CN1966678A - 突变的木糖异构酶及其基因和用途 - Google Patents
突变的木糖异构酶及其基因和用途 Download PDFInfo
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Abstract
本发明公开了突变的木糖异构酶及其基因和用途。该木糖异构酶的氨基酸序列为SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.7所示。该木糖异构酶比野生酶活性有较大提高，可用于高果糖浆的生产。突变木糖异构酶的基因可用于构建利用木糖的重组菌。
Description
技术领域
本发明属于酶的基因工程和酶生化工程领域。本发明涉及活性提高的突变木糖异构酶，本发明还涉及编码该突变木糖异构酶的基因。
背景技术
木糖异构酶(Xylose Isomerase，XI)(EC 5.3.1.5)又称葡萄糖异构酶，可以催化D-木糖到D-木酮糖及D-葡萄糖到D-果糖的异构化反应，在微生物体内糖的代谢过程中起着重要的作用，并已经用于高果糖浆的工业生产[Bhosale SH，Rao MB and Deshpande VV.Molecular and Industrial Aspects of Glucose Isomerase.Microbiol.Rev.，1996，60：280-300]。来自Thermus thermophilus HB8的木糖异构酶[Zeikus JG，Vieille C，SavchenkoA.Thermozymes：biotechnology and structure function relationships.Extremophiles，1998，2(3)：179-183]，最适反应温度约为85℃，具有极高的稳定性，以及对有机溶剂、去污剂和变性剂的较强抗性，已被广泛应用到食品、医药等各个行业的工业生产中。由于高温会导致葡萄糖异构过程副产物的形成，因此高果糖浆生产中主要反应温度控制在60-70℃。目前国内主要采用的是来自链霉菌的葡萄糖异构酶，但其稳定性不够理想，导致果糖产量不高，生产工艺复杂。因此， 提高Thermus thermophilus HB8葡萄糖异构酶60-70℃附近的催化活性对于高果糖浆的工业生产具有重要意义。
近年来，随着能源危机和环境污染的日益严重，对广泛存在于可再生的木质纤维素中的木糖利用也成为研究热点之一。国内外科研人员一直致力于构建能够发酵木糖的重组酵母。迄今为止，已经报道了九种不同菌属中的XI在酿酒酵母中进行表达，但除来源于Thermus Thermophilus，Thermoanaerobacter Tengcongensis MB4T和Piromyces sp.的木糖异构酶基因之外，都没有表达出活性。Thermus thermophilus XI在木糖发酵乙醇方面的应用潜力及其极端的热稳定性引起了国内外的极大关注[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.，1996，62：4648-4651]，但由于Thermus thermophilus XI在常温下活性较低导致重组菌对木糖的利用并不理想。选择在常温下高活力的木糖异构酶已经成为这一领域的主要研究方向之一[沈煜，王颖，鲍晓明，曲音波.酿酒酵母木糖发酵酒精途径工程的研究进展.生物工程学报，2003，19(5)：636-640.]。美国专利(US6475768)报道通过对Thermus thermophilus HB8的木糖异构酶进行随机PCR突变，获得三种常温下活性提高的木糖异构酶。近年来，国内在发酵木糖生产乙醇重组酵母构建方面也取得了可喜的进展，通过在酿酒酵母工业菌株中表达Thermus thermophilus木糖异构酶，建立了木糖代谢途径(WANG Y.，SHI W.L.，LIU X.Y.，et al.BiotechnologyLetters，2004，26(11)：885-890)，但由于木糖异构酶活性较低使得重组菌对木糖的利用并不理想。
定点突变是蛋白质工程广泛采用的有效技术。它是根据酶的结构、功能和作用机理等信息，在基因水平上精确改变酶分子中的特定氨基酸残基，使酶的性质最佳化。利用生物信息学工具进行蛋白质的设计，并结合定点突变技术提高Thermus thermophilus HB8中木糖异构酶的活性，尚未见有关文献报道。
发明内容
本发明的目的是提供一种突变的木糖异构酶。该突变的木糖异构酶与野生菌Thermus thermophilus HB8中的木糖异构酶相比，活性有所提高。
本发明另一个目的是提供编码上述突变木糖异构酶的基因(核苷酸序列)。这些基因可用于构建利用木糖的重组菌。
本发明再一个目的是提供上述突变木糖异构酶在高果糖浆生产中的应用。
上述突变木糖异构酶的氨基酸序列中至少有一个氨基酸不同于野生菌Thermusthermophilus HB8中木糖异构酶的氨基酸序列(Swiss-Prot entry：P26997)。
本发明的目的可以通过下列措施来达到：
一种突变的木糖异构酶，其氨基酸序列为SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.7所示。
这些序列分别表示原木糖异构酶氨基酸序列(即野生型Thermus thermophilus HB8xylA基因所表达的氨基酸序列)的91位的天冬酰胺残基以及375位的天冬氨酸残基分别被天冬氨酸残基和甘氨酸残基取代，91位的天冬酰胺残基被天冬氨酸残基取代，91位的天冬酰胺残基以及355位的赖氨酸残基分别被天冬氨酸残基和丙氨酸残基取代，91位的天冬酰胺残基以及144位的缬氨酸残基分别被天冬氨酸残基和丙氨酸残基取代。
编码所述的突变木糖异构酶的核苷酸序列。
所述的核苷酸序列，其序列为SEQ ID NO.2、SEQ ID NO.4、SEQ ID NO.6或SEQ IDNO.8所示。
含有权利要求2所述核苷酸序列的重组载体。常用载体如质粒、噬菌体等。
所述的重组载体，该表达载体为含有上述核苷酸序列的重组质粒。该重组质粒为pET-22b(+)-xylA-N91D/D375G、pET-22b(+)-xylA-N91D、pET-22b(+)-xylA-N91D/V144A、pET-22b(+)-xylA-N91D/K355A。
导入上述核苷酸序列或重组载体的转化体，其宿主为细菌或酵母菌。所述细菌常用大肠杆菌。
所述的木糖异构酶在高果糖浆生产中的应用。
本发明的有益效果：
本发明提供的突变型木糖异构酶活性有所提高。本发明还提供了该突变型木糖异构酶基因序列，这些序列可用于构建利用木糖的重组菌。本发明所提供的突变木糖异构酶可用于高果糖浆的生产。
附图说明
图1是重组质粒pET-22b(+)-xylA构建图。
图2是表达产物的SDS-PAGE分析。图中1、2、3、4分别代表含有突变质粒pET-22b(+)-xylA-N91D/D375G、pET-22b(+)-xylA-N91D、pET-22b(+)-xylA-N91D/V144A、pET-22b(+)-xylA-N91D/K355A的重组大肠杆菌Rosseta中的表达产物。
图3是不同温度下本发明突变型木糖异构酶活性比较。
具体实施方式
以下通过实施例对本发明作进一步的阐述，但不限制本发明。
说明：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位氨基酸为Asp，本发明中类似的表述均为此方式。
实施例中未注明具体条件的实验方法，基本上都按照Sambrook，J等人编著的《分子克隆实验指南(第3版)》(Molecular Cloning：A Laboratory Manual，3rded.黄培堂等译，科学出版社.2002.8)中所述的条件及方法或按照材料提供商所建议的条件及方法进行，其它没有详细描述的技术相应于本领域人员来说是熟知的标准方法。
本发明的材料：本申请中采用的未特别限定来源的微生物、质粒或其他表达载体以及培养基均有商品供应或以别的途径能为公众所得，它们仅作举例，对本发明不是唯一的，可分别用其它适合的工具和生物材料来代替。
实施例1：野生型Thermus thermophilus HB8木糖异构酶表达重组质粒的构建、筛选与鉴定。
嗜热栖热菌(Thermus thermophilus HB8)购于美国菌种保藏中心(ATCC)，ATCC编号为27634。载体pET-22b(+)购于Novagen公司。
酵母粉4g/L，蛋白胨8g/L，NaCl 2.0g/L，将pH调至7.0，高压蒸气灭菌121℃，15min。将Thermus thermophilus HB8接种于上述培养基中，置75℃摇床，200rpm，培养12-16小时。8000rpm，离心15min，收集菌体。
运用基因组提取试剂盒(上海华舜生物工程有限公司)，提取Thermus Thermophilus基因组。以Thermus Thermophilus HB8总DNA为模板，使用如下PCR引物(上海博亚公司合成)：
正向引物：5’-CCATATGTACGAACCAAAACCGGAACATCGCTTTACCTTT-3’(SEQ ID NO.9)
反向引物：5’-AAGGAAAAAAGCGGCCGCTCAACCACGCACACCCAGGAG-3’(SEQ ID NO.10)
PCR扩增木糖异构酶基因xylA。PCR循环参数为：95℃，2min；94℃，30s；70℃，50s；68℃，1.5min，共进行35个循环。
用DNA回收试剂盒(Takara公司)对扩增出的基因进行回收纯化。用限制性内切酶NdeI和NotI双酶切基因xylA和载体pET-22b(+)，回收后将上述双酶切过的基因xylA及pET-22b(+)载体连接，运用常规的基因工程技术将连接液转化到大肠杆菌DH5α感受态细胞，提取质粒。运用上述引物进行扩增，鉴定基因xylA确定连接到表达载体pET-22b(+)上。经鉴定过的重组质粒为pET-22b(+)-xylA。构建过程如图1所示。
实施例2：木糖异构酶基因的定点突变
根据多点突变试剂盒(QuikChange Multi Site-Directed Mutagenesis Kit，购自STRATAGENE公司)的要求，设计N91D引物，5’-ATGGTCACCGCC
GACCTCTTCTCCGAC-3’(SEQ ID NO.11)以pET-22b(+)-xylA质粒为模板进行PCR，将Asn91突变为Asp91。按照多点突变试剂盒说明书，用限制性内切酶Dpn I消化原始质粒模板pET-22b(+)-xylA，将PCR产物转化高效率的感受态细胞。运用常规的基因工程技术对转化后的感受态细胞进行培养，提取质粒进行测序鉴定，得到含有突变基因的重组质粒pET-22b(+)-xylA-N91D。
运用D375G引物5’-TGGAACGCCTG
GGCCAGCTGGCGGTG-3’(SEQ ID NO.12)，以pET-22b(+)-xylA-N91D质粒为模板进行PCR，将氨基酸残基Asp375突变为Gly375。其它步骤同上，即可得到同时具有N91D和D375G两个突变位点的重组质粒pET-22b(+)-xylA-N91D/D375G。
运用V144A引物5’-AGGGAGCTGAG
GCGGAGGCCACGGGC-3’(SEQ ID NO.13)以pET-22b(+)-xylA-N91D质粒为模板进行PCR，将氨基酸残基Val144突变为Ala144。按上述步骤操作，即可获得同时具有N91D和V144A两个突变位点的重组质粒pET-22b(+)-xylA-N91D/V144A。
运用K355A引物5’-CGAAGCCCTC
GCGCGGGCGGAGCTTCC-3’(SEQ ID NO.14)以pET-22b(+)-xylA-N91D质粒为模板进行PCR，将氨基酸残基Lys355突变为Ala355。按上述步骤操作，即可获得同时具有N91D和K355A两个突变位点的重组质粒pET-22b(+)-xylA-N91D/K355A。
实施例3：突变的木糖异构酶在大肠杆菌Rosseta中的表达
分别将实施例2制备的含有突变基因的重组质粒转化到商业化的表达宿主大肠杆菌Rosetta(DE3)中，挑取单菌落到含75μg/mL氨苄青霉素和34μg/ml氯霉素的LB培养液，37℃振荡培养过夜，然后按2％(v/v)接种量接种到含有75μg/mL氨苄青霉素和34μg/mL氯霉素的LB培养液中，37℃培养至OD600约为0.6时，加入IPTG至终浓度0.9mM，诱导表达7h，取一定体积菌液8000rpm，4℃，离心15min收集菌体。表达产物的SDS-PAGE分析见图2。
实施例4：木糖异构酶活性测定方法
1.突变木糖异构酶的纯化
分别取10mL实施例3中诱导后的培养液8000rpm，4℃，离心15min收集菌体，用无菌水洗涤两次后，菌体重悬于0.5mL pH7.550mM Tris-HCl缓冲液中，冰浴中超声破碎细胞，12,000rpm，4℃离心10min取上清即为粗酶液。将粗酶液置于70℃水浴，加热40min，12,000rpm，4℃离心10min可去除绝大部分杂蛋白。取上清后，再通过常规蛋白纯化方法进行纯化，得到氨基酸序列分别为SEQ ID NO.1、SEQ ID NO.3、SEQ IDNO.5、SEQ ID NO.7所示的纯化的突变木糖异构酶。
2.酶活力测定
木糖异构酶活力测定采用两步法，第一步：分别取纯化的突变木糖异构酶50μL，1.5M D-木糖50μL，30mM MgCl2 50μL，在不同温度下反应15min，冰浴终止反应；第二步：取10μL上述反应液，加入1μL(0.08U/μL)山梨醇脱氢酶，3μL 25mM NADH，186μLpH7.550mM Tris-HCl缓冲液，加入96孔微培养板。使用酶标仪在25℃下检测OD340的光吸收变化。酶活力单位(U)定义为每分钟催化产生1μmol木酮糖所需的酶量。通过ΔA340/min的变化根据标准曲线方程Y＝2805.81357X(其中Y：mΔA340/min；X：木酮糖的浓度，单位mol/L)确定木糖异构酶的活力，结果见图3，结果显示，在N91D突变的基础上，引入D375G，K355A或V144A突变可以使得木糖异构酶活力提高。这些木糖异构酶在60-70℃附近的催化活性较好，既可提高高果糖浆的生产效率，又能减少由于高温导致葡萄糖异构过程副产物的形成，适用于高果糖浆的生产。
序列表
<110>南京工业大学
<120>突变的木糖异构酶及其基因和用途
<160>14
<210>1
<211>387
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<213>人工序列
<220>
<223>突变的木糖异构酶(N91D/D375G)氨基酸序列
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Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
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Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg
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Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu
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Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg
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Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys
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Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala
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Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser
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Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu
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Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val
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Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala
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Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala
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Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu
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Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
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Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg
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Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu
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Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu
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Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255
Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu
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Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe
275 280 285
Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala
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Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315
Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala
320 325 330
Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345
Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro
350 355 360
Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Gly
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Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly
380 385
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<220>
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<222>(1)…(1164)
<223>突变的木糖异构酶(N91D/D375G)核苷酸序列
<400>2
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Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
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act gtg ggc aat gtg ggc cgt gat ccc ttc ggg gac gcg gtt cgg 90
Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg
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Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu
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Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg
50 55 60
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Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys
65 70 75
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Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala
80 85 90
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Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser
95 100 105
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Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu
110 115 120
acc atg gac ctg ggg gca gag ctt ggg gcc gag atc tac gtg gtc 405
Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val
125 130 135
tgg ccg ggc cgg gag gga gct gag gtg gag gcc acg ggc aag gcc 450
Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala
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cgg aag gtc tgg gac tgg gtg cgg gag gcg ctg aac ttc atg gcc 495
Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala
155 160 165
gcc tac gcc gag gac cag gga tac ggg tac cgg ttt gcc ctc gag 540
Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu
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ccc aag cct aac gag ccc cgg ggg gac att tac ttc gcc acc gtg 585
Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
185 190 195
ggg agc atg ctc gcc ttt att cat acc ctg gac cgg ccc gag cgc 630
Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg
200 205 210
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Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu
215 220 225
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Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu
230 235 240
ttc cac att gac ctc aac gac caa cgg atg agc cgg ttt gac cag 765
Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255
gac ctc cgc ttc ggc tcg gag aac ctc aag gcg gcc ttt ttc ctg 810
Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu
260 265 270
gtg gac ctc ctg gaa agc tcc ggc tac cag ggc ccc cgc cac ttt 855
Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe
275 280 285
gac gcc cac gcc ctg cgt acc gag gac gaa gaa ggg gtt tgg gcc 900
Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala
290 295 300
ttc gcc cga ggc tgc atg cgt acc tac ctg atc tta aag gaa agg 945
Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315
gct gaa gcc ttc cgc gag gat ccc gag gtc aag gag ctt ctt gcc 990
Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala
320 325 330
gct tac tat caa gaa gat cct gcg gcc ttg gcc ctt ttg ggc ccc 1035
Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345
tac tcc cgc gag aag gcc gaa gcc ctc aag cgg gcg gag ctt ccc 1080
Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro
350 355 360
ctc gag gcc aag cgg cgc cgg ggt tat gcc ctg gaa cgc ctg ggc 1125
Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Gly
365 370 375
cag ctg gcg gtg gag tac ctc ctg ggg gtg cgg ggg tga 1164
Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly
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<210>3
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<212>PRT
<213>人工序列
<220>
<223>突变的木糖异构酶(N91D)氨基酸序列
<400>3
Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg
20 25 30
Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu
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Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg
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Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys
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Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala
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Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser
95 100 105
Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu
110 115 120
Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val
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Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala
140 145 150
Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala
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Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu
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Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
185 190 195
Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg
200 205 210
Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu
215 220 225
Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu
230 235 240
Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255
Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu
260 265 270
Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe
275 280 285
Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala
290 295 300
Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315
Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala
320 325 330
Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345
Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro
350 355 360
Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp
365 370 375
Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly
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<210>4
<211>1164
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)…(1164)
<223>突变的木糖异构酶(N91D)核苷酸序列
<400>4
gtg tac gag ccc aaa ccg gag cac agg ttt acc ttt ggc ctt tgg 45
Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
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Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg
20 25 30
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Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu
35 40 45
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Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg
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Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys
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Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala
80 85 90
gac ctc ttc tcc gac cct gct ttc aag gac ggg gcc ttc acg agc 315
Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser
95 100 105
ccg gac cct tgg gtt cgg gcc tat gcc ttg cgg aag agc ctg gag 360
Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu
110 115 120
acc atg gac ctg ggg gca gag ctt ggg gcc gag atc tac gtg gtc 405
Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val
125 130 135
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Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala
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Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala
155 160 165
gcc tac gcc gag gac cag gga tac ggg tac cgg ttt gcc ctc gag 540
Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu
170 175 180
ccc aag cct aac gag ccc cgg ggg gac att tac ttc gcc acc gtg 585
Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
185 190 195
ggg agc atg ctc gcc ttt att cat acc ctg gac cgg ccc gag cgc 630
Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg
200 205 210
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Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu
215 220 225
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Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu
230 235 240
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Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255
gac ctc cgc ttc ggc tcg gag aac ctc aag gcg gcc ttt ttc ctg 810
Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu
260 265 270
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Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe
275 280 285
gac gcc cac gcc ctg cgt acc gag gac gaa gaa ggg gtt tgg gcc 9O0
Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala
290 295 300
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Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315
gct gaa gcc ttc cgc gag gat ccc gag gtc aag gag ctt ctt gcc 990
Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala
320 325 330
gct tac tat caa gaa gat cct gcg gcc ttg gcc ctt ttg ggc ccc 1035
Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345
tac tcc cgc gag aag gcc gaa gcc ctc aag cgg gcg gag ctt ccc 1080
Tyr Ser Arg GLu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro
350 355 360
ctc gag gcc aag cgg cgc cgg ggt tat gcc ctg gaa cgc ctg gac 1125
Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp
365 370 375
cag ctg gcg gtg gag tac ctc ctg ggg gtg cgg ggg tga 1164
Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly
380 385
<210>5
<211>387
<212>PRT
<213>人工序列
<220>
<223>突变的木糖异构酶(N91D/K355A)氨基酸序列
<400>5
Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg
20 25 30
Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu
35 40 45
Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg
50 55 60
Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys
65 70 75
Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala
80 85 90
Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser
95 100 105
Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu
110 115 120
Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val
125 130 135
Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala
140 145 150
Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala
155 160 165
Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu
170 175 180
Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
185 190 195
Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg
200 205 210
Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu
215 220 225
Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu
230 235 240
Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255
Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu
260 265 270
Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe
275 280 285
Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala
290 295 300
Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315
Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala
320 325 330
Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345
Tyr Ser Arg Glu Lys Ala Glu Ala Leu Ala Arg Ala Glu Leu Pro
350 355 360
Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp
365 370 375
Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly
380 385
<210>6
<211>1164
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)…(1164)
<223>突变的木糖异构酶(N91D/K355A)核苷酸序列
<400>6
gtg tac gag ccc aaa ccg gag cac agg ttt acc ttt ggc ctt tgg 45
Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
act gtg ggc aat gtg ggc cgt gat ccc ttc ggg gac gcg gtt cgg 90
Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg
20 25 30
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Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu
35 40 45
ggg gcc tac ggg gta aac ctt cac gac gag gac ctg atc ccg cgg 180
Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg
50 55 60
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Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys
65 70 75
aag gct ctc gat gaa acc ggc ctc aag gtc ccc atg gtc acc gcc 270
Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala
80 85 90
gac ctc ttc tcc gac cct gct ttc aag gac ggg gcc ttc acg agc 315
Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser
95 100 105
ccg gac cct tgg gtt cgg gcc tat gcc ttg cgg aag agc ctg gag 360
Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu
110 115 120
acc atg gac ctg ggg gca gag ctt ggg gcc gag atc tac gtg gtc 405
Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val
125 130 135
tgg ccg ggc cgg gag gga gct gag gtg gag gcc acg ggc aag gcc 450
Trp Pro Gly Arg Glu Gly Ala Glu Val Glu Ala Thr Gly Lys Ala
140 145 150
cgg aag gtc tgg gac tgg gtg cgg gag gcg ctg aac ttc atg gcc 495
Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala
155 160 165
gcc tac gcc gag gac cag gga tac ggg tac cgg ttt gcc ctc gag 540
Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu
170 175 180
ccc aag cct aac gag ccc cgg ggg gac att tac ttc gcc acc gtg 585
Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
185 190 195
ggg agc atg ctc gcc ttt att cat acc ctg gac cgg ccc gag cgc 630
Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg
200 205 210
ttc ggc ctg aac ccc gag ttc gcc cac gag acc atg gcc ggg ctt 675
Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu
215 220 225
aac ttt gtc cac gcc gtg gcc cag gct ctc gac gcc ggg aag ctt 720
Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu
230 235 240
ttc cac att gac ctc aac gac caa cgg atg agc cgg ttt gac cag 765
Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255
gac ctc cgc ttc ggc tcg gag aac ctc aag gcg gcc ttt ttc ctg 810
Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu
260 265 270
gtg gac ctc ctg gaa agc tcc ggc tac cag ggc ccc cgc cac ttt 855
Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe
275 280 285
gac gcc cac gcc ctg cgt acc gag gac gaa gaa ggg gtt tgg gcc 900
Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala
290 295 300
ttc gcc cga ggc tgc atg cgt acc tac ctg atc tta aag gaa agg 945
Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315
gct gaa gcc ttc cgc gag gat ccc gag gtc aag gag ctt ctt gcc 990
Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala
320 325 330
gct tac tat caa gaa gat cct gcg gcc ttg gcc ctt ttg ggc ccc 1035
Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345
tac tcc cgc gag aag gcc gaa gcc ctc gcg cgg gcg gag ctt ccc 1080
Tyr Ser Arg Glu Lys Ala Glu Ala Leu Ala Arg Ala Glu Leu Pro
350 355 360
ctc gag gcc aag cgg cgc cgg ggt tat gcc ctg gaa cgc ctg gac 1125
Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp
365 370 375
cag ctg gcg gtg gag tac ctc ctg ggg gtg cgg ggg tga 1164
Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly
380 385
<210>7
<211>387
<212>PRT
<213>人工序列
<220>
<223>突变的木糖异构酶(N91D/V144A)氨基酸序列
<400>7
Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg
20 25 30
Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu
35 40 45
Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg
50 55 60
Gly Thr Pro Pro Gln Glu Arg Asp Gln Ile Val Arg Arg Phe Lys
65 70 75
Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala
80 85 90
Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser
95 100 105
Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu
110 115 120
Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val
125 130 135
Trp Pro Gly Arg Glu Gly Ala Glu Ala Glu Ala Thr Gly Lys Ala
140 145 150
Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala
155 160 165
Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu
170 175 180
Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
185 190 195
Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg
200 205 210
Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu
215 220 225
Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu
230 235 240
Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255
Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu
260 265 270
Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe
275 280 285
Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala
290 295 300
Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315
Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala
320 325 330
Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345
Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro
350 355 360
Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp
365 370 375
Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly
380 385
<210>8
<211>1164
<212>DNA
<213>人工序列
<220>
<221>CDS
<222>(1)…(1164)
<223>突变的木糖异构酶(N91D/V144A)核苷酸序列
<400>8
gtg tac gag ccc aaa ccg gag cac agg ttt acc ttt ggc ctt tgg 45
Met Tyr Glu Pro Lys Pro Glu His Arg Phe Thr Phe Gly Leu Trp
1 5 10 15
act gtg ggc aat gtg ggc cgt gat ccc ttc ggg gac gcg gtt cgg 90
Thr Val Gly Asn Val Gly Arg Asp Pro Phe Gly Asp Ala Val Arg
20 25 30
gag agg ctg gac ccg gtt tac gtg gtt cat aag ctg gcg gag ctt 135
Glu Arg Leu Asp Pro Val Tyr Val Val His Lys Leu Ala Glu Leu
35 40 45
ggg gcc tac ggg gta aac ctt cac gac gag gac ctg atc ccg cgg 180
Gly Ala Tyr Gly Val Asn Leu His Asp Glu Asp Leu Ile Pro Arg
50 55 60
ggc 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 Lys
65 70 75
aag gct ctc gat gaa acc ggc ctc aag gtc ccc atg gtc acc gcc 270
Lys Ala Leu Asp Glu Thr Gly Leu Lys Val Pro Met Val Thr Ala
80 85 90
gac ctc ttc tcc gac cct gct ttc aag gac ggg gcc ttc acg agc 315
Asp Leu Phe Ser Asp Pro Ala Phe Lys Asp Gly Ala Phe Thr Ser
95 100 105
ccg gac cct tgg gtt cgg gcc tat gcc ttg cgg aag agc ctg gag 360
Pro Asp Pro Trp Val Arg Ala Tyr Ala Leu Arg Lys Ser Leu Glu
110 115 120
acc atg gac ctg ggg gca gag ctt ggg gcc gag atc tac gtg gtc 405
Thr Met Asp Leu Gly Ala Glu Leu Gly Ala Glu Ile Tyr Val Val
125 130 135
tgg ccg ggc cgg gag gga gct gag gcg gag gcc acg ggc aag gcc 450
Trp Pro Gly Arg Glu Gly Ala Glu Ala Glu Ala Thr Gly Lys Ala
140 145 150
cgg aag gtc tgg gac tgg gtg cgg gag gcg ctg aac ttc atg gcc 495
Arg Lys Val Trp Asp Trp Val Arg Glu Ala Leu Asn Phe Met Ala
155 160 165
gcc tac gcc gag gac cag gga tac ggg tac cgg ttt gcc ctc gag 540
Ala Tyr Ala Glu Asp Gln Gly Tyr Gly Tyr Arg Phe Ala Leu Glu
170 175 180
ccc aag cct aac gag ccc cgg ggg gac att tac ttc gcc acc gtg 585
Pro Lys Pro Asn Glu Pro Arg Gly Asp Ile Tyr Phe Ala Thr Val
185 190 195
ggg agc atg ctc gcc ttt att cat acc ctg gac cgg ccc gag cgc 630
Gly Ser Met Leu Ala Phe Ile His Thr Leu Asp Arg Pro Glu Arg
200 205 210
ttc ggc ctg aac ccc gag ttc gcc cac gag acc atg gcc ggg ctt 675
Phe Gly Leu Asn Pro Glu Phe Ala His Glu Thr Met Ala Gly Leu
215 220 225
aac ttt gtc cac gcc gtg gcc cag gct ctc gac gcc ggg aag ctt 720
Asn Phe Val His Ala Val Ala Gln Ala Leu Asp Ala Gly Lys Leu
230 235 240
ttc cac att gac ctc aac gac caa cgg atg agc cgg ttt gac cag 765
Phe His Ile Asp Leu Asn Asp Gln Arg Met Ser Arg Phe Asp Gln
245 250 255
gac ctc cgc ttc ggc tcg gag aac ctc aag gcg gcc ttt ttc ctg 810
Asp Leu Arg Phe Gly Ser Glu Asn Leu Lys Ala Ala Phe Phe Leu
260 265 270
gtg gac ctc ctg gaa agc tcc ggc tac cag ggc ccc cgc cac ttt 855
Val Asp Leu Leu Glu Ser Ser Gly Tyr Gln Gly Pro Arg His Phe
275 280 285
gac gcc cac gcc ctg cgt acc gag gac gaa gaa ggg gtt tgg gcc 900
Asp Ala His Ala Leu Arg Thr Glu Asp Glu Glu Gly Val Trp Ala
290 295 300
ttc gcc cga ggc tgc atg cgt acc tac ctg atc tta aag gaa agg 945
Phe Ala Arg Gly Cys Met Arg Thr Tyr Leu Ile Leu Lys Glu Arg
305 310 315
gct gaa gcc ttc cgc gag gat ccc gag gtc aag gag ctt ctt gcc 990
Ala Glu Ala Phe Arg Glu Asp Pro Glu Val Lys Glu Leu Leu Ala
320 325 330
gct tac tat caa gaa gat cct gcg gcc ttg gcc ctt ttg ggc ccc 1035
Ala Tyr Tyr Gln Glu Asp Pro Ala Ala Leu Ala Leu Leu Gly Pro
335 340 345
tac tcc cgc gag aag gcc gaa gcc ctc aag cgg gcg gag ctt ccc 1080
Tyr Ser Arg Glu Lys Ala Glu Ala Leu Lys Arg Ala Glu Leu Pro
350 355 360
ctc gag gcc aag cgg cgc cgg ggt tat gcc ctg gaa cgc ctg gac 1125
Leu Glu Ala Lys Arg Arg Arg Gly Tyr Ala Leu Glu Arg Leu Asp
365 370 375
cag ctg gcg gtg gag tac ctc ctg ggg gtg cgg ggg tga 1164
Gln Leu Ala Val Glu Tyr Leu Leu Gly Val Arg Gly
380 385
<210>9
<211>40
<212>DNA
<213>人工序列
<220>
<223>上游引物
<400>9
ccatatgtac gaaccaaaac cggaacatcg ctttaccttt 40
<210>10
<211>39
<212>DNA
<213>人工序列
<220>
<223>下游引物
<400>10
aaggaaaaaa gcggccgctc aaccacgcac acccaggag 39
<210>11
<211>27
<212>DNA
<213>人工序列
<220>
<223>N91D引物
<400>11
atggtcaccg ccgacctctt ctccgac 27
<210>12
<211>26
<212>DNA
<213>人工序列
<220>
<223>D375G引物
<400>12
tggaacgcct gggccagctg gcggtg 26
<210>13
<211>26
<212>DNA
<213>人工序列
<220>
<223>V144A引物
<400>13
agggagctga ggcggaggcc acgggc 26
<210>14
<211>27
<212>DNA
<213>人工序列
<220>
<223>K355A引物
<400>14
cgaagccctc gcgcgggcgg agcttcc 27
Claims (9)
1、突变的木糖异构酶，其氨基酸序列为SEQ ID NO.1、SEQ ID NO.3、SEQ ID NO.5或SEQ ID NO.7所示。
2、编码权利要求1所述的突变木糖异构酶的核苷酸序列。
3、根据权利要求2所述的核苷酸序列，其序列为SEQ ID NO.2、SEQ ID NO.4、SEQID NO.6或SEQ ID NO.8所示。
4、含有权利要求2所述核苷酸序列的重组载体。
5、根据权利要求4所述的重组载体，其特征在于该表达载体为含有权利要求2所述核苷酸序列的重组质粒。
6、根据权利要求4所述的重组载体，其特征在于该重组质粒为pET-22b(+)-xylA-N91D/D375G、pET-22b(+)-xylA-N91D、pET-22b(+)-xylA-N91D/V144A、pET-22b(+)-xylA-N91D/K355A。
7、导入权利要求2所述核苷酸序列或权利要求4所述表达载体的转化体，其宿主为细菌或酵母菌。
8、根据权利要求7所述的转化体，其特征在于所述细菌为大肠杆菌。
9、权利要求1所述的木糖异构酶在高果糖浆生产中的应用。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698839B (zh) * | 2008-12-26 | 2012-05-23 | 北京大学 | 尿苷二磷酸木糖异构酶及其编码基因与应用 |
| WO2012113120A1 (zh) * | 2011-02-22 | 2012-08-30 | 山东大学 | 一种编码木糖异构酶的核酸分子及其编码的木糖异构酶 |
| CN103710329A (zh) * | 2013-12-24 | 2014-04-09 | 山西天骄食业有限公司 | 一种基因工程技术制备共表达重组酶的方法 |
| CN109468305A (zh) * | 2017-12-29 | 2019-03-15 | 吉林中粮生化有限公司 | 木糖异构酶突变体、编码该酶的dna分子、导入该dna分子的重组菌株及它们的应用 |
| CN111235139A (zh) * | 2018-11-29 | 2020-06-05 | 国投生物科技投资有限公司 | 木糖异构酶及其编码基因和制备方法、载体和宿主细胞以及他们的应用 |
| CN111235138A (zh) * | 2018-11-29 | 2020-06-05 | 国投生物科技投资有限公司 | 木糖异构酶及其编码基因和制备方法、载体和宿主细胞以及他们的应用 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0351029B1 (en) * | 1988-07-15 | 2002-03-06 | Genencor International, Inc. | Novel glucose isomerase enzymes and their use |
| EP1264883B1 (en) * | 1990-01-04 | 2010-04-14 | Danisco US Inc. | Glucose Isomerases having altered substrate specificity |
| SE9901298D0 (sv) * | 1999-04-09 | 1999-04-09 | Forskarpatent I Syd Ab | Xylose isomerase with improved kinetic properties |
| CN1884508A (zh) * | 2006-06-29 | 2006-12-27 | 南京工业大学 | 一种突变的木糖异构酶及其基因 |
-
2006
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101698839B (zh) * | 2008-12-26 | 2012-05-23 | 北京大学 | 尿苷二磷酸木糖异构酶及其编码基因与应用 |
| WO2012113120A1 (zh) * | 2011-02-22 | 2012-08-30 | 山东大学 | 一种编码木糖异构酶的核酸分子及其编码的木糖异构酶 |
| US8586336B2 (en) | 2011-02-22 | 2013-11-19 | Shandong University | Nucleic acid molecule encoding xylose isomerase and xylose isomerase encoded by the nucleic acid molecule |
| CN103710329A (zh) * | 2013-12-24 | 2014-04-09 | 山西天骄食业有限公司 | 一种基因工程技术制备共表达重组酶的方法 |
| CN109468305A (zh) * | 2017-12-29 | 2019-03-15 | 吉林中粮生化有限公司 | 木糖异构酶突变体、编码该酶的dna分子、导入该dna分子的重组菌株及它们的应用 |
| CN109468305B (zh) * | 2017-12-29 | 2021-11-16 | 吉林中粮生化有限公司 | 木糖异构酶突变体、编码该酶的dna分子、导入该dna分子的重组菌株及它们的应用 |
| CN111235139A (zh) * | 2018-11-29 | 2020-06-05 | 国投生物科技投资有限公司 | 木糖异构酶及其编码基因和制备方法、载体和宿主细胞以及他们的应用 |
| CN111235138A (zh) * | 2018-11-29 | 2020-06-05 | 国投生物科技投资有限公司 | 木糖异构酶及其编码基因和制备方法、载体和宿主细胞以及他们的应用 |
| CN111235139B (zh) * | 2018-11-29 | 2021-08-20 | 国投生物科技投资有限公司 | 木糖异构酶及其编码基因和制备方法、载体和宿主细胞以及他们的应用 |
| CN111235138B (zh) * | 2018-11-29 | 2021-08-24 | 国投生物科技投资有限公司 | 木糖异构酶及其编码基因和制备方法、载体和宿主细胞以及他们的应用 |
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|---|---|
| CN100445376C (zh) | 2008-12-24 |
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