JP5413949B2 - ドライイースト製造用組成物 - Google Patents
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Description
一倍体パン酵母である3346 株および3347 株におけるMPR遺伝子のコピー数を判定するために、パルスフィールドゲル電気泳動を行い、次いでMPR1遺伝子をプローブとして用いてサザン分析を行った(図2)。パルスフィールドゲル電気泳動は以前に記載されたようにして、いくらか改変を加えて行った(J. Bacteriol. 182:4249-4256)。最初の切り替え時間間隔を50秒とし、最後の切り替え時間間隔を75秒とした。総泳動時間は30時間であった。サザンブロッティング分析のために用いたプローブは、以前に記載されているMPR1遺伝子のコード領域を含む1.6 kb Bgl II-Mlu I 断片とした(J. Bacteriol. 182:4249-4256)。結果は明らかに、両方の株 3346 および3347がX染色体に1つのMPR遺伝子を有していることを示した。一方、Σ1278b 株およびMB329-17C 株は2コピーのMPR遺伝子を有し(XIV染色体上にMPR1およびX染色体上にMPR2)、S288C 株はMPR遺伝子を有さない。LD1014 株はMPR1およびMPR2遺伝子の両方の遺伝子破壊株であり、これをネガティブコントロールとして用いた。パン酵母におけるMPR遺伝子をMPR1と命名した。パン酵母におけるMPR遺伝子のヌクレオチド配列を配列決定したところ、推定アミノ酸配列(配列番号1)は、Σ1278b 株のMpr2と全く同一であった(データ示さず)。
本実施例で用いたすべての酵母株はS. cerevisiae 一倍体株、3346−ura3および3347−ura3由来のものであった。本実施例にて用いたすべての株の関連する遺伝子型を表1に列挙する。変異型Mpr1を発現するパン酵母株を構築するために、変異型Mpr1の発現のためのプラスミド、pRS406−K63RおよびpRS406−F65Lを構築した。pRS406-K63Rは、URA3マーカーをもつYIp型のプラスミドpRS406のマルチクローニングサイト内に含まれるKpnIサイトに、変異型MPR1遺伝子の上流、コーディング領域、下流領域を含む約5.5 kbのKpnI断片を挿入して作製した。pRS406-F65Lは変異型MPR1遺伝子のORFを開始コドン側にHindIIIサイト、終止コドン側にSac Iサイトを付加した下記プライマーでPCRにて増幅し、pRS406のマルチクローニングサイト内のhindIIIとSac Iに挿入した。
開始コドン側 5'-GGCCAAGCTTAGATGGATGCGGAATC-3'(配列番号2)
終止コドン側 5'-CCCCGAGCTCTGTCTATGATTATTCCATGG-3'(配列番号3)
pMPR1Uは以下のとおり作製したプラスミドである。MPR1遺伝子の上流、下流域を含む約5.4 kbのSau3AI断片をpYES2(インビトロジェン)ベクターのBamHIサイトに挿入したプラスミドpMH1を作製した。pMH1の1.6 kbのBglII-MluI断片を取り除き、そこにURA3遺伝子、プロモーター、ターミネーターを含む1.2 kbのHindIII断片をベクター、インサートとも平滑化したのちライゲーションして作製した。
ストレス条件下でのパン酵母におけるMPR1遺伝子の寄与を評価するために、乾燥ストレス下でのmpr1-破壊二倍体パン酵母の細胞生存率および細胞内ROSレベルを試験した。
パン製造中の発酵プロセスにおけるMPR1遺伝子の関与を調べるために、生地中の△mpr1株の乾燥ストレス耐性をアッセイした。
以前の研究により、本発明者らは酵素機能が改良された変異型Mpr1を単離し、K63RまたはF65L突然変異がそれぞれMpr1の抗酸化活性または熱安定性の上昇を示すことを見いだしている(国際公開2008/007475公報)。それゆえ、パン酵母における元のMpr1の代わりに変異型Mpr1を発現させることを試み、WT、K63R、F65L 株の発酵能を評価した。
Claims (8)
- 配列番号1に示される野生型Mpr1アセチルトランスフェラーゼ、あるいは、配列番号1に示される野生型Mpr1アセチルトランスフェラーゼのアミノ酸配列の、Lys63がArgに置換された、または、Phe65がLeuに置換された、変異型アセチルトランスフェラーゼMpr1のいずれかをコードする遺伝子を含むパン酵母を含む、ドライイースト製造用組成物。
- 配列番号1に示される野生型Mpr1アセチルトランスフェラーゼのアミノ酸配列の、Lys63がArgに置換された、または、Phe65がLeuに置換された、変異型アセチルトランスフェラーゼMpr1のいずれかをコードする遺伝子を含むパン酵母を含む、ドライイースト製造用組成物。
- パン酵母が二倍体である請求項1または2に記載のドライイースト製造用組成物。
- パン酵母が、野生型γグルタミン酸リン酸化酵素遺伝子のAsp154位がAsnで置換されてなるプロリン多産性型酵母である、請求項1〜3のいずれかに記載のドライイースト製造用組成物。
- 請求項1〜4のいずれかのドライイースト製造用組成物を用いることを含む、ドライイーストの製造方法。
- 請求項5の方法により製造されたドライイースト。
- 請求項6のドライイーストを用いることを含む、パン生地の製造方法。
- パン生地が冷凍パン生地である。請求項7の製造方法。
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