JP5702714B2 - 新規キメラアルファアミラーゼ変異体 - Google Patents
新規キメラアルファアミラーゼ変異体 Download PDFInfo
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- JP5702714B2 JP5702714B2 JP2011507544A JP2011507544A JP5702714B2 JP 5702714 B2 JP5702714 B2 JP 5702714B2 JP 2011507544 A JP2011507544 A JP 2011507544A JP 2011507544 A JP2011507544 A JP 2011507544A JP 5702714 B2 JP5702714 B2 JP 5702714B2
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- amylase
- chimeric
- amys
- amino acid
- starch
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- 108010075550 termamyl Proteins 0.000 description 1
- 210000003813 thumb Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 102000003601 transglutaminase Human genes 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 101150110790 xylB gene Proteins 0.000 description 1
- 229930010764 α-maltose Natural products 0.000 description 1
- 229930028731 β-maltose Natural products 0.000 description 1
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Description
A.定義及び略語
1.1定義
B.略語
AmyL:バチルスリケニフォルミス(Bacillus licheniformisαアミラーゼ
AmyQ:バチルスアミロリケファシエンス(Bacillus amyloliquefaciens)αアミラーゼ
AmyS:バチルスステアロセレモフィリス(Bacillus stearothermophilus)αアミラーゼ
AAU:アルファアミラーゼ単位
ATCC:アメリカンタイプカルチャーコレクション
cDNA:相補DNA
C.F.R.:連邦規制基準
CFU:コロニー形成単位
DE:デキストロース当量
DEAE:ジエチルアミノエタノール
DNA:デオキシリボ核酸
DNS:3,5−ニジトロサリチル酸
DPn:nサブユニットを有する重合度の程度
ds:乾燥固形分
EC:酵素分類についての酵素委員会
EEC:欧州経済共同体
EDTA:エチレンジアミン四酢酸
EGTA:エチレングリコール四酢酸
FDA:食品医薬品局
FAO:食糧農業機関
GLP:医薬品安全性試験実施基準
GMP:適正製造基準
GRAS:一般に安全と認められる(食品)、安全食品認定
HFCS:フルクトース高含量コーンシロップ
HPLC:高圧液体クロマトグラフィー
HS:多糖類(DPn、n>3)
JECFA:FAO/WHO合同食品添加物専門家会議Kb:キロベース
kJ:キロジュール
LAT:バチルスリケニフォルミス(B.licheniformis)αアミラーゼ
LU:液化単位
mRNA:メッセンジャーリボヌクレオチド
mg:ミリグラム
mL:ミリリットル
mt:メートルトン(1000kg)
N:ノルマル(Normal)
NTIS:技術情報局
PCR:ポリメラーゼチェーンリアクション
PEG:ポリエチレングリコール
ppm:百万分率
RO:逆浸透
RT−PCR:逆転写ポリメラーゼチェーンリアクション
SB:塩架橋
SDS−PAGE:ドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動
SGA:高品質穀物アミラーゼ(Superior Grain Amylase)
SKBU/gds:乾燥固形分1グラム当たりのαアミラーゼ単位。αアミラーゼ1単位は1XSSC、0.15M NaCl、0.015Mクエン酸ナトリウム、pH7.0でのアッセイ条件において、1時間に1.0gの限界デキストリン基質をデキストリン化する。
WHO:世界保健機構
w/v:重量/容量
w/w:重量/重量
μg:マイクログラム
μL:マイクロリットル
第一の側面において、約480−515アミノ酸残基のキメラポリペプチドを提供する。このキメラポリペプチドはAmyLアミラーゼのN末端部分の約180以上の連続的なアミノ酸残基を含むアミノ末端ドメインと、AmySアミラーゼのカルボキシ末端部分を含むカルボキシドメインとを含む。このキメラポリペプチドはAmyLアミラーゼ又はAmySアミラーゼのいずれかの1次アミノ酸配列を有してはいない。しかしながら、このキメラポリペプチドは少なくともAmySアミラーゼと比較して高められた熱安定性を有している。
D.キメラアミラーゼを含む組成物
(a)前述のキメラポリペプチドであって、約480乃至515アミノ酸残基長を有し、AmyLのN末端部分の約180以上の連続するアミノ酸残基を含むアミノ末端ドメイン及びAmySアミラーゼのカルボキシ末端部分を含むカルボキシ末端ドメインを有し、少なくともAmySアミラーゼよりも、高い熱安定性を有する、キメラポリペプチド;
(b)約475乃至502アミノ酸残基長の熱安定性キメラαアミラーゼであって、AmyLアミラーゼのN末端部分からの連続するアミノ酸配列を含むN末端部分及びAmySアミラーゼのC末端部分からの連続的なアミノ酸配列を含むC末端ドメインを含み、前述のキメラアミラーゼはAmyLアミラーゼよりも特異活性が高く、95℃における熱安定性がAmySアミラーゼよりも高い、熱安定性キメラアミラーゼ、及び
(c)(a)のキメラポリペプチドと(b)の熱安定キメラアミラーゼの組合せ。
E.キメラアミラーゼの特徴付け
F.キメラαアミラーゼをコードしているポリヌクレオチド
(a)前述のキメラポリペプチドであって、約480乃至515アミノ酸残基長を有し、AmyLのN末端部分の約180以上の連続するアミノ酸残基を含むアミノ末端ドメイン及びAmySアミラーゼのカルボキシ末端部分を含むカルボキシ末端ドメインを有し、少なくともAmySアミラーゼよりも、高い熱安定性を有する、キメラポリペプチド、
(b)約475乃至502アミノ酸残基長の熱安定性キメラαアミラーゼであって、AmyLアミラーゼのN末端部分からの連続するアミノ酸配列を含むN末端部分及びAmySアミラーゼのC末端部分からの連続的なアミノ酸配列を含むC末端ドメインを含み、前述のキメラアミラーゼはAmyLアミラーゼよりも特異活性が高く、95℃における熱安定性がAmySアミラーゼよりも高い、熱安定性キメラアミラーゼ。
(a)前述のキメラポリペプチドであって、約480乃至515アミノ酸残基長を有し、AmyLのN末端部分の約180以上の連続するアミノ酸残基を含むアミノ末端ドメイン及びAmySアミラーゼのカルボキシ末端部分を含むカルボキシ末端ドメインを有し、少なくともAmySアミラーゼよりも、高い熱安定性を有する、キメラポリペプチド、
(b)約475乃至502アミノ酸残基長の熱安定性キメラαアミラーゼであって、AmyLアミラーゼのN末端部分からの連続するアミノ酸配列を含むN末端部分及びAmySアミラーゼのC末端部分からの連続的なアミノ酸配列を含むC末端ドメインを含み、前述のキメラアミラーゼはAmyLアミラーゼよりも特異活性が高く、95℃における熱安定性がAmySアミラーゼよりも高い、熱安定性キメラアミラーゼ、又は(c)これらの任意の組合せ、及び
このスラリーを組成物と一緒にデンプンスラリーを液化するのに充分な温度及び/又は時間インキュベートする工程を含む。
(配列番号39)
(配列番号40)
このアッセイはメガザイムエンドαアミラーゼキットK−CERA08/05(AOAC法2002.1)(メガザイムインターナショナル、アイルランド)の公式な方法の変法である。試薬バイアルは、非還元エンドブロックp−ニトロフェニルマルトヘパトシド(BPNPG7、54.5mg)及び熱安定性アルファグルコシダーゼ(pH6.0、125U)この基質を含む。各アッセイのために、1つのバイアルの全体の含量を蒸留水の10.0mlに溶解した。30mlアッセイ緩衝液(50mMりんご酸Na、2.6mMCaCl2、50mMNaCl、0.002%トライトンX−100、pH6.7)をこのバイアル溶液に添加して、10mlアリコートを使用前まで凍結させた。緩衝液中の0.97ml基質溶液をキュベット(好ましくはマスクしたもの)へ添加した。このキュベットをホルダー中に置き、ブランク読みを得た。10μL酵素サンプルをキュベットに添加して、アッセイを開始した。1分当りの吸光度を400nm又は410nmで測定し、この値を希釈及びタンパク質濃度について補正した。残余活性%をAmyS対照を100%としてモニタリングした後、各キメラ酵素について報告した。
8gNaOHを300mlの水に溶解し、5gの3乃至5ジニトロサリチル酸をこの溶液に添加し、溶解し、必要に応じて加熱する。150g酒石酸カリウムナトリウムをこの溶液に添加して、総量を500mLにした。
4%のデンプン基質溶液(100mlの水に5.33gポテト基質を溶解)。ワーキング基質溶液のために、1部の緩衝液を3部のデンプン溶液に添加した。基質溶液は毎日新たに調製した。
AmyL及びAmyS酵素由来の第一キメラαアミラーゼの熱安定性スクリーニング
結果を図2に示す。AmyL及びAmySの熱安定性について知られているもの又は信じられていることに基づいて、一部位交差での予想はAmyL由来のアミノ酸残基の数が多くなると、キメラの熱安定性が大きくなるということである。しかしながら、図2からわかるように、AmyL由来のアミノ酸残基の数が最も低いキメラアミラーゼが最も熱安定性があるというおどろくべき事実が発見された。興味深いことに、AmyL由来の初めの187残基を含むキメラアミラーゼは187位におけるたった一つのアミノ酸残基の違いにのみにも関わらず、熱安定性が高くなかった。
キメラ分子の第一セットについての熱安定性スクリーニングの結果を図3に示す。おどろくべきことに、2つのアミノ酸残基の置換はキメラの熱安定性を改善した。特定の理論に拘束されることを意図しないけれども、S187D及びS188T変異は活性部位又は酵素の全体三次構造を安定させる塩架橋を形成するのを助け、それゆえ熱安定性が高くなるようだ。一方、他に関するS187D変異体はAmyLアミノ酸配列は熱に対して安定化されているけれども、本明細書のキメラアミラーゼに関しては、S178D変異タンパク質幾つかのに存在するAsp残基は分子のAmyS部分からの1つ以上のアミノ酸残基と相互作用して、その結果熱安定性が増す。従って、例えば、AmyL配列由来の200、202、及び208残基を有するキメラアミラーゼは塩架橋又は他の安定化構造が含まれているので、良い熱安定性を提供する。
より長いAmyl配列を有する塩架橋安定化キメラの取込
キメラアミラーゼの第二セットについての熱安定性スクリーニングの結果を図4に示す。この図に示されるように、キメラアミラーゼのそれぞれは試験条件下で優れた熱安定性を示した。これらの第三世代キメラ酵素は良好な熱安定性を示さなかった第一世代酵素よりも30乃至40%以上のAmyL配列を有し、特にAmyL及びAmySの両方の好適な性質を有するキメラを作ることができる。これらの酵素は、デンプン分解、HFCS生成、でサイジング、及びクリーニング等の熱安定性アミラーゼが近年用いられている全ての製品に有用である。それらの高められた特異活性及び高い熱安定性に依存して、デンプンスラリーの最終粘度だけでなく、低いピーク粘度を提供するので、デンプンの液化工程に特に有用である。
特異活性を比較した結果を酵素1mg/mlが1秒間に生成スルグルコースmg/mlとして図5に示した。これらの3つのキメラアミラーゼは高温においてAmyLアミラーゼと比較したときに、有意に高い特異活性を示した。
図6は全粒コーン基質の粘度低下が試験されたアミラーゼキメラのいくつかについて、観察されたことを示している。タンパク質安定性で見られた結果と一致して(図2)、ハイブリッド186は最終粘度においてAmyS対照に匹敵する性能を示した。キメラ228は高温に置ける熱安定性が良好でなかった(図2)ことと一致して粘度低下を示さなかった。塩架橋キメラ202SB及び228SBの両方はこのアッセイにおいて粘度低下を示した。塩架橋キメラ228SBのケースにおいて、ピーク粘度はAmyS対照と同じであり、最終粘度はAmyS対象に見られるものよりも低かった(図6)。この事は明らかに、性能における利点を示している。
Claims (51)
- 480乃至515アミノ酸残基の長さを有するキメラポリペプチドであって、
このキメラポリペプチドは
(a)AmyLアミラーゼのN末端部分の186位までの連続しているアミノ酸残基を含むアミノ末端ドメイン及び(c)AmySアミラーゼのカルボキシ末端部分を含むカルボキシ末端ドメインを有し、
ただし、キメラポリペプチドは野生型AmyLアミラーゼ又はAmySではなく、このキメラポリペプチドはAmySアミラーゼよりも高い熱安定性を有する、キメラポリペプチド。 - 前記AmyLアミラーゼが配列番号1のアミノ酸配列を有する、請求項1のキメラポリペプチド。
- AmySアミラーゼが配列番号2で定義されるアミノ酸配列を有する、請求項1のキメラポリペプチド。
- キメラポリペプチドが配列番号1又は2ではない場合に、配列番号1乃至17のいずれかと少なくとも95%の配列同一性を有する請求項1のキメラポリペプチド。
- 95℃で30分インキュベーションした後にその活性が少なくとも50%残っているα-アミラーゼの触媒活性を含む請求項1のキメラポリペプチド。
- 95℃で60分インキュベーションした後に、少なくとも60%の触媒活性が残っている請求項5のキメラポリペプチド。
- 95℃で60分インキュベーションした後に少なくとも80%の触媒活性が残っている、請求項6のキメラポリペプチド。
- AmyLアミラーゼよりも高い特異活性を有するα-アミラーゼの触媒活性含む請求項1のキメラポリペプチド。
- N末端部分がAmyLアミラーゼのN末端部分由来の186位までの連続したアミノ酸配列及びC末端部分がAmySアミラーゼのC末端部分由来の連続したアミノ酸配列を含み、このキメラα-アミラーゼはAmyLアミラーゼよりも高い特異活性、AmySアミラーゼよりも95℃において高い熱安定性、及び475乃至520残基長の一次アミノ酸配列を有する、N末端部分とC末端部分とを含む熱安定性キメラα-アミラーゼ。
- デンプンスラリーの液化工程に用いられた時に、比較可能な液化工程において、AmySアミラーゼのデンプンスラリーと等しいピーク粘度低下を示し、比較可能な液化工程においてAmyLアミラーゼと同様のデンプンスラリーの最終粘度にまで減らす、請求項9のキメラアミラーゼ。
- 95℃で30分インキュベートした後のその特異活性が少なくとも50%残っている、請求項10のキメラポリペプチド。
- 95℃で60分インキュベートした後のその特異活性が少なくとも80%残っている、請求項11のキメラポリペプチド。
- AmyLアミラーゼが配列番号1の配列を有し、AmySが配列番号2の配列を有する、請求項10のキメラポリペプチド。
- キメラアミラーゼが配列番号1又は2ではない場合、配列番号1乃至17のいずれかに少なくとも95%の配列同一性を有する請求項12のキメラポリペプチド。
- AmyLアミラーゼのN末端部分の186位までの連続的なアミノ酸残基を含むアミノ末端ドメイン及びAmySアミラーゼのカルボキシ末端部分を含むカルボキシ末端ドメインを有し、AmySアミラーゼに対して少なくとも高められた熱安定性を有する、480乃至515アミノ酸残基の長さを有するキメラポリペプチドを含む組成物。
- 更に1つ以上の追加的なポリペプチドを含む請求項15の組成物。
- 前記1つ以上の追加的なポリペプチドが酵素である、請求項16の組成物。
- 1つ以上の追加的な洗剤又は洗浄剤を含む請求項15の組成物。
- 食品又は食品加工に用いられる、請求項15の組成物。
- 請求項15の組成物を含む食品グレードの凍結乾燥組成物。
- AmylLのN末端部分の186位までの連続的なアミノ酸残基を含むアミノ末端ドメイン及びAmySアミラーゼのカルボキシ末端を含むカルボキシ末端ドメインを有し、少なくともAmySアミラーゼに対して高められた熱安定性を有し、480乃至515アミノ酸残基の長さのキメラポリペプチドをコードしているポリヌクレオチド。
- 前記ポリペプチドが配列番号1又は2ではない場合、配列番号1乃至17のいずれかと加えて少なくとも95%同一なポリペプチドをコードする、請求項21のポリヌクレオチド。
- コドン使用が微生物又は植物においてキメラポリペプチドを発現するのに最適化されている、請求項22のポリヌクレオチド。
- 請求項23のポリヌクレオチドを含むベクター。
- ベクターが発現ベクターである、請求項24のベクターを含むバクテリア細胞。
- 請求項21のポリヌクレオチドを発現している宿主細胞。
- 宿主細胞がバクテリア又は植物である、請求項26の宿主細胞。
- 食品加工剤の生成に利用可能な微生物由来の請求項27の宿主細胞。
- バチルスリケニフォルミス、バチルススブチリス、又はバチルスステアロセレモフィリスである、請求項26の宿主細胞。
- 宿主細胞が植物であり、前記植物がエタノール生成に用いられる請求項27の宿主細胞。
- キメラポリペプチド又は熱安定性α-アミラーゼを含む組成物を生成する方法であって、
バチルスリケニフォルミス、バチルススブチリス、及びバチルスステアロセレモフィリスからなる群より選択される宿主細胞をもちいて、タンパク質が発現される発酵工程を用いて、
前記タンパク質はAmyLアミラーゼのN末端部分の186位までの連続するアミノ酸残基を含むN末端部分及びAmySアミラーゼのカルボキシ末端部分を含むカルボキシ末端ドメインを有し、少なくともAmySに対して高められた熱安定性を有し、480乃至515アミノ酸残基の長さを有するキメラポリペプチドを含み、
少なくとも発現されたポリペプチドを部分的に生成し、従って、組成物を生成する、方法。 - 発酵プロセスがフェドバッチ発酵プロセスである、請求項31の方法。
- 更に発現されたポリペプチドを精製してin vitroアッセイにおいて遺伝毒性の可能性がなく、急性毒性もなく、ヒトを除く動物投与試験において順慢性的である、精製組成物を生成する工程を含む、請求項31の方法。
- 精製された組成物は40ppmを超えない総重金属を含み、標準的な試験により、5ppmを超えないヒ素を含み、10ppmを超えない鉛を含み、5x104の総生菌を含み(CFU/g)、30コリフォームズ(CFU/g)未満、及び検出不可能なサルモネラ、マイコトキシン、又は抗バクテリア活性を有しない、請求項33の方法。
- 組成物が1つ以上のα-アミラーゼ活性を有する、請求項31の方法。
- デンプンスラリーを液化する方法であって、
デンプンを含むスラリーを調製する工程、
液化に使用可能な温度にスラリーを加熱する工程、
このスラリーに、
AmyLアミラーゼのN末端部分の186位までの連続的なアミノ酸残基を含むN末端ドメイン及びAmySアミラーゼのカルボキシ末端部分を含む、カルボキシ末端部分を有し、少なくともAmySアミラーゼに対して高められた熱安定性を有し、480乃至515アミノ酸残渣の長さのキメラポリペプチドを含む組成物を添加する工程、及び
このスラリーを組成物と一緒にインキュベートしてデンプンスラリーを液化するのに十分な時間及び温度で、デンプンスラリーを液化する工程を含む、方法。 - 組成物の添加が比較可能な液化工程に用いられるAmySの添加によると同程度にスラリーのピーク粘度を減らし、比較可能な液化において用いられるAmyLアミラーゼの添加と同程度にこのスラリーの最終粘度を減らす、請求項36の方法。
- 温度が少なくとも80℃乃至100℃である、請求項37の方法。
- スラリーが乾燥重量ベースで15乃至40%のデンプンを含む、請求項36の方法。
- 液化が発酵の一部である、請求項39の方法。
- 発酵が食品、食品添加物、燃料、又は燃料添加物を生成するために用いられる、請求項40の方法。
- 燃料又は燃料添加剤がアルコールである、請求項41の方法。
- アルコールがエタノールである、請求項42の方法。
- 除去されるべきデンプンを含む表面を提供する工程、この表面に請求項15の組成物をデンプンを除去するのに十分な時間及び温度、接触させる工程を含む、デンプンを除去して表面を洗浄する方法。
- 組成物がプロテアーゼ、リパーゼ、追加的なアミラーゼ、又はこれらの組み合わせを含む、請求項44の方法。
- 接触工程の前にデンプン残渣をすすぐ、又は取り除く工程を更に含む、請求項45の方法。
- 接触工程の温度が少なくとも50乃至100℃の間の温度である、請求項44の方法。
- デンプン又はデンプン誘導体を含むコーティングに接触させたことのある不織布を処理する方法であって、この不織布を請求項15の組成物を含む溶液に、この不織布からコーティングの大部分を除去するのに十分な時間及び条件下で接触させる工程を含む、請求項45の方法。
- 前記不織布が布製品である請求項48の方法。
- 接触工程が室温の大気圧よりも高い圧力で行われる、請求項48の方法。
- スラリーの液化を促進するキットであって、前記キットは
AmyLアミラーゼのN末端部分の186位までの連続的なアミノ酸残基を含むアミノ末端ドメイン及びAmySアミラーゼのカルボキシ末端部分を含むカルボキシ末端ドメインを有し、少なくともAmySアミラーゼに対して高い熱安定性を有し、480乃至515アミノ酸残基の長さのキメラポリペプチド及び
デンプンスラリーの液化におけるキットの説明書を含む、キット。
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PCT/US2009/041498 WO2009134670A2 (en) | 2008-04-30 | 2009-04-23 | New chimeric alpha-amylase variants |
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JP2011520432A (ja) | 2011-07-21 |
WO2009134670A2 (en) | 2009-11-05 |
US20140106409A1 (en) | 2014-04-17 |
CA2722889A1 (en) | 2009-11-05 |
CN102016044B (zh) | 2014-11-26 |
MX2010011721A (es) | 2010-11-30 |
US20110097778A1 (en) | 2011-04-28 |
WO2009134670A3 (en) | 2010-04-15 |
US9303254B2 (en) | 2016-04-05 |
CN102016044A (zh) | 2011-04-13 |
EP2283136A2 (en) | 2011-02-16 |
BRPI0910547A2 (pt) | 2015-08-04 |
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