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METHOD FOR PRODUCING PHOSPHITE DEHYDROGENASE PROTEIN AND USE THEREOF meetings

Foreign code F130007353
File No. S2011-0649-C0
Posted date May 14, 2013
Country WIPO
International application number 2012JP060293
International publication number WO 2012147556
Date of international filing Apr 16, 2012
Date of international publication Nov 1, 2012
Priority data
  • P2011-098670 (Apr 26, 2011) JP
Title METHOD FOR PRODUCING PHOSPHITE DEHYDROGENASE PROTEIN AND USE THEREOF meetings
Abstract In order to provide a phosphite dehydrogenase protein simultaneously having an improvement in both the properties of solubility and heat stability, a gene that codes for the protein, a method for producing the protein, and a use thereof, a phosphite dehydrogenase protein having a specific amino acid sequence and a gene coding for the protein are used.
Outline of related art and contending technology BACKGROUND ART
(PtxD) quality is phosphorous acid, and a protein having a part of the bacteria, NAD+ dependent or NADP+ -dependent oxidation of phosphorous acid, the enzyme is NADH or NADPH. Or less, NAD+ dependent or NADP+ -dependent oxidation of the phosphorous acid to a reaction formula described.
The chemical reaction is, a substance producing reactions in a living body is extremely important in the function as a cofactor NADH or NADPH can be produced efficiently but attention, the reactions use the industrially is not yet obtained. That is, at present, using phosphorous acid industrially mass-produce NADH or NADPH is not yet in the situation where (for example, see non-patent document 1 and 2). Conventionally, the production of NADH and NADPH, formate dehydrogenase, glucose dehydrogenase and alcohol dehydrogenase and the like have been used. However, these enzymes are, because of the use of reactive-rich substrate, as a result, the reaction system while having a problem of unstable. In addition, these enzymes, which results in a product rich in reactivity, as a result, the reaction system while having a problem of unstable. In addition, a main reason for the unstable reaction system, the variation of the pH. On the other hand, phosphite dehydrogenase, both substrate and product are less reactive with, the substrate has the advantage of being inexpensive, can be used industrially if, instead of the enzyme described above can be widely used are a possibility. Specifically, in the case of a phosphite dehydrogenase, phosphite and phosphoric acid for both the buffer action, it is possible to stabilize the reaction system is possible.
Industrially mass-produce NADH and NADPH in order, a large amount of phosphorous acid quality is required. Therefore, conventionally, a heterologous host such as E. coli wild-type bio-quality phosphite after the forced expression and purifying the protein quality of a large amount of phosphorous acid to obtain are attempts have been made.
However, in the art, wild-type phosphite in quality when forcibly expressed such as E. coli, many of the phosphorous acid to the aqueous solution and insoluble , recovery impossible. Therefore, even when the forced expression high solubility to an aqueous solution of a phosphorous acid quality is demanded.
In addition, industrially mass-produce NADH and NADPH when elevated in temperature of the reaction system, and has high thermal stability phosphite quality needs to be used. However, conventional phosphorous acid of a wild-type protein is a low thermal stability (specifically, a number of enzymes in 40 °C deactivation). Therefore, retains high activity even under the high phosphorous acid capable quality is demanded.
In such a situation, the above properties improved screening variants of phosphite dehydrogenase attempt has been made.
For example, the non-patent document 3, has been introduced mutation phosphite by forced expression in E. coli the quality, of the variants contained in the soluble fraction to increase the amount of phosphorous acid disclosed technique. In addition, non-patent document 3 is described from the data, in the soluble fraction of quality phosphite type variants contained an increasing amount of the reason, the solubility of the protein was due to the rise, the expression level of the protein was due to the rise or not cannot be determined. In addition, the non-patent document 4 and 5, and has high thermal stability of the variant quality phosphite disclosed.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 以下の(a)または(b)に記載のタンパク質。
(a)配列番号1のアミノ酸配列からなるタンパク質。
(b)配列番号1のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸からなり、かつ、亜リン酸デヒドロゲナーゼ活性を有するタンパク質。

[請求項2]
 請求項1に記載のタンパク質をコードする遺伝子。

[請求項3]
 以下の(c)または(d)のDNAからなる、請求項2に記載の遺伝子。
(c)配列番号2の塩基配列からなるDNA。
(d)配列番号2の塩基配列からなるDNAと相補的な塩基配列とストリンジェントな条件下でハイブリダイズし、かつ、亜リン酸デヒドロゲナーゼタンパク質をコードするDNA。

[請求項4]
 請求項1に記載のタンパク質を宿主内で発現させる工程と、上記宿主内で発現させたタンパク質を溶液中に可溶化させる工程と、を有する亜リン酸デヒドロゲナーゼタンパク質の製造方法。

[請求項5]
 上記発現させる工程は、請求項2または3に記載の遺伝子を含むベクターを宿主へ導入する工程を包含する請求項4に記載の亜リン酸デヒドロゲナーゼタンパク質の製造方法。

[請求項6]
 上記可溶化させる工程は、上記タンパク質を発現している宿主を、0(w/v)または0(w/v)よりも多く0.1%(w/v)以下の界面活性剤、および、0(w/v)または0(w/v)よりも多く50mM以下のNaClの少なくとも一方を含有する溶液中で破砕する工程を包含することを特徴とする請求項4または5に記載の亜リン酸デヒドロゲナーゼタンパク質の製造方法。

[請求項7]
 上記界面活性剤は、Tween-20またはTriton-X100であることを特徴とする請求項6に記載の亜リン酸デヒドロゲナーゼタンパク質の製造方法。

[請求項8]
 上記タンパク質を発現している宿主、または、上記タンパク質を発現している宿主の破砕物を加熱する工程を包含することを特徴とする請求項4~7の何れか1項に記載の亜リン酸デヒドロゲナーゼタンパク質の製造方法。

[請求項9]
 上記加熱する工程は、上記タンパク質を発現している宿主、または、上記タンパク質を発現している宿主の破砕物を、40℃~50℃へ加熱する工程であることを特徴とする請求項8に記載の亜リン酸デヒドロゲナーゼタンパク質の製造方法。

[請求項10]
 請求項1に記載のタンパク質、または、請求項4~9の何れか1項に記載の方法によって製造される亜リン酸デヒドロゲナーゼタンパク質を酵素として用いて、HPO 3 2-と、NAD またはNADP と、H 2Oとを反応させることを特徴とするNADHまたはNADPHの製造方法。

[請求項11]
 上記反応は、40℃~50℃にて行われることを特徴とする請求項10に記載のNADHまたはNADPHの製造方法。

[請求項12]
 上記反応は、亜ヒ酸塩、硝酸塩、硫酸塩またはNaClの存在下で行われることを特徴とする請求項10または11に記載のNADHまたはNADPHの製造方法。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • Hiroshima University
  • Inventor
  • KURODA, Akio
  • HIROTA, Ryuichi
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT QA RO RS RU RW SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ MD RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

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