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METHOD FOR PRODUCING PHOSPHITE DEHYDROGENASE PROTEIN AND USE THEREOF 新技術説明会

外国特許コード F130007353
整理番号 S2011-0649-C0
掲載日 2013年5月14日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2012JP060293
国際公開番号 WO 2012147556
国際出願日 平成24年4月16日(2012.4.16)
国際公開日 平成24年11月1日(2012.11.1)
優先権データ
  • 特願2011-098670 (2011.4.26) JP
発明の名称 (英語) METHOD FOR PRODUCING PHOSPHITE DEHYDROGENASE PROTEIN AND USE THEREOF 新技術説明会
発明の概要(英語) 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.
従来技術、競合技術の概要(英語) 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.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • Hiroshima University
  • 発明者(英語)
  • KURODA, Akio
  • HIROTA, Ryuichi
国際特許分類(IPC)
指定国 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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