來自枯草芽孢桿菌的變體α-淀粉酶及其使用方法

專利名稱：來自枯草芽孢桿菌的變體α-淀粉酶及其使用方法
技術領域：
本發明提供來自枯草芽孢桿菌(Bacillus subtilis)的α -淀粉酶(AmyE)、其變 體、編碼其的核酸、和包括該核酸的宿主細胞。公開了使用AmyE或其變體的方法，包括淀粉 的液化和/或糖化。此種方法可以產生對于乙醇生產或高果糖玉米糖漿生產有用的糖。在 某些情況下，所述淀粉酶可以在低PH下、在不存在鈣的情況下和/或在不存在葡糖淀粉酶 的情況下使用。
背景技術：
谷物、谷類和蔬菜淀粉例如玉米淀粉廣泛用于產品例如糖漿和生物燃料的工業制 造中。例如，高果糖玉米糖漿(HFCQ是玉米糖漿的加工形式，具有高果糖含量和與糖相當 的甜度，這使得HFCS可以作為代糖在軟飲料和其他加工食品中使用。HFCS生產目前是十億 美元的工業。乙醇作為生物燃料的生產也是增長中的工業。糖漿和生物燃料可以通過催化淀粉分解成葡萄糖的酶促過程由淀粉生產。這種酶 促過程一般涉及一系列酶催化的反應(1)液化α -淀粉酶(EC 3. 2. 1. 1)首先催化淀粉懸浮液降解為麥芽糖糊精，所 述淀粉懸浮液可以包含30-40% w/w干固形物(ds)。α-淀粉酶是內切水解酶，催化內部 a-l，4_D-糖苷鍵的隨機斷裂。因為液化一般在高溫例如90-100°C進行，所以熱穩定的 α-淀粉酶例如來自芽孢桿菌屬物種(Bacillus sp.)的α -淀粉酶優選用于這個步驟。目 前用于這個步驟的α-淀粉酶，例如來自地衣芽孢桿菌(B. licheniformis)、解淀粉芽孢桿 菌(B. amyloliquefaciens)和嗜熱脂肪芽孢桿菌(B. stearothermophilus)的 α -淀粉酶 (AmyS)，不產生顯著量的葡萄糖。相反，所得到的液化物(Iiquefact)具有低葡萄糖當量 (DE)，并且包含麥芽糖和具有高聚合度(DPn)的糖。(2)糖化葡糖淀粉酶和/或生麥芽糖α -淀粉酶催化在液化后形成的麥芽糖 糊精的非還原末端的水解，釋放D-葡萄糖、麥芽糖和異麥芽糖。糖化作用產生富含葡萄 糖或高麥芽糖的糖漿。在前面一種情況下，葡糖淀粉酶一般在酸性條件下在升高的溫 度，例如60°C，pH 4.3催化糖化。在這個過程中使用的葡糖淀粉酶一般得自真菌，例如 在OPTIDEX L400中使用的黑曲霉(Aspergillus niger)葡糖淀粉酶或灰腐質霉 (Humicola grisea)葡糖淀粉酶。脫支酶例如支鏈淀粉酶可以幫助糖化作用。生麥芽糖α-淀粉酶可替代地可以催化糖化作用，以形成高麥芽糖糖漿。生麥芽 糖α -淀粉酶一般具有比葡糖淀粉酶更高的最適PH和更低的最適溫度，并且生麥芽糖淀粉 酶一般需要Ca2+。目前用于這種應用的生麥芽糖α-淀粉酶包括枯草芽孢桿菌α-淀粉酶、 植物淀粉酶和來自米曲霉(Aspergillus oryzae)的α -淀粉酶，CLARASE L的活性組分。用于產生各種產物的示例性糖化反應描述如下
權利要求
1.用于在淀粉轉化工藝中液化和糖化淀粉的方法，其包括使淀粉底物與AmyE多肽接觸，以形成反應混合物用于液化和糖化該反應混合物中的 淀粉底物以產生葡萄糖，其中所述液化和糖化在相同反應混合物中進行，無需PH調整。
2.權利要求1的方法，其中所述糖化在缺乏具有葡糖淀粉酶活性的另外多肽的情況下 執行。
3.權利要求1的方法，其中所述液化在適于葡糖淀粉酶多肽的活性的PH執行。
4.權利要求3的方法，其中所述pH是5.0或更低。
5.權利要求3的方法，其中所述pH是4.5或更低。
6.權利要求3的方法，其中所述pH是4.0或更低。
7.權利要求1的方法，其中在所述淀粉底物與所述AmyE多肽接觸前，將具有葡糖淀粉 酶活性的另外多肽加入所述反應混合物中。
8.權利要求1的方法，其中在所述淀粉底物與所述AmyE多肽接觸后，將具有葡糖淀粉 酶活性的另外多肽加入所述反應混合物中。
9.權利要求1的方法，其中在所述淀粉底物與所述AmyE多肽接觸的同時，將具有葡糖 淀粉酶活性的另外多肽加入所述反應混合物中。
10.權利要求1的方法，其進一步包括使通過液化和糖化產生的葡萄糖發酵以生產醇。
11.權利要求1的方法，其中所述醇是乙醇。
12.權利要求11的方法，其中所述糖化和發酵的至少部分在相同反應混合物中同時發生。
13.權利要求1的方法，其進一步包括使通過液化和糖化產生的葡萄糖與葡萄糖異構 酶接觸以生產果糖。
14.權利要求1的方法，其中外源鈣不加入所述反應混合物中。
15.權利要求1的方法，其中所述反應混合物中的鈣濃度小于約8ppm。
16.權利要求14的方法，其進一步包括在不減少反應混合物中的鈣量的情況下使通過 液化和糖化產生的葡萄糖與葡萄糖異構酶接觸以產生果糖。
17.權利要求1的方法，其中所述AmyE多肽與SEQID NO 1的氨基酸序列具有至少 80%氨基酸序列同一性。
18.權利要求1的方法，其中所述AmyE多肽與SEQID NO 1的氨基酸序列具有至少 90%氨基酸序列同一性。
19.權利要求1的方法，其中所述AmyE多肽包括C末端淀粉結合結構域的缺失。
20.權利要求1的方法，其中所述AmyE多肽是具有α_淀粉酶活性和改善酶性能的至 少一種改變的特性的變體多肽，所述變體多肽包括與選自AmyE (SEQ ID NO 1)或AmyE截短變體(SEQ ID NO 2)的親本α -淀粉酶多肽 具有至少60%氨基酸序列同一性的氨基酸序列，和在選自下組的一個或多個位置的修飾:1，2，3，4，5，6，7，8，9，10，11，12，13，14，15，16， 17，18，19，20，21，22，23，24，25，26，27，28，29，30，31，32，33，34，35，36，37，38，39，40，41， 42，43，44，45，46，47，48，49，50，51，52，53，54，55，56，57，58，59，60，61，62，63，64，65，66， 67，68，69，70，71，72，73，74，75，76，77，78，79，80，81，82，83，84，85，86，87，88，89，90，91，.92,93,94,95,96,97,98,99,100，101，102，103，104，105，106，107，108，109，110，111，112，.113，114，115，116，117，118，119，121，122，123，124，125，126，127，128，129，130，131，132，.134，135，136，137，138，139，140，141，142，143，144，145，146，147，148，149，150，151，152，.153，154，155，156，157，158，159，160，161，162，163，164，165，166，167，168，169，170，171，.172，173，174，175，176，177，178，179，180，181，182，183，184，185，186，187，188，189，190，.191，192，193，194，195，196，197，198，199，200，201，202，203，204，205，206，207，208，209，.210,211,212,213，214，215，216，217，218，219，220，221，222，223，224，225，226，227,228,.229,230,231,232，233，234，235，236，237，238，239，240，241，242，243，244，245，246，247，.248，249，250，251，252，253，254，255，256，257，258，259，260，261，262，263，264，265，267，.268,270,271,272，273，274，275，276，277，278，279，280，281，282，283，284，285，286,287,.288，289，290，291，292，293，294，295，296，297，298，299，300，301，302，303，304，305,307,.308,309,310,311，312，313，314，315，316，317，318，319，320，321，322，323，324，325,326,.327，328，329，330，331，332，333，334，335，336，337，338，339，340，341，342，343，344，345，.346，347，348，349，350，351，352，353，354，355，356，357，358，359，360，361，362，363，364，.365，366，367，368，369，370，371，372，373，374，375，376，377，378，379，380，381，382,383,.384，385，386，387，388，389，390，391，392，393，394，395，396，397，398，399，400，401,402,.403,404,405,406，407，408，409，410，411，412，413，414，415，416，417，418，419，420,421,.422，423，424，和 425，其中所述修飾產生對于改善酶性能的至少一種特性具有大于1.0的性能指數(PI)的 變體多肽。
21.權利要求20的方法，其中所述修飾是選自以下的在一個或多個位置處親本多肽中 存在的一個或多個氨基酸殘基置換為不同的氨基酸殘基1A, 1C, ID, IE, IF, 1G, 1H, IK, 1M, IN, 1Q, 1R, IS, IT, IV, Iff, 1Y, 2A, 2C, 2D, 2E, 2F, 2G, 2H, 21，2K，2L,2M,2N,2P,2Q,2R,2S,2V,2ff, Ti, 3C,3D,3E,3F,3G,3H，31，3K,3L,3M,3N,3P,3Q, 3R,3S,3V,3ff,3Y,4C,4D，4E，4F,4G,4H,41,4K,4L,4M,4N,4Q,4S,4T,4V,4ff,4Y,5A,5C,5D, 5E,5F,5G,5H,51，5K，5L,5N,5R,5V,5ff,5Y,6C,6D,6E,6H,6K,6L,6Μ,6Ν,6P,6Q,6R,6S,6T, 6V,6ff,7A,7C,7D,7E,7F,7G,7H，71，7L,7M,7N,7P,7Q,7R,7S,7T,7ff, Ti, 8A,8C,8E,8F,8G, 8H，81，8K,8L,8M,8N,8P,8Q,8R,8T,8V,8ff,8Y,9A,9C,9D,9E,9F,9H，91，9K,9M,9N,9P,9R, 9S，9T，9V，9W，9Y，10A,101，10L，10M,ΙΟΝ,10P,10Q,10S,10V，11A，1IF,11G，11H，11M，11S， 11V, llff, 11Y, 121，12M，12V, 13A, 13C, 13D, 13E, 13F, 13G, 131，13L, 13M, 13Q, 13T, 13V, 13W, 13Y, 14C, 14F, 14G,14M,14N,14S,14T,14V,15A,15F,16A,16D,16E,16F,16G, 16H, 161，16L， 16M,16Q,16S,16T,16V,17A,17F,171，17M,17Q,17Y,18A,18C,18D,18E,18G,18H,18M, 18N, 18Q,18R,18T,19A,19C,19H,19L,19M,19N,19S,19W,19Y，20A，20C，20D，20F，20G，20H，201， 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36F,36H,361，36K，36L36M,36N,36Q,36R，36S，36T,36Y,37L,.37M,37N,37V,38A,38C,38D,38E,38H,38L,38M,38N,38P,38V,39A,39C，39I，39L,39M,39N,.39P,39S,39V,40A,棚，40M,40N,40P,40Q,40T,40V,40W,41A,41C,41E，41G，41N,41S,41V,.42A,42L,42M,42P,42V,43A,43G,43H,43L,43M,43Q，43S,43T,43V,44A，44C，44D,44E,44F,.44G,44H,441，44K,44L,44M,44N,44P,44R,44S,44T,44V,44W,44Y,45A，45C，45F,45G,45H,.451，45L,45M,45N,45P,45Q，45S,45T,45Y,46A,46C,46D,46E,46F,46H，46I，46L,46M,46N,.46Q,46R,46S,46T,46V,46W,46Y,47A,47C,47D,47F,47G,47H,471，47K，47L，47N,47P,47R,.47S,47T,47V,47Y,48A,48C,48D,48E,48F,48H,481，48K,48L,48N,48P，48S，48T,48V,48W,.49A,49C,49D,49F,49G,49H,491，49K,49L,49P,49Q,49R,49S,49T,49V, 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422S，422V，422W,422Y,423A,423D,423G,423H,4231，423K,423P,423Q,423R,423T,423V, 423W,424D,424E,424G,4241，424K,424M,424N,424Q,424R,424S,424T，424V，424W,424Y, 425A,4251，425K,425L,425M,425S,425T,425V,425W,和 425Y。
22.權利要求20的方法，其中所述修飾為位置153上存在的氨基酸殘基置換為N、K或 F，并且與所述親本多肽比較，所述變體多肽顯示將麥芽糖和麥芽七糖底物轉化為葡萄糖的 能力增加。
23.權利要求20的方法，其中所述修飾是選自L142F、L142G、L142Q、L142S、L142W、 L142Y、A214I、A214V、S245Y、Q126F、Q126L、Q126P、Q126V、S131L 和 S254I 的置換。
24.權利要求20的方法，其中所述修飾是選自W60L、W60M、W60N、I100F、I100M、S105M、 S105W、G207A、T270A、T270E、T270L、T270N、T270V 和 T279A 的置換。
25.用于減少面包陳化的方法，其包括將足以減少陳化的AmyE多肽量加入面包面團 中，其中所述減少的陳化是相對于在用缺乏AmyE多肽的等價面包面團時觀察到的陳化量而言的。
全文摘要
本發明提供來自枯草芽孢桿菌的α-淀粉酶(AmyE)、其變體、編碼其的核酸、和包括該核酸的宿主細胞。公開使用AmyE或其變體的方法，包括淀粉的液化和/或糖化。這些方法可以產生對于乙醇生產或高果糖玉米糖漿生產有用的糖。在某些情況下，該淀粉酶可以在低pH、在缺乏鈣的情況下和/或在缺乏葡糖淀粉酶的情況下使用。
文檔編號C12N15/00GK102131924SQ200980129863
公開日2011年7月20日 申請日期2009年6月5日 優先權日2008年6月6日
發明者A·R·托波扎達, A·肖, C·A·里夸德特, D·A·埃斯特爾, L·華萊士, M·J·佩珀森, R·金, S·D·鮑爾, S·W·拉默, S-K·李, W·A·奎瓦斯 申請人:丹尼斯科美國公司