CN109486793B - 一种蔗糖水解酶突变体及其制备方法与应用 - Google Patents

一种蔗糖水解酶突变体及其制备方法与应用 Download PDF

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CN109486793B
CN109486793B CN201811425686.3A CN201811425686A CN109486793B CN 109486793 B CN109486793 B CN 109486793B CN 201811425686 A CN201811425686 A CN 201811425686A CN 109486793 B CN109486793 B CN 109486793B
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吴敬
宿玲恰
郭志勇
李玲玲
姚锴琳
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Abstract

本发明公开了一种蔗糖水解酶,属于基因工程和酶工程领域。本发明对来源于Xanthomonas axonopodis pv.glycines、Janthinobacterium agaricidamnosum NBRC 102515、Caulobacter crescentus NA1000 CB15的蔗糖水解酶进行改造,分别对其第271位或第279位或第281位的丝氨酸残基进行定点突变,获得的单突变体酶的转苷活性较野生型蔗糖水解酶升高。这一发明有助于对于糖苷水解酶转苷和水解机理的研究,亦可应用于糖苷水解酶工业生产多聚糖。

Description

一种蔗糖水解酶突变体及其制备方法与应用
技术领域
本发明涉及一种蔗糖水解酶突变体及其制备方法与应用,属于基因工程和酶工程领域。
背景技术
蔗糖水解酶(SH,EC 3.2.1.–),属于GH13糖苷水解酶家族,是一个很强的水解酶。几乎没有转苷能力,可将蔗糖分子几乎等比例地水解为葡萄糖和果糖分子。蔗糖水解酶含有5个结构域(A、B、B'、C和N),其中A、B和B'-结构域构成了蔗糖水解酶的催化核心。
现有的蔗糖水解酶大多水解能力很强,转苷能力相对比较弱。研究水解和转苷的决定机制一直是一个热门的话题。目前,已经有许多关于水解和转苷的报道,但大多集中在供体和受体位点,能够显著改变水解和转苷平衡的的方法目前报道的还很少。因此,本发明通过单点突变即显著提高了蔗糖水解酶的转苷能力,说明此位点对蔗糖水解酶的水解和转苷作用很重要。进而该位点可以为其他糖苷水解酶的水解和转苷的改造提供参考和借鉴。
发明内容
本发明所要解决的一个技术问题是提供一种蔗糖水解酶的突变体,对Caulobacter crescentus NA1000 CB15来源的蔗糖水解酶的第271位丝氨酸进行突变;
或,对Janthinobacterium agaricidamnosum NBRC 102515来源的蔗糖水解酶的第279位丝氨酸进行突变;
或,对Xanthomonas axonopodis pv.glycines来源的蔗糖水解酶的第281位丝氨酸进行突变;
上述突变位点与蔗糖水解酶的转苷作用和水解作用相关。
在本发明的一种实施方式中,所述来源于Caulobacter crescentus NA1000 CB15蔗糖水解酶的氨基酸序列如SEQ ID NO.1所示,所述来源于Janthinobacteriumagaricidamnosum NBRC 102515蔗糖水解酶的氨基酸序列如SEQ ID NO.2所示,所述来源于Xanthomonas axonopodis pv.glycines蔗糖水解酶的氨基酸序列如SEQ ID NO.3所示。
在本发明的一种实施方式中,所述突变是将氨基酸序列如SEQ ID NO.1所示的第271位丝氨酸残基变为丙氨酸残基,突变体命名为S271A;
或,所述突变是将氨基酸序列如SEQ ID NO.2所示的第279的丝氨酸残基变为丙氨酸残基,突变体命名为S279A;
或,所述突变是将氨基酸序列如SEQ ID NO.3所示的第281的丝氨酸残基变为丙氨酸残基,突变体命名为S281A。
编码所述蔗糖水解酶突变体的基因。
携带所述蔗糖水解酶突变体的基因的载体。
携带所述蔗糖水解酶突变体的基因的重组细胞。
本发明所要解决的另一个技术问题是提供一种蔗糖水解酶的突变体的制备方法,包括如下步骤:
(1)在蔗糖水解酶氨基酸序列的基础上确定突变位点;设计定点突变的突变引物,以携带蔗糖水解酶基因的载体为模板进行定点突变;构建含突变体的质粒载体;
(2)将突变体质粒转化进宿主细胞;
(3)挑选阳性克隆进行发酵培养,并分别纯化蔗糖水解酶突变体S271A、S281A、S279A。
所述质粒载体为pUC系列,pET系列,或pGEX中的任意一种。
所述宿主细胞为细菌和真菌细胞,其也为本发明的保护范围。
所述的细菌为革兰氏阴性菌或革兰氏阳性菌。
所述的蔗糖水解酶突变体在生产多聚糖中的应用。
有益效果:
本发明对来源于Caulobacter crescentus NA1000 CB15的蔗糖水解酶第271位的丝氨酸残基进行定点突变,来源于Janthinobacterium agaricidamnosum NBRC 102515的蔗糖水解酶第279位的丝氨酸残基进行定点突变,来源于Xanthomonas axonopodispv.glycines的蔗糖水解酶第281位的丝氨酸残基进行定点突变,获得的单突变体酶的转苷活性较野生型蔗糖水解酶均得到提高。在最优的酶转化条件下,蔗糖水解酶突变体转苷活性较野生酶水解活性最大提高10倍。因此,本发明提供的蔗糖水解酶突变体S271A、S279A、S281A可以应用于糖苷水解酶工业生产多聚糖。
附图说明
图1野生酶以及突变体的水解率、异构率、聚合率和转苷率的HPLC检测结果。
具体实施方式
下述实施例中所涉及的培养基和计算方法如下:
LB固体培养基:5g/L酵母粉,10g/L蛋白胨,5g/L NaCl,2%琼脂粉。
LB液体培养基:5g/L酵母粉,10g/L蛋白胨,5g/L NaCl。
水解率={(生成的葡萄糖含量)/(消耗的蔗糖含量-生成的果糖含量)}*100%
异构率={(异构产物含量)/(消耗的蔗糖含量-生成的果糖含量)}*100%
聚合率={(聚合产物含量)/(消耗的蔗糖含量-生成的果糖含量)}*100%
转苷率=异构率+聚合率
具体实施方式
实施例1:重组菌构建
根据NCBI上登录号为YP_002516566.2的蔗糖水解酶基因序列采用化学合成法合成含有蔗糖水解酶的CcSH基因,登录号为CDG80999.1的蔗糖水解酶基因序列采用化学合成法合成含有蔗糖水解酶的JaSH基因,登录号为AAQ93678.1的蔗糖水解酶基因序列采用化学合成法合成含有蔗糖水解酶的XaSH基因。将CcSH基因、JaSH基因和XaSH基因分别与pET-24a(+)质粒用NdeI和HindⅢ双酶切,酶切产物割胶回收后,再用T4连接酶连接,连接产物转化E.coli JM109感受态细胞,得到重组细胞。将重组细胞经37℃培养8h,挑取转化子在LB液体培养基(含30mg/L卡纳霉素)中震荡培养,提取质粒,酶切验证后分别得到表达质粒CcSH/pET-24a(+)、JaSH/pET-24a(+)、XaSH/pET-24a(+)。
将质粒CcSH/pET-24a(+)、JaSH/pET-24a(+)、XaSH/pET-24a(+)分别转化E.coliBL21(DE3)宿主菌,涂布LB平板(含30mg/L卡纳霉素),37℃培养8h,挑单菌落到LB液体培养基(含30mg/L卡纳霉素)中,37℃培养过夜,保存于甘油管中。
挑取重组菌E.coli J BL21(DE3)/CcSH/pET-24a(+)、E.coli BL21(DE3)/JaSH/pET-24a(+)、E.coli J BL21(DE3)/XaSH/pET-24a(+),于LB液体培养基(含30μg/mL卡纳霉素)生长8~10h,按5%接种量将种子发酵液接到TB培养基(含30μg/mL卡纳霉素)中,当600nm处的光密度达到0.6时,加入0.4mM异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)进行诱导,在25℃摇床中培养24h后,将发酵液于4℃、8000rpm离心20min除菌体,收集离心上清液得到粗酶液。
实施例2:蔗糖水解酶突变体的制备
(1)蔗糖水解酶单突变的制备
根据蔗糖水解酶的CcSH基因序列,设计并合成引入S271A突变的引物,利用快速PCR技术,以携带编码野生型蔗糖水解酶的基因的质粒CcSH/pET-24a(+)为模板,对蔗糖水解酶的CcSH基因序列进行定点突变,测定DNA编码序列,鉴别出第271位Ser密码子变成Ala密码子的基因,得到蔗糖水解酶单突变S271A。
根据蔗糖水解酶的JaSH基因序列,设计并合成引入S279A突变的引物,利用快速PCR技术,以携带编码野生型蔗糖水解酶的基因的质粒JaSH/pET-24a(+)为模板,对蔗糖水解酶的JaSH基因序列进行定点突变,测定DNA编码序列,鉴别出第279位Ser密码子变成Ala密码子的基因,得到蔗糖水解酶单突变S279A。
根据蔗糖水解酶的XaSH基因序列,设计并合成引入S281A突变的引物,利用快速PCR技术,以携带编码野生型蔗糖水解酶的基因的质粒XaSH/pET-24a(+)为模板,对蔗糖水解酶的XaSH基因序列进行定点突变,测定DNA编码序列,鉴别出第281位Ser密码子变成Ala密码子的基因,得到蔗糖水解酶单突变S281A。
引入S271A突变的定点突变引物为:
核苷酸序列为SEQ ID NO.4的正向引物:
5’-GGCTTTCGCTTAGATGCCGCACCGTTTCTGTGG-3’(下划线为突变碱基)
核苷酸序列为SEQ ID NO.5的反向引物:
5’-CCACAGAAACGGTGCGGCATCTAAGCGAAAGCC-3’(下划线为突变碱基)
引入S279A突变的定点突变引物为:
核苷酸序列为SEQ ID NO.6的正向引物:
5’-GTGTTTCGCTTAGATGCAACCGCCTTTCTG-3’(下划线为突变碱基)
核苷酸序列为SEQ ID NO.7的反向引物:
5’-CAGAAAGGCGGTTGCATCTAAGCGAAACAC-3’(下划线为突变碱基)
引入S281A突变的定点突变引物为:
核苷酸序列为SEQ ID NO.8的正向引物:
5’-GGCATTTCGTCTGGATGCAACAGCGTATCTGTG-3’(下划线为突变碱基)
核苷酸序列为SEQ ID NO.9的反向引物:
5’-CACAGATACGCTGTTGCATCCAGACGAAATGCC-3’(下划线为突变碱基)
PCR反应体系均为:5×PS buffer 10μL,dNTPs Mix(2.5mM)4μL,正向引物(10μM)1μL,反向引物(10μM)1μL,模板DNA 1μL,PrimerStar HS(5U/μL)0.5μL,加入双蒸水至50μL。
PCR扩增条件为:94℃预变性4min;随后30个循环(98℃10s,55℃5s,72℃8min);72℃继续延伸10min。
PCR产物经DpnⅠ消化,转化大肠杆菌JM109感受态,感受态细胞在LB固体培养基(含30μg/mL卡纳霉素)培养过夜后,挑克隆于LB液体培养基(含30μg/mL卡纳霉素)中培养后提取质粒,所有突变质粒均测序正确,得到的重组菌命名为E.coli JM109/CcSH/pET-24a(+)(S271A)、E.coli JM109/JaSH/pET-24a(+)(S279A)、E.coli JM109/XaSH/pET-24a(+)(S281A)。
测序正确的突变体,从甘油管接种至LB培养基,过夜培养,提取质粒,将质粒转化表达宿主大肠杆菌BL21(DE3)感受态细胞,得到的重组菌命名为E.coli J BL21(DE3)/CcSH/pET-24a(+)(S271A)、E.coli BL21(DE3)/JaSH/pET-24a(+)(S279A)、E.coli J BL21(DE3)/XaSH/pET-24a(+)(S281A)。
(2)突变体酶的发酵与纯化
分别挑取重组菌E.coli J BL21(DE3)/CcSH/pET-24a(+)(S271A)、E.coli BL21(DE3)/JaSH/pET-24a(+)(S279A)、E.coli J BL21(DE3)/XaSH/pET-24a(+)(S281A),于LB液体培养基(含30μg/mL卡纳霉素)生长8~10h,按5%接种量将种子发酵液接到TB培养基(含30μg/mL卡纳霉素)中,当600nm处的光密度达到0.6时,加入0.4mM异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)进行诱导,在25℃摇床中培养24h后,将发酵液于4℃、8000rpm离心20min除菌体,收集离心上清液得到粗酶液。
实施例3:粗酶液的浓缩
将实施例1和2中获得的酶液边搅拌边缓慢加入浓度为相对于酶液质量分数20%的硫酸铵,搅拌至硫酸铵溶解,在4℃条件下静置8~10小时沉淀蛋白。混合物经离心(8000rpm,10min)收集沉淀,再用最小体积的50mM KH2PO4-Na2HPO4缓冲液(pH 7.0)复溶,复溶后经过再次离心除去固形物,收集上清透析后获得浓缩酶液。
实施例4:HPLC检测水解和转苷产物的产量
在反应器中加入100mM蔗糖作为底物,加入6U/ml酶活的实例3中获得的突变体的浓缩酶液,和野生型加酶量保持一致。在30℃,150rpm的水浴摇床中反应24小时后取样,终止反应后,过膜过滤并进行HPLC分析。
色谱条件如下:Agilent 1200HPLC色谱仪,Agilent自动进样器,Agilent氨基柱5mm,(4.6mm×250mm)示差折光检测器;流动相为080%乙腈,20%超纯水,流速0.8mL min-1;柱温35℃。
HPLC检测结果见图1,其中水解率表示生成的葡萄糖含量,异构率表示生产的异构产物(异构产物是松二糖和海藻酮糖)的含量,聚合率表示生产的聚合产物(聚合产物是麦芽寡糖)的含量,转苷率包括异构率加聚合率。
结果数值见表1,蔗糖水解酶的突变体转苷率大幅度增加,其中突变体S281A的转苷率增加幅度最大。是野生型的转苷率的11倍左右,水解率降低至野生型的34%左右;突变体S279A的转苷率是野生型的转苷率的10倍左右,水解率降低至野生型的59%左右;突变体S271A的转苷率是野生型的转苷率的8倍左右,水解率降低至野生型的49%左右。这说明突变位点是蔗糖水解酶糖苷和水解功能的关键位点,对于糖苷水解酶的糖苷和水解性质的研究具有重要意义。
表1野生酶以及突变体的水解率、异构率、聚合率和转苷率
Figure BDA0001881554610000051
Figure BDA0001881554610000061
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种蔗糖水解酶突变体及其制备方法与应用
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 609
<212> PRT
<213> Caulobacter crescentus NA1000 CB15
<400> 1
Met Ile Ser Thr Ala Ser Ile Pro Thr Pro Tyr Asp Asp Ala Val Gln
1 5 10 15
Ala Arg Phe Ala Ala Leu Trp Pro Ile Val Glu Ser Arg Phe Ala Lys
20 25 30
Leu Tyr Gly Ala Asp Ala Arg Gly Pro Ala Val Leu Glu Arg Leu Lys
35 40 45
Thr Asn Leu Leu Lys Ala Ala His Ala Arg Pro Glu Pro Leu Arg Ala
50 55 60
Leu Asp Ala Ala Arg Ala Ala Asp Pro Ala Trp Leu His Ala Pro Gly
65 70 75 80
Gln Thr Ala Tyr Thr Phe Tyr Val Asp Arg Phe Ala Gly Asp Leu Asn
85 90 95
Gly Val Arg Gly Lys Leu Asp Tyr Leu Thr Glu Leu Gly Val Arg Trp
100 105 110
Leu His Pro Leu Pro Leu Leu Glu Pro Arg Pro Gly Asp Ser Asp Gly
115 120 125
Gly Phe Ala Val Ala Asp Tyr Arg Lys Val Asp Pro Arg Leu Gly Thr
130 135 140
Ile Asp Asp Leu Glu Ala Leu Ala Gly Asp Leu Arg Gln Arg Asp Met
145 150 155 160
Gly Leu Ile Leu Asp Val Val Cys Asn His Thr Ala Arg Glu His Ala
165 170 175
Trp Ala Ala Lys Ala Arg Ala Gly Asp Pro Ala Tyr Arg Asp Tyr Tyr
180 185 190
Ile Val Leu Pro Asp Ala Gln Ser Ala Ala Ala Arg Asp Arg Glu Leu
195 200 205
Ile Asp Val Phe Pro Asp Thr Ala Pro Gly Ser Phe Thr Tyr Asp Ala
210 215 220
Ala Met Gly Gly Tyr Val Trp Thr Thr Phe Tyr Pro Phe Gln Trp Asp
225 230 235 240
Leu Asn Tyr Ala Asn Pro Ala Val Phe Ala Glu Met Leu Glu Val Leu
245 250 255
Ile Phe Leu Ala Ala Lys Gly Ala Gln Gly Phe Arg Leu Asp Ser Ala
260 265 270
Pro Phe Leu Trp Lys Gln Ala Gly Thr Thr Cys Arg Asn Leu Pro Gln
275 280 285
Thr Tyr Glu Ile Val Glu Ala Trp Arg Ala Ala Leu Ser Ile Val Ala
290 295 300
Pro Gly Val Val Leu Leu Ala Glu Ala Ile Glu Ser Val Glu Asp Val
305 310 315 320
Leu Pro Phe Phe Gly Gly Glu Glu Ser Gly Cys Asn Leu Ala Tyr Asn
325 330 335
Asn Val Val Met Thr Ala Leu Trp Ala Ala Leu Ala Asp Gly Asp Ala
340 345 350
Val Ile Ala Arg Arg Cys Leu Ala Val Ala Ala Arg Lys Pro Ala Gln
355 360 365
Gly Ala Trp Leu Asn Tyr Val Arg Cys His Asp Asp Leu Ile Trp Asn
370 375 380
Ala Leu Ala Ala Tyr Ala Pro Ala Ser Asp Leu Arg Arg Trp Ser Asn
385 390 395 400
Ala Tyr Gly Asn Gly Glu Gly Phe Ser Arg Gly Arg Ala Phe Gln Thr
405 410 415
Ala Glu Gly Gly Val Pro Ser Thr Asn Gly Met Ala Ala Ala Leu Ala
420 425 430
Gly Leu Thr Ala Asp Ala Asp Gly Asp Cys Leu Gly Ala Arg Arg Leu
435 440 445
Arg Leu Leu Tyr Gly Ile Ile His Ala Leu Asp Gly Trp Pro Leu Ile
450 455 460
Tyr Met Gly Asp Glu Ile Gly Leu Asp Asn Asp Glu Ala Tyr Gln Asp
465 470 475 480
Asp Pro Leu Arg Ala Gly Asp Gly Arg Trp Leu His Arg Pro Gln Met
485 490 495
Asp Trp Ser Leu Ala Glu Arg Arg Gly Glu Ala Gly Ala Leu Gln Ala
500 505 510
Asp Leu Phe Ala Thr Phe Ala Arg Leu Gly Gln Arg Ala Arg Arg Leu
515 520 525
Ala Thr Leu Gly Val Ala Gly Pro Ala Arg Pro Val Glu Val Ser Ser
530 535 540
Pro Ala Val Leu Ala Phe Leu Arg Asp Glu Gly Ala Arg Pro Phe Leu
545 550 555 560
Cys Val Ala Asn Val Ser Asp Ala Pro Gln Asp Phe Glu Leu Pro Pro
565 570 575
Ala Phe Ala Gln Gly Ala Glu Asp Val Leu Asp Gly Ala Pro Ser Pro
580 585 590
Ala Gly Ala Val Ser Leu Pro Pro Tyr Gly Ile Thr Trp Leu Val Ala
595 600 605
Arg
<210> 2
<211> 619
<212> PRT
<213> Janthinobacterium agaricidamnosum NBRC 102515
<400> 2
Met Met Ser Glu Lys Thr Pro Leu Glu Cys Leu Leu Ala Ala Met Pro
1 5 10 15
Val His Leu Gln Thr Glu Ala Ala Arg Arg Tyr Thr Arg Gln Glu Pro
20 25 30
Val Leu Phe Glu Arg Leu Gly Arg Leu Tyr Gly Gly Arg Ala Asp Phe
35 40 45
Leu Pro Trp Phe Cys Asp Leu Met Glu Ser Val Gly Arg Leu His Ala
50 55 60
Ala Arg Pro Ala Ala Leu Leu Arg Leu Asp Ala Glu Arg Leu Ala Arg
65 70 75 80
Pro Asp Trp Phe Ser Ser Gln Trp Met Leu Gly Tyr Ser Ala Tyr Val
85 90 95
Gln Arg Phe Gly Gly Ser Leu Asn Gly Val Ala Gln Arg Ile Pro His
100 105 110
Leu Arg Glu Leu Gly Val Thr Tyr Leu His Leu Leu Pro Phe Leu Arg
115 120 125
Pro Arg Ala Gly Glu Ser Asp Gly Gly Phe Ala Val Ala Ser Phe Asp
130 135 140
Glu Val Asp Pro Glu Leu Gly Ser Asn Ala Asp Leu Glu Ser Leu Thr
145 150 155 160
Ala Gln Leu Arg Asp Ala Gly Ile Ser Leu Cys Ser Asp Leu Ile Leu
165 170 175
Asn His Val Ala Asp Asp His Ala Trp Ala Leu Gly Ala Lys Asn Gly
180 185 190
Asp Pro Leu Leu Arg Glu Phe Phe His Thr Phe Pro Asp Arg Ala Met
195 200 205
Pro Asp Arg Tyr Glu Ala Thr Leu Gly Gln Ile Phe Pro Gln Ala Ala
210 215 220
Pro Gly Asn Phe Thr Phe Asp Ala Ser Leu Gln Arg Trp Val Trp Thr
225 230 235 240
Thr Phe Tyr Pro Tyr Gln Trp Asp Leu Asn Tyr Ala Asn Pro Ala Val
245 250 255
Phe Ser Ala Met Met Ser Ala Met Leu Gly Leu Ala Asn Arg Gly Val
260 265 270
Glu Val Phe Arg Leu Asp Ser Thr Ala Phe Leu Trp Lys Arg Glu Gly
275 280 285
Thr Asp Ser Met Asn Gln Pro Glu Ala His Met Leu Leu Gln Ala Met
290 295 300
Arg Ala Ile Val Glu Ile Ala Ala Pro Gly Val Leu Met Lys Ala Glu
305 310 315 320
Ala Ile Val Pro Thr Pro Lys Leu Pro Ala Tyr Phe Gly Ser Asp Ala
325 330 335
Ala Pro Glu Cys His Ile Ala Tyr His Ser Ser Leu Met Ala Ala Gly
340 345 350
Trp Gly Ala Leu Ala Glu Gln Asp Thr Gly Leu Leu Arg Glu Val Val
355 360 365
Lys Ala Thr Pro Pro Leu Pro Pro Ala Ala Cys Trp Leu Ser Tyr Val
370 375 380
Arg Cys His Asp Asp Ile Gly Trp Asn Val Leu Leu Asn Glu Ala Gly
385 390 395 400
Ala Asp Gly Pro Ala Arg Leu Ala Arg Ile Ala Ala Phe Phe Ala Gly
405 410 415
Ala Asn Gly Ser Phe Ala Arg Gly Ala Ala Phe Gln Ser Ser Ser Ala
420 425 430
Asp Lys Ala His Gly Ser Asn Gly Met Ala Ala Ser Leu Thr Gly Leu
435 440 445
Glu Ser Ala Ala Asp Ala Gly Glu Arg Asp Leu Ala Leu Arg Arg Leu
450 455 460
Leu Leu Leu His Gly Leu Ala Leu Ser Phe Gly Ala Leu Pro Met Leu
465 470 475 480
Tyr Met Gly Asp Glu Leu Gly Met Thr Asn Asp Tyr Ser Tyr Ser Arg
485 490 495
Arg Ala Asp Arg Ala Met Asp Ser Arg Trp Leu Gln Arg Pro Leu Phe
500 505 510
Asp Asp Thr Leu Leu Ala Trp Arg Tyr Asp Arg Ser Ser Val Val Gly
515 520 525
Arg Leu Phe Ser Gly Leu Arg Gln Leu Ile Glu Leu Arg Arg Arg His
530 535 540
Glu Ala Leu Ala Ala Asp Ala Pro Arg Gln Leu Leu Ala Ser Gly Asp
545 550 555 560
Ala Ala Val Leu Ala Leu Met Arg Gly Pro Arg Phe Leu Asn Leu Ser
565 570 575
Asn Phe Gly Ala Arg Ser Val Ala Cys Asp Leu Pro Gln Gly Gly Trp
580 585 590
Arg Asp Gly Leu Ser Gly Val Glu Leu Ser Gly Lys Val Phe Leu Gln
595 600 605
Pro Trp Ala Met Leu Trp Leu Glu Arg Thr Thr
610 615
<210> 3
<211> 644
<212> PRT
<213> Xanthomonas axonopodis pv. glycines
<400> 3
Met Ser Thr Cys Pro Ile Asp Pro Pro Ala Leu Arg Ala Ala Phe Ala
1 5 10 15
Gly Pro Leu Asp Pro Gln His Ala Glu Val Leu Leu Ser Arg Tyr Asp
20 25 30
Gln His Ala Ser Arg Leu Leu Asp Ala Leu His Ala Leu Tyr Gly Gln
35 40 45
Arg Ala Asp Tyr Ala Ser Trp Leu Ala Gln Trp Leu Gly Glu Val Gly
50 55 60
Asp Ile Ala Arg Gln Arg Pro Gln Ala Leu Gln Thr Leu Asp Ser Thr
65 70 75 80
Arg His Ala Gly Trp Phe Gly Gln Pro His Met Leu Gly Tyr Ser Ala
85 90 95
Tyr Ala Asp Arg Phe Ala Gly Thr Leu Gln Gly Val Ala Glu Arg Val
100 105 110
Pro Tyr Leu Gln Glu Leu Gly Val Arg Tyr Leu His Leu Leu Pro Phe
115 120 125
Leu Arg Ala Arg Ala Gly Asp Asn Asp Gly Gly Phe Ala Val Ser Asp
130 135 140
Tyr Gly Gln Val Glu Pro Ser Leu Gly Ser Asn Asp Asp Leu Val Ala
145 150 155 160
Leu Thr Ser Arg Leu Arg Glu Ala Gly Ile Ser Leu Cys Ala Asp Phe
165 170 175
Val Leu Asn His Thr Ala Asp Asp His Ala Trp Ala Gln Ala Ala Arg
180 185 190
Ala Gly Asp Ala Arg Tyr Leu Asp Tyr Tyr His His Phe Ala Asp Arg
195 200 205
Thr Val Pro Asp Arg Tyr Glu Ala Thr Leu Gly Gln Val Phe Pro His
210 215 220
Thr Ala Pro Gly Asn Phe Thr Trp Val Asp Asp Thr Ala Gln Trp Met
225 230 235 240
Trp Thr Thr Phe Tyr Pro Tyr Gln Trp Asp Leu Asn Trp Ser Asn Pro
245 250 255
Ala Val Phe Gly Asp Met Ala Leu Ala Met Leu Arg Leu Ala Asn Leu
260 265 270
Gly Val Glu Ala Phe Arg Leu Asp Ser Thr Ala Tyr Leu Trp Lys Arg
275 280 285
Ile Gly Thr Asp Cys Met Asn Gln Ser Glu Ala His Thr Leu Leu Val
290 295 300
Ala Leu Arg Ala Val Thr Asp Ile Val Ala Pro Ala Val Val Met Lys
305 310 315 320
Ala Glu Ala Ile Val Pro Met Thr Gln Leu Pro Pro Tyr Phe Gly Ser
325 330 335
Gly Val Asp Glu Gly His Glu Cys His Leu Ala Tyr His Ser Thr Leu
340 345 350
Met Ala Ala Gly Trp Ser Ala Leu Ala Leu Gln Arg Gly Asp Ile Leu
355 360 365
His Asn Val Ile Ala His Ser Pro Pro Leu Pro Arg His Cys Ala Trp
370 375 380
Leu Ser Tyr Val Arg Cys His Asp Asp Ile Gly Trp Asn Val Leu Gln
385 390 395 400
His Glu Ala Cys Gly Asn Ala Ala Gln Pro Pro Phe Ser Leu Arg Asp
405 410 415
Val Ala Arg Phe Tyr Ala Asn Ala Val Pro Gly Ser Tyr Ala Arg Gly
420 425 430
Glu Ser Phe Gln Ser Ser Gly Asp Gly Val His Gly Thr Asn Gly Met
435 440 445
Ala Ala Ala Leu Ala Gly Ile Gln Ala Ala Gln Glu Ala Gly Asp Ala
450 455 460
Ala Ala Leu Ala Val Ala Val Asp Arg Leu Val Leu Leu Tyr Ala Ile
465 470 475 480
Ala Leu Ala Met Pro Gly Val Pro Leu Ile Tyr Met Gly Asp Glu Leu
485 490 495
Ala Met Val Asn Asp Pro Gly Tyr Arg Asp Asp Pro His Arg Gln His
500 505 510
Glu Gly Arg Trp Leu His Arg Pro Ala Met Asp Trp Gln Leu Ala Ala
515 520 525
Gln Arg His Asp Ala Lys Ser Leu Ser Gly Thr Val Tyr Arg Arg Leu
530 535 540
Arg Gly Leu Ile Arg Gln Arg Ala Ala Leu Gly Ala Leu Ala Ala Asp
545 550 555 560
Gln Ala Leu Ala Ser Ile Ala Leu Asn Asp Pro Arg Val Phe Ala Leu
565 570 575
Thr Arg Gly Asp Ser Phe Ile Ala Leu His Asn Phe Ser Asp Gln Leu
580 585 590
Leu Asp Val Glu Leu Ala Ala Ile Gly Val Asp Gly Trp Thr Leu Leu
595 600 605
Ala Ile Asp Asp Ala Ile Gly Gly Ala Ala Ala Arg Gly Asp Gly Ser
610 615 620
Ile Val Leu Pro Pro Tyr Gly Val Arg Trp Leu Gln Arg Gly Thr Glu
625 630 635 640
His Ala Pro Glu
<210> 4
<211> 33
<212> DNA
<213> 人工合成
<400> 4
ggctttcgct tagatgccgc accgtttctg tgg 33
<210> 5
<211> 33
<212> DNA
<213> 人工合成
<400> 5
ccacagaaac ggtgcggcat ctaagcgaaa gcc 33
<210> 6
<211> 30
<212> DNA
<213> 人工合成
<400> 6
gtgtttcgct tagatgcaac cgcctttctg 30
<210> 7
<211> 30
<212> DNA
<213> 人工合成
<400> 7
cagaaaggcg gttgcatcta agcgaaacac 30
<210> 8
<211> 33
<212> DNA
<213> 人工合成
<400> 8
ggcatttcgt ctggatgcaa cagcgtatct gtg 33
<210> 9
<211> 33
<212> DNA
<213> 人工合成
<400> 9
cacagatacg ctgttgcatc cagacgaaat gcc 33

Claims (8)

1.一种蔗糖水解酶突变体,其特征在于,所述蔗糖水解酶突变体与氨基酸序列如SEQID NO.1所示的蔗糖水解酶相比,第271位丝氨酸残基突变为了丙氨酸残基。
2.编码权利要求1所述蔗糖水解酶突变体的基因。
3.携带权利要求2所述基因的载体。
4.携带权利要求2所述基因的重组细胞。
5.制备权利要求1所述蔗糖水解酶突变体的方法,其特征在于,包括如下步骤:
(1)在蔗糖水解酶氨基酸序列上确定突变位点;设计定点突变的突变引物,以携带蔗糖水解酶基因的载体为模板进行定点突变;构建含突变体的质粒载体;
(2)将突变体质粒转化进宿主细胞;
(3)挑选阳性克隆进行发酵培养,并纯化蔗糖水解酶突变体。
6.根据权利要5所述的方法,其特征在于,所述质粒载体为pUC系列,pET系列,或pGEX中的任意一种。
7.根据权利要求5所述的方法,其特征在于,所述宿主细胞为细菌或真菌细胞;所述的细菌为革兰氏阴性菌或革兰氏阳性菌。
8.权利要求1所述的蔗糖水解酶突变体在生产多聚糖中的应用。
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