專利名稱：突變的木糖異構(gòu)酶及其基因和用途的制作方法
技術(shù)領(lǐng)域：
本發(fā)明屬于酶的基因工程和酶生化工程領(lǐng)域。本發(fā)明涉及活性提高的突變木糖異構(gòu)酶，本發(fā)明還涉及編碼該突變木糖異構(gòu)酶的基因。
背景技術(shù)：
木糖異構(gòu)酶(Xylose Isomerase，XI)(EC 5.3.1.5)又稱葡萄糖異構(gòu)酶，可以催化D-木糖到D-木酮糖及D-葡萄糖到D-果糖的異構(gòu)化反應(yīng)，在微生物體內(nèi)糖的代謝過程中起著重要的作用，并已經(jīng)用于高果糖漿的工業(yè)生產(chǎn)[Bhosale SH，Rao MB and Deshpande VV.Molecular and Industrial Aspects of Glucose Isomerase.Microbiol.Rev.，1996，60280-300]。來自Thermus thermophilus HB8的木糖異構(gòu)酶[Zeikus JG，Vieille C，SavchenkoA.Thermozymesbiotechnology and structure function relationships.Extremophiles，1998，2(3)179-183]，最適反應(yīng)溫度約為85℃，具有極高的穩(wěn)定性，以及對有機溶劑、去污劑和變性劑的較強抗性，已被廣泛應(yīng)用到食品、醫(yī)藥等各個行業(yè)的工業(yè)生產(chǎn)中。由于高溫會導(dǎo)致葡萄糖異構(gòu)過程副產(chǎn)物的形成，因此高果糖漿生產(chǎn)中主要反應(yīng)溫度控制在60-70℃。目前國內(nèi)主要采用的是來自鏈霉菌的葡萄糖異構(gòu)酶，但其穩(wěn)定性不夠理想，導(dǎo)致果糖產(chǎn)量不高，生產(chǎn)工藝復(fù)雜。因此， 提高Thermus thermophilus HB8葡萄糖異構(gòu)酶60-70℃附近的催化活性對于高果糖漿的工業(yè)生產(chǎn)具有重要意義。
近年來，隨著能源危機和環(huán)境污染的日益嚴(yán)重，對廣泛存在于可再生的木質(zhì)纖維素中的木糖利用也成為研究熱點之一。國內(nèi)外科研人員一直致力于構(gòu)建能夠發(fā)酵木糖的重組酵母。迄今為止，已經(jīng)報道了九種不同菌屬中的XI在釀酒酵母中進(jìn)行表達(dá)，但除來源于Thermus Thermophilus，Thermoanaerobacter Tengcongensis MB4T和Piromyces sp.的木糖異構(gòu)酶基因之外，都沒有表達(dá)出活性。Thermus thermophilus XI在木糖發(fā)酵乙醇方面的應(yīng)用潛力及其極端的熱穩(wěn)定性引起了國內(nèi)外的極大關(guān)注[Walfridsson M，Bao X，Anderlund M，et al.Ethanolic fermentation of xylose with Saccharomyces cerevisiaeharboring the Thermus thermophilus xylA gene，which expresses an active xylose(glucose)isomerase.Appl.Environ.Microbiol.，1996，624648-4651]，但由于Thermus thermophilus XI在常溫下活性較低導(dǎo)致重組菌對木糖的利用并不理想。選擇在常溫下高活力的木糖異構(gòu)酶已經(jīng)成為這一領(lǐng)域的主要研究方向之一[沈煜，王穎，鮑曉明，曲音波.釀酒酵母木糖發(fā)酵酒精途徑工程的研究進(jìn)展.生物工程學(xué)報，2003，19(5)636-640.]。美國專利(US6475768)報道通過對Thermus thermophilus HB8的木糖異構(gòu)酶進(jìn)行隨機PCR突變，獲得三種常溫下活性提高的木糖異構(gòu)酶。近年來，國內(nèi)在發(fā)酵木糖生產(chǎn)乙醇重組酵母構(gòu)建方面也取得了可喜的進(jìn)展，通過在釀酒酵母工業(yè)菌株中表達(dá)Thermus thermophilus木糖異構(gòu)酶，建立了木糖代謝途徑(WANG Y.，SHI W.L.，LIU X.Y.，et al.BiotechnologyLetters，2004，26(11)885-890)，但由于木糖異構(gòu)酶活性較低使得重組菌對木糖的利用并不理想。
定點突變是蛋白質(zhì)工程廣泛采用的有效技術(shù)。它是根據(jù)酶的結(jié)構(gòu)、功能和作用機理等信息，在基因水平上精確改變酶分子中的特定氨基酸殘基，使酶的性質(zhì)最佳化。利用生物信息學(xué)工具進(jìn)行蛋白質(zhì)的設(shè)計，并結(jié)合定點突變技術(shù)提高Thermus thermophilus HB8中木糖異構(gòu)酶的活性，尚未見有關(guān)文獻(xiàn)報道。

發(fā)明內(nèi)容
本發(fā)明的目的是提供一種突變的木糖異構(gòu)酶。該突變的木糖異構(gòu)酶與野生菌Thermus thermophilus HB8中的木糖異構(gòu)酶相比，活性有所提高。
本發(fā)明另一個目的是提供編碼上述突變木糖異構(gòu)酶的基因(核苷酸序列)。這些基因可用于構(gòu)建利用木糖的重組菌。
本發(fā)明再一個目的是提供上述突變木糖異構(gòu)酶在高果糖漿生產(chǎn)中的應(yīng)用。
上述突變木糖異構(gòu)酶的氨基酸序列中至少有一個氨基酸不同于野生菌Thermusthermophilus HB8中木糖異構(gòu)酶的氨基酸序列(Swiss-Prot entryP26997)。
本發(fā)明的目的可以通過下列措施來達(dá)到一種突變的木糖異構(gòu)酶，其氨基酸序列為SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.7所示。
這些序列分別表示原木糖異構(gòu)酶氨基酸序列(即野生型Thermus thermophilus HB8xylA基因所表達(dá)的氨基酸序列)的91位的天冬酰胺殘基以及375位的天冬氨酸殘基分別被天冬氨酸殘基和甘氨酸殘基取代，91位的天冬酰胺殘基被天冬氨酸殘基取代，91位的天冬酰胺殘基以及355位的賴氨酸殘基分別被天冬氨酸殘基和丙氨酸殘基取代，91位的天冬酰胺殘基以及144位的纈氨酸殘基分別被天冬氨酸殘基和丙氨酸殘基取代。
編碼所述的突變木糖異構(gòu)酶的核苷酸序列。
所述的核苷酸序列，其序列為SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6或SEQ IDNO.8所示。
含有權(quán)利要求2所述核苷酸序列的重組載體。常用載體如質(zhì)粒、噬菌體等。
所述的重組載體，該表達(dá)載體為含有上述核苷酸序列的重組質(zhì)粒。該重組質(zhì)粒為pET-22b(+)-xylA-N91D/D375G、pET-22b(+)-xylA-N91D、pET-22b(+)-xylA-N91D/V144A、pET-22b(+)-xylA-N91D/K355A。
導(dǎo)入上述核苷酸序列或重組載體的轉(zhuǎn)化體，其宿主為細(xì)菌或酵母菌。所述細(xì)菌常用大腸桿菌。
所述的木糖異構(gòu)酶在高果糖漿生產(chǎn)中的應(yīng)用。
本發(fā)明的有益效果本發(fā)明提供的突變型木糖異構(gòu)酶活性有所提高。本發(fā)明還提供了該突變型木糖異構(gòu)酶基因序列，這些序列可用于構(gòu)建利用木糖的重組菌。本發(fā)明所提供的突變木糖異構(gòu)酶可用于高果糖漿的生產(chǎn)。


圖1是重組質(zhì)粒pET-22b(+)-xylA構(gòu)建圖。
圖2是表達(dá)產(chǎn)物的SDS-PAGE分析。圖中1、2、3、4分別代表含有突變質(zhì)粒pET-22b(+)-xylA-N91D/D375G、pET-22b(+)-xylA-N91D、pET-22b(+)-xylA-N91D/V144A、pET-22b(+)-xylA-N91D/K355A的重組大腸桿菌Rosseta中的表達(dá)產(chǎn)物。
圖3是不同溫度下本發(fā)明突變型木糖異構(gòu)酶活性比較。
具體實施例方式
以下通過實施例對本發(fā)明作進(jìn)一步的闡述，但不限制本發(fā)明。
說明D375G表示氨基酸序列第375位的D被G取代，Asp375表示氨基酸序列第375位氨基酸為Asp，本發(fā)明中類似的表述均為此方式。
實施例中未注明具體條件的實驗方法，基本上都按照Sambrook，J等人編著的《分子克隆實驗指南(第3版)》(Molecular CloningA Laboratory Manual，3rded.黃培堂等譯，科學(xué)出版社.2002.8)中所述的條件及方法或按照材料提供商所建議的條件及方法進(jìn)行，其它沒有詳細(xì)描述的技術(shù)相應(yīng)于本領(lǐng)域人員來說是熟知的標(biāo)準(zhǔn)方法。
本發(fā)明的材料本申請中采用的未特別限定來源的微生物、質(zhì)?；蚱渌磉_(dá)載體以及培養(yǎng)基均有商品供應(yīng)或以別的途徑能為公眾所得，它們僅作舉例，對本發(fā)明不是唯一的，可分別用其它適合的工具和生物材料來代替。
實施例1野生型Thermus thermophilus HB8木糖異構(gòu)酶表達(dá)重組質(zhì)粒的構(gòu)建、篩選與鑒定。
嗜熱棲熱菌(Thermus thermophilus HB8)購于美國菌種保藏中心(ATCC)，ATCC編號為27634。載體pET-22b(+)購于Novagen公司。
酵母粉4g/L，蛋白胨8g/L，NaCl 2.0g/L，將pH調(diào)至7.0，高壓蒸氣滅菌121℃，15min。將Thermus thermophilus HB8接種于上述培養(yǎng)基中，置75℃搖床，200rpm，培養(yǎng)12-16小時。8000rpm，離心15min，收集菌體。
運用基因組提取試劑盒(上海華舜生物工程有限公司)，提取Thermus Thermophilus基因組。以Thermus Thermophilus HB8總DNA為模板，使用如下PCR引物(上海博亞公司合成)正向引物5’-CCATATGTACGAACCAAAACCGGAACATCGCTTTACCTTT-3’(SEQ ID NO.9)反向引物5’-AAGGAAAAAAGCGGCCGCTCAACCACGCACACCCAGGAG-3’(SEQ ID NO.10)PCR擴增木糖異構(gòu)酶基因xylA。PCR循環(huán)參數(shù)為95℃，2min；94℃，30s；70℃，50s；68℃，1.5min，共進(jìn)行35個循環(huán)。
用DNA回收試劑盒(Takara公司)對擴增出的基因進(jìn)行回收純化。用限制性內(nèi)切酶NdeI和NotI雙酶切基因xylA和載體pET-22b(+)，回收后將上述雙酶切過的基因xylA及pET-22b(+)載體連接，運用常規(guī)的基因工程技術(shù)將連接液轉(zhuǎn)化到大腸桿菌DH5α感受態(tài)細(xì)胞，提取質(zhì)粒。運用上述引物進(jìn)行擴增，鑒定基因xylA確定連接到表達(dá)載體pET-22b(+)上。經(jīng)鑒定過的重組質(zhì)粒為pET-22b(+)-xylA。構(gòu)建過程如圖1所示。
實施例2木糖異構(gòu)酶基因的定點突變根據(jù)多點突變試劑盒(QuikChange Multi Site-Directed Mutagenesis Kit，購自STRATAGENE公司)的要求，設(shè)計N91D引物，5’-ATGGTCACCGCCGACCTCTTCTCCGAC-3’(SEQ ID NO.11)以pET-22b(+)-xylA質(zhì)粒為模板進(jìn)行PCR，將Asn91突變?yōu)锳sp91。按照多點突變試劑盒說明書，用限制性內(nèi)切酶Dpn I消化原始質(zhì)粒模板pET-22b(+)-xylA，將PCR產(chǎn)物轉(zhuǎn)化高效率的感受態(tài)細(xì)胞。運用常規(guī)的基因工程技術(shù)對轉(zhuǎn)化后的感受態(tài)細(xì)胞進(jìn)行培養(yǎng)，提取質(zhì)粒進(jìn)行測序鑒定，得到含有突變基因的重組質(zhì)粒pET-22b(+)-xylA-N91D。
運用D375G引物5’-TGGAACGCCTGGGCCAGCTGGCGGTG-3’(SEQ ID NO.12)，以pET-22b(+)-xylA-N91D質(zhì)粒為模板進(jìn)行PCR，將氨基酸殘基Asp375突變?yōu)镚ly375。其它步驟同上，即可得到同時具有N91D和D375G兩個突變位點的重組質(zhì)粒pET-22b(+)-xylA-N91D/D375G。
運用V144A引物5’-AGGGAGCTGAGGCGGAGGCCACGGGC-3’(SEQ ID NO.13)以pET-22b(+)-xylA-N91D質(zhì)粒為模板進(jìn)行PCR，將氨基酸殘基Val144突變?yōu)锳la144。按上述步驟操作，即可獲得同時具有N91D和V144A兩個突變位點的重組質(zhì)粒pET-22b(+)-xylA-N91D/V144A。
運用K355A引物5’-CGAAGCCCTCGCGCGGGCGGAGCTTCC-3’(SEQ ID NO.14)以pET-22b(+)-xylA-N91D質(zhì)粒為模板進(jìn)行PCR，將氨基酸殘基Lys355突變?yōu)锳la355。按上述步驟操作，即可獲得同時具有N91D和K355A兩個突變位點的重組質(zhì)粒pET-22b(+)-xylA-N91D/K355A。
實施例3突變的木糖異構(gòu)酶在大腸桿菌Rosseta中的表達(dá)分別將實施例2制備的含有突變基因的重組質(zhì)粒轉(zhuǎn)化到商業(yè)化的表達(dá)宿主大腸桿菌Rosetta(DE3)中，挑取單菌落到含75μg/mL氨芐青霉素和34μg/ml氯霉素的LB培養(yǎng)液，37℃振蕩培養(yǎng)過夜，然后按2％(v/v)接種量接種到含有75μg/mL氨芐青霉素和34μg/mL氯霉素的LB培養(yǎng)液中，37℃培養(yǎng)至OD600約為0.6時，加入IPTG至終濃度0.9mM，誘導(dǎo)表達(dá)7h，取一定體積菌液8000rpm，4℃，離心15min收集菌體。表達(dá)產(chǎn)物的SDS-PAGE分析見圖2。
實施例4木糖異構(gòu)酶活性測定方法1.突變木糖異構(gòu)酶的純化分別取10mL實施例3中誘導(dǎo)后的培養(yǎng)液8000rpm，4℃，離心15min收集菌體，用無菌水洗滌兩次后，菌體重懸于0.5mL pH7.550mM Tris-HCl緩沖液中，冰浴中超聲破碎細(xì)胞，12,000rpm，4℃離心10min取上清即為粗酶液。將粗酶液置于70℃水浴，加熱40min，12,000rpm，4℃離心10min可去除絕大部分雜蛋白。取上清后，再通過常規(guī)蛋白純化方法進(jìn)行純化，得到氨基酸序列分別為SEQ ID NO.1、SEQ ID NO.3、SEQ IDNO.5、SEQ ID NO.7所示的純化的突變木糖異構(gòu)酶。
2.酶活力測定木糖異構(gòu)酶活力測定采用兩步法，第一步分別取純化的突變木糖異構(gòu)酶50μL，1.5M D-木糖50μL，30mM MgCl250μL，在不同溫度下反應(yīng)15min，冰浴終止反應(yīng)；第二步取10μL上述反應(yīng)液，加入1μL(0.08U/μL)山梨醇脫氫酶，3μL 25mM NADH，186μLpH7.550mM Tris-HCl緩沖液，加入96孔微培養(yǎng)板。使用酶標(biāo)儀在25℃下檢測OD340的光吸收變化。酶活力單位(U)定義為每分鐘催化產(chǎn)生1μmol木酮糖所需的酶量。通過ΔA340/min的變化根據(jù)標(biāo)準(zhǔn)曲線方程Y＝2805.81357X(其中YmΔA340/min；X木酮糖的濃度，單位mol/L)確定木糖異構(gòu)酶的活力，結(jié)果見圖3，結(jié)果顯示，在N91D突變的基礎(chǔ)上，引入D375G，K355A或V144A突變可以使得木糖異構(gòu)酶活力提高。這些木糖異構(gòu)酶在60-70℃附近的催化活性較好，既可提高高果糖漿的生產(chǎn)效率，又能減少由于高溫導(dǎo)致葡萄糖異構(gòu)過程副產(chǎn)物的形成，適用于高果糖漿的生產(chǎn)。
序列表<110>南京工業(yè)大學(xué)<120>突變的木糖異構(gòu)酶及其基因和用途<160>14<210>1<211>387<212>PRT<213>人工序列<220>
<223>突變的木糖異構(gòu)酶(N91D/D375G)氨基酸序列<400>1Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp1 5 10 15Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg20 25 3OGlu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu35 40 45Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg50 55 6OGly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys65 70 75Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala80 85 90Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser95 100 105Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu110 115 120Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val125 130 135Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala140 145 150Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala155 160 165Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu170 175 180Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val185 190 195Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg200 205 210Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu215 220 225
Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu230 235 240Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln245 250 255Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu260 265 270Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe275 280 285Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala290 295 300Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg305 310 315Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala320 325 330Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro335 340 345Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro350 355 360Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Gly365 370 375Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly380 385<210>2<211>1164<212>DNA<213>人工序列<220>
<221>CDS<222>(1)…(1164)<223>突變的木糖異構(gòu)酶(N91D/D375G)核苷酸序列<400>2gtg tac gag ccc aaa ccg gag cac agg ttt acc ttt ggc ctt tgg45Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp1 5 10 15act gtg ggc aat gtg ggc cgt gat ccc ttc ggg gac gcg gtt cgg90Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg20 25 30gag agg ctg gac ccg gtt tac gtg gtt cat aag ctg gcg gag ctt135Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu35 40 45ggg gcc tac ggg gta aac ctt cac gac gag gac ctg atc ccg cgg180
Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg50 55 60ggc acg cct cct cag gag cgg gac cag atc gtg agg cgc ttc aag 225Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys65 70 75aag gct ctc gat gaa acc ggc ctc aag gtc ccc atg gtc acc gcc 270Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala80 85 90gac ctc ttc tcc gac cct gct ttc aag gac ggg gcc ttc acg agc 315Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser95 100 105ccg gac cct tgg gtt cgg gcc tat gcc ttg cgg aag agc ctg gag 360Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu110 115 120acc atg gac ctg ggg gca gag ctt ggg gcc gag atc tac gtg gtc 405Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val125 130 135tgg ccg ggc cgg gag gga gct gag gtg gag gcc acg ggc aag gcc 450Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala140 145 150cgg aag gtc tgg gac tgg gtg cgg gag gcg ctg aac ttc atg gcc 495Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala155 160 165gcc tac gcc gag gac cag gga tac ggg tac cgg ttt gcc ctc gag 540Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu170 175 180ccc aag cct aac gag ccc cgg ggg gac att tac ttc gcc acc gtg 585Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val185 190 195ggg agc atg ctc gcc ttt att cat acc ctg gac cgg ccc gag cgc 630Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg200 205 210ttc ggc ctg aac ccc gag ttc gcc cac gag acc atg gcc ggg ctt 675Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu215 220 225aac ttt gtc cac gcc gtg gcc cag gct ctc gac gcc ggg aag ctt 720Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu230 235 240
ttc cac att gac ctc aac gac caa cgg atg agc cgg ttt gac cag 765Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln245 250 255gac ctc cgc ttc ggc tcg gag aac ctc aag gcg gcc ttt ttc ctg 810Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu260 265 270gtg gac ctc ctg gaa agc tcc ggc tac cag ggc ccc cgc cac ttt 855Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe275 280 285gac gcc cac gcc ctg cgt acc gag gac gaa gaa ggg gtt tgg gcc 900Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala290 295 300ttc gcc cga ggc tgc atg cgt acc tac ctg atc tta aag gaa agg 945Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg305 310 315gct gaa gcc ttc cgc gag gat ccc gag gtc aag gag ctt ctt gcc 990Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala320 325 330gct tac tat caa gaa gat cct gcg gcc ttg gcc ctt ttg ggc ccc 1035Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro335 340 345tac tcc cgc gag aag gcc gaa gcc ctc aag cgg gcg gag ctt ccc 1080Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro350 355 360ctc gag gcc aag cgg cgc cgg ggt tat gcc ctg gaa cgc ctg ggc 1125Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Gly365 370 375cag ctg gcg gtg gag tac ctc ctg ggg gtg cgg ggg tga 1164Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly380 385<210>3<211>387<212>PRT<213>人工序列<220>
<223>突變的木糖異構(gòu)酶(N91D)氨基酸序列<400>3Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
1 5 10 15Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg20 25 30Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu35 40 45Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg50 55 60Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys65 70 75Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala80 85 90Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser95 100 105Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu110 115 120Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val125 130 135Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala140 145 150Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala155 160 165Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu170 175 180Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val185 190 195Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg200 205 210Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu215 220 225Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu230 235 240Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln245 250 255Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu260 265 270Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe275 280 285Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala290 295 300Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg305 310 315Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala320 325 330Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro350 355 360Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp365 370 375Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly380 385<210>4<211>1164<212>DNA<213>人工序列<220>
<221>CDS<222>(1)…(1164)<223>突變的木糖異構(gòu)酶(N91D)核苷酸序列<400>4gtg tac gag ccc aaa ccg gag cac agg ttt acc ttt ggc ctt tgg 45Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp1 5 10 15act gtg ggc aat gtg ggc cgt gat ccc ttc ggg gac gcg gtt cgg 90Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg20 25 30gag agg ctg gac ccg gtt tac gtg gtt cat aag ctg gcg gag ctt 135Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu35 40 45ggg gcc tac ggg gta aac ctt cac gac gag gac ctg atc ccg cgg 180Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg50 55 60ggc acg cct cct cag gag cgg gac cag atc gtg agg cgc ttc aag 225Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys65 70 75aag gct ctc gat gaa acc ggc ctc aag gtc ccc atg gtc acc gcc 270Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala80 85 90gac ctc ttc tcc gac cct gct ttc aag gac ggg gcc ttc acg agc 315Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser95 100 105ccg gac cct tgg gtt cgg gcc tat gcc ttg cgg aag agc ctg gag 360Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu110 115 120
acc atg gac ctg ggg gca gag ctt ggg gcc gag atc tac gtg gtc405Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val125 130 135tgg ccg ggc cgg gag gga gct gag gtg gag gcc acg ggc aag gcc450Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala140 145 150cgg aag gtc tgg gac tgg gtg cgg gag gcg ctg aac ttc atg gcc495Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala155 160 165gcc tac gcc gag gac cag gga tac ggg tac cgg ttt gcc ctc gag540Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu170 175 180ccc aag cct aac gag ccc cgg ggg gac att tac ttc gcc acc gtg585Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val185 190 195ggg agc atg ctc gcc ttt att cat acc ctg gac cgg ccc gag cgc630Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg200 205 210ttc ggc ctg aac ccc gag ttc gcc cac gag acc atg gcc ggg ctt675Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu215 220 225aac ttt gtc cac gcc gtg gcc cag gct ctc gac gcc ggg aag ctt720Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu230 235 240ttc cac att gac ctc aac gac caa cgg atg agc cgg ttt gac cag765Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln245 250 255gac ctc cgc ttc ggc tcg gag aac ctc aag gcg gcc ttt ttc ctg810Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu260 265 270gtg gac ctc ctg gaa agc tcc ggc tac cag ggc ccc cgc cac ttt855Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe275 280 285gac gcc cac gcc ctg cgt acc gag gac gaa gaa ggg gtt tgg gcc9O0Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala290 295 300ttc gcc cga ggc tgc atg cgt acc tac ctg atc tta aag gaa agg945Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315gct gaa gcc ttc cgc gag gat ccc gag gtc aag gag ctt ctt gcc990Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala320 325 330gct tac tat caa gaa gat cct gcg gcc ttg gcc ctt ttg ggc ccc1035Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro335 340 345tac tcc cgc gag aag gcc gaa gcc ctc aag cgg gcg gag ctt ccc1080Tyr Ser Arg GLu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro350 355 360ctc gag gcc aag cgg cgc cgg ggt tat gcc ctg gaa cgc ctg gac1125Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp365 370 375cag ctg gcg gtg gag tac ctc ctg ggg gtg cgg ggg tga1164Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly380 385<210>5<211>387<212>PRT<213>人工序列<220>
<223>突變的木糖異構(gòu)酶(N91D/K355A)氨基酸序列<400>5Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp1 5 10 15Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg20 25 30Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu35 40 45Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg50 55 60Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys65 70 75Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala80 85 90Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser95 100 105Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu110 115 120Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val125 130 135
Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala140 145 150Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala155 160 165Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu170 175 180Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val185 190 195Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg200 205 210Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu215 220 225Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu230 235 240Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln245 250 255Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu260 265 270Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe275 280 285Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala290 295 300Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg305 310 315Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala320 325 330Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro335 340 345Tyr Ser Arg Glu Lys Ala Glu Ala Leu Ala Arg Ala Glu Leu Pro350 355 360Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp365 370 375Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly380 385<210>6<211>1164<212>DNA<213>人工序列<220>
<221>CDS<222>(1)…(1164)<223>突變的木糖異構(gòu)酶(N91D/K355A)核苷酸序列<400>6
gtg tac gag ccc aaa ccg gag cac agg ttt acc ttt ggc ctt tgg45Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp1 5 10 15act gtg ggc aat gtg ggc cgt gat ccc ttc ggg gac gcg gtt cgg90Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg20 25 30gag agg ctg gac ccg gtt tac gtg gtt cat aag ctg gcg gag ctt135Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu35 40 45ggg gcc tac ggg gta aac ctt cac gac gag gac ctg atc ccg cgg180Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg50 55 60ggc acg cct cct cag gag cgg gac cag atc gtg agg cgc ttc aag225Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys65 70 75aag gct ctc gat gaa acc ggc ctc aag gtc ccc atg gtc acc gcc270Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala80 85 90gac ctc ttc tcc gac cct gct ttc aag gac ggg gcc ttc acg agc315Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser95 100 105ccg gac cct tgg gtt cgg gcc tat gcc ttg cgg aag agc ctg gag360Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu110 115 120acc atg gac ctg ggg gca gag ctt ggg gcc gag atc tac gtg gtc405Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val125 130 135tgg ccg ggc cgg gag gga gct gag gtg gag gcc acg ggc aag gcc450Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala140 145 150cgg aag gtc tgg gac tgg gtg cgg gag gcg ctg aac ttc atg gcc495Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala155 160 165gcc tac gcc gag gac cag gga tac ggg tac cgg ttt gcc ctc gag540Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu170 175 180ccc aag cct aac gag ccc cgg ggg gac att tac ttc gcc acc gtg585Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
185 190 195ggg agc atg ctc gcc ttt att cat acc ctg gac cgg ccc gag cgc630Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg200 205 210ttc ggc ctg aac ccc gag ttc gcc cac gag acc atg gcc ggg ctt675Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu215 220 225aac ttt gtc cac gcc gtg gcc cag gct ctc gac gcc ggg aag ctt720Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu230 235 240ttc cac att gac ctc aac gac caa cgg atg agc cgg ttt gac cag765Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln245 250 255gac ctc cgc ttc ggc tcg gag aac ctc aag gcg gcc ttt ttc ctg810Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu260 265 270gtg gac ctc ctg gaa agc tcc ggc tac cag ggc ccc cgc cac ttt855Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe275 280 285gac gcc cac gcc ctg cgt acc gag gac gaa gaa ggg gtt tgg gcc900Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala290 295 300ttc gcc cga ggc tgc atg cgt acc tac ctg atc tta aag gaa agg945Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg305 310 315gct gaa gcc ttc cgc gag gat ccc gag gtc aag gag ctt ctt gcc990Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala320 325 330gct tac tat caa gaa gat cct gcg gcc ttg gcc ctt ttg ggc ccc1035Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro335 340 345tac tcc cgc gag aag gcc gaa gcc ctc gcg cgg gcg gag ctt ccc1080Tyr Ser Arg Glu Lys Ala Glu Ala Leu Ala Arg Ala Glu Leu Pro350 355 360ctc gag gcc aag cgg cgc cgg ggt tat gcc ctg gaa cgc ctg gac1125Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp365 370 375cag ctg gcg gtg gag tac ctc ctg ggg gtg cgg ggg tga1164
Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly380 385<210>7<211>387<212>PRT<213>人工序列<220>
<223>突變的木糖異構(gòu)酶(N91D/V144A)氨基酸序列<400>7Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp1 5 10 15Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg20 25 30Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu35 40 45Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg50 55 60Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys65 70 75Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala80 85 90Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser95 100 105Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu110 115 120Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val125 130 135Trp Pro Gly Arg Glu Gly Ala Glu Ala Glu Ala Thr Gly Lys Ala140 145 150Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala155 160 165Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu170 175 180Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val185 190 195Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg200 205 210Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu215 220 225Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu230 235 240Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu260 265 270Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe275 280 285Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala290 295 300Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg305 310 315Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala320 325 330Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro335 340 345Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro350 355 360Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp365 370 375Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly380 385<210>8<211>1164<212>DNA<213>人工序列<220>
<221>CDS<222>(1)…(1164)<223>突變的木糖異構(gòu)酶(N91D/V144A)核苷酸序列<400>8gtg tac gag ccc aaa ccg gag cac agg ttt acc ttt ggc ctt tgg 45Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp1 5 10 15act gtg ggc aat gtg ggc cgt gat ccc ttc ggg gac gcg gtt cgg 90Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg20 25 30gag agg ctg gac ccg gtt tac gtg gtt cat aag ctg gcg gag ctt 135Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu35 40 45ggg gcc tac ggg gta aac ctt cac gac gag gac ctg atc ccg cgg 180Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg50 55 60ggc acg cct cct cag gag cgg gac cag atc gtg agg cgc ttc aag 225
Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys65 70 75aag gct ctc gat gaa acc ggc ctc aag gtc ccc atg gtc acc gcc 270Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala80 85 90gac ctc ttc tcc gac cct gct ttc aag gac ggg gcc ttc acg agc 315Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser95 100 105ccg gac cct tgg gtt cgg gcc tat gcc ttg cgg aag agc ctg gag 360Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu110 115 120acc atg gac ctg ggg gca gag ctt ggg gcc gag atc tac gtg gtc 405Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val125 130 135tgg ccg ggc cgg gag gga gct gag gcg gag gcc acg ggc aag gcc 450Trp Pro Gly Arg Glu Gly Ala Glu Ala Glu Ala Thr Gly Lys Ala140 145 150cgg aag gtc tgg gac tgg gtg cgg gag gcg ctg aac ttc atg gcc 495Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala155 160 165gcc tac gcc gag gac cag gga tac ggg tac cgg ttt gcc ctc gag 540Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu170 175 180ccc aag cct aac gag ccc cgg ggg gac att tac ttc gcc acc gtg 585Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val185 190 195ggg agc atg ctc gcc ttt att cat acc ctg gac cgg ccc gag cgc 630Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg200 205 210ttc ggc ctg aac ccc gag ttc gcc cac gag acc atg gcc ggg ctt 675Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu215 220 225aac ttt gtc cac gcc gtg gcc cag gct ctc gac gcc ggg aag ctt 720Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu230 235 240ttc cac att gac ctc aac gac caa cgg atg agc cgg ttt gac cag 765Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln245 250 255
gac ctc cgc ttc ggc tcg gag aac ctc aag gcg gcc ttt ttc ctg810Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu260 265 270gtg gac ctc ctg gaa agc tcc ggc tac cag ggc ccc cgc cac ttt855Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe275 280 285gac gcc cac gcc ctg cgt acc gag gac gaa gaa ggg gtt tgg gcc900Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala290 295 300ttc gcc cga ggc tgc atg cgt acc tac ctg atc tta aag gaa agg945Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg305 310 315gct gaa gcc ttc cgc gag gat ccc gag gtc aag gag ctt ctt gcc990Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala320 325 330gct tac tat caa gaa gat cct gcg gcc ttg gcc ctt ttg ggc ccc1035Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro335 340 345tac tcc cgc gag aag gcc gaa gcc ctc aag cgg gcg gag ctt ccc1080Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro350 355 360ctc gag gcc aag cgg cgc cgg ggt tat gcc ctg gaa cgc ctg gac1125Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp365 370 375cag ctg gcg gtg gag tac ctc ctg ggg gtg cgg ggg tga1164Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly380 385<210>9<211>40<212>DNA<213>人工序列<220>
<223>上游引物<400>9ccatatgtac gaaccaaaac cggaacatcg ctttaccttt 40<210>10<211>39<212>DNA
<213>人工序列<220>
<223>下游引物<400>10aaggaaaaaa gcggccgctc aaccacgcac acccaggag 39<210>11<211>27<212>DNA<213>人工序列<220>
<223>N91D引物<400>11atggtcaccg ccgacctctt ctccgac 27<210>12<211>26<212>DNA<213>人工序列<220>
<223>D375G引物<400>12tggaacgcct gggccagctg gcggtg 26<210>13<211>26<212>DNA<213>人工序列<220>
<223>V144A引物<400>13agggagctga ggcggaggcc acgggc 26<210>14<211>27<212>DNA<213>人工序列<220>
<223>K355A引物<400>14cgaagccctc gcgcgggcgg agcttcc 2權(quán)利要求
1.突變的木糖異構(gòu)酶，其氨基酸序列為SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.7所示。
2.編碼權(quán)利要求1所述的突變木糖異構(gòu)酶的核苷酸序列。
3.根據(jù)權(quán)利要求2所述的核苷酸序列，其序列為SEQ ID NO.2、SEQ ID NO.4、SEQID NO.6或SEQ ID NO.8所示。
4.含有權(quán)利要求2所述核苷酸序列的重組載體。
5.根據(jù)權(quán)利要求4所述的重組載體，其特征在于該表達(dá)載體為含有權(quán)利要求2所述核苷酸序列的重組質(zhì)粒。
6.根據(jù)權(quán)利要求4所述的重組載體，其特征在于該重組質(zhì)粒為pET-22b(+)-xylA-N91D/D375G、pET-22b(+)-xylA-N91D、pET-22b(+)-xylA-N91D/V144A、pET-22b(+)-xylA-N91D/K355A。
7.導(dǎo)入權(quán)利要求2所述核苷酸序列或權(quán)利要求4所述表達(dá)載體的轉(zhuǎn)化體，其宿主為細(xì)菌或酵母菌。
8.根據(jù)權(quán)利要求7所述的轉(zhuǎn)化體，其特征在于所述細(xì)菌為大腸桿菌。
9.權(quán)利要求1所述的木糖異構(gòu)酶在高果糖漿生產(chǎn)中的應(yīng)用。
全文摘要
本發(fā)明公開了突變的木糖異構(gòu)酶及其基因和用途。該木糖異構(gòu)酶的氨基酸序列為SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.7所示。該木糖異構(gòu)酶比野生酶活性有較大提高，可用于高果糖漿的生產(chǎn)。突變木糖異構(gòu)酶的基因可用于構(gòu)建利用木糖的重組菌。
文檔編號C12N15/61GK1966678SQ20061009799
公開日2007年5月23日 申請日期2006年11月27日 優(yōu)先權(quán)日2006年11月27日
發(fā)明者嚴(yán)明, 許偉, 許琳, 丁莉, 歐陽平凱 申請人:南京工業(yè)大學(xué)