JPH03251185A - Production of heat-resistant beta-amylase - Google Patents
Production of heat-resistant beta-amylaseInfo
- Publication number
- JPH03251185A JPH03251185A JP4847790A JP4847790A JPH03251185A JP H03251185 A JPH03251185 A JP H03251185A JP 4847790 A JP4847790 A JP 4847790A JP 4847790 A JP4847790 A JP 4847790A JP H03251185 A JPH03251185 A JP H03251185A
- Authority
- JP
- Japan
- Prior art keywords
- plasmid
- amylase
- gene
- thermostable
- cell wall
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010019077 beta-Amylase Proteins 0.000 title claims abstract description 58
- 238000004519 manufacturing process Methods 0.000 title claims description 12
- 239000013612 plasmid Substances 0.000 claims abstract description 77
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 12
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 12
- 239000004382 Amylase Substances 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 3
- 241000193764 Brevibacillus brevis Species 0.000 claims abstract 11
- 108090000623 proteins and genes Proteins 0.000 claims description 45
- 210000002421 cell wall Anatomy 0.000 claims description 27
- 239000012634 fragment Substances 0.000 claims description 9
- 241001468159 Thermoanaerobacterium thermosulfurigenes Species 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 6
- 108010065511 Amylases Proteins 0.000 abstract description 4
- 102000013142 Amylases Human genes 0.000 abstract description 4
- 235000019418 amylase Nutrition 0.000 abstract description 4
- 229940041514 candida albicans extract Drugs 0.000 abstract description 3
- 238000007796 conventional method Methods 0.000 abstract description 3
- 239000012138 yeast extract Substances 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 abstract 1
- 230000002463 transducing effect Effects 0.000 abstract 1
- 108091008146 restriction endonucleases Proteins 0.000 description 9
- 238000013519 translation Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
- 239000012506 Sephacryl® Substances 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- BZSALXKCVOJCJJ-IPEMHBBOSA-N (4s)-4-[[(2s)-2-acetamido-3-methylbutanoyl]amino]-5-[[(2s)-1-[[(2s)-1-[[(2s,3r)-1-[[(2s)-1-[[(2s)-1-[[2-[[(2s)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-2-oxoethyl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-hydroxy Chemical compound CC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCC)C(=O)N[C@@H](CCCC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(=O)N[C@H](C(N)=O)CC1=CC=CC=C1 BZSALXKCVOJCJJ-IPEMHBBOSA-N 0.000 description 1
- 101000651036 Arabidopsis thaliana Galactolipid galactosyltransferase SFR2, chloroplastic Proteins 0.000 description 1
- 241000272201 Columbiformes Species 0.000 description 1
- 102100040870 Glycine amidinotransferase, mitochondrial Human genes 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101000893303 Homo sapiens Glycine amidinotransferase, mitochondrial Proteins 0.000 description 1
- 101000804764 Homo sapiens Lymphotactin Proteins 0.000 description 1
- 102100035304 Lymphotactin Human genes 0.000 description 1
- 108700010674 N-acetylVal-Nle(7,8)- allatotropin (5-13) Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- WCDYMMVGBZNUGB-ORPFKJIMSA-N [(2r,3r,4s,5r,6r)-6-[[(1r,3r,4r,5r,6r)-4,5-dihydroxy-2,7-dioxabicyclo[4.2.0]octan-3-yl]oxy]-3,4,5-trihydroxyoxan-2-yl]methyl 3-hydroxy-2-tetradecyloctadecanoate Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](COC(=O)C(CCCCCCCCCCCCCC)C(O)CCCCCCCCCCCCCCC)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H]2OC[C@H]2O1 WCDYMMVGBZNUGB-ORPFKJIMSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- -1 katyonyx Substances 0.000 description 1
- 125000003071 maltose group Chemical group 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 229930028731 β-maltose Natural products 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、耐熱性β−アミラーゼ遺伝子を導入したプラ
スミド、このプラスミドをバチルス プレビスに導入し
た形質転換体及びこの形質転換体を用いた耐熱性β−ア
ミラーゼの製造方法に関する。[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a plasmid into which a heat-stable β-amylase gene is introduced, a transformant into which this plasmid is introduced into Bacillus plebis, and a heat-stable product using this transformant. The present invention relates to a method for producing β-amylase.
β−アミラーゼは、デンプンに作用してその非還元末端
からマルトース単位にα−１，４−グルコシド結合を切
断し、β−マルトースを生成する酵素である。β-Amylase is an enzyme that acts on starch to cleave α-1,4-glucoside bonds from its non-reducing end to maltose units, producing β-maltose.
現在、工業的には、約６０°Ｃで使用可能な大豆由来の
β−アミラーゼが主に使用されている。Currently, soybean-derived β-amylase, which can be used at about 60°C, is mainly used industrially.
それに対して、本発明者らは、より高い温度で安定な耐
熱性β−アミラーゼについて研究し、耐熱性β−アミラ
ーゼの遺伝子の塩基配列を解明した〔特開平１−２１８
５８９号〕。In contrast, the present inventors conducted research on thermostable β-amylase that is stable at higher temperatures, and elucidated the base sequence of the gene for thermostable β-amylase [JP-A-1-218
No. 589].
本発明の目的は、上記耐熱性β−アミラーゼの遺伝子を
用いて、耐熱性β〜アミラーゼを効率良く生産すること
である。An object of the present invention is to efficiently produce thermostable β-amylase using the above gene for thermostable β-amylase.
本発明の目的は、より具体的には、耐熱性β−アミラー
ゼを効率良く生産することができる、耐熱性β−アミラ
ーゼの遺伝子を含むプラスミド、このプラスミドを導入
した微生物及びこの微生物を用いた耐熱性β−アミラー
ゼの製造方法を提供することにある。More specifically, the purpose of the present invention is to provide a plasmid containing a gene for heat-stable β-amylase that can efficiently produce heat-stable β-amylase, a microorganism into which this plasmid is introduced, and a heat-stable microorganism using this microorganism. An object of the present invention is to provide a method for producing a β-amylase.
本発明は、（ａ）枯草菌に生育し得るプラスミドに耐熱
性β−アミラーゼを導入したプラスミド、（ｂｌこのプ
ラスミドをバチルス プレビス（Ｂａｃｉｌｌｕｓ ｂ
ｒｅｖｉｓ） （以下、Ｂ、プレビスと略記することが
ある）に導入した形質転換体、及び（Ｃ）この形質転換
体を培養し、培養物から耐熱性β−アミラーゼを採取す
る耐熱性β−アミラーゼの製造方法に関する。The present invention provides (a) a plasmid in which heat-stable β-amylase has been introduced into a plasmid that can grow in Bacillus subtilis, (bl) this plasmid, and
revis) (hereinafter sometimes abbreviated as B, previs), and (C) the transformant is cultured and the thermostable β-amylase is collected from the culture. Relating to a manufacturing method.
以下本発明をさらに説明する。The present invention will be further explained below.
（ａ）プラスミド
本発明のプラスミドは、枯草菌又はＢ、プレビスに生育
し得るプラスミドに耐熱性β−アミラーゼを導入したプ
ラスミドである。枯草菌に生育し得るプラスミドとして
は、ｐＵＢｌｌｏ、ｐｃ１９４、ｐＥ１９４等を例示で
きる。(a) Plasmid The plasmid of the present invention is a plasmid in which thermostable β-amylase is introduced into a plasmid that can grow in Bacillus subtilis or B. plebis. Examples of plasmids that can grow in Bacillus subtilis include pUBllo, pc194, and pE194.
本発明のプラスミドは、上記枯草菌に生育し得るプラス
ミドに耐熱性β−アミラーゼを導入したプラスミドに、
Ｂ、プレビスの細胞壁タンパク質遺伝子の一部または全
部をさらに導入したプラスミドも包含する。Ｂ、プレビ
スの細胞壁タンパク質遺伝子の一部または全部を導入し
た枯草菌に生育し得るプラスミドとしては、プラスミド
ｐＮＵ２００を例示できる。The plasmid of the present invention is a plasmid in which heat-stable β-amylase is introduced into the plasmid capable of growing in Bacillus subtilis.
It also includes a plasmid into which part or all of the B. plebis cell wall protein gene has been further introduced. Plasmid pNU200 is an example of a plasmid that can grow in Bacillus subtilis into which part or all of the B. plebis cell wall protein gene has been introduced.
プラスミドｐＮＵ２００は、枯草菌のプラスミドである
ｐＵＢ １１０とＢ、プレビスの細胞壁タンパク質遺伝
子の５°位制御領域を含む多コピープラスミドであり、
鵜高重三、日本農芸化学会誌６１６６９ （１９８７）
及びＴ、カワズ（Ｋａｗａｚｕ）ら、Ｊ、Ｂａｃｔｅｒ
ｉｏｌ、、 １６９． １５６４（１９８７）に記載
の方法により得ることができる。Plasmid pNU200 is a multicopy plasmid containing the Bacillus subtilis plasmids pUB 110 and B, and the 5° regulatory region of the cell wall protein gene of Bacillus plebis.
Shigezo Udaka, Journal of the Japanese Society of Agricultural Chemistry 61669 (1987)
and T., Kawazu et al., J., Bacter.
iol,, 169. 1564 (1987).
一方、耐熱性β−アミラーゼ遺伝子は、クロストリジラ
ム サーモサルフロゲネス（Ｃｌｏｓｔｒｉｄｉｕｍゲ
ネスと略記することがある）ＡＴＣＣ３３７３３から得
られ、例えばプラスミドｐＮＫ１に含まれている。ｐＮ
Ｋｌを例えば制限酵素ＭｆｌＩで切断することにより、
耐熱性β−アミラーゼ遺伝子を含むＤＮＡ断片を得るこ
とができる。尚、プラスミドｐＮＫ１は、特開平１−２
１８５８９号に記載の方法により得ることができる。On the other hand, the thermostable β-amylase gene is obtained from Clostridium thermosulfologenes (sometimes abbreviated as Clostridium) ATCC33733, and is contained in, for example, plasmid pNK1. pN
For example, by cleaving Kl with the restriction enzyme MflI,
A DNA fragment containing the thermostable β-amylase gene can be obtained. In addition, plasmid pNK1 is disclosed in Japanese Patent Application Laid-open No. 1-2
It can be obtained by the method described in No. 18589.
本発明のプラスミドの１例として、上記プラスミドｐＮ
ＫｌのＭｆｌＩ−ＭｆｌＩ約３．６Ｋｂ断片の一部また
は全部を切出し、プラスミドｐＮＵ２００（７）Ｂａｍ
ＨＩ部位を連結することにより得られるプラスミドを挙
げられる。このようなプラスミドの内、ｐＮＫｌのＭｆ
ｌＩ−ＭｆｌＩ約３．６Ｋｂ断片の全部を含むプラスミ
ドを、以下ｐＭＭ１と呼ぶ。As an example of the plasmid of the present invention, the above plasmid pN
Part or all of the MflI-MflI approximately 3.6 Kb fragment of Kl was excised, and plasmid pNU200(7) Bam
Examples include plasmids obtained by ligating HI sites. Among these plasmids, Mf of pNKl
The plasmid containing the entire lI-MflI approximately 3.6 Kb fragment is hereinafter referred to as pMM1.
さらに本発明は、細胞壁タンパク質遺伝子の内、５８６
塩基以降の塩基を欠く細胞壁タンパク質遺伝子を含むプ
ラスミドｐＮＵ２００の細胞壁タンパク質遺伝子の直後
に耐熱性β−アミラーゼ構造遺伝子を連結したプラスミ
ドも包含する。このプラスミドを、以下ｐＭＭ２と呼ぶ
。Furthermore, the present invention provides that among cell wall protein genes, 586
It also includes a plasmid in which a thermostable β-amylase structural gene is linked immediately after the cell wall protein gene of plasmid pNU200, which contains the cell wall protein gene lacking the bases after the base. This plasmid is hereinafter referred to as pMM2.
さらに本発明は、細胞壁タンパク質遺伝子の内、５１６
塩基以降の塩基を欠く細胞壁タンパク質遺伝子を含むプ
ラスミドｐＮＵ２００の細胞壁タンパク質遺伝子の直後
に耐熱性β−アミラーゼのシグナル遺伝子及び構造遺伝
子を連結したプラスミドも包含する。このプラスミドを
、以下ｐＭＭ３と呼ぶ。Furthermore, the present invention provides that 516 of the cell wall protein genes
Also included is a plasmid in which the signal gene and structural gene of thermostable β-amylase are linked immediately after the cell wall protein gene of plasmid pNU200, which contains the cell wall protein gene lacking the bases after the base. This plasmid is hereinafter referred to as pMM3.
９ＭＭ２及びｐ Ｍ Ｍ ３は、以下のようにして調製
できる。9MM2 and pMM3 can be prepared as follows.
細胞壁タンパク質遺伝子のシグナル切断部位以前とβ−
アミラーゼのシグナル切断部位以後を合成りＮＡを用い
て結合させることにより、９ＭＭ２を構築することがで
きる。Before signal cleavage site of cell wall protein gene and β-
9MM2 can be constructed by synthesizing the region after the signal cleavage site of amylase and linking it using NA.
また、両遺伝子の翻訳開始部位に制限酵素ＢｓｐＨＩの
認識部位を導入後、５ｐｅｌ−Ｂｓｐ旧、ＢｓｐＨｌ−
３ｐｈｌ断片をｐ Ｍ Ｍ ２の５ｐｅｌ −５ｐｈ１
部位に挿入することにより、ｐＭＭ３を構築することが
できる。In addition, after introducing the recognition site of the restriction enzyme BspHI into the translation start site of both genes, 5pel-Bsp old, BspHI-
3phl fragment to pM M2 5pel -5ph1
pMM3 can be constructed by inserting into the site.
（ｂ）上記プラスミドを導入した形質転換本発明は、上
記プラスミドを８．プレビスに導入した形質転換体も包
含する。Ｂ、プレビスとしては、例えば、４７に株（Ｆ
ＥＲＭ ＢＰ−２３０８）、４７株（ＦＥＲＭ Ｐ
−７２２４）、ＨＯ１４株（ＦＥＲＭ Ｐ−８８９１
）、８１４０２株（ＦＥＲＭ Ｐ−８８９２）、ＨＯ
２３株（Ｆ Ｅ ＲＭＰ−８８９３）、８１４４株等を
挙げることができる。(b) Transformation into which the above-mentioned plasmid has been introduced In the present invention, the above-mentioned plasmid is introduced into 8. It also includes transformants introduced into Previs. B. As a previs, for example, 47 stocks (F
ERM BP-2308), 47 stocks (FERM P
-7224), HO14 strain (FERM P-8891
), 81402 strain (FERM P-8892), HO
Examples include 23 strains (FERMP-8893) and 8144 strains.
さらに、本発明のプラスミドは、Ｂ、プレビス以外に、
枯草菌やその他、このプラスミドが生育し得る細菌中に
導入することもできる。そのような細菌とができる。Furthermore, the plasmid of the present invention, in addition to B. previs,
It can also be introduced into Bacillus subtilis and other bacteria in which this plasmid can grow. Such bacteria can.
本発明のプラスミドのＢ、プレビス等への導入は、Ｊ、
Ｂａｃｔｅｒ ｉｏ １． 、１５６ ：１１３０−
１１３４（１９８３）に記載の方法により行うことがで
きる。The plasmid of the present invention can be introduced into B, Previs, etc.
Bacterio 1. , 156:1130-
1134 (1983).
（Ｃ）耐熱性 −アミラーゼの製造方法さらに、本発明
は、上記本発明のプラスミドをＢ、プレビス等に導入し
た形質転換体を培養し、培養物から耐熱性β−アミラー
ゼを採取する耐熱性β−アミラーゼの製造方法も包含す
る。(C) Thermostable - Method for producing amylase Furthermore, the present invention provides thermostable β-amylase in which a transformant in which the plasmid of the present invention is introduced into B, Previs, etc. is cultured, and thermostable β-amylase is collected from the culture. - Also includes a method for producing amylase.
本発明の形質転換体の培養は、例えばトリプトン、酵母
抽出物、カッオニキス等を含む培養液中、３０〜３７℃
、５〜６日間行うことが適当である。The transformant of the present invention can be cultured at 30 to 37°C in a culture solution containing, for example, tryptone, yeast extract, blackberry extract, etc.
, it is appropriate to carry out the treatment for 5 to 6 days.
培養物からの耐熱性β−アミラーゼの採取は、常法、例
えば硫安沈澱法等により行うことができる。Thermostable β-amylase can be collected from the culture by a conventional method, such as ammonium sulfate precipitation.
さらに、得られた耐熱性β−アミラーゼは、さらに菌体
外酵素取得の常法により、精製することができる。Furthermore, the obtained thermostable β-amylase can be further purified by a conventional method for obtaining extracellular enzymes.
本発明の形質転換体を用いる耐熱性β−アミラーゼの製
造方法によれば、大量に耐熱性β−アミラーゼを製造す
ることができる。According to the method for producing thermostable β-amylase using the transformant of the present invention, thermostable β-amylase can be produced in large quantities.
以下、本発明を実施例によりさらに説明する。 The present invention will be further explained below with reference to Examples.
ただし、本発明は、これらの実施例に限定されるもので
はない。However, the present invention is not limited to these examples.
実施例１ ｐＭＭｌの調製及び培養
鵜高重三、日本農芸化学会誌６１．６６９（１９８７）
に記載の方法により枯草菌プラスミドｐＵＢＩＩＯとＢ
、プレビスの細胞壁タンパク質遺伝子とも含むプラスミ
ドｐＮ０２００を得た。Example 1 Preparation and culture of pMMl Shigezo Udaka, Journal of the Japanese Society of Agricultural Chemistry 61.669 (1987)
Bacillus subtilis plasmids pUBIIO and B by the method described in
Plasmid pN0200, which also contains the cell wall protein gene of P. plevis, was obtained.
一方、特開平１−２１８５８９号に記載の方法により耐
熱性β−アミラーゼ遺伝子を含むプラスミドｐＮＫ１を
得た。このプラスミドｐＮＫ１（ＤＭｆｌＩ−ＭｆｌＩ
約３．６Ｋｂ断片を切出し、上記プラスミドｐＮＵ２０
０（７）ＢａｍＨＩ部位に連結し、プラスミドｐＭＭ１
（８４６６ｂｐ）を得た。On the other hand, plasmid pNK1 containing a thermostable β-amylase gene was obtained by the method described in JP-A-1-218589. This plasmid pNK1 (DMflI-MflI
An approximately 3.6 Kb fragment was excised and used as the above plasmid pNU20.
0(7) into the BamHI site to create plasmid pMM1.
(8466bp) was obtained.
プラスミドｐＭＭ１の制限酵素地図を第４図に示し、Ｄ
ＮＡ塩基配列の一部を第１図に示す。第４図中のＭＷＰ
は、細胞壁タンパク質遺伝子を示す。尚、第１図中、１
〜９６塩基までが細胞壁タンパク質遺伝子であり、９７
〜３７４４塩基がβ−アミラーゼ遺伝子であり、３７４
５塩基以後がｐＮＵ２００の遺伝子である。さらに、６
６０〜６６２塩基に翻訳開始点「ＡＴＧ」があり、７５
５塩基までがシグナル配列である。２３１３〜２３１５
塩基に翻訳終止点ｒＴＡＡＪがある。The restriction enzyme map of plasmid pMM1 is shown in FIG.
A part of the NA base sequence is shown in FIG. MWP in Figure 4
indicates a cell wall protein gene. In addition, in Figure 1, 1
~96 bases are cell wall protein genes, and 97
~3744 bases are the β-amylase gene;
The portion after the 5th base is the gene of pNU200. Furthermore, 6
There is a translation start point "ATG" at bases 60 to 662, and 75
Up to 5 bases are the signal sequence. 2313-2315
There is a translation termination point rTAAJ at the base.
プラスミドｐＭＭｌをＢ、プレビス４７−５Ｑ（日、プ
レビス４７株のウラシル要求株）に導入し、トリプトン
、酵母抽出物、カッオニキス、ブドウ糖を含む培養液（
ｐＨ７，０）中、３７°Ｃ１３日間培養した。培養終了
後、培養液上清をＤＮＳ法により測定したところ、７．
０７０／−の耐熱性β−アミラーゼを得た。Plasmid pMMl was introduced into B, Previs 47-5Q (Japanese, uracil auxotrophic strain of Previs 47 strain), and culture medium containing tryptone, yeast extract, katyonyx, and glucose (
The cells were cultured at 37°C for 13 days in pH 7.0). After the culture was completed, the culture supernatant was measured by the DNS method, and the result was 7.
070/- thermostable β-amylase was obtained.
比較のため、上記プラスミドｐＮＫ１をＢ、プレビス４
７−５Ｑ株に導入して同様の条件で培養した。その結果
、耐熱性β−アミラーゼの生産量は、１．６４１Ｊ／−
であった。For comparison, the above plasmid pNK1 was used as B and Previs4.
It was introduced into the 7-5Q strain and cultured under the same conditions. As a result, the production amount of thermostable β-amylase was 1.641J/-
Met.
実施例２ ９ＭＭ２の調製及び培養
プラスミドｐＮ０２００の細胞壁タンパク質遺伝子のシ
グナル直後にプラスミドｐＮＫ１の耐熱性β−アミラー
ゼ遺伝子の構造部分を連結するプラスミド（９ＭＭ２）
を、以下のようにして構築した。Example 2 Preparation and cultivation of 9MM2 Plasmid (9MM2) in which the structural part of the thermostable β-amylase gene of plasmid pNK1 is linked immediately after the cell wall protein gene signal of plasmid pN0200
was constructed as follows.
Ｂ、プレビスの細胞壁タンパク質遺伝子のＮｃｏｌ以後
、β−アミラーゼ遺伝子のＡｖａＩＩ部位迄ＤＮＡ合成
した。次いで、Ａｖａｌｌ−ＭｆｌＩ断片と結合し、Ａ
ｐａＬＩ−３ｐｈｌ断片を切出し、ｐＭＭｌの相当部位
と置換することにより９ＭＭ２ （７７３１ｂｐ）を得
た。B, DNA was synthesized from the Ncol site of the cell wall protein gene of Plebis to the AvaII site of the β-amylase gene. Then, it is ligated with the Avall-MflI fragment, and A
The paLI-3phl fragment was excised and replaced with the corresponding site in pMMl to obtain 9MM2 (7731 bp).
プラスミドｐＭＭ２の制限酵素地図を第５図に示し、Ｄ
ＮＡ塩基配列の一部を第２図に示す。第５図中のＭＷＰ
は、細胞壁タンパク質遺伝子を示す。尚、第２図中、５
１７〜５８５塩基までが細胞壁タンパク質遺伝子のシグ
ナル配列であり、５８６塩基以後がβ−アミラーゼ遺伝
子である。さらに、５１７〜５１９塩基に翻訳開始点「
ＡＴＧ」があり、２１４３〜２１４５塩基に翻訳終止点
ｒＴＡＡ」がある。The restriction enzyme map of plasmid pMM2 is shown in FIG.
A part of the NA base sequence is shown in FIG. MWP in Figure 5
indicates a cell wall protein gene. In addition, in Figure 2, 5
The 17th to 585th bases are the signal sequence of the cell wall protein gene, and the 586th and subsequent bases are the β-amylase gene. Furthermore, the translation start point “
ATG" and a translation end point "rTAA" at bases 2143 to 2145.
得られた９ＭＭ２を実施例１と同様にしてＢ、２レビス
４７−５Ｑ株に導入し、ＴＺＵ＋５０ｍＭＭＥＳ＋グル
コース２％を含む培養液（ｐＨ６，０）中、３７°Ｃ１
７日間培養し、培養液液中の耐熱性β−アミラーゼ量を
経時的に測定した。その結果を表１に示す。The obtained 9MM2 was introduced into the B, 2 Levis 47-5Q strain in the same manner as in Example 1, and incubated at 37°C in a culture medium (pH 6,0) containing TZU + 50mMMES + 2% glucose.
The cells were cultured for 7 days, and the amount of heat-stable β-amylase in the culture solution was measured over time. The results are shown in Table 1.
これとは別に、９ＭＭ２をＢ、プレビス−４７に株、３
１株（Ｂ、プレビスＨ１０２、ＦＥＲＭ ＢＰ−１０
８７）、３１Ｍ株（３１株のミュータント）にも同様に
して導入し、培養した。結果を表１に示す。Separately, 9MM2 was placed in B, Previs-47, and 3
1 strain (B, Previs H102, FERM BP-10
87) and 31M strain (mutant of 31 strains) were similarly introduced and cultured. The results are shown in Table 1.
さらに９ＭＭ２を導入したＢ、プレビス４７に株を、Ｔ
３±１００ｍＭ ＭＥＳ＋グルコース２％を含む培養液
（ｐＨ６，０）を用いて培養した結果、耐熱性β−アミ
ラーゼの生産量は２６００ Ｕ／１ｎｌであった。この
値は、タンパク質量に換算すると約１．７ｇ／ｌに相当
する。In addition, 9MM2 was introduced into B, the stock was placed in Previs 47, and T
As a result of culturing using a culture medium (pH 6.0) containing 3±100 mM MES + 2% glucose, the production amount of thermostable β-amylase was 2600 U/1 nl. This value corresponds to approximately 1.7 g/l in terms of protein amount.
得られた耐熱性β−アミラーゼをセファクリル（５ｅｐ
ｈａｃｒｙ ｌ ）Ｓ−２００で精製した結果比活性ハ
、１６３４０７ｍｇタンパク質であり、Ｃ，サーモサル
フロゲネスの耐熱性β−アミラーゼを同様にして精製し
た酵素の比活性、１５１５０７ｍｇタンパク質とほぼ同
じであった。The obtained thermostable β-amylase was treated with Sephacryl (5ep
As a result of purification using S-200, the specific activity was 163,407 mg protein, which was almost the same as the specific activity of the enzyme purified in the same manner as heat-stable β-amylase of C. thermosulfurogenes, 151,507 mg protein. .
表 １
シス法により、各開始コドン（ＡＴＧ）を含む部分の塩
基配列をＴＣＡＴＧＡ、即ちＢｓｐ旧部位に変更した。Table 1 Using the cis method, the base sequence of the portion containing each initiation codon (ATG) was changed to TCATGA, that is, the Bsp old site.
ＭＷＰ： ↑ 実施例３ ｐＭＭ３の調製及び培養 ｐＭＭ３を以下のように調製した。MWP: ↑ Example 3 Preparation and culture of pMM3 pMM3 was prepared as follows.
プラスミドｐＮＵ２００の細胞壁タンパク質遺伝子（Ｍ
ＷＰ）及びプラスミドｐＮＫｌの耐熱性β−アミラーゼ
遺伝子（β−ａｍ）の各開始コドン直前に、新しく制限
酵素の認識部位を導入し、後に続く部分をつなぎかえた
。即ち、以下に示すように、サイト−ブイレフテッド
ミュータジエネ次に、Ｂｓｐ旧切断後、両配列をつなぎ
かえることにより開始コドン以降は、Ｃ、サーモサルフ
ロゲ＋２の配列とし、それ以前は、細胞壁タンパク質遺
伝子（ＭＷＰ）であるプラスミドｐＭＭ３ （７７５８
Ｍ）を構築した。Cell wall protein gene (M
A new restriction enzyme recognition site was introduced immediately before each start codon of the thermostable β-amylase gene (β-am) of WP) and plasmid pNKl, and the subsequent portions were reconnected. That is, as shown below, the site-blefted
Mutagiene Next, after Bsp old cleavage, both sequences are reconnected, so that after the start codon, the sequence is C, thermosulfroge +2, and before that, the cell wall protein gene (MWP), plasmid pMM3 (7758
M) was constructed.
プラスミドｐＭＭ３の制限酵素地図を第６図に示し、Ｄ
ＮＡ塩基配列の一部を第３図に示す。第６図中のＭＷＰ
は、細胞壁タンパク質遺伝子を示す。尚、第３図中、１
〜５１６塩基が細胞壁タンパク質遺伝子であり、５１７
〜６１２塩基がβ−アミラーゼ遺伝子のシグナル配列で
あり、６１３塩基以後がβ−アミラーゼ構造遺伝子であ
る。さらに、５１７〜５１９塩基に翻訳開始点「ＡＴＧ
」があり、２１７０〜２１７２塩基に翻訳終止点ｒＴＡ
ＡＪがある。The restriction enzyme map of plasmid pMM3 is shown in FIG.
A part of the NA base sequence is shown in FIG. MWP in Figure 6
indicates a cell wall protein gene. In addition, in Figure 3, 1
~516 bases are cell wall protein genes, 517
The 612th base is the signal sequence of the β-amylase gene, and the 613th and subsequent bases are the β-amylase structural gene. Furthermore, the translation start point “ATG” is located at bases 517 to 519.
”, and there is a translation end point rTA between bases 2170 and 2172.
There is A.J.
得られたｐＭＭ３をＢ、プレビス４７に株に導入し、Ｔ
３＋ １００ｍＭ ＭＥＳ＋グルコース２％を含む培養
液（ｐＨ６，０）中、３７℃、７日間培養し、培養液液
中の耐熱性β−アミラーゼ量を経時的に測定した。その
結果を表２に示す。The obtained pMM3 was introduced into strains B and Previs 47, and T
The cells were cultured at 37° C. for 7 days in a culture medium (pH 6,0) containing 3+ 100 mM MES + 2% glucose, and the amount of thermostable β-amylase in the culture medium was measured over time. The results are shown in Table 2.
表 ２
その結果、耐熱性β−アミラーゼの生産量は約５００Ｕ
／−であった。得られた耐熱性β−アミラーゼをセファ
クリル（５ｅｐｈａｃｒｙｌ）Ｓ−２００で精製した結
果鳩活性は、ｌ ５１７０７ｍｇタンパク質であり、Ｃ
，サーモサルフロゲネスの耐熱性β−アミラーゼの比活
性、１５１５０７ｍｇタンパク質とほぼ同じであった。Table 2 As a result, the production amount of thermostable β-amylase was approximately 500 U.
It was /-. The obtained thermostable β-amylase was purified with Sephacryl S-200, and the pigeon activity was 151,707 mg protein.
, the specific activity of thermostable β-amylase of S. thermosulfurogenes was almost the same as that of 151,507 mg protein.
さらに、Ｃ，サーモサルフロゲネス由来の耐熱性β−ア
ミラーゼタンパク質、及び実施例１〜３で得たｐＭＭ１
〜３由来の耐熱性β−アミラーゼタンパク質の５ＤＳ−
ＰＡＧＥを測定した結果、全て同一の箇所に一本のバン
ドが得られた。Furthermore, thermostable β-amylase protein derived from C. thermosulfurogenes, and pMM1 obtained in Examples 1 to 3
5DS- of thermostable β-amylase protein derived from ~3
As a result of PAGE measurement, one band was obtained at the same location in all cases.
さらに、実施例２及び３で得たｐＭＭ２及び３由来の耐
熱性β−アミラーゼの活性の温度依存性を測定した結果
、いずれの耐熱性β−アミラーゼも約７０℃に最適温度
を有していた。Furthermore, as a result of measuring the temperature dependence of the activity of the thermostable β-amylases derived from pMM2 and 3 obtained in Examples 2 and 3, it was found that both thermostable β-amylases had an optimum temperature at about 70°C. .
第１図は、９〜１Ｍ１のＤＮＡ塩基配列の一部を示す。
第２図は、ｐＭＭ２のＤＮＡ塩基配列の一部を示す。第
３図は、ｐＭＭ３のＤＮＡ塩基配列の一部を示す。第４
図は、ｐＭＭｌの制限酵素地図を示す。第５図は、ｐＭ
Ｍ２の制限酵素地図を示す。第６図は、ｐＭＭ３の制限
酵素地図を示す。
第
１図−その
０
ＣＣＡＡＴＧＧＣＴＴ
０
ＴＣＧＣＡＧＣＡＧＡ
０
ＡＧＡＡＧＣＡＧＣＡ
０
ＡＣＴＡＣＴＡＣＡＧ
０
ＣＴＣＣＡＡＡＡＡＴ
０
ＧＧＡＣＧＣＴＧＡＴ
０
ＡＴＧＧＡＡＡＡＡＡ
０
ＣＣＧＴＡＡＡＡＣＧ
０
ＴＣＴＧＧＡＡＧＣＴ
００
ＣＴＴＧＧＣＧＧＡＴ
１０
ＣＴＴＡＧＴＴＴＡＧ
２０
ＧＴＧＴＧＴＴＴＴＴ
３０
ＡＴＡＣＴＡＴＧＧＣ
４０
ＧＣＡＴＴＡＡＡＡＧ
５０
ＧＡＡＴＡＣＣＧＡＣ
６０
ＡＴＣＴＴＴＡＧＡＴ
７０
ＧＡＡＧＣＡＧＣＴＴ
８０
ＴＡＡＴＡＧＡＴＧＧ
９０
ＴＴＧＴＡＧＴＡＧＡ
００
ＴＴＴＡＧＡＡＴＣＴ
１０
ＡＣＴＧＧＡＡＴＡＴ
２０
ＣＡＴＡＣＴＧＣＣＡ
３０
ＴＴＡＴＴＡＡＡＴＣ
４０
ＣＴＡＣＴＡＣＧＡＴ
５０
ＣＡＣＣＣＴＴＧＣＡ
６０
ＧＴＴＴＴＧＧＡＴＡ
７０
ＴＴＡＴＧＴＧＧＡＴ
８０
ＡＴＧＧＡＡＴＧＡＣ
９０
ＴＡＣＴＴＡＴＴＧＣ
００
ＣＡＴＣＴＴＴＡＧＴ’
１０
ＣＡＴＡＡＡＣＡＡＧ
２０
ＴＣＧＧＴＴＣＣＡＧ
３０
ＧＡＣＧＣＴＴＣＣＡ
４０
ＣＴＡＡＴＧＡＴＴＴ
５０
ＴＴＴＡＣＴＴＣＴＴ
６０
ＴＡＧＣＣＡＧＴＡＣ
７０
ＡＣＡＡＡＧＣＡＧＴ
８０
ＧＧＡＡＴＣＴＣＧＧ
９０
ＴＡＴＧＧＣＡＧＧＡ
００
ＣＴＧＡＣＡＡＴＡＧ
弔
１図〜その
１０
ＣＡＡＴＴＴＴＡＣＣ
２０
ＴＧＴＴＧＴＡＡＴＴ
３０
ＴＴＣＴＡＣＴＴＣＴ
４０
ＴＧＧＣＡＣＡＧＡＧ
５０
ＡＡＡＡＴＴＡＧＴＣ
６０
ＡＣＡＧＣＴＡＴＡＡ
７０
ＴＡＧＣＴＧＧＴＧＣ
８０
ＴＧＴＡＡＡＧＣＡＧ
９０
ＴＡＧＧＴＡＡＡＧＣ
００
ＡＡＡＡＴＴＡＴＡＧ
１０
ＴＴＡＧＧＡＧＧＴＧ
２０
ＡＴＡＴＴＡＡＡＡＴ
３０
ＡＧＡＡＡＴＴＴＧＡ
４０
ＴＡＧＡＡＡＣＴＧＡ
５０
ＡＴＡＴＡＡＴＴＡＴ
６０
ＴＧＡＡＡＧＧＧＧＴ
７０
ＧＧＧＡＡＣＴＡＴＴ
８０
ＧＧＣＡＡＡＡＴＡＡ
９０
ＴＡＴＣＴＴＡＴＴＡ
００
ＡＡＡＴＴＴＴＴＧＧ
１０
ＣＧＧＣＡＡＡＡＡＡ
２０
ＴＡＧＴＧＡＴＴＡＴ
３０
ＡＴＴＡＴＴＴＴＴＡ
４０
ＴＣＧＴＡＴＧＡＴＧ
５０
ＧＧＡＧＧＧＡＡＡＡ
６０
ＴＡＡＡＡＡＴＡＧＡ
７０
ＴＧＡＴＴＧＧＡＧＣ
８０
ＴＴＴＴＡＡＡＡＧＧ
９０
ＴＴＧＧＧＴＣＡＡＡ
００
ＡＡＴＴＧＴＴＴＴＴ
１０
ＧＡＣＡＴＴＧＴＴＡ
？２０
ＡＣＧＧＣＡＴＣＡＴ
３０
ＴＡＡＴＴＴＴＴＧＣ
４０
ＡＴＣＴＴＣＴＡＴＡ
５０
ＧＴＡＡＣＴＧＣＴＡ
） ７９０ ８００Ｔ
ＴＴＧＴＡＡＴＧＧＧ ＴＣＣＡＴＴＡＧＡＡ５８４
−
第１図
その５
１８６０ １８７０
ＴＣＣＡＡＣＡＡＣＡ ＡＴＣＡＡＧＣＴＴＡ８８０
ＴＧＴＧＡＡＴＴＧＴ
８９０
ＧＣＡＡＡＴＧＡＡＴ
９００
ＴＡＡＣＡＧＧＡＴＡ
９１０
ＴＡＡＴＴＴＴＴＣＴ
９２０
ＧＧＡＴＴＴＡＣＡＣ
９３０
ＴＴＴＴＡＡＧＡＣＴ
９４０
ＴＴＣＧＡＡＴＡＴＴ
９５０
ＧＴＡＡＡＴＡＧＴＧ
９６０
ＡＴＧＧＡＴＣＴＧＴ
９７０
ＧＡＣＡＴＣＡＧＡＧ
９８０
ＡＴＧＧＣＴＣＣＴＴ
９９０
ＴＴＧＴＡＡＴＴＡＡ
０００
ＴＡＴＡＧＴＴＡＣＡ
０１０
ＣＴＡＡＣＧＣＣＴＡ
０２０
ＡＣＧＧＴＡＣＧＡＴ
０３０
ＡＣＣＡＧＴＴＡＣＡ
０４０
ＴＴＴＡＣＡＡＴＡＡ
０５０
ＡＣＡＡＴＧＣＧＡＣ
０６０
ＡＡＣＴＴＡＴＴＡＴ
２０７０ ２０８０
ＧＧＡＣＡＡＡＡＴＧ ＴＡＴＡＴＡＴＴＧＴ０９０
ＴＧＧＴＡＧＴＡＣＡ
１００
ＴＣＴＧＡＴＣＴＴＧ
２１１０ ２１２０
ＧＡＡＡＴＴＧＧＡＡ ＴＡＣＡＡＣＣＴＡＴ１３０
ＧＣＣＣＧＴＧＧＴＣ
１４０
ＣＴＧＣＡＴＣＡＴＧ
１５０
ＣＣＣＴＡＡＴＴＡＴ
１６０
ＣＣＴＡＣＴＴＧＧＡ
１７０
ＣＡＡＴＡＡＣＧＣＴ
１８０
ＴＡＡＴＣＴＡＴＴＡ
１９０
ＣＣＴＧＧＴＧＡＧＣ
２００
ＡＧＡＴＡＣＡＧＴＴ
２２１０ ２２２０
ＴＡＡＡＧＣＴＧＴＡ ＡＡＡＡＴＴＧＡＴＡ２３０
ＧＴＴＣＡＧＧＡＡＡ
２４０
ＴＧＴＡＡＣＴＴＧＧ
２５０
ＧＡＡＧＧＴＧＧＣＴ
２６０
ＣＧＡＡＴＣＡＴＡＣ
２７０
ＴＴＡＴＡＣＴＧＴＧ
２２８０ ２２９０
ＣＣＧＡＣＡＴＣＴＧ ＧＧＡＣＴＧＧＴＡＧ３００
ＴＧＴＣＡＣＣＡＴＴ
３１０
ＡＣＡＴＧＧＣＡＡＡ
３２０
ＡＴＴＡＡＴＣＡＡＴ
３３０
ＡＡＡＡＴＧＴＴＡＣ
２３４０２３５０
ＡＣＡＴＡＧＡＡＣＡ ＡＡＴＴＧＴＡＡＡＣ３６０
ＡＣＴＧＧＡＡＴＡＴ
３７０
ＡＴＴＣＣＧＧＴＧＴ
３８０
ＴＴＴＴＴＴＧＴＡＴ
３９０
ＡＴＴＡＴＧＧＧＣＧ
４００
ＴＴＴＡＡＴＧＴＴＡ
第１図
その６
４１０
ＡＡＡＡＴＡＡＴＡＧ
４２０
ＴＧＴＴＴＴＧＡＴＴ
４３０
ＴＴＡＴＴＡＡＡＡＡ
４４０
ＧＴＴＴＧＧＡＧＧＴ
４５０
ＡＡＧＡＧＡＴＧＡＧ
４６０
ＴＡＡＡＡＡＡＧＴＴ
４７０
ＧＧＴＡＴＴＣＣＡＡ
４８０
ＡＡＧＧＧＣＴＴＴＴ
４９０
ＡＴＡＣＴＡＣＡＡＣ
５００
ＴＴＴＴＡＴＣＣＴＡ
５１０
ＴＧＴＧＧＡＡＡＡＣ
５２０
ＡＴＴＴＴＴＴＧＡＡ
５３０
ＧＡＡＣＴＧＧＧＴＧ
５４０
ＣＴＧＡＡＧＴＧＧＴ
５５０
ＴＡＣＴＴＣＣＡＧＴ
５６０
ＧＡＴＡＣＡＴＧＴＡ
５７０
ＡＡＡＡＡＡＴＡＡＴ
５８０
ＴＧＡＴＧＡＴＧＧＣ
５９０
ＡＴＣＡＡＧＡＣＴＴ
６００
ＧＣＧＴＧＧＡＴＧＡ
６１０
ＡＡＣＣＴＧＣＣＴＴ
６２０
ＣＣＴＧＴＣＡＡＧＡ
２６３０ ２６４０
ＣＡＴＴＴＡＴＧＧＧ ＴＣＡＴＧＴＧＡＴＴ６５０
ＧＡＴＴＴＧＡＡＧＧ
６６０
ＡＡＡＡＧＧＧＴＧＴ
６７０
ＴＧＡＴＴＡＴＡＴＡ
６８０
ＴＴＴＧＴＴＣＣＡＡ
６９０
ＧＡＧＴＣＡＴＡＡＧ
７００
ＣＧＴＡＧＡＡＡＧＧ
７１０
ＣＧＴＡＧＡＴＡＴＣ
７２０
ＴＡＴＧＣＴＣＡＡＡ
７３０
ＡＴＴＴＴＴＧＧＧＴ
７４０
ＴＴＧＣＣＴＧＡＣＴ
７５０
ＴＡＧＴＧＡＧＡＡＡ
７６０
ＴＣＴＴＡＴＴＴＣＴ
７７０
ＧＡＴＴＴＧＣＣＡＣ
２７８０２７９０
ＡＡＡＴＡＡＴＡＧＡ ＴＡＴＧＡＡＧＡＴＴ８００
ＧＡＴＴＡＣＴＡＴＣ
８１０
ＧＴＧＧＡＧＡＡＧＡ
２８２０ ２８３０
ＧＴＴＴＡＴＧＧＡＡ ＡＧＡＧＡＡＡＴＴＴ８４０
ＴＧＡＧＡＧＴＴＧＧ
８５０
ＣＡＡＧＣＴＧＴＴＴ
８６０
ＧＴＴＧＡＴＡＧＣＡ
８７０
ＣＡＡＧＴＡＡＡＡＴ
８８０
ＴＡＡＡＧＡＣＧＣＡ
８９０
ＴＡＣＧＡＡＡＡＡＴ
９００
ＣＴＴＴＡＡＡＡＡＧ
５８６−
第１図−そのＴ
２９１０ ２９２０
ＧＣＡＡＡＧＧＡＣＴ ＴＴＴＧＡＡＴＴＣＡ２９３
０ ２９４０
ＴＴＡＴＴＡＧＡＧＡ ＡＧＧＡＴＴＴＴＣＡ９５０
ＡＡＴＡＣＡＧＡＡＧ
９６０
ＣＡＡＴＡＡＡＡＡＴ
９７０
ＴＡＴＧＧＡＡＧＧＧ
９８０
ＡＡＡＡＡＡＴＴＡＧ
９９０
ＡＣＧＣＡＡＡＴＡＣ
０００
ＡＡＡＡＧＧＧＧＡＴ
０１０
ＴＴＡＡＡＡＡＴＡＧ
０２０
ＣＴＴＴＡＴＴＡＧＣ
０３０
ＣＣＡＴＴＣＣＴＡＴ
０４０
ＧＡＣＡＴＡＡＴＧＧ
０５０
ＡＴＧＡＴＴＡＴＴＴ
０６０
ＡＴＣＴＡＴＧＧＧＡ
０７０
ＴＴＧＡＴＡＡＡＴＡ
０８０
ＧＧＣＴＴＡＡＧＡＡ
０９０
ＴＡＴＧＧＧＡＧＣＴ
１００
ＴＡＴＧＴＧＣＴＴＡ
１１０
ＣＡＡＣＡＡＡＴＡＴ
１２０
ＧＡＴＡＧＡＡＡＧＡ
１３０
ＧＡＴＡＡＡＡＴＴＧ
１４０
ＡＡＡＡＴＧＧＴＧＣ
１５０
ＣＡＧＣＡＡＡＣＴＴ
１６０
ＣＡＡＡＡＡＧＡＴＴ
１７０
ＴＡＴＴＴＴＧＧＡＣ
１８０
ＴＴＡＴＧＧＡＡＧＧ
１９０
ＧＡＴＡＴＴＴＴＡＧ
２００
ＧＴＧＣＡＧＧＴＡＡ
２１０
ＡＴＡＴＴＴＴＡＴＣ
２２０
ＧＡＧＡＧＣＡＡＡＧ
２３０
ＡＡＧＴＧＧＡＣＧＧ
２４０
ＧＧＴＡＡＴＡＴＣＴ
２５０
ＧＴＴＴＣＴＧＣＴＴ
２６０
ＴＴＧＧＡＴＧＴＧＧ
２７０
ＧＣＣＡＧＡＣＴＣＴ
２８０
ＡＴＣＡＣＴＧＡＴＧ
２９０
ＡＣＣＴＴＴＴＡＧＡ
３００
ＡＡＧＧＧＡＴＴＡＣ
３１０
ＡＡＧＡＧＡＧＡＴＧ
３２０
ＧＴＧＧＧＡＴＴＣＣ
３３０
ＡＴＴＴＡＴＧＣＣＣ
３４０
ＡＴＡＡＣＣＡＴＴＧ
３５０
ＡＴＧＡＡＣＡＴＡＣ
３６０
ＴＧＧＴＧＡＧＧＣＡ
３７０
ＧＧＴＴＴＡＡＡＣＴ
３８０
ＡＣＣＴＡＧＧＴＴＡ
３９０
ＧＡＡＧＣＣＴＴＴＡ
４００
ＴＧＧＡＴＴＴＧＴＴ
第１図−その８
４１０
ＡＡＧＡＴＧＧＣＣＡ
４２０
ＣＡＡＧＧＡＧＡＧＡ
４３０
ＴＴＡＴＧＧＣＡＴＧ
４４０
ＡＡＡＡＴＡＡＣＡＴ
４５０
ＡＴＣＣＡＣＡＴＡＴ
４６０
ＧＧＧＴＴＣＧＴＴＡ
４７０
ＡＡＴＡＴＧＡＴＡＴ
４８０
ＴＡＡＡＧＡＣＧＡＴ
４９０
ＧＴＴＴＧＡＡＧＧＡ
５００
ＡＴＣＡＡＴＧＴＡＧ
５１０
ＡＡＧＴＴＧＴＧＧＡ
５２０
ＧＣＣＧＣＣＡＣＣＴ
５３０
ＧＴＧＡＣＴＡＡＴＡ
３５４０ ３５５０
ＧＧＡＣＴＣＴＡＴ、ＣＡＡＴＴＧＧＡＧＴＡ５６０
ＡＡＡＴＡＴＴＣＡＣ
５７０
ＣＴＧＡＧＴＴＴＧＴ
５８０
ＣＴＧＴＣＴＴＣＣＡ
５９０
ＴＡＴＡＡＧＡＴＡＡ
６００
ＡＴＣＴＧＧＧＧＡＡ
６１０
ＴＴＴＴＡＴＡＧＡＡ
６２０
ＧＣＡＣＴＴＧＡＡＡ
６３０
ＡＣＧＧＴＧＣＴＧＡ
６４０
ＴＡＣＴＡＴＡＡＴＡ
６５０
ＡＴＧＴＴＧＧＧＴＧ
６６０
ＧＴＡＴＡＧＧＧＣＣ
６７０
ＴＴＧＣＡＧＧＴＴＣ
６８０
ＧＧＴＴＡＴＴＡＴＧ
６９０
ＧＡＣＡＡＧＣＡＣＡ
７００
ＡＡＧＡＧＡＡＡＣＡ
７１０
ＴＴＡＡＴＴＧＡＴＣ
７２０
ＴＴＧＧＡＴＡＣＡＡ
７３０
ＧＴＴＴＡＡＴＡＴＧ
７４０
ＡＣＴＡＡＴＡＴＴＡ
７５０
ＧＡＴＣＣＡＣＡＧＧ
７６０
ＡＣＧＧＧＴＧＴＧＧ
７７０
ＴＣＧＣＣＡＴＧＡＴ
７８０
ＣＧＣＧＴＡＧＴＣＧ
７９０
ＡＴＡＧＴＧＧＣＴＣ
８００
ＣＡＡＧＴＡＧＣＧＡ
８１０
ＡＧＣＧＡＧＣＡＧＧ
８２０
ＡＣＴＧＧＧＣＧＧＣ
８３０
ＧＧＣＣＡＡＡＧＣＧ
８４０
ＧＴＣＧＧＡＣＡＧＴ
８５０
ＧＣＴＣＣＧＡＧＡＡ
８６０
ＧＧ
第２図
その
０
ＡＧＡＴＣＴＡＡＡＧ
０
ＣＴＡＴＣＣＴＧＴＣ
０
ＴＴＡＣＡＡＣＴＴＧ
０
ＧＣＴＧＴＴＧＴＡＡ
０
ＡＣＴＴＴＧＡＡＡＡ
０
ＴＧＣＡＴＴＡＧＧＡ
０
ＡＡＴＴＡＡＣＣＴＡ
０
ＡＴＴＣＡＡＧＣＡＡ
０
ＧＡＴＴＡＴＧＡＧＧ
００
ＴＴＴＴＧＡＡＣＣＡ
１０
ＡＡＴＴＧＧＡＡＡＡ
２０
ＡＧＧＴＴＣＡＧＴＣ
３０
ＧＴＧＡＣＡＧＣＣＣ
４０
ＧＣＣＡＴＡＴＧＴＣ
５０
ＣＣＣＴＡＴＡＡＴＡ
６０
ＣＧＧＡＴＴＧＴＧＧ
７０
ＣＧＧＡＴＧＴＣＡＣ
８０
ＴＴＣＧＴＡＣＡＴＡ
９０
ＡＴＧＧＡＣＡＧＧＴ
００
ＧＡＡＴＡＡＣＧＡＡ
１０
ＣＣＡＣＧＡＡＡＡＡ
２０
ＡＡＣＴＴＴＡＡＡＴ
３０
ＴＴＴＴＴＴＣＧＡＡ
４０
ＧＧＣＧＣＣＧＣＡＡ
５０
ＣＴＴＴＴＧＡＴＴＣ
６０
ＧＣＴＣＡＧＧＣＧＴ
７０
ＴＴＡＡＴＡＧＧＡＴ
８０
ＧＴＣＡＣＡＣＧＡＡ
９０
ＡＡＡＣＧＧＧＧＡＡ
００
ＴＴＧＴＧＴＡＡＡＡ
１０
ＡＡＧＡＴＴＣＡＣＧ
２０
ＡＡＴＴＣＴＡＧＣＡ
３０
ＧＴＴＧＴＧＴＴＡＣ
４０
ＡＣＴＡＧＴＧＡＴＴ
５０
ＧＴＴＧＣＡＴＴＴＴ
６０
ＡＣＡＣＡＡＴＡＣＴ
７０
ＧＡＡＴＡＴＡＣＴＡ
８０
ＧＡＧＡＴＴＴＴＴＡ
９０
ＡＣＡＣＡＡＡＡＡＧ
００
ＣＧＡＧＧＣＴＴＴＣ
第２図−その２
１０
ＣＴＧＣＧＡＡＡＧＧ
２０
ＡＧＧＴＧＡＣＡＣＧ
３０
ＣＧＣＴＴＧＣＡＧＧ
４０
ＡＴＴＣＧＧＧＣＴＴ
５０
ＴＡＡＡＡＡＧＡＡＡ
６０
ＧＡＴＡＧＡＴＴＡＡ
７０
ＣＡＡＣＡＡＡＴＡＴ
８０
ＴＣＣＣＣＡＡＧＡＡ
９０
ＣＡＡＴＴＴＧＴＴＴ
００
ＡＴＡＣＴＡＧＡＧＧ
１０
ＡＧＧＡＧＡＡＣＡＣ
２０
ＡＡＧＧＴＴＡＴＧＡ
３０
ＡＡＡＡＧＧＴＣＧＴ
４０
ＴＡＡＣＡＧＴＧＴＡ
５０
ＴＴＧＧＣＴＡＧＴＧ
６０
ＣＡＣＴＣＧＣＡＣＴ
７０
ＴＡＣＴＧＴＴＧＣＴ
８０
ＣＣＣＡＴＧＧＣＴＴ
９０
ＴＣＧＣＴＡＧＣＡＴ
００
ＣＧＣＴＣＣＡＡＡＣ
１０
ＴＴＣＡＡＡＧＴＴＴ
２０
ＴＣＧＴＴＡＴＧＧＧ
３０
ＴＣＣＡＴＴＡＧＡＡ
４０
ＡＡＡＧＴＣＡＣＡＧ
５０
ＡＴＴＴＴＡＡＴＧＣ
６０
ＡＴＴＣＡＡＡＧＡＴ
７０
ＣＡＡＴＴＧＡＴＡＡ
８０
ＣＴＴＴＡＡＡＧＡＡ
９０
ＴＡＡＴＧＧＴＧＴＴ
００
ＴＡＴＧＧＴＡＴＡＡ
１０
ＣＡＡＣＡＧＡＴＡＴ
２０
ＴＴＧＧＴＧＧＧＧＣ
３０
ＴＡＴＧＴＴＧＡＡＡ
４０
ＡＴＧＣＡＧＧＴＧＡ
５０
ＡＡＡＴＣＡＡＴＴＴ
６０
７０
８０
９０
００
第２図−その３
８１０
ＧＡＡＧＴＧＧＧＴＴ
２０
ＣＣＡＡＴＡＡＴＧＴ
３０
ＣＡＡＣＧＣＡＴＧＣ
４０
ＣＴＧＴＧＧＡＧＧＴ
５０
ＡＡＴＧＴＴＧＧＴＧ
６０
ＡＴＡＣＡＧＴＡＡＡ
７０
ＴＡＴＡＣＣＴＡＴＴ
８０
ＣＣＧＴＣＡＴＧＧＧ
９０
ＴＡＴＧＧＡＣＡＡＡ
００
ＡＧＡＴＡＣＣＣＡＡ
１０
ＧＡＴＡＡＴＡＴＧＣ
２０
ＡＧＴＡＴＡＡＧＧＡ
３０
ＴＧＡＡＧＣＣＧＧＡ
４０
ＡＡＴＴＧＧＧＡＴＡ
５０
ＡＴＧＡＡＧＣＡＧＴ
６０
ＡＡＧＴＣＣＡＴＧＧ
７０
ＴＡＴＴＣＴＧＧＣＴ
８０
ＴＡＡＣＣＣＡＡＣＴ
９０
ＣＴＡＴＡＡＴＧＡＡ
０００
ＴＴＴＴＡＴＴＣＡＴ
０１０
ＣＴＴＴＴＧＣＡＴＣ
０２０
ＡＡＡＴＴＴＴＡＧＣ
０３０
ＡＧＣＴＡＴＡＡＡＧ
０４０
ＡＴＡＴＡＡＴＴＡＣ
０５０
ＴＡＡＡＡＴＡＴＡＴ
０６０
ＡＴＡＴＣＴＧＧＡＧ
０７０
ＧＣＣＣＴＴＣＴＧＧ
０８０
ＡＧＡＡＴＴＡＡＧＡ
０９０
ＴＡＴＣＣＴＴＣＡＴ
１００
ＡＴＡＡＴＣＣＴＴＣ
１１０
ＧＣＡＴＧＧＡＴＧＧ
１２０
ＡＣＡＴＡＴＣＣＴＧ
１３０
ＧＡＣＧＴＧＧＣＴＣ
１４０
ＧＣＴＧＣＡＧＴＧＣ
１５０
ＴＡＴＡＧＴＡＡＡＧ
１６０
ＣＧＧＣＴＡＴＡＡＣ
１７０
ＡＡＧＴＴＴＴＣＡＡ
１８０
ＡＡＴＧＣＴＡＴＧＡ
１９０
ＡＧＴＣＴＡＡＡＴＡ
２００
ＴＧＧＡＡＣＴＡＴＡ
２１０
ＧＣＡＧＣＡＧＴＴＡ
２２０
ＡＴＡＧＴＧＣＡＴＧ
２３０
ＧＧＧＴＡＣＡＡＧＣ
２４０
ＣＴＡＡＣＴＧＡＴＴ
２５０
ＴＴＴＣＴＣＡＡＡＴ
２６０
ＴＡＧＴＣＣＡＣＣＴ
２７０
ＡＣＡＧＡＴＧＧＴＧ
２８０
ＡＴＡＡＴＴＴＣＴＴ
２９０
ＴＡＣＡＡＡＴＧＧＴ
３００
ＴＡＴＡＡＡＡＣＴＡ
第２図
その４
３１０
ＣＴＴＡＴＧＧＴＡＡ
１３２０ １３３０
ＴＧＡＣＴＴＴＴＴＧ ＡＣＡＴＧＧＴＡＴＣ３４０
ＡＡＡＧＴＧＴＴＴＴ
３５０
ＧＡＣＴＡＡＴＧＡＧ
３６０
ＴＴＡＧＣＣＡＡＴＡ
３７０
ＴＴＧＣＴＴＣＴＧＴ
３８０
ＡＧＣＴＣＡＴＡＧＣ
１３９゜
ＴＧＣＴＴＴＧＡＴＣ
４００
ＣＡＧＴＡＴＴＴＡＡ
４１０
ＴＧＴＴＣＣＡＡＴＡ
４２０
ＧＧＡＧＣＡＡＡＡＡ
４３０
ＴＡＧＣＴＧＧＡＧＴ
４４０
ＧＣＡＴＴＧＧＣＴＡ
４５０
ＴＡＴＡＡＴＡＧＴＣ
４６０
ＣＧＡＣＡＡＴＧＣＣ
４７０
ＡＣＡＴＧＣＴＧＣＡ
４８０
ＧＡＡＴＡＴＴＧＴＧ
４９０
ＣＣＧＧＴＴＡＴＴＡ
５００
ＴＡＡＴＴＡＴＡＧＣ
１５１０１５２０
ＡＣＧＣＴＡＣＴＣＧ ＡＴＣＡＡＴＴＴＡＡ５３０
ＧＧＣＡＴＣＴＡＡＴ
５４０
ＣＴＴＧＣＴＡＴＧＡ
５５０
ＣＡＴＴＴＡＣＡＴＧ
５６０
ＴＣＴＴＧＡＡＡＴＧ
５７０
ＧＡＴＧＡＴＴＣＴＡ
５８０
ＡＴＧＣＡＴＡＴＧＴ
５９０
ＡＡＧＴＣＣＡＴＡＴ
６００
ＴＡＴＴＣＴＧＣＡＣ
６１０
ＣＴＡＴＧＡＣＧＴＴ
６２０
ＡＧＴＣＣＡＴＴＡＴ
６３０
ＧＴＡＧＣＴＡＡＴＣ
６４０
ＴＴＧＣＴＡＡＴＡＡ
６５０
ＴＡＡＡＧＧＴＡＴＡ
１６６０ １６７０
ＧＴＣＣＡＣＡＡＴＧ ＧＡＧＡＡＡＡＴＧＣ６８０
ＴＴＴＧＧＣＴＡＴＡ
６９０
ＴＣＣＡＡＣＡＡＣＡ
７００
ＡＴＣＡＡＧＣＴＴＡ
７１０
ＴＧＴＧＡＡＴＴＧＴ
７２０
ＧＣＡＡＡＴＧＡＡＴ
７３０
ＴＡＡＣＡＧＧＡＴＡ
７４０
ＴＡＡＴＴＴＴＴＣＴ
７５０
ＧＧＡＴＴＴＡＣＡＣ
７６０
ＴＴＴＴＡＡＧＡＣＴ
７７０
ＴＴＣＧＡＡＴＡＴＴ
１７８０ １７９０
ＧＴＡＡＡＴＡＧＴＧ ＡＴＧＧＡＴＣＴＧＴ８００
ＧＡＣＡＴＣＡＧＡＧ
８１０
１８２゜
１日３０
８４０
８５０
第２図−その５
１８６０
ＡＣＣＡＧＴＴＡＣＡ
８７０
ＴＴＴＡＣＡＡＴＡＡ
８８０
ＡＣＡＡＴＧＣＧＡＣ
８９０
ＡＡＣＴＴＡＴＴＡＴ
９００
ＧＧＡＣＡＡＡＡＴＧ
９１０
ＴＡＴＡＴＡＴＴＧＴ
９２０
ＴＧＧＴＡＧＴＡＣＡ
９３０
ＴＣＴＧＡＴＣＴＴＧ
９４０
ＧＡＡＡＴＴＧＧＡＡ
９５０
ＴＡＣＡＡＣＣＴＡＴ
９６０
ＧＣＣＣＧＴＧＧＴＣ
９７０
ＣＴＧＣＡＴＣＡＴＧ
９８０
ＣＣＣＴＡＡＴＴＡＴ
９９０
ＣＣＴＡＣＴＴＧＧＡ
０００
ＣＡＡＴＡＡＣＧＣＴ
０１０
ＴＡＡＴＣＴＡＴＴＡ
０２０
ＣＣＴＧＧＴＧＡＧＣ
０３０
ＡＧＡＴＡＣＡＧＴＴ
０４０
ＴＡＡＡＧＣＴＧＴＡ
０５０
ＡＡＡＡＴＴＧＡＴＡ
０６０
ＧＴＴＣＡＧＧＡＡＡ
０７０
ＴＧＴＡＡＣＴＴＧＧ
０８０
ＧＡＡＧＧＴＧＧＣＴ
０９０
ＣＧＡＡＴＣＡＴＡＣ
１００
ＴＴＡＴＡＣＴＧＴＧ
１１０
ＣＣＧＡＣＡＴＣＴＧ
１２０
ＧＧＡＣＴＧＧＴＡＧ
１３０
ＴＧＴＣＡＣＣＡＴＴ
１４０
ＡＣＡＴＧＧＣＡＡＡ
１５０
ＡＴＴＡＡＴＣＡＡＴ
１６０
ＡＡＡＡＴＧＴＴＡＣ
１７０
ＡＣＡＴＡＧＡＡＣＡ
１８０
ＡＡＴＴＧＴＡＡＡＣ
１９０
ＡＣＴＧＧＡＡＴＡＴ
２００
ＡＴＴＣＣＧＧＴＧＴ
２１０
ＴＴＴＴＴＴＧＴＡＴ
２２０
ＡＴＴＡＴＧＧＧＣＧ
２３０
ＴＴＴＡＡＴＧＴＴＡ
２４０
ＡＡＡＡＴＡＡＴＡＧ
２５０
ＴＧＴＴＴＴＧＡＴＴ
２６０
ＴＴＡＴＴＡＡＡＡＡ
２７０
ＧＴＴＴＧＧＡＧＧＴ
２８０
ＡＡＧＡＧＡＴＧＡＧ
２９０
ＴＡＡＡＡＡＡＧＴＴ
３００
ＧＧＴＡＴＴＣＣＡＡ
３１０
ＡＡＧＧＧＣＴＴＴＴ
３２０
ＡＴＡＣＴＡＣＡＡＣ
３３０
ＴＴＴＴＡＴＣＣＴＡ
３４０
ＴＧＴＧＧＡＡＡＡＣ
３５０
ＡＴＴＴＴＴＴＧＡＡ
第２図−その６
２３６０ ２３７０
ＧＡＡＣＴＧＧＧＴＧ ＣＴＧＡＡＧＴＧＧＴ３８０
ＴＡＣＴＴＣＣＡＧＴ
２３９０ ２４００
ＧＡＴＡＣＡＴＧＴＡ ＡＡＡＡＡＡＴＡＡＴ４１０
ＴＧＡＴＧＡＴＧＧＣ
４２０
ＡＴＣＡＡＧＡＣＴＴ
２４３０ ２４４０
ＧＣＧＴＧＧＡＴＧＡ ＡＡＣＣＴＧＣＣＴＴ２４５
゜
ＣＣＴＧＴＣＡＡＧＡ
４６０
ＣＡＴＴＴＡＴＧＧＧ
４７０
ＴＣＡＴＧＴＧＡＴＴ
２４８０ ２４９０
ＧＡＴＴＴＧＡＡＧＧ ＡＡＡＡＧＧＧＴＧＴ５００
ＴＧＡＴＴＡＴＡＴＡ
５１０
ＴＴＴＧＴＴＣＣＡＡ
２５２０ ２５３０
ＧＡＧＴＣＡＴＡＡＧ ＣＧＴＡＧＡＡＡＧＧ５４０
ＣＧＴＡＧＡＴＡＴＣ
５５０
ＴＡＴＧＣＴＣＡＡＡ
５６０
ＡＴＴＴＴＴＧＧＧＴ
５７０
ＴＴＧＣＣＴＧＡＣＴ
５８０
ＴＡＧＴＧＡＧＡＡＡ
５９０
ＴＣＴＴＡＴＴＴＣＴ
６００
ＧＡＴＴＴＧＣＣＡＣ
６１０
ＡＡＡＴＡＡＴＡＧＡ
６２０
ＴＡＴＧＡＡＧＡＴＴ
６３０
ＧＡＴＴＡＣＴＡＴＣ
６４０
ＧＴＧＧＡＧＡＡＧＡ
６５０
ＧＴＴＴＡＴＧＧＡＡ
６６０
ＡＧＡＧＡＡＡＴＴＴ
２６７０ ２６８０
ＴＧＡＧＡＧＴＴＧＧ ＣＡＡＧＣＴＧＴＴＴ６９０
ＧＴＴＧＡＴＡＧＣＡ
７００
ＣＡＡＧＴＡＡＡＡＴ
７１０
ＴＡＡＡＧＡＣＧＣＡ
７２０
ＴＡＣＧＡＡＡＡＡＴ
２７３０ ２７４０
ＣＴＴＴＡＡＡＡＡＧ ＧＣＡＡＡＧＧＡＣＴ７５０
ＴＴＴＧＡＡＴＴＣＡ
２７６０ ２７７０ ２７８０Ｔ
ＴＡＴＴＡＧＡＧＡ ＡＧＧＡＴＴＴＴＣＡ ＡＡＴＡ
ＣＡＧＡＡＧ７９０
ＣＡＡＴＡＡＡＡＡＴ
８００
ＴＡＴＧＧＡＡＧＧＧ
８４０
＝ＴＡＡＡＡＡＴＡＧ
２８５゜
ＣＴＴＴＡＴＴＡＧＣ
８６０
８７０
８８０
８９０
９００
第２図−その７
２９１０ ２９２０
ＧＧＣＴＴＡＡＧＡＡ ＴＡＴＧＧＧＡＧＣＴ９３０
ＴＡＴＧＴＧＣＴＴＡ
Ａ
９４０
第３図−
１０
ＣＴＧＣＧＡＡＡＧＧ
２０
ＡＧＧＴＧＡＣＡＣＧ
３０
ＣＧＣＴＴＧＣＡＧＧ
４０
ＡＴＴＣＧＧＧＣＴＴ
５０
ＴＡＡＡＡＡＧＡＡＡ
６０
ＧＡＴＡＧＡＴＴＡＡ
７０
ＣＡＡＣＡＡＡＴＡＴ
８０
ＴＣＣＣＣＡＡＧＡＡ
９０
ＣＡＡＴＴＴＧＴＴＴ
００
ＡＴＡＣＴＡＧＡＧＧ
１０
ＡＧＧＡＧＡＡＣＡＣ
２０
ＡＡＧＧＴＣＡＴＧＡ
３０
ＴＴＧＧＡＧＣＴＴＴ
４０
ＴＡＡＡＡＧＧＴＴＧ
５０
ＧＧＴＣＡＡＡＡＡＴ
６０
ＴＧＴＴＴＴＴＧＡＣ
７０
ＡＴＴＧＴＴＡＡＣＧ
８０
ＧＣＡＴＣＡＴＴＡＡ
９０
ＴＴＴＴＴＧＣＡＴＣ
００
ＴＴＣＴＡＴＡＧＴＡ
１０
ＡＣＴＧＣＴＡＡＴＧ
２０
ＣＡＡＧＣＡＴＡＧＣ
３０
ＡＣＣＡＡＡＴＴＴＣ
４０
ＡＡＡＧＴＴＴＴＴＧ
５０
ＴＡＡＴＧＧＧＴＣＣ
６０
ＡＴＴＡＧＡＡＡＡＡ
７０
ＧＴＣＡＣＡＧＡＴＴ
８０
ＴＴＡＡＴＧＣＡＴＴ
９０
ＣＡＡＡＧＡＴＣＡＡ
００
ＴＴＧＡＴＡＡＣＴＴ
１０
ＴＡＡＡＧＡＡＴＡＡ
２０
ＴＧＧＴＧＴＴＴＡＴ
３０
ＧＧＴＡＴＡＡＣＡＡ
４０
ＣＡＧＡＴＡＴＴＴＧ
５０
ＧＴＧＧＧＧＣＴＡＴ
６０
ＧＴＴＧＡＡＡＡＴＧ
７０
ＣＡＧＧＴＧＡＡＡＡ
８０
ＴＣＡＡＴＴＴＧＡＣ
９０
ＴＧＧＡＧＴＴＡＴＴ
００
ＡＴＡＡＧＡＣＡＴＡ
５９１−
第３図−その３
８１０ ８２０ ８３０
８４０ ８５０ＴＧＣＴＧＡＴＡＣＣＧ
ＴＡＣＧＣＧＣＴＧ ＣＧＧＧＡＴＴＧＡＡ ＧＴＧＧ
ＧＴＴＣＣＡ ＡＴＡＡＴＧＴＣＡＡ６０
ＣＧＣＡＴＧＣＣＴＧ
７０
ＴＧＧＡＧＧＴＡＡＴ
８０
ＧＴＴＧＧＴＧＡＴＡ
９０
ＣＡＧＴＡＡＡＴＡＴ
００
ＡＣＣＴＡＴＴＣＣＧ
１０
ＴＣＡＴＧＧＧＴＡＴ
９２０ ９３０
ＧＧＡＣＡＡＡＡＧＡ ＴＡＣＣＣＡＡＧＡＴ４０
ＡＡＴＡＴＧＣＡＧＴ
５０
ＡＴＡＡＧＧＡＴＧＡ
６０
ＡＧＣＣＧＧＡＡＡＴ
９７０ ９８０ ９９０ＴＧ
ＧＧＡＴＡＡＴＧ ＡＡＧＣＡＧＴＡＡＧ ＴＣＣＡＴ
ＧＧＴＡＴ０００
ＴＣＴＧＧＣＴＴＡＡ
０１０
ＣＣＣＡＡＣＴＣＴＡ
０２０
ＴＡＡＴＧＡＡＴＴＴ
０３０
ＴＡＴＴＣＡＴＣＴＴ
０４０
ＴＴＧＣＡＴＣＡＡＡ
１０５゜
ＴＴＴＴＡＧＣＡＧＣ
０６０
ＴＡＴＡＡＡＧＡＴＡ
０７０
ＴＡＡＴＴＡＣＴＡＡ
１０８０ １０９０
ＡＡＴＡＴＡＴＡＴＡ ＴＣＴＧＧＡＧＧＣＣ１００
ＣＴＴＣＴＧＧＡＧＡ
１１０
ＡＴＴＡＡＧＡＴＡＴ
１１２０ １１３０
ＣＣＴＴＣＡＴＡＴＡ ＡＴＣＣＴＴＣＧＣＡ１４０
ＴＧＧＡＴＧＧＡＣＡ
１５０
ＴＡＴＣＣＴＧＧＡＣ
１６０
ＧＴＧＧＣＴＣＧＣＴ
１７０
ＧＣＡＧＴＧＣＴＡＴ
１８０
ＡＧＴＡＡＡＧＣＧＧ
１１９０ １２００
ＣＴＡＴＡＡＣＡＡＧ ＴＴＴＴＣＡＡＡＡＴ１２１
０ １２２０ １２３０
１２４０ＧＣＴＡＴＧＡＡＧＴ ＣＴＡＡＡＴＡＴ
ＧＧ ＡＡＣＴＡＴＡＧＣＡ ＧＣＡＧＴＴＡＡＴＡ２
５０
ＧＴＧＣＡＴＧＧＧＧ
１２６０ １２７０
ＴＡＣＡＡＧＣＣＴＡ ＡＣＴＧＡＴＴＴＴＴ２８０
ＣＴＣＡＡＡＴＴＡＧ
２９０
ＴＣＣＡＣＣＴＡＣＡ
３００
ＧＡＴＧＧＴＧＡＴＡ
３１０
ＡＴＴＴＣＴＴＴＡＣ
３２０
ＡＡＡＴＧＧＴＴＡＴ
３３０
ＡＡＡＡＣＴＡＣＴＴ
３４０
ＡＴＧＧＴＡＡＴＧＡ
３５０
ＣＴＴＴＴＴＧＡＣＡ
第３図−その４
３６０
ＴＧＧＴＡＴＣＡＡＡ
３７０
ＧＴＧＴＴＴＴＧＡＣ
３８０
ＴＡＡＴＧＡＧＴＴＡ
３９０
ＧＣＣＡＡＴＡＴＴＧ
４００
ＣＴＴＣＴＧＴＡＧＣ
４１０
ＴＣＡＴＡＧＣＴＧＣ
４２０
ＴＴＴＧＡＴＣＣＡＧ
４３０
ＴＡＴＴＴＡＡＴＧＴ
４４０
ＴＣＣＡＡＴＡＧＧＡ
４５０
ＧＣＡＡＡＡＡＴＡＧ
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ＧＴＧＣＴＴＡＣＡFIG. 1 shows a part of the DNA base sequence of 9 to 1M1. FIG. 2 shows a part of the DNA base sequence of pMM2. FIG. 3 shows a part of the DNA base sequence of pMM3. Fourth
The figure shows the restriction enzyme map of pMMl. Figure 5 shows pM
A restriction enzyme map of M2 is shown. FIG. 6 shows a restriction enzyme map of pMM3. Figure 1 - Part 0 CCAATGGCTT 0 TCGCAGCAGA 0 AGAAGCAGCA 0 ACTACTACAG 0 CTCCAAAAT 0 GGACGCTGAT 0 ATGGAAAAAAA 0 CCGTAAACG 0 TCTGGAAGCT 00 CTTGGCGGAT 10 CTTAGTTTAG 20 GTGTGTTTTT 30 ATACTATGGC 40 GCATTAAAG 50 GAATACCGAC 60 ATCTTTAGAT 70 GAAGCAGCTT 80 TAATAGAT GG 90 TTGTAGTAGA 00 TTTAGAATCT 10 ACTGGAATAT 20 CATACTGCCA 30 TTATTAAATC 40 CTACTACGAT 50 CACCCTTGCA 60 GTTTTGGATA 70 TTATGTGGAT 80 ATGGAATGAC 90 TACTTATTGC 00 CATCTTTAGT' 10 CATAAACA AG 20 TCGGTTCCAG 30 GACGCTTCCA 40 CTAATGATTT 50 TTTACTTCTT 60 TAGCCAGTAC 70 ACAAAGCAGT 80 GGAATCTCGG 90 TATGGCAGGA 00 CTGAC AATAG Funeral Figure 1 - Part 10 CAATTTTACC 20 TGTTGTAATT 30 TTCTACTTCT 40 TGGCACAGAG 50 AAAATTAGTC 60 ACAGCTATAA 70 TAGCTGGTGC 80 TGTAAAGCAG 90 TAGGTAAAGC 00 AAAATTATAG 10 TTAGGAGGTG 20 ATATTAAAAT 30 AGAAATTTG A 40 TAGAAAACTGA 50 ATATAATTAT 60 TGAAAGGGGT 70 GGGAACTATT 80 GGCAAATAA 90 TATCTTATTA 00 AAATTTTGG 10 CGGCAAAAAA 20 TAGTGA TTAT 30 ATTATTTTTA 40 TCGTATGATG 50 GGAGGGAAA 60 TAAAAAATAGA 70 TGATTGGAGC 80 TTTTAAAGG 90 TTGGGTCAA 00 AATTGTTTTT 10 GACATTGTTA? 20 ACGGCATCAT 30 TAATTTTTGC 40 ATCTTCTATA 50 GTAACTGCTA) 790 800T
TTGTAATGGG TCCATTAGAA584
- Figure 1 Part 5 1860 1870 TCCAACAACA ATCAAGCTTA880 TGTGAATTGT 890 GCAAATGAAT 900 TAACAGGATA 910 TAATTTTCT 920 GGATTTACAC 930 TTTTA AGACT 940 TTCGAATATT 950 GTAAATAGTG 960 ATGGATCTGT 970 GACATCAGAG 980 ATGGCTCCTT 990 TTGTATTAA 000 TATAGTTACA 010 CTAACGCC TA 020 ACGGTACGAT 030 ACCAGTTACA 040 TTTACAATAA 050 ACAATGCGAC 060 AACTTATTAT 2070 2080 GGACAAAATG TATATATTGT090 TGGTAGTACA 100 TCTGATCTTG 2110 2120 GAAATTGGAA TACAACCTAT130 GCCCGTGGTC 140 CTGCATCATG 150 CCCTAATTAT 160 C CTACTTGGA 170 CAATAACGCT 180 TAATCTATTA 190 CCTGGTGAGC 200 AGATACAGTT 2210 2220 TAAAGCTGTA AAAATTGATA230 GTTCAGGAAA 240 TGT AACTTGG 250 GAAGGTGGCT 260 CGAATCATAC 270 TTATACTGTG 2280 2290 CCGACATCTG GGACTGGTAG300 TGTCACCATT 310 ACATGGCAAA 320 ATTAA TCAAT 330 AAAATGTTAC 23402350 ACATAGAACA AATTGTAAAC360 ACTGGAATAT 370 ATTCCGGTGT 380 TTTTTTGTAT 390 ATTATGGGCG 400 TTTAATGTTA Figure 1 Part 6 410 AAAATAATAG 420 TGTTTTGATT 430 TTATTAAAAAA 440 GTTTGGAGGT 450 AAGAGATGAG 460 TAAAAAAGTT 470 GGTATTCCAA 480 AAGGGCTTTT 490 ATACTACAAC 500 TTTTATCCTA 510 TGTGGAAAAC 520 ATTTTTTGAA 530 GAACTGGGTG 540 CTGAAGTGGT 550 TACTTCCAGT 560 GATACATGTA 570 AAAAAAATAAT 580 TGATGATGGC 590 ATCAAGACTT 600 GCGTGGATGA 610 AACCTGCCTT 620 CCTGTCAAGA 2630 2640 CATTTATGGG TCATGTGATT650 GATTTGAAGG 660 AAAAGGGTGT 670 TGATTATATA 680 TTTGTTCCAA 690 GAGTCATAAG 700 CGTAGAAAGG 710 CGTAGATATC 720 TATGCTCAAA 730 ATTTTTGGGT 740 TTGCCTGACT 750 TAGTGAGAAA 760 TCTTATTTCT 770 GATTTGCCAC 27802790 AAATAATAGA TATGAAGATT800 GATTACTATC 810 GTGGAGAAGA 2820 2830 GTTTATGGAA AGAGAAAATTT840 TGAGAGTTGG 850 CAAGCTGTTT 860 GTTGATAGCA 870 CAAGTAAAAT 880 TAAAGACGCA 890 TACGAAAAAT 900 CTTTAAAAG 586- Figure 1 - Part T 2910 2920 GCAAAGGACT TTTGAATTCA293
0 2940 TTATTAGAGA AGGATTTTCA950 AATACAGAAG 960 CAATAAAAAT 970 TATGGAAGGG 980 AAAAAAATTAG 990 ACGCAAATAC 000 AAAAGGGGAT 010 TTAAAATAG 020 CTTTATTAGC 030 CCATTCCTAT 040 GACATAATG 050 ATGATTATTT 060 ATCTATGGGA 070 TTGATAAATA 080 GGCTTAAGAA 090 TA TGGGAGCT 100 TATGTGCTTA 110 CAACAAATAT 120 GATAGAAAGA 130 GATAAAATTG 140 AAAATGGTGC 150 CAGCAAAACTT 160 CAAAAAAGATT 170 TATTT TGGAC 180 TTATGGAAGG 190 GATATTTTAG 200 GTGCAGGTAA 210 ATATTTTATC 220 GAGAGCAAAG 230 AAGTGGACGG 240 GGTAATATCT 250 GTTTCTGCTT 260 T TGGATGTGG 270 GCCAGACTCT 280 ATCACTGATG 290 ACCTTTTAGA 300 AAGGGATTAC 310 AAGAGAGATG 320 GTGGGATTCC 330 ATTTATGCC 340 ATAA CCATTG 350 ATGAACATAC 360 TGGTGAGGCA 370 GGTTTAAAACT 380 ACCTAGGTTA 390 GAAGCCTTA 400 TGGATTTGTT Figure 1 - Part 8 410 AAGATGGCCA 420 CAAGGAGAGA 430 TTATGGCATG 440 AAAATAACAT 450 ATCCACATAT 460 GGGTTCGTTA 470 AATATGATAT 480 TAAAGACGAT 490 GTTTGAAGGA 500 ATCAATGTAG 510 AAGTTGTGGA 520 GCCGCCACCT 530 GTGACTAATA 3540 3550 GGACTCTAT, CAATTGGAGTA560 AAATATTCAC 5 70 CTGAGTTTGT 580 CTGTCTTCCA 590 TATAAGATAA 600 ATCTGGGGAA 610 TTTTATAGAA 620 GCACTTGAAA 630 ACGGTGCTGA 640 TACTATAATA 650 ATGTTGGGTG 660 GTATAGGGCC 670 TTGCAGGTTC 680 GGTTATTATG 690 GACAAGCACA 700 AAGAGAAACA 710 TTAATTGATC 720 TTGGATACAA 730 G TTTAATATG 740 ACTAATATTA 750 GATCCACAGG 760 ACGGGTGTGG 770 TCGCCATGAT 780 CGCGTAGTCG 790 ATAGTGGCTC 800 CAAGTAGCGA 810 AGCG AGCAGG 820 ACTGGGCGGC 830 GGCCAAAGCG 840 GTCGGACAGT 850 GCTCCGAGAA 860 GG Figure 2 Part 0 AGATCTAAAG 0 CTATCCTGTC 0 TTACAACTTG 0 GCTGTTGTAA 0 ACTTTGAAAA 0 TGCATTAGGA 0 AATTAACCTA 0 ATTCAAGCAA 0 GATTATGAGG 00 TTTTGAACCA 10 AA TTGGAAAA 20 AGGTTCAGTC 30 GTGACAGCCC 40 GCCATATGTC 50 CCCTATAATA 60 CGGATTGTGG 70 CGGATGTCAC 80 TTCGTACATA 90 ATGGACAGGT 00 GAATAACGAA 10 CCACGAAAAA 20 AACTTTAAAT 30 TTTTTTCGAA 40 GGCGCCGCAA 50 CTTTTGATTC 60 GCTCAGGCGT 70 TTAATAGGAT 80 GTCACACGAA 90 AAACGGGGAA 00 TTGTGTAAAA 10 AAGATTCACG 20 AATTCTAGCA 30 GTTGTGTTAC 40 ACTAGTGATT 50 GTTGCATTT T 60 ACACAATACT 70 GAATATACTA 80 GAGATTTTTA 90 ACACAAAAAG 00 CGAGGCTTTC Figure 2 - Part 2 10 CTGCGAAAGG 20 AGGTGACACG 30 CGCTTGCAGG 40 ATTCGGGCTT 50 TAAAAAAGAAA 60 GATAGATTAA 70 CAACAAAATAT 80 TCCCCAAGAA 90 CAATTTGTT 00 ATACTAGGG 10 AGGAGAACAC 20 AAGGTTATGA 30 AAAAGGTCGT 40 TAACAGTGTA 50 TTGGCTAGTG 60 CACTCGCACT 70 TACTGTTGC T 80 CCCATGGCTT 90 TCGCTAGCAT 00 CGCTCCAAAC 10 TTCAAGTTT 20 TCGTTATGGG 30 TCCATTAGAA 40 AAAGTCACAG 50 ATTTTAATG 60 ATTCAA AGAT 70 CAATTGATAA 80 CTTTAAAGAA 90 TAATGGTGTT 00 TATGGTATAA 10 CAACAGATAT 20 TTGGTGGGGC 30 TATGTTGAAA 40 ATGCAGGTGA 50 AAATCAATTT 60 70 80 90 00 Figure 2 - Part 3 810 GAAGTGGGT 20 CCAATAATGT 30 CAACGCATGC 40 CTGTGGAGGT 50 AATGTTGGTG 60 A TACAGTAAA 70 TATACCTATT 80 CCGTCATGGG 90 TATGGACAAA 00 AGATACCCAA 10 GATAATATGC 20 AGTATAAGGA 30 TGAAGCCGGA 40 AATTGGGATA 5 0 ATGAAGCAGT 60 AAGTCCATGG 70 TATTCTGGCT 80 TAACCCAACT 90 CTATAATGAA 000 TTTTATTCAT 010 CTTTTGCATC 020 AAATTTTAGC 030 AGCTATAAAAG 040 ATATAATTAC 050 TAAAATATAT 060 ATATCTGGAG 07 0 GCCCTTCTG 080 AGAATTAAGA 090 TATCCTTCAT 100 ATAATCCTTC 110 GCATGGATGG 120 ACATATCCTG 130 GACGTGGCTC 140 GCTGCAGGTGC 150 T ATAGTAAG 160 CGGCTATAAC 170 AAGTTTCAA 180 AATGCTATGA 190 AGTCTAAATA 200 TGGAACTATA 210 GCAGCAGTTTA 220 ATAGTGCATG 230 GGGT ACAAGC 240 CTAACTGATT 250 TTTCTCAAAT 260 TAGTCCACCT 270 ACAGATGGTG 280 ATAATTTCTT 290 TACAAATGGT 300 TATAAAAACTA Figure 2 Part 4 310 CTTATGG TAA 1320 1330 TGACTTTTTG ACATGGTATC340 AAAGTGTTTT 350 GACTAATGAG 360 TTAGCCAATA 370 TTGCTTCTGT 380 AGCTCATAGC 139゜TGCTTTG ATC 400 CAGTATTTAA 410 TGTTCCAATA 420 GGAGCAAAA 430 TAGCTGGAGT 440 GCATTGGCTA 450 TATAATAGTC 460 CGACAATGCC 470 ACATGCTGCA 480 GAATATTGTG 490 CCGGTTATTA 500 TAATTATAGC 15101520 ACGCTACTCG ATCAATTTAA530 GGCATCTAAT 540 C TTGCTATGA 550 CATTTACATG 560 TCTTGAAATG 570 GATGATTCTA 580 ATGCATATGT 590 AAGTCCATAT 600 TATTCTGCAC 610 CTATGACGTT 620 AGTC CATTAT 630 GTAGCTAATC 640 TTGCTAATAA 650 TAAAGGTATA 1660 1670 GTCCACATG GAGAAAATGC680 TTTGGCTATA 690 TCCAAACAACA 700 ATCAAG CTTA 710 TGTGAATTGT 720 GCAAATGAAT 730 TAACAGGATA 740 TAATTTTCT 750 GGATTTACAC 760 TTTTAAGACT 770 TTCGAATATT 1780 1790 GTAAATAGTG ATGGATCTGT800 GACATCAGAG 810 182° 1 day 30 840 850 Figure 2 - Part 5 1860 ACCAGTTACA 870 TTTACAATAA 880 ACAATGCGAC 890 AACTTATTAT 900 GGACAAAATG 910 TATATATTGT 920 TGGTAGTA 930 TCTGATCTTG 940 GAAATTGGAA 950 TACAACCTAT 960 GCCCGTGGTC 970 CTGCATCATG 980 CCCTAATTAT 990 CCTACTTGGA 000 CAATAACGCT 010 TAATCTATTA 020 CCTGGTGAGC 030 AGATACAGTT 040 T AAAGCTGTA 050 AAAATTGATA 060 GTTCAGGAAA 070 TGTAACTTGG 080 GAAGGTGGCT 090 CGAATCATAC 100 TTATACTGTG 110 CCGACATCTG 120 GGAC TGGTAG 130 TGTCACCATT 140 ACATGGCAAA 150 ATTAATCAAT 160 AAATGTTAC 170 ACATAGAACA 180 AATTGTAAAC 190 ACTGGAATAT 200 ATTCCGG TGT 210 TTTTTTGTAT 220 ATTATGGGCG 230 TTTAATGTTA 240 AAAATAATAG 250 TGTTTTGATT 260 TTATTAAAAAA 270 GTTTGGAGGT 280 AAGAGATGAG 290 T AAAAAAGTT 300 GGTATTCCAA 310 AAGGGCTTTT 320 ATACTACAAC 330 TTTTATCCTA 340 TGTGGAAAAC 350 ATTTTTTGAA Figure 2 - Part 6 2360 2370 GAACTGG GTG CTGAAGTGGT380 TACTTCCAGT 2390 2400 GATACATGTA AAAAAAATAAT410 TGATGATGGC 420 ATCAAGACTT 2430 2440 GCGTGGATGA AACCTGCCTT245
゜CCTGTCAAGA 460 CATTTATGGG 470 TCATGTGATT 2480 2490 GATTTGAAGG AAAAGGGGTGT500 TGATTATATA 510 TTTGTTCCAA 2520 2530 GAGTCA TAAG CGTAGAAAGG540 CGTAGATATC 550 TATGCTCAAA 560 ATTTTTGGGGT 570 TTGCCTGACT 580 TAGTGAGAAA 590 TCTTATTTCT 600 GATTTGCCAC 610 AAATAATAGA 620 TATGAAGATT 630 GATTACTATC 640 GTGGAGAAGA 650 GTTTATGGAA 660 AGAGAAAATTT 2670 2680 TGAGAGTTGG CAAGCTGTTT690 G TTGATAGCA 700 CAAGTAAAAT 710 TAAAGACGCA 720 TACGAAAAAT 2730 2740 CTTTAAAAG GCAAAGGACT750 TTTGAATTCA 2760 2770 2780T
TATTAGAGAGAGGATTTTCA AATA
CAGAAG790 CAATAAAAAT 800 TATGGAAGGG 840 = TAAAAAATAG 285°CTTTATTAGC 860 870 880 890 900 Figure 2 - Part 7 2910 2920 GGCTTAAGAA TATGGG AGCT930 TATGTGCTTA A 940 Figure 3 - 10 CTGCGAAAGG 20 AGGTGAACACG 30 CGCTTGCAGG 40 ATTCGGGCTT 50 TAAAAAAGAAA 60 GATAGATTAA 70 CAACAAA TAT 80 TCCCCAAGAA 90 CAATTTTGTTT 00 ATACTAGAGG 10 AGGAGAACAC 20 AAGGTCATGA 30 TTGGAGCTT 40 TAAAAGGTTG 50 GGTCAAAAAAT 60 TGTTTTTGAC 70 ATTGTTAACG 80 GCATCA TTAA 90 TTTTTGCATC 00 TTCTATAGTA 10 ACTGCTAATG 20 CAAGCATAGC 30 ACCAAATTTC 40 AAAGTTTTTG 50 TAATGGGTCC 60 ATTAGAAAAAA 70 GTC ACAGATT 80 TTAATGCATT 90 CAAAGATCAA 00 TTGATAACTT 10 TAAAGAATAA 20 TGGTGTTTAT 30 GGTATAACAA 40 CAGATATTTG 50 GTGGGGCTAT 60 GTTGAAATG 70 CAGGTGAAAA 80 TCAATTTGAC 90 TGGAGTTATT 00 ATAAGACATA 591- Figure 3 - Part 3 810 820 830
840 850TGCTGATAACCG
TACGCGCTG CGGGATTGAA GTGG
GTTCCA ATAATGTCAA60 CGCATGCCTG 70 TGGAGGTAAT 80 GTTGGTGATA 90 CAGTAAATAT 00 ACCTATTCCG 10 TCATGGGTAT 920 930 GGACAAAAGA T ACCCAAGAT40 AATATGCAGT 50 ATAAGGATGA 60 AGCCGGAAAT 970 980 990TG
GGATAATG AAGCAGTAAG TCCAT
GGTAT000 TCTGGCTTAA 010 CCCAAACTCTA 020 TAATGAATTT 030 TATTCATCTT 040 TTGCATCAAA 105゜TTTTAGCAGC 060 TATAAAAGATA 070 TAATT ACTAA 1080 1090 AATATATATA TCTGGAGGCC100 CTTCTGGAGA 110 ATTAAGATAT 1120 1130 CCTTCATATA ATCCTTCGCA140 TGGATGGACA 150 TATCCT GGAC 160 GTGGCTCGCT 170 GCAGTGCTAT 180 AGTAAAGCGG 1190 1200 CTATAACAAG TTTTCAAAT121
0 1220 1230
1240GCTATGAAGTCTAAAATAT
GG AACTATAGCA GCAGTTATAATA2
50 GTGCATGGGG 1260 1270 TACAAGCCTA ACTGATTTTT280 CTCAAATTAG 290 TCCACCTACA 300 GATGGTGATA 310 ATTTCTTTAC 320 AAATGGTTAT 3 30 AAAACTACTT 340 ATGGTAATGA 350 CTTTTTGACA Figure 3 - Part 4 360 TGGTATCAAA 370 GTGTTTTGAC 380 TAATGAGTTA 390 GCCAATATTG 400 CTTCTGTAG C 410 TCATAGCTGC 420 TTTGATCCAG 430 TATTTAATGT 440 TCCAATAGGA 450 GCAAAAAATAG 460 CTGGAGTGCA 470 TTGGCTATAT 480 AATAGTCCGA 490 CAATGCACA 500 TGCTGCAGAA 510 TATTGTGCCG 520 GTTATTATAA 153゜TTATAGCACG 540 CTACTCGATC 550 AATTTAAGGC 560 ATCTAATCTT 570 GCTATGACAT 580 TTACATGTCT 590 TGAAATGGAT 600 GATTCTAATG 610 CATATGTAAG 620 TCCATATTAT 630 TC TGCACCTA 640 TGACGTTAGT 650 CCATTATGTA 660 GCTAATCTTG 670 CTAATAATAA 680 AGGTATAGTC 690 CACAATGGAG 700 AAAATGCTTT 710 GGCTA TATCC 720 AACAACAATC 730 AAGCTTATGT 740 GAATTGTGCA 750 AATGAATTAA 760 CAGGATATAA 770 TTTTTCTGGA 780 TTTACACTTT 790 TAAGACTTTC 8 00 GAATATTGTA Figure 3 - Part 6 2410 TTCCAGTGAT 420 ACATGTAAAA 430 AAATAATTGA 440 TGATGGCATC 450 AAGACTTGCG 460 TGGATGAAAC 470 CTGCCTTC CT 480 GTCAAGACAT 490 TTATGGGTCA 500 TGTGATTGAT 510 TTGAAGGAAA 520 AGGGTGTTGA 530 TTATATATTT 540 GTTCCAAGAG 550 TCATAAGCGT 560 AGAAAGGCGT 570 AGATATCTAT 580 GCTCAAATT 590 TTTGGGTTTG 600 CCTGACTTAG 610 TGAGAAATCT 6 20 TATTTCTGAT 630 TTGCCACAAA 640 TAATAGATAT 650 GAAGATTGAT 660 TACTATCGTG 670 GAGAAGAGTT 680 TATGGAAAGA 690 GAAATTTTGA 700 GAGTTGGCAA 710 GCTGTTTGTT 720 GATAGCACAA 730 GTAAAATTAA 740 AGACGC:ATAC 750 GAAAAAATCTT 760 TAAAAAAGGCA 770 AAGGACTTT 780 GAATTCATTA 790 TTAGAGAAGG 800 ATTTTCAAAT 810 ACAGAAGCAA 820 TAAAATTAT 830 GGAAGGGAAA 840 AAATTAGACG 850 CAAATACAAAT 3rd Figure-Part 7 2910 ATTATTTATC 292゜TATGGGATTG 930 ATAAAATAGGC 940 TTAAGAATAT 950 GGGAGCTTAT 960 GTGCTTACA
Claims (11)
ラスミドに耐熱性β−アミラーゼ遺伝子を導入したプラ
スミド。(1) A plasmid in which a thermostable β-amylase gene is introduced into a plasmid that can grow on Bacillus subtilis or Bacillus brevis.
の一部または全部をさらに導入した請求項１記載のプラ
スミド。(2) The plasmid according to claim 1, further comprising a part or all of a Bacillus brevis cell wall protein gene introduced therein.
の一部または全部を導入した枯草菌に生育し得るプラス
ミドが、プラスミドｐＮＵ２００であり、耐熱性β−ア
ミラーゼ遺伝子が￥クロスト￥￥リジウムサーモサルフ
ロゲネス￥のβ−アミラーゼ遺伝子である請求項１又は
２記載のプラスミド。(3) Plasmid pNU200 is a plasmid that can grow in Bacillus subtilis into which part or all of the cell wall protein genes of Bacillus brevis have been introduced, and the thermostable β-amylase gene is The plasmid according to claim 1 or 2, which is the β-amylase gene.
−アミラーゼ遺伝子がプラスミドｐＮＫ１のＭｆｌＩ−
ＭｆｌＩ約３．６Ｋｂ断片の一部または全部である請求
項３記載のプラスミド。(4) β of Clostridium thermosulfurogenes
-Amylase gene is MflI of plasmid pNK1-
The plasmid according to claim 3, which is part or all of the approximately 3.6 Kb fragment of MflI.
プラスミドｐＮＫ１（ＤＭｆｌＩ−ＭｆｌＩ約３．６Ｋ
ｂ断片の一部または全部を連結した請求項４記載のプラ
スミド。(5) Plasmid pNU200 (7) Plasmid pNK1 (DMflI-MflI approximately 3.6K
5. The plasmid according to claim 4, wherein part or all of the b fragments are ligated.
子の５８６塩基以降を欠くものであり、この細胞壁タン
パク質遺伝子の直後に￥クロストリ￥￥ジウムサーモサ
ルフロゲネス￥のβ−アミラーゼ構造遺伝子を連結した
ものである請求項４記載のプラスミド。(6) A claim in which the plasmid pNU200 lacks the 586th base and subsequent bases of the cell wall protein gene, and the β-amylase structural gene of \ Clostri \\ Zium thermosulfologenes \ is ligated immediately after the cell wall protein gene. The plasmid described in 4.
子の５１６塩基以降を欠くものであり、この細胞壁タン
パク質遺伝子の直後に￥クロストリ￥￥ジウムサーモサ
ルフロゲネス￥のβ−アミラーゼのシグナル遺伝子及び
構造遺伝子を連結したものである請求項４記載のプラス
ミド。(7) Plasmid pNU200 lacks the 516th base onward of the cell wall protein gene, and the signal gene and structural gene of β-amylase of Clostri ￥ Zium thermosulfurogenes ￥ are linked immediately after this cell wall protein gene. The plasmid according to claim 4, which is
￥バチルスブレビス￥に導入した形質転換体。(8) A transformant obtained by introducing the plasmid according to any one of claims 1 to 7 into Bacillus brevis.
７Ｋ株である請求項８記載の形質転換体。(9) ￥ Bacillus brevis ￥ ￥ Bacillus brevis ￥4
The transformant according to claim 8, which is a 7K strain.
ら耐熱性β−アミラーゼを採取する耐熱性β−アミラー
ゼの製造方法。(10) A method for producing thermostable β-amylase, which comprises culturing the transformant according to claim 8 and collecting thermostable β-amylase from the culture.
請求項１０記載の製造方法。(11) The production method according to claim 10, wherein the transformant is the transformant according to claim 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4847790A JPH03251185A (en) | 1990-02-28 | 1990-02-28 | Production of heat-resistant beta-amylase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4847790A JPH03251185A (en) | 1990-02-28 | 1990-02-28 | Production of heat-resistant beta-amylase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03251185A true JPH03251185A (en) | 1991-11-08 |
Family
ID=12804469
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4847790A Pending JPH03251185A (en) | 1990-02-28 | 1990-02-28 | Production of heat-resistant beta-amylase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03251185A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6506595B2 (en) | 1998-03-31 | 2003-01-14 | Itoham Foods Inc. | DNAs encoding new fusion proteins and processes for preparing useful polypeptides through expression of the DNAs |
| WO2006004067A1 (en) * | 2004-07-06 | 2006-01-12 | Kaneka Corporation | Process for producing protein a-like protein with use of brevibacillus genus bacaterium |
-
1990
- 1990-02-28 JP JP4847790A patent/JPH03251185A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6506595B2 (en) | 1998-03-31 | 2003-01-14 | Itoham Foods Inc. | DNAs encoding new fusion proteins and processes for preparing useful polypeptides through expression of the DNAs |
| WO2006004067A1 (en) * | 2004-07-06 | 2006-01-12 | Kaneka Corporation | Process for producing protein a-like protein with use of brevibacillus genus bacaterium |
| JPWO2006004067A1 (en) * | 2004-07-06 | 2008-04-24 | 株式会社カネカ | Method for producing protein A-like protein using Brevibacillus bacteria |
| JP2010246569A (en) * | 2004-07-06 | 2010-11-04 | Kaneka Corp | Method for producing protein a-like protein with use of genus brevibacillus bacaterium |
| US8597908B2 (en) | 2004-07-06 | 2013-12-03 | Kaneka Corporation | Process for producing protein A-like protein with use of Brevibacillus genus bacterium |
| JP2014064589A (en) * | 2004-07-06 | 2014-04-17 | Kaneka Corp | Method for producing protein a-like protein with use of genus brevibacillus bacterium |
| US8889389B2 (en) | 2004-07-06 | 2014-11-18 | Kaneka Corporation | Process for producing protein A-like protein with use of Brevibacillus genus bacterium |
| JP2016063844A (en) * | 2004-07-06 | 2016-04-28 | 株式会社カネカ | Production method of protein a-like protein using brevibacillus bacteria |
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