專利名稱：葡萄糖異構(gòu)酶突變體的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及分子生物學與生物技術(shù)領(lǐng)域，具體地說，涉及利用基因突變技術(shù)制備高活力、或高活力并耐熱的葡萄糖異構(gòu)酶突變體的方法、所獲得的突變體及其應(yīng)用。
背景技術(shù)：
葡萄糖異構(gòu)酶(Glucose isomerase，E.C.5.3.1.5，簡稱GI)、或稱木糖異構(gòu)酶(Xyloseisomerase)是戊糖發(fā)酵途徑的關(guān)鍵酶。該酶是最重要的酶制劑之一(Kaneko，et al.，BiosciBiotechnol Biochem，64940-947，2000)。食品工業(yè)使用該酶制備果葡糖漿。
酶法生產(chǎn)果葡糖漿，產(chǎn)品中果糖的含量取決于反應(yīng)的溫度。溫度越高，異構(gòu)反應(yīng)越趨向于果糖的生成。目前商用葡萄糖異構(gòu)酶主要取自密蘇里游動放線菌(Actinoplanesmissouriensis)、凝結(jié)芽孢桿菌(Bacillus coagulans)、或鏈霉菌(Streptomyces)。這些葡萄糖異構(gòu)酶在高溫(如高于65℃)下穩(wěn)定性差。因此，目前工業(yè)上在60℃左右制備果葡糖漿，產(chǎn)物中果糖的含量也就較低，一般不超過44％。含更高濃度果糖的果葡糖漿通常依賴層析分離方法生產(chǎn)，由此增加了生產(chǎn)成本。
世界各地的科學家對從耐熱菌中篩選并通過蛋白質(zhì)工程的方法培育耐熱、高催化活性的葡萄糖異構(gòu)酶進行了許多研究。如J.G.Zeikus及其合作者從Thermoanaerobacteriumsaccharolyticum、Thermotoga neapolitana等數(shù)種耐高溫菌中分離并研究了耐高溫葡萄糖異構(gòu)酶(見Lee et al.，Journal of General Microbiology，1391227-1234，1993；Vieille et al.，MethodsEnzymology，330215-24，2001；Lee et al.，Journal of General Microbiology，1391241-1243，1993；Scriprapundh et al.，Protein Engineering，13259-265，2000；Scriprapundh et al.，Protein Engineering，16683-690，2003；Zeikus et al.，US Patent NO 5,656,497)。這些或其它來源的耐高溫葡萄糖異構(gòu)酶之耐熱性能均不如人意，且活力較低，尚未見用于工業(yè)化生產(chǎn)。因此，目前仍存在高活力、或高活力并耐熱的葡萄糖異構(gòu)酶的需求。

發(fā)明內(nèi)容
本發(fā)明利用遺傳工程和蛋白質(zhì)工程技術(shù)對來源于T.saccharolyticum的葡萄糖異構(gòu)酶進行改良，獲得了一系列高催化活性的葡萄糖異構(gòu)酶突變體，適宜生產(chǎn)含高果糖的果葡糖漿。
本發(fā)明的目的在于提供高催化活性的葡萄糖異構(gòu)酶突變體。本發(fā)明的目的還在于提供使用本發(fā)明所述的葡萄糖異構(gòu)酶突變體直接生產(chǎn)果糖含量等于或高于55％的果葡糖漿。本發(fā)明的目的還在于提供使用本發(fā)明所述的葡萄糖異構(gòu)酶突變體生產(chǎn)果糖含量低于55％的果葡糖漿。
為實現(xiàn)本發(fā)明的上述目的，本發(fā)明人進行了大量深入的實驗，通過對T.saccharolyticum葡萄糖異構(gòu)酶基因進行定點突變，隨后在MacConkey培養(yǎng)基上篩選，從而取得一系列高催化活性、或具高催化活性并耐熱的葡萄糖異構(gòu)酶突變體。具體而言，利用本領(lǐng)域已知的技術(shù)，構(gòu)建含有親本葡萄糖異構(gòu)酶基因的載體質(zhì)粒，然后設(shè)定定點突變的位點以及突變后的氨基酸種類，再合成適當?shù)囊?，以所述的含親本葡萄糖異構(gòu)酶基因的載體質(zhì)粒為模板，PCR擴增DNA片段、裝配所擴增的DNA片段以及PCR擴增全長突變基因。通過將該全長突變基因克隆到適當?shù)妮d體上并轉(zhuǎn)化適當?shù)乃拗骷毎?，?jīng)培養(yǎng)篩選出具有葡萄糖異構(gòu)酶活性的陽性克隆。最后從陽性克隆中提取質(zhì)粒DNA，進行DNA序列測定分析，以確定引入的突變。
在本發(fā)明制備葡萄糖異構(gòu)酶突變體的方法中，可采用任何適當?shù)妮d體。例如，適用的載體包括但不限于原核表達載體pGEMT-Easy，pRSET和pET21；包括但不限于真核表達載體pYD1和pYES2/GS；包括但不限于克隆載體pUC18/19和pBluscript-SK。
在本發(fā)明制備葡萄糖異構(gòu)酶突變體的方法中，所獲得的葡萄糖異構(gòu)酶突變體基因可以在原核細胞或真核細胞胞內(nèi)表達，也可采用本領(lǐng)域已知的任何其它適當方法實現(xiàn)在原核細胞或真核細胞胞外表達。
在本發(fā)明制備葡萄糖異構(gòu)酶突變體的方法中，所述載體的微生物宿主細胞為原核細胞或真核細胞。所述原核微生物包括但不限于大腸桿菌、凝結(jié)芽孢桿菌、枯草芽胞桿菌、巨大芽胞桿菌(如巨大芽胞桿菌BP931)、T.saccharolyticum和鏈霉菌(如Streptomyces diastaticusM1033)。所述真核微生物包括但不限于釀酒酵母和畢赤巴斯德酵母(如畢赤巴斯德酵母GS115/9891)。
本發(fā)明獲得了一種葡萄糖異構(gòu)酶突變體，其特征在于以序列表中序列2所示氨基酸序列為參考序列，至少存在一個氨基酸的差異，并且以D-葡萄糖為底物其具有比親本至少高出50-150％、優(yōu)選至少高出150-250％、更優(yōu)選至少高出250％的葡萄糖異構(gòu)酶催化活性。優(yōu)選的是，以序列表中的序列2為參考序列，具有至少一個選自第139位的色氨酸到其他19個天然氨基酸、第182位的精氨酸到其他19個天然氨基酸、第187位的苯丙氨酸到其他19個天然氨基酸和第299位的蘇氨酸到其他19個天然氨基酸的突變，并且以D-葡萄糖為底物其具有比親本高出至少50％的葡萄糖異構(gòu)酶催化活性。更優(yōu)選所述第139位的色氨酸突變?yōu)橘嚢彼?Lys)、或絲氨酸(Ser)、或半胱氨酸(Cys)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或天冬酰胺(Asn)、或苯丙氨酸(Phe)；所述第182位的精氨酸突變?yōu)楦彼?Pro)、或絲氨酸(Ser)、或丙氨酸(Ala)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或纈氨酸(Val)；所述第187位的苯丙氨酸突變?yōu)楦拾彼?Gly)、或絲氨酸(Ser)、或丙氨酸(Ala)、或脯氨酸(Pro)；和/或所述第299位的蘇氨酸突變?yōu)楫惲涟彼?Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或蛋氨酸(Met)、或谷氨酸(Glu)、或谷氨酰胺(Gln)。最優(yōu)選的是，本發(fā)明的葡萄糖異構(gòu)酶突變體包含下文表2中所列的那些突變的氨基酸序列。
這些突變體具有高的催化活性、并耐熱和對較低pH具有耐受性。例如，在本發(fā)明獲得的一系列突變體中，一個具有四個點突變的突變體MGI-4的比活性較親本高651％，且在80℃反應(yīng)24小時后仍保持50％或以上的活力，且在pH5.0處的活性相當于其在最佳pH(pH7.0)時的活性的80％。另一個具有三個點突變的突變體MGI-3的比活性較親本高412％，且在80℃反應(yīng)16小時后仍保持50％或以上的活力，且在pH5.0處的活性相當于其在最佳pH(pH7.0)時的活性的70％。
本發(fā)明獲得的高催化活性或高催化活性并耐熱的葡萄糖異構(gòu)酶突變體可用于直接生產(chǎn)果糖含量等于或高于55％的果葡糖漿，或用于生產(chǎn)果糖含量低于55％的果葡糖漿。所述的葡萄糖異構(gòu)酶突變體可以以未經(jīng)純化粗酶形式使用，也可以是經(jīng)部分純化的或完全純化的形式。如果需要，還可利用本領(lǐng)域已知的固化技術(shù)將本發(fā)明葡萄糖異構(gòu)酶突變體制成固相酶或固相細胞形式的固化酶。
定義本申請文本中所用的術(shù)語“親本”系指來自Thermoanaerobacterium saccharolyticum ATCC49915的葡萄糖異構(gòu)酶，其核苷酸序列如序列1所示，氨基酸序列如序列2所示。本發(fā)明中親本基因的核苷酸序列與公布的T.saccharolyticum葡萄糖異構(gòu)酶的基因序列(Lee et al.，Journal of General Microbiology，1391227-1234，1993；GenBank L09699)相比有兩個核苷酸的差異，即與基因庫(GenBank L09699)的核苷酸序列相比，本發(fā)明中親本基因第241-242位為GC，相應(yīng)的第81位氨基酸序列為丙氨酸(Ala)，GenBankL09699對應(yīng)處、第241-242位核苷酸序列為CG，相應(yīng)的第81位氨基酸序列為精氨酸(Arg)。
本申請文本中所用的術(shù)語“參考序列”，當其為核苷酸序列時，系指序列表中的序列1，當其為氨基酸序列時，是指序列表中的序列2。在將參考序列和突變的葡萄糖異構(gòu)酶序列進行排序比較時，可以手工進行，也可以用計算機進行(目前有許多可供利用的計算機軟件，例如CLUSTALW，AMAS，DIALIGN程序等)。
本申請文本中所用的術(shù)語“位點”或“第x位”是指當本發(fā)明的葡萄糖異構(gòu)酶突變體的序列與參考序列的排序?qū)Ρ冗_最大同源性時，被比較的葡萄糖異構(gòu)酶突變體的序列與參考序列不匹配的各核苷酸或氨基酸所對應(yīng)的參考序列中的核苷酸或氨基酸位置。
本申請文本所用的術(shù)語“葡萄糖異構(gòu)酶突變體”是指一種以序列表中序列2所示氨基酸序列為參考序列，在第139位、第182位、第187位、第299位具有至少一個氨基酸的差異，并且以D-葡萄糖為底物生成果糖時其具有比親本高出至少50％的葡萄糖異構(gòu)酶催化活性的酶。因此，在本專利申請中，所述葡萄糖異構(gòu)酶突變體包括具有序列4所示氨基酸序列的突變體、序列4的保守取代形式、增加或缺失一個或幾個氨基酸的形式、氨基端截斷的形式、羧基端截斷的形式、以及序列4的部分或全部串聯(lián)重復(fù)形式。
在本申請文本所用的氨基酸三字母或單字母表達方式，采用IUPAC規(guī)定的氨基酸代碼(Eur.J.Biochem.，1389-37，1984)。
附圖的簡要說明

圖1葡萄糖異構(gòu)酶突變體MGI-4聚丙烯酰胺凝膠電泳圖。其中從左至右的四條泳道依次為蛋白分子量標準、粗提蛋白、牛血清白蛋白、部分純化的葡萄糖異構(gòu)酶突變體MGI-4。(粗提蛋白、部分純化的葡萄糖異構(gòu)酶突變體MGI-4的制備見實例10)圖2葡萄糖異構(gòu)酶突變體MGI-4在80℃轉(zhuǎn)化D-葡萄糖至果糖之轉(zhuǎn)化率。其中黑色柱代表葡萄糖剩余量，黑色柱下方的白色柱代表果糖的生成量。該圖顯示出在反應(yīng)的頭兩個小時內(nèi)果糖的生成量幾乎與反應(yīng)時間呈線性關(guān)系增長，在隨后的兩個小時內(nèi)，果糖生成量的速度減緩。
圖3親本葡萄糖異構(gòu)酶與葡萄糖異構(gòu)酶突變體在80℃的熱穩(wěn)定。其中TS表示親本葡萄糖異構(gòu)酶，MGI-2，MGI-3和MGI-4分別代表具有兩個、三個和四個突變位點的葡萄糖異構(gòu)酶突變體，具體請參見實施例6-8。
圖4pH對親本葡萄糖異構(gòu)酶及葡萄糖異構(gòu)酶突變體的影響。其中TS表示親本葡萄糖異構(gòu)酶，MGI-2，MGI-3和MGI-4分別代表具有兩個、三個和四個突變位點的葡萄糖異構(gòu)酶突變體，具體請參見實施例6-8。
具體實施例方式下列實施例僅用于說明本發(fā)明而不應(yīng)視為限定本發(fā)明的范圍。實施例中未注明具體條件者，按常規(guī)條件或制造商建議的條件進行。
實施例1親本基因的擴增及pGEMT-TS的構(gòu)建根據(jù)基因庫(GenBank L09699)基因序列設(shè)計引物T1和T2(見表1)。利用引物對T1和T2從T.saccharolyticum ATCC 49915(購自ATCC，USA)中擴增葡萄糖異構(gòu)酶親本基因。
擴增條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1，400nM引物T2，1.5U Pfu DNA聚合酶(Promega，USA)，用接種環(huán)挑取少許T.saccharolyticum菌體，再用無菌水調(diào)反應(yīng)體積至50μl。
PCR擴增反應(yīng)程序為95℃ 3分鐘，40圈循環(huán)95℃ 50秒、50℃ 30秒和72℃ 1分鐘，最后72℃ 10分鐘。將擴增的產(chǎn)物(長約1.5kb)連接至載體pGEMT-Easy，得質(zhì)粒pGEMT-TS。經(jīng)DNA測序確定親本葡萄糖異構(gòu)酶的核苷酸序列為序列1，相應(yīng)的氨基酸序列為序列2。與基因庫(GenBank L09699)的核苷酸序列相比，本發(fā)明中親本基因第241-242位為GC，相應(yīng)的第81位氨基酸序列為丙氨酸(Ala)。GenBankL09699對應(yīng)處、第241-242位核苷酸序列為CG，相應(yīng)的第81位氨基酸序列為精氨酸(Arg)。
實施例2葡萄糖異構(gòu)酶位點139之定點突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989和White et al.(PCR Protocolcurrentmethods and applications.Totowa，N.J.Humana Press，1993)之描述。
以質(zhì)粒pGEMT-TS(見實施例1)為模板，設(shè)計引物對139FF和139FR(見表1)，將親本氨基酸序列中第139位點的Trp(W)突變?yōu)镻he(F)，獲得突變體MGI-W139F。引物對T1和T2見利用引物對T1和139FR，擴增T1FR片段；利用引物對139FF和T2，擴增FFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物139FR或400nM引物139FF和400nM引物T2，1.5U Pfu DNA聚合酶(Promega，USA)，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit(QIAGEN，German)回收，得到T1FR片段和FFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段與20ng FFT2片段，用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquickExtraction Gel Kit回收，得到全長突變基因MGI-W139F。將MGI-W139F與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-W139F。將質(zhì)粒pGEMT-MGI-W139F轉(zhuǎn)入感受態(tài)細菌細胞E.coliHB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-W139F DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-W139F氨基酸序列見序列表序列5。
按照類似步驟構(gòu)建突變體MGI-W139K、MGI-W139S、MGI-W139C、MGI-W139I、MGI-W139T和MGI-W139N，所用引物見表1。所得諸突變體氨基酸序列見序列表序列6-11。
實施例3葡萄糖異構(gòu)酶位點182之定點突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrentmethods and applications.Totowa，N.J.Humana Press，1993)之描述。
以質(zhì)粒pGEMT-TS(見實施例1)為模板，設(shè)計引物對182AF和182AR(見表1)，將親本氨基酸序列中第182位點的Arg(R)突變?yōu)锳la(A)，獲得突變體MGI-R182A。引物T1和T2見利用引物對T1和182AR，擴增T1AR片段；利用引物對182AF和T2，擴增AFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM (NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物182AR或400nM引物182AF和400nM引物T2，1.5U Pfu DNA聚合酶，20ngpGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1AR片段和AFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1AR片段與20ng AFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI-R182A。將MGI-R182A與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-R182A。將質(zhì)粒pGEMT-MGI-R182A轉(zhuǎn)入感受態(tài)細菌細胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-R182A DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-R182A的氨基酸序列見序列表序列12。
按照類似步驟構(gòu)建突變體MGI-R182P、MGI-R182S、MGI-R182I、MGI-R182T和MGI-R182V，所用引物見表1。所得諸突變體的氨基酸序列見序列表序列13-17。
實施例4葡萄糖異構(gòu)酶位點187之定點突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrentmethods and applications.Totowa，N.J.Humana Press，1993)之描述。
以質(zhì)粒pGEMT-TS(見實施例1)為模板，設(shè)計引物對187SF和187SR(見表1)，將親本氨基酸序列中第187位點的Phe(F)突變?yōu)镾er(S)，獲得突變體MGI-F187S。引物T1和T2見實施例1。
利用引物對T1和187SR，擴增T1SR片段；利用引物對187SF和T2，擴增SFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物187SR或400nM引物T2和400nM引物187SF，1.5U Pfu DNA聚合酶，20ngpGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1SR片段和SFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1SR片段與20ng SFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI-F187S。將MGI-F187S與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-F187S。將質(zhì)粒pGEMT-MGI-F187S轉(zhuǎn)入感受態(tài)細菌細胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-F187S DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-F187S的氨基酸序列見序列表序列18。
按照類似步驟構(gòu)建突變體MGI-F187G、MGI-F187P和MGI-F187A，所用引物見表1。所得諸突變體的氨基酸序列見序列表序列19-21。
實施例5葡萄糖異構(gòu)酶位點299之定點突變定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrentmethods and applications.Totowa，N.J.Humana Press，1993)之描述。
以質(zhì)粒pGEMT-TS(見實施例1)為模板，設(shè)計引物對299QF和299QR(見表1)，將親本氨基酸序列中第299位點的Thr(T)突變?yōu)镚ln(Q)，獲得突變體MGI-T299Q。引物T1和T2見利用引物對T1和299QR，擴增T1QR片段；利用引物對299QF和T2，擴增QFT2片段。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物299QR或400nM引物299QF和400nM引物T2，1.5U Pfu DNA聚合酶，20ngpGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到T1QR片段和QFT2片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1QR片段與20ng QFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI-T299Q。將MGI-T299Q與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-T299Q。將質(zhì)粒pGEMT-MGI-T299Q轉(zhuǎn)入感受態(tài)細菌細胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-T299Q DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-T299Q的氨基酸序列見序列表序列22。
按照類似步驟構(gòu)建突變體MGI-T299I、MGI-T299Y、MGI-T299C、MGI-T299M和MGI-T299E，所用引物見表1。所得諸突變體的氨基酸序列見序列表序列23-27。
實施例6葡萄糖異構(gòu)酶雙突變組合MGI-2、MGI-2AQ和MGI-2FQ之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrentmethods and applications.Totowa，N.J.Humana Press，1993)之描述。
按實施例2擴增和回收T1FR片段，按實施例3擴增和回收AFT2片段。用引物對139FF(如實施例2)和182AR(如實施例3)擴增和回收FFAR片段。FFAR片段擴增反應(yīng)條件為20mMTris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 139FF和400nM 182AR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到FFAR片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段，20ng FFAR片段和20ng AFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick DNA回收試劑盒，得到全長突變基因MGI-2。將MGI-2與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-2。將質(zhì)粒pGEMT-MGI-2轉(zhuǎn)入感受態(tài)細菌細胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-2 DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-2的氨基酸序列見序列表序列28。
按照類似步驟構(gòu)建雙突變體MGI-2AQ和MGI-2FQ。突變體MGI-2AQ所用引物對為T1和182AR、182AF和299QR、299QF和T2(見表1)，所得突變體序列含有R182A和T299Q的雙突變。突變體MGI-2FQ所用引物對為T1和139AR、139AF和299QR、299Q和FT2(見表1)，所得突變體序列含有T299Q和W139F，所得突變體的氨基酸序列見序列表序列29-30。
實施例7葡萄糖異構(gòu)酶三突變組合MGI-3之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrentmethods and applications.Totowa，N.J.Humana Press，1993)之描述。
按實施例2擴增和回收T1FR片段，按實施例5擴增和回收QFT2片段，按實施例6擴增和回收FFAR片段。用引物對182AF(如實施例3)和299QR(如實施例5)擴增和回收AFQR片段。AFQR片段擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM182AF和400nM 299QR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到AFQR片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段，20ngFFAR片段，20ng AFQR片段和20ng QFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI-3。將MGI-3與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-3。將質(zhì)粒pGEMT-MGI-3轉(zhuǎn)入感受態(tài)細菌細胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-3 DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-3序列含有W139F、R182A和T299Q的三突變。MGI-3的氨基酸序列見序列表序列31。
實施例8葡萄糖異構(gòu)酶四突變組合MGI-4之構(gòu)建定點突變技術(shù)參考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrentmethods and applications.Totowa，N.J.Humana Press，1993)之描述。
按實施例2擴增和回收T1FR片段，按實施例5擴增和回收QFT2片段，按實施例6擴增和回收FFAR片段。用引物對182AF(如實施例3)和187SR(如實施例4)擴增AFSR片段。AFSR片段擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mMMgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM182AF和400nM 187SR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到AFSR片段。用引物對187SF(如實施例4)和299QR(如實施例5)擴增SFQR片段。SFQR片段擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mMMgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM187SF和400nM 299QR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃ 3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到SFQR片段。然后擴增全長基因。擴增反應(yīng)條件為20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段，20ngFFAR片段，20ng AFSR片段，20ng SFQR片段和20ng QFT2片段，再用無菌水調(diào)反應(yīng)體積至50μl。PCR擴增反應(yīng)程序為95℃ 3分鐘，35圈循環(huán)95℃ 50秒、52℃ 30秒和72℃3分鐘，最后72℃ 5分鐘。經(jīng)1％瓊脂糖膠電泳分離并用QIAquick Extraction Gel Kit回收，得到全長突變基因MGI-4。將MGI-4與載體pGEMT-Easy連接，得質(zhì)粒pGEMT-MGI-4。將質(zhì)粒pGEMT-MGI-3轉(zhuǎn)入感受態(tài)細菌細胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上篩選出具葡萄糖異構(gòu)酶活性的克隆。從克隆中提取質(zhì)粒pGEMT-MGI-4 DNA，經(jīng)DNA測序確定引入的點突變無誤。MGI-4序列含有W139F、R182A、F187S和T299Q的四突變。MGI-4的氨基酸序列見序列表序列32。
擴增親本葡萄糖異構(gòu)酶(親本)與實施例1-8所葡萄糖異構(gòu)酶突變體所用的引物如下表1所列
表1

實施例9親本葡萄糖異構(gòu)酶的提取與純化葡萄糖異構(gòu)酶的提取與純化主要參考Lee et al.，Journal of General Microbiology，1391227-1234(1993)。
將含親本葡萄糖異構(gòu)酶基因的質(zhì)粒pGEMT-TS轉(zhuǎn)化感受態(tài)細菌細胞E.coli HB101，在MacConkey平板(含1％D-木糖和50mg/L氨芐青霉素)上、37℃培養(yǎng)36小時。接種單個克隆在5ml LB液體培養(yǎng)基(含50mg/L氨芐青霉素)中培養(yǎng)16小時。離心收集菌體，并懸浮于1ml20mM磷酸鈉緩沖液(pH6.5)中，加CoCl2和MgCl2至終濃度分別為250μM和5mM。然后用超聲波裂解細菌細胞。離心(10℃，17,800g，15分鐘)并收集上清液為粗提蛋白。粗提蛋白經(jīng)80℃熱處理10分鐘后，離心(10℃，17,800g，15分鐘)去沉淀，即為部分純化的葡萄糖異構(gòu)酶，可用于酶活性的測定和制備果葡糖漿。
實施例10葡萄糖異構(gòu)酶突變體的提取與純化葡萄糖異構(gòu)酶突變體MGI-4的提取與純化與實施例9同，惟所用質(zhì)粒為pGEMT-MGI-4。部分純化的葡萄糖異構(gòu)酶見圖1。其它葡萄糖異構(gòu)酶突變體也照實施例9所述提取與純化。
實施例11親本葡萄糖異構(gòu)酶活性的測定配底物溶液A含1.0M的D-葡萄糖、20mM磷酸鈉緩沖溶液(pH6.5)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至6.5。取底物溶液A 90μl，然后加入10μl按實施例9制備的葡萄糖異構(gòu)酶。反應(yīng)于80℃進行10分鐘。將反應(yīng)物置冰上以終止反應(yīng)。反應(yīng)產(chǎn)物D-果糖通過半胱氨酸-咔唑法測定。測定方法參見Dische et al.，J.Biol.Chem，192583-587，1951)以及Nakamura，Agr.Biol.Chem.32701-706，1968)。用CoomassiePlusProtein Assay Reagent Kit(PIERCE，USA)并結(jié)合SDS聚丙烯酰胺凝膠電泳測定酶蛋白濃度。一單位酶比活性定義為在上述條件下每分鐘轉(zhuǎn)化一微摩爾D-葡萄糖為果糖所需之酶量。下列表2顯示親本葡萄糖異構(gòu)酶之比活性。
實施例12葡萄糖異構(gòu)酶突變體活性的測定葡萄糖異構(gòu)酶突變體MGI-4活性測定與實施例11同。其它葡萄糖異構(gòu)酶突變體的活性也照實施例11所述測定。下表2顯示諸葡萄糖異構(gòu)酶突變體與親本葡萄糖異構(gòu)酶(親本)比活性之差異。
表2親本葡萄糖異構(gòu)酶與葡萄糖異構(gòu)酶突變體比活性之差異

實施例13D-葡萄糖轉(zhuǎn)化為果糖轉(zhuǎn)化率之測定主要參考Kaneko et al.，Biosci Biotechnol Biochem，64940-947，2000。
配底物溶液B含50％(W/V)D-葡萄糖、20mM磷酸鈉緩沖溶液(pH6.5)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至6.5。取底物溶液B 60μl，然后加入40μl按實施例10制備的葡萄糖異構(gòu)酶突變體MGI-4。80℃反應(yīng)4小時后，加入100μl 20％三氯醋酸終止反應(yīng)，離心(10℃，17,800g，15分鐘)并收集上清液。取10μl上清液稀釋100倍后，用HPLC測定產(chǎn)生的果糖含量。HPLC測定用色譜柱為μBONDAPAK NH2 SS COL 3.9X300(Waters，USA)，流動相為乙腈-水(85∶15)，流速0.5毫升/分鐘。探測器為ELSD 500。進樣量為10μl。圖2顯示葡萄糖異構(gòu)酶突變體MGI-4將D-葡萄糖轉(zhuǎn)化至果糖之轉(zhuǎn)化率。
實施例14親本葡萄糖異構(gòu)酶熱穩(wěn)定性測定置按實施例9獲得的部分純化的親本葡萄糖異構(gòu)酶于7管1.5ml離心管中。每管離心管分別加入200μl酶液，并加入200μl礦物油。將離心管置于80℃水浴中，0小時、2小時、4小時、8小時、16小時、32小時、72小時后分別取出一管酶液，離心(10℃，17,800g，20分鐘)后，取上清液，按實施例11所述、測定葡萄糖異構(gòu)酶殘余蛋白和殘余比活性。圖3顯示親本葡萄糖異構(gòu)酶在80℃的熱穩(wěn)定。
實施例15葡萄糖異構(gòu)酶突變體熱穩(wěn)定性測定葡萄糖異構(gòu)酶突變體MGI-2、MGI-3、或MGI-4熱穩(wěn)定性測定與實施例14同。圖3顯示葡萄糖異構(gòu)酶突變體MGI-2、MGI-3、或MGI-4在80℃的熱穩(wěn)定。如圖所示，親本葡萄糖異構(gòu)酶的活性在80℃的半衰期為4.0小時；MGI-2的活性在80℃的半衰期為4.4小時；MGI-3的活性在80℃的半衰期為21小時；MGI-4的活性在80℃的半衰期為25.5小時。
實施例16pH對親本葡萄糖異構(gòu)酶的影響配制底物溶液C含1.0M的D-葡萄糖、20mM醋酸鈉緩沖溶液(pH4.0)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至4.0。配制底物溶液D含1.0M的D-葡萄糖、20mM醋酸鈉緩沖溶液(pH4.5)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至4.5。配制底物溶液E含1.0M的D-葡萄糖、20mM醋酸鈉緩沖溶液(pH5.0)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至5.0。配制底物溶液F含1.0M的D-葡萄糖、20mM醋酸鈉緩沖溶液(pH5.5)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至5.5。配制底物溶液G含1.0M的D-葡萄糖、20mM磷酸鈉緩沖溶液(pH6.0)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至6.0。配制底物溶液H含1.0M的D-葡萄糖、20mM磷酸鈉緩沖溶液(pH6.5)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至6.5。配制底物溶液I含1.0M的D-葡萄糖、20mM磷酸鈉緩沖溶液(pH7.0)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至7.0。配制底物溶液J含1.0M的D-葡萄糖、20mM磷酸鈉緩沖溶液(pH7.5)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至7.5。配制底物溶液K含1.0M的D-葡萄糖、20mM磷酸鈉緩沖溶液(pH8.0)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至8.0。配制底物溶液L含1.0M的D-葡萄糖、20mM Tris-HCl緩沖溶液(pH8.5)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至8.5。配制底物溶液M含1.0M的D-葡萄糖、20mM Tris-HCL緩沖溶液(pH9.0)、終濃度為250μM之CoCl2和終濃度為5mM之MgCl2，調(diào)pH至9.0。取反應(yīng)管11管，每管100μl反應(yīng)液包括按實施例9制備的D-葡萄糖異構(gòu)酶10μl以及底物溶液C、或底物溶液D、或底物溶液E、或底物溶液F、或底物溶液G、或底物溶液H、或底物溶液I、或底物溶液J、或底物溶液K、或底物溶液L、或底物溶液M 90μl。反應(yīng)于80℃進行10分鐘。將反應(yīng)物置冰上以終止反應(yīng)。反應(yīng)產(chǎn)物D-果糖的測定與實施例11同。圖4顯示pH對親本葡萄糖異構(gòu)酶的影響。
實施例17pH對葡萄糖異構(gòu)酶突變體的影響pH對葡萄糖異構(gòu)酶突變體MGI-2、MGI-3、或MGI-4的影響的測定與實施例16同。圖4顯示pH對葡萄糖異構(gòu)酶突變體MGI-2、MGI-3、或MGI-4的影響。以親本葡萄糖異構(gòu)酶在最佳pH(pH7.0)的比活性為100％，葡萄糖異構(gòu)酶突變體MGI-2、MGI-3、和MGI-4在pH5.0-9.0的范圍內(nèi)均保有大部分活性。如在pH5.0處，葡萄糖異構(gòu)酶突變體MGI-2的相對比活性為365％，為其在最佳pH(pH7.0)的相對比活性的72％；在pH5.0處，葡萄糖異構(gòu)酶突變體MGI-3的相對比活性是370％，為其在最佳pH(pH7.0)的相對比活性的70％；在pH5.0處，葡萄糖異構(gòu)酶突變體MGI-4的相對比活性為600％，為其在最佳pH(pH7.0)的相對比活性的80％。
實施例18親本葡萄糖異構(gòu)酶生化常數(shù)測定配制底物溶液N含磷酸鈉-MgCl2-CoCl2緩沖溶液[20mM磷酸鈉(pH6.5)、250μMCoCl2、5mM MgCl2]和2MD-葡萄糖，調(diào)pH至6.5。用同樣的磷酸鈉-MgCl2-CoCl2緩沖溶液將底物溶液N稀釋至含D-葡萄糖1.8M、1.6M、1.4M、1.2M、1.0M、0.8M、0.6M、0.4M、0.2M、0.1M、0.05M和0.025M。取10μl部分純化的親本葡萄糖異構(gòu)酶(見實施例9)與90μl不同濃度的底物溶液N在65℃或80℃反應(yīng)10分鐘。然后，按米-曼(Michaelis-Menten)方程并采用Lineweaver-Burk雙倒數(shù)作圖法，測定米氏常數(shù)km、飽和底物濃度下的最大反應(yīng)速率Vmax與轉(zhuǎn)換數(shù)Kcat。其結(jié)果如下表3所列。
實施例19葡萄糖異構(gòu)酶突變體MGI-4生化常數(shù)之測定葡萄糖異構(gòu)酶突變體MGI-4生化常數(shù)測定與實施例18同。表3顯示葡萄糖異構(gòu)酶突變體MGI-4與親本葡萄糖異構(gòu)酶生化常數(shù)之比較。
表3親本葡萄糖異構(gòu)酶(親本)與葡萄糖異構(gòu)酶突變體MGI-4(MGI-4)之生化常數(shù)

實施例20 固相化葡萄糖異構(gòu)酶突變體MGI-4主要參考Ge，et al.，Appl.Biochem.Biotechnol.6957-69，1998。具體步驟是取固相酶載體氯化聚苯乙烯乙基胺(成都化工研究所提供)顆粒100克，加10mM磷酸鹽緩沖液(pH8.0)1升和部分提純之葡萄糖異構(gòu)酶突變體MGI-4 8克(按實施例10所述)，室溫(22℃)攪拌(60～120轉(zhuǎn)/分)反應(yīng)18小時。抽濾收集固相酶，以水洗滌三次，得固相酶107克。固相酶活力測定如實施例11所述，惟所用酶為如上制備的固相酶0.01克，測得該固相酶的活性為820單位/克。
實施例21固定化含葡萄糖異構(gòu)酶突變體MGI-4的細胞在含50mg/L氨芐青霉素的LB培養(yǎng)液中過夜生長攜有pGEMT-MGI-4的E.coli HB101至OD600為7。離心收集菌體細胞。取菌體細胞10克，加3％海藻酸鈉溶液20克，充分攪拌混合，然后將菌體細胞從管徑約0.5mm的針頭滴入500ml 2％氯化鈣溶液中，室溫反應(yīng)1小時，用蒸餾水浸泡洗滌3次，每次約半小時，得固定化細胞約30克。固定化細胞活力測定如實施例11所述，惟所用酶為如上制備的固定化細胞0.01克，測得固定化細胞的活性為370單位/克。
本發(fā)明不受上述具體文字描述的限制，本發(fā)明可在權(quán)利要求書所概括的范圍內(nèi)做各種改變。這些改變均在本發(fā)明的范圍之內(nèi)。
序列表<110>百瑞全球有限公司(BioRight Worldwide Company Ltd.)<120>葡萄糖異構(gòu)酶突變體<130>002FPI0007C1<150>CN 200410042773.2<151>2004-05-26<160>32<170>PatentIn version 3.2<210>1<211>1320<212>DNA<213>Thermoanaerobacterium saccharolyticum<400>1atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaataat60ccttattcct ttaaatttta caatccagag gaagtaatcg atggcaagac gatggaggag120catctccgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt180ggcaaggcta ctatgcaaag accatggaac cactacacag atcctatgga tatagcgaaa240gcaagggtag aagcagcatt tgagtttttt gataagataa atgcaccttt cttctgcttc300catgataggg atattgcccc tgaaggagat actcttagag agacaaacaa aaacttagat360acaatagttg ctatgataaa ggattactta aagaccagca agacaaaagt tttgtggggt420accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct480gacgtttttg catattctgc agcgcaagtc aaaaaagccc ttgagattac taaggagctt540ggccgcgaaa actacgtatt ttggggtgga agagaagggt acgagacgct tctcaataca600gatatggagt tagagcttga taactttgca agatttttgc acatggctgt tgactatgca660aaggaaatcg gctttgaagg tcagttcttg attgagccga agccaaagga gcctacaaaa720catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgaccttgac780aaatatttca aagtaaatat cgaagcaaac catgcgacat tggcattcca cgacttccaa840catgagctaa gatacgccag aataaacggt gtattaggat caattgacgc aaatacaggc900gacatgcttt tgggatggga tacggaccag ttccctacag atatacgcat gacaacgctt960gctatgtatg aagtcataaa gatgggtgga tttgacaaag gtggccttaa ctttgatgca1020aaagtaagac gtgcttcatt tgagccagaa gatcttttct taggtcacat agcaggaatg1080gatgcttttg caaaaggctt taaagttgct tacaagcttg tgaaagatgg cgtatttgac1140aagttcatcg aagaaagata cgcaagctac aaagaaggca ttggcgctga tattgtaagc1200ggtaaagctg acttcaagag ccttgaaaag tatgcattag agcacagcca gattgtaaac1260aaatcaggca gacaagagct attagaatca atcctaaatc agtatttgtt tgcagaataa1320<210>2<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>2Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>3<211>1320<212>DNA<213>Thermoanaerobacterium saccharolyticum<220>
<221>misc_feature<222>(415)..(417)<223>″n″代表a，t，g或c，并且″nnn″代表編碼除色氨酸之外的任何氨基酸的密碼子。
<220>
<221>misc_feature<222>(544)..(546)<223>″n″代表a，t，g或c，并且″nnn″代表編碼除精氨酸之外的任何氨基酸的密碼子。
<220>
<221>misc_feature<222>(559)..(561)<223>″n″代表a，t，g或c，并且″nnn″代表編碼除苯丙氨酸之外的任何氨基酸的密碼子。
<220>
<221>misc_feature<222>(895)..(897)<223>″n″代表a，t，g或c，并且″nnn″代表編碼除蘇氨酸之外的任何氨基酸的密碼子。
<400>3atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaataat60ccttattcct ttaaatttta caatccagag gaagtaatcg atggcaagac gatggaggag120catctccgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt180ggcaaggcta ctatgcaaag accatggaac cactacacag atcctatgga tatagcgaaa240gcaagggtag aagcagcatt tgagtttttt gataagataa atgcaccttt cttctgcttc300catgataggg atattgcccc tgaaggagat actcttagag agacaaacaa aaacttagat360acaatagttg ctatgataaa ggattactta aagaccagca agacaaaagt tttgnnnggt420accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct480gacgtttttg catattctgc agcgcaagtc aaaaaagccc ttgagattac taaggagctt540ggcnnngaaa actacgtann ntggggtgga agagaagggt acgagacgct tctcaataca600gatatggagt tagagcttga taactttgca agatttttgc acatggctgt tgactatgca660aaggaaatcg gctttgaagg tcagttcttg attgagccga agccaaagga gcctacaaaa720
catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgaccttgac780aaatatttca aagtaaatat cgaagcaaac catgcgacat tggcattcca cgacttccaa840catgagctaa gatacgccag aataaacggt gtattaggat caattgacgc aaatnnnggc900gacatgcttt tgggatggga tacggaccag ttccctacag atatacgcat gacaacgctt960gctatgtatg aagtcataaa gatgggtgga tttgacaaag gtggccttaa ctttgatgca1020aaagtaagac gtgcttcatt tgagccagaa gatcttttct taggtcacat agcaggaatg1080gatgcttttg caaaaggctt taaagttgct tacaagcttg tgaaagatgg cgtatttgac1140aagttcatcg aagaaagata cgcaagctac aaagaaggca ttggcgctga tattgtaagc1200ggtaaagctg acttcaagag ccttgaaaag tatgcattag agcacagcca gattgtaaac1260aaatcaggca gacaagagct attagaatca atcctaaatc agtatttgtt tgcagaataa1320<210>4<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<220>
<221>MISC_FEATURE<222>(139)..(139)<223>Xaa代表除色氨酸之外的任何氨基酸殘基。
<220>
<221>MISC_FEATURE<222>(182)..(182)<223>Xaa代表除精氨酸之外的任何氨基酸殘基。
<220>
<221>MISC_FEATURE<222>(187)..(187)<223>Xaa代表除苯丙氨酸之外的任何氨基酸殘基。
<220>
<221>MISC_FEATURE<222>(299)..(299)<223>Xaa代表除蘇氨酸之外的任何氨基酸殘基。
<400>4Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Xaa Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Xaa Glu Asn Tyr Val Xaa Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Xaa Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu
435<210>5<211>439<2l2>PRT<213>Thermoanaerobacterium saccharolyticum<400>5Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>6<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>6Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Lys Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430
Asn Gln Tyr Leu Phe Ala Glu435<210>7<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>7Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Ser Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>8<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>8Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Cys Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430
Asn Gln Tyr Leu Phe Ala Glu435<210>9<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>9Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Ile Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>10<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>10Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Thr Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430
Asn Gln Tyr Leu Phe Ala Glu435<210>11<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>11Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Asn Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>12<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>12Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>13<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>13Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Pro Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>14<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>14Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ser Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>15<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>15Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ile Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>16<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>16Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Thr Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>17<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>17Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Val Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>18<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>18Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>19<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>19Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Gly Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>20<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>20Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Pro Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>21<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>21Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Ala Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>22<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>22Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>23<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>23Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Ile Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>24<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>24Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Tyr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>25<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>25Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Cys Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>26<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>26Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Met Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>27<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>27Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Glu Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>28<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>28Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>29<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>29Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>30<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>30Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>31<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>31Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>32<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>32Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu43權(quán)利要求
1.一種葡萄糖異構(gòu)酶突變體，其特征在于以序列表中的序列2為參考序列，在第139位、第182位、第187位、第299位具有至少一個突變，并且以D-葡萄糖為底物其具有比親本高出至少50％的葡萄糖異構(gòu)酶催化活性。
2.權(quán)利要求1所述的葡萄糖異構(gòu)酶突變體，其中第139位的色氨酸突變?yōu)橘嚢彼?Lys)、或絲氨酸(Ser)、或半胱氨酸(Cys)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或天冬酰胺(Asn)、或苯丙氨酸(Phe)。
3.權(quán)利要求2所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第139位的Xaa代表苯丙氨酸(Phe)、賴氨酸(Lys)、絲氨酸(Ser)、半胱氨酸(Cys)、異亮氨酸(Ile)、蘇氨酸(Thr)、或天冬酰胺(Asn)的氨基酸序列。
4.權(quán)利要求3所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第139位的Xaa代表異亮氨酸(Ile)、絲氨酸(Ser)、半胱氨酸(Cys)、或天冬酰胺(Asn)的氨基酸序列。
5.權(quán)利要求4所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第139位的Xaa代表絲氨酸(Ser)、半胱氨酸(Cys)、或天冬酰胺(Asn)的氨基酸序列。
6.權(quán)利要求1所述的葡萄糖異構(gòu)酶突變體，其中第182位的精氨酸突變?yōu)楦彼?Pro)、或絲氨酸(Ser)、或丙氨酸(Ala)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或纈氨酸(Val)。
7.權(quán)利要求6所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第182位的Xaa代表丙氨酸(Ala)、脯氨酸(Pro)、絲氨酸(Ser)、異亮氨酸(Ile)、蘇氨酸(Thr)、或纈氨酸(Val)的氨基酸序列。
8.權(quán)利要求7所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第182位的Xaa代表異亮氨酸(Ile)、絲氨酸(Ser)、或纈氨酸(Val)的氨基酸序列。
9.權(quán)利要求8所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第182位的Xaa代表異亮氨酸(Ile)、或纈氨酸(Val)的氨基酸序列。
10.權(quán)利要求1所述的葡萄糖異構(gòu)酶突變體，其中第187位的苯丙氨酸突變?yōu)楦拾彼?Gly)、或絲氨酸(Ser)、或丙氨酸(Ala)、或脯氨酸(Pro)。
11.權(quán)利要求10所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第187位的Xaa代表絲氨酸(Ser)、甘氨酸(Gly)、脯氨酸(Pro)、或丙氨酸(Ala)的氨基酸序列。
12.權(quán)利要求11所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第187位的Xaa代表脯氨酸(Pro)的氨基酸序列。
13.權(quán)利要求1所述的葡萄糖異構(gòu)酶突變體，其中第299位的蘇氨酸突變?yōu)楫惲涟彼?Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或蛋氨酸(Met)、或谷氨酸(Glu)、或谷氨酰胺(Gln)。
14.權(quán)利要求13的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第299位的Xaa代表谷氨酰胺(Gln)、異亮氨酸(Ile)、酪氨酸(Tyr)、半胱氨酸(Cys)、蛋氨酸(Met)、或谷氨酸(Glu)的氨基酸序列。
15.權(quán)利要求14的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第299位的Xaa代表半胱氨酸(Cys)、或谷氨酸(Glu)的氨基酸序列。
16.權(quán)利要求1所述的葡萄糖異構(gòu)酶突變體，其特征還在于以說明書所附的序列2為參考序列，具有至少兩個選自第139位的色氨酸到其他19個天然氨基酸、第182位的精氨酸到其他19個天然氨基酸、第187位的苯丙氨酸到其他19個天然氨基酸、第299位的蘇氨酸到其他19個天然氨基酸的突變。
17.權(quán)利要求16所述的葡萄糖異構(gòu)酶突變體，其中第139位的色氨酸突變?yōu)橘嚢彼?Lys)、或絲氨酸(Ser)、或半胱氨酸(Cys)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或天冬酰胺(Asn)、或苯丙氨酸(Phe)。
18.權(quán)利要求16所述的葡萄糖異構(gòu)酶突變體，其中第182位的精氨酸突變?yōu)楦彼?Pro)、或絲氨酸(Ser)、或丙氨酸(Ala)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或纈氨酸(Val)。
19.權(quán)利要求16所述的葡萄糖異構(gòu)酶突變體，其中第187位的苯丙氨酸突變?yōu)楦拾彼?Gly)、或絲氨酸(Ser)、或丙氨酸(Ala)、或脯氨酸(Pro)。
20.權(quán)利要求16所述的葡萄糖異構(gòu)酶突變體，其中第299位的蘇氨酸突變?yōu)楫惲涟彼?Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或蛋氨酸(Met)、或谷氨酸(Glu)、或谷氨酰胺(Gln)。
21.權(quán)利要求16所述的葡萄糖異構(gòu)酶突變體，其特征在于具有說明書中所附的序列4中第139位的Xaa代表苯丙氨酸(Phe)同時第182位的Xaa代表丙氨酸(Ala)的氨基酸序列(序列29)，或者序列4中第182位的Xaa代表丙氨酸(Ala)同時第299位的Xaa代表谷氨酰胺(Gln)的氨基酸序列(序列30)、或者序列4中第139位的Xaa代表苯丙氨酸(Phe)同時第299位的Xaa代表谷氨酰胺(Gln)的氨基酸序列(序列31)。
22.權(quán)利要求16所述的葡萄糖異構(gòu)酶突變體，其特征還在于以說明書所附的序列2為參考序列，具有至少三個選自第139位的色氨酸到其他19個天然氨基酸、第182位的精氨酸到其他19個天然氨基酸、第187位的苯丙氨酸到其他19個天然氨基酸、第299位的蘇氨酸到其他19個天然氨基酸的突變。
23.權(quán)利要求22所述的葡萄糖異構(gòu)酶突變體，其中第139位的色氨酸突變?yōu)橘嚢彼?Lys)、或絲氨酸(Ser)、或半胱氨酸(Cys)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或天冬酰胺(Asn)、或苯丙氨酸(Phe)。
24.權(quán)利要求22所述的葡萄糖異構(gòu)酶突變體，其中第182位的精氨酸突變?yōu)楦彼?Pro)、或絲氨酸(Ser)、或丙氨酸(Ala)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或纈氨酸(Val)。
25.權(quán)利要求22所述的葡萄糖異構(gòu)酶突變體，其中第187位的苯丙氨酸突變?yōu)楦拾彼?Gly)、或絲氨酸(Ser)、或丙氨酸(Ala)、或脯氨酸(Pro)。
26.權(quán)利要求22所述的葡萄糖異構(gòu)酶突變體，其中第299位的蘇氨酸突變?yōu)楫惲涟彼?Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或蛋氨酸(Met)、或谷氨酸(Glu)、或谷氨酰胺(Gln)。
27.權(quán)利要求22所述的葡萄糖異構(gòu)酶突變體，其特征在于具有序列4中第139位的Xaa代表苯丙氨酸(Phe)、第182位的Xaa代表丙氨酸(Ala)同時第299位的Xaa代表谷氨酰胺(Gln)的氨基酸序列(序列32)。
28.權(quán)利要求22所述的葡萄糖異構(gòu)酶突變體，特征還在于以說明書所附的序列2為參考序列，具有第139位的色氨酸到其他19個天然氨基酸、第182位的精氨酸到其他19個天然氨基酸、第187位的苯丙氨酸到其他19個天然氨基酸、第299位的蘇氨酸到其他19個天然氨基酸的突變。
29.權(quán)利要求28所述的葡萄糖異構(gòu)酶突變體，其中第139位的色氨酸突變?yōu)橘嚢彼?Lys)、或絲氨酸(Ser)、或半胱氨酸(Cys)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或天冬酰胺(Asn)、或苯丙氨酸(Phe)。
30.權(quán)利要求28所述的葡萄糖異構(gòu)酶突變體，其中第182位的精氨酸突變?yōu)楦彼?Pro)、或絲氨酸(Ser)、或丙氨酸(Ala)、或異亮氨酸(Ile)、或蘇氨酸(Thr)、或纈氨酸(Val)。
31.權(quán)利要求28所述的葡萄糖異構(gòu)酶突變體，其中第187位的苯丙氨酸突變?yōu)楦拾彼?Gly)、或絲氨酸(Ser)、或丙氨酸(Ala)、或脯氨酸(Pro)。
32.權(quán)利要求28所述的葡萄糖異構(gòu)酶突變體，其中第299位的蘇氨酸突變?yōu)楫惲涟彼?Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或蛋氨酸(Met)、或谷氨酸(Glu)、或谷氨酰胺(Gln)。
33.權(quán)利要求28所述的葡萄糖異構(gòu)酶突變體，其特征在于具有序列4中第139位的Xaa代表苯丙氨酸(Phe)、第182位的Xaa代表丙氨酸(Ala)、第187位的Xaa代表絲氨酸(Ser)同時第299位的Xaa代表谷氨酰胺(Gln)的氨基酸序列(序列33)。
34.權(quán)利要求1-33任意一項所述的葡萄糖異構(gòu)酶突變體的應(yīng)用，其中所述的葡萄糖異構(gòu)酶突變體用于轉(zhuǎn)化D-葡萄糖至果糖。
35.權(quán)利要求34所述的葡萄糖異構(gòu)酶突變體的應(yīng)用，其中所述的應(yīng)用為制備果葡糖漿。
36.權(quán)利要求35所述的應(yīng)用，其中所述的果葡糖漿的果糖含量為至少55重量％。
37.一種DNA，其含有編碼權(quán)利要求1-33任意一項所述的葡萄糖異構(gòu)酶突變體的核苷酸序列。
全文摘要
本發(fā)明披露利用基因突變技術(shù)改良的一系列耐熱高催化活性Thermoanaerobacterium saccharolyticum葡萄糖異構(gòu)酶突變體。這些葡萄糖異構(gòu)酶突變體可用于直接產(chǎn)生果糖含量等于或高于55重量％的果葡糖漿，或用于產(chǎn)生果糖含量低于55重量％的果葡糖漿。
文檔編號C12N9/92GK1702172SQ20041004786
公開日2005年11月30日 申請日期2004年6月16日 優(yōu)先權(quán)日2004年5月26日
發(fā)明者王駿, 傅榮昭, 沈冬, 金彩科, 劉兆明, 陳軍明 申請人:百瑞全球有限公司