CN113583997A - 一种磷酸酶突变体及在催化麦芽糊精制备甘露糖中的应用 - Google Patents

一种磷酸酶突变体及在催化麦芽糊精制备甘露糖中的应用 Download PDF

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CN113583997A
CN113583997A CN202110847558.3A CN202110847558A CN113583997A CN 113583997 A CN113583997 A CN 113583997A CN 202110847558 A CN202110847558 A CN 202110847558A CN 113583997 A CN113583997 A CN 113583997A
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郑仁朝
汤晓玲
王文豪
郑裕国
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Abstract

本发明公开了一种磷酸酶突变体及在催化麦芽糊精制备甘露糖中的应用，所述突变体是将SEQ ID NO.1所示氨基酸的第175位、179位或195位进行单点突变获得的。本发明以含磷酸酶突变体编码基因的工程菌发酵培养获得的湿菌体经超声破碎提取的酶液为催化剂，以麦芽糊精为底物，联合葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶、甘露糖6‑磷酸异构酶，添加MgCl2和磷酸盐，以pH为7.5～8.5的缓冲液为反应介质构成反应体系，制备甘露糖。本发明磷酸酶突变体F179M通过体外一锅法以麦芽糊精为底物生产甘露糖，转化率达到60‑70％，产物甘露糖与副产物果糖、葡萄糖比例为11:1:1。

Description

一种磷酸酶突变体及在催化麦芽糊精制备甘露糖中的应用
(一)技术领域
本发明涉及酶工程技术领域，具体涉及一种大西洋嗜热菌(Thermosiphoatlanticus)来源的对甘露糖-6-磷酸特异性提高的磷酸酶突变体及其在体外多酶合成甘露糖中应用。
(二)背景技术
体外多酶合成系统是模仿体内代谢途径，在体外组合一系列酶及辅酶构建生化反应途径，催化底物高效合成产物的新型生物制造方法。通过酶元件、多酶复合体的体外设计与组装调控，构建崭新的人工生物合成途径，重塑自然生产线，具有途径多样、高度可控、高效率和高原子经济性等显著优势，对变革化学品的制造模式产生具有重要意义。近年来，体外多酶合成体系在肌醇等功能性糖醇化学品合成中得到了重要应用。
从麦芽糊精出发，构建体外多酶合成体系是生产甘露糖的重要方法。在此途径中，磷酸酶作为催化甘露糖-6-磷酸去磷酸化合成甘露糖的关键酶，其催化性能直接影响甘露糖合成效率。但是目前发现的多个磷酸酶，均有着广泛的底物谱，即可催化各种磷酸单糖如葡萄糖-6-磷酸、果糖-6-磷酸的去磷酸化，导致在催化甘露糖6-磷酸生产甘露糖过程中产生大量的副产物，产率降低，导致后续产物分离纯化困难。因此，磷酸酶底物专一性的调控对高纯度甘露糖的合成具有重要意义。本发明筛选挖掘了大西洋嗜热菌(Thermosiphoatlanticus)来源磷酸酶(M6PP)，并通过基因工程技术，对该酶进行了改造，对其底物专一性进行调控，以获得理想突变体，对甘露糖6-磷酸具有严格的专一性，实现甘露糖的体外高效合成。
(三)发明内容
本发明目的是提供一种大西洋嗜热菌(T.atlanticus)来源的底物专一性提高的磷酸酶突变体，重组菌及在催化麦芽糊精制备甘露糖中的应用。本发明的磷酸酶突变体可以在60℃下体外联合多酶催化麦芽糊精合成甘露糖，具有高度的底物特异性，副产物大量减少，目标产物产率提高。
本发明采用的技术方案是：
本发明提供一种磷酸酶突变体，所述突变体是将SEQ ID NO.1所示氨基酸序列(核苷酸序列如SEQ ID NO.2)的第175位、179位或195位进行单位点突变获得的；优选突变体为下列之一：第175位丝氨酸Ser突变为天冬酰胺Asn(S175N，氨基酸序列如SEQ ID NO：3所示，核苷酸序列如SEQ ID NO：7所示)，第179位苯丙氨酸Phe突变为甲硫氨酸Met(F179M，氨基酸序列如SEQ ID NO：4所示，核苷酸序列如SEQ ID NO：8所示)、第179位苯丙氨酸Phe突变为半胱氨酸Cys(F179C，氨基酸序列如SEQ ID NO：5所示，核苷酸序列如SEQ ID NO：9所示)、第195位酪氨酸Tyr突变为亮氨酸Leu(Y195L，氨基酸序列如SEQ ID NO：6所示，核苷酸序列如SEQ ID NO：10所示)。
SEQ ID NO.1：
MVFVFDLDGTLLKKDNTISPNMVALIKRLDKNNHRIVFASGRMLISIRKIVEKYFQKMFPIIAYNGAMVFIPNKGIIFEKTLDFQTSKEIIELLRNKNIHRQAYINDELFSEEDNENIKFYSRHAGVEYKVVEDLIELIKHKNSTKLLAIDSPMKLDKLKEELENLNLNAEIFKSMNIFLDIVPKDVNKAIALKYLLKTLKAEHEKLIVFGDNHNDIPLFKFADFSIAVGNAVTELKKIADFVSKTNDEDGVYYALTEKFPEFLKE。
SEQ ID NO.2：
ATGGTGTTCGTTTTTGACCTGGATGGTACCCTGCTGAAGAAAGACAACACCATCAGCCCGAACATGGTGGCGCTGATTAAGCGTCTGGATAAAAACAACCACCGTATCGTTTTCGCGAGCGGCCGTATGCTGATCAGCATTCGTAAGATTGTGGAAAAATACTTCCAGAAGATGTTTCCGATCATTGCGTATAACGGTGCGATGGTTTTTATCCCGAACAAAGGCATCATTTTCGAGAAGACCCTGGACTTTCAGACCAGCAAAGAGATCATTGAACTGCTGCGTAACAAGAACATCCACCGTCAAGCGTACATTAACGACGAACTGTTCAGCGAGGAAGATAACGAGAACATCAAATTTTACAGCCGTCACGCGGGTGTGGAATATAAGGTGGTTGAGGATCTGATCGAACTGATTAAGCACAAAAACAGCACCAAACTGCTGGCGATCGACAGCCCGATGAAGCTGGATAAGCTGAAAGAGGAACTGGAAAACCTGAACCTGAACGCGGAGATCTTCAAAAGCATGAACATCTTTCTGGACATTGTGCCGAAAGATGTTAACAAGGCGATTGCGCTGAAATATCTGCTGAAGACCCTGAAAGCGGAGCACGAAAAGCTGATCGTTTTCGGTGACAACCACAACGATATTCCGCTGTTCAAATTTGCGGACTTTAGCATCGCGGTGGGCAACGCGGTTACCGAACTGAAGAAAATTGCGGATTTCGTGAGCAAGACCAACGACGAGGATGGCGTTTACTATGCGCTGACCGAGAAATTCCCGGAATTTCTGAAGGAGTAA。
任何对SEQ ID NO.1所示氨基酸序列中氨基酸经过缺失、插入或者替换一个或几个氨基酸且具有磷酸酶活性的，仍属于本发明的保护范围。
本发明还涉及磷酸酶突变体编码基因构建的重组载体及重组基因工程菌。
本发明对用于构建磷酸酶突变体重组载体的载体没有限制，只要其可以在原核和/或真核细胞的各种宿主细胞中保持其复制或自主复制即可，所述载体可为本领域常规的各种载体，如各种质粒、噬菌体或病毒载体等，优选pET-28b(+)。
引入编码本发明的磷酸酶突变体DNA的宿主细胞没有限制，只要满足重组表达载体可以稳定自我复制且所携带的本发明的磷酸酶突变体基因可以有效表达。例如大肠杆菌、枯草芽孢杆菌、酵母菌、放线菌、曲霉菌，以及动物细胞和高等植物细胞。本发明优选大肠杆菌，更优选E.coli BL21(DE3)。含磷酸酶突变体基因的重组菌是将目的基因插入质粒pET28b(+)上的BamHI和HindIII之间，转化于E.coli BL21(DE3)宿主细胞中，即为工程菌。
本发明提供一种磷酸酶突变体在体外多酶催化麦芽糊精制备甘露糖中的应用，所述应用为：将含磷酸酶突变体编码基因的工程菌发酵培养获得的湿菌体用缓冲液(优选100mM HEPES缓冲液(pH 7.2))重悬后超声破碎、一次离心，上清液在60-80℃水浴中热处理10-30min(优选70℃、20min)后二次离心的上清液为催化剂，以麦芽糊精(De＝4-7)为底物，联合葡聚糖磷酸化酶α-GP、葡萄糖磷酸变位酶PGM、葡萄糖磷酸异构酶PGI和甘露糖6-磷酸异构酶MPI，添加MgCl2和磷酸盐，以pH为7.5～8.5的缓冲液为反应介质构成反应体系，在50～70℃条件下反应8～48h(优选60-70℃反应12h)，冰浴终止反应，获得含甘露糖的反应液，反应液分离纯化，获得甘露糖。
所述反应体系中，底物加入终浓度为5-50g/L，优选10g/L；所述磷酸酶突变体加入终浓度以蛋白含量计为20-200mg/L，优选50mg/L；所述葡聚糖磷酸化酶α-GP加入终浓度以蛋白含量计为100-300mg/L，优选200mg/L；所述葡萄糖磷酸变位酶PGM加入终浓度以蛋白含量计为10-100mg/L，优选50mg/L；所述葡萄糖磷酸异构酶PGI加入终浓度以蛋白含量计为10-150mg/L，优选50mg/L；甘露糖6-磷酸异构酶MPI加入终浓度以蛋白含量计为10-150mg/L，优选90mg/L；所述MgCl2加入终浓度为5-50mM，优选5mM；所述磷酸盐加入终浓度为5-50mM，优选10mM。所述磷酸盐为Na2HPO4。
所述催化剂按如下方法制备：将磷酸酶突变体基因连接在pET-28b(+)载体上，连接位点为BamHI和HindIII，获得表达载体pET-28b(+)-M6PPM(M6PP的突变体)；将表达载体转化至E.coli BL21(DE3)中，获得含磷酸酶突变体基因的工程菌；工程菌接种在LB液体培养基，37℃，180rpm条件下培养12h后，将菌液以体积浓度10％接种量转接至LB培养基，加卡那霉素至终浓度50mg/L，37℃、180rpm培养至OD600为0.6-0.8时，降温至28℃，加IPTG至终浓度0.1mM，诱导表达12h；上述培养液在8,000×g下离心10min，弃上清，沉淀用pH 7.2、100mM的HEPES缓冲液重悬，即为菌体悬液；菌体悬液超声破碎(60W，持续2s，间歇4s，连续破碎15min)，获得的细胞破碎液在12000×g下离心10min，上清液在60-80℃水浴中热处理10-30min(优选70℃、20min)，4℃、12000×g离心10min，上清即为催化剂。
所述葡聚糖磷酸化酶α-GP、葡萄糖磷酸变位酶PGM、葡萄糖磷酸异构酶PGI和甘露糖6-磷酸异构酶MPI是以含相应酶编码基因的工程菌发酵培养获得的菌体经超声破碎、热处理后的上清液形式加入，其中葡聚糖磷酸化酶α-GP来源于海栖热袍菌(Thermotogamaritima MSB8,GenBank：AHD18925.1)，其氨基酸序列如SEQ ID NO：11所示，催化麦芽糊精转化为葡萄糖1-磷酸；葡萄糖磷酸变位酶PGM来源于嗜热古生菌(Thermococcuskodakarensis KOD1，GenBank：BAD42440.1)，氨基酸序列如SEQ ID NO：12所示，催化葡萄糖1-磷酸转化为葡萄糖6-磷酸；葡萄糖磷酸异构酶PGI来源于嗜热栖热菌(Thermusthermophilus HB8，GenBank:BAD70100.1)，氨基酸序列如SEQ ID NO：13所示，催化葡萄糖6-磷酸转化为果糖6-磷酸；甘露糖6-磷酸异构酶MPI来源于嗜热脱氮芽孢杆菌(GeobaciIlus thermodenitrificans，GenBank：ATO37551.1)，氨基酸序列如SEQ ID NO：14所示，该酶催化果糖6-磷酸转化为甘露糖6-磷酸；磷酸酶(M6PP)来源于大西洋嗜热菌(T.atlanticus，GenBank:WP_073073090.1)，催化甘露糖6-磷酸去磷酸化生成甘露糖，通过蛋白质工程手段对磷酸酶进行改造后，获得突变体。由于这些酶均为嗜热微生物来源，可通过热处理进行纯化。菌体破碎后离心，取上清液在60-80℃水浴中热处理10-30min(优选70℃、20min)，离心收集上清。目标蛋白存在于上清中，宿主产生的其它蛋白受热变性沉淀，即得初步纯化酶液。
所述葡聚糖磷酸化酶α-GP、葡萄糖磷酸变位酶PGM、葡萄糖磷酸异构酶PGI和甘露糖6-磷酸异构酶MPI的上清液按如下方法制备：分别将编码相应酶的目标基因连接到pET-28b(+)载体上，连接位点均为BamHI和HindIII，获得相应的表达载体pET-28b(+)-α-GP，pET-28b(+)-PGM，pET-28b(+)-PGI，pET-28b(+)-MPI，pET-28b(+)-M6PP。将表达载体分别转化至E.coli BL21(DE3)中，获得相应的重组菌。重组菌在LB液体培养基，37℃，180rpm条件下培养12h后，将菌液以10％(v/v)接种量转接至LB培养基，加卡那霉素至终浓度50mg/L，37℃、180rpm培养至OD600为0.6-0.8时，降温至28℃，加IPTG至终浓度0.1mM，诱导表达12h。上述培养液在8,000×g下离心10min，弃上清，沉淀用pH 7.2、100mM的HEPES缓冲液重悬，即为菌体悬液。菌体悬液超声破碎(60W，持续2s，间歇4s，连续破碎15min)，获得的细胞破碎液在12000×g下离心10min。上清液在60-80℃水浴中热处理10-30min(优选70℃、20min)，4℃、12000×g离心10min，上清液即为酶液。
本发明获得的4个突变体S175N、F179M、F179C、Y195L通过体外一锅法催化麦芽糊精的反应主产物为甘露糖，副产物为葡萄糖、果糖。野生型产物中甘露糖、果糖与葡萄糖比例为1:1:1，突变体F175N产物中甘露糖、果糖与葡萄糖比例为7:2:1，突变体F179M产物中甘露糖、果糖与葡萄糖比例为11:1:1，突变体F179C产物中甘露糖、果糖与葡萄糖比例为5:3.5:1，突变体Y195L产物中甘露糖、果糖与葡萄糖比例为4:2:1。
与现有技术相比，本发明的有益效果主要体现在：本发明提供了一种底物专一性显著提高的磷酸酶突变体，有利于定向生成目的产物甘露糖。构建的突变体F179M通过体外一锅法以麦芽糊精为底物生产甘露糖，转化率达到60％以上，产物中甘露糖与副产物果糖、葡萄糖比例为11:1:1。底物专一性调控后的磷酸酶突变体在体外多酶系统合成功能性糖醇中具有重要工业应用潜力。
(四)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。
各实施例中所用相同名称的材料或试剂如无特别说明即为相同。实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为实施本发明时对生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。
实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。
本发明实施例所用麦芽糊精De＝4-7。
实施例1、磷酸酶基因序列的获得和载体构建
通过NCBI、KEGG数据库挖掘获得来源于大西洋嗜热菌(T.atlanticus，GenBank:WP_073073090.1)的多肽序列(氨基酸序列如SEQ ID NO：1所示)，该序列未被注释，尚不明确其功能。在不改变该多肽氨基酸序列的前提下，将上述野生型基因的密码子替换为大肠杆菌偏好的密码子，经密码子优化后的基因记为磷酸酶M6PP基因，其基因序列如SEQ IDNO：2所示。
将SEQ ID NO：2所示基因序列连接于pET-28b(+)载体，位于酶切位点BamHI和HindIII之间，重组质粒命名为pET-28b(+)-M6PP。
实施例2、野生型磷酸酶M6PP的表达和纯化
(1)重组菌构建：将实施例1重组质粒pET-28b(+)-M6PP转化于E.coli BL21(DE3)感受态细胞中，获得野生型重组菌E.coli BL21(DE3)/pET-28b(+)-M6PP，作为野生型对照。
(2)重组菌培养：挑取步骤(1)重组菌至10mL LB液体培养基中，于37℃、200rpm培养12h；将10mL培养液转接至100mL LB培养基中，加卡那霉素至终浓度50mg/L，37℃、180rpm培养至OD600为0.6-0.8时，降温至28℃，加IPTG至终浓度0.1mM，诱导表达12h。上述培养液在8000×g下离心10min，弃上清，沉淀用HEPES缓冲液(pH 7.2)重悬，即为菌悬液。
(3)粗酶液制备：步骤(2)菌悬液在4℃冰浴下，超声破碎(60W，持续2s，间歇4s，连续破碎15min)，获得细胞破碎液，12000×g离心10min。上清液在80℃水浴下热处理20分钟，4℃、12000×g离心10min，取上清即为磷酸酶M6PP酶液。
实施例3、α-GP、PGM、PGI，MPI酶液的制备
采用实施例2方法，分别来源于海栖热袍菌(Thermotoga maritima MSB8)的葡聚糖磷酸化酶α-GP(GenBank：AHD18925.1，其氨基酸序列如SEQ ID NO：11所示)、来源于嗜热古生菌(Thermococcus kodakarensis KOD1)的葡萄糖磷酸变位酶PGM(GenBank：BAD42440.1，氨基酸序列如SEQ ID NO：12所示)、来源于嗜热栖热菌(Thermusthermophilus HB8)的葡萄糖磷酸异构酶PGI(GenBank:BAD70100.1，氨基酸序列如SEQ IDNO：13所示)；来源于嗜热脱氮芽孢杆菌(GeobaciIlus thermodenitrificans)的甘露糖6-磷酸异构酶MPI(GenBank：ATO37551.1，氨基酸序列如SEQ ID NO：14所示)，连接到pET-28b(+)载体上，连接位点均为BamHI和HindIII，获得相应的表达载体，然后分别转化至E.coliBL21(DE3)，采用实施例2方法，分别制备α-GP酶液、PGM酶液、PGI酶液，MPI酶液，采用采用BCA蛋白含量检测试剂盒检测目标蛋白含量，分别为2.1mg/mL，1.8mg/mL，0.9mg/mL和1.7mg/mL。
实施例4、含磷酸酶M6PP突变体编码基因的重组表达载体及重组菌的构建
以含有SEQ ID NO：2所示核苷酸序列的pET-28b(+)-M6PP重组质粒为模板，对第175、179和195位设计饱和突变引物，序列如表1。
表1第175、179和195位饱和突变引物序列
Figure BDA0003181237150000071
注：N＝A/G/C/T，K＝G/T，M＝A/C。
采用全质粒扩增PCR法构建突变体质粒(反应体系如表2所示，反应条件如表3所示)，获得突变序列。
表2 PCR反应体系
Figure BDA0003181237150000072
表3 PCR反应条件
Figure BDA0003181237150000073
PCR产物经过凝胶电泳检验，然后在50μL的PCR产物中加入1μL的Dpn I限制性内切酶对模板质粒进行消化，37℃下孵化2h。吸取15μL酶切产物转化至E.coli BL21(DE3)中，得到含有相应突变体的重组大肠杆菌，涂布于含50mg/L卡那霉素的LB平板，37℃下培养过夜，获得单菌落培养物。
实施例5、磷酸酶M6PP突变体的筛选
1、初筛
将实施例3获得的单菌落接种至含LB培养基的96孔板，37℃培养14h后，获得种子液。取200μL种子液转接至新的无菌96深孔板中，且每个孔中均含有600μLLB液体培养基(含有终浓度为50μg/mL的卡那霉素、终浓度为0.1mM的IPTG)，放置于28℃、150r/min条件下培养12h后，于8000×g，4℃条件下离心20min，弃上清。用400μL HEPES缓冲液(pH7.2，50mM)重悬各孔中的菌体，-80℃冷冻40min，37℃融解20min，反复4次，80℃热处理20min，8000×g离心20min，去沉淀后获得突变体酶液。同样条件下，制备野生型重组菌E.coli BL21(DE3)/pET-28b(+)-M6PP酶液作为对照酶液。
在96孔反应板中添加200μL反应液：170μL突变体酶液，10μL MgCl2水溶液(反应液中终浓度为5mM)，20μL甘露糖-6-磷酸水溶液(反应液中终浓度为1mM)，于70℃反应3min，冰浴终止。取50μL以上反应液至含有150μL显色液的96孔标准透明板中，放置于37℃保温5min，利用酶标仪检测OD655的吸光值。显色液组成为0.12g/mL抗坏血酸(溶剂1M HCl水溶液)与0.02g/mL钼酸铵水溶液以体积比2:1混合。同样条件下，以对照酶液替换突变体酶液，检测对照组吸光值。
2、复筛
从步骤1的96孔板中挑取与对照组相比吸光值OD655显著提高的菌株9株，分别接种于5mL LB培养基中(含50μg/mL的卡那霉素)，37℃，200r/min条件下培养8h。培养液按体积浓度2％的接种量接种至含有50mL LB液体培养基的250mL锥形瓶中，37℃、180rpm培养至OD600为0.6-0.8时，降温至28℃，加IPTG至终浓度0.1mM，诱导表达12h。上述培养液在8,000×g下离心10min，弃上清，沉淀用pH 7.2的HEPES缓冲液重悬。菌体悬液超声破碎(60W，持续2s，间歇4s，连续破碎15min)，获得细胞破碎液，在12000×g下离心10min。上清液在70℃水浴下热处理20分钟，4℃、12000×g离心10min，取上清液，得复筛M6PP突变体酶液，采用BCA蛋白含量检测试剂盒检测目标蛋白含量，为1.1-1.2mg/mL。
在10mL HEPES缓冲液(100mM，pH 7.2)中加入10g/L麦芽糊精，20mM Na2HPO4，5mMMgCl2，以蛋白含量计200mg/L实施例3方法制备的α-GP酶液(所制备的酶含量为2.1mg/mL)，以蛋白含量计50mg/L实施例3方法制备的PGM酶液(所制备的酶含量为1.8mg/mL)，以蛋白含量计50mg/L实施例3方法制备的PGI酶液(所制备的酶含量为0.9mg/mL)，以蛋白含量计90mg/L实施例3方法制备的MPI酶液(所制备的酶含量为1.7mg/mL)和以蛋白含量计50mg/L上述复筛M6PP突变体酶液(所制备的酶含量为1.1-1.2mg/mL)。反应液于70℃反应2、4、8、12和24h后直接取样，12000×g离心5min后，进行HPLC检测。根据HPLC所检测甘露糖标准品峰面积与含量建立标准曲线，并根据实际检测的样品峰面积，计算甘露糖含量。
液相色谱仪为安捷伦HPLC 1260II(RID)，检测器采用示差折光检测器(RID)，色谱柱为Hi-Plex Ca,7.7mm×300mm，8μm，以H2O为流动相，进样量为5μL，检测器温度为35℃，柱温80℃，流速0.25mL/min。与野生型对照，根据甘露糖产量的提高，筛选获得有益突变体M6PP-S175N(SEQ ID NO：1第175位丝氨酸Ser突变为天冬酰胺Asn，氨基酸序列如SEQ IDNO：3所示，核苷酸序列如SEQ ID NO：7所示)、M6PP-F179M(SEQ ID NO：1第179位苯丙氨酸Phe突变为甲硫氨酸Met，氨基酸序列如SEQ ID NO：4所示，核苷酸序列如SEQ ID NO：8所示)、M6PP-F179C(SEQ ID NO：1第179位苯丙氨酸Phe突变为半胱氨酸Cys，氨基酸序列如SEQID NO：5所示，核苷酸序列如SEQ ID NO：9所示)、M6PP-Y195L(SEQ ID NO：1第195位酪氨酸Tyr突变为亮氨酸Leu，氨基酸序列如SEQ ID NO：6所示，核苷酸序列如SEQ ID NO：10所示)，即相应的磷酸酶突变体工程菌为E.coliBL21(DE3)/pET-28b(+)-M6PP-S175N、E.coli BL21(DE3)/pET-28b(+)-M6PP-F179M、E.coli BL21(DE3)/pET-28b(+)-M6PP-F179C。
实施例6、体外一锅法多酶催化麦芽糊精合成甘露糖
多酶催化体系中酶的来源包括(1)葡聚糖磷酸化酶α-GP，该酶催化麦芽糊精转化为葡萄糖1-磷酸，(2)葡萄糖磷酸变位酶PGM，该酶催化葡萄糖1-磷酸转化为葡萄糖6-磷酸；(3)葡萄糖磷酸异构酶PGI，该酶催化葡萄糖6-磷酸转化为果糖6-磷酸；(4)甘露糖6-磷酸异构酶MPI，该酶催化果糖6-磷酸转化为甘露糖6-磷酸；(5)磷酸酶M6PP及其突变体，该酶催化甘露糖6-磷酸脱磷酸生成甘露糖。
多酶催化反应体系(10mL)组成为：100mM HEPES缓冲液(pH 7.2)，5mM MgCl2，10mMNa2HPO4，10g/L麦芽糊精，添加实施例3方法制备的α-GP酶液、PGM酶液、PGI酶液，MPI酶液和实施例5方法制备的磷酸酶M6PP突变体(M6PP-S175N、M6PP-F179M、M6PP-F179C、M6PP-Y195L)酶液的浓度以蛋白含量计分别为200mg/L、50mg/L、50mg/L、90mg/L和50mg/L。在60℃催化反应12小时，通过实施例5所述HPLC法测定反应液中甘露糖含量。
同样条件下，添加实施例5方法制备的对照酶液替换磷酸酶M6PP突变体酶液，测定野生型产物甘露糖含量。
野生型产物中甘露糖、果糖与葡萄糖质量比为1:1:1，甘露糖质量产率为13.3％，突变体M6PP-F175N产物中甘露糖、果糖与葡萄糖质量比为7:2:1，但转化率不高，甘露糖质量产率为26.5％，果糖和葡萄糖质量产率为7.5％和4％；突变体M6PP-F179M产物中甘露糖、果糖与葡萄糖质量比为11:1:1，甘露糖质量产率为63％，果糖和葡萄糖质量产率为5.7％；突变体M6PP-F179C产物中甘露糖、果糖与葡萄糖质量比为5:3.5:1，甘露糖质量产率为26％，果糖和葡萄糖质量产率为18％和5％；突变体M6PP-Y195L产物中甘露糖、果糖与葡萄糖质量比为4:1:2，甘露糖质量产率为32％，果糖和葡萄糖质量产率为8％和16％。
表4野生型磷酸酶及其突变体对体外多酶合成甘露糖的影响
Figure BDA0003181237150000101
本发明不受上述具体文字描述的限制。本发明可在权利要求书所概括的范围内作各种改变，这些改变均在本发明的范围之内。
序列表
<110> 浙江工业大学
<120> 一种磷酸酶突变体及在催化麦芽糊精制备甘露糖中的应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 266
<212> PRT
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 1
Met Val Phe Val Phe Asp Leu Asp Gly Thr Leu Leu Lys Lys Asp Asn
1 5 10 15
Thr Ile Ser Pro Asn Met Val Ala Leu Ile Lys Arg Leu Asp Lys Asn
20 25 30
Asn His Arg Ile Val Phe Ala Ser Gly Arg Met Leu Ile Ser Ile Arg
35 40 45
Lys Ile Val Glu Lys Tyr Phe Gln Lys Met Phe Pro Ile Ile Ala Tyr
50 55 60
Asn Gly Ala Met Val Phe Ile Pro Asn Lys Gly Ile Ile Phe Glu Lys
65 70 75 80
Thr Leu Asp Phe Gln Thr Ser Lys Glu Ile Ile Glu Leu Leu Arg Asn
85 90 95
Lys Asn Ile His Arg Gln Ala Tyr Ile Asn Asp Glu Leu Phe Ser Glu
100 105 110
Glu Asp Asn Glu Asn Ile Lys Phe Tyr Ser Arg His Ala Gly Val Glu
115 120 125
Tyr Lys Val Val Glu Asp Leu Ile Glu Leu Ile Lys His Lys Asn Ser
130 135 140
Thr Lys Leu Leu Ala Ile Asp Ser Pro Met Lys Leu Asp Lys Leu Lys
145 150 155 160
Glu Glu Leu Glu Asn Leu Asn Leu Asn Ala Glu Ile Phe Lys Ser Met
165 170 175
Asn Ile Phe Leu Asp Ile Val Pro Lys Asp Val Asn Lys Ala Ile Ala
180 185 190
Leu Lys Tyr Leu Leu Lys Thr Leu Lys Ala Glu His Glu Lys Leu Ile
195 200 205
Val Phe Gly Asp Asn His Asn Asp Ile Pro Leu Phe Lys Phe Ala Asp
210 215 220
Phe Ser Ile Ala Val Gly Asn Ala Val Thr Glu Leu Lys Lys Ile Ala
225 230 235 240
Asp Phe Val Ser Lys Thr Asn Asp Glu Asp Gly Val Tyr Tyr Ala Leu
245 250 255
Thr Glu Lys Phe Pro Glu Phe Leu Lys Glu
260 265
<210> 2
<211> 801
<212> DNA
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 2
atggtgttcg tttttgacct ggatggtacc ctgctgaaga aagacaacac catcagcccg 60
aacatggtgg cgctgattaa gcgtctggat aaaaacaacc accgtatcgt tttcgcgagc 120
ggccgtatgc tgatcagcat tcgtaagatt gtggaaaaat acttccagaa gatgtttccg 180
atcattgcgt ataacggtgc gatggttttt atcccgaaca aaggcatcat tttcgagaag 240
accctggact ttcagaccag caaagagatc attgaactgc tgcgtaacaa gaacatccac 300
cgtcaagcgt acattaacga cgaactgttc agcgaggaag ataacgagaa catcaaattt 360
tacagccgtc acgcgggtgt ggaatataag gtggttgagg atctgatcga actgattaag 420
cacaaaaaca gcaccaaact gctggcgatc gacagcccga tgaagctgga taagctgaaa 480
gaggaactgg aaaacctgaa cctgaacgcg gagatcttca aaagcatgaa catctttctg 540
gacattgtgc cgaaagatgt taacaaggcg attgcgctga aatatctgct gaagaccctg 600
aaagcggagc acgaaaagct gatcgttttc ggtgacaacc acaacgatat tccgctgttc 660
aaatttgcgg actttagcat cgcggtgggc aacgcggtta ccgaactgaa gaaaattgcg 720
gatttcgtga gcaagaccaa cgacgaggat ggcgtttact atgcgctgac cgagaaattc 780
ccggaatttc tgaaggagta a 801
<210> 3
<211> 266
<212> PRT
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 3
Met Val Phe Val Phe Asp Leu Asp Gly Thr Leu Leu Lys Lys Asp Asn
1 5 10 15
Thr Ile Ser Pro Asn Met Val Ala Leu Ile Lys Arg Leu Asp Lys Asn
20 25 30
Asn His Arg Ile Val Phe Ala Ser Gly Arg Met Leu Ile Ser Ile Arg
35 40 45
Lys Ile Val Glu Lys Tyr Phe Gln Lys Met Phe Pro Ile Ile Ala Tyr
50 55 60
Asn Gly Ala Met Val Phe Ile Pro Asn Lys Gly Ile Ile Phe Glu Lys
65 70 75 80
Thr Leu Asp Phe Gln Thr Ser Lys Glu Ile Ile Glu Leu Leu Arg Asn
85 90 95
Lys Asn Ile His Arg Gln Ala Tyr Ile Asn Asp Glu Leu Phe Ser Glu
100 105 110
Glu Asp Asn Glu Asn Ile Lys Phe Tyr Ser Arg His Ala Gly Val Glu
115 120 125
Tyr Lys Val Val Glu Asp Leu Ile Glu Leu Ile Lys His Lys Asn Ser
130 135 140
Thr Lys Leu Leu Ala Ile Asp Ser Pro Met Lys Leu Asp Lys Leu Lys
145 150 155 160
Glu Glu Leu Glu Asn Leu Asn Leu Asn Ala Glu Ile Phe Lys Asn Met
165 170 175
Asn Ile Phe Leu Asp Ile Val Pro Lys Asp Val Asn Lys Ala Ile Ala
180 185 190
Leu Lys Tyr Leu Leu Lys Thr Leu Lys Ala Glu His Glu Lys Leu Ile
195 200 205
Val Phe Gly Asp Asn His Asn Asp Ile Pro Leu Phe Lys Phe Ala Asp
210 215 220
Phe Ser Ile Ala Val Gly Asn Ala Val Thr Glu Leu Lys Lys Ile Ala
225 230 235 240
Asp Phe Val Ser Lys Thr Asn Asp Glu Asp Gly Val Tyr Tyr Ala Leu
245 250 255
Thr Glu Lys Phe Pro Glu Phe Leu Lys Glu
260 265
<210> 4
<211> 266
<212> PRT
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 4
Met Val Phe Val Phe Asp Leu Asp Gly Thr Leu Leu Lys Lys Asp Asn
1 5 10 15
Thr Ile Ser Pro Asn Met Val Ala Leu Ile Lys Arg Leu Asp Lys Asn
20 25 30
Asn His Arg Ile Val Phe Ala Ser Gly Arg Met Leu Ile Ser Ile Arg
35 40 45
Lys Ile Val Glu Lys Tyr Phe Gln Lys Met Phe Pro Ile Ile Ala Tyr
50 55 60
Asn Gly Ala Met Val Phe Ile Pro Asn Lys Gly Ile Ile Phe Glu Lys
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Thr Leu Asp Phe Gln Thr Ser Lys Glu Ile Ile Glu Leu Leu Arg Asn
85 90 95
Lys Asn Ile His Arg Gln Ala Tyr Ile Asn Asp Glu Leu Phe Ser Glu
100 105 110
Glu Asp Asn Glu Asn Ile Lys Phe Tyr Ser Arg His Ala Gly Val Glu
115 120 125
Tyr Lys Val Val Glu Asp Leu Ile Glu Leu Ile Lys His Lys Asn Ser
130 135 140
Thr Lys Leu Leu Ala Ile Asp Ser Pro Met Lys Leu Asp Lys Leu Lys
145 150 155 160
Glu Glu Leu Glu Asn Leu Asn Leu Asn Ala Glu Ile Phe Lys Ser Met
165 170 175
Asn Ile Met Leu Asp Ile Val Pro Lys Asp Val Asn Lys Ala Ile Ala
180 185 190
Leu Lys Tyr Leu Leu Lys Thr Leu Lys Ala Glu His Glu Lys Leu Ile
195 200 205
Val Phe Gly Asp Asn His Asn Asp Ile Pro Leu Phe Lys Phe Ala Asp
210 215 220
Phe Ser Ile Ala Val Gly Asn Ala Val Thr Glu Leu Lys Lys Ile Ala
225 230 235 240
Asp Phe Val Ser Lys Thr Asn Asp Glu Asp Gly Val Tyr Tyr Ala Leu
245 250 255
Thr Glu Lys Phe Pro Glu Phe Leu Lys Glu
260 265
<210> 5
<211> 266
<212> PRT
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 5
Met Val Phe Val Phe Asp Leu Asp Gly Thr Leu Leu Lys Lys Asp Asn
1 5 10 15
Thr Ile Ser Pro Asn Met Val Ala Leu Ile Lys Arg Leu Asp Lys Asn
20 25 30
Asn His Arg Ile Val Phe Ala Ser Gly Arg Met Leu Ile Ser Ile Arg
35 40 45
Lys Ile Val Glu Lys Tyr Phe Gln Lys Met Phe Pro Ile Ile Ala Tyr
50 55 60
Asn Gly Ala Met Val Phe Ile Pro Asn Lys Gly Ile Ile Phe Glu Lys
65 70 75 80
Thr Leu Asp Phe Gln Thr Ser Lys Glu Ile Ile Glu Leu Leu Arg Asn
85 90 95
Lys Asn Ile His Arg Gln Ala Tyr Ile Asn Asp Glu Leu Phe Ser Glu
100 105 110
Glu Asp Asn Glu Asn Ile Lys Phe Tyr Ser Arg His Ala Gly Val Glu
115 120 125
Tyr Lys Val Val Glu Asp Leu Ile Glu Leu Ile Lys His Lys Asn Ser
130 135 140
Thr Lys Leu Leu Ala Ile Asp Ser Pro Met Lys Leu Asp Lys Leu Lys
145 150 155 160
Glu Glu Leu Glu Asn Leu Asn Leu Asn Ala Glu Ile Phe Lys Asn Met
165 170 175
Asn Ile Cys Leu Asp Ile Val Pro Lys Asp Val Asn Lys Ala Ile Ala
180 185 190
Leu Lys Tyr Leu Leu Lys Thr Leu Lys Ala Glu His Glu Lys Leu Ile
195 200 205
Val Phe Gly Asp Asn His Asn Asp Ile Pro Leu Phe Lys Phe Ala Asp
210 215 220
Phe Ser Ile Ala Val Gly Asn Ala Val Thr Glu Leu Lys Lys Ile Ala
225 230 235 240
Asp Phe Val Ser Lys Thr Asn Asp Glu Asp Gly Val Tyr Tyr Ala Leu
245 250 255
Thr Glu Lys Phe Pro Glu Phe Leu Lys Glu
260 265
<210> 6
<211> 266
<212> PRT
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 6
Met Val Phe Val Phe Asp Leu Asp Gly Thr Leu Leu Lys Lys Asp Asn
1 5 10 15
Thr Ile Ser Pro Asn Met Val Ala Leu Ile Lys Arg Leu Asp Lys Asn
20 25 30
Asn His Arg Ile Val Phe Ala Ser Gly Arg Met Leu Ile Ser Ile Arg
35 40 45
Lys Ile Val Glu Lys Tyr Phe Gln Lys Met Phe Pro Ile Ile Ala Tyr
50 55 60
Asn Gly Ala Met Val Phe Ile Pro Asn Lys Gly Ile Ile Phe Glu Lys
65 70 75 80
Thr Leu Asp Phe Gln Thr Ser Lys Glu Ile Ile Glu Leu Leu Arg Asn
85 90 95
Lys Asn Ile His Arg Gln Ala Tyr Ile Asn Asp Glu Leu Phe Ser Glu
100 105 110
Glu Asp Asn Glu Asn Ile Lys Phe Tyr Ser Arg His Ala Gly Val Glu
115 120 125
Tyr Lys Val Val Glu Asp Leu Ile Glu Leu Ile Lys His Lys Asn Ser
130 135 140
Thr Lys Leu Leu Ala Ile Asp Ser Pro Met Lys Leu Asp Lys Leu Lys
145 150 155 160
Glu Glu Leu Glu Asn Leu Asn Leu Asn Ala Glu Ile Phe Lys Ser Met
165 170 175
Asn Ile Phe Leu Asp Ile Val Pro Lys Asp Val Asn Lys Ala Ile Ala
180 185 190
Leu Lys Leu Leu Leu Lys Thr Leu Lys Ala Glu His Glu Lys Leu Ile
195 200 205
Val Phe Gly Asp Asn His Asn Asp Ile Pro Leu Phe Lys Phe Ala Asp
210 215 220
Phe Ser Ile Ala Val Gly Asn Ala Val Thr Glu Leu Lys Lys Ile Ala
225 230 235 240
Asp Phe Val Ser Lys Thr Asn Asp Glu Asp Gly Val Tyr Tyr Ala Leu
245 250 255
Thr Glu Lys Phe Pro Glu Phe Leu Lys Glu
260 265
<210> 7
<211> 801
<212> DNA
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 7
atggtgttcg tttttgacct ggatggtacc ctgctgaaga aagacaacac catcagcccg 60
aacatggtgg cgctgattaa gcgtctggat aaaaacaacc accgtatcgt tttcgcgagc 120
ggccgtatgc tgatcagcat tcgtaagatt gtggaaaaat acttccagaa gatgtttccg 180
atcattgcgt ataacggtgc gatggttttt atcccgaaca aaggcatcat tttcgagaag 240
accctggact ttcagaccag caaagagatc attgaactgc tgcgtaacaa gaacatccac 300
cgtcaagcgt acattaacga cgaactgttc agcgaggaag ataacgagaa catcaaattt 360
tacagccgtc acgcgggtgt ggaatataag gtggttgagg atctgatcga actgattaag 420
cacaaaaaca gcaccaaact gctggcgatc gacagcccga tgaagctgga taagctgaaa 480
gaggaactgg aaaacctgaa cctgaacgcg gagatcttca aaaatatgaa catctttctg 540
gacattgtgc cgaaagatgt taacaaggcg attgcgctga aatatctgct gaagaccctg 600
aaagcggagc acgaaaagct gatcgttttc ggtgacaacc acaacgatat tccgctgttc 660
aaatttgcgg actttagcat cgcggtgggc aacgcggtta ccgaactgaa gaaaattgcg 720
gatttcgtga gcaagaccaa cgacgaggat ggcgtttact atgcgctgac cgagaaattc 780
ccggaatttc tgaaggagta a 801
<210> 8
<211> 801
<212> DNA
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 8
atggtgttcg tttttgacct ggatggtacc ctgctgaaga aagacaacac catcagcccg 60
aacatggtgg cgctgattaa gcgtctggat aaaaacaacc accgtatcgt tttcgcgagc 120
ggccgtatgc tgatcagcat tcgtaagatt gtggaaaaat acttccagaa gatgtttccg 180
atcattgcgt ataacggtgc gatggttttt atcccgaaca aaggcatcat tttcgagaag 240
accctggact ttcagaccag caaagagatc attgaactgc tgcgtaacaa gaacatccac 300
cgtcaagcgt acattaacga cgaactgttc agcgaggaag ataacgagaa catcaaattt 360
tacagccgtc acgcgggtgt ggaatataag gtggttgagg atctgatcga actgattaag 420
cacaaaaaca gcaccaaact gctggcgatc gacagcccga tgaagctgga taagctgaaa 480
gaggaactgg aaaacctgaa cctgaacgcg gagatcttca aaagcatgaa catcatgctg 540
gacattgtgc cgaaagatgt taacaaggcg attgcgctga aatatctgct gaagaccctg 600
aaagcggagc acgaaaagct gatcgttttc ggtgacaacc acaacgatat tccgctgttc 660
aaatttgcgg actttagcat cgcggtgggc aacgcggtta ccgaactgaa gaaaattgcg 720
gatttcgtga gcaagaccaa cgacgaggat ggcgtttact atgcgctgac cgagaaattc 780
ccggaatttc tgaaggagta a 801
<210> 9
<211> 801
<212> DNA
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 9
atggtgttcg tttttgacct ggatggtacc ctgctgaaga aagacaacac catcagcccg 60
aacatggtgg cgctgattaa gcgtctggat aaaaacaacc accgtatcgt tttcgcgagc 120
ggccgtatgc tgatcagcat tcgtaagatt gtggaaaaat acttccagaa gatgtttccg 180
atcattgcgt ataacggtgc gatggttttt atcccgaaca aaggcatcat tttcgagaag 240
accctggact ttcagaccag caaagagatc attgaactgc tgcgtaacaa gaacatccac 300
cgtcaagcgt acattaacga cgaactgttc agcgaggaag ataacgagaa catcaaattt 360
tacagccgtc acgcgggtgt ggaatataag gtggttgagg atctgatcga actgattaag 420
cacaaaaaca gcaccaaact gctggcgatc gacagcccga tgaagctgga taagctgaaa 480
gaggaactgg aaaacctgaa cctgaacgcg gagatcttca aaagcatgaa catctgtctg 540
gacattgtgc cgaaagatgt taacaaggcg attgcgctga aatatctgct gaagaccctg 600
aaagcggagc acgaaaagct gatcgttttc ggtgacaacc acaacgatat tccgctgttc 660
aaatttgcgg actttagcat cgcggtgggc aacgcggtta ccgaactgaa gaaaattgcg 720
gatttcgtga gcaagaccaa cgacgaggat ggcgtttact atgcgctgac cgagaaattc 780
ccggaatttc tgaaggagta a 801
<210> 10
<211> 801
<212> DNA
<213> 大西洋嗜热菌(Thermosipho atlanticus)
<400> 10
atggtgttcg tttttgacct ggatggtacc ctgctgaaga aagacaacac catcagcccg 60
aacatggtgg cgctgattaa gcgtctggat aaaaacaacc accgtatcgt tttcgcgagc 120
ggccgtatgc tgatcagcat tcgtaagatt gtggaaaaat acttccagaa gatgtttccg 180
atcattgcgt ataacggtgc gatggttttt atcccgaaca aaggcatcat tttcgagaag 240
accctggact ttcagaccag caaagagatc attgaactgc tgcgtaacaa gaacatccac 300
cgtcaagcgt acattaacga cgaactgttc agcgaggaag ataacgagaa catcaaattt 360
tacagccgtc acgcgggtgt ggaatataag gtggttgagg atctgatcga actgattaag 420
cacaaaaaca gcaccaaact gctggcgatc gacagcccga tgaagctgga taagctgaaa 480
gaggaactgg aaaacctgaa cctgaacgcg gagatcttca aaagcatgaa catctttctg 540
gacattgtgc cgaaagatgt taacaaggcg attgcgctga aacttctgct gaagaccctg 600
aaagcggagc acgaaaagct gatcgttttc ggtgacaacc acaacgatat tccgctgttc 660
aaatttgcgg actttagcat cgcggtgggc aacgcggtta ccgaactgaa gaaaattgcg 720
gatttcgtga gcaagaccaa cgacgaggat ggcgtttact atgcgctgac cgagaaattc 780
ccggaatttc tgaaggagta a 801
<210> 11
<211> 835
<212> PRT
<213> 海栖热袍菌(Thermotoga maritima)
<400> 11
Met Leu Leu Lys Glu Thr Ser Leu Arg Gly Gly Glu Ile Val Leu Glu
1 5 10 15
Lys Leu Pro Glu Asn Leu Lys Glu Leu Glu Ser Leu Ala Tyr Asn Leu
20 25 30
Trp Trp Ser Trp Ser Arg Pro Ala Gln Arg Leu Trp Arg Met Ile Asp
35 40 45
Ser Glu Lys Trp Glu Glu His Arg Asn Pro Val Lys Ile Leu Arg Glu
50 55 60
Val Ser Lys Glu Arg Leu Glu Glu Leu Ser Lys Asp Glu Asp Phe Ile
65 70 75 80
Ala Leu Tyr Glu Leu Thr Leu Glu Arg Phe Thr Asp Tyr Met Glu Arg
85 90 95
Glu Asp Thr Trp Phe Asn Val Asn Tyr Pro Glu Trp Asp Glu Lys Ile
100 105 110
Val Tyr Met Cys Met Glu Tyr Gly Leu Thr Lys Ala Leu Pro Ile Tyr
115 120 125
Ser Gly Gly Leu Gly Ile Leu Ala Gly Asp His Leu Lys Ser Ala Ser
130 135 140
Asp Leu Gly Leu Pro Leu Ile Ala Val Gly Leu Leu Tyr Lys His Gly
145 150 155 160
Tyr Phe Thr Gln Gln Ile Asp Ser Asp Gly Arg Gln Ile Glu Ile Phe
165 170 175
Pro Glu Tyr Asp Ile Glu Glu Leu Pro Met Lys Pro Leu Arg Asp Glu
180 185 190
Asp Gly Asn Gln Val Ile Val Glu Val Pro Ile Asp Asn Asp Thr Val
195 200 205
Lys Ala Arg Val Phe Glu Val Gln Val Gly Arg Val Lys Leu Tyr Leu
210 215 220
Leu Asp Thr Asp Phe Glu Glu Asn Glu Asp Arg Phe Arg Lys Ile Cys
225 230 235 240
Asp Tyr Leu Tyr Asn Pro Glu Pro Asp Val Arg Val Ser Gln Glu Ile
245 250 255
Leu Leu Gly Ile Gly Gly Met Lys Leu Leu Lys Thr Leu Lys Ile Lys
260 265 270
Pro Gly Val Ile His Leu Asn Glu Gly His Pro Ala Phe Ser Ser Leu
275 280 285
Glu Arg Ile Lys Ser Tyr Met Glu Glu Gly Tyr Ser Phe Thr Glu Ala
290 295 300
Leu Glu Ile Val Arg Gln Thr Thr Val Phe Thr Thr His Thr Pro Val
305 310 315 320
Pro Ala Gly His Asp Arg Phe Pro Phe Asp Phe Val Glu Lys Lys Leu
325 330 335
Thr Lys Phe Phe Glu Gly Phe Glu Ser Lys Glu Leu Leu Met Asn Leu
340 345 350
Gly Lys Asp Glu Asp Gly Asn Phe Asn Met Thr Tyr Leu Ala Leu Arg
355 360 365
Thr Ser Ser Phe Ile Asn Gly Val Ser Lys Leu His Ala Asp Val Ser
370 375 380
Arg Arg Met Phe Lys Asn Val Trp Lys Gly Val Pro Val Glu Glu Ile
385 390 395 400
Pro Ile Glu Gly Ile Thr Asn Gly Val His Met Gly Thr Trp Ile Asn
405 410 415
Arg Glu Met Arg Lys Leu Phe Asp Arg Tyr Leu Gly Arg Val Trp Arg
420 425 430
Glu His Thr Asp Leu Glu Gly Ile Trp Tyr Gly Val Asp Arg Ile Pro
435 440 445
Asp Glu Glu Leu Trp Glu Ala His Leu Asn Ala Lys Lys Arg Phe Ile
450 455 460
Asp Tyr Ile Arg Glu Ser Ile Lys Arg Arg Asn Glu Arg Leu Gly Ile
465 470 475 480
Asn Glu Pro Leu Pro Glu Ile Ser Glu Asn Val Leu Ile Ile Gly Phe
485 490 495
Ala Arg Arg Phe Ala Thr Tyr Lys Arg Ala Val Leu Leu Phe Ser Asp
500 505 510
Leu Glu Arg Leu Lys Arg Ile Val Asn Asn Ser Glu Arg Pro Val Tyr
515 520 525
Ile Val Tyr Ala Gly Lys Ala His Pro Arg Asp Glu Gly Gly Lys Glu
530 535 540
Phe Leu Arg Arg Ile Tyr Glu Val Ser Gln Met Pro Asp Phe Lys Asn
545 550 555 560
Lys Ile Ile Val Leu Glu Asn Tyr Asp Ile Gly Met Ala Arg Leu Met
565 570 575
Val Ser Gly Val Asp Val Trp Leu Asn Asn Pro Arg Arg Pro Met Glu
580 585 590
Ala Ser Gly Thr Ser Gly Met Lys Ala Ala Ala Asn Gly Val Leu Asn
595 600 605
Ala Ser Val Tyr Asp Gly Trp Trp Val Glu Gly Tyr Asn Gly Arg Asn
610 615 620
Gly Trp Val Ile Gly Asp Glu Ser Val Leu Pro Glu Thr Glu Ala Asp
625 630 635 640
Asp Pro Lys Asp Ala Glu Ala Leu Tyr Glu Leu Leu Glu Asn Glu Ile
645 650 655
Ile Pro Thr Tyr Tyr Glu Asn Arg Glu Lys Trp Ile Phe Met Met Lys
660 665 670
Glu Ser Ile Lys Ser Val Ala Pro Lys Phe Ser Thr Thr Arg Met Leu
675 680 685
Lys Glu Tyr Thr Glu Lys Phe Tyr Ile Lys Gly Leu Val Asn Arg Glu
690 695 700
Trp Leu Glu Arg Arg Glu Asn Val Glu Lys Ile Gly Ala Trp Lys Glu
705 710 715 720
Arg Ile Leu Lys Asn Trp Glu Asn Val Ser Ile Glu Arg Ile Val Leu
725 730 735
Glu Asp Ser Lys Ser Val Glu Val Thr Val Lys Leu Gly Asp Leu Thr
740 745 750
Pro Asn Asp Val Ile Val Glu Leu Val Ala Gly Arg Gly Glu Gly Met
755 760 765
Glu Asp Leu Glu Val Trp Lys Val Ile His Ile Arg Arg Tyr Arg Lys
770 775 780
Glu Asn Asp Leu Phe Val Tyr Thr Tyr Thr Asn Gly Val Leu Gly His
785 790 795 800
Leu Gly Ser Pro Gly Trp Phe Tyr Ala Val Arg Val Ile Pro Tyr His
805 810 815
Pro Arg Leu Pro Ile Lys Phe Leu Pro Glu Val Pro Val Val Trp Lys
820 825 830
Lys Val Leu
835
<210> 12
<211> 456
<212> PRT
<213> 嗜热古生菌(Thermococcus kodakarensis )
<400> 12
Met Gly Lys Leu Phe Gly Thr Phe Gly Val Arg Gly Ile Ala Asn Glu
1 5 10 15
Glu Ile Thr Pro Glu Phe Ala Leu Lys Ile Gly Met Ala Phe Gly Thr
20 25 30
Leu Leu Lys Arg Glu Gly Arg Glu Arg Pro Leu Val Val Val Gly Arg
35 40 45
Asp Thr Arg Val Ser Gly Glu Met Leu Lys Asp Ala Leu Ile Ser Gly
50 55 60
Leu Leu Ser Thr Gly Cys Asp Val Ile Asp Val Gly Ile Ala Pro Thr
65 70 75 80
Pro Ala Ile Gln Trp Ala Thr Asn His Phe Asn Ala Asp Gly Gly Ala
85 90 95
Val Ile Thr Ala Ser His Asn Pro Pro Glu Tyr Asn Gly Ile Lys Leu
100 105 110
Leu Glu Pro Asn Gly Met Gly Leu Lys Lys Glu Arg Glu Ala Ile Val
115 120 125
Glu Glu Leu Phe Phe Ser Glu Asp Phe His Arg Ala Lys Trp Asn Glu
130 135 140
Ile Gly Glu Leu Arg Lys Glu Asp Ile Ile Lys Pro Tyr Ile Glu Ala
145 150 155 160
Ile Lys Asn Arg Val Asp Val Glu Ala Ile Lys Lys Arg Arg Pro Phe
165 170 175
Val Val Val Asp Thr Ser Asn Gly Ala Gly Ser Leu Thr Leu Pro Tyr
180 185 190
Leu Leu Arg Glu Leu Gly Cys Lys Val Val Ser Val Asn Ala His Pro
195 200 205
Asp Gly His Phe Pro Ala Arg Asn Pro Glu Pro Asn Glu Glu Asn Leu
210 215 220
Lys Gly Phe Met Glu Ile Val Lys Ala Leu Gly Ala Asp Phe Gly Val
225 230 235 240
Ala Gln Asp Gly Asp Ala Asp Arg Ala Val Phe Ile Asp Glu Asn Gly
245 250 255
Arg Phe Ile Gln Gly Asp Lys Thr Phe Ala Leu Val Ala Asp Ala Val
260 265 270
Leu Arg Glu Asn Gly Gly Gly Leu Leu Val Thr Thr Ile Ala Thr Ser
275 280 285
Asn Leu Leu Asp Asp Ile Ala Lys Arg Asn Gly Ala Lys Val Met Arg
290 295 300
Thr Lys Val Gly Asp Leu Ile Val Ala Arg Ala Leu Leu Glu Asn Asn
305 310 315 320
Gly Thr Ile Gly Gly Glu Glu Asn Gly Gly Val Ile Phe Pro Asp Phe
325 330 335
Val Leu Gly Arg Asp Gly Ala Met Thr Thr Ala Lys Ile Val Glu Ile
340 345 350
Phe Ala Lys Ser Gly Lys Lys Phe Ser Glu Leu Ile Asp Glu Leu Pro
355 360 365
Lys Tyr Tyr Gln Phe Lys Thr Lys Arg His Val Glu Gly Asp Arg Lys
370 375 380
Ala Ile Val Ala Lys Val Ala Glu Leu Ala Glu Lys Lys Gly Tyr Lys
385 390 395 400
Ile Asp Thr Thr Asp Gly Thr Lys Ile Ile Phe Asp Asp Gly Trp Val
405 410 415
Leu Val Arg Ala Ser Gly Thr Glu Pro Ile Ile Arg Ile Phe Ser Glu
420 425 430
Ala Lys Ser Glu Glu Lys Ala Arg Glu Tyr Leu Glu Leu Gly Ile Lys
435 440 445
Leu Leu Glu Glu Ala Leu Lys Gly
450 455
<210> 13
<211> 415
<212> PRT
<213> 嗜热栖热菌(Thermus thermophilus)
<400> 13
Met Leu Arg Leu Asp Thr Arg Phe Leu Pro Gly Phe Pro Glu Ala Leu
1 5 10 15
Ser Arg His Gly Pro Leu Leu Glu Glu Ala Arg Arg Arg Leu Leu Ala
20 25 30
Lys Arg Gly Glu Pro Gly Ser Met Leu Gly Trp Met Asp Leu Pro Glu
35 40 45
Asp Thr Glu Thr Leu Arg Glu Val Arg Arg Tyr Arg Glu Ala Asn Pro
50 55 60
Trp Val Glu Asp Phe Val Leu Ile Gly Ile Gly Gly Ser Ala Leu Gly
65 70 75 80
Pro Lys Ala Leu Glu Ala Ala Phe Asn Glu Ser Gly Val Arg Phe His
85 90 95
Tyr Leu Asp His Val Glu Pro Glu Pro Ile Leu Arg Leu Leu Arg Thr
100 105 110
Leu Asp Pro Arg Lys Thr Leu Val Asn Ala Val Ser Lys Ser Gly Ser
115 120 125
Thr Ala Glu Thr Leu Ala Gly Leu Ala Val Phe Leu Lys Trp Leu Lys
130 135 140
Ala His Leu Gly Glu Asp Trp Arg Arg His Leu Val Val Thr Thr Asp
145 150 155 160
Pro Lys Glu Gly Pro Leu Arg Ala Phe Ala Glu Arg Glu Gly Leu Lys
165 170 175
Ala Phe Ala Ile Pro Lys Glu Val Gly Gly Arg Phe Ser Ala Leu Ser
180 185 190
Pro Val Gly Leu Leu Pro Leu Ala Phe Ala Gly Ala Asp Leu Asp Ala
195 200 205
Leu Leu Met Gly Ala Arg Lys Ala Asn Glu Thr Ala Leu Ala Pro Leu
210 215 220
Glu Glu Ser Leu Pro Leu Lys Thr Ala Leu Leu Leu His Leu His Arg
225 230 235 240
His Leu Pro Val His Val Phe Met Val Tyr Ser Glu Arg Leu Ser His
245 250 255
Leu Pro Ser Trp Phe Val Gln Leu His Asp Glu Ser Leu Gly Lys Val
260 265 270
Asp Arg Gln Gly Gln Arg Val Gly Thr Thr Ala Val Pro Ala Leu Gly
275 280 285
Pro Lys Asp Gln His Ala Gln Val Gln Leu Phe Arg Glu Gly Pro Leu
290 295 300
Asp Lys Leu Leu Ala Leu Val Ile Pro Glu Ala Pro Leu Glu Asp Val
305 310 315 320
Glu Ile Pro Glu Val Glu Gly Leu Glu Ala Ala Ser Tyr Leu Phe Gly
325 330 335
Lys Thr Leu Phe Gln Leu Leu Lys Ala Glu Ala Glu Ala Thr Tyr Glu
340 345 350
Ala Leu Ala Glu Ala Gly Gln Arg Val Tyr Ala Leu Phe Leu Pro Glu
355 360 365
Val Ser Pro Tyr Ala Val Gly Trp Leu Met Gln His Leu Met Trp Gln
370 375 380
Thr Ala Phe Leu Gly Glu Leu Trp Glu Val Asn Ala Phe Asp Gln Pro
385 390 395 400
Gly Val Glu Leu Gly Lys Val Leu Thr Arg Lys Arg Leu Ala Gly
405 410 415
<210> 14
<211> 320
<212> PRT
<213> 嗜热脱氮芽孢杆菌(GeobaciIlus thermodenitrificans)
<400> 14
Met His Gln Glu Pro Ile Phe Leu Thr Pro Val Phe Gln Glu Arg Ile
1 5 10 15
Trp Gly Gly Thr Lys Leu Ala Glu Arg Phe Gly Tyr Asp Ile Pro Ser
20 25 30
Ser Gln Thr Gly Glu Cys Trp Ala Val Ser Ala His Pro His Gly Gln
35 40 45
Thr Val Val Ala Arg Gly Pro Phe Gln Gly Met Thr Leu Gly Gln Leu
50 55 60
Trp Glu Glu Arg Arg Asp Leu Phe Gly Asn Phe Pro Ser Asp Arg Phe
65 70 75 80
Pro Leu Leu Thr Lys Ile Leu Asp Ala Asn Ala Asp Leu Ser Val Gln
85 90 95
Val His Pro Asp Asp Asp Tyr Ala Lys Thr Asn Glu Gly Gly Glu Leu
100 105 110
Gly Lys Thr Glu Cys Trp Tyr Ile Ile Asp Cys Lys Pro Gly Ala Gln
115 120 125
Leu Ile Tyr Gly His Tyr Ala Gln Thr Lys Glu Glu Leu Arg Ala Met
130 135 140
Met Glu Ala Gly Glu Trp Asp Arg Leu Leu Arg Lys Val Pro Ile His
145 150 155 160
Pro Gly Asp Phe Phe Tyr Val Pro Ser Gly Thr Ile His Ala Leu Cys
165 170 175
Glu Gly Thr Leu Val Leu Glu Thr Gln Gln Ser Ser Asp Thr Thr Tyr
180 185 190
Arg Val Tyr Asp Tyr Asp Arg Val Asp Ser Gln Gly Arg Lys Arg Glu
195 200 205
Leu His Leu Glu Lys Ala Ile Asp Val Thr Thr Val Pro His Arg Asp
210 215 220
Thr Asp Val Gln Pro His Val Ala Asn Ile Pro Gly Ala Thr Val Thr
225 230 235 240
Thr Phe Val Glu Gly Asp Tyr Phe Gly Val Gln Lys Trp His Val His
245 250 255
Gly Glu Ala Glu Trp Glu Gln Thr Lys Pro Phe Leu Ile Val Ser Ile
260 265 270
Leu Gln Gly Glu Gly Glu Leu Val His Gly Glu Arg Thr Tyr Pro Ile
275 280 285
Arg Gln Gly Asp His Phe Ile Leu Pro His Gln Phe Gly Arg Phe Ala
290 295 300
Ile Arg Gly Thr Leu Glu Ala Ile Ala Ser Trp Pro Arg Thr Ser Lys
305 310 315 320

Claims (10)

1.一种磷酸酶突变体，其特征在于所述突变体是将SEQ ID NO.1所示氨基酸序列的第175位、179位或195位进行单位点突变获得的。
2.如权利要求1所述突变体，其特征在于所述突变体是将SEQ ID NO.1所示氨基酸序列突变为下列之一：第175位丝氨酸突变为天冬酰胺、第179位苯丙氨酸突变为甲硫氨酸或半胱氨酸、第195位酪氨酸突变为亮氨酸。
3.一种权利要求1所述磷酸酶突变体的编码基因。
4.一种权利要求3所述磷酸酶突变体编码基因构建的重组基因工程菌。
5.一种权利要求1所述磷酸酶突变体在催化麦芽糊精生产甘露糖中的应用。
6.如权利要求5所述的应用，其特征在于所述的应用为：将含磷酸酶突变体编码基因的工程菌发酵培养获得的湿菌体用缓冲液重悬后超声破碎、离心，上清液在60-80℃水浴中热处理10-30min后再次离心的上清液作为催化剂，以麦芽糊精为底物，添加MgCl2和磷酸盐，联合葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶和甘露糖6-磷酸异构酶，以pH为7.5～8.5的缓冲液为反应介质构成反应体系，在50～70℃条件下反应8～48h，冰浴终止反应，获得含甘露糖的反应液，反应液分离纯化，获得甘露糖。
7.如权利要求6所述的应用，其特征在于所述反应体系中，底物加入终浓度为5-50g/L，所述催化剂加入终浓度以蛋白含量计为20-200μg/mL；所述葡聚糖磷酸化酶加入终浓度为100-300μg/mL；所述葡萄糖磷酸变位酶加入终浓度为10-100μg/mL；所述葡萄糖磷酸异构酶加入终浓度为10-150μg/mL；甘露糖6-磷酸异构酶加入终浓度为10-150μg/mL；所述MgCl2加入终浓度为5-50mM；所述磷酸盐加入终浓度为5-50mM。
8.如权利要求6所述的应用，其特征在于所述催化剂按如下步骤制备：将含磷酸酶突变体编码基因的工程菌接种至含50mg/L卡那霉素的LB液体培养基中，37℃，180rpm条件下培养12h后，将菌液以体积浓度10％接种量转接至新的含50mg/L卡那霉素的LB培养基，37℃、180rpm培养至OD600为0.6-0.8时，降温至28℃，加IPTG至终浓度0.1mM，诱导表达12h；将诱导培养菌液离心，弃上清，沉淀用pH 7.2的HEPES缓冲液重悬，即为菌体悬液；菌体悬液在60W下超声破碎15min，持续2s，间歇4s，获得细胞破碎液，离心，上清液在60-80℃水浴中热处理10-30min，离心后的上清液即为酶液。
9.如权利要求6所述的应用，其特征在于所述葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶和甘露糖6-磷酸异构酶均以含相应酶编码基因的重组菌发酵培养获得的湿菌体用缓冲液重悬，超声破碎、离心，上清液在60-80℃水浴中热处理10-30min后再次离心的上清液形式加入。
10.如权利要求9所述的应用，其特征在于所述上清液按如下方法制备：分别构建含葡聚糖磷酸化酶编码基因，葡萄糖磷酸变位酶编码基因，葡萄糖磷酸异构酶编码基因和甘露糖6-磷酸异构酶的重组大肠杆菌工程菌，分别接种至LB液体培养基中，37℃、200r/min培养12h后；将菌液以体积浓度10％的接种量转接至含终浓度50mg/L卡那霉素的LB培养基中，37℃、180r/min培养至OD600为0.6-0.8时，降温至28℃，加IPTG至终浓度0.02mM，28℃诱导表达12h；将诱导培养菌液离心；弃上清，沉淀用pH 7.2、100mM的HEPES缓冲液重悬，即为菌体悬液；菌体悬液采用超声破碎仪，在4℃冰浴、超声功率60W、超声破碎2s、间歇4s的条件下，超声连续破碎10-30min，获得细胞破碎液，离心，上清在60-80℃水浴下热处理10-30min，离心，取上清液。
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CN112760305A (zh) * 2021-01-25 2021-05-07 浙江工业大学 一种栖热腔菌磷酸酶突变体及其应用
CN114606213A (zh) * 2022-01-28 2022-06-10 浙江工业大学 一种多聚磷酸激酶突变体、工程菌及其应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112760305A (zh) * 2021-01-25 2021-05-07 浙江工业大学 一种栖热腔菌磷酸酶突变体及其应用
CN112760305B (zh) * 2021-01-25 2022-04-29 浙江工业大学 一种栖热腔菌磷酸酶突变体及其应用
CN114606213A (zh) * 2022-01-28 2022-06-10 浙江工业大学 一种多聚磷酸激酶突变体、工程菌及其应用
CN114606213B (zh) * 2022-01-28 2024-05-03 浙江工业大学 一种多聚磷酸激酶突变体、工程菌及其应用

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