JP2010227023A - Method for producing 2-deoxy-scyllo-inosose by nadh/nad ratio control - Google Patents
Method for producing 2-deoxy-scyllo-inosose by nadh/nad ratio control Download PDFInfo
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- JP2010227023A JP2010227023A JP2009078714A JP2009078714A JP2010227023A JP 2010227023 A JP2010227023 A JP 2010227023A JP 2009078714 A JP2009078714 A JP 2009078714A JP 2009078714 A JP2009078714 A JP 2009078714A JP 2010227023 A JP2010227023 A JP 2010227023A
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Abstract
Description
本発明は、２−デオキシ−シロ−イノソース合成酵素を用いて２−デオキシ−シロ−イノソースを製造する方法においてＮＡＤＨ／ＮＡＤ比を制御することを特徴とする２−デオキシ−シロ−イノソースの製造方法に関する。 The present invention relates to a method for producing 2-deoxy-siro-inosose, wherein the NADH / NAD ratio is controlled in a method for producing 2-deoxy-siro-inosose using 2-deoxy-siro-inosose synthase. About.
現在、カテコールなどの芳香族化合物の多くは、石油を原料として生産されているが、石油資源の枯渇問題や二酸化炭素排出量の削減などの観点から、バイオマスを利用した環境調和型の新規製造プロセスの開発が求められている。 At present, most of aromatic compounds such as catechol are produced using petroleum as a raw material. However, from the viewpoint of depletion of petroleum resources and reduction of carbon dioxide emissions, environmentally friendly new manufacturing processes using biomass. Development is required.
一方、２−デオキシ−シロ−イノソース（以下、ＤＯＩ）が、産業上有用とされる芳香族化合物（カテコール等）へと変換できることが見出され（非特許文献１）、さらには、このＤＯＩを、バイオマスの構成要素の一つであるグルコースから合成できることが報告された（非特許文献１）。本ＤＯＩ製造プロセスで重要となるのがグルコース−６−リン酸からＤＯＩへと変換する２−デオキシ−シロ−イノソース合成酵素（以下、ＤＯＩ合成酵素）であり、ＤＯＩは上記芳香族化合物への変換のみならず各種有用化合物の中間体ともなり得ることから、ＤＯＩ合成酵素は非常に大きな注目を浴びている。 On the other hand, it was found that 2-deoxy-siro-inosose (hereinafter DOI) can be converted into an industrially useful aromatic compound (catechol etc.) (Non-Patent Document 1). It has been reported that it can be synthesized from glucose, which is one of the components of biomass (Non-patent Document 1). What is important in this DOI production process is 2-deoxy-siro-inosose synthase (hereinafter referred to as DOI synthase) that converts glucose-6-phosphate to DOI, and DOI is converted to the above aromatic compound. In addition, DOI synthase has attracted much attention because it can be an intermediate for various useful compounds.
ＤＯＩ合成酵素はブチロシン生産菌であるＢａｃｉｌｌｕｓ ｃirculans に属する微生物から１９９７年に単離精製（非特許文献２）され、続いて、その遺伝子配列も公開されている（特許文献１）。この他にもＳｔｒｅｐｔｏａｌｌｏｔｅｉｃｈｕｓ ｈｉｎｄｕｓｔａｎｕｓ ＪＣＭ３２６８株（非特許文献３）由来のＤＯＩ合成酵素などがこれまでに発見されている。 DOI synthase was isolated and purified in 1997 from a microorganism belonging to Bacillus circulans, which is a butyrosine-producing bacterium (Non-patent Document 2), and its gene sequence was also disclosed (Patent Document 1). In addition to this, DOI synthase derived from Streptoallotichichus Hindustanus JCM3268 (Non-patent Document 3) has been discovered so far.
しかしながら、前記ＤＯＩ合成酵素の安定性と比活性などの各種特性は、満足のいくものではなく、また、数千トン規模の工業化製法に向けたＤＯＩ発酵生産に関する研究開発、特に細胞内代謝物の制御に関するものはほとんどなされてこなかった。
本発明は、従来法と比較して、ＤＯＩ発酵生産性を飛躍的に向上させうる細胞内代謝産物制御技術を提供することを解決すべき課題とする。
However, the various properties such as the stability and specific activity of the DOI synthase are not satisfactory, and the research and development related to the DOI fermentation production for the industrial production process of several thousand tons, especially the intracellular metabolites Little has been done about control.
This invention makes it the subject which should be solved to provide the intracellular metabolite control technique which can improve DOI fermentation productivity dramatically compared with the conventional method.
これらの課題を解決するため鋭意検討を重ねた結果、本発明者らは、ＤＯＩ合成酵素の活性が反応系中のＮＡＤＨ／ＮＡＤ比に予想し得ない領域で影響を受けることを見出し、従来法と比較して、ＤＯＩ発酵生産性を飛躍的に向上させうるＤＯＩ生産技術を見出し、本発明を完成するに至った。 As a result of extensive studies to solve these problems, the present inventors have found that the activity of DOI synthase is influenced by the NADH / NAD ratio in the reaction system in an unpredictable region. As a result, the present inventors have found a DOI production technique capable of dramatically improving DOI fermentation productivity and completed the present invention.
すなわち、本発明は以下の［１］〜［７］に示すＤＯＩ製造方法に関するものである。
［１］ ２−デオキシ−シロ−イノソース合成酵素又は２−デオキシ−シロ−イノソース合成酵素を発現可能な微生物を利用してグルコース−６−リン酸から２−デオキシ−シロ−イノソースを製造する方法において、反応系におけるＮＡＤＨ／ＮＡＤ比を１．４以下に制御することを特徴とする、２−デオキシ−シロ−イノソースの製造方法。
［２］ グルコースを構成成分とする多糖類あるいはグルコースを含有する原料を用いることを特徴とする、［１］に記載の方法。
［３］ ２−デオキシ−シロ−イノソース合成酵素が、安定温度範囲が少なくとも４６℃まで安定である耐熱性酵素である、［１］又は［２］に記載の方法。
［４］ ２−デオキシ−シロ−イノソース合成酵素が、ｐＨ６．０〜 ８．０で安定な酵素である、［１］から［３］の何れか記載の方法。
［５］ ２−デオキシ−シロ−イノソース合成酵素が下記の特性を有する酵素である、［１］又は［２］に記載の方法。
（１）作用：本酵素は、グルコース−６−リン酸を２−デオキシ−シロ−イノソースへと変換する機能を有する。
（２）安定温度範囲：少なくとも４６℃まで安定である。
（３）補酵素としてＮＡＤ+を利用。
（４）至適ｐＨ範囲：７．０〜 ７．７
（５）安定ｐＨ範囲：少なくとも６．０〜 ８．０の範囲で安定である。
（６）至適温度範囲：５５ 〜 ７０ ℃
（７）分子量：３９０００〜４２０００
（８）補因子：Ｃｏ2+イオンの添加により活性向上。
（９）比活性：１．０μｍｏｌ／ｍｉｎ／ｍｇ以上（反応温度６５℃）。
［６］ ２−デオキシ−シロ−イノソース合成酵素が、配列番号２、４、６、８、１０又は１２に記載のアミノ酸配列を有する２−デオキシ−シロ−イノソース合成酵素である、［１］から［２］の何れか記載の方法。
［７］ ２−デオキシ−シロ−イノソース合成酵素が、配列番号２、４、６、８、１０又は１２で示されるアミノ酸配列において１個または複数個のアミノ酸の欠失、付加、及び／又は置換を含み、配列番号２、４、６、８、１０又は１２で示されるアミノ酸配列に対して８０％以上の相同性を有して且つ、高温安定性及び／又は広範囲ｐH安定性を持つ２−デオキシ−シロ−イノソース合成酵素である、［１］から［５］の何れか記載の方法。
That is, this invention relates to the DOI manufacturing method shown in the following [1]-[7].
[1] In a method for producing 2-deoxy-siro-inosose from glucose-6-phosphate using a microorganism capable of expressing 2-deoxy-siro-inosose synthase or 2-deoxy-siro-inosose synthase A method for producing 2-deoxy-siro-inosose, wherein the NADH / NAD ratio in the reaction system is controlled to 1.4 or less.
[2] The method according to [1], wherein a polysaccharide comprising glucose or a raw material containing glucose is used.
[3] The method according to [1] or [2], wherein the 2-deoxy-siro-inosose synthase is a thermostable enzyme having a stable temperature range of up to at least 46 ° C.
[4] The method according to any one of [1] to [3], wherein the 2-deoxy-siro-inosose synthase is an enzyme that is stable at pH 6.0 to 8.0.
[5] The method according to [1] or [2], wherein the 2-deoxy-siro-inosose synthase is an enzyme having the following characteristics.
(1) Action: This enzyme has a function of converting glucose-6-phosphate into 2-deoxy-siro-inosose.
(2) Stable temperature range: Stable up to at least 46 ° C.
(3) Use NAD + as a coenzyme.
(4) Optimal pH range: 7.0 to 7.7
(5) Stable pH range: Stable at least in the range of 6.0 to 8.0.
(6) Optimal temperature range: 55 to 70 ° C
(7) Molecular weight: 39000-42000
(8) Cofactor: Activity is improved by adding Co 2+ ions.
(9) Specific activity: 1.0 μmol / min / mg or more (reaction temperature 65 ° C.).
[6] From [1], the 2-deoxy-siro-inosose synthase is a 2-deoxy-siro-inosose synthase having the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, or 12. The method according to any one of [2].
[7] 2-deoxy-siro-inosose synthase is a deletion, addition, and / or substitution of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10, or 12. And has 80% or more homology to the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12, and has high temperature stability and / or broad pH stability 2- The method according to any one of [1] to [5], which is deoxy-siro-inosose synthase.
本発明によると、菌体あたりのＤＯＩ発酵生産性の向上に寄与することから、短時間で大量のＤＯＩを合成することができる。また、触媒となる菌体量の低減も可能となり、原料利用効率を向上させることもできる。 According to the present invention, since it contributes to the improvement of DOI fermentation productivity per bacterial cell, a large amount of DOI can be synthesized in a short time. In addition, the amount of bacterial cells serving as a catalyst can be reduced, and the raw material utilization efficiency can be improved.
以下、本発明について具体的に説明する。
[ＤＯＩ合成酵素]
本発明を実施するための形態（以下、本実施の形態という）のＤＯＩ合成酵素は、下記の特性を有し、
（１）作用：本酵素は、グルコース−６−リン酸をＤＯＩへと変換する機能を有する。
さらには、下記に示す一部またはすべての特性を有する。
（２）安定温度範囲：少なくとも４６℃まで安定である。
（３）補酵素としてＮＡＤ+を利用。
（４）至適ｐＨ範囲：７．０〜 ７．７
（５）安定ｐＨ範囲：少なくとも６．０〜 ８．０の範囲で安定である。
（６）至適温度範囲：５５ 〜 ７０ ℃
（７）分子量：３９０００〜４２０００
（８）補因子：Ｃｏ2+イオンの添加により活性向上。
（９）比活性：１．０μｍｏｌ／ｍｉｎ／ｍｇ以上（反応温度６５℃）。
Hereinafter, the present invention will be specifically described.
[DOI synthase]
The DOI synthase in the form for carrying out the present invention (hereinafter referred to as the present embodiment) has the following characteristics:
(1) Action: This enzyme has a function of converting glucose-6-phosphate to DOI.
Further, it has some or all of the following characteristics.
(2) Stable temperature range: Stable up to at least 46 ° C.
(3) Use NAD + as a coenzyme.
(4) Optimal pH range: 7.0 to 7.7
(5) Stable pH range: Stable at least in the range of 6.0 to 8.0.
(6) Optimal temperature range: 55 to 70 ° C
(7) Molecular weight: 39000-42000
(8) Cofactor: Activity is improved by adding Co 2+ ions.
(9) Specific activity: 1.0 μmol / min / mg or more (reaction temperature 65 ° C.).
配列番号２、４、６、８、１０及び１２に従来のＤＯＩ合成酵素と比較して、耐熱性やpH安定性などの面で優れた新規ＤＯＩ合成酵素のアミノ酸配列を例示するが、このアミノ酸配列からなるタンパク質がＤＯＩ合成酵素活性を有する限り、当該アミノ酸配列において少なくとも１個のアミノ酸に欠失、置換、付加等の変異が生じてもよい。例えば、配列番号２、４、６、８、１０又は１２に示されるアミノ酸配列に対して８０％以上、好ましくは８５％以上、さらに好ましくは９０％以上、さらに好ましくは９５％以上、さらに好ましくは９９％以上の相同性の範囲内で、配列番号２、４、６、８、１０又は１２で表されるアミノ酸配列の１個または複数個（例えば１から５０個、好ましくは１から３０個、さらに好ましくは１から２０個、さらに好ましくは１から１０個、さらに好ましくは１から５個、さらに好ましくは１から３個））のアミノ酸の欠失、付加、及び／又は置換してもよい。
ＤＯＩ合成酵素中のアミノ酸に欠失、置換、付加等の変異を生じさせる方法としては、ＰＣＲ法、エラープローンＰＣＲ法、ＤＮＡシャッフリング法やキメラ酵素を作製する手法等の公知の方法が利用できる。
上記に、新規ＤＯＩ合成酵素を示したが、もちろん本発明は従来のＤＯＩ合成酵素を用いても良い。
ＤＯＩ合成酵素活性を測定する方法としては、例えば、１０００ｍＭグルコース−６−リン酸溶液２０μＬ、１００ｍＭＮＡＤ+溶液５０μＬ、１００ｍＭ塩化コバルト六水和物溶液５０μＬを含むｐＨ７．０の１５０ｍＭＢｉｓ−Ｔｒｉｓ緩衝液９００μＬに適当な濃度のＤＯＩ合成酵素液１００μＬを混合し、５〜６０分間程度反応させた後、酵素を失活させ、ＤＯＩを定量する方法などが挙げられる。
酵素の至適温度範囲あるいは至適ｐＨ範囲は、反応温度あるいは反応ｐＨを変化させて酵素活性を測定すればよい。さらにＤＯＩ合成酵素を種々のｐＨ条件下又は温度条件下に一定時間さらした後に酵素活性を測定することにより、安定ｐＨ範囲及び安定温度範囲を調べることができる。例えば、各ｐH条件におけるＤＯＩ合成酵素の評価には、Ｂｉｓ−Ｔｒｉｓ緩衝液（ｐＨ５．５〜８．０）およびＴｒｉｓ緩衝液（ｐＨ７．４〜８．０）を用いることができる。
至適ｐＨ範囲とは、最大活性を１００とするときの活性値が７０以上の範囲であり、安定ｐＨ範囲とは最大活性を１００とするときの活性値が７０以上の範囲である。また、至適温度範囲とは、最大活性を１００とするときの活性値が５０以上の範囲であり、安定温度範囲は最大活性を１００とするときの活性値が５０以上の範囲である。
本実施の形態におけるＤＯＩ合成酵素の高温安定性とは、安定温度範囲が４６℃以下、より好ましくは、５０℃以下、さらにより好ましくは６０℃以下、さらに特に好ましくは９５℃以下まで安定なことである。高温安定性を有する２−デオキシ−シロ−イノソース合成酵素としては、例えば、５０℃で１時間インキュベートした後の残存酵素活性（最大活性を１００とする相対活性）が５０以上である酵素を選抜することができる。
本実施の形態における広範囲ｐＨ安定性を持つＤＯＩ合成酵素は、安定ｐＨ範囲が少なくともｐＨ６．０〜７．０、好ましくはｐＨ６．０〜７．４、より好ましくはｐＨ６．０〜７．７、より好ましくはｐＨ６．０〜８．０、特により好ましくはｐＨ４．０〜９．０である特性を有する。
SEQ ID NOs: 2, 4, 6, 8, 10 and 12 illustrate amino acid sequences of a novel DOI synthetase which is superior in terms of heat resistance and pH stability compared to conventional DOI synthetases. As long as the protein comprising the sequence has DOI synthetase activity, at least one amino acid in the amino acid sequence may be mutated such as deletion, substitution, addition and the like. For example, 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably, relative to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12. One or more of the amino acid sequences represented by SEQ ID NO: 2, 4, 6, 8, 10 or 12 (for example, 1 to 50, preferably 1 to 30, within a range of homology of 99% or more, More preferably, 1 to 20, more preferably 1 to 10, more preferably 1 to 5, more preferably 1 to 3)) amino acid deletions, additions, and / or substitutions may be made.
Known methods such as a PCR method, an error-prone PCR method, a DNA shuffling method, and a method for producing a chimeric enzyme can be used as a method for causing mutations such as deletion, substitution and addition in amino acids in DOI synthase.
Although a novel DOI synthetase has been shown above, of course, the present invention may use a conventional DOI synthetase.
As a method for measuring DOI synthase activity, for example, 900 μL of 150 mM Bis-Tris buffer pH 7.0 containing 20 μL of 1000 mM glucose-6-phosphate solution, 50 μL of 100 mM NAD + solution, and 50 μL of 100 mM cobalt chloride hexahydrate solution was used. Examples include a method of mixing 100 μL of a DOI synthetase solution having an appropriate concentration and reacting for about 5 to 60 minutes, then inactivating the enzyme, and quantifying DOI.
The optimum temperature range or optimum pH range of the enzyme may be determined by changing the reaction temperature or reaction pH and measuring the enzyme activity. Furthermore, the stable pH range and the stable temperature range can be examined by measuring the enzyme activity after exposing the DOI synthase to various pH conditions or temperature conditions for a certain period of time. For example, Bis-Tris buffer (pH 5.5 to 8.0) and Tris buffer (pH 7.4 to 8.0) can be used for evaluation of DOI synthase under each pH condition.
The optimum pH range is a range where the activity value is 70 or more when the maximum activity is 100, and the stable pH range is a range where the activity value is 70 or more when the maximum activity is 100. The optimum temperature range is a range where the activity value is 50 or more when the maximum activity is 100, and the stable temperature range is a range where the activity value is 50 or more when the maximum activity is 100.
The high temperature stability of the DOI synthase in the present embodiment means that the stable temperature range is 46 ° C. or lower, more preferably 50 ° C. or lower, even more preferably 60 ° C. or lower, and even more preferably 95 ° C. or lower. It is. As the 2-deoxy-siro-inosose synthase having high temperature stability, for example, an enzyme having a residual enzyme activity (relative activity with a maximum activity of 100) after incubation at 50 ° C. for 1 hour is selected. be able to.
The DOI synthase having a wide range of pH stability in the present embodiment has a stable pH range of at least pH 6.0 to 7.0, preferably pH 6.0 to 7.4, more preferably pH 6.0 to 7.7, More preferably, it has a property of pH 6.0 to 8.0, particularly preferably pH 4.0 to 9.0.
[ＤＯＩ合成酵素遺伝子]
本実施の形態のＤＯＩ合成酵素遺伝子は、メタゲノムや微生物から単離・抽出した天然のものでも良く、また、その塩基配列に従ってＰＣＲ法、人工合成法等の公知の方法によって合成したものでも良い。また、新規ＤＯＩ酵素キメラ遺伝子の作成には、既知ＤＯＩ合成酵素遺伝子を組み合わせてもよく、例示される遺伝子としてはＰａｅｎｉｂａｃｉｌｌｕｓ ｓｐ．ＮＢＲＣ１３１５７株、Ｓｔｒｅｐｔｏａｌｌｏｔｅｉｃｈｕｓ ｈｉｎｄｕｓｔａｎｕｓ ＪＣＭ３２６８、Ｓｔｒｅｐｔｏｍｙｃｅｓ ｆｒａｄｉａｅ ＮＢＲＣ１２７７３株などに由来するＤＯＩ合成酵素遺伝子が挙げられる。
[DOI synthase gene]
The DOI synthase gene of the present embodiment may be a natural gene isolated or extracted from a metagenome or a microorganism, or may be synthesized by a known method such as a PCR method or an artificial synthesis method according to its base sequence. In addition, a known DOI synthase gene may be combined in the preparation of a novel DOI enzyme chimera gene. Examples of such genes include Paenibacillus sp. Examples thereof include DOI synthase genes derived from the NBRC13157 strain, Streptoallotichus Hindustanus JCM3268, Streptomyces fradiae NBRC12773 strain, and the like.
配列番号１、３、５、７、９及び１１に新規ＤＯＩ合成酵素遺伝子の塩基配列を例示するが、もちろん本発明は従来のＤＯＩ合成酵素遺伝子を用いても良い。 The nucleotide sequences of the novel DOI synthetase genes are exemplified in SEQ ID NOs: 1, 3, 5, 7, 9, and 11. Of course, the present invention may use conventional DOI synthetase genes.
[組み換えベクター及び形質転換体]
本実施の形態の組換えベクターは、プラスミド等の公知のベクターに本発明の遺伝子を連結（挿入）して得ることができる。前記ベクターは宿主中で複製可能なものであれば特に限定されず、例えばプラスミドＤＮＡ、ファージＤＮＡ等が挙げられる。
[Recombinant vectors and transformants]
The recombinant vector of this embodiment can be obtained by linking (inserting) the gene of the present invention to a known vector such as a plasmid. The vector is not particularly limited as long as it can replicate in the host, and examples thereof include plasmid DNA and phage DNA.
前記プラスミドDNAとしては、大腸菌由来のプラスミド（例えば pBR322, pBR325, pUC18, pUC119, pTrcHis, pBlueBacHis 等）、枯草菌由来のプラスミド（例えば pUB110, pTP5 等）、酵母由来のプラスミド（例えば YEp13, YEp24, YCp50, pYE52 等）などが、ファージ DNAとしてはλファージ等が挙げられる。 Examples of the plasmid DNA include plasmids derived from E. coli (eg, pBR322, pBR325, pUC18, pUC119, pTrcHis, pBlueBacHis, etc.), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5 etc.), and plasmids derived from yeast (eg, YEp13, YEp24, YCp50). , pYE52, etc.), and phage DNA includes λ phage.
前記ベクターへの本発明の遺伝子の挿入は、まず、精製されたDNAを適当な制限酵素で切断し、ベクターDNAの適当な制限酵素部位またはマルチクローニングサイトに挿入してベクターに連結する方法が採用される。 For insertion of the gene of the present invention into the vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into an appropriate restriction enzyme site or a multicloning site of the vector DNA, and then ligated to the vector. Is done.
宿主内で外来遺伝子を発現させるためには、構造遺伝子の前に、適当なプロモーターを配置させる必要がある。前記プロモーターは特に限定されず、宿主内で機能することが知られている任意のものを用いることができる。なおプロモーターについては、後述する形質転換体において、宿主ごとに詳述する。また、必要であればエンハンサー等のシスエレメント、スプライシングシグナル、ポリA付加シグナル、リボソーム結合配列（SD配列）、ターミネーター配列等を配置させてもよい。 In order to express a foreign gene in a host, it is necessary to arrange an appropriate promoter before the structural gene. The promoter is not particularly limited, and any promoter known to function in the host can be used. The promoter will be described in detail for each host in the transformant described later. If necessary, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a ribosome binding sequence (SD sequence), a terminator sequence and the like may be arranged.
本実施の形態の形質転換体は、本発明の組換えベクターを目的遺伝子が発現しうるように宿主中に導入することによって得ることができる。ここで宿主としては、本発明のDNAを発現できるものであれば特に限定されず、例えば、エッシェリヒア・コリ（Escherichia coli）等のエッシェリヒア属、バチルス・ズブチリス（Bacillus subtilis）等のバチルス属、シュードモナス・プチダ（Pseudomonas putida）等のシュードモナス属、リゾビウム・メリロテイ（Rhizobium meliloti）等のリゾビウム属に属する細菌、またサッカロミセス・セルビシエ（Saccharomyces cerevisiae）等の酵母、その他COS細胞、CHO細胞等の動物細胞、あるいはSf19、Sf21等の昆虫細胞を挙げることができる。 The transformant of the present embodiment can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, the host is not particularly limited as long as it can express the DNA of the present invention. For example, Escherichia such as Escherichia coli, Bacillus subtilis such as Bacillus subtilis, Bacteria belonging to the genus Pseudomonas such as Pseudomonas putida, Rhizobium meliloti, such as Rhizobium, yeast such as Saccharomyces cerevisiae, other animal cells such as COS cells, CHO cells, or Sf19 And insect cells such as Sf21.
また、本実施の形態の形質転換体として利用する宿主は、グルコース−6−リン酸をグルコース、フルクトース、ガラクトース、キシロースなどの単糖類から合成する機能を有するものが好ましい。また、必要に応じて、二つ以上の単糖が連結した多糖類を分解する機能を有することも好ましい。 In addition, the host used as the transformant of the present embodiment preferably has a function of synthesizing glucose-6-phosphate from monosaccharides such as glucose, fructose, galactose, and xylose. Moreover, it is also preferable that it has a function which decomposes | disassembles the polysaccharide which two or more monosaccharide connected as needed.
大腸菌等の細菌を宿主とする場合は、本発明の組換えベクターが各細菌中で自律複製可能であるとともにプロモーター、リボゾーム結合配列、本発明遺伝子、転写終結配列により構成されていることが望ましい。また、プロモーターを制御する遺伝子が含まれていても良い。大腸菌としてはエッシェリヒア・コリ（E. coli）K12、DH1、DH10B（Invitrogen社）、BL21-CodonPlus(DE3)-RIL（ストラタジーン社）、TOP10F等が挙げられ、枯草菌としてはバチルス・ズブチリス（B. subtilis）MI114、207-21 等が挙げられる。 When a bacterium such as Escherichia coli is used as a host, it is desirable that the recombinant vector of the present invention is capable of autonomous replication in each bacterium and is composed of a promoter, a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Moreover, the gene which controls a promoter may be contained. Examples of Escherichia coli include Escherichia coli K12, DH1, DH10B (Invitrogen), BL21-CodonPlus (DE3) -RIL (Stratagene), TOP10F, and Bacillus subtilis (B subtilis) MI114, 207-21 and the like.
プロモーターとしては大腸菌等の宿主で機能するものであれば特に限定されず、例えばgapAプロモーター、gadAプロモーター、trpプロモーター、lacプロモーター、PLプロモーター、PRプロモーター等の大腸菌由来のプロモーターや、T7プロモーター等のファージ由来のプロモーターを用いることができる。 The promoter is not particularly limited as long as it functions in a host such as Escherichia coli. For example, a promoter derived from Escherichia coli such as gapA promoter, gadA promoter, trp promoter, lac promoter, PL promoter, PR promoter, or phage such as T7 promoter. Origin promoters can be used.
細菌への組換えベクターの導入方法は、細菌にDNAを導入できる方法であれば特に限定されないが、例えばカルシウムイオンを用いる方法（Cohen, SN et al. Proc. Natl. Acad. Sci. USA, 69 : 2110 (1972)）、エレクトロポレーション法等が挙げられる。 The method for introducing a recombinant vector into bacteria is not particularly limited as long as it can introduce DNA into bacteria. For example, a method using calcium ions (Cohen, SN et al. Proc. Natl. Acad. Sci. USA, 69 : 2110 (1972)), electroporation method and the like.
酵母を宿主とする場合は、例えばサッカロミセス・セルビシエ（S. cerevisiae）、ピヒア・パストリス（Pichia pastoris）等が用いられる。この場合、プロモーターとしては酵母で発現しうるもの、例えばgal1プロモーター、gal10プロモーター、ヒートショックタンパク質プロモーター、MFα 1プロモーター、PHO5プロモーター、AOXプロモーター等を挙げることができる。
When yeast is used as a host, for example, S. cerevisiae, Pichia pastoris, and the like are used. In this case, examples of promoters that can be expressed in yeast include gal1 promoter, gal10 promoter, heat shock protein promoter,
酵母への組換えベクターの導入方法としては、例えばエレクトロポレーション法（Becker, D.M. et al. : Methods. Enzymol., 194 : 180 (1990)）、スフェロプラスト法（Hinnen, A. et al. : Proc Natl. Acad. Sci. USA, 75 : 1929 (1978)）、酢酸リチウム法（Itoh, H. : J. Bacteriol., 153 : 163 (1983)）等を挙げることができる。 Examples of methods for introducing a recombinant vector into yeast include the electroporation method (Becker, DM et al .: Methods. Enzymol., 194: 180 (1990)) and the spheroplast method (Hinnen, A. et al. : Proc Natl. Acad. Sci. USA, 75: 1929 (1978)), lithium acetate method (Itoh, H .: J. Bacteriol., 153: 163 (1983)) and the like.
[ＤＯＩ生産法]
本実施の形態のＤＯＩ生産法では、反応系におけるＮＡＤＨ／ＮＡＤ比を１．４以下に制御することを特徴とする。なお、本実施の形態で言う反応系とは、細胞内でもよいし、インビトロでもよい。ＤＯＩ合成酵素を発現可能な微生物を利用する場合、本実施の形態のＤＯＩ生産法では、反応系（細胞内）のＮＡＤＨ／ＮＡＤ比を制御することを除いては、ＤＯＩ合成酵素産生微生物を常法に従って培養して得られる培養物からＤＯＩを採取することでえられる。
[DOI production method]
The DOI production method of the present embodiment is characterized in that the NADH / NAD ratio in the reaction system is controlled to 1.4 or less. The reaction system referred to in this embodiment may be in a cell or in vitro. When a microorganism capable of expressing DOI synthase is used, the DOI production method of the present embodiment normally uses a DOI synthase-producing microorganism except that the NADH / NAD ratio in the reaction system (intracellular) is controlled. It is obtained by collecting DOI from a culture obtained by culturing according to the method.
本実施の形態における反応系（細胞内）のＮＡＤＨ／ＮＡＤ比の制御は、菌の培養開始と同時に行っても良いが、より好ましくは、菌の生育およびＤＯＩ合成酵素の発現などの効率性を考慮し、ＤＯＩの生成濃度が２ｇ／Ｌに到達してから開始する。また、ＤＯＩの最終到達濃度が５０ｇ／Ｌを超えるようなＤＯＩ製造プロセスにおいては、ＤＯＩの生成濃度が３０ｇ／Ｌに到達してから反応系（細胞内）のＮＡＤＨ／ＮＡＤ比の制御を開始してもよい。 The control of the NADH / NAD ratio in the reaction system (intracellular) in the present embodiment may be performed simultaneously with the start of culturing of the bacterium, but more preferably, the efficiency of growth of the bacterium and expression of DOI synthase is improved. Considering, start when the DOI production concentration reaches 2 g / L. In the DOI production process in which the final concentration of DOI exceeds 50 g / L, control of the NADH / NAD ratio of the reaction system (intracellular) is started after the DOI production concentration reaches 30 g / L. May be.
反応系（細胞内）のＮＡＤＨ／ＮＡＤ比は、好ましくは１．４以下、より好ましくは１．０以下、さらにより好ましくは０．５以下、さらにより好ましくは０．１以下、特により好ましくは０．０２以下である。 The NADH / NAD ratio of the reaction system (intracellular) is preferably 1.4 or less, more preferably 1.0 or less, even more preferably 0.5 or less, even more preferably 0.1 or less, and particularly preferably 0.02 or less.
反応系（細胞内）のＮＡＤＨ／ＮＡＤ比は、制御開始から少なくとも１．４以下に１分間保てばよく、より好ましくは1時間、より好ましくは１０時間、さらにより好ましくは２４時間、特により好ましくは４８時間保てばよい。 The NADH / NAD ratio in the reaction system (intracellular) may be kept at least 1.4 or less from the start of control for 1 minute, more preferably 1 hour, more preferably 10 hours, even more preferably 24 hours, particularly more Preferably, it may be kept for 48 hours.
本実施の形態における反応系（細胞内）のＮＡＤＨ／ＮＡＤ比を１．４以下に保つ効果的な手法としては、常法に従えばよく、例えば、非特許文献（Metabolic Engineering Volume 7, Issue 2, March 2005, Pages 104-115）に知られているピルビン酸キナーゼ関連遺伝子欠損が利用でき、本手法を用いれば反応系（細胞内）のＮＡＤＨ／ＮＡＤ比が０．０１０にできることが知られている。本発明における反応系（細胞内）のＮＡＤＨ／ＮＡＤ比の測定は、非特許文献（Metabolic Engineering Volume 7, Issue 2, March 2005, Pages 104-115）に記載の手法などで行うことができる。
As an effective technique for maintaining the NADH / NAD ratio of the reaction system (intracellular) in the present embodiment at 1.4 or less, a conventional method may be followed. For example, non-patent literature (
また、本実施の形態における反応系（細胞内）のＮＡＤＨ／ＮＡＤ比を１．４以下に保つその他の効果的な手法としては、非特許文献（Appl Microbiol Biotechnol. 2004 Apr;64(3):367-75. Epub 2003 Dec 12.）に記載のグルコースを炭素源とした嫌気（微好気）条件下での培養技術を利用することができる。本技術を用いれば、好気条件で、反応系（細胞内）のＮＡＤＨ／ＮＡＤ比が１．４４にも関わらず、これを嫌気培養することにより０．１３まで低下させることができる。 In addition, as another effective technique for maintaining the NADH / NAD ratio of the reaction system (intracellular) in the present embodiment at 1.4 or less, non-patent literature (Appl Microbiol Biotechnol. 2004 Apr; 64 (3): 367-75. Epub 2003 Dec 12.), the culture technique under anaerobic (microaerobic) conditions using glucose as a carbon source can be used. If this technique is used, even if the NADH / NAD ratio of the reaction system (intracellular) is 1.44 under aerobic conditions, it can be reduced to 0.13 by anaerobic culture.
培養に用いる炭素源としては、形質転換体が資化できるものであればグルコースを構成成分とする多糖類を直接用いてもよく、より好ましくは、単糖類もしくはでんぷんや米ぬかや廃糖蜜などの多糖類を含む原料に由来する単糖類が挙げられ、具体的にはＤ−グルコースなどが挙げられる。また、プロモーターの発現が誘導型である場合には、適時誘導物質を添加すればよい。炭素源以外の栄養源としては、窒素源、無機塩類、その他有機栄養源を含む培地を使用できる。培養温度はその菌の生育可能な温度であれば良い。培養時間には特に制限はないが、１日から７日程度でよい。その後、得られた培養菌体又は培養上清液からＤＯＩを回収すればよい。 As the carbon source used for the culture, a polysaccharide comprising glucose as a constituent may be directly used as long as the transformant can assimilate, and more preferably a monosaccharide or starch, rice bran or molasses. Examples include monosaccharides derived from raw materials containing saccharides, and specifically D-glucose. In addition, when the expression of the promoter is inducible, an inducer may be added in a timely manner. As a nutrient source other than the carbon source, a medium containing a nitrogen source, inorganic salts, and other organic nutrient sources can be used. The culture temperature may be any temperature at which the bacteria can grow. The culture time is not particularly limited, but may be about 1 to 7 days. Then, what is necessary is just to collect | recover DOI from the obtained cultured microbial cell or culture supernatant liquid.
炭素源としては、Ｄ−グルコース、ガラクトース、マルトース、サッカロース、トレハロース等の糖類や油脂類や脂肪酸類さらにはｎ−パラフィン等を用いることができる。油脂としては、例えば、ナタネ油、ヤシ油、パーム油、パーム核油などがあげられる。脂肪酸としてはヘキサン酸、オクタン酸、デカン酸、ラウリン酸、オレイン酸、パルミチン酸、リノール酸、リノレン酸、ミリスチン酸などの飽和・不飽和脂肪酸、あるいはこれら脂肪酸のエステルや塩など脂肪酸誘導体などがあげられる。 As the carbon source, saccharides such as D-glucose, galactose, maltose, saccharose, and trehalose, fats and oils, fatty acids, and n-paraffin can be used. Examples of the fats and oils include rapeseed oil, coconut oil, palm oil, and palm kernel oil. Examples of fatty acids include saturated and unsaturated fatty acids such as hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, and myristic acid, and fatty acid derivatives such as esters and salts of these fatty acids. It is done.
窒素源としては、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、リン酸アンモニウムなどのアンモニウム塩の他、ペプトン、肉エキス、酵母エキスなどが挙げられる。無機塩類としては、例えば、リン酸水素ナトリウム、リン酸二水素カリウム、塩化マグネシウム、硫酸マグネシウム、塩化ナトリウム、塩化コバルトなどが挙げられる。 Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, yeast extract, and the like. Examples of inorganic salts include sodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, sodium chloride, cobalt chloride and the like.
その他の有機栄養源としては、アミノ酸類、例えば、アデニン、ヒスチジン、ロイシン、ウラシル、トリプトファンなどが挙げられる。 Other organic nutrient sources include amino acids such as adenine, histidine, leucine, uracil, tryptophan and the like.
生成するＤＯＩの確認は、非特許文献（Journal of Biotechnology 129, 502-509 (2007)）に記載の方法などを用いることができる。 For confirmation of the DOI to be generated, a method described in a non-patent document (Journal of Biotechnology 129, 502-509 (2007)) can be used.
本実施の形態において、ＤＯＩの培養液からの回収は，例えば、次のような方法が使用できる。培養終了後、培養液から遠心分離器や濾過装置などで菌体を除き、培養上清液を得る。この培養上清液に対してさらに濾過処理を行い、菌体等の固形物を除き、その濾液にイオン交換樹脂を添加し、蒸留水で溶出を行う。ＩＣＰ発光分析、ｐＨなどを測定しながら不純物を含まないフラクションを分取して、その水溶液の溶媒を取り除くことでＤＯＩを回収することができる。得られたＤＯＩの分析は、例えば、高速液体クロマトグラフィーや核磁気共鳴法などにより行う。 In this Embodiment, the following methods can be used for the collection | recovery from the culture solution of DOI, for example. After completion of the culture, the cells are removed from the culture solution with a centrifuge or a filtration device to obtain a culture supernatant. The culture supernatant is further filtered to remove solids such as bacterial cells, and an ion exchange resin is added to the filtrate, followed by elution with distilled water. While measuring ICP emission analysis, pH, etc., fractions containing no impurities are collected, and DOI can be recovered by removing the solvent of the aqueous solution. The obtained DOI is analyzed by, for example, high performance liquid chromatography or nuclear magnetic resonance.
本実施の形態のインビトロ下でのＤＯＩ製造温度は、反応速度や酵素の安定性の点から、好ましくは１０〜９５℃、より好ましくは２０〜７０℃であり、さらに好ましくは３０〜６０℃である。反応ｐＨは広範囲で調整可能であり、酵素の安定性の点から、好ましくはｐＨ２．０〜１０．０、より好ましくはｐＨ４．０〜８．０、さらに好ましくは６．０〜８．０である。 The in vitro DOI production temperature of the present embodiment is preferably 10 to 95 ° C, more preferably 20 to 70 ° C, and further preferably 30 to 60 ° C from the viewpoint of reaction rate and enzyme stability. is there. The reaction pH can be adjusted over a wide range, and is preferably pH 2.0 to 10.0, more preferably pH 4.0 to 8.0, and still more preferably 6.0 to 8.0 from the viewpoint of enzyme stability. is there.
しかしながら、本発明は以上の各種条件に限定されるものではなく、適宜選択することができる。 However, the present invention is not limited to the above various conditions, and can be appropriately selected.
以下、実施例により具体的に説明するが、本発明はこれらの実施例により何ら限定されるものではない。 Hereinafter, although an example explains concretely, the present invention is not limited at all by these examples.
[実施例１]
（１）染色体ＤＮＡの調製
常法に従ってＰａｅｎｉｂａｃｉｌｌｕｓ ｓｐ．ＮＢＲＣ１３１５７株の染色体ＤＮＡを調製した。
Ｐａｅｎｉｂａｃｉｌｌｕｓ ｓｐ．ＮＢＲＣ１３１５７株をＮＲ寒天プレート（１％ Ｂａｃｔｏ Ｔｒｙｐｔｏｎｅ、０．２％ Ｙｅａｓｔ Ｅｘｔｒａｃｔ、１％ エルリッヒカツオエキス、１．５％ Ｂａｃｔｏ Ａｇａｒ、ｐＨ７．０）で、３０℃、１日間培養してコロニーを形成させた。その１白金耳を、ＮＲ培地（１％ Ｂａｃｔｏ Ｔｒｙｐｔｏｎｅ、０．２％ Ｙｅａｓｔ Ｅｘｔｒａｃｔ、１％ エルリッヒカツオエキス、ｐＨ７．０）３０ｍＬを１５０ｍＬ三角フラスコに分注したものに接種して、３０℃、１８０ｒｐｍで１日間培養した。この培養液を、４℃で、１，２０００ｇ×１分間遠心分離して上清を除去し、菌体を回収した。
得られた菌体をＬｙｓｉｓバッファー（５０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ ８．０）、２０ｍＭ ＥＤＴＡ、５０ｍＭ グルコース）に懸濁し、よく洗浄した。遠心分離して菌体を回収した後、Ｌｙｓｉｓバッファーに再懸濁し、これにリゾチームを加え３７℃で４５分間インキュベートした。次いでＳＤＳとＲＮａｓｅを添加し、３７℃で４５分間インキュベートした。その後Ｐｒｏｔｅｉｎａｓｅ Ｋを添加し、５０℃で６０分間穏やかに振盪した。ここで得られた溶液をフェノール−クロロホルム、クロロホルムで処理後、エタノール沈殿し、析出した核酸をガラスピペットに巻きつけて回収した。この核酸を７０％エタノールで洗浄後、乾燥し、ＴＥに再溶解した。この操作により、約１００μｇの染色体ＤＮＡを調製した。
[Example 1]
(1) Preparation of chromosomal DNA Paenibacillus sp. Chromosomal DNA of NBRC13157 strain was prepared.
Paenibacillus sp. NBRC13157 strain was cultured on NR agar plate (1% Bacto Tryptone, 0.2% Yeast Extract, 1% Erlich Skipjack extract, 1.5% Bacto Agar, pH 7.0) for 1 day to form colonies. It was. The 1 platinum ear was inoculated into a 30 mL NR medium (1% Bacto Tryptone, 0.2% Yeast Extract, 1% Erlich skipjack extract, pH 7.0) dispensed into a 150 mL Erlenmeyer flask, 30 ° C., 180 rpm For 1 day. This culture solution was centrifuged at 12,000 g × 1 minute at 4 ° C., the supernatant was removed, and the cells were collected.
The obtained cells were suspended in Lysis buffer (50 mM Tris-HCl (pH 8.0), 20 mM EDTA, 50 mM glucose) and washed thoroughly. The cells were collected by centrifugation, then resuspended in Lysis buffer, added with lysozyme, and incubated at 37 ° C. for 45 minutes. Then SDS and RNase were added and incubated at 37 ° C. for 45 minutes. Proteinase K was then added and gently shaken at 50 ° C. for 60 minutes. The solution obtained here was treated with phenol-chloroform and chloroform and then precipitated with ethanol, and the precipitated nucleic acid was collected by winding it around a glass pipette. The nucleic acid was washed with 70% ethanol, dried and redissolved in TE. By this operation, about 100 μg of chromosomal DNA was prepared.
（２）ＤＯＩ合成酵素遺伝子の単離
上記（１）において調製した染色体ＤＮＡからＤＯＩ合成酵素遺伝子を増幅するためのＰＣＲプライマーとして、センスプライマーは配列番号１５の配列を有するオリゴＤＮＡを、アンチセンスプライマーは配列番号１６の配列を有するオリゴＤＮＡをそれぞれ合成した。
ここで得られたＰＣＲプライマーを用い、上記（１）において調製した染色体ＤＮＡを鋳型としてＰＣＲ法によるＤＯＩ合成酵素遺伝子の増幅を行い、１１０７塩基対からなるＰＣＲ産物を取得した。
(2) Isolation of DOI synthase gene As a PCR primer for amplifying the DOI synthase gene from the chromosomal DNA prepared in (1) above, the sense primer is an oligo DNA having the sequence of SEQ ID NO: 15; Respectively synthesized oligo DNAs having the sequence of SEQ ID NO: 16.
The PCR primer obtained here was used to amplify the DOI synthase gene by PCR using the chromosomal DNA prepared in (1) above as a template, and a PCR product consisting of 1107 base pairs was obtained.
ここで得られたＰＣＲ産物の遺伝子配列をＤＮＡシークエンサーで解析することで確認し、配列番号１３の塩基配列を有する既知ＤＯＩ合成酵素ｂｔｒＣ遺伝子を得た。また、配列番号１４には、既知ＤＯＩ合成酵素ＢｔｒＣのアミノ酸配列を示した。 The gene sequence of the PCR product obtained here was confirmed by analyzing with a DNA sequencer, and a known DOI synthase btrC gene having the base sequence of SEQ ID NO: 13 was obtained. SEQ ID NO: 14 shows the amino acid sequence of known DOI synthase BtrC.
（３）ＤＯＩ合成酵素遺伝子の発現プラスミドベクターの構築及び形質転換
上記（２）で取得したＰＣＲ産物の平滑末端化、リン酸化を行い、ｐＵＣ１９に大腸菌由来のｇａｐＡプロモーター、ＳＤ配列、ターミネーターを連結したプラスミドにライゲーションした。このプラスミドベクターには大腸菌中で外来遺伝子として連結された遺伝子を効率的に転写できるｇａｐＡプロモーターが導入されており、グルコースを含む培地で組換え微生物を培養した場合においてもＤＯＩ合成酵素遺伝子を効率的に発現・製造させることができる。
ここで得られたプラスミドベクターを、塩化カルシウム法で調製した大腸菌ＪＭ１０９株のコンピテントセルにヒートショック法で形質転換し、組換え微生物を作製した。
(3) Construction and transformation of DOI synthase gene expression plasmid vector The PCR product obtained in (2) above was blunted and phosphorylated, and E. coli-derived gapA promoter, SD sequence, and terminator were ligated to pUC19. Ligated to the plasmid. This plasmid vector is introduced with a gapA promoter capable of efficiently transcribing a gene linked as a foreign gene in E. coli. Even when a recombinant microorganism is cultured in a medium containing glucose, the DOI synthase gene is efficiently expressed. Can be expressed and produced.
The plasmid vector obtained here was transformed into a competent cell of Escherichia coli JM109 prepared by the calcium chloride method by the heat shock method to produce a recombinant microorganism.
（４）耐熱性ＤＯＩ合成酵素遺伝子の取得
上記（３）で取得したプラスミドベクターに対し、常法による変異導入を行うことで、配列番号１（ＤＯＩＳ−１）、配列番号３（ＤＯＩＳ−２）、配列番号５（ＤＯＩＳ−３）、配列番号７（ＤＯＩＳ−４）に記載の耐熱性ＤＯＩ合成酵素遺伝子を得ることができる。また、これらの遺伝子配列に対応するそれぞれのアミノ酸配列を配列番号２、４、６及び８に示した。ここで取得したＤＯＩ合成酵素遺伝子（ＤＯＩＳ−１）についても（３）と同様に形質転換体の取得を行った。
(4) Acquisition of heat-resistant DOI synthase gene SEQ ID NO: 1 (DOIS-1) and SEQ ID NO: 3 (DOIS-2) are obtained by introducing mutations into the plasmid vector obtained in (3) above by a conventional method. , SEQ ID NO: 5 (DOIS-3) and SEQ ID NO: 7 (DOIS-4) can be obtained. The amino acid sequences corresponding to these gene sequences are shown in SEQ ID NOs: 2, 4, 6, and 8, respectively. For the DOI synthase gene (DOIS-1) obtained here, a transformant was obtained in the same manner as in (3).
（５）広範囲ｐＨ安定型ＤＯＩ合成酵素遺伝子の取得
上記（３）で取得したプラスミドベクターに対し、常法による変異導入を行うことで、配列番号９（ＤＯＩＳ−５）、配列番号１１（ＤＯＩＳ−６）、に記載の広範囲ｐＨ安定型ＤＯＩ合成酵素遺伝子を得ることができる。また、これらの遺伝子配列に対応するそれぞれのアミノ酸配列を配列番号１０及び１２に示した。
(5) Acquisition of wide-range pH-stable DOI synthetase gene SEQ ID NO: 9 (DOIS-5), SEQ ID NO: 11 (DOIS- 6), a wide-range pH stable DOI synthase gene can be obtained. The amino acid sequences corresponding to these gene sequences are shown in SEQ ID NOs: 10 and 12, respectively.
[実施例２]
［精製酵素の取得］
実施例１で作製したＤＯＩ合成酵素（ＤＯＩＳ−１）の形質転換体を１００ｍｇ／Ｌのアンピシリンを含むＬＢプレートで、３７℃、一日間培養して、コロニーを形成させた。
次いで１００ｍｇ／Ｌのアンピシリンを含むＬＢ培地３０ｍＬを１５０ｍＬ容の三角フラスコに入れ、上記プレートからコロニーを白金耳で植菌し、３７℃で、３〜８時間、ＯＤ（６００ｎｍ）が０．５程度になるまで１８０ｒｐｍで回転振盪培養を行い、これを本培養の前培養液とした。
[Example 2]
[Acquisition of purified enzyme]
The transformant of DOI synthetase (DOIS-1) prepared in Example 1 was cultured at 37 ° C. for 1 day on an LB plate containing 100 mg / L ampicillin to form colonies.
Next, 30 mL of LB medium containing 100 mg / L ampicillin is placed in a 150 mL Erlenmeyer flask, and colonies are inoculated from the above plate with a platinum loop, and OD (600 nm) is about 0.5 at 37 ° C. for 3 to 8 hours. Until this time, rotary shaking culture was performed at 180 rpm, and this was used as a pre-culture solution for main culture.
５００ｍＬ容の三角フラスコ３６本に、２ｇ／Ｌのグルコースと１００ｍｇ／Ｌのアンピシリンを含むＬＢ培地を１００ｍＬずつ入れ、それぞれの三角フラスコに０．５ｍLの前培養液を添加し、３７℃で、１６時間、１８０ｒｐｍで回転振盪培養を行った。
次いでこの培養液を、４℃で、１０，０００ｇ×３０分間遠心分離して上清を除去し、０．２ｍｇ／Ｌ塩化コバルト六水和物を含む５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）で数回洗浄しながら菌体を回収した。回収した菌体は、−８０℃で凍結保存した。
100 mL of LB medium containing 2 g / L glucose and 100 mg / L ampicillin was added to 36 500 mL Erlenmeyer flasks, and 0.5 mL of the preculture was added to each Erlenmeyer flask. Rotational shaking culture was performed for 180 hours at 180 rpm.
Next, the culture solution was centrifuged at 4 ° C. for 10,000 g × 30 minutes to remove the supernatant, and 50 mM Tris-HCl buffer (pH 7.7) containing 0.2 mg / L cobalt chloride hexahydrate. The cells were collected while washing several times. The collected cells were stored frozen at -80 ° C.
この凍結保存菌体を０．２ｍｇ／Ｌ塩化コバルト六水和物を含む５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）で懸濁し、リゾチーム（Ｓｉｇｍａ社製 卵白由来）を１８０ｍｇとデオキシリボヌクレアーゼＩ（ＷａＫｏ社製 ウシ由来 組換え体溶液）を６０μＬ添加して、３７℃で、５時間、１２０ｒｐｍで振盪し、菌体破砕を行った。
菌体破砕後の溶液を、４℃で、１０，０００ｇ×３０分間遠心分離して菌体残渣を除去し、上清を回収した。
The cryopreserved cells were suspended in 50 mM Tris-HCl buffer (pH 7.7) containing 0.2 mg / L cobalt chloride hexahydrate, 180 mg of lysozyme (derived from Sigma egg white) and deoxyribonuclease I (Wako). 60 μL of a bovine-derived recombinant solution (manufactured by Co., Ltd.) was added and shaken at 120 rpm for 5 hours at 37 ° C. to disrupt the cells.
The solution after disrupting the cells was centrifuged at 10,000 g × 30 minutes at 4 ° C. to remove cell residues, and the supernatant was collected.
この上清に硫酸アンモニウムを加えて３０％飽和とし、４℃でしばらく攪拌した後に、生じた沈殿を、４℃で、１０，０００ｇ×３０分間遠心分離して除去し、上清を回収した。
この上清にさらに硫酸アンモニウムを加えて４０．０％飽和とし、４℃でしばらく攪拌した後に、生じた沈殿を、４℃で、１０，０００ｇ×３０分間遠心分離して上清を除去し、沈殿を回収した。次いでこの沈殿を０．２ｍｇ／Ｌ塩化コバルト六水和物を含む５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）に溶解させた。
Ammonium sulfate was added to the supernatant to make it 30% saturated, and after stirring at 4 ° C. for a while, the resulting precipitate was removed by centrifugation at 10,000 g × 30 minutes at 4 ° C., and the supernatant was recovered.
To this supernatant, ammonium sulfate was further added to achieve 40.0% saturation. After stirring at 4 ° C. for a while, the resulting precipitate was centrifuged at 4 ° C. for 10,000 g × 30 minutes to remove the supernatant, and the precipitate was precipitated. Was recovered. Next, this precipitate was dissolved in 50 mM Tris-HCl buffer (pH 7.7) containing 0.2 mg / L cobalt chloride hexahydrate.
この溶解液を４℃で、平均分画分子量１０，０００の限外濾過膜を用いて濃縮し、０．２ｍｇ／Ｌ塩化コバルト六水和物を含む５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）を加え、再度、濃縮するという脱塩操作を２〜３回繰り返した。
上記で得られた酵素溶液を、５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）で平衡化した「ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＦＦ」（商標）（ＧＥヘルスケア バイオサイエンス株式会社）に吸着させた後、０〜０．４Ｍの塩化ナトリウムを含有する５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）の濃度勾配法によって酵素を溶出させた。
The lysate was concentrated at 4 ° C. using an ultrafiltration membrane having an average molecular weight cut-off of 10,000, and 50 mM Tris-HCl buffer (pH 7.7) containing 0.2 mg / L cobalt chloride hexahydrate. The desalting operation of adding and concentrating again was repeated 2-3 times.
The enzyme solution obtained above was adsorbed on “DEAE Sepharose FF” (trademark) (GE Healthcare Biosciences) equilibrated with 50 mM Tris-HCl buffer (pH 7.7). The enzyme was eluted by a gradient method of 50 mM Tris-HCl buffer (pH 7.7) containing 4 M sodium chloride.
上記で溶出した活性画分を集め、平均分画分子量１０，０００の限外濾過膜を用いて濃縮し、１０重量％の硫酸アンモニウムを含有する５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）で懸濁し、１０重量％の硫酸アンモニウムを含有する５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）で平衡化した「ＨｉＴｒａｐ Ｐｈｅｎｙｌ ＦＦ （ｈｉｇｈ ｓｕｂ）」（商標）（ＧＥヘルスケア バイオサイエンス株式会社）に吸着させた後、１０〜０重量％の硫酸アンモニウムを含有する５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）の濃度勾配法によって酵素を溶出させた。
上記で溶出した活性画分を集め、平均分画分子量１０，０００の限外濾過膜を用いて濃縮し、５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）で平衡化した「ＭｏｎｏＱ ５／５０ ＧＬ」（商標）（ＧＥヘルスケア バイオサイエンス株式会社）に吸着させた後、０〜０．２Ｍの塩化ナトリウムを含有する５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）の濃度勾配法によって酵素を溶出させた。
The active fractions eluted above are collected, concentrated using an ultrafiltration membrane with an average molecular weight cut off of 10,000, and suspended in 50 mM Tris-HCl buffer (pH 7.7) containing 10 wt% ammonium sulfate. After adsorbing to “HiTrap Phenyl FF (high sub)” (trademark) (GE Healthcare Biosciences) equilibrated with 50 mM Tris-HCl buffer (pH 7.7) containing 10 wt% ammonium sulfate The enzyme was eluted by a concentration gradient method of 50 mM Tris-HCl buffer (pH 7.7) containing 10 to 0% by weight of ammonium sulfate.
The active fraction eluted above was collected, concentrated using an ultrafiltration membrane with an average molecular weight cut off of 10,000, and equilibrated with 50 mM Tris-HCl buffer (pH 7.7) “
上記で溶出した活性画分を集め、平均分画分子量１０，０００の限外濾過膜を用いて濃縮し、０．２ｍｇ／Ｌ塩化コバルト六水和物と０．１ＭＮａＣｌを含む５０ｍＭ Ｔｒｉｓ−ＨＣｌ緩衝液（ｐＨ７．７）で平衡化した「ＨｉＬｏａｄ １６／６０ Ｓｕｐｅｒｄｅｘ ２００」（商標）（ＧＥヘルスケア バイオサイエンス株式会社）に充填した後、同様の緩衝液で溶出を行った。
上記で溶出した活性画分を集め、平均分画分子量１０，０００の限外濾過膜を用いて濃縮し、精製ＤＯＩ合成酵素（ＤＯＩＳ−１）を得た。
The active fraction eluted above was collected, concentrated using an ultrafiltration membrane with an average molecular weight cut off of 10,000, and 50 mM Tris-HCl buffer containing 0.2 mg / L cobalt chloride hexahydrate and 0.1 M NaCl. After loading in “HiLoad 16/60 Superdex 200” (trademark) (GE Healthcare Bioscience Co., Ltd.) equilibrated with the solution (pH 7.7), elution was performed with the same buffer.
The active fractions eluted above were collected and concentrated using an ultrafiltration membrane having an average fractional molecular weight of 10,000 to obtain purified DOI synthase (DOIS-1).
上記、一連の精製酵素取得実験と同様にして、精製ＤＯＩ合成酵素（ＤＯＩＳ−２）、精製ＤＯＩ合成酵素（ＤＯＩＳ−３）、精製ＤＯＩ合成酵素（ＤＯＩ−４）、精製ＤＯＩ合成酵素（ＤＯＩ−５）および精製ＤＯＩ合成酵素（ＤＯＩ−６）、精製既知ＤＯＩ合成酵素（ＢｔｒＣ）を取得した。 In the same manner as in the above series of purified enzyme acquisition experiments, purified DOI synthetase (DOIS-2), purified DOI synthetase (DOIS-3), purified DOI synthetase (DOI-4), and purified DOI synthetase (DOI- 5) and purified DOI synthase (DOI-6) and purified known DOI synthase (BtrC) were obtained.
[実施例３]
［新規ＤＯＩ合成酵素の評価］
実施例２で得られた精製ＤＯＩ合成酵素を用いて、その作用に関する実験を行った。
[Example 3]
[Evaluation of novel DOI synthase]
Using the purified DOI synthase obtained in Example 2, an experiment on its action was conducted.
（１）至適ｐＨ範囲
各ｐＨにおける活性測定では、反応液に精製ＤＯＩ合成酵素（ＤＯＩＳ−１）を適量添加し、グルコース−６−リン酸二ナトリウム塩（オリエンタル酵母製）が２０ｍＭ、ＮＡＤ＋（オリエンタル酵母製）が５ｍＭ、塩化コバルト六水和物が５ｍＭ、各種緩衝液が１００ｍＭとなるようにして反応液を調整した。緩衝液としては、Ｂｉｓ−Ｔｒｉｓ緩衝液（ｐＨ６．０〜７．７）およびＴｒｉｓ緩衝液（ｐＨ７．４〜８．０） を使用した。反応温度は３０℃とし、生成するＤＯＩを定量することで活性を測定した。最大活性を１００とする相対活性を求め、この結果を図１に示す。本発明酵素における至適ｐＨ範囲は、ｐＨ７．０〜 ７．７であった。
(1) Optimal pH range In the activity measurement at each pH, an appropriate amount of purified DOI synthase (DOIS-1) was added to the reaction solution, and glucose-6-phosphate disodium salt (made by Oriental Yeast) was 20 mM, NAD + ( The reaction solution was adjusted so that (oriental yeast) was 5 mM, cobalt chloride hexahydrate was 5 mM, and various buffers were 100 mM. Bis-Tris buffer (pH 6.0 to 7.7) and Tris buffer (pH 7.4 to 8.0) were used as buffers. The reaction temperature was 30 ° C., and the activity was measured by quantifying the generated DOI. The relative activity with the maximum activity of 100 was determined, and the results are shown in FIG. The optimum pH range for the enzyme of the present invention was pH 7.0 to 7.7.
（２）安定ｐＨ範囲
ｐＨ５．５〜８．０の範囲の１００ｍＭの緩衝液を用いて、精製ＤＯＩ合成酵素（ＤＯＩＳ−１）を各ｐＨで３０℃、６０分間インキュベートし、その残存酵素活性を測定した。インキュベートに用いた緩衝液としては、Ｂｉｓ−Ｔｒｉｓ緩衝液（ｐＨ５．５〜８．０）およびＴｒｉｓ緩衝液（ｐＨ７．４〜８．０）を使用した。
残存酵素活性測定は、グルコース−６−リン酸二ナトリウム塩（オリエンタル酵母製）が２０ｍＭ、ＮＡＤ＋（オリエンタル酵母製）が５ｍＭ、塩化コバルト六水和物が５ｍＭとなる１００ｍＭＢｉｓ−Ｔｒｉｓ緩衝液（ｐＨ７．０）中で、反応温度３０℃で実施した。最大活性を１００とする相対活性を求め、この結果を図２に示した。本酵素の安定ｐＨ範囲は、ｐＨ６．０〜 ８．０であり、非常に幅広いｐＨ範囲で安定であった。精製ＤＯＩ合成酵素（ＤＯＩＳ−２）および精製ＤＯＩ合成酵素（ＤＯＩＳ−３）も同様の結果を示した。
(2) Stable pH range Purified DOI synthase (DOIS-1) is incubated at 30 ° C for 60 minutes at each pH using a 100 mM buffer in the range of pH 5.5 to 8.0. It was measured. Bis-Tris buffer (pH 5.5 to 8.0) and Tris buffer (pH 7.4 to 8.0) were used as the buffer used for the incubation.
Residual enzyme activity was measured by measuring 100 mM Bis-Tris buffer (pH 7.5) with glucose-6-phosphate disodium salt (produced by Oriental Yeast) at 20 mM, NAD + (produced by Oriental Yeast) at 5 mM, and cobalt chloride hexahydrate at 5 mM. In 0) at a reaction temperature of 30 ° C. The relative activity with the maximum activity of 100 was determined, and the results are shown in FIG. The stable pH range of this enzyme was pH 6.0 to 8.0 and was stable in a very wide pH range. Purified DOI synthase (DOIS-2) and purified DOI synthase (DOIS-3) showed similar results.
（３）至適温度範囲
反応温度１０〜７０℃の各反応温度条件において、グルコース−６−リン酸二ナトリウム塩（オリエンタル酵母製）が２０ｍＭ、ＮＡＤ＋（オリエンタル酵母製）が５ｍＭ、塩化コバルト六水和物が５ｍＭとなる１００ｍＭＢｉｓ−Ｔｒｉｓ緩衝液（ｐＨ７．０）中で、精製ＤＯＩ合成酵素（ＤＯＩＳ−１）の酵素活性測定を実施した。最大活性を１００とする相対活性を求め、この結果を図３に示す。至適温度範囲は５５〜７０℃であり、反応温度６５℃における比活性は１．８μｍｏｌ（ＤＯＩ）／ｍｉｎ／ｍｇ（酵素）と非常に高い値を示した。
(3) Optimum temperature range In each reaction temperature condition of reaction temperature 10-70 degreeC, glucose-6-phosphate disodium salt (product made from oriental yeast) is 20 mM, NAD + (product made from oriental yeast) is 5 mM,
（４）安定温度範囲
精製ＤＯＩ合成酵素（ＤＯＩＳ−１）を添加して且つ、ＮＡＤ＋（オリエンタル酵母製）が５ｍＭ、塩化コバルト六水和物が５ｍＭとなるように調整した１００ｍＭＢｉｓ−Ｔｒｉｓ緩衝液（ｐＨ７．０）を、温度範囲２５〜６０℃の各温度で１時間インキュベートした後、それぞれの残存酵素活性を測定した。
残存酵素活性測定は、グルコース−６−リン酸二ナトリウム塩（オリエンタル酵母製）が２０ｍＭ、ＮＡＤ＋（オリエンタル酵母製）が５ｍＭ、塩化コバルト六水和物が５ｍＭとなる１００ｍＭＢｉｓ−Ｔｒｉｓ緩衝液（ｐＨ７．０）中で、反応温度３０℃で実施した。最大活性を１００とする相対活性を求め、この結果を図４に示した。精製ＤＯＩ合成酵素（ＤＯＩＳ−１）の安定温度範囲は５０℃以下であり、精製ＤＯＩ合成酵素（ＤＯＩＳ−２）および精製ＤＯＩ合成酵素（ＤＯＩＳ−３）も同様の結果を示した。これらの高温安定性は、既知酵素にはない新規特性であった。
(4) Stable temperature range 100 mM Bis-Tris buffer solution to which purified DOI synthase (DOIS-1) was added and adjusted so that NAD + (made by Oriental Yeast) was 5 mM and cobalt chloride hexahydrate was 5 mM ( pH 7.0) was incubated at each temperature in the temperature range of 25 to 60 ° C. for 1 hour, and then the residual enzyme activity was measured.
Residual enzyme activity was measured by measuring 100 mM Bis-Tris buffer (pH 7.5) with glucose-6-phosphate disodium salt (produced by Oriental Yeast) at 20 mM, NAD + (produced by Oriental Yeast) at 5 mM, and cobalt chloride hexahydrate at 5 mM. In 0) at a reaction temperature of 30 ° C. The relative activity with the maximum activity of 100 was determined, and the results are shown in FIG. The stable temperature range of purified DOI synthase (DOIS-1) was 50 ° C. or lower, and purified DOI synthase (DOIS-2) and purified DOI synthase (DOIS-3) showed similar results. Their high temperature stability was a novel property not found in known enzymes.
また、安定化剤として作用することが知られているＮＡＤ＋およびコバルトイオン非存在化でインキュベートした安定温度範囲試験を実施したところ、精製ＤＯＩ合成酵素（ＤＯＩＳ−１）のインキュベート温度４６℃における相対活性は３８、インキュベート温度４２℃における相対活性は８９、インキュベート温度３７℃における相対活性は１００であった。 In addition, when a stable temperature range test was performed in the absence of NAD + and cobalt ions, which are known to act as a stabilizer, the relative activity of purified DOI synthase (DOIS-1) at an incubation temperature of 46 ° C. 38, the relative activity at an incubation temperature of 42 ° C. was 89, and the relative activity at an incubation temperature of 37 ° C. was 100.
（５）分子量
分離ゲル濃度が１０％の「レディーゲルＪ」（商標）（日本バイオ・ラッド ラボラトリーズ株式会社）を用いるＳＤＳ−ポリアクリルアミドゲル電気泳動により、分子量を求めたところ、精製ＤＯＩ合成酵素（ＤＯＩＳ−１）の分子量は約４０，０００であり、アミノ酸配列から推定される分子量４０，６５６とほぼ一致した。
(5) Molecular weight When molecular weight was determined by SDS-polyacrylamide gel electrophoresis using “Ready Gel J” (trademark) (Nippon Bio-Rad Laboratories, Inc.) having a separation gel concentration of 10%, purified DOI synthase ( The molecular weight of DOIS-1) was about 40,000, which almost coincided with the molecular weight of 40,656 estimated from the amino acid sequence.
[比較例１]
［既知ＤＯＩ合成酵素の評価］
配列番号１４に記載のアミノ酸配列を有する精製既知ＤＯＩ合成酵素（ＢｔｒＣ）に関して、実施例３−（４）安定温度範囲と同様にして以下実験を行った。
精製既知ＤＯＩ合成酵素（ＢｔｒＣ）を添加して且つ、ＮＡＤ＋（オリエンタル酵母製）が５ｍＭ、塩化コバルト六水和物が５ｍＭとなるように調整した１００ｍＭＢｉｓ−Ｔｒｉｓ緩衝液（ｐＨ７．０）を、温度範囲２５〜６０℃の各温度で１時間インキュベートした後、それぞれの残存酵素活性を測定した。
[Comparative Example 1]
[Evaluation of known DOI synthase]
With respect to purified known DOI synthase (BtrC) having the amino acid sequence of SEQ ID NO: 14, the following experiment was conducted in the same manner as in Example 3- (4) stable temperature range.
A purified 100 mM Bis-Tris buffer (pH 7.0) prepared by adding purified known DOI synthase (BtrC) and adjusting NAD + (oriental yeast) to 5 mM and cobalt chloride hexahydrate to 5 mM, After incubation for 1 hour at each temperature in the range 25-60 ° C., the remaining enzyme activity was measured.
残存酵素活性測定は、グルコース−６−リン酸二ナトリウム塩（オリエンタル酵母製）が２０ｍＭ、ＮＡＤ＋（オリエンタル酵母製）が５ｍＭ、塩化コバルト六水和物が５ｍＭとなる１００ｍＭＢｉｓ−Ｔｒｉｓ緩衝液（ｐＨ７．０）中で、反応温度３０℃で実施した。最大活性を１００とする相対活性を求め、この結果を図５に示した。精製既知ＤＯＩ合成酵素（ＢｔｒＣ）の安定温度範囲は４２℃以下であり、４６℃での相対活性は２３、５０℃での相対活性は０と著しく低い熱安定性を示した。 Residual enzyme activity was measured by measuring 100 mM Bis-Tris buffer (pH 7.5) with glucose-6-phosphate disodium salt (produced by Oriental Yeast) at 20 mM, NAD + (produced by Oriental Yeast) at 5 mM, and cobalt chloride hexahydrate at 5 mM. In 0) at a reaction temperature of 30 ° C. The relative activity with the maximum activity of 100 was determined, and the results are shown in FIG. The stable temperature range of purified known DOI synthase (BtrC) was 42 ° C. or less, the relative activity at 46 ° C. was 23, and the relative activity at 50 ° C. was 0, indicating a very low thermal stability.
また、安定化剤として作用することが知られているＮＡＤ＋およびコバルトイオン非存在化でインキュベートした安定温度範囲試験を実施したところ、精製既知ＤＯＩ合成酵素（ＢｔｒＣ）のインキュベート温度４２℃および４６℃における相対活性は０、インキュベート温度３７℃における相対活性は１４であった。本実験からも既存酵素が著しく低い熱安定性を有することが示された。 In addition, when a stable temperature range test was performed in the absence of NAD + and cobalt ions, which are known to act as stabilizers, the incubation temperature of purified known DOI synthase (BtrC) at 42 ° C. and 46 ° C. The relative activity was 0, and the relative activity at an incubation temperature of 37 ° C. was 14. This experiment also showed that the existing enzyme has a significantly lower thermostability.
配列番号１４に記載のアミノ酸配列を有する精製既知ＤＯＩ合成酵素（ＢｔｒＣ）に関して、実施例３−（２）安定ｐＨ範囲と同様にして以下実験を行った。
精製既知ＤＯＩ合成酵素（ＢｔｒＣ）を各ｐＨで３０℃、６０分間インキュベートし、その残存酵素活性を測定した。インキュベートに用いた緩衝液としては、Ｂｉｓ−Ｔｒｉｓ緩衝液（ｐＨ５．５〜７．０）およびＴｒｉｓ緩衝液（ｐＨ７．４〜８．０）を使用した。
残存酵素活性測定は、グルコース−６−リン酸二ナトリウム塩（オリエンタル酵母製）が２０ｍＭ、ＮＡＤ＋（オリエンタル酵母製）が５ｍＭ、塩化コバルト六水和物が５ｍＭとなる１００ｍＭＢｉｓ−Ｔｒｉｓ緩衝液（ｐＨ７．０）中で、反応温度３０℃で実施した。最大活性を１００とする相対活性を求めたところ、ｐH６における相対活性は２７と非常に低い値であった。
With respect to the purified known DOI synthase (BtrC) having the amino acid sequence shown in SEQ ID NO: 14, the following experiment was conducted in the same manner as in Example 3- (2) stable pH range.
Purified known DOI synthase (BtrC) was incubated at 30 ° C. for 60 minutes at each pH, and the residual enzyme activity was measured. Bis-Tris buffer (pH 5.5-7.0) and Tris buffer (pH 7.4-8.0) were used as the buffer used for incubation.
Residual enzyme activity was measured by measuring 100 mM Bis-Tris buffer (pH 7.5) with glucose-6-phosphate disodium salt (produced by Oriental Yeast) at 20 mM, NAD + (produced by Oriental Yeast) at 5 mM, and cobalt chloride hexahydrate at 5 mM. In 0) at a reaction temperature of 30 ° C. When the relative activity with the maximum activity of 100 was determined, the relative activity at
[実施例４]
［ＮＡＤＨ／ＮＡＤ比の酵素活性依存性］
ＮＡＤ＋およびＮＡＤＨを表１と表２に記載の濃度となるように調整し且つ、グルコース−６−リン酸二ナトリウム塩（オリエンタル酵母製）が２０ｍＭ、塩化コバルト六水和物が５ｍＭとなる１００ｍＭＢｉｓ−Ｔｒｉｓ緩衝液（ｐＨ７．０）中で、精製ＤＯＩ合成酵素（ＤＯＩＳ−１）の酵素活性測定を実施した。ＮＡＤＨ／ＮＡＤ比が０となる際の活性を１００とするときの活性値をそれぞれ求め、この結果を表１および表２に示した。
表１および表２に示すように、ＮＡＤＨ／ＮＡＤ比にＤＯＩ合成酵素活性は非常に大きな影響を受けることが明らかとなった。
[Example 4]
[Dependence of NADH / NAD ratio on enzyme activity]
NAD + and NADH were adjusted to the concentrations shown in Tables 1 and 2, and glucose-6-phosphate disodium salt (manufactured by Oriental Yeast) was 20 mM, and cobalt chloride hexahydrate was 5 mM. The enzyme activity of purified DOI synthase (DOIS-1) was measured in Tris buffer (pH 7.0). The activity values when the activity at the NADH / NAD ratio of 0 was taken as 100 were determined, and the results are shown in Tables 1 and 2.
As shown in Tables 1 and 2, it was revealed that the DOI synthase activity is greatly influenced by the NADH / NAD ratio.
本発明を用いることにより、利用価値の高いＤＯＩを効率的な製造プロセスを用いて生産することができる。 By using the present invention, a highly useful DOI can be produced using an efficient manufacturing process.
Claims (7)
（１）作用：本酵素は、グルコース−６−リン酸を２−デオキシ−シロ−イノソースへと変換する機能を有する。
（２）安定温度範囲：少なくとも４６℃まで安定である。
（３）補酵素としてＮＡＤ+を利用。
（４）至適ｐＨ範囲：７．０〜 ７．７
（５）安定ｐＨ範囲：少なくとも６．０〜 ８．０の範囲で安定である。
（６）至適温度範囲：５５ 〜 ７０ ℃
（７）分子量：３９０００〜４２０００
（８）補因子：Ｃｏ2+イオンの添加により活性向上。
（９）比活性：１．０μｍｏｌ／ｍｉｎ／ｍｇ以上（反応温度６５℃）。 The method according to claim 1 or 2, wherein the 2-deoxy-siro-inosose synthase is an enzyme having the following characteristics.
(1) Action: This enzyme has a function of converting glucose-6-phosphate into 2-deoxy-siro-inosose.
(2) Stable temperature range: Stable up to at least 46 ° C.
(3) Use NAD + as a coenzyme.
(4) Optimal pH range: 7.0 to 7.7
(5) Stable pH range: Stable at least in the range of 6.0 to 8.0.
(6) Optimal temperature range: 55 to 70 ° C
(7) Molecular weight: 39000-42000
(8) Cofactor: Activity is improved by adding Co 2+ ions.
(9) Specific activity: 1.0 μmol / min / mg or more (reaction temperature 65 ° C.).
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