp CGG ACC CAT GGC TTC CTC GAC GGC GCC GTG CGG ACG CGT GAG GCC 2518 Arg His Gly Phe Leu Asp Gly Ala Val Arg Thr Arg Glu Ala ACC AGG ATT GAG CCG CGG TTC GCT GAG GCC ATG AAG ATC ATG GTG 2563 Thr Arg Ile Glu Pro Arg Phe Ala Glu Ala Met Lys Ile Met Val CCG CAG CTG ACC AAC GCC GAG TAC CAG GCG GTG GCG GGC TGC GGA 2608 Pro Gln Leu Thr Asn Ala Glu Tyr Gln Ala Val Ala Gly Cys Gly ATG ATC ATC TCG GCC GTC GAG AAC CAG GAG CTC CGT CAG GGC TAC 2653 Met Ile Ile Ser Ala Val Glu Asn Gln Glu Leu Arg Gln Gly Tyr GCC GCT CAG ATG CTC GAT GAG GTG CGG CAC GCG CAG CTC GAG ATG 2698 Ala Ala Gln Met Leu Asp Glu Val Arg His Ala Gln Leu Glu Met ACG CTA CGC AAC TAC TAC GCG AAG CAC TGG TGC GCC TCC GGC 2743 Thr Leu Arg Asn Tyr Tyr Ala Lys His Trp Cys Asp Pro Ser Gly TTC GAC ATC GGT CAG CGC GGC CTG TAC CAG CAC CCC GCG GGG CTG 2788 Phe Asp Ile Gly Gln Arg Gly Leu Tyr Gln His Pro Ala Gly Leu GTG TCC ATC GGC GAG TTC CAG CAC TTC AAT ACT GGT GAC CCG CTT 2833 Val Ser Ile Gly Glu Phe Gln His Phe Asn Thr Gly Asp Pro Leu GAC GTC ATC ATC GAT CTC AAC ATC GTG GCC GAG ACG GCG TTC ACG 2878 Asp Val Ile Ile Asp Leu Asn Ile Val Ala Glu Thr Ala Phe Thr AAC ATC CTG CTG GTG GCC ACT CCA CAG GTC GCC GTG GCC AAC GGG 2923 Asn Ile Leu Leu Val Ala Thr Pro Gln Val Ala Val Ala Asn Gly GAC AAC GCG ATG GCC AGC GTG TTC CTC TCG ATC CAG TCG GAC GAG 2968 Asp Asn Ala Met Ala Ser Val Phe Leu Ser Ile Gln Ser Asp Glu GCC AGG CAC ATG GCC AAC GGG TAC GGC TCG GTC ATG GCG CTG CTG 3013 Ala Arg His Met Ala Asn Gly Tyr Gly Ser Val Met Ala Leu Leu GAG AAC GAG GAC AAC CTC CCG CTG CTC AAC CAG TCT CTC GAT CGG 3058 Glu Asn Glu Asp Asn Leu Pro Leu Leu Asn Gln Ser Leu Asp Arg CAC TTC TGG CGT GCC CAC AAG GCC TTG GAC AAC GCG GTC GGA TGG 3103 His Phe Trp Arg Ala His Lys Ala Leu Asp Asn Ala Val Gly Trp TGT TCG GAG TAT GGC GCC CGC AAG CGG CCA TGG AGC TAC AAG GCC 3148 Cys Ser Glu Tyr Gly Ala Arg Lys Arg Pro Trp Ser Tyr Lys Ala CAG TGG GAG GAA TGG GTC GTC GAC GAC TTC GTG GGC GGC TAC ATC 3193 Gln Trp Glu Glu Trp Val Val Asp Asp Phe Val Gly Gly Tyr Ile GAC CGA CTC AGC GAG TTC GGC GTT CAG GCT CCG GCC TGC CTT GGC 3238 Asp Arg Leu Ser Glu Phe Gly Val Gln Ala Pro Ala Cys Leu Gly GCG GCC GCC GAC GAG GTC AAG TGG TCG CAC CAC ACG CTC GGT CAG 3283 Ala Ala Ala Ala Asp Glu Val Lys Trp Ser His His Thr Leu Gly Gln GTG CTG TCG GCG GTG TGG CCG CTG AAC TTC TGG CGC TCG GAC GCC 3328 Val Leu Ser Ala Val Trp Pro Leu Asn Phe Trp Arg Ser Asp Ala ATG GGA CCG GCG GAC TTC GAG TGG TTC GAG AAC CAC TAC CCG GGC 3373 Met Gly Pro Ala Asp Phe Glu Trp Phe Glu Asn His Tyr Pro Gly TGG AGC GCG GCC TAC CAG GGT TAC TGG GAG GGC TAC AAG GCG CTC 3418 Trp Ser Ala Ala Tyr Gln Gly Tyr Trp Glu Gly Tyr Lys Ala Leu GCC GAC CCA GCA GGC GGA CGC ATC ATG CTC CAG GAG CTG CCG GGT 3463 Ala Asp Pro Ala Gly Gly Arg Ile Met Leu Gln Glu Leu Pro Gly CTG CCG CCG ATG TGT CAG GTG TGC CAG GTG CCG TGC GTG ATG CCG 3508 Leu Pro Pro Met Cys Gln Val Cys Gln Val Pro Cys Val Met Pro CGG CTG GAT ATG AAC GCC GCG CGG ATC ATC GAG TTC GAG GGG CAG 3553 Arg Leu Asp Met Asn Ala Ala Arg Ile Ile Glu Phe Glu Gly Gln AAA ATC GCG CTG TGC AGC GAA CCC TGC CAG CGG ATC TTC ACC AAC 3598 Lys Ile Ala Cys Ser Glu Pro Cys Gln Arg Ile Phe Thr Asn TGG CCG GAG GCG TAC CGC CAC CGC AAG CAA TAC TGG GCC CGC TAC 3643 Trp Pro Glu Ala Tyr Arg His Arg Lys Gln Tyr Trp Ala Arg Tyr CAC GGA TGG GAC CTG GCG GAC GTC ATC GTT GAT CTC GGC TAC ATC 3688 His Gly Trp Asp Leu Ala Asp Val Ile Val Asp Leu Gly Tyr Ile CGC CCG GAC GGC AAG ACC CTC ATC GGC CAG CCG CTG CTC GAG ATG 3733 Arg Pro Asp Gly Lys Thr Leu Ile Gly Gln Pro Leu Leu Glu Met GAG CGG CTG TGG ACC ATC GAC GAC ATC CGG GCC CTT CAG TAC GAA 3778 Glu Arg Leu Trp Thr Ile Asp Asp Ile Arg Ala Leu Gln Tyr Glu GTC AAG GAC CCG TTG CAG GAG GCG TG ATG ACG ATC AAT GTG 3822 Val Lys Asp Pro Leu Gln Glu Ala * * (amoD) Met Thr Thr Ile Asn Val CAG CCC TTC TCA CAC GAG TAC TCG TGC GAG GAC GGC GAG AGC CTC 3867 Gln Pro Phe Ser His Glu Tyr Ser Cys Glu Asp Gly Glu Ser Leu CTC GAC GGC GCC CTG CGC AAC AGC CTG CTC CTG AAG TAC GGG TGC 3912 Leu Asp Gly Ala Leu Arg Asn Ser Leu Leu Leu Lys Tyr Gly Cys AAG CAC GGG GGC TGC GGG ACC TGC AAG GTC CGG CTG CTC GAC G957 Lys His Gly Gly Cys Gly Thr Cys Lys Val Arg Leu Leu Asp Gly GAC GTA GAG GAA CCC GGG TCG TCG TTC GCG CTG ACG CCG GAG GAC 4002 Asp Val Glu Glu Pro Gly Ser Ser Phe Ala Leu Thr Pro Glu Asp CGC GAG AAC GAC G TG ATC CTC GCG TGC GCC AGC GTG CCG CTG GAA 4047 Arg Glu Asn Asp Val Ile Leu Ala Cys Ala Ser Val Pro Leu Glu CCG TGC ACC ATC GAC GTC GAG CCG AGC GGC CTC ACG GAG GAG GAG 4092 Pro Cys Thr Ile Asp Val Glu Pro Ser Gly Leu Thr Glu Glu Glu TTC TTC TCG GGC GAC ACC TCG CGC GAG TTC CAG ACG GTC GTG GGC 4137 Phe Phe Ser Gly Asp Thr Ser Arg Glu Phe Gln Thr Val Val Gly GGT GTC GAG TTT CTC ACG GCG GAC ATC GCC CGG GTC CGG CTC CGG 4182 Gly Val Glu Phe Leu Thr Ala Asp Ile Ala Arg Val Arg Leu Arg CTA GAG CCG GGC GAG GAG ATC GCC TTC ACC GCC GGT CAG TTC GTC 4227 Leu Glu Pro Gly Glu Glu Ile Ala Phe Thr Ala Gly Gln Phe Val AAC GTC GAG GTG CCG GGC ACG GGT CTG CTG CGG ACC TTC TCG CTG 4272 Asn Val Glu Val Pro Gly Thr Gly Leu Leu Arg Thr Phe Ser Leu GCA AAC GCC CCT GAC GAC CCG TCA GTG GTG GAG CTG ATC TGC AAG 4317 Ala Asn Ala Pro Asp Asp Pro Ser Val Val Glu Leu Ile Cys Lys CTC TAC CCG GAT GGC CTC TTC TCC CGC TTC CTG AGG GAC GAG GCT 4362 Leu Tyr Pro Asp Gly Leu Phe Ser Arg Phe Leu Arg Asp Glu Ala GCC CCG GGC ACG C CG GTC CGG GTG TTC GGG CCG TAT GGT CAG CTC 4407 Ala Pro Gly Thr Pro Val Arg Val Phe Gly Pro Tyr Gly Gln Leu AAG ATC CGC TTG TCC CAC CGG CCG ATC CTG ATG ATC GCC GGT GGG 4452 Lys Ile Arg Leu Ser His Arg Pro Ile Leu Met Ile Ala Gly Gly TCC GGT CTC GCC CCG CTG CTC TCG ATG CTG CGA GAC TTG GCC GCC 4497 Ser Gly Leu Ala Pro Leu Leu Ser Met Leu Arg Asp Leu Ala Ala AAG AAG TGC GAC CGG CCG GTC TCG ATG TTC TTC GGC GCA CGC AGC 4542 Lys Lys Cys Asp Arg Pro Val Ser Met Phe Phe Gly Ala Arg Ser GTC GAC GAC CTG TAC CTC ATC GAG GAG ATC CGC GAG ATC GGC GAG 4587 Val Asp Asp Leu Tyr Leu Ile Glu Glu Ile Arg Glu Ile Gly Glu TCG CTA GCC GAT TTC GAG TTC ATC CCC GTG CTC TCG GAG TCG TCG 4632 Ser Leu Ala Asp Phe Glu Phe Ile Pro Val Leu Ser Glu Ser Ser CCA GCC GAC TGG CAC GGC GAG ACG GGC ATG GTC ACC GAC GCC TTG 4677 Pro Ala Asp Trp His Gly Glu Thr Gly Met Val Thr Asp Ala Leu CTG CGG TGG CGC GCC GAA CTG GCG CAT GAC GTC TAC CTG TGC GGG 4722 Leu Arg Trp Arg Ala Glu Leu Ala His Asp Val Tyr Leu Cys Gly CCG CCA CCC ATG A TC GAC GCC GCT GTG CCG CTG CTC GTC GAG CGG 4767 Pro Pro Pro Met Ile Asp Ala Ala Val Pro Leu Leu Val Glu Arg GGC GTG CGC CCA CGC AAC ATC TAC TAC GAC GCA TTC ACC CCA GCT 4812 Gly Val Arg Pro Arg Asn Ile Tyr Tyr Asp Ala Phe Thr Pro Ala GCT CAG GTA GTC GTC GTC TGA TGG TGC ATA TCC GAT TGG GCG GCC 4857 Ala Gln Val Val Val Val * GGT ACC GGC GGG GTT AGG GCA GGG TAA TCG GCC GCG ATA GAG GCA 4902 GCC AGC AGA GAT GAT GCG CGT GCA AAG CGA AGC TTC GAC TCA GTT 4947 GAC ACC TTC GGC AAT GTG CTG TGC GAG CAC GAT AAC GGT GTG TTA 4992 CGG GTG AGG CTC AAT CGC ACC GGT GCC CTG AGC GCA ATC AAT TCA 5037 GCT ATG GCT GAT GAT TTG CGG GAA GCG TGG GTG TGG GTG CGC AGC 5082 CAA ACC GGA ATT CAT TCA ATC GTC ATC TCG GCT TCG CGG CCG GAG 5127 TCG TTT TTG CAT CGG TTT TGA TCC GGC CGA CCC GCC GGA GCC GAT 5172 GTT CGA TCG CCCCAT GAA GGA ATG CGG CGT CGA TCA ACG 5217 CGT CAT AGT CGC GGT GAA CGG CAT CGC GTG CCG CAT CCT GAG CGC 5262 CGG CGA ACC GAC ACT GCG TAA GCC GGT GAC GGC GGA ACA GGC GAT 5307 GGA GTC GGG CTT GGT TCG GG A GGT CGT ACC GCT TGC ATC GGC TGC 5352 GTT GTC GAG CGA ATA GCT CAG GCG GCA GGA CAT CGT ATA GGG ATT 5397 CAG GTG GGT AGT TGA TCT TCC GCG TGT CGA TGT GTG ACG GGC CGG 5442 ATC TGG CCT ACG AGC ATC ATC AAG CGT GAC CTT CGG CCC ACA 5487 ACA TCG TGC CGT CCG CGA CCG GTG CCG ACC GTG ATC TCG GCG AAG 5532 TCC TGC CGG AGT TCG CGG GGC GGT TCC ACA GCG TTG GGG GTC CCT 5577 CGG CTA CAC CTC GGC GTC CTC CGT CGA CAT CAT CGG TGA 5622 CCC ACA CTC CTG CGC GGT GTT CGG CAA CAT GGT GCA CGA CGG CCA 5667 GGT GAA CCT TCG GCT GGT ACG ACA ACG AAT GGG CGA TGC CAA TCG 5712 GTT GCT CGA CCT CGT CGG GGT CAT GGC TGT CTG ACT AGG 5757 CGG CCT GTT CGT GCC GGC CGC AAC ATC CAG CCC GTG AGG AGT TGC 5802 GTA CCG GTC GGC GAG AAA CTC CAC GAC CCG GGC AAC CTC GTC GGG 5847 CTC ACC GAG TCG GCC GAC TGG GAT CTT CGC GAT GTC CC AGC GAG 5892 CGC CTC CTT GGG CAC CGC AGC AAC TAT CTC GGT GGA GAT GTA GCC 5937 AGG CGG AAC GGA ATT GAC GGT GAT GCC CTT GCG GGC GGT CTC CTG 5982 GGC CAG GGT CTG TGT CCT TAT CTC TTG TCG ACC GAA CAC GGT GTG 6027 CGG TCG AGT TGG TCG GTT TGA TCA GGC GGG AAC GCG GAA TAG GAG 6072 GGC TTC GCC CTG ACC GCC GCC GCC GCA CAG GCC GGC CGC CCC GAC 6117 GCC GCC GCC GCG GCG GGC CAG CTC GAG CAC GAG ATG CAG CGC 6162 CCG GGC GCC GGA GGC GCC GAC CGG GTG CCC CAT GGC GAT CGC GCC 6207 GCC GTT GAC GTT GAC GTT GTC GGG GCC GAT CTG CAG GTC GCG CAT 6252 GGA TTG AAT AGC GAC GGC CGC GAA TGC CTC GTT GAT CTC GTC CAG CAC ATC CAT GGC GGA GAG TCC TTC GCG GCG CAG CGC CCT GGC 6342 GAT CGC GTT GGA GGG TTG CGA CAG CAG CGA GGG ATC C 6379

【００３２】配列番号：5 配列の長さ：12 配列の型 ：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 1 2 3 4 5 6 7 8 9 10 11 12 subunit 1 Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu ACI ACI GAA GCI ACI GTI GCI CGI CCI GTI GA G ASEQ ID NO: 5 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide 1 2 3 4 5 6 7 8 9 10 11 12 subunit 1 Thr Thr Glu Ala Thr Val Ala Arg Pro Val Glu Leu ACI ACI GAA GCI ACI GTI GCI CGI CCI GTI GA GA

【００３３】配列番号：6 配列の長さ：12 配列の型 ：アミノ酸 トポロジー：直鎖状 配列の種類：ペプチド 1 2 3 4 5 6 7 8 9 10 11 12 subunit 2 Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys AAT CCI ACI CAI CTI CAT GAI AA C T CSEQ ID NO: 6 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide 1 2 3 4 5 6 7 8 9 10 11 12 subunit 2 Ala Ser Asn Pro Thr Gln Leu His Glu Lys Ser Lys AAT CCI ACI CAI CTI CAT GAI AA CTC

【図面の簡単な説明】[Brief description of the drawings]

【図１】本発明の実施例に示したベクター pDBB-1 の制
限酵素地図である。FIG. 1 is a restriction map of a vector pDBB-1 shown in Examples of the present invention.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ Ｃ１２Ｒ 1:19） Ｃ１２Ｒ 1:365 (Ｃ１２Ｎ 9/02 Ｃ１２Ｎ 15/00 Ａ Ｃ１２Ｒ 1:19） (Ｃ１２Ｎ 15/09 Ｃ１２Ｒ 1:365） (Ｃ１２Ｐ 17/02 Ｃ１２Ｒ 1:19） (56)参考文献 Ｊ Ｇｅｎ Ｍｉｃｒｏｂｉｏｌ, 1991，Ｖｏｌ．137，ｐ．2555−60 Ｊ Ｂａｃｔｅｒｉｏｌ，1992，Ｖｏ ｌ．174，ｐ．3275−3281 (58)調査した分野(Int.Cl.7，ＤＢ名) C12N 9/02 C12N 15/31 C12N 15/53 C12P 17/02 ＢＩＯＳＩＳ／ＷＰＩ（ＤＩＡＬＯＧ) ＰｕｂＭｅｄ ＳｗｉｓｓＰｒｏｔ／ＰＩＲ／ＧｅｎｅＳ ｅｑ ＧｅｎＢａｎｋ／ＥＭＢＬ／ＤＤＢＪ／Ｇ ｅｎｅＳｅｑ──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 7 Identification symbol FI C12R 1:19) C12R 1: 365 (C12N 9/02 C12N 15/00 A C12R 1:19) (C12N 15/09 C12R 1: 365) (C12P 17/02 C12R 1:19) (56) References J Gen Microbiol, 1991, Vol. 137, p. 2555-60 J Bacteriol, 1992, Vol. 174, p. 3275-3281 (58) Fields surveyed (Int. Cl. 7 , DB name) C12N 9/02 C12N 15/31 C12N 15/53 C12P 17/02 BIOSIS / WPI (DIALOG) PubMed SwissProt / PIR / GeneSeqGenBank / EMBL / DDBJ / GeneSeq

Claims (7)

(57)【特許請求の範囲】(57) [Claims] 【請求項１】 下記式１及び式２のアミノ酸配列を含
み、アルケンに酸素を添加する能力を有するモノオキシ
ゲナーゼ。 【式１】 【式１のつづき】 【式２】 【式２のつづき】 【式２のつづき】 1. A monooxygenase comprising an amino acid sequence represented by the following formulas 1 and 2, and having an ability to add oxygen to an alkene. (Equation 1) [Continuation of Equation 1] (Equation 2) [Continuation of Equation 2] [Continuation of Equation 2] 【請求項２】 請求項１記載のモノオキシゲナーゼのア
ミノ酸配列をコードする塩基配列を含むことを特徴とす
るアルケンモノオキシゲナーゼ遺伝子。Wherein alkene monooxygenase gene which comprises a nucleotide sequence encoding the amino acid sequence of the monooxygenase according to claim 1, wherein. 【請求項３】 下記式３のアミノ酸配列を含み、NADH補
酵素からモノオキシゲナーゼに電子を伝達する能力を有
するレダクダーゼ。 【式３】 【式３のつづき】 3. A comprises the amino acid sequence of formula 3, Redakudaze having the ability to transfer electrons to the monooxygenase from NADH coenzyme. (Equation 3) [Continuation of Equation 3] 【請求項４】 請求項３記載のレダクダーゼのアミノ酸
配列をコードする塩基配列を含むことを特徴とするアル
ケンモノオキシゲナーゼ遺伝子。4. alkene monooxygenase gene which comprises a nucleotide sequence encoding the amino acid sequence of Redakudaze of claim 3, wherein. 【請求項５】 下記式４の塩基配列を含有することを特
徴とするアルケンモノオキシゲナーゼ遺伝子。 【式４】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 【式４のつづき】 〔式中（amoA)、（amoB)、(amoC)、（amoD)は、DNAの塩
基配列に対応するアミノ酸配列を示し、また＊は翻訳の
終止コドンを示す〕5. The alkene monooxygenase gene characterized by containing the nucleotide sequence under following formula 4. (Equation 4) [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Continuation of Equation 4] [Wherein (amoA), (amoB), (amoC), and (amoD) indicate the amino acid sequence corresponding to the DNA base sequence, and * indicates the translation termination codon. 【請求項６】 請求項５記載のアルケンモノオキシゲナ
ーゼ遺伝子で形質転換した微生物。6. A microorganism transformed with the alkene monooxygenase gene according to claim 5. 【請求項７】 請求項６記載の形質転換微生物を用いて
アルケンをエポキシ化することを特徴とするアルケンの
エポキシ化方法。7. A method for epoxidizing an alkene, comprising epoxidizing the alkene using the transformed microorganism according to claim 6.
JP10517193A 1993-04-06 1993-04-06 Alkene monooxygenase, gene encoding the same, transformed microorganism and alkene epoxidation method Expired - Fee Related JP3330670B2 (en)

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EP1106699A1 (en) * 1999-11-18 2001-06-13 Eidgenössische Technische Hochschule Zürich Biocatalytic epoxidation of vinylaromatic compounds

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Ｊ Ｂａｃｔｅｒｉｏｌ，１９９２，Ｖｏｌ．１７４，ｐ．３２７５−３２８１
Ｊ Ｇｅｎ Ｍｉｃｒｏｂｉｏｌ，１９９１，Ｖｏｌ．１３７，ｐ．２５５５−６０

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