CN111019918A - 一种糖基转移酶突变体及其应用 - Google Patents

一种糖基转移酶突变体及其应用 Download PDF

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CN111019918A
CN111019918A CN201911140138.0A CN201911140138A CN111019918A CN 111019918 A CN111019918 A CN 111019918A CN 201911140138 A CN201911140138 A CN 201911140138A CN 111019918 A CN111019918 A CN 111019918A
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蔡萍
王三永
卫娜
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徐勇
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Abstract

本发明公开了一种糖基转移酶的突变体及其编码基因，还公开了含有上述基因的重组质粒和工程菌株，以及上述糖基转移酶的突变体、编码基因、重组质粒或工程菌在甜味剂甜菊醇糖苷糖基化修饰中的应用。该糖基转移酶的突变体的转糖基活性较之野生型显著提高。在甜味剂甜菊糖开发应用领域具有极大的潜力。

Description

一种糖基转移酶突变体及其应用
技术领域
本发明属于生物酶技术领域，具体涉及一种糖基转移酶突变体及其应用。
背景技术
甜味剂是一类对食品的加工特性和口感起重要作用的食品添加剂，随着全世界肥胖症、糖尿病、心血管疾病和龋齿的发病率不断增加，人们对绿色健康甜味剂的需求日趋增长。甜菊糖苷是从菊科植物甜叶菊中分离提取到的零卡路里天然高倍甜味剂，其甜度为蔗糖的300倍，具有甜度高、热值低、营养全、功效多等特点，被誉为继蔗糖、阿斯巴甜之后的“第三代糖源”。
甜菊糖苷是一类多种甜味成分组成的混合物，它们具有相同的苷元四环二萜类化合物甜菊醇(Steviol)，C19和C13位上连接不同数量的葡萄糖基或鼠李糖基，从而形成甜度、味质、生物活性等性能迥异的衍生物如甜菊苷、莱鲍迪苷A-F、杜尔可苷A和甜茶苷，其化学结构如下所示：
Figure BDA0002280679650000011
Figure BDA0002280679650000012
虽然甜菊糖苷是一种高倍甜味剂，但存在后苦涩味这一缺点，严重限制了其在食品、饮料等对口感要求较高的领域中的应用。而引起甜菊糖苷后苦涩味的本质原因是其内在分子结构引起的，甜菊糖苷中的R1和R2基团上连接糖基数量越多口感越好，但其混合物中存在部分甜菊醇，甜菊醇具有一定的苦涩味，影响了整个甜菊糖苷的口感，又很难通过分离将甜菊醇去除，而通过糖基转移酶可以向甜菊醇的R1和R2位置引入糖基，使甜菊醇转化为甜度更好且没有苦涩味的其他类型的甜菊糖苷化合物。即苷元甜菊醇没有任何甜味，仅有苦涩味，而这一缺点可以通过酶修饰甜菊醇糖苷的糖基部分来改善或消除。
糖基转移酶(glycosyl transferase)是负责催化糖基化反应的酶类，它们将活性糖基从糖基供体转移到糖基受体，并形成糖苷键，是一类重要的转化反应，可应用于甜菊醇糖苷的性质改善。
来源于Streptococcus devriesei的一种糖基转移酶应用于甜菊醇糖苷糖基化修饰的催化反应尚未见报道，但该酶的催化活性相对较低，提高其催化活性势在必行。
随着对酶序列、结构和功能关系认知的逐步进展，利用计算机辅助设计进化酶学性质的方法也日益成熟。利用计算机模拟的方法，鉴定与识别该糖基转移酶催化活性中心直接相关的重要氨基酸以及有可能提高酶催化活性的靶标氨基酸残基，可以为糖基转移酶突变体的性质改善提供了快速的指导，本发明拟在这个方面进行深入的研究。
发明内容
本发明的目的在于提供一种糖基转移酶的突变体及其编码基因。
本发明的目的还在于提供含有上述基因的重组质粒和工程菌株。
本发明的最后一个目的在于提供上述糖基转移酶的突变体、编码基因、重组质粒或工程菌在甜味剂甜菊醇糖苷糖基化修饰中的应用。
因此，本发明的上述第一个目的可以通过以下技术方案来实现的：一种糖基转移酶的突变体，所述糖基转移酶突变体是将氨基酸序列如SEQ ID NO.2所示的糖基转移酶的第590位Gln氨基酸突变为Glu得到的，所述糖基转移酶突变体的氨基酸序列如SEQ ID NO.4所示。
其中氨基酸序列如SEQ ID NO.2所示的糖基转移酶的碱基序列如SEQ ID NO.1所示。
编码上述糖基转移酶突变体的基因，其碱基序列如SEQ ID NO.3所示。
本发明的上述第二个目的是通过以下技术方案来实现的：携带具有编码序列为SEQ ID NO.4的糖基转移酶突变体基因的重组质粒，所述重组质粒的载体可以为pET载体、pGEX载体或pUC载体，优选所述的重组质粒为pET-Q590E，其中Q590E为本发明具体实施方式中构建的糖基转移酶的突变体。
本发明还涉及一种工程菌株，其携带上述重组质粒。
优选的，所述的工程菌株为重组表达菌株E.coliBL21-Q590E，重组质粒转化进宿主大肠杆菌BL21所得。
本发明的上述最后一个目的是通过以下技术方案来实现的：上述糖基转移酶的突变体、编码糖基转移酶的突变体基因、所述重组质粒、所述工程菌株在甜味剂甜菊醇糖苷糖基化修饰中的应用。
优选的，所述的糖基转移酶突变体在催化甜菊醇糖苷糖基化反应中的应用时，以蔗糖为糖基供体，该突变体的催化活性是野生型的5～6倍。
作为本发明的一种优选的实施方式，该突变体的催化活性是野生型的大约5.3倍。
本发明具有以下优点：
(1)本发明运用同源建模，分子对接的计算机模拟技术，对糖基转移酶进行理性设计，成功构建了Q590E突变体；
(2)本发明中的糖基转移酶的突变体在甜菊醇糖苷糖基化反应的催化效率上是野生型酶的大约5-6倍；
(3)本发明中的糖基转移酶的突变体在甜味剂甜菊糖开发应用领域具有极大的潜力。
具体实施方式
下面结合具体实施例进一步说明本发明的应用方法。下述实施例仅用于示例性说明，不能理解为对本发明的限制。除非特别说明，下述实施例中使用的试剂原料为常规市购或商业途径获得的生试剂原料，使用的实验仪器均为实验室常规仪器，除非特别说明，下述实施例中使用的方法和设备为本领域常规使用的方法和设备。
实施例1：野生型糖基转移酶的获得
在NCBI上公布的糖基转移酶的基因序列SEQ ID NO.1(GenBank：NZ_KE384084.1)，在其起始密码子ATG前增加TCTAGA(XbaⅠ酶切位点)，在其终止密码子TGA后增加GCGGCCGC(NotⅠ酶切位点)，添加酶切位点的野生型基因序列由南京金斯瑞生物科技股份有限公司合成获得。之后用限制性内切酶XbaⅠ和NotⅠ(Invitrogen公司)对糖基转移酶的基因进行双酶切，同时，用限制性内切酶XbaⅠ和NotⅠ对pET-22b(+)载体(Invitrogen公司)进行双酶切，使用凝胶纯化试剂盒将酶切产物纯化，并用T4连接酶将上述两个酶切产物连接，将连接产物转化入E.coliDH5α感受态细胞中，提取质粒，酶切鉴定，PCR鉴定后送测序确定表达载体构建成功，命名为pET-GT。重组质粒转化E.coli BL21，获得重组表达菌株E.coliBL21-GT。所述糖基转移酶的氨基酸序列为SEQ ID NO.2(GENBANK:WP_027976201.1)
PCR鉴定引物-F:5’-TCTAGAATGGAAACGAAACTACG-3’(下划线为Xba Ⅰ酶切位点)
PCR鉴定引物-S:5’-GCGGCCGCCTATAAGTAGCGTGCAATAC-3’(下划线为Not Ⅰ酶切位点)。
实施例2：糖基转移酶突变体的获得
为了提高来源于Streptococcus devriesei的糖基转移酶催化甜菊醇糖苷糖基化修饰反应的活性，本发明通过将来源于Streptococcus devriesei的糖基转移酶序列进行三维结构模拟以及与底物的对接分析，利用理性设计的方法在该糖基转移酶与底物结合位点选择关键位点进行定点突变，获得了该糖基转移酶的突变体。
其中理性设计是通过计算机模拟来预测酶的三维结构，对酶和底物的结合位点以及催化机理进行分析，从而选择关键位点进行定点突变，并对突变后的结构进行模拟，评价突变位点对酶的影响，通过计算机模拟来筛选突变位点，由此来指导突变位点的选择。对应的非理性设计则不需要了解酶的结构及催化机理，直接通过实验构建大量随机突变库，从随机突变库中筛选活性提高的酶，相比较而言，理性设计大大提高了实验的成功率，并且减少了大量的实验筛选工作，提高了效率。
利用全质粒PCR方法构建突变体载体，以质粒pET-GT为模板，结合两端突变引物，利用重叠延伸PCR技术，扩增出含有突变位点的糖基转移酶质粒，突变位点的选择采用理性设计的方法，通过酶与底物结构构象的分析，动力学模拟等分析研究发现590位残基突变能改善酶与底物的结合，故此选择590位进行定点突变实验验证，并获得了与理性设计预测相符的结果，PCR体系如下：
Figure BDA0002280679650000041
突变引物如下：
Prime-F 5’-GACAGTGAAGTTCAGACGGTTATTGCCAAG-3’
Prime-S 5’-GTTGGCAATAACCGTTTCAACTTCACTGTC-3’
获得的糖基转移酶突变体Q590E，其氨基酸序列如SEQ ID NO.4所示，编码基因的核苷酸序列如SEQ ID NO.3所示。
PCR扩增程序：预变性98℃3min，循环设定：变性98℃15s，退火56℃15s，延伸72℃8min，20个循环，最后延伸：72℃10min；反应完成后，对产物进行回收，将消化后的PCR产物转化至E.coli DH5α感受态细胞中，涂板培养过夜，挑取阳性克隆子进行测序验证，确定成功获得突变体表达载体。经过测序鉴定后，重组质粒转化E.coli BL21菌株，获得糖基转移酶突变体的重组表达菌株E.coliBL21-Q590E。
实施例3：野生型E.coli BL21-GT与突变体工程菌E.coli BL21-Q590E的表达及催化活性分析
用重组质粒pET-GT和Q590E分别转化表达宿主E.coliBL21，获得的重组表达菌株E.coliBL21-GT和突变体重组表达菌株E.coliBL21-Q590E。按照1％接种量分别将野生型和突变体菌液接种到100mL LB液体培养基中(加入100μg/mL的氨苄青霉素)，37℃，220rpm培养至OD600＝0.6～0.8左右，加入终浓度为0.8mM的诱导剂IPTG，28℃诱导4h。
在4℃，8000rpm离心10min收集菌体，每100ml原始发酵液加入20mL乙酸钠缓冲液(20mM,pH5.4)进行重悬，利用超声细胞破碎仪对重悬菌体进行破碎，总超声时间10min，工作6s，间隔6s，功率400w，超声过程始终保持冰浴。破碎后，4℃，12000rpm离心10min，收集上清即获得野生型粗酶液和突变体粗酶液。
酶活性的测定：在37℃下，在20mM乙酸钠(pH5.4)缓冲溶液中，加入50mM底物甜菊醇糖苷，100mM蔗糖作为糖基供体，添加0.1mg/mL的野生型酶(E.coliBL21-GT)或突变体(E.coliBL21-Q590E)分别进行酶活性测定。每隔30s取100μL样品并通过50μL 1M浓度的NaOH孵育来终止反应。将灭活的样品进行稀释，取10μL稀释样品分别与己糖激酶和葡萄糖-6-磷酸脱氢酶反应，监测NADP的还原来测定反应液中葡萄糖和果糖浓度，其中果糖浓度对应于总酶活性，葡萄糖浓度对应于水解活性，通过从总活性中减去水解活性来得到转糖基活性。将一个单位(U)的酶定义为37℃下于含有20mM乙酸钠(pH5.4)、100mM蔗糖的反应混合物中每分钟产生1μmol单糖所需的酶的量。经过检测，突变体的比活性为12.1U/mg，野生型糖基转移酶的活性约为2.3U/mg，突变体的转糖基活性较之野生型显著提高。
本发明不局限于上述特定的实施方案范围内，上述实施方案仅仅是为了能够对本发明的使用过程进行详细地说明，而且有相等功能的生产方法和技术细节也属于本发明内容的一部分。事实上，本领域技术人员根据前文的描述，就能够根据各自需要找到不同的调整方案，这些调整都应在本文所附的权利要求书的范围内。
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<120> 一种糖基转移酶突变体及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4398
<212> DNA
<213> 糖基转移酶(glycosyl transferase)
<400> 1
atggaaacga aactacgtta taaaatgcgc aaagtgaaaa aacactgggt gactgtcgcc 60
attgcatcca gcttgctgac tttgggcagt gcggcgctgg atgtgtctgc atcagcagat 120
acagaacagg cagcgaataa tcaggtggtc gtcactcagc aaagtcaaag tgagaataca 180
gagcagactt ctgcagccga tcagcctgcc gcagagagca gccaaacaca ggctgctgaa 240
cagcaggcac cggtcactga acaggaatct cagcagcctc aagagcagag caccgaaacg 300
gttactgaag ctccagagga tcaggtgcaa agccaaagca gcactgctca agaaaccact 360
gagaaggcag taacagctcc ggctcaggaa caagcggcaa gctctgagac aactgcaaga 420
gcagctaaag ttcagacctc tggagaacgt gtaacagccg ctcagacagc cgaacaaaat 480
caaacaaaaa acttgagtga tgctgattta tcagctattc caaatgttaa aaagattgat 540
ggcaagtatt attatattga ctcagacggt caggtcaaaa agaacttcgc cttaacagtt 600
gacggtcaaa cccttttctt tgataaagaa acaggggctc tgtcagatac ggcacagtat 660
cagtttgctc aggggctgac atctttaaac aatgagtaca caccgcacaa ccagattgtt 720
aatttggaga agaacagcct tgaaactgtt gataattatg tcacagcgga ttcttggtac 780
cgtcctaaag atatcctgaa agacggcaag acttggacag catcaacaga aaatgatctg 840
cgtccgctgc tcatgtcttg gtggcctgat aagcaaactc aggtatctta tttgaactac 900
atgaatgagc agggattagg tacaggaaca acctatacgg ctgacagcag tcaggaaagt 960
ctgaacttag ctgctcaaaa tgttcaggag aagattgaaa gcaagatttc gcaaacccag 1020
cagacgcagt ggctgcgtga cattatcaac agttttgtca aaacgcagtc taattggaat 1080
attgagaccg aatctgatac ttcagccggc gaaaaggacc atctgcaggg cggtgctttg 1140
ctttatgaaa acagcgataa aacaccgtat gcaaattctg attaccgttt gctgaaccgc 1200
acaccaacca gccagaccgg tgagcaaaag tatttcgatg ataattctat gggtggctac 1260
gaattcctcc tagctaatga tattgataat tcaaatcccg ttgttcaggc agagcagctc 1320
aactggcttc attatttaat gaactatggt tctattgtcg ctaatgaccc tgaagctaat 1380
tttgacggcg ttcgggtcga tgcggttgat aatgtcaatg ctgatttgct gcagattgcc 1440
tcagattatt tcaaggctta ttacggcgta gataagagcg agaaaaatgc ccttgatcac 1500
ctgtctatct tagaagcttg gtcagacaat gatccgcagt ataataaaga tacttacgga 1560
gcacagctgc cgattgacaa taagcttcgt ctgtcactcc tgtatgctct ggcacgtcct 1620
cttgaaaaag atgcggttga taaaagcaat gtccgcagtg gcttggaatc tgtcattaca 1680
aacagcttga ataaccgttc agctgagggc aaaaacagcg aacgcatggc caattatatc 1740
tttatccggg ctcatgacag tgaagttcag acggttattg ccaagatcat caaggataat 1800
atcaatccta atacagatgg tttgaccttt acgctggacg agctcaagca agccttcaag 1860
atttacaatg aagatatgcg tcaggctaat aagaaataca cacaatccaa tattccgaca 1920
gcctatgctt tgatgctctc caataaggat tccatcaccc gtgtttacta tggggatatg 1980
tataccgacg acggccaata catggctact aagtcaccgt actacgatgc tattgaggcc 2040
ttgatgaagg ctcgcatcaa gtatgcagcc ggcggtcagg atatgaagat tacctatgtt 2100
gagggtgata aggccaatat ggattgggat tataccggcg tcttaacatc tgtgcgctac 2160
ggtacaggag ctaatgaagc tactgacagc ggcaatgaag ccactaagac ccaaggtatg 2220
gctgttatta ccagcaataa cccaagtctg aaactcaatg ccaatgataa agtagttgtg 2280
aacatggggg cagctcataa aaaccaagag taccgtccgc tcatcttaac aactcaggac 2340
ggacttgcag cctatgcatc tgatgatgct gctaaagcct tctaccgcaa aacaaatgaa 2400
aatggcgaac tcatttttga tgcgtcagat attcaaggtt atcttaatcc gcaagtatca 2460
ggctatctgg ctgtttgggt gccggtcggt gccagcgaca gccaagatgt tcgtgtagca 2520
gccagtgaca aggccaatgc tgacggacag gtttacaatg cttcaagtgc tctggattca 2580
cagttaatct atgaaggttt ttcaaatttc caagattttg tcactaacga ttcagaatac 2640
acaaataaga agattgcaca aaacgttgat ctcttcaaat catggggtgt gacgtccttt 2700
gaaatggcac cgcagtacgt gtcatctgaa gacggttcct tccttgattc aatcatttta 2760
aacggctatt cctttgaaga ccgttacgat cttgccatga gtaagaacaa taagtacggt 2820
tctcagcagg atatgatcaa tgcccttaaa gccctgcacc gcaatggtat tcaagtcatt 2880
gccgactggg taccggatca aatttacaat ctgccgggca gagaagtcgt tacagcgacc 2940
cgcgttaatg actatggtga ctatcgccat gattcggaaa ttaaggattc gctttatctg 3000
gctaatacca agaccaatgg cgatgactat caggctaaat acggcggcgc tttcttagac 3060
gagctggcag ccaagtatcc tagcatcttt gaacgtactc aaatttcaaa cggtcaaaaa 3120
ttggatccga gtgaaaagat tacccaatgg caggctaagt atttcaatgg taccaatatt 3180
ttgggacgcg gtgtcggcta tgtccttaaa gatgcggcca gcgataagta ctttacgctg 3240
gatggcaagg aaacctatct gccaaaacag ctgactaacc aagaagcttt aacaggcttt 3300
gtcaatgacg gcagcggcgt gaccttctat tcaagcagcg gctatcaggc taaaaacagc 3360
tttgttcaag atgccaaagg caactggtat tatttcgata aagatggtca tatggtctat 3420
ggccagcagc atttgaacaa tgaagtacag tacttcctgc ctaacggtgt tcagctgcgt 3480
gaagctatct tagaaaatgc cgacggcact aaaaattatt ttggccgcct gggcaatcgc 3540
ttcagcaatg actattactc ttttgaaaag gataccaaat ggcgttactt tgatgaaaac 3600
ggcgtgatgg cagtaggtct gaaaacgatc aatggcaata cacagtactt tgatcaaaac 3660
ggctatcaag tcaagggtga atggataaca gatgctgatg gcaagaaacg ctattttgat 3720
gatggttccg gaaatatggc tgttaatcgt tttgctaatg acagcaatgg tgattggtat 3780
tacctcggag cagacggcac agcgcaaacc ggcagtcaaa ctattgacgg taaatcttat 3840
tactttgatg aaaacggcaa acaggtcaaa ggaaagatta tcacggctaa tgatggtaaa 3900
ctctactatt atctcgcaga ttccggagag ctggcgcaca atatttttgc aacagacagt 3960
caaaataact ggtattactt tggctcagac ggcacagcag taacaggcag ccagacgatt 4020
aatggacaaa acctttattt tgccaatgac ggcaagcagg ttaaaggtga ctttgtcaat 4080
gaaaacggca aaatccgcta ttaccatgct gactcaggcc aactgcaaac caatcgcttt 4140
gaagctgata aagacggcaa ttggtactat ctgggttcag acggcacagc gctgacaggc 4200
agtcagtaca tcaataatca gcgtctcttc tttaccagag aaggtaagca ggttaaaggt 4260
gatgttgcct acgatggctg gggactgctt cgctattacg acaaagacag cggcagcatg 4320
gtctataata gacttgtaac cttggctaat ggcagaagaa tcggcattaa ccgttggggt 4380
attgcacgct acttatag 4398
<210> 2
<211> 1465
<212> PRT
<213> 糖基转移酶(glycosyl transferase)
<400> 2
Met Glu Thr Lys Leu Arg Tyr Lys Met Arg Lys Val Lys Lys His Trp
1 5 10 15
Val Thr Val Ala Ile Ala Ser Ser Leu Leu Thr Leu Gly Ser Ala Ala
20 25 30
Leu Asp Val Ser Ala Ser Ala Asp Thr Glu Gln Ala Ala Asn Asn Gln
35 40 45
Val Val Val Thr Gln Gln Ser Gln Ser Glu Asn Thr Glu Gln Thr Ser
50 55 60
Ala Ala Asp Gln Pro Ala Ala Glu Ser Ser Gln Thr Gln Ala Ala Glu
65 70 75 80
Gln Gln Ala Pro Val Thr Glu Gln Glu Ser Gln Gln Pro Gln Glu Gln
85 90 95
Ser Thr Glu Thr Val Thr Glu Ala Pro Glu Asp Gln Val Gln Ser Gln
100 105 110
Ser Ser Thr Ala Gln Glu Thr Thr Glu Lys Ala Val Thr Ala Pro Ala
115 120 125
Gln Glu Gln Ala Ala Ser Ser Glu Thr Thr Ala Arg Ala Ala Lys Val
130 135 140
Gln Thr Ser Gly Glu Arg Val Thr Ala Ala Gln Thr Ala Glu Gln Asn
145 150 155 160
Gln Thr Lys Asn Leu Ser Asp Ala Asp Leu Ser Ala Ile Pro Asn Val
165 170 175
Lys Lys Ile Asp Gly Lys Tyr Tyr Tyr Ile Asp Ser Asp Gly Gln Val
180 185 190
Lys Lys Asn Phe Ala Leu Thr Val Asp Gly Gln Thr Leu Phe Phe Asp
195 200 205
Lys Glu Thr Gly Ala Leu Ser Asp Thr Ala Gln Tyr Gln Phe Ala Gln
210 215 220
Gly Leu Thr Ser Leu Asn Asn Glu Tyr Thr Pro His Asn Gln Ile Val
225 230 235 240
Asn Leu Glu Lys Asn Ser Leu Glu Thr Val Asp Asn Tyr Val Thr Ala
245 250 255
Asp Ser Trp Tyr Arg Pro Lys Asp Ile Leu Lys Asp Gly Lys Thr Trp
260 265 270
Thr Ala Ser Thr Glu Asn Asp Leu Arg Pro Leu Leu Met Ser Trp Trp
275 280 285
Pro Asp Lys Gln Thr Gln Val Ser Tyr Leu Asn Tyr Met Asn Glu Gln
290 295 300
Gly Leu Gly Thr Gly Thr Thr Tyr Thr Ala Asp Ser Ser Gln Glu Ser
305 310 315 320
Leu Asn Leu Ala Ala Gln Asn Val Gln Glu Lys Ile Glu Ser Lys Ile
325 330 335
Ser Gln Thr Gln Gln Thr Gln Trp Leu Arg Asp Ile Ile Asn Ser Phe
340 345 350
Val Lys Thr Gln Ser Asn Trp Asn Ile Glu Thr Glu Ser Asp Thr Ser
355 360 365
Ala Gly Glu Lys Asp His Leu Gln Gly Gly Ala Leu Leu Tyr Glu Asn
370 375 380
Ser Asp Lys Thr Pro Tyr Ala Asn Ser Asp Tyr Arg Leu Leu Asn Arg
385 390 395 400
Thr Pro Thr Ser Gln Thr Gly Glu Gln Lys Tyr Phe Asp Asp Asn Ser
405 410 415
Met Gly Gly Tyr Glu Phe Leu Leu Ala Asn Asp Ile Asp Asn Ser Asn
420 425 430
Pro Val Val Gln Ala Glu Gln Leu Asn Trp Leu His Tyr Leu Met Asn
435 440 445
Tyr Gly Ser Ile Val Ala Asn Asp Pro Glu Ala Asn Phe Asp Gly Val
450 455 460
Arg Val Asp Ala Val Asp Asn Val Asn Ala Asp Leu Leu Gln Ile Ala
465 470 475 480
Ser Asp Tyr Phe Lys Ala Tyr Tyr Gly Val Asp Lys Ser Glu Lys Asn
485 490 495
Ala Leu Asp His Leu Ser Ile Leu Glu Ala Trp Ser Asp Asn Asp Pro
500 505 510
Gln Tyr Asn Lys Asp Thr Tyr Gly Ala Gln Leu Pro Ile Asp Asn Lys
515 520 525
Leu Arg Leu Ser Leu Leu Tyr Ala Leu Ala Arg Pro Leu Glu Lys Asp
530 535 540
Ala Val Asp Lys Ser Asn Val Arg Ser Gly Leu Glu Ser Val Ile Thr
545 550 555 560
Asn Ser Leu Asn Asn Arg Ser Ala Glu Gly Lys Asn Ser Glu Arg Met
565 570 575
Ala Asn Tyr Ile Phe Ile Arg Ala His Asp Ser Glu Val Gln Thr Val
580 585 590
Ile Ala Lys Ile Ile Lys Asp Asn Ile Asn Pro Asn Thr Asp Gly Leu
595 600 605
Thr Phe Thr Leu Asp Glu Leu Lys Gln Ala Phe Lys Ile Tyr Asn Glu
610 615 620
Asp Met Arg Gln Ala Asn Lys Lys Tyr Thr Gln Ser Asn Ile Pro Thr
625 630 635 640
Ala Tyr Ala Leu Met Leu Ser Asn Lys Asp Ser Ile Thr Arg Val Tyr
645 650 655
Tyr Gly Asp Met Tyr Thr Asp Asp Gly Gln Tyr Met Ala Thr Lys Ser
660 665 670
Pro Tyr Tyr Asp Ala Ile Glu Ala Leu Met Lys Ala Arg Ile Lys Tyr
675 680 685
Ala Ala Gly Gly Gln Asp Met Lys Ile Thr Tyr Val Glu Gly Asp Lys
690 695 700
Ala Asn Met Asp Trp Asp Tyr Thr Gly Val Leu Thr Ser Val Arg Tyr
705 710 715 720
Gly Thr Gly Ala Asn Glu Ala Thr Asp Ser Gly Asn Glu Ala Thr Lys
725 730 735
Thr Gln Gly Met Ala Val Ile Thr Ser Asn Asn Pro Ser Leu Lys Leu
740 745 750
Asn Ala Asn Asp Lys Val Val Val Asn Met Gly Ala Ala His Lys Asn
755 760 765
Gln Glu Tyr Arg Pro Leu Ile Leu Thr Thr Gln Asp Gly Leu Ala Ala
770 775 780
Tyr Ala Ser Asp Asp Ala Ala Lys Ala Phe Tyr Arg Lys Thr Asn Glu
785 790 795 800
Asn Gly Glu Leu Ile Phe Asp Ala Ser Asp Ile Gln Gly Tyr Leu Asn
805 810 815
Pro Gln Val Ser Gly Tyr Leu Ala Val Trp Val Pro Val Gly Ala Ser
820 825 830
Asp Ser Gln Asp Val Arg Val Ala Ala Ser Asp Lys Ala Asn Ala Asp
835 840 845
Gly Gln Val Tyr Asn Ala Ser Ser Ala Leu Asp Ser Gln Leu Ile Tyr
850 855 860
Glu Gly Phe Ser Asn Phe Gln Asp Phe Val Thr Asn Asp Ser Glu Tyr
865 870 875 880
Thr Asn Lys Lys Ile Ala Gln Asn Val Asp Leu Phe Lys Ser Trp Gly
885 890 895
Val Thr Ser Phe Glu Met Ala Pro Gln Tyr Val Ser Ser Glu Asp Gly
900 905 910
Ser Phe Leu Asp Ser Ile Ile Leu Asn Gly Tyr Ser Phe Glu Asp Arg
915 920 925
Tyr Asp Leu Ala Met Ser Lys Asn Asn Lys Tyr Gly Ser Gln Gln Asp
930 935 940
Met Ile Asn Ala Leu Lys Ala Leu His Arg Asn Gly Ile Gln Val Ile
945 950 955 960
Ala Asp Trp Val Pro Asp Gln Ile Tyr Asn Leu Pro Gly Arg Glu Val
965 970 975
Val Thr Ala Thr Arg Val Asn Asp Tyr Gly Asp Tyr Arg His Asp Ser
980 985 990
Glu Ile Lys Asp Ser Leu Tyr Leu Ala Asn Thr Lys Thr Asn Gly Asp
995 1000 1005
Asp Tyr Gln Ala Lys Tyr Gly Gly Ala Phe Leu Asp Glu Leu Ala Ala
1010 1015 1020
Lys Tyr Pro Ser Ile Phe Glu Arg Thr Gln Ile Ser Asn Gly Gln Lys
1025 1030 1035 1040
Leu Asp Pro Ser Glu Lys Ile Thr Gln Trp Gln Ala Lys Tyr Phe Asn
1045 1050 1055
Gly Thr Asn Ile Leu Gly Arg Gly Val Gly Tyr Val Leu Lys Asp Ala
1060 1065 1070
Ala Ser Asp Lys Tyr Phe Thr Leu Asp Gly Lys Glu Thr Tyr Leu Pro
1075 1080 1085
Lys Gln Leu Thr Asn Gln Glu Ala Leu Thr Gly Phe Val Asn Asp Gly
1090 1095 1100
Ser Gly Val Thr Phe Tyr Ser Ser Ser Gly Tyr Gln Ala Lys Asn Ser
1105 1110 1115 1120
Phe Val Gln Asp Ala Lys Gly Asn Trp Tyr Tyr Phe Asp Lys Asp Gly
1125 1130 1135
His Met Val Tyr Gly Gln Gln His Leu Asn Asn Glu Val Gln Tyr Phe
1140 1145 1150
Leu Pro Asn Gly Val Gln Leu Arg Glu Ala Ile Leu Glu Asn Ala Asp
1155 1160 1165
Gly Thr Lys Asn Tyr Phe Gly Arg Leu Gly Asn Arg Phe Ser Asn Asp
1170 1175 1180
Tyr Tyr Ser Phe Glu Lys Asp Thr Lys Trp Arg Tyr Phe Asp Glu Asn
1185 1190 1195 1200
Gly Val Met Ala Val Gly Leu Lys Thr Ile Asn Gly Asn Thr Gln Tyr
1205 1210 1215
Phe Asp Gln Asn Gly Tyr Gln Val Lys Gly Glu Trp Ile Thr Asp Ala
1220 1225 1230
Asp Gly Lys Lys Arg Tyr Phe Asp Asp Gly Ser Gly Asn Met Ala Val
1235 1240 1245
Asn Arg Phe Ala Asn Asp Ser Asn Gly Asp Trp Tyr Tyr Leu Gly Ala
1250 1255 1260
Asp Gly Thr Ala Gln Thr Gly Ser Gln Thr Ile Asp Gly Lys Ser Tyr
1265 1270 1275 1280
Tyr Phe Asp Glu Asn Gly Lys Gln Val Lys Gly Lys Ile Ile Thr Ala
1285 1290 1295
Asn Asp Gly Lys Leu Tyr Tyr Tyr Leu Ala Asp Ser Gly Glu Leu Ala
1300 1305 1310
His Asn Ile Phe Ala Thr Asp Ser Gln Asn Asn Trp Tyr Tyr Phe Gly
1315 1320 1325
Ser Asp Gly Thr Ala Val Thr Gly Ser Gln Thr Ile Asn Gly Gln Asn
1330 1335 1340
Leu Tyr Phe Ala Asn Asp Gly Lys Gln Val Lys Gly Asp Phe Val Asn
1345 1350 1355 1360
Glu Asn Gly Lys Ile Arg Tyr Tyr His Ala Asp Ser Gly Gln Leu Gln
1365 1370 1375
Thr Asn Arg Phe Glu Ala Asp Lys Asp Gly Asn Trp Tyr Tyr Leu Gly
1380 1385 1390
Ser Asp Gly Thr Ala Leu Thr Gly Ser Gln Tyr Ile Asn Asn Gln Arg
1395 1400 1405
Leu Phe Phe Thr Arg Glu Gly Lys Gln Val Lys Gly Asp Val Ala Tyr
1410 1415 1420
Asp Gly Trp Gly Leu Leu Arg Tyr Tyr Asp Lys Asp Ser Gly Ser Met
1425 1430 1435 1440
Val Tyr Asn Arg Leu Val Thr Leu Ala Asn Gly Arg Arg Ile Gly Ile
1445 1450 1455
Asn Arg Trp Gly Ile Ala Arg Tyr Leu
1460 1465
<210> 3
<211> 4398
<212> DNA
<213> 糖基转移酶(glycosyl transferase)
<400> 3
atggaaacga aactacgtta taaaatgcgc aaagtgaaaa aacactgggt gactgtcgcc 60
attgcatcca gcttgctgac tttgggcagt gcggcgctgg atgtgtctgc atcagcagat 120
acagaacagg cagcgaataa tcaggtggtc gtcactcagc aaagtcaaag tgagaataca 180
gagcagactt ctgcagccga tcagcctgcc gcagagagca gccaaacaca ggctgctgaa 240
cagcaggcac cggtcactga acaggaatct cagcagcctc aagagcagag caccgaaacg 300
gttactgaag ctccagagga tcaggtgcaa agccaaagca gcactgctca agaaaccact 360
gagaaggcag taacagctcc ggctcaggaa caagcggcaa gctctgagac aactgcaaga 420
gcagctaaag ttcagacctc tggagaacgt gtaacagccg ctcagacagc cgaacaaaat 480
caaacaaaaa acttgagtga tgctgattta tcagctattc caaatgttaa aaagattgat 540
ggcaagtatt attatattga ctcagacggt caggtcaaaa agaacttcgc cttaacagtt 600
gacggtcaaa cccttttctt tgataaagaa acaggggctc tgtcagatac ggcacagtat 660
cagtttgctc aggggctgac atctttaaac aatgagtaca caccgcacaa ccagattgtt 720
aatttggaga agaacagcct tgaaactgtt gataattatg tcacagcgga ttcttggtac 780
cgtcctaaag atatcctgaa agacggcaag acttggacag catcaacaga aaatgatctg 840
cgtccgctgc tcatgtcttg gtggcctgat aagcaaactc aggtatctta tttgaactac 900
atgaatgagc agggattagg tacaggaaca acctatacgg ctgacagcag tcaggaaagt 960
ctgaacttag ctgctcaaaa tgttcaggag aagattgaaa gcaagatttc gcaaacccag 1020
cagacgcagt ggctgcgtga cattatcaac agttttgtca aaacgcagtc taattggaat 1080
attgagaccg aatctgatac ttcagccggc gaaaaggacc atctgcaggg cggtgctttg 1140
ctttatgaaa acagcgataa aacaccgtat gcaaattctg attaccgttt gctgaaccgc 1200
acaccaacca gccagaccgg tgagcaaaag tatttcgatg ataattctat gggtggctac 1260
gaattcctcc tagctaatga tattgataat tcaaatcccg ttgttcaggc agagcagctc 1320
aactggcttc attatttaat gaactatggt tctattgtcg ctaatgaccc tgaagctaat 1380
tttgacggcg ttcgggtcga tgcggttgat aatgtcaatg ctgatttgct gcagattgcc 1440
tcagattatt tcaaggctta ttacggcgta gataagagcg agaaaaatgc ccttgatcac 1500
ctgtctatct tagaagcttg gtcagacaat gatccgcagt ataataaaga tacttacgga 1560
gcacagctgc cgattgacaa taagcttcgt ctgtcactcc tgtatgctct ggcacgtcct 1620
cttgaaaaag atgcggttga taaaagcaat gtccgcagtg gcttggaatc tgtcattaca 1680
aacagcttga ataaccgttc agctgagggc aaaaacagcg aacgcatggc caattatatc 1740
tttatccggg ctcatgacag tgaagtggag acggttattg ccaagatcat caaggataat 1800
atcaatccta atacagatgg tttgaccttt acgctggacg agctcaagca agccttcaag 1860
atttacaatg aagatatgcg tcaggctaat aagaaataca cacaatccaa tattccgaca 1920
gcctatgctt tgatgctctc caataaggat tccatcaccc gtgtttacta tggggatatg 1980
tataccgacg acggccaata catggctact aagtcaccgt actacgatgc tattgaggcc 2040
ttgatgaagg ctcgcatcaa gtatgcagcc ggcggtcagg atatgaagat tacctatgtt 2100
gagggtgata aggccaatat ggattgggat tataccggcg tcttaacatc tgtgcgctac 2160
ggtacaggag ctaatgaagc tactgacagc ggcaatgaag ccactaagac ccaaggtatg 2220
gctgttatta ccagcaataa cccaagtctg aaactcaatg ccaatgataa agtagttgtg 2280
aacatggggg cagctcataa aaaccaagag taccgtccgc tcatcttaac aactcaggac 2340
ggacttgcag cctatgcatc tgatgatgct gctaaagcct tctaccgcaa aacaaatgaa 2400
aatggcgaac tcatttttga tgcgtcagat attcaaggtt atcttaatcc gcaagtatca 2460
ggctatctgg ctgtttgggt gccggtcggt gccagcgaca gccaagatgt tcgtgtagca 2520
gccagtgaca aggccaatgc tgacggacag gtttacaatg cttcaagtgc tctggattca 2580
cagttaatct atgaaggttt ttcaaatttc caagattttg tcactaacga ttcagaatac 2640
acaaataaga agattgcaca aaacgttgat ctcttcaaat catggggtgt gacgtccttt 2700
gaaatggcac cgcagtacgt gtcatctgaa gacggttcct tccttgattc aatcatttta 2760
aacggctatt cctttgaaga ccgttacgat cttgccatga gtaagaacaa taagtacggt 2820
tctcagcagg atatgatcaa tgcccttaaa gccctgcacc gcaatggtat tcaagtcatt 2880
gccgactggg taccggatca aatttacaat ctgccgggca gagaagtcgt tacagcgacc 2940
cgcgttaatg actatggtga ctatcgccat gattcggaaa ttaaggattc gctttatctg 3000
gctaatacca agaccaatgg cgatgactat caggctaaat acggcggcgc tttcttagac 3060
gagctggcag ccaagtatcc tagcatcttt gaacgtactc aaatttcaaa cggtcaaaaa 3120
ttggatccga gtgaaaagat tacccaatgg caggctaagt atttcaatgg taccaatatt 3180
ttgggacgcg gtgtcggcta tgtccttaaa gatgcggcca gcgataagta ctttacgctg 3240
gatggcaagg aaacctatct gccaaaacag ctgactaacc aagaagcttt aacaggcttt 3300
gtcaatgacg gcagcggcgt gaccttctat tcaagcagcg gctatcaggc taaaaacagc 3360
tttgttcaag atgccaaagg caactggtat tatttcgata aagatggtca tatggtctat 3420
ggccagcagc atttgaacaa tgaagtacag tacttcctgc ctaacggtgt tcagctgcgt 3480
gaagctatct tagaaaatgc cgacggcact aaaaattatt ttggccgcct gggcaatcgc 3540
ttcagcaatg actattactc ttttgaaaag gataccaaat ggcgttactt tgatgaaaac 3600
ggcgtgatgg cagtaggtct gaaaacgatc aatggcaata cacagtactt tgatcaaaac 3660
ggctatcaag tcaagggtga atggataaca gatgctgatg gcaagaaacg ctattttgat 3720
gatggttccg gaaatatggc tgttaatcgt tttgctaatg acagcaatgg tgattggtat 3780
tacctcggag cagacggcac agcgcaaacc ggcagtcaaa ctattgacgg taaatcttat 3840
tactttgatg aaaacggcaa acaggtcaaa ggaaagatta tcacggctaa tgatggtaaa 3900
ctctactatt atctcgcaga ttccggagag ctggcgcaca atatttttgc aacagacagt 3960
caaaataact ggtattactt tggctcagac ggcacagcag taacaggcag ccagacgatt 4020
aatggacaaa acctttattt tgccaatgac ggcaagcagg ttaaaggtga ctttgtcaat 4080
gaaaacggca aaatccgcta ttaccatgct gactcaggcc aactgcaaac caatcgcttt 4140
gaagctgata aagacggcaa ttggtactat ctgggttcag acggcacagc gctgacaggc 4200
agtcagtaca tcaataatca gcgtctcttc tttaccagag aaggtaagca ggttaaaggt 4260
gatgttgcct acgatggctg gggactgctt cgctattacg acaaagacag cggcagcatg 4320
gtctataata gacttgtaac cttggctaat ggcagaagaa tcggcattaa ccgttggggt 4380
attgcacgct acttatag 4398
<210> 4
<211> 1465
<212> PRT
<213> 糖基转移酶(glycosyl transferase)
<400> 4
Met Glu Thr Lys Leu Arg Tyr Lys Met Arg Lys Val Lys Lys His Trp
1 5 10 15
Val Thr Val Ala Ile Ala Ser Ser Leu Leu Thr Leu Gly Ser Ala Ala
20 25 30
Leu Asp Val Ser Ala Ser Ala Asp Thr Glu Gln Ala Ala Asn Asn Gln
35 40 45
Val Val Val Thr Gln Gln Ser Gln Ser Glu Asn Thr Glu Gln Thr Ser
50 55 60
Ala Ala Asp Gln Pro Ala Ala Glu Ser Ser Gln Thr Gln Ala Ala Glu
65 70 75 80
Gln Gln Ala Pro Val Thr Glu Gln Glu Ser Gln Gln Pro Gln Glu Gln
85 90 95
Ser Thr Glu Thr Val Thr Glu Ala Pro Glu Asp Gln Val Gln Ser Gln
100 105 110
Ser Ser Thr Ala Gln Glu Thr Thr Glu Lys Ala Val Thr Ala Pro Ala
115 120 125
Gln Glu Gln Ala Ala Ser Ser Glu Thr Thr Ala Arg Ala Ala Lys Val
130 135 140
Gln Thr Ser Gly Glu Arg Val Thr Ala Ala Gln Thr Ala Glu Gln Asn
145 150 155 160
Gln Thr Lys Asn Leu Ser Asp Ala Asp Leu Ser Ala Ile Pro Asn Val
165 170 175
Lys Lys Ile Asp Gly Lys Tyr Tyr Tyr Ile Asp Ser Asp Gly Gln Val
180 185 190
Lys Lys Asn Phe Ala Leu Thr Val Asp Gly Gln Thr Leu Phe Phe Asp
195 200 205
Lys Glu Thr Gly Ala Leu Ser Asp Thr Ala Gln Tyr Gln Phe Ala Gln
210 215 220
Gly Leu Thr Ser Leu Asn Asn Glu Tyr Thr Pro His Asn Gln Ile Val
225 230 235 240
Asn Leu Glu Lys Asn Ser Leu Glu Thr Val Asp Asn Tyr Val Thr Ala
245 250 255
Asp Ser Trp Tyr Arg Pro Lys Asp Ile Leu Lys Asp Gly Lys Thr Trp
260 265 270
Thr Ala Ser Thr Glu Asn Asp Leu Arg Pro Leu Leu Met Ser Trp Trp
275 280 285
Pro Asp Lys Gln Thr Gln Val Ser Tyr Leu Asn Tyr Met Asn Glu Gln
290 295 300
Gly Leu Gly Thr Gly Thr Thr Tyr Thr Ala Asp Ser Ser Gln Glu Ser
305 310 315 320
Leu Asn Leu Ala Ala Gln Asn Val Gln Glu Lys Ile Glu Ser Lys Ile
325 330 335
Ser Gln Thr Gln Gln Thr Gln Trp Leu Arg Asp Ile Ile Asn Ser Phe
340 345 350
Val Lys Thr Gln Ser Asn Trp Asn Ile Glu Thr Glu Ser Asp Thr Ser
355 360 365
Ala Gly Glu Lys Asp His Leu Gln Gly Gly Ala Leu Leu Tyr Glu Asn
370 375 380
Ser Asp Lys Thr Pro Tyr Ala Asn Ser Asp Tyr Arg Leu Leu Asn Arg
385 390 395 400
Thr Pro Thr Ser Gln Thr Gly Glu Gln Lys Tyr Phe Asp Asp Asn Ser
405 410 415
Met Gly Gly Tyr Glu Phe Leu Leu Ala Asn Asp Ile Asp Asn Ser Asn
420 425 430
Pro Val Val Gln Ala Glu Gln Leu Asn Trp Leu His Tyr Leu Met Asn
435 440 445
Tyr Gly Ser Ile Val Ala Asn Asp Pro Glu Ala Asn Phe Asp Gly Val
450 455 460
Arg Val Asp Ala Val Asp Asn Val Asn Ala Asp Leu Leu Gln Ile Ala
465 470 475 480
Ser Asp Tyr Phe Lys Ala Tyr Tyr Gly Val Asp Lys Ser Glu Lys Asn
485 490 495
Ala Leu Asp His Leu Ser Ile Leu Glu Ala Trp Ser Asp Asn Asp Pro
500 505 510
Gln Tyr Asn Lys Asp Thr Tyr Gly Ala Gln Leu Pro Ile Asp Asn Lys
515 520 525
Leu Arg Leu Ser Leu Leu Tyr Ala Leu Ala Arg Pro Leu Glu Lys Asp
530 535 540
Ala Val Asp Lys Ser Asn Val Arg Ser Gly Leu Glu Ser Val Ile Thr
545 550 555 560
Asn Ser Leu Asn Asn Arg Ser Ala Glu Gly Lys Asn Ser Glu Arg Met
565 570 575
Ala Asn Tyr Ile Phe Ile Arg Ala His Asp Ser Glu Val Glu Thr Val
580 585 590
Ile Ala Lys Ile Ile Lys Asp Asn Ile Asn Pro Asn Thr Asp Gly Leu
595 600 605
Thr Phe Thr Leu Asp Glu Leu Lys Gln Ala Phe Lys Ile Tyr Asn Glu
610 615 620
Asp Met Arg Gln Ala Asn Lys Lys Tyr Thr Gln Ser Asn Ile Pro Thr
625 630 635 640
Ala Tyr Ala Leu Met Leu Ser Asn Lys Asp Ser Ile Thr Arg Val Tyr
645 650 655
Tyr Gly Asp Met Tyr Thr Asp Asp Gly Gln Tyr Met Ala Thr Lys Ser
660 665 670
Pro Tyr Tyr Asp Ala Ile Glu Ala Leu Met Lys Ala Arg Ile Lys Tyr
675 680 685
Ala Ala Gly Gly Gln Asp Met Lys Ile Thr Tyr Val Glu Gly Asp Lys
690 695 700
Ala Asn Met Asp Trp Asp Tyr Thr Gly Val Leu Thr Ser Val Arg Tyr
705 710 715 720
Gly Thr Gly Ala Asn Glu Ala Thr Asp Ser Gly Asn Glu Ala Thr Lys
725 730 735
Thr Gln Gly Met Ala Val Ile Thr Ser Asn Asn Pro Ser Leu Lys Leu
740 745 750
Asn Ala Asn Asp Lys Val Val Val Asn Met Gly Ala Ala His Lys Asn
755 760 765
Gln Glu Tyr Arg Pro Leu Ile Leu Thr Thr Gln Asp Gly Leu Ala Ala
770 775 780
Tyr Ala Ser Asp Asp Ala Ala Lys Ala Phe Tyr Arg Lys Thr Asn Glu
785 790 795 800
Asn Gly Glu Leu Ile Phe Asp Ala Ser Asp Ile Gln Gly Tyr Leu Asn
805 810 815
Pro Gln Val Ser Gly Tyr Leu Ala Val Trp Val Pro Val Gly Ala Ser
820 825 830
Asp Ser Gln Asp Val Arg Val Ala Ala Ser Asp Lys Ala Asn Ala Asp
835 840 845
Gly Gln Val Tyr Asn Ala Ser Ser Ala Leu Asp Ser Gln Leu Ile Tyr
850 855 860
Glu Gly Phe Ser Asn Phe Gln Asp Phe Val Thr Asn Asp Ser Glu Tyr
865 870 875 880
Thr Asn Lys Lys Ile Ala Gln Asn Val Asp Leu Phe Lys Ser Trp Gly
885 890 895
Val Thr Ser Phe Glu Met Ala Pro Gln Tyr Val Ser Ser Glu Asp Gly
900 905 910
Ser Phe Leu Asp Ser Ile Ile Leu Asn Gly Tyr Ser Phe Glu Asp Arg
915 920 925
Tyr Asp Leu Ala Met Ser Lys Asn Asn Lys Tyr Gly Ser Gln Gln Asp
930 935 940
Met Ile Asn Ala Leu Lys Ala Leu His Arg Asn Gly Ile Gln Val Ile
945 950 955 960
Ala Asp Trp Val Pro Asp Gln Ile Tyr Asn Leu Pro Gly Arg Glu Val
965 970 975
Val Thr Ala Thr Arg Val Asn Asp Tyr Gly Asp Tyr Arg His Asp Ser
980 985 990
Glu Ile Lys Asp Ser Leu Tyr Leu Ala Asn Thr Lys Thr Asn Gly Asp
995 1000 1005
Asp Tyr Gln Ala Lys Tyr Gly Gly Ala Phe Leu Asp Glu Leu Ala Ala
1010 1015 1020
Lys Tyr Pro Ser Ile Phe Glu Arg Thr Gln Ile Ser Asn Gly Gln Lys
1025 1030 1035 1040
Leu Asp Pro Ser Glu Lys Ile Thr Gln Trp Gln Ala Lys Tyr Phe Asn
1045 1050 1055
Gly Thr Asn Ile Leu Gly Arg Gly Val Gly Tyr Val Leu Lys Asp Ala
1060 1065 1070
Ala Ser Asp Lys Tyr Phe Thr Leu Asp Gly Lys Glu Thr Tyr Leu Pro
1075 1080 1085
Lys Gln Leu Thr Asn Gln Glu Ala Leu Thr Gly Phe Val Asn Asp Gly
1090 1095 1100
Ser Gly Val Thr Phe Tyr Ser Ser Ser Gly Tyr Gln Ala Lys Asn Ser
1105 1110 1115 1120
Phe Val Gln Asp Ala Lys Gly Asn Trp Tyr Tyr Phe Asp Lys Asp Gly
1125 1130 1135
His Met Val Tyr Gly Gln Gln His Leu Asn Asn Glu Val Gln Tyr Phe
1140 1145 1150
Leu Pro Asn Gly Val Gln Leu Arg Glu Ala Ile Leu Glu Asn Ala Asp
1155 1160 1165
Gly Thr Lys Asn Tyr Phe Gly Arg Leu Gly Asn Arg Phe Ser Asn Asp
1170 1175 1180
Tyr Tyr Ser Phe Glu Lys Asp Thr Lys Trp Arg Tyr Phe Asp Glu Asn
1185 1190 1195 1200
Gly Val Met Ala Val Gly Leu Lys Thr Ile Asn Gly Asn Thr Gln Tyr
1205 1210 1215
Phe Asp Gln Asn Gly Tyr Gln Val Lys Gly Glu Trp Ile Thr Asp Ala
1220 1225 1230
Asp Gly Lys Lys Arg Tyr Phe Asp Asp Gly Ser Gly Asn Met Ala Val
1235 1240 1245
Asn Arg Phe Ala Asn Asp Ser Asn Gly Asp Trp Tyr Tyr Leu Gly Ala
1250 1255 1260
Asp Gly Thr Ala Gln Thr Gly Ser Gln Thr Ile Asp Gly Lys Ser Tyr
1265 1270 1275 1280
Tyr Phe Asp Glu Asn Gly Lys Gln Val Lys Gly Lys Ile Ile Thr Ala
1285 1290 1295
Asn Asp Gly Lys Leu Tyr Tyr Tyr Leu Ala Asp Ser Gly Glu Leu Ala
1300 1305 1310
His Asn Ile Phe Ala Thr Asp Ser Gln Asn Asn Trp Tyr Tyr Phe Gly
1315 1320 1325
Ser Asp Gly Thr Ala Val Thr Gly Ser Gln Thr Ile Asn Gly Gln Asn
1330 1335 1340
Leu Tyr Phe Ala Asn Asp Gly Lys Gln Val Lys Gly Asp Phe Val Asn
1345 1350 1355 1360
Glu Asn Gly Lys Ile Arg Tyr Tyr His Ala Asp Ser Gly Gln Leu Gln
1365 1370 1375
Thr Asn Arg Phe Glu Ala Asp Lys Asp Gly Asn Trp Tyr Tyr Leu Gly
1380 1385 1390
Ser Asp Gly Thr Ala Leu Thr Gly Ser Gln Tyr Ile Asn Asn Gln Arg
1395 1400 1405
Leu Phe Phe Thr Arg Glu Gly Lys Gln Val Lys Gly Asp Val Ala Tyr
1410 1415 1420
Asp Gly Trp Gly Leu Leu Arg Tyr Tyr Asp Lys Asp Ser Gly Ser Met
1425 1430 1435 1440
Val Tyr Asn Arg Leu Val Thr Leu Ala Asn Gly Arg Arg Ile Gly Ile
1445 1450 1455
Asn Arg Trp Gly Ile Ala Arg Tyr Leu
1460 1465

Claims (8)

1.一种糖基转移酶的突变体，其特征在于，所述糖基转移酶突变体是将氨基酸序列如SEQ ID NO.2所示的糖基转移酶的第590位Gln氨基酸突变为Glu得到的，所述糖基转移酶突变体的氨基酸序列如SEQ ID NO.4所示。
2.编码权利要求1所述糖基转移酶突变体的基因，其碱基序列如SEQ ID NO.3所示。
3.携带权利要求2所述基因的重组质粒。
4.如权利要求3所述的重组质粒，其特征在于，所述重组质粒的载体为pET载体、pGEX载体或pUC载体。
5.一种工程菌株，其特征在于，携带权利要求3或4所述的重组质粒。
6.如权利要求5所述的工程菌株，其特征在于，所述工程菌株为E.coliBL21-Q590E。
7.权利要求1所述糖基转移酶的突变体、权利要求2所述基因、权利要求3或4中所述重组质粒、权利要求5或6中所述工程菌株在甜味剂甜菊醇糖苷糖基化修饰中的应用。
8.如权利要求7所述的应用，其特征在于，所述突变体的催化效率是野生型的5～6倍。
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