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ALGINATE LYASE AND METHOD USING ENZYME FOR PRODUCING UNSATURATED URONIC ACID MONOSACCHARIDE meetings

Foreign code F170009261
File No. (S2016-0651-N0)
Posted date Oct 24, 2017
Country WIPO
International application number 2017JP013871
International publication number WO 2017175694
Date of international filing Apr 3, 2017
Date of international publication Oct 12, 2017
Priority data
  • P2016-075364 (Apr 4, 2016) JP
Title ALGINATE LYASE AND METHOD USING ENZYME FOR PRODUCING UNSATURATED URONIC ACID MONOSACCHARIDE meetings
Abstract [Problem] To provide a novel alginate lyase and a method for producing an unsaturated uronic acid monosaccharide using the enzyme.
[Solution] The present invention is achieved by a polypeptide that has an alginate lyase activity and that has an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence sharing at least 90% homology with said sequence. This polypeptide is used in a method for producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH), the method comprising: (a) an enzyme addition step for bringing said polypeptide into contact with an alginic acid containing a uronic portion; and (b) an enzymatic action step for keeping the mixture of the alginic acid and the polypeptide at a temperature at which exo-type alginate lyase activity remains active.
Outline of related art and contending technology BACKGROUND ART
The kind of the aquatic plants in the world can be seen from the ratio of the amount of production, 700 million tons in 68.6% occupied by the brown algae, the configuration of the polysaccharide is alginate is most often, in about 30%. Is an edible brown algae such as Laminaria Undaria and is used as a part but, for the most part do not use the resources. Brown algae is, arable land and fresh water, without the need for fertilizer, exists naturally in a gel, terrestrial biomass compared to the enzyme reaction can be conducted easily has attracted attention because of their advantages. In particular country Japan are surrounded by the sea, the marine area leading Japan from deconvolution methods, the use of the brown algae as long as it can also be made of a stable source of feedstock material. In addition, the configuration of the polysaccharides include brown algae and is largest in the alginic acid from, the use of the alginate has become an important issue. C5 Β-D - alginic acid epimer mannuronic acid and guluronic acid 2 α-L - one uronic acid glycosidically bound by a linear polymer (Fig. 1), β-D - mannuronic acid is combined with poly (M) block, poly α-L - guluronic acid bound (g) block, mannuronic acid and α-L - β-D - guluronic acid are alternately bound to poly (M, g) are mixed in the fraction of the block 3.
So far, can be metabolized by alginic acid has been found and several types of microorganisms, these microorganisms having alginate lyase. Alginic acid lyase by β-elimination is polysaccharide lyases(PLs) belonging to the glycoside bond. Current, alginate lyase is PL5, PL6, PL7, PL14, PL15, PL17, PL18 family is divided into. In addition, as a truncated, saturated polymer to decompose the unsaturated oligosaccharides oligosaccharides end type with a, unsaturated monomer or an unsaturated monosaccharide sugar polymer is decomposed into exo type 2 of known type. At present, PL5 and family PL7 endo, exo type PL17 family and PL15 thereof. Exo-type alginate lyase unsaturated monosaccharide is generated in the degradation by non-enzymatic reaction by the unsaturated double bonds are cleaved by ring-opening, becomes 4-deoxy-L-erythro-5-hexoseulose uronic acid(DEH). In some marine microorganisms and metabolism DEH fermenting microorganism is modified by reducing the assimilate lactic acid or succinic acid and the like of the organic acids, biofuels such as bioethanol can be made. For this reason, a method of producing DEH efficiently have been studied (Patent Document 1, 2, non-patent document 1-5).
Scope of claims (In Japanese)[請求項1]
配列番号1に示されるアミノ酸配列又は当該アミノ酸配列と少なくとも90%以上の相同性を有するアミノ酸配列を備えるものであって、エキソ型アルギン酸リアーゼ活性を有するポリペプチド。
[請求項2]
請求項1に記載のポリペプチドをコードするDNA。
[請求項3]
請求項2に記載のDNAを請求項1に記載のポリペプチドを発現可能な状態で有する発現用ベクター。
[請求項4]
(a)ウロン酸部分を含むアルギン酸に対し、請求項1に記載のポリペプチドを接触させる酵素添加工程と、
(b)前記アルギン酸とポリペプチドの混合物を前記エキソ型アルギン酸リアーゼ活性が働く温度で保持する酵素作用工程と、
を備える4-deoxy-L-erythro-5-hexoseulose uronic acid(DEH)の製造システム。
[請求項5]
配列番号2に示されるアミノ酸配列又は当該アミノ酸配列と少なくとも90%以上の相同性を有するアミノ酸配列を備えるものであって、エンド型アルギン酸リアーゼ活性を有するポリペプチド。
[請求項6]
請求項5に記載のポリペプチドをコードするDNA。
[請求項7]
請求項6に記載のDNAを請求項5に記載のポリペプチドを発現可能な状態で有する発現用ベクター。
[請求項8]
(c)ウロン酸部分を含むアルギン酸に対し、請求項1に記載のポリペプチド及びエンド型アルギン酸リアーゼ活性を有するポリペプチドの二種類のポリペプチドを接触させる複数酵素添加工程と、
(d)前記アルギン酸と二種類のポリペプチドの混合物を前記アルギン酸リアーゼ活性が働く温度で保持する複数酵素作用工程と、
を備えるDEHの製造システム。
[請求項9]
前記エンド型アルギン酸リアーゼ活性を有するポリペプチドが、請求項5に記載のポリペプチドである請求項8に記載のDEHの製造システム。
[請求項10]
陰イオン分析用カラムと、移動相として一種類のギ酸含有アンモニウム緩衝液とを有するHPLC(高速液体クロマトグラフ)部を備えるDEH測定用LC-MS(液体クロマトグラフ質量分析計)装置。
[請求項11]
HPLC(高速液体クロマトグラフィー)において陰イオン分析用カラムを使用し、移動相として一種類のギ酸含有ギ酸アンモニウム緩衝液を用いるLC/MS(液体クロマトグラフィー質量分析)を用いたDEHの測定方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • MIE UNIVERSITY
  • Inventor
  • SHIBATA Toshiyuki
  • MIYAKE Hideo
  • MURASE Yoshihiro
  • TANAKA Reiji
  • MORI Tetsushi
  • TAKEYAMA Haruko
  • TAKAHASHI Mami
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA 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 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 KM ML MR NE SN ST TD TG
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