JP2023554112A - 熱安定性に優れたアルロースエピマー化酵素変異体、その製造方法およびこれを用いたアルロースの製造方法 - Google Patents
熱安定性に優れたアルロースエピマー化酵素変異体、その製造方法およびこれを用いたアルロースの製造方法 Download PDFInfo
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Abstract
Description
A77S
*順方向プライマー塩基配列(5’→3’)
AATACGACCTGAGCAGCGACGATCCGGCGGTG
*逆方向プライマー塩基配列(5’→3’)
GATCGTCGCTGCTCAGGTCGTATTTGGCCTCCA
前記アルロース生産用組成物の一例は、配列番号5のアミノ酸配列からなるアルロースエピマー化酵素変異体W29K/A77S/G216S/M234Iを含む。また、前記アルロース生産用組成物の他の例は、配列番号5のアミノ酸配列からなるアルロースエピマー化酵素変異体をコードするポリヌクレオチドに形質転換されるか、または前記ポリヌクレオチドを含む組換えベクターにより形質転換された組換え菌株、該組換え菌株の培養物または前記組換え菌株の破砕物を含む。このとき、前記アルロース生産用組成物は、好ましくはマンガンイオン、ニッケルイオンおよびコバルトイオンからなる群から選択される1種以上をさらに含むことができ、より好ましくは、マンガンイオンまたはニッケルイオンをさらに含むことができる。本発明による新規なアルロースエピマー化酵素変異体は、金属イオンにより活性化が調節される金属酵素(metalloenzyme)の特性を示し、前記酵素による反応をマンガンイオンまたはニッケルイオンのような特定の金属イオンの存在下で行うことにより、アルロースの生産収率を増加させることができる。
本発明の出願人は、フラボニフラクター・プラウティ(Flavonifractor plautii)に由来する野生型D-アルロース3-エピマー化酵素の果糖のアルロースへの転換率および熱安定性を向上させるために、野生型D-アルロース3-エピメラーゼのアミノ酸配列のうち、29番目の位置に存在するトリプトファン(Trp)がリジン(Lys)に置換され、216番目の位置に存在するグリシン(Gly)がセリン(Ser)に置換され、同時に234番目の位置に存在するメチオニン(Met)がイソロイシン(Ile)に置換された新規なD-アルロースエピマー化酵素変異体W29K/G216S/M234Iについて、以前に特許出願した[大韓民国公開特許公報第10-2021-0132405号(2021.11.04)]。前記フラボニフラクター・プラウティ(Flavonifractor plautii)に由来する野生型D-アルロース3-エピマー化酵素は、配列番号1のアミノ酸配列からなり、野生型D-アルロース3-エピマー化酵素を暗号化するポリヌクレオチド断片は、配列番号2の塩基配列からなる。また、前記D-アルロースエピマー化酵素変異体W29K/G216S/M234Iは、配列番号3のアミノ酸配列からなり、D-アルロースエピマー化酵素変異体W29K/G216S/M234Iのアミノ酸配列を暗号化するポリヌクレオチド断片は、配列番号4の塩基配列からなる。
D-アルロースエピマー化酵素変異体W29K/G216S/M234Iのポリヌクレオチド(配列番号4)に基づくオーバーラップ伸長ポリメラーゼチェーン鎖反応法(overlap extension polymerase chain reaction)を用いて、酵素変異体4種(W29K/A77S/G216S/M234I、W29K/E173A/G216S/M234I、W29K/W209L/G216S/M234I、W29K/G216S/M234I/Q240D)のアミノ酸配列を暗号化するポリヌクレオチド断片を作製した。
下記表3の組成(培地1L基準)を有するタンパク質発現用培地150mlが収容された1L容量のフラスコに実施例2で作製した組換え大腸菌1mlを接種し、振とう培養器で32℃の温度条件および140rpmの振とう条件を維持しながら24時間培養した。培地に含まれた1%ラクトース(Lactose)でアルロースエピマー化酵素変異体の過剰発現を誘導した。
本発明の出願人が以前特許出願したD-アルロースエピマー化酵素変異体W29K/G216S/M234I[大韓民国公開特許公報第10-2021-0132405号(2021.11.04)]および本発明にて新たに作製したD-アルロースエピマー化酵素変異体4種(W29K/A77S/G216S/M234I、W29K/E173A/G216S/M234I、W29K/W209L/G216S/M234I、W29K/G216S/M234I/Q240D)の果糖のアルロースの転換率および熱安定性を確認するために、酵素を高温に一定時間晒した後、果糖のアルロースへの転換活性が低下する程度を解析した。具体的には、大韓民国公開特許公報第10-2021-0132405号(2021.11.04)の実施例3で得られたW29K/G216S/M234Iの精製酵素液(酵素濃度0.3mg/ml)および本発明の実施例3で得られた精製酵素液(酵素濃度0.3mg/ml)を62℃の恒温水槽で0hr、0.5hr、1hr、1.5hrおよび2hr保管して熱処理した。その後、10%(w/w)果糖および1mM硫酸マンガン(MnSO4)の金属イオンが含まれた50mM PIPES緩衝溶液(pH7.0)に熱処理された精製酵素液を酵素濃度が75μg/mlとなるように添加し、振とう恒温水槽(VS-1205SW1、ビジョン科学)を用いて、62℃の温度条件および120rpmの振とう条件で10分間反応させた。それから、反応生成液の温度を4℃まで下げて反応を停止させ、16,600×gおよび4℃の条件で遠心分離して上澄み液を回収した。その後、糖分析カラム(Benson、米国)が取り付けられた高性能液体クロマトグラフィー(HPLC)システム(SP930Dpump、ヨンリン機器;MIDASオートサンプラー、Spark Holland社製)および示差屈折率検出器(2414 refractive index detector、Waters社)を用いて、上澄み液中のアルロース濃度および果糖濃度を測定し、測定した結果から果糖のアルロースへの転換率を計算した後、転換率を酵素活性の指標として用いた。
Claims (10)
- 配列番号5のアミノ酸配列からなるアルロースエピマー化酵素変異体。
- 請求項1に記載のアルロースエピマー化酵素変異体をコードするポリヌクレオチド。
- 前記ポリヌクレオチドは、配列番号9の塩基配列からなることを特徴とする、請求項2に記載のポリヌクレオチド。
- 請求項2に記載のポリヌクレオチドを含む、組換えベクター。
- 請求項2に記載のポリヌクレオチド、請求項3に記載のポリヌクレオチド、または請求項4に記載の組換えベクターのいずれかによって形質転換された組換え菌株。
- 請求項5に記載の組換え菌株を培養し、アルロースエピマー化酵素変異体を発現させる工程;および
前記アルロースエピマー化酵素変異体が発現された組換え菌株の破砕物からアルロースエピマー化酵素変異体を分離する工程を含む、アルロースエピマー化酵素変異体の製造方法。 - 請求項1に記載のアルロースエピマー化酵素変異体を含む、アルロース生産用組成物。
- 請求項5に記載の組換え菌株、該組換え菌株の培養物または前記組換え菌株の破砕物を含む、アルロース生産用組成物。
- 果糖を請求項1に記載のアルロースエピマー化酵素変異体または前記アルロースエピマー化酵素変異体を含む組成物と反応させる工程を含む、アルロースの製造方法。
- 果糖を請求項5に記載の組換え菌株、該組換え菌株の培養物、前記組換え菌株の破砕物、またはそれらのうちのいずれか1つ以上を含む組成物と反応させる工程を含む、アルロースの製造方法。
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KR101203856B1 (ko) | 2011-08-24 | 2012-11-21 | 씨제이제일제당 (주) | 열 안정성이 향상된 사이코스 에피머화 효소 변이체 및 이를 이용한 사이코스의 연속적 생산 |
CN104769125B (zh) * | 2012-08-10 | 2018-10-23 | 株式会社三养社 | 阿洛酮糖差向异构酶及使用其的用于转变为阿洛酮糖的组合物 |
KR101318422B1 (ko) | 2013-04-09 | 2013-10-15 | 주식회사 삼양제넥스 | D-사이코스 에피머화 효소, 및 이를 이용하는 사이코스 생산방법 |
KR101455759B1 (ko) | 2013-04-23 | 2014-10-28 | 씨제이제일제당(주) | 사이코스 에피머화 효소 변이체 및 이를 이용한 사이코스의 제조 방법 |
KR101473918B1 (ko) | 2014-05-28 | 2014-12-17 | 대상 주식회사 | 사이코스 에피머화 효소, 이의 제조방법 및 이를 이용한 사이코스의 제조방법 |
KR101695830B1 (ko) * | 2014-10-30 | 2017-01-12 | 주식회사 삼양사 | 사이코스 에퍼머화 효소의 발현 시스템 및 이를 이용한 사이코스의 생산 |
RU2727903C1 (ru) * | 2016-11-16 | 2020-07-24 | СиДжей ЧеилДжеданг Корпорейшн | Новая d-псикозо-3-эпимераза и способ получения d-псикозы с ее использованием |
KR102187354B1 (ko) * | 2017-06-02 | 2020-12-04 | 주식회사 삼양사 | 사이코스 에피머화 효소 및 이를 이용한 사이코스의 제조 방법 |
BR112020014345B1 (pt) | 2018-01-24 | 2023-09-26 | Matsutani Chemical Industry Co., Ltd | Cetose 3-epimerase variante e método para produzir d- psicose |
KR102254411B1 (ko) | 2019-12-19 | 2021-05-24 | 대상 주식회사 | 알룰로스 에피머화 효소 변이체, 이의 제조방법 및 이를 이용한 알룰로스의 제조방법 |
KR102448351B1 (ko) | 2020-04-27 | 2022-09-28 | 대상 주식회사 | 알룰로스 에피머화 효소 변이체, 이의 제조방법 및 이를 이용한 알룰로스의 제조방법 |
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