CN113174385B - 一种具有高活力和高转化率的蔗糖异构酶突变体及应用 - Google Patents
一种具有高活力和高转化率的蔗糖异构酶突变体及应用 Download PDFInfo
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- CN113174385B CN113174385B CN202110394774.7A CN202110394774A CN113174385B CN 113174385 B CN113174385 B CN 113174385B CN 202110394774 A CN202110394774 A CN 202110394774A CN 113174385 B CN113174385 B CN 113174385B
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- NMXLJRHBJVMYPD-IPFGBZKGSA-N trehalulose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@]1(O)CO[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 NMXLJRHBJVMYPD-IPFGBZKGSA-N 0.000 description 1
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Abstract
本发明公开了一种具有高活力和高转化率蔗糖异构酶突变体及应用，属于基因工程技术领域。该突变体包括突变体Y246L、H287R、H481P，属于基因工程技术领域。本发明通过设计定点突变引物，对基因进行定点突变，获得编码突变体的基因，并在大肠杆菌表达系统中进行表达，得到的蔗糖异构酶突变体活力和转化率显著提高。本发明制备的蔗糖异构酶突变体是一种更高效的生物催化剂，在异麦芽酮糖的工业生产中应用可以提高生产效率，减少生产成本。
Description
技术领域
本发明涉及一种具有高活力和高转化率的蔗糖异构酶突变体及应用，属于基因工程技术领域。
背景技术
异麦芽酮糖(isomaltulose)又称帕拉金糖，分子式为C12H22O11，是一种功能性二糖，属蔗糖同分异构体，为还原性糖，可被加氢还原。异麦芽酮糖晶体呈正交晶体，含水时异麦芽酮糖晶体的相对分子质量变为360.32，熔点为120-128℃，25℃时水溶性为250g/L；与蔗糖相比甜度自然且甜度为其52％，因而可作为新型甜味剂替代蔗糖。
美国FDA批准异麦芽酮糖作为安全型食品添加剂，无摄入量限制。其所具有的理化性质和生理功能也使得异麦芽酮糖能够替代蔗糖广泛的应用于功能饮料，减肥保健品，糖尿病人食品中等。我国糖尿病患者，肥胖综合症患者数以千万计，因而成为异麦芽酮糖工业化生产的市场保证。目前，全球已有40多个国家和地区已开始使用异麦芽酮糖来替代蔗糖作为食品添加剂，其中欧美与日本使用最多，2009年消耗量就已经达到100000t。而就我国而言，异麦芽酮糖需求量大，而国内异麦芽酮糖的工业化生产起步晚产能低，因而所需产品主要依赖德国和日本的进口，其中该糖的市场售价为1.5万元/t。
异麦芽酮糖在自然界极少，只少量的存在于蜂蜜和甘蔗汁中，因而直接的提取方法不可用。目前，经文献报道的异麦芽酮糖合成方法主要有：化学合成法、植物体内转化法、微生物细胞转化法、蔗糖异构酶转化法。化学合成法成本高且污染大，植物体内转化法会导致植物生产停滞，而微生物细胞转化法由于发酵液成分复杂，导致后期分离纯化困难。故目前生产异麦芽酮糖的最佳方式为蔗糖异构酶转化法。
蔗糖异构酶，又称α-葡糖基转移酶，能够将蔗糖转化成异麦芽酮糖和海藻酮糖，并生成少量单糖的一种酶。蔗糖异构酶的催化机理：亲核攻击作用蔗糖使α1-β1糖苷键断裂及果糖基的取向；异构化或水解的发生。蔗糖异构化过程即蔗糖异构酶催化蔗糖转化的两种去向如图4所示。
用蔗糖异构酶来实现异麦芽酮糖的高产率，高纯度，低成本的生产始终是蔗糖异构酶研究的方向。在用蔗糖异构酶生产异麦芽酮糖的过程中，酶的利用率低和不可避免生成的副产物是两个重要的不利因素，且因为这两个因素使得异麦芽酮糖的生产成本维持在较高水平，而高成本也限制了异麦芽酮糖替代蔗糖的步伐。与单糖副产物的产生相比，在使用蔗糖异构酶的过程中，产生蔗糖的另一种同分异构体——海藻酮糖，由于其和异麦芽酮糖结构的相似性，使得下游分离纯化的难度提高，一般工业上分离海藻酮糖与异麦芽酮糖使用重结晶的方法。更高的异麦芽酮糖转化率意味着在生产过程中，可以使异麦芽酮糖的产率更高，或生产纯度更高的异麦芽酮糖产品。与此同时，提高蔗糖异构酶的酶活，可以提高生产效率，减少生产成本。
为了获得更高效的生物催化剂，筛选新的并且可能具有高效率生成异麦芽酮糖的蔗糖异构酶是当前的关键任务。
发明内容
发明目的：为了克服现有技术中存在的不足，本发明提供一种具有高活力和高转化率的蔗糖异构酶突变体、工程菌及应用，通过定点突变的办法，对筛选到的蔗糖异构酶进行分子改造，从而进一步提高其催化活性并得到更适合于工业应用的蔗糖异构酶。
技术方案：本发明采用的技术方案如下。
本发明的第一个目的是，提供一种具有高活力和高转化率的蔗糖异构酶突变体，该蔗糖异构酶突变体的氨基酸序列如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8其中任意一种所示。
在本发明的一种实施方式中，所述蔗糖异构酶突变体是通过将出发氨基酸序列如SEQ ID NO.2所示的蔗糖异构酶经以下任意一种定位突变方式获得：
Ⅰ.第246位氨基酸由酪氨酸突变成亮氨酸，得突变体Y246L；
Ⅱ.第287位氨基酸由组氨酸突变成精氨酸，得突变体H287R；
Ⅲ.第481位氨基酸由组氨酸突变成脯氨酸，得突变体H481P。
本发明的另一个目的是，提供一种编码蔗糖异构酶突变体的基因，该基因的核苷酸序列包括以下任意一种：
a)SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.7其中任意一种所示的碱基序列；
b)编码由如SEQ ID NO.4、SEQ ID NO.6、SEQ ID NO.8其中任意一种所示的氨基酸序列或其无义突变序列组成的蛋白质。
在本发明的一种实施方式中，该基因的核苷酸序列由SEQ ID NO.1所示的模板核苷酸序列通过PCR扩增得到，所述PCR扩增对应的正向引物分别如SEQ ID NO.9、SEQ IDNO.11、SEQ ID NO.13所示，对应的反向引物分别如SEQ ID NO.10、SEQ ID NO.12、SEQ IDNO.14所示。
本发明的另一个目的是，提供一种用于编码蔗糖异构酶突变体的重组表达质粒，含有以上所述的核苷酸序列，表达载体包括pET-28a(+)。
本发明的另一个目的是，提供一种高效表达蔗糖异构酶的工程菌株，所述工程菌株能代谢产生上述的蔗糖异构酶突变体，或整合有上述的蔗糖异构酶突变体的基因，或含有上述的重组表达质粒。
在本发明的一种实施方式中，所述工程菌株以包括大肠杆菌、谷氨酸棒杆菌在内的宿主微生物为感受态细胞。
在本发明的一种实施方式中，所述宿主为大肠杆菌(Escherichia coli)BL21(DE3)。
本发明的另一个目的是，提供一种蔗糖异构酶突变体粗酶液，挑选上述的工程菌株中的阳性单克隆菌株进行发酵培养，经分离、纯化得蔗糖异构酶突变体发酵液，再经过滤后得所述蔗糖异构酶突变体粗酶液。
在本发明的一种实施方式中，所述发酵培养的条件为：发酵温度37℃、发酵时间16-18h；所述纯化的方法为镍离子亲和层析。
本发明的另一个目的是，提供上述的高效表达蔗糖异构酶的工程菌株或上述的蔗糖异构酶突变体粗酶液的应用，具体包括作为生物催化剂，用于催化异麦芽酮糖合成。
在本发明的一种实施方式中，在所述催化异麦芽酮糖合成的反应体系中，底物浓度为400g/L，加酶量25U/g，pH为4-7，温度为30-55℃。
在本发明的一种实施方式中，在所述催化异麦芽酮糖合成的反应体系中，pH为5.5，温度为40℃。
本发明的有益效果：
(1)本发明通过设计定点突变引物，对基因进行定点突变，获得编码突变体的基因，并在大肠杆菌表达系统中进行表达，得到的蔗糖异构酶突变体活力和转化率显著提高；
(2)本发明制备的蔗糖异构酶突变体是一种更高效的生物催化剂，在异麦芽酮糖的工业生产中应用可以提高生产效率，减少生产成本，在制备异麦芽酮糖过程中，底物的转化率有效提高至89.5％，相较于野生株，提高了15％以上。
(3)本发明制备的蔗糖异构酶突变体作为生物催化剂，在异麦芽酮糖的工业生产中，底物浓度提高至400g/L，适用于工业化大规模生产，具有广阔的实际生产应用前景。
附图说明
图1为蔗糖异构酶突变体Y246L反应产物的鉴定结果图；
图2为温度对蔗糖异构酶与其突变体Y246L、H287R、H481P催化反应的影响；
图3为pH对蔗糖异构酶与其突变体Y246L、H287R、H481P催化反应的影响；
图4为蔗糖异构化过程即蔗糖异构酶催化蔗糖转化的两种去向示意图。
具体实施方式
本发明以SEQ ID NO.1所示编码的pal-2基因的核苷酸序列为模板，以序列如SEQID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14所示的引物，进行PCR，即得到编码的246位氨基酸由酪氨酸突变成亮氨酸的突变体Y246L基因序列、编码的287位氨基酸由组氨酸突变成精氨酸的突变体H287R基因序列以及编码的481位氨基酸由组氨酸突变成脯氨酸的突变体H481P基因序列；将得到的重组基因序列，分别连接到pET-28a(+)表达载体中，得到重组质粒pET-28a(+)-Y246L、pET-28a(+)-H287R、pET-28a(+)-H481P，将重组质粒转化到大肠杆菌BL21(DE3)，挑选阳性单克隆进行发酵培养。
本发明所使用的原始基因序列SEQ ID NO.1来源于Raoultella terrigena，该菌种在GenBank中基因组编号是LR595855.1，而该酶基因的编号为VUC84579.1，基因全长为1800个核苷酸，如SEQ ID NO.1所示。该基因所编译的蛋白质编号是ACL40859.1，共有599个氨基酸，如SEQ ID NO.2所示。
以下对本发明的优选实施例进行说明，应当理解实施例是为了更好地解释本发明，不用于限制本发明。
实施例1
一种具有高活力和高转化率的蔗糖异构酶突变体Y246L，其制备方法的具体步骤如下：
(1)以携带蔗糖异构酶基因的载体pET-28a(+)-pal-2为模板进行定点突变构建突变质粒pET-28a(+)-Y246L，突变引物如下所示(下划线为突变点)：
正向突变引物如SEQ ID NO.9所示，其中CGTTGCG，下划线为突变碱基；
反向突变引物如SEQ ID NO.10所示，其中GCAACGC，下划线为突变碱基。
(2)进行PCR扩增，其条件为：95℃预变性3min，95℃变性30s，60℃退火l min，68℃延伸5min，进行35个循环，最后68℃保温10min。PCR扩增产物经琼脂糖凝胶电泳检测，割胶回收纯化。反应体系如表1所示。
表1蔗糖异构酶突变体Y246L的PCR扩增反应体系
(3)纯化后的PCR扩增产物经限制性内切酶BamHI和XhoI酶切后连接至载体pET-28a(+)，并转化至大肠杆菌DH5α感受态细胞中，培养后挑取单克隆菌株进行发酵，提取突变质粒pET-28a(+)-Y246L进行测序，核苷酸序列如SEQ ID NO.3所示，编码的氨基酸序列如SEQ ID NO.4所示。
(4)将测序正确的突变质粒pET-28a(+)-Y246L转化至大肠杆菌BL21(DE3)细胞，挑取单克隆于含30μg/mL卡那霉素的LB液体培养基中发酵培养，后转接入含30μg/mL卡那霉素的LB液体培养基37℃扩大培养和诱导得到发酵液。发酵液于4℃、8000rpm离心10min，收集菌体后超声波破碎，离心后上清液过水系膜，获得粗酶液。利用镍离子亲和层析柱纯化粗酶液，收集的酶液4℃条件下进行透析，透析完成后将目的蛋白溶液放在低温冷藏。纯化后pal-2的突变体酶Y246L经电泳验证达到电泳纯。
实施例2
一种具有高活力和高转化率的蔗糖异构酶突变体H287R，其制备方法的具体步骤如下：
(1)以携带蔗糖异构酶基因的载体pET-28a(+)-pal-2为模板进行定点突变构建突变质粒pET-28a(+)-H287R，突变引物如下所示(下划线为突变点)：
正向突变引物如SEQ ID NO.11所示，其中GTCGCGT，下划线为突变碱基；
反向突变引物如SEQ ID NO.12所示，其中ACGCGAC，下划线为突变碱基。
(2)进行PCR扩增，其条件为：95℃预变性3min，95℃变性30s，60℃退火l min，68℃延伸5min，进行35个循环，最后68℃保温10min。PCR扩增产物经琼脂糖凝胶电泳检测，割胶回收纯化。反应体系如表2所示。
表2蔗糖异构酶突变体H287R的PCR扩增反应体系
(3)纯化后的PCR扩增产物经限制性内切酶BamHI和XhoI酶切后连接至载体pET-28a(+)，并转化至大肠杆菌DH5α感受态细胞中，培养后挑取单克隆菌株进行发酵，提取突变质粒pET-28a(+)-H287R进行测序，核苷酸序列如SEQ ID NO.5所示，编码的氨基酸序列如SEQ ID NO.6所示。
(4)将测序正确的突变质粒pET-28a(+)-H287R转化至大肠杆菌BL21(DE3)细胞，挑取单克隆于含30μg/mL卡那霉素的LB液体培养基中发酵培养，后转接入含30μg/mL卡那霉素的LB液体培养基37℃扩大培养和诱导得到发酵液。发酵液于4℃、8000rpm离心10min，收集菌体后超声波破碎，离心后上清液过水系膜，获得粗酶液。利用镍离子亲和层析柱纯化粗酶液，收集的酶液4℃条件下进行透析，透析完成后将目的蛋白溶液放在低温冷藏。纯化后pal-2的突变体酶H287R经电泳验证达到电泳纯。
实施例3
一种具有高活力和高转化率的蔗糖异构酶突变体H481P，其制备方法的具体步骤如下：
(1)以携带蔗糖异构酶基因的载体pET-28a(+)-pal-2为模板进行定点突变构建突变质粒pET-28a(+)-H481P，突变引物如下所示(下划线为突变点)：
正向突变引物如SEQ ID NO.13所示，其中TCCCGGT，下划线为突变碱基；
反向突变引物如SEQ ID NO.14所示，其中AGGGCCA，下划线为突变碱基。
(2)进行PCR扩增，其条件为：95℃预变性3min，95℃变性30s，60℃退火l min，68℃延伸5min，进行35个循环，最后68℃保温10min。PCR扩增产物经琼脂糖凝胶电泳检测，割胶回收纯化。反应体系如表3所示。
表3蔗糖异构酶突变体H481P的PCR扩增反应体系
(3)纯化后的PCR扩增产物经限制性内切酶BamHI和XhoI酶切后连接至载体pET-28a(+)，并转化至大肠杆菌DH5α感受态细胞中，培养后挑取单克隆菌株进行发酵，提取突变质粒pET-28a(+)-H481P进行测序，核苷酸序列如SEQ ID NO.7所示，编码的氨基酸序列如SEQ ID NO.8所示。
(4)将测序正确的突变质粒pET-28a(+)-H481P转化至大肠杆菌BL21(DE3)细胞，挑取单克隆于含30μg/mL卡那霉素的LB液体培养基中发酵培养，后转接入含30μg/mL卡那霉素的LB液体培养基37℃扩大培养和诱导得到发酵液。发酵液于4℃、8000rpm离心10min，收集菌体后超声波破碎，离心后上清液过水系膜，获得粗酶液。利用镍离子亲和层析柱纯化粗酶液，收集的酶液4℃条件下进行透析，透析完成后将目的蛋白溶液放在低温冷藏。纯化后pal-2的突变体酶H481P经电泳验证达到电泳纯。
为更好的解释本发明的显著效果，添加对比例如下：
对比例1
按常规方法制备蔗糖异构酶，不经任何基因突变。具体方法如下：
(1)将来源于Raoultella terrigena微生物的编码蔗糖异构酶基因在大肠杆菌工程菌中进行分子克隆表达。
(2)克隆所用质粒载体为pET-28a(+)，构建的重组表达质粒命名为pET-28a(+)-pal-2。宿主为E.coli DH5α，主要是用于质粒的扩增和抽提。将质粒pET-28a(+)-pal-2转化到E.coli BL21(DE3)细胞中，用于蔗糖异构酶的表达。质粒pET-28a(+)-pal-2转化到大肠杆菌BL21(DE3)细胞中后，将50μL细胞液体均匀涂布在含有50μg/mLkana抗生素的LB固体平板培养基上，37℃培养16小时后从平板中挑选阳性单克隆进行发酵培养。离心收集菌体，并进行超声破碎。破碎后的上清通过镍离子亲和层析纯化，纯化后进行透析，得到蔗糖异构酶。
实施例及对比例的产品特性对比
1、蔗糖异构酶与其突变体Y246L、H287R、H481P酶活测定
酶活测定方法：将100μL稀释后的酶液加入到900μL含有20％蔗糖的柠檬酸-磷酸氢二钠缓冲液(50mmol/L，PH6.0)中，30℃条件下反应15min，加热10min终止反应。4℃条件下12000r离心10min。离心上清液经0.22μm滤膜过滤到液相瓶中，高效液相配备氨基柱Shodex HILICpak VG-50 4E(4.6mmI.D.x 250mm)色谱柱和示差折光显示器用于检测异麦芽酮糖。1U酶活定义为在pH6.0，30℃条件下反应，每分钟生成1μmol异麦芽酮糖所需要的酶量。酶活比较：蔗糖异构酶在最适反应条件下的比酶活定义为100％，而突变体相对酶活是基于蔗糖异构酶酶活，即用突变体比酶活除以蔗糖异构酶酶活得到比酶活来计算。测定结果如下表4：
表4实施例及对比例产品酶活对比
结果表明，蔗糖异构酶的原始酶活为286.35U/mg，突变体酶活均有较大程度提高，其中突变体Y246L的酶活相较于原始酶活提高了27.5％。说明通过本发明制备的蔗糖异构酶突变体有利于提升蔗糖异构酶酶活。
2、温度对pal-2与其突变体Y246L、H287R、H481P催化反应的影响
将100μL稀释后的酶液加入到900μL含有20％蔗糖的50mmol/L柠檬酸-磷酸氢二钠缓冲液中，在PH6.0条件下分别在20℃、30℃、40℃、50℃、60℃条件下反应15min，沸水浴10min终止反应。4℃条件下12000r离心10min，反应液经过0.22μm滤膜过滤到液相瓶中，高效液相配备氨基柱Shodex HILICpak VG-50 4E(4.6mmI.D.x 250mm)色谱柱和示差折光显示器用于检测异麦芽酮糖。将结果中最高酶活的一组定义为相对酶活的100％，如附图2所示，对于pal-2与其突变体Y246L、H287R、H481P，均是在温度为40℃时，具有最好的催化活性。
3、pH对pal-2与其突变体Y246L、H287R、H481P催化反应的影响
将100μL稀释后的酶液加入到900μL含有20％蔗糖的50mmol/L柠檬酸-磷酸氢二钠缓冲液中，在40℃温度条件下，分别设置pH为3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0，反应15min，沸水浴10min终止反应。4℃条件下12000r离心10min，反应液经过0.22μm滤膜过滤到液相瓶中，高效液相配备氨基柱Shodex HILICpak VG-50 4E(4.6mmI.D.x250mm)色谱柱和示差折光显示器用于检测异麦芽酮糖。将结果中最高酶活的一组定义为相对酶活的100％。附图3所示，对于pal-2与其突变体Y246L、H287R、H481P，均是在pH 5.5时，具有最好的催化活性。
4、蔗糖异构酶与其突变体Y246L、H287R、H481P最大转化率
测定实施例和对比例产品在制备异麦芽酮糖过程中的转化率，转化条件为pH5.5、40℃、加酶量25U/g、底物浓度400g/L、转化时间3h。如图1所示为蔗糖异构酶突变体Y246L的反应产物的鉴定结果图，结果证实了异麦芽酮糖的成功转化；此外，蔗糖异构酶突变体H287R和H481P也具有与蔗糖异构酶突变体Y246L的反应产物相似的鉴定结果，证实了异麦芽酮糖的成功转化。各实施例的最大转化率结果如表5所示：
表5实施例及对比例产品最大转化率对比
可以发现与蔗糖异构酶酶相比，Y246L、H287R和H481P的最大转化率均提高了15％以上。蔗糖异构酶突变体不仅酶活得到提高，而且对异麦芽酮糖的生产也起到了一定的促进作用，说明蔗糖异构酶突变体具有更好的应用性能。
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。
<110> 苏州朗邦营养科技有限公司
<120> 一种具有高活力和高转化率的蔗糖异构酶突变体及应用
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accgataacg gctatgatat cagcaactac cagcagatca tgaaagaata cggcaccatg 360
gcagactttg acaccctgat tgcggaaatg aaaaaacgta acatgcgtct gatgattgat 420
gttgttatca accacacctc tgatcgtcac ccgtggttta tccagtctaa atccggcaaa 480
aacaacccgt accgtgatta ctacttctgg cgcgatggta aagataacca gccgccgaac 540
aactatccgt ccttcttcgg cggctctgcg tggcagaaag atgcgaaaag cggccagtac 600
tacctgcact acttcgcacg tcagcagccg gacctgaact gggataaccc gaaagttcgt 660
gaagatctgt atgcgatgct gcgcttctgg ctggataaag gcgtgtctgg catgcgtttc 720
gataccgttg cgacctattc caaaatccca ggttttccgg atctgacccc ggaacagcag 780
aaaaactttg cgcagcgcat accatgggtc cgaacatcca ccagtatatc caggaaatga 840
accgtgaagt gctgtcccac tatgacgtgg caaccgcagg tgaaatcttc ggtgttccgc 900
tggatcgtag ctctcagttc tttgaccgcc gccgtcatga actgaacatg gccttcatgt 960
tcgacttgat tcgtttggac cgtgatagca acgaacgttg gcgccaccgt ccgtggaccc 1020
tgtcccagtt tcgtcagatt gtgagcaaaa tggataccac cgttggtgaa tacggttgga 1080
acaccttctt tctggataac catgataacc cgcgtgcggt tagccacttt ggtgatgatt 1140
ctccgcagtg gcgtgaagct tctgcgaaag cactggccac cgtgaccctg acgcagcgtg 1200
cgaccccgtt catctaccag ggttccgaac tgggcatgac caactatccg ttcaaaaaac 1260
tgtctgaatt tgacgatatc gaagtgaaag gtttttggca tgattacgtt gaatctggta 1320
aagttaccgc ggctgaattc ctggataacg tgcgtctgac ctctcgtgat aacagccgca 1380
ccccgttcca gtgggatgat agccagaacg cgggtttcac cagcggtaaa ccgtggttcc 1440
acgtaaaccc gaactacgta cagattaacg cggcacgtga actgaccgaa aacaaatctg 1500
tgctgaacta ttataaacag atgatccacc tgcgtcacga actgccggct ctggtgtacg 1560
gtgcgtacca ggatctgaac ccgcaggata acaacgtata tgcttatact cgcaccctgg 1620
ataacgaacg ctatctggtt gtggttaact tcaaagaaca gccagttcgt tacgcgctgc 1680
cggacaacga tgctatcgaa caggtgatga tcgaaaccaa ccagcagagc gataccgcga 1740
aagaatccac cgcgatcgca ctgagcccgt ggcaggcggg cgtttataaa ctgcgttaa 1799
<210> 6
<211> 599
<212> PRT
<213> 蔗糖异构酶突变体H287R
<400> 6
Met Ser Phe Ile Lys Arg Arg Thr Ala Ala Ala Leu Ala Leu Ser Ser
1 5 10 15
Leu Met Met Thr Trp Ala Cys Pro Ser Leu Ser Ala Thr Pro Ser Phe
20 25 30
Ala Asp Asp Ile Asp Val His Lys Glu Asn Thr Phe Pro Ala Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Ile Arg Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ser Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Ser Asn Tyr Gln Gln
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Ala Asp Phe Asp Thr Leu Ile Ala
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Arg His Pro Trp Phe Ile Gln Ser Lys Ser Gly Lys
145 150 155 160
Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Asn
165 170 175
Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Ala Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Arg Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Glu Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Met Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Gly Phe Pro Asp Leu Thr
245 250 255
Pro Glu Gln Gln Lys Asn Phe Ala Gln Gln Tyr Thr Met Gly Pro Asn
260 265 270
Ile His Gln Tyr Ile Gln Glu Met Asn Arg Glu Val Leu Ser Arg Tyr
275 280 285
Asp Val Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Arg Ser
290 295 300
Ser Gln Phe Phe Asp Arg Arg Arg His Glu Leu Asn Met Ala Phe Met
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asn Glu Arg Trp Arg His
325 330 335
Arg Pro Trp Thr Leu Ser Gln Phe Arg Gln Ile Val Ser Lys Met Asp
340 345 350
Thr Thr Val Gly Glu Tyr Gly Trp Asn Thr Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Ser Pro Gln Trp
370 375 380
Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Val Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Lys Leu Ser Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp His Asp Tyr Val Glu Ser Gly Lys Val Thr Ala Ala Glu Phe Leu
435 440 445
Asp Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asp Asp Ser Gln Asn Ala Gly Phe Thr Ser Gly Lys Pro Trp Phe
465 470 475 480
His Val Asn Pro Asn Tyr Val Gln Ile Asn Ala Ala Arg Glu Leu Thr
485 490 495
Glu Asn Lys Ser Val Leu Asn Tyr Tyr Lys Gln Met Ile His Leu Arg
500 505 510
His Glu Leu Pro Ala Leu Val Tyr Gly Ala Tyr Gln Asp Leu Asn Pro
515 520 525
Gln Asp Asn Asn Val Tyr Ala Tyr Thr Arg Thr Leu Asp Asn Glu Arg
530 535 540
Tyr Leu Val Val Val Asn Phe Lys Glu Gln Pro Val Arg Tyr Ala Leu
545 550 555 560
Pro Asp Asn Asp Ala Ile Glu Gln Val Met Ile Glu Thr Asn Gln Gln
565 570 575
Ser Asp Thr Ala Lys Glu Ser Thr Ala Ile Ala Leu Ser Pro Trp Gln
580 585 590
Ala Gly Val Tyr Lys Leu Arg
595
<210> 7
<211> 1800
<212> DNA
<213> 蔗糖异构酶突变体H481P
<400> 7
atgagcttca tcaaacgtcg taccgcggca gctctggcgc tgagctccct gatgatgacc 60
tgggcgtgcc cgtctctgag cgcgaccccg agcttcgcgg acgatatcga tgttcacaaa 120
gaaaacacct tcccggcgtg gtggaaagaa gctgttttct accaggtgta cccgcgttct 180
ttcaaagata ccaacggcga tggcatcggt gatattcgcg gcatcatcga aaaactggat 240
tacctgaaat ccctgggcat cgacgcgatc tggatcaacc cgcactacga cagcccgaac 300
accgataacg gctatgatat cagcaactac cagcagatca tgaaagaata cggcaccatg 360
gcagactttg acaccctgat tgcggaaatg aaaaaacgta acatgcgtct gatgattgat 420
gttgttatca accacacctc tgatcgtcac ccgtggttta tccagtctaa atccggcaaa 480
aacaacccgt accgtgatta ctacttctgg cgcgatggta aagataacca gccgccgaac 540
aactatccgt ccttcttcgg cggctctgcg tggcagaaag atgcgaaaag cggccagtac 600
tacctgcact acttcgcacg tcagcagccg gacctgaact gggataaccc gaaagttcgt 660
gaagatctgt atgcgatgct gcgcttctgg ctggataaag gcgtgtctgg catgcgtttc 720
gataccgttg cgacctattc caaaatccca ggttttccgg atctgacccc ggaacagcag 780
aaaaactttg cgcagcagta taccatgggt ccgaacatcc accagtatat ccaggaaatg 840
aaccgtgaag tgctgtccca ctatgacgtg gcaaccgcag gtgaaatctt cggtgttccg 900
ctggatcgta gctctcagtt ctttgaccgc cgccgtcatg aactgaacat ggccttcatg 960
ttcgacttga ttcgtttgga ccgtgatagc aacgaacgtt ggcgccaccg tccgtggacc 1020
ctgtcccagt ttcgtcagat tgtgagcaaa atggatacca ccgttggtga atacggttgg 1080
aacaccttct ttctggataa ccatgataac ccgcgtgcgg ttagccactt tggtgatgat 1140
tctccgcagt ggcgtgaagc ttctgcgaaa gcactggcca ccgtgaccct gacgcagcgt 1200
gcgaccccgt tcatctacca gggttccgaa ctgggcatga ccaactatcc gttcaaaaaa 1260
ctgtctgaat ttgacgatat cgaagtgaaa ggtttttggc atgattacgt tgaatctggt 1320
aaagttaccg cggctgaatt cctggataac gtgcgtctga cctctcgtga taacagccgc 1380
accccgttcc agtgggatga tagccagaac gcgggtttca ccagcggtaa accgtggttc 1440
ccggtaaacc cgaactacgt acagattaac gcggcacgtg aactgaccga aaacaaatct 1500
gtgctgaact attataaaca gatgatccac ctgcgtcacg aactgccggc tctggtgtac 1560
ggtgcgtacc aggatctgaa cccgcaggat aacaacgtat atgcttatac tcgcaccctg 1620
gataacgaac gctatctggt tgtggttaac ttcaaagaac agccagttcg ttacgcgctg 1680
ccggacaacg atgctatcga acaggtgatg atcgaaacca accagcagag cgataccgcg 1740
aaagaatcca ccgcgatcgc actgagcccg tggcaggcgg gcgtttataa actgcgttaa 1800
<210> 8
<211> 599
<212> PRT
<213> 蔗糖异构酶突变体H481P
<400> 8
Met Ser Phe Ile Lys Arg Arg Thr Ala Ala Ala Leu Ala Leu Ser Ser
1 5 10 15
Leu Met Met Thr Trp Ala Cys Pro Ser Leu Ser Ala Thr Pro Ser Phe
20 25 30
Ala Asp Asp Ile Asp Val His Lys Glu Asn Thr Phe Pro Ala Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Ile Arg Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ser Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Ser Asn Tyr Gln Gln
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Ala Asp Phe Asp Thr Leu Ile Ala
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Arg His Pro Trp Phe Ile Gln Ser Lys Ser Gly Lys
145 150 155 160
Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Asn
165 170 175
Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Ala Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Arg Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Glu Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Met Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Gly Phe Pro Asp Leu Thr
245 250 255
Pro Glu Gln Gln Lys Asn Phe Ala Gln Gln Tyr Thr Met Gly Pro Asn
260 265 270
Ile His Gln Tyr Ile Gln Glu Met Asn Arg Glu Val Leu Ser His Tyr
275 280 285
Asp Val Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Arg Ser
290 295 300
Ser Gln Phe Phe Asp Arg Arg Arg His Glu Leu Asn Met Ala Phe Met
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asn Glu Arg Trp Arg His
325 330 335
Arg Pro Trp Thr Leu Ser Gln Phe Arg Gln Ile Val Ser Lys Met Asp
340 345 350
Thr Thr Val Gly Glu Tyr Gly Trp Asn Thr Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Ser Pro Gln Trp
370 375 380
Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Val Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Lys Leu Ser Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp His Asp Tyr Val Glu Ser Gly Lys Val Thr Ala Ala Glu Phe Leu
435 440 445
Asp Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asp Asp Ser Gln Asn Ala Gly Phe Thr Ser Gly Lys Pro Trp Phe
465 470 475 480
Pro Val Asn Pro Asn Tyr Val Gln Ile Asn Ala Ala Arg Glu Leu Thr
485 490 495
Glu Asn Lys Ser Val Leu Asn Tyr Tyr Lys Gln Met Ile His Leu Arg
500 505 510
His Glu Leu Pro Ala Leu Val Tyr Gly Ala Tyr Gln Asp Leu Asn Pro
515 520 525
Gln Asp Asn Asn Val Tyr Ala Tyr Thr Arg Thr Leu Asp Asn Glu Arg
530 535 540
Tyr Leu Val Val Val Asn Phe Lys Glu Gln Pro Val Arg Tyr Ala Leu
545 550 555 560
Pro Asp Asn Asp Ala Ile Glu Gln Val Met Ile Glu Thr Asn Gln Gln
565 570 575
Ser Asp Thr Ala Lys Glu Ser Thr Ala Ile Ala Leu Ser Pro Trp Gln
580 585 590
Ala Gly Val Tyr Lys Leu Arg
595
<210> 9
<211> 42
<212> DNA
<213> 人工序列
<400> 9
gtttcgatac cgttgcgacc ctgtccaaaa tcccaggttt tc 42
<210> 10
<211> 42
<212> DNA
<213> 人工序列
<400> 10
gaaaacctgg gattttggac agggtcgcaa cggtatcgaa ac 42
<210> 11
<211> 31
<212> DNA
<213> 人工序列
<400> 11
gtgaagtgct gtcgcgttat gacgtggcaa c 31
<210> 12
<211> 31
<212> DNA
<213> 人工序列
<400> 12
gttgccacgt cataacgcga cagcacttca c 31
<210> 13
<211> 32
<212> DNA
<213> 人工序列
<400> 13
gtaaaccgtg gttcccggta aacccgaact ac 32
<210> 14
<211> 32
<212> DNA
<213> 人工序列
<400> 14
gtagttcggg tttaccggga accacggttt ac 32
Claims (11)
1.一种具有高活力和高转化率的蔗糖异构酶突变体，其特征在于，氨基酸序列如SEQID NO.4、SEQ ID NO.6、SEQ ID NO.8其中任意一种所示。
2.根据权利要求1所述的具有高活力和高转化率的蔗糖异构酶突变体，其特征在于，所述蔗糖异构酶突变体是通过将出发氨基酸序列如SEQ ID NO.2所示的蔗糖异构酶经以下任意一种定位突变方式获得：
Ⅰ.第246位氨基酸由酪氨酸突变成亮氨酸，得突变体Y246L；
Ⅱ.第287位氨基酸由组氨酸突变成精氨酸，得突变体H287R；
Ⅲ.第481位氨基酸由组氨酸突变成脯氨酸，得突变体H481P。
3.一种编码蔗糖异构酶突变体的基因，其特征在于，编码由如SEQ ID NO.4、SEQ IDNO.6、SEQ ID NO.8其中任意一种所示的氨基酸序列。
4.根据权利要求3所述的一种编码蔗糖异构酶突变体的基因，其特征在于，该基因的核苷酸序列以SEQ ID NO.1所示编码的pal-2基因的核苷酸序列为模板，通过PCR扩增得到。
5.一种用于编码蔗糖异构酶突变体的重组表达质粒，其特征在于，含有权利要求3所述的基因，表达载体包括pET-28a(+)。
6.一种高效表达蔗糖异构酶的工程菌株，其特征在于，所述工程菌株能代谢产生权利要求1或2所述的蔗糖异构酶突变体，或整合有权利要求3或4所述的蔗糖异构酶突变体的基因，或含有权利要求5所述的重组表达质粒。
7.根据权利要求6所述的高效表达蔗糖异构酶的工程菌株，其特征在于，所述工程菌株以包括大肠杆菌、谷氨酸棒杆菌在内的宿主微生物为感受态细胞。
8.一种蔗糖异构酶突变体粗酶液，其特征在于，挑选如权利要求6所述的工程菌株进行发酵培养，经分离、纯化得蔗糖异构酶突变体粗酶液。
9.根据权利要求6或7所述的高效表达蔗糖异构酶的工程菌株，或根据权利要求8所述的蔗糖异构酶突变体粗酶液的应用，其特征在于，作为生物催化剂，用于催化异麦芽酮糖合成。
10.根据权利要求9所述的应用，其特征在于，在所述催化异麦芽酮糖合成的反应体系中，底物浓度为400g/L，加酶量为25U/g，pH为4-7，温度为30-55℃。
11.根据权利要求10所述的应用，其特征在于，pH为5.5，温度为40℃。
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