JP6814737B2 - 酵素重合ゲル化デキストラン - Google Patents
酵素重合ゲル化デキストラン Download PDFInfo
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- JP6814737B2 JP6814737B2 JP2017543312A JP2017543312A JP6814737B2 JP 6814737 B2 JP6814737 B2 JP 6814737B2 JP 2017543312 A JP2017543312 A JP 2017543312A JP 2017543312 A JP2017543312 A JP 2017543312A JP 6814737 B2 JP6814737 B2 JP 6814737B2
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- C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
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- C—CHEMISTRY; METALLURGY
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- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Description
配列表の正式な写しは、164キロバイトのサイズを有して本明細書と同時に提出される、2015年11月5日に作成された20151105_CL6294USNP_SequenceListing.txtのファイル名を備えるASCIIフォーマットの配列表としてEFS−Webを介して電子的に提出される。このASCIIフォーマット文献に含有される配列表は、本明細書の一部であり、これにより全体として参照により本明細書に組み込まれる。
(i)1位および6位で連結した約87〜93重量%のグルコース;
(ii)1位および3位で連結した約0.1〜1.2重量%のグルコース;
(iii)1位および4位で連結した約0.1〜0.7重量%のグルコース;
(iv)1位、3位および6位で連結した約7.7〜8.6重量%のグルコース;および
(v)
(a)1位、2位および6位、または
(b)1位、4位および6位で連結した約0.4〜1.7重量%のグルコースを含むデキストランであって、
デキストランの重量平均分子量(Mw)は約5,000万〜2億Da(ダルトン)であり、デキストランのz平均慣性半径は約200〜280nmであり、デキストランは任意選択的にロイコノストック・メセンテロイデス(Leuconostoc mesenteroides)グルコシルトランスフェラーゼ酵素の生成物ではないデキストランに関する。
(i)1位および6位で連結した約87〜93重量%のグルコース;
(ii)1位および3位で連結した約0.1〜1.2重量%のグルコース;
(iii)1位および4位で連結した約0.1〜0.7重量%のグルコース;
(iv)1位、3位および6位で連結した約7.7〜8.6重量%のグルコース;および
(v)
(a)1位、2位および6位、または
(b)1位、4位および6位で連結した約0.4〜1.7重量%のグルコース
を含むデキストランを含む組成物に関する。
標)、LUMA FAST(商標)、および LIPOMAX(商標)(Genencor);LIPEX(登録商標)、LIPOLASE(登録商標)および LIPOLASE(登録商標)ULTRA (Novozymes);およびLIPASE P(商標)「Amano」(Amano Pharmaceutical Co.Ltd、日本)が含まれる。
書、国際公開第2012059336A1号パンフレット、米国特許第6730646B1、国際公開第2008087426A1号パンフレット、国際公開第2010116139A1号パンフレット、および 国際公開第2012104613A1号パンフレットに開示されている。
1)少なくとも600g/Lのバルク密度を有する顆粒として調製された洗剤組成物であって、約7〜12重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約1〜4重量%のアルコールエトキシスルフェート(例えば、C12〜18アルコール、1〜2エチレンオキシド[EO])もしくはアルキルスルフェート(例えば、C16〜18);約5〜9重量%のアルコールエトキシレート(例えば、C14〜15アルコール);約14〜20重量%の炭酸ナトリウム;約2〜6重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約15〜22重量%のゼオライト(例えば、NaAlSiO4);約0〜6重量%の硫酸ナトリウム;約0〜15重量%のクエン酸ナトリウム/クエン酸;約11〜18重量%の過ホウ酸ナトリウム;約2〜6重量%のTAED;約2重量%までの本明細書のデキストラン;約0〜3重量%の他のポリマー(例えば、マレイン酸/アクリル酸コポリマー、PVP、PEG);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、石鹸泡抑制剤、香料、蛍光増白剤、光漂白剤)を含む洗剤組成物。
2)少なくとも600g/Lのバルク密度を有する顆粒として調製された洗剤組成物であって、約6〜11重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約1〜3重量%のアルコールエトキシスルフェート(例えば、C12〜18アルコール、1〜2エチレンオキシド[EO])もしくはアルキルスルフェート(例えば、C16〜18);約5〜9重量%のアルコールエトキシレート(例えば、C14〜15アルコール);約15〜21重量%の炭酸ナトリウム;約1〜4重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約24〜34重量%のゼオライト(例えば、NaAlSiO4);約4〜10重量%の硫酸ナトリウム;約0〜15重量%のクエン酸ナトリウム/クエン酸;約11〜18重量%の過ホウ酸ナトリウム;約2〜6重量%のTAED;約2重量%までの本明細書のデキストラン;約1〜6重量%の他のポリマー(例えば、マレイン酸/アクリル酸コポリマー、PVP、PEG);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、石鹸泡抑制剤、香料、蛍光増白剤、光漂白剤)を含む洗剤組成物。
3)少なくとも600g/Lのバルク密度を有する顆粒として調製された洗剤組成物であって、約5〜9重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約7〜14重量%のアルコールエトキシスルフェート(例えば、C12〜18アルコール、7EO);約1〜3重量%の脂肪酸(例えば、C16〜22脂肪酸)としての石鹸;約10〜17重量%の炭酸ナトリウム;約3〜9重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約23〜33重量%のゼオライト(例えば、NaAlSiO4);約0〜4重量%の硫酸ナトリウム;約8〜16重量%の過ホウ酸ナトリウム;約2〜8重量%のTAED;約0〜1重量%のホスホン酸塩(例えば、EDTMPA);約2重量%までの本明細書のデキストラン;約0〜3重量%の他のポリマー(例えば、マレイン酸/アクリル酸コポリマー、PVP、PEG);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、石鹸泡抑制剤、香料、蛍光増白剤)を含む洗剤組成物。
4)少なくとも600g/Lのバルク密度を有する顆粒として調製された洗剤組成物であって、約8〜12重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約10〜25重量%のアルコールエトキシレート(例えば、C12〜18アルコール、7EO);約14〜22重量%の炭酸ナトリウム;約1〜5重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約25〜35重量%のゼオライト(例えば、NaAlSiO4);約0〜10重量%の硫酸ナトリウム;約8〜16重量%の過ホウ酸ナトリウム;約2〜8重量%のTAED;約0〜1重量%のホスホン酸塩(例えば、EDTMPA);約2重量%までの本明細書のデキストラン;約1〜3重量%の他のポリマー(例えば、マレイン酸/アクリル酸コポリマー、PVP、PEG);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、石鹸泡抑制剤、香料)を含む洗剤組成物。
5)水性液体洗剤組成物であって、約15〜21重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約12〜18重量%のアルコールエトキシレート(例えば、C12〜18アルコール、7EO;もしくはC12〜15アルコール、5EO);約3〜13重量%の脂肪酸(例えば、オレイン酸)としての石鹸;約0〜13重量%のアルケニルコハク酸(C12〜14);約8〜18重量%のアミノエタノール;約2〜8重量%のクエン酸;約0〜3重量%のホスホン酸塩;約2重量%までの本明細書のデキストラン;約0〜3重量%の他のポリマー(例えば、PVP、PEG);約0〜2重量%のホウ酸塩約0〜3重量%のエタノール;約8〜14重量%のプロピレングリコール;任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、分散剤、石鹸泡抑制剤、香料、蛍光増白剤)を含む水性液体洗剤組成物。
6)水性構造化液体洗剤組成物であって、約15〜21重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約3〜9重量%のアルコールエトキシレート(例えば、C12〜18アルコール、7EO;もしくはC12〜15アルコール、5EO);約3〜10重量%の脂肪酸(例えば、オレイン酸)としての石鹸;約14〜22重量%のゼオライト(例えば、NaAlSiO4);約9〜18重量%のクエン酸カリウム;約0〜2重量%のホウ酸塩;約2重量%までの本明細書のデキストラン;約0〜3重量%の他のポリマー(例えば、PVP、PEG);約0〜3重量%のエタノール;固定化ポリマー(例えば、ラウリルメタクリレート/アクリル酸コポリマー、モル比25:1、MW3800);約0〜5重量%のグリセロール;任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、分散剤、石鹸泡抑制剤、香料、蛍光増白剤)を含む水性構造化液体洗剤組成物。
7)少なくとも600g/Lのバルク密度を有する顆粒として調製された洗剤組成物であって、約5〜10重量%の高級アルコール硫酸エステル塩;約3〜9重量%のエトキシル化脂肪酸モノエタノールアミド;約0〜3重量%の脂肪酸としての石鹸;約5〜10重量%の炭酸ナトリウム;約1〜4重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約20〜40重量%のゼオライト(例えば、NaAlSiO4);約2〜8重量%の硫酸ナトリウム;約12〜18重量%の過ホウ酸ナトリウム;約2〜7重量%のTAED;約2重量%までの本明細書のデキストラン;約1〜5重量%の他のポリマー(例えば、マレイン酸/アクリル酸コポリマー、PEG);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、蛍光増白剤、石鹸泡抑制剤、香料)を含む洗剤組成物。
8)顆粒として調製された洗剤組成物であって、約8〜14重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約5〜11重量%のエトキシル化脂肪酸モノエタノールアミド;約0〜3重量%の脂肪酸としての石鹸;約4〜10重量%の炭酸ナトリウム;約1〜4重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約30〜50重量%のゼオライト(例えば、NaAlSiO4);約3〜11重量%の硫酸ナトリウム;約5〜12重量%のクエン酸ナトリウム;約2重量%までの本明細書のデキストラン;約1〜5重量%の他のポリマー(例えば、PVP、マレイン酸/アクリル酸コポリマー、PEG);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、石鹸泡抑制剤、香料)を含む洗剤組成物。
9)顆粒として調製された洗剤組成物であって、約6〜12重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約1〜4重量%の非イオン性界面活性剤;約2〜6重量%の脂肪酸としての石鹸;約14〜22重量%の炭酸ナトリウム;約18〜32重量%のゼオライト(例えば、NaAlSiO4);約5〜20重量%の硫酸ナトリウム;約3〜8重量%のクエン酸ナトリウム;約4〜9重量%の過ホウ酸ナトリウム;約1〜5重量%の漂白活性化剤(例えば、NOBSもしくはTAED);約2重量%までの本明細書のデキストラン;約1〜5重量%の他のポリマー(例えば、ポリカルボキシレートもしくはPEG);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、蛍光増白剤、香料)を含む洗剤組成物。
10)水性液体洗剤組成物であって、約15〜23重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約8〜15重量%のアルコールエトキシスルフェート(例えば、C12〜15アルコール、2〜3EO);約3〜9重量%のアルコールエトキシレート(例えば、C12〜15アルコール、7EO;もしくはC12〜15アルコール、5EO);約0〜3重量%の脂肪酸(例えば、ラウリン酸)としての石鹸;約1〜5重量%のアミノエタノール;約5〜10重量%のクエン酸ナトリウム;約2〜6重量%のヒドロトロープ(例えば、トルエンスルホン酸ナトリウム);約0〜2重量%のホウ酸塩;約1重量%までの本明細書のデキストラン;約1〜3重量%のエタノール;約2〜5重量%のプロピレングリコール;任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、分散剤、香料、蛍光増白剤)を含む水性液体洗剤組成物。
11)水性液体洗剤組成物であって、約20〜32重量%の直鎖アルキルベンゼンスルホネート(酸として計算された);約6〜12重量%のアルコールエトキシレート(例えば、C12〜15アルコール、7EO;もしくはC12〜15アルコール、5EO);約2〜6重量%のアミノエタノール;約8〜14重量%のクエン酸;約1〜3重量%のホウ酸塩;約2重量%までの本明細書のデキストラン;約1〜3重量%のエタノール;約2〜5重量%のプロピレングリコール;約0〜3重量%の他のポリマー(例えば、マレイン酸/アクリル酸コポリマー;約0〜3重量%のエタノール;固定化ポリマー、例えばラウリルメタクリレート/アクリル酸コポリマー);約3〜8重量のグリセロール;任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、ヒドロトープ、分散剤、香料、蛍光増白剤)を含む水性液体洗剤組成物。
12)少なくとも600g/Lのバルク密度を有する顆粒として調製された洗剤組成物であって、約25〜40重量%のアニオン性界面活性剤(例えば、直鎖アルキルベンゼンスルホネート、アルキルスルフェート、α−オレフィンスルホン酸、α−スルホ脂肪酸メチルエステル、アルカンスルホン酸塩、石鹸);約1〜10重量%の非イオン性界面活性剤(アルコールエトキシレート;約8〜25重量%の炭酸ナトリウム;約5〜15重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約15〜28重量%のゼオライト(NaAlSiO4);約0〜20重量%の過ホウ酸ナトリウム;約0〜5重量%の漂白活性化剤(例えば、TAEDもしくはNOBS);約2重量%までの本明細書のデキストラン;任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、香料、蛍光増白剤)を含む洗剤組成物。
13)上記の(1)〜(12)に記載されているが、直鎖アルキルベンゼンスルホネートの全部もしくは一部がC12〜C18アルキルスルフェートで置換されている洗剤組成物。
14)少なくとも600g/Lのバルク密度を有する顆粒として調製された洗剤組成物であって、約9〜15重量%のC12〜C18アルキルスルフェート;約3〜6重量%のアルコールエトキシレート;約1〜5重量%のポリヒドロキシアルキル脂肪酸アミド;約10〜20重量%のゼオライト(例えば、NaAlSiO4);約10〜20重量%の層状二ケイ酸塩(例えば、Hoechst社製のSK56);約3〜12重量%の炭酸ナトリウム;約0〜6重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約4〜8重量%のクエン酸ナトリウム;約13〜22重量%の過炭酸ナトリウム;約3〜8重量%のTAED;約2重量%までの本明細書のデキストラン;約0〜5重量%の他のポリマー(例えば、ポリカルボキシレートおよびPVP);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜5重量%の微量の成分(例えば、蛍光増白剤、光漂白剤、香料、石鹸泡抑制剤)を含む洗剤組成物。
15)少なくとも600g/Lのバルク密度を有する顆粒として調製された洗剤組成物であって、約4〜8重量%のC12〜C18アルキルスルフェート;約11〜15重量%のアルコールエトキシレート;約1〜4重量%の石鹸;約35〜45重量%のゼオライトMAPもしくはゼオライトA;約2〜8重量%の炭酸ナトリウム;約0〜4重量%の可溶性ケイ酸塩(例えば、Na2O 2SiO2);約13〜22重量%の過炭酸ナトリウム;約1〜8重量%のTAED;約3重量%までの本明細書のデキストラン;約0〜3重量%の他のポリマー(例えば、ポリカルボキシレートおよびPVP);任意選択的に約0.0001〜0.1重量%の酵素(純粋酵素タンパク質として計算された);および約0〜3重量%の微量の成分(例えば、蛍光増白剤、ホスホン酸塩、香料)を含む洗剤組成物。16)上記の(1)〜(15)に記載した洗剤調製物であって、追加の成分または既に特定した漂白系の代替物のいずれかとして安定化もしくはカプセル封入過酸を含有する洗剤調製物。
17)上記の(1)、(3)、(7)、(9)および(12)に記載した洗剤組成物であって、過ホウ酸塩が過炭酸塩と置換されている洗剤組成物。
(18)上記の(1)、(3)、(7)、(9)、(12)、(14)および(15)に記載した洗剤組成物であって、追加してマンガン系触媒を含有する洗剤組成物。マンガン系触媒は、例えば、参照により本明細書に組み込まれるHage et al.(1994,Nature 369:637−639)によって記載された化合物の1つである。19)非水性洗剤液体として調製された洗剤組成物であって、液体非イオン性界面活性剤(例えば、直鎖アルコキシル化第一級アルコール)、ビルダー系(TAED、ホスホン酸塩)、本明細書のデキストラン、任意選択的に酵素およびアルカリを含む洗剤組成物。洗剤は、さらにアニオン性界面活性剤および/または漂白剤系を含むことができる。
1.デキストランを含む組成物であって、デキストランが:
(i)1位および6位で連結した約87〜93重量%のグルコース;
(ii)1位および3位で連結した約0.1〜1.2重量%のグルコース;
(iii)1位および4位で連結した約0.1〜0.7重量%のグルコース;
(iv)1位、3位および6位で連結した約7.7〜8.6重量%のグルコース;および(v)
(a)1位、2位および6位、または
(b)1位、4位および6位で連結した約0.4〜1.7重量%のグルコースを含み、デキストランの重量平均分子量(Mw)は約5,000万〜2億Daであり、デキストランのz平均旋回半径は約200〜280nmであり、デキストランは任意選択的にロイコノストック・メセンテロイデス(Leuconostoc mesenteroides)グルコシルトランスフェラーゼ酵素の生成物ではないデキストランを含む組成物。
2.デキストランは:
(i)1位および6位で連結した約89.5〜90.5重量%のグルコース;
(ii)1位および3位で連結した約0.4〜0.9重量%のグルコース;
(iii)1位および4位で連結した約0.3〜0.5重量%のグルコース;
(iv)1位、3位および6位で連結した約8.0〜8.3重量%のグルコース;および
(v)
(a)1位、2位および6位、または
(b)1位、4位および6位で連結した約0.7〜1.4重量%のグルコースを含む、実施形態1の組成物。
3.デキストランは、分岐鎖構造内で一緒に連結した鎖(長鎖)を含み、ここで、その鎖は長さが類似であり、実質的にα−1,6−グルコシド結合を含む、実施形態1または2の組成物。
4.鎖の平均長は、約10〜50モノマー単位である、実施形態1、2または3の組成物。
5.デキストランは、配列番号1、配列番号2、配列番号5、配列番号9、配列番号13、または配列番号17と少なくとも90%同一であるアミノ酸配列を含むグルコシルトランスフェラーゼ酵素の生成物である、実施形態1、2、3または4の組成物。
6.本組成物は、少なくとも約25cPの粘度を有する水性組成物である、実施形態1、2、3、4または5の組成物。
7.デキストランのMwは、約8,000万〜1億2,000万Daである実施形態1、2、3、4、5または6の組成物。
8.デキストランのz平均旋回半径は、約230〜250nmである実施形態1、2、3、4、5、6または7の組成物。
9.本組成物は、食品、パーソナルケア製品、医薬製品、家庭用製品または工業製品の形態にある、実施形態1、2、3、4、5、6、7または8の組成物。
10.本組成物は、菓子類の形態にある、実施形態9の組成物。
11.水性組成物の粘度を増加させるための方法であって:実施形態1〜8のいずれかに記載のデキストランを水性組成物と接触させる工程を含み、水性組成物の粘度は接触させる工程の前の水性組成物の粘度と比較してデキストランによって増加させられる方法。
12.材料を処理する方法であって、材料を実施形態1〜8のいずれかに記載のデキストランを含む水性組成物と接触させる工程を含む方法。
13.水、スクロースおよび配列番号1、配列番号2、配列番号5、配列番号9、配列番号13、または配列番号17と少なくとも90%同一であるアミノ酸配列を含む酵素反応であって、グルコシルトランスフェラーゼ酵素は、実施形態1〜8のいずれかに記載のデキストランを合成する酵素反応。
14.デキストランを生成するための方法であって、
a)少なくとも水、スクロースおよび配列番号1、配列番号2、配列番号5、配列番号9、配列番号13、または配列番号17と少なくとも90%同一であるアミノ酸配列を接触させる工程であって、実施形態1〜8のいずれかに記載のデキストランが合成される接触させる工程;およびと少なくとも90%同一であるアミノ酸配列を接触させる工程であって、実施形態1〜8のいずれかに記載のデキストランが合成される接触させる工程;および
b)任意選択的に、工程(a)で生成されたデキストランを単離する工程を含む方法。15.本方法において生成されるデキストランの粘度は、工程(a)におけるスクロースの量を減少させることによって増加する、実施形態14の方法。
枯草菌(Bacillus subtilis)中のグルコシルトランスフェラーゼ酵素のクローニングおよび発現
実施例3〜6において使用した各グルコシルトランスフェラーゼは、以下のように調製した。
酵素ストック液は、各gtfのために10mLの分子グレードのH2Oを凍結乾燥酵素粉末を含有する各50mLの円錐形遠心管内に加えることによって作成した。
大腸菌(E.coli)中でのグルコシルトランスフェラーゼ(0768)の発現および粗活性酵素溶解物の生成
本実施例は、大腸菌(E.coli)中での成熟グルコシルトランスフェラーゼ(gtf)酵素の発現について記載する。大腸菌(E.coli)発現菌株の粗細胞溶解物を生成すると、スクロースの存在下でゲル生成物形成活性を証明した。
スクロースとGtf0768との反応およびゲル化デキストラン反応生成物の分析
本実施例では、実施例1に提供した結果を補足する、水、スクロースおよびgtf0768を含む追加の反応物について記載する。さらに、本実施例は、gtf0768によって合成されたゲル化生成物のグリコシド結合分析を提供し、この生成物がデキストランの1つのタイプであることを証明している。
− スクロース(Sigma製品番号S−9378)。
− リン酸ナトリウムバッファーストック(200mM)(pH5.5):したがって、リン酸ナトリウム一塩基性一水和物(Sigma製品番号S9638)およびリン酸ナトリウムに塩基性七水和物(Sigma製品番号S9390)を使用して250mLの水溶液を調製する。
− Gtf0768酵素溶液(実施例1において調製した細胞溶解物)。
2.72gのリン酸ナトリウムバッファーストック(pH5.5)、100g/Lのスクロースおよび2mLのgtf0768酵素溶液を含有する1,000mLの反応を調製した。この反応を26℃で20時間撹拌すると粘性になった。gtf酵素は、10分間にわたり80℃で反応を加熱することによって脱活性化した。次に粘性生成物を沈降させるために、脱活性化粘性反応を3Lの100%メタノールと混合した。白色沈降物が形成されるので、これを次に濾過し、120mLの100%メタノールを用いた4回の洗浄を実施した。固体生成物を72時間にわたりオーブン内の真空下で室温で乾燥させた。
グルコシルトランスフェラーゼ(2919)の発現およびゲル化デキストラン生成物を生成するためのそれらの使用
本実施例は、枯草菌(B.subtilis)中での成熟ワイセラ・シバリア(Weissella cibaria)グルコシルトランスフェラーゼ(gtf)酵素の発現について記載する。さらに、本実施例は、この酵素が、使用された場合に水およびスクロースを含有する反応であるデキストランのようなゲル化生成物を生成することを証明している。
グルコシルトランスフェラーゼ(2918)の発現およびゲル化デキストラン生成物を生成するためのそれらの使用
本実施例は、枯草菌(B.subtilis)中での成熟ラクトバシラス ファーメンタム(Lactobacillus fermentum)グルコシルトランスフェラーゼ(gtf)酵素の発現について記載する。さらに、本実施例は、この酵素が、使用された場合に水およびスクロースを含有する反応であるデキストランのようなゲル化生成物を生成することを証明している。
グルコシルトランスフェラーゼ(2920)の発現およびゲル化デキストラン生成物を生成するためのそれらの使用
本実施例は、枯草菌(B.subtilis)中での成熟ストレプトコッカス・ソブリヌス(Streptococcus sobrinus)グルコシルトランスフェラーゼ(gtf)酵素の発現について記載する。さらに、本実施例は、この酵素が、使用された場合に水およびスクロースを含有する反応であるデキストランのようなゲル化生成物を生成することを証明している。
グルコシルトランスフェラーゼ(2921)の発現およびゲル化デキストラン生成物を生成するためのそれらの使用
本実施例は、枯草菌(B.subtilis)中での成熟ストレプトコッカス・ダウネイ(Streptococcus downei)グルコシルトランスフェラーゼ(gtf)酵素の発現について記載する。さらに、本実施例は、この酵素が、使用された場合に水およびスクロースを含有する反応であるデキストランのようなゲル化生成物を生成することを証明している。
市販で入手できるデキストラン分解酵素を使用したデキストランの生成
本実施例は、水およびスクロースを含む反応物中で市販で入手できるデキストラン分解酵素を使用したデキストランの合成について記載する。これで生成されたデキストランは、実施例8において、実施例1〜6で合成されたゲル化デキストラン生成物に比較して分析した。
− スクロース(Sigma製品番号S−9378)。400g/Lのストック液を調製した。
− リン酸ナトリウムバッファーストック(200mM)(pH5.5):したがって、リン酸ナトリウム一塩基性一水和物(Sigma製品番号S9638)およびリン酸ナトリウムに塩基性七水和物(Sigma製品番号S9390)を使用して250mLの水溶液を調製する。
− ロイコノストック・メセンテロイデス(Leuconostoc mesenteroides)由来のデキストラン分解酵素、凍結乾燥粉末、≧100単位/mgのタンパク質(Sigma社製品番号D9909)。
デキストランサンプルの粘度
本実施例では、実施例1〜7で生成されたデキストランポリマーの粘度、ならびに市販起源から得られたデキストランの粘度を測定することについて記載する。粘度測定は、様々な剪断速度で実施した。
gtf0768により合成されたデキストランのまた別の生成および分析
本実施例は、実施例2に加えて、水、スクロースの量およびgtf0768を含むまた別の反応について記載する。さらに、本実施例は、gtf0768によって合成されたゲル化生成物の追加の結合分析および分子量分析を提供し、この生成物がデキストランの1つのタイプであることを証明している。
− スクロース(Sigma製品番号S−9378)。
− リン酸ナトリウムバッファーストック(1M、pH6.5、Teknovaカタログ番号S0276)。
− Gtf0768酵素溶液(実施例1において調製した細胞溶解物)。
20mMのリン酸ナトリウムバッファー(バッファーは1Mストック(pH6.5)からのddH2Oを用いて希釈した)、100g/Lのスクロースおよび0.1mLのgtf0768酵素溶液を含有する50mLの反応を調製した。この反応を100rpmで26℃のインキュベーターシェーカー内(Innova,Model 4000)で43時間にわたり振とうした;反応は、約24時間後に粘性になった。
(i)1位および6位でのみ連結した約87〜93重量%のグルコース;
(ii)1位および3位でのみ連結した約0.1〜1.2重量%のグルコース;
(iii)1位および4位でのみ連結した約0.1〜0.7重量%のグルコース;
(iv)1位、3位および6位でのみ連結した約7.7〜8.6重量%のグルコース;および
(v)(a)1位、2位および6位、または(b)1位、4位および6位でのみ連結した約0.4〜1.7重量%のグルコース
を含むことを指摘している。
gtf0768によって合成されたデキストランを含む製剤
本実施例は、gtf0768のデキストラン生成物を含む製剤について開示する。この製剤は、所定の消費者製品(例えば、ローションなどのパーソナルケア組成物)に粘度を提供するために一般に使用される所定の化合物(キサンタンゴム、Carbopol(登録商標))を含む製剤と比較して優れた感覚的特徴(もしくは「感触」)を有することが証明された。
懸濁粒子を備えるデキストランを含むクレンジング剤
本実施例は、gtf0768のデキストラン生成物を含むクレンジング剤について開示する。ホホバエステルビーズはこの組成物中に懸濁させることができたが、これはデキストランが分散剤として機能できることを指摘した。
1.デキストランを含む組成物であって、前記デキストランは、
(i)1位および6位で連結した約87〜93重量%のグルコース;
(ii)1位および3位で連結した約0.1〜1.2重量%のグルコース;
(iii)1位および4位で連結した約0.1〜0.7重量%のグルコース;
(iv)1位、3位および6位で連結した約7.7〜8.6重量%のグルコース;および
(v)
(a)1位、2位および6位、または
(b)1位、4位および6位で連結した約0.4〜1.7重量%のグルコースを含み、
前記デキストランの重量平均分子量(Mw)は約5,000万〜2億Daであり、前記デキストランのz平均旋回半径は約200〜280nmであり、前記デキストランは任意選択的にロイコノストック・メセンテロイデス(Leuconostoc mesenteroides)グルコシルトランスフェラーゼ酵素の生成物ではないデキストランを含む組成物。
2.前記デキストランは、
(i)1位および6位で連結した約89.5〜90.5重量%のグルコース;
(ii)1位および3位で連結した約0.4〜0.9重量%のグルコース;
(iii)1位および4位で連結した約0.3〜0.5重量%のグルコース;
(iv)1位、3位および6位で連結した約8.0〜8.3重量%のグルコース;および
(v)
(a)1位、2位および6位、または
(b)1位、4位および6位で連結した約0.7〜1.4重量%のグルコースを含む上記1に記載の組成物。
3.前記デキストランは、分岐鎖構造内で一緒に連結した鎖を含み、ここで、前記鎖は長さが類似であり、実質的にα−1,6−グルコシド結合を含む、上記1に記載の組成物。4.前記鎖の平均長は、約10〜50モノマー単位である、上記3に記載の組成物。
5.前記デキストランは、配列番号1、配列番号2、配列番号5、配列番号9、配列番号13、または配列番号17と少なくとも90%同一であるアミノ酸配列を含むグルコシルトランスフェラーゼ酵素の生成物である、上記1に記載の組成物。
6.前記組成物は、少なくとも約25cPの粘度を有する水性組成物である、上記1に記載の組成物。
7.前記デキストランのMwは、約8,000万〜1億2,000万Daである上記1に記載の組成物。
8.前記デキストランのz平均旋回半径は、約230〜250nmである上記1に記載の組成物。
9.前記組成物は、食品、パーソナルケア製品、医薬製品、家庭用製品または工業製品の形態にある、上記1に記載の組成物。
10.前記組成物は、菓子類の形態にある、上記1に記載の組成物。
11.水性組成物の粘度を増加させるための方法であって、
上記1に記載のデキストランを前記水性組成物と接触させる工程を含み、前記水性組成物の粘度は前記接触させる工程の前の前記水性組成物の粘度と比較して前記デキストランによって増加させられる方法。
12.材料を処理する方法であって、材料を上記1に記載のデキストランを含む水性組成物と接触させる工程を含む方法。
13.水、スクロースおよび配列番号1、配列番号2、配列番号5、配列番号9、配列番号13、または配列番号17と少なくとも90%同一であるアミノ酸配列を含む、もしくはそれらからなるグルコシルトランスフェラーゼ酵素を含む酵素反応であって、
前記グルコシルトランスフェラーゼ酵素は上記1に記載のデキストランを合成する酵素反応。
14.デキストランを生成するための方法であって、
a)水、スクロースおよび配列番号1、配列番号2、配列番号5、配列番号9、配列番号13、または配列番号17と少なくとも90%同一であるアミノ酸配列を含む、もしくはそれらからなるグルコシルトランスフェラーゼ酵素を接触させる工程であって、それにより上記1に記載のデキストランが生成される工程;および
b)任意選択的に、工程(a)で生成されたデキストランを単離する工程を含む方法。15.前記方法において製造されるデキストランの粘度は、工程(a)におけるスクロースの量を減少させることによって増加する、上記14に記載の方法。
Claims (7)
- デキストランを含む組成物であって、前記デキストランは、
(i)1位および6位で連結した87〜91.5重量%のグルコース;
(ii)1位および3位で連結した0.1〜1.2重量%のグルコース;
(iii)1位および4位で連結した0.1〜0.7重量%のグルコース;
(iv)1位、3位および6位で連結した7.7〜8.6重量%のグルコース;および
(v)
(a)1位、2位および6位、または
(b)1位、4位および6位で連結した0.4〜1.7重量%のグルコースを含み、
前記デキストランの重量平均分子量(Mw)は5,000万〜2億Daであり、前記デキストランのz平均旋回半径は200〜280nmである組成物。 - 前記デキストランは、
(i)1位および6位で連結した89.5〜90.5重量%のグルコース;
(ii)1位および3位で連結した0.4〜0.9重量%のグルコース;
(iii)1位および4位で連結した0.3〜0.5重量%のグルコース;
(iv)1位、3位および6位で連結した8.0〜8.3重量%のグルコース;および
(v)
(a)1位、2位および6位、または
(b)1位、4位および6位で連結した0.7〜1.4重量%のグルコースを含む、請求項1に記載の組成物。 - 請前記組成物は、少なくとも25cPの粘度を有する水性組成物である、請求項1に記載の組成物。
- 前記デキストランのMwは、8,000万〜1億2,000万Daであり、z平均旋回半径は、230〜250nmである請求項1に記載の組成物。
- 前記組成物は、食品、パーソナルケア製品、医薬製品、家庭用製品または工業製品の形態にある、請求項1〜4のいずれか1項に記載の組成物。
- 水性組成物の粘度を増加させるための方法であって、前記方法は、
請求項1、2または4のいずれか1項に記載のデキストランを前記水性組成物と接触させる工程を含み、前記水性組成物の粘度は前記接触させる工程の前の前記水性組成物の粘度と比較して前記デキストランによって増加させられる方法。 - 材料を処理する方法であって、材料を請求項1、2または4のいずれか1項に記載のデキストランを含む水性組成物と接触させる工程を含む方法。
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PCT/US2015/059261 WO2016073732A1 (en) | 2014-11-05 | 2015-11-05 | Enzymatically polymerized gelling dextrans |
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