JP6675519B2 - D型アミノ酸脱水素酵素 - Google Patents
D型アミノ酸脱水素酵素 Download PDFInfo
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- JP6675519B2 JP6675519B2 JP2019535714A JP2019535714A JP6675519B2 JP 6675519 B2 JP6675519 B2 JP 6675519B2 JP 2019535714 A JP2019535714 A JP 2019535714A JP 2019535714 A JP2019535714 A JP 2019535714A JP 6675519 B2 JP6675519 B2 JP 6675519B2
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Description
項1.
下記(a)及び(b)の特徴を有する酵素:
(a)D−アミノ酸を可逆的に脱水素する活性を有する
(b)配列番号2のアミノ酸配列との同一性が80%以上であるアミノ酸配列を有するポリペプチドの6量体である。
項2.
2−オキソブタン二酸からD−アスパラギン酸を合成する活性を有する、項1に記載の酵素。
項3.
更に下記特徴(c)を有する項1又は2に記載の酵素:
(c)NADH及びNADPHの両方を補酵素として利用可能である。
項4.
更に下記特徴(d)を有する項1〜3のいずれかに記載の酵素:
(d)meso−ジアミノピメリン酸を基質とし、NAD+を補酵素とする場合のNAD+に対するKm値が30mM以下である。
項5.
更に下記特徴(e)を有する項1〜4のいずれかに記載の酵素:
(e)meso−ジアミノピメリン酸を基質とする場合の至適活性pHが10.5である。
項6.
更に下記特徴(f)を有する項1に記載の酵素:
(f)meso−ジアミノピメリン酸を基質とする場合の至適活性温度が55℃である。
項7.
配列番号2のアミノ酸配列との同一性が80%以上であるアミノ酸配列において、Asp94Ser、Met154Leu、Val158Gly、Thr173Ile、Arg183Met、及びHis229Asnからなる群より選択される1つ以上のアミノ酸置換を有する、項1又は2に記載の酵素。
項8.
項1〜7のいずれかの酵素をコードするポリヌクレオチド。
項9.
項8に記載のポリヌクレオチドを組み込んだベクター。
項10.
項9に記載のベクターを含む形質転換体。
項11.
項10に記載の形質転換体を培養することを含む、項1〜7のいずれかに記載の酵素の製造方法。
項12.
項1〜7のいずれかに記載の酵素を2−オキソ酸に作用させ、D−アミノ酸を製造する方法。
D−アミノ酸+NAD(P)++H2O⇔2−オキソ酸+NH4 ++NAD(P)H+H+
D型アミノ酸脱水素酵素遺伝子は、公知の遺伝子クローニング技術を用いて取得することができる。例えば、GenBank等の公知のデータベースを検索することによって取得可能な配列情報を基に遺伝子を合成して取得できる。
上記実施例1で得られた発現ベクターを利用して、E. coli BL21(DE3)株を形質転換した。これを、抗生物質アンピシリン(最終濃度 100mg/L)を含むLB培地(500 mL)に接種し、A600=0.6程度になるまで37℃で振とう培養し、その後、イソプロピル−β−D(−)−ガラクトピラノシド(和光純薬社製)を最終濃度で0.1 mMとなるように加え、37℃でさらに6時間振とう培養した。
上記実施例2で取得したD型アミノ酸脱水素酵素について、補酵素依存性を評価した。前記酵素の補酵素依存性は、酵素の触媒反応に起因した活性染色法により評価した。
実施例2で得られたD型アミノ酸脱水素酵素について、最適pHを評価した。前記酵素の活性は、酵素の触媒反応で生成するNADPHを波長340 nmの吸光度の増大を測定することに定量し、これを指標として、酵素活性を求めることにより測定した。
D:酵素希釈率
6.22:340 nmにおけるNADPHのミリモル分子吸光係数 (L・mmol−1・cm−1)
C:タンパク濃度 (mg/mL)
d:光路長 (1cm)
所定温度(50、55、60、65、70、75、又は80℃)で加温した反応溶液に1.25 mM NADP+を添加し、直ちに吸光度の増大を測定した以外は実施例4と同様にして吸光度を測定し、相対活性を算出した。図6に、測定結果を示す。この結果から至適活性温度は、約55℃であることが確認された。
実施例2で精製したD型アミノ酸脱水素酵素を、10 mMリン酸緩衝液(pH 7.2)中で、様々な温度条件(50、55、60、65、又は70℃)下で30分間熱処理し、氷中に5分間静置後の残存活性を確認した。酵素活性は、実施例4に記載の方法で、meso−ジアミノピメリン酸を基質として利用した場合のNADPHの生成に起因した340 nmにおける吸光度の増大により評価した。50℃での処理における活性を100%として、その他の温度での処理後の残存活性を相対活性として算出した。
実施例2で精製したD型アミノ酸脱水素酵素を、100 mMの各緩衝液(pH 1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0、9.5、10.0、10.5、11.0、又は11.3)中で、50℃で30分間熱処理し、氷中に5分間静置後の残存活性を確認した。酵素活性は、実施例4に記載の方法で、meso−ジアミノピメリン酸を基質として利用した場合のNADPHの生成に起因した340 nmにおける吸光度の増大により評価した。pH 9.0での処理における活性を100%として、その他のpHでの処理後の残存活性を相対活性として算出した。
実施例2で得られたD型アミノ酸脱水素酵素について、meso−ジアミノピメリン酸を基質に、NADP+またはNAD+を補酵素に用いて活性の測定を行い、速度論的解析を行った。
精製したD型アミノ酸脱水素酵素(濃度10.64 mg/mL)溶液と、0.2 M 塩化カリウム、20%w/vポリエチレングリセロール3,350から成る結晶化溶液を同量ずつ(各々0.5 μL)混合した。96穴プレート(ハンプトン・リサーチ社)を使用して、上記の結晶化溶液50 μLを母液とし、シッティングドロップ法での蒸気拡散を用いて、20℃にて静置した。1日後に結晶が析出し、3日後には測定可能な大きさ(1.5×1.0×1.0 mm程度)の結晶に成長した(図9)。
D型アミノ酸脱水素酵素の結晶は、常温測定ではX線損傷により結晶が劣化し、徐々に分解能が下がるため、低温条件下での測定を行った。結晶を、30%のグリセロールを含む結晶化溶液に移した後、90Kの窒素ガスを吹き付け、急速冷却した。X線回折装置 MX300HEdetector(Raynonix社製)を用いて、2.30Å分解能のX線回折データを収集し、結晶学的パラメーターを決定した。空間群はC2、格子定数は、a=132.88Å、b=100.45Å、c=83.27Å、α=90°、β=110.01°、γ=90°となった。非対称単位に6つの分子が含まれると仮定すれば、結晶の水分含有率は54.1%となった。
得られたX線回折強度データと、実施例10で取得したD型アミノ酸脱水素酵素の三次元構造座標を用いて、プログラムPHASERによる分子置換法を行った。Symbiobacterium thermophilum由来のmeso−DAPDHの三次元構造座標をサーチモデルとして分子置換法の計算を行った。50.0Åから2.30Å分解能までのX線回折強度データを用いた計算の結果、1種類の有意な解が得られた。
T. lipolytica由来のD型アミノ酸脱水素酵素のアミノ酸配列に対して、6種類の変異(Asp94Ser、Met154Leu、Val158Gly、Thr173Ile、Arg183Met、His229Asn)が導入された変異酵素のポリペプチドをコードするDNAを合成により取得した。これを鋳型に利用して、タカラバイオ社製の「PrimeSTAR Max DNA Polymerase」を使用し、PCRにより当該酵素の遺伝子を増幅した。PCRは、製造業者の指示に従って実行した。PCR反応液は、以下のプライマーを各0.3 μM、上記の鋳型DNAを50 ng含んで調製した。
5’−TTAAACCAGTTGGCGGATGATTTCATCCGG−3’(配列番号10)
5’−GCTGGCCGTGAATATCATAGCTATCCACGG−3’(配列番号12)
上記実施例2で取得したD型アミノ酸脱水素酵素について、光学活性を評価した。なお酵素の光学活性は、酵素の触媒反応に起因した活性染色法により評価した。より詳細には、適量の酵素溶液を、ディスクゲル電気泳動に供した。泳動後のゲルを、200 mM リン酸種緩衝液(pH8.0)、10 mM D−アラニンまたはL−アラニン、0.1 mM 2−(4−ヨードフェニル)−3−(4−ニトロフェニル)−5−フェニル−2H−テトラゾリウム塩化物 (INT)(同仁化学社製)、 0.04 mM 1−メトキシ−5−メチルフェナジニウムメチル硫酸塩(PMS)(同仁化学社製)及び1.25 mMのNADP+を含む反応液に浸して、50℃で30分間保温した。この反応液中の2−(4−ヨードフェニル)−3−(4−ニトロフェニル)−5−フェニル−2H−テトラゾリウム塩化物が還元されて、水溶性ホルマザンを生じる。反応式を以下に示す。尚、下記の反応式では、D型アミノ酸脱水素酵素を「meso−DAPDH」と表記する。
実施例2及び12で得られた各種酵素のD−アミノ酸合成活性の測定を行い、変異導入が、D−アミノ酸の合成活性に及ぼす影響を検討した。前記酵素の活性は、酵素の触媒反応で減少するNADPHまたはNADHを波長340 nmの吸光度の減少を測定することに定量し、これを指標として、酵素活性を求めることにより測定した。より詳細には、適量の酵素溶液を、5 mM 2−オキソ酸、0.1 mM NAD(P)H、200 mM 塩化アンモニウムを含む200 mMのグリシン緩衝液(pH9.5)中で混合することにより反応液を調製した。続いて、この反応液中のNAD(P)HからNAD(P)+への変化に伴う340 nmの吸光度の減少を反応温度50℃で測定することにより活性測定を行った。吸光度は、紫外可視分光光度計 UV−1800(SHIMADZU社製)により測定した。得られた吸光度変化と実施例4で使用した式と同じ式を利用して酵素活性を測定し、使用した酵素のタンパク質量と酵素希釈率から酵素の比活性を算出した。表3に、各酵素のD−アミノ酸合成活性をそれぞれ示す。
Claims (9)
- 下記(a)及び(b)の特徴を有する酵素:
(a)D−アミノ酸を可逆的に脱水素する活性を有する
(b)配列番号2のアミノ酸配列との同一性が90%以上であるアミノ酸配列を有するポリペプチドの6量体である。 - 2−オキソブタン二酸からD−アスパラギン酸を合成する活性を有する、請求項1に記載の酵素。
- 更に下記特徴(c)を有する請求項1又は2に記載の酵素:
(c)NADH及びNADPHの両方を補酵素として利用可能である。 - 更に下記特徴(d)を有する請求項1〜3のいずれかに記載の酵素:
(d)meso−ジアミノピメリン酸を基質とし、NAD+を補酵素とする場合のNAD+に対するKm値が30mM以下である。 - 更に下記特徴(e)を有する請求項1〜4のいずれかに記載の酵素:
(e)meso−ジアミノピメリン酸を基質とする場合の至適活性pHが10.5である。 - 更に下記特徴(f)を有する請求項1〜5のいずれかに記載の酵素:
(f)meso−ジアミノピメリン酸を基質とする場合の至適活性温度が55度である。 - 配列番号2のアミノ酸配列との同一性が90%以上であるアミノ酸配列において、Asp94Ser、Met154Leu、Val158Gly、Thr173Ile、Arg183Met、及びHis229Asnからなる群より選択される1つ以上のアミノ酸置換を有する、請求項1〜3のいずれかに記載の酵素。
- 請求項1に記載の酵素をコードするポリヌクレオチドを組み込んだベクターを含む形質転換体を培養することを含む、請求項1〜5のいずれかに記載の酵素の製造方法。
- 請求項1〜7のいずれかに記載の酵素をD−アミノ酸に作用させ、2−オキソ酸を製造する方法。
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