CN113604454B - 一种磷酸酶突变体及在催化麦芽糊精制备果糖中的应用 - Google Patents

一种磷酸酶突变体及在催化麦芽糊精制备果糖中的应用 Download PDF

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CN113604454B
CN113604454B CN202110847681.5A CN202110847681A CN113604454B CN 113604454 B CN113604454 B CN 113604454B CN 202110847681 A CN202110847681 A CN 202110847681A CN 113604454 B CN113604454 B CN 113604454B
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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体外多酶催化麦芽糊精制备果糖中的应用，所述突变体是将SEQ ID NO.1所示氨基酸的第173位、175位或179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，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，制备果糖，转化率达到70％以上，产物中果糖与副产物葡萄糖比例为11:1。底物专一性调控后的磷酸酶突变体在体外多酶合成系统生产果糖的绿色合成中具有重要工业应用潜力。

Description

一种磷酸酶突变体及在催化麦芽糊精制备果糖中的应用
(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)来源的对果糖-6-磷酸特异性提高的磷酸酶突变体及其在体外多酶合成果糖中应用。
(二)背景技术
果糖广泛用于食品、医药和化学工业等领域，其高效生产受到广泛关注。淀粉先酶解制备葡萄糖，再经葡萄糖异构酶异构化是目前工业化生产果糖的主要方法。但是，受反应热力学控制，该工艺仅能获得葡萄糖和果糖的混合物，需进一步通过复杂的色谱分离才能制备高纯度果糖。体外生物合成系统由于其途径多样性、高度可控性和高效性等优势，将对化学品、能源和材料生产模式产生重大变革。它通过酶元件的设计合成、多酶复合体的组装调控，构建崭新的人工生物合成途径用于目标化学品的高效合成，成为新一代生物制造模式。
以淀粉、麦芽糊精等为原料，构建由α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、葡萄糖磷酸异构酶和磷酸酶组成的体外多酶合成体系是绿色、高效合成高纯度果糖的重要方法。其中，磷酸酶是催化果糖-6-磷酸去磷酸化合成果糖的关键酶，其催化性能决定果糖的合成效率与纯度。但是，目前已知的磷酸酶底物谱广，可催化葡萄糖-1-磷酸、葡萄糖-6-磷酸等多种磷酸单糖去磷酸化，导致果糖生产过程副产物积累，下游分离纯化成本升高。因此，磷酸酶底物专一性的调控对高纯度果糖的合成具有重要意义。本发明筛选挖掘了大西洋嗜热菌(T.atlanticus)来源磷酸酶(M6PP)，并通过基因工程技术，对该酶进行改造，对其底物专一性进行调控以获得理想突变体，实现果糖的体外高效合成。
(三)发明内容
本发明的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，包含突变体的重组菌及其应用。本发明的磷酸酶突变体可以在60℃下，高选择性催化多酶合成体系的中间产物果糖-6-磷酸合成果糖，副产物大幅减少，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所示氨基酸序列的第173位、175位或179位进行单点突变获得的。优选突变体是将SEQ ID NO.1所示氨基酸序列进行下列之一突变：第173位苯丙氨酸Phe突变为半胱氨酸Cys(F173C，氨基酸序列SEQ IDNO.3，核苷酸序列SEQ ID NO：7)，第175位丝氨酸Ser突变为缬氨酸Val(S175V，氨基酸序列SEQ ID NO.4，核苷酸序列SEQ ID NO：8)，第179位苯丙氨酸Phe突变为苏氨酸Thr(F179T，氨基酸序列SEQ ID NO.5，核苷酸序列SEQ ID NO：9)或第179位苯丙氨酸Phe突变为丙氨酸Ala(F179A，氨基酸序列SEQ ID NO.6，核苷酸序列SEQ ID NO：10)。
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。
本发明还包括磷酸酶突变体编码基因构建的重组载体以及重组载体转化制备的重组基因工程菌。
本发明对用于构建磷酸酶突变体重组载体的基础载体没有限制，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，所述基础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。
本发明还提供一种磷酸酶突变体在体外多酶催化麦芽糊精制备果糖中的应用，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(优选70℃、20min)后再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和葡萄糖磷酸异构酶PGI，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，冰浴终止反应，获得含果糖的反应液，反应液分离纯化，获得果糖。
所述反应体系中，底物终浓度为5-50g/L，优选10g/L。所添加的磷酸酶突变体终浓度以蛋白含量计为50-200mg/L，优选150mg/L；葡聚糖磷酸化酶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加入终浓度以蛋白含量计为50-200mg/L，优选100mg/L；所述葡萄糖磷酸异构酶加入终浓度以蛋白含量计为10-150mg/L，优选50mg/L；所述MgCl2加入终浓度为5-50mM，优选5mM；所述磷酸盐为Na2HPO4，加入终浓度为5-50mM，优选10mM。
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℃水浴下热处理20分钟)，离心后得上清即为催化剂。
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是以含所述酶的相应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Thermotoga maritima 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T.kodakansis KOD1(GenBank：BAD42440.1)，其氨基酸序列如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T.thermophilus HB8(GenBank:BAD70100.1)，其氨基酸序列如SEQ ID NO：13所示，催化葡萄糖6-磷酸转化为果糖6-磷酸；磷酸酶突变体是通过分子改造来源于大西洋嗜热菌(T.atlanticus，GenBank:WP_073073090.1)的磷酸酶(M6PP)获得的，催化果糖6-磷酸去磷酸化生成果糖。由于这些酶均为嗜热微生物来源，在高温下稳定，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，即得初步纯化的酶液。
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。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℃水浴下热处理20分钟)，离心后得上清液即为酶液。
本发明获得的突变体F173C、S175V、F179T、F179A通过体外一锅法催化麦芽糊精的反应主产物为果糖，副产物为葡萄糖。野生型磷酸酶(M6PP)催化产物中果糖和葡萄糖比例约为1:1，突变体F173C产物中果糖与葡萄糖比例为4:1，突变体S175V产物中果糖与葡萄糖比例为3:1，突变体F179T产物中果糖与葡萄糖比例为11:1，突变体F179A产物中果糖与葡萄糖比例为3:1。
与现有技术相比，本发明的有益效果主要体现在：本发明提供了对果糖-6-磷酸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果糖。本发明构建的突变体F179T通过体外一锅法以麦芽糊精为底物生产果糖，产物中果糖与副产物葡萄糖的比例为11:1(果糖的含量高达76％)。底物专一性调控后的磷酸酶突变体在体外多酶合成系统生产高纯度果糖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。
本发明所述麦芽糊精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)的多肽序列(GenBank:WP_073073090.1)，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，将上述基因的密码子替换为大肠杆菌偏好(高频使用)的密码子，经密码子优化后的磷酸酶(M6PP)基因核苷酸序列如SEQ ID NO：2所示，编码蛋白的氨基酸序列如SEQID NO：1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E.coli BL21(DE3)感受态细胞中，获得野生型重组菌。
(2)重组菌培养：挑取步骤(1)野生型重组菌至10mL LB液体培养基中，于37℃、200rpm培养至OD600为0.6-0.8；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，取上清液即为野生型磷酸酶M6PP酶液，采用BCA蛋白含量检测试剂盒检测目标蛋白含量，蛋白含量为1.2mg/mL。
实施例3 α-GP、PGM、PGI酶液的制备
采用实施例2方法，分别将来源于海栖热袍菌(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)，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.coli BL21(DE3)，采用实施例2方法，分别制备α-GP酶液、PGM酶液、PGI酶液，采用BCA蛋白含量检测试剂盒检测目标蛋白含量，分别为α-GP酶液：2.1mg/mL；PGM酶：1.8mg/mL；PGI酶液：0.9mg/mL。
实施例4 磷酸酶M6PP突变体库的构建
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，设计第173、175和179位氨基酸残基定点饱和突变引物，引物如表1。
表1 第173、175和179位饱和突变引物序列
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
表3 PCR反应条件
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，并涂布于含50μg/mL卡那霉素的LB固体培养基，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4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600μL LB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8000r/min，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，8000rpm离心20min，去沉淀后获得得突变体酶液。同样条件下，制备野生型重组菌E.coliBL21(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果糖-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。显色液由0.12g/ml抗坏血酸溶液(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显著提高的菌株12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。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，离心后得M6PP突变体酶液，BCA蛋白含量检测试剂盒检测目标蛋白含量，蛋白含量为1.0-1.4mg/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0.1g/L实施例3方法制备的α-GP酶液，以蛋白含量计0.05g/L实施例3方法制备的PGM酶液，以蛋白含量计0.015g/L实施例3方法制备的PGI酶液和以蛋白含量计0.135g/L M6PP突变体酶液。反应液于70℃反应2、4、8、12和24h后直接取样，12000×g离心5min后，取上清通过HPLC检测样品峰面积，根据HPLC所检测果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，计算果糖含量。同样条件下，以实施例2方法制备的野生型M6PP酶液为对照。
所用液相色谱仪为安捷伦HPLC 1260II(RID)，色谱柱为Hi-Plex Ca，7.7mm×300mm，8μm，以H2O为流动相，进样量为5μL，检测器采用示差折光检测器(RID)，温度为35℃，柱温80℃，流速0.25mL/min。与野生型对照，根据果糖产量的提高，筛选获得有益突变体M6PP-F173C酶液(蛋白含量为1.13mg/mL，氨基酸序列SEQ ID NO.3，核苷酸序列SEQ ID NO：7)、M6PP-S175V酶液(蛋白含量为1.35mg/mL，氨基酸序列SEQ ID NO.4，核苷酸序列SEQ IDNO：8)、M6PP-F179T酶液(蛋白含量为1.4mg/mL，氨基酸序列SEQ ID NO.5，核苷酸序列SEQID NO：9)和M6PP-F179A酶液(蛋白含量为1.21mg/mL，氨基酸序列SEQ ID NO.6，核苷酸序列SEQ ID NO：10)，即相应的磷酸酶突变体工程菌为E.coli BL21(DE3)/pET-28b(+)-M6PP-F173C、E.coli BL21(DE3)/pET-28b(+)-M6PP-S175V、E.coli BL21(DE3)/pET-28b(+)-M6PP-F179T、E.coli BL21(DE3)/pET-28b(+)-M6PP-F179A。
实施例6 体外多酶一锅法催化麦芽糊精合成果糖
多酶催化体系包括(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)磷酸酶M6PP突变体，该酶催化果糖6-磷酸脱磷酸生成果糖。
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、实施例3方法制备的PGM酶液、实施例3方法制备的PGI酶液和实施例5方法制备的磷酸酶M6PP突变体酶液(F173C、S175V、F179T、F179A)浓度以蛋白含量计分别为200mg/L、100mg/L、50mg/L和150mg/mL。在60℃催化反应12h，通过实施例5所述HPLC法测定反应液中果糖含量。同样条件下，以实施例2方法制备的野生型磷酸酶M6PP酶液为对照。
如表4所示，以野生型磷酸酶为催化剂时，产物中果糖与葡萄糖质量比为1:1，果糖质量产率为17％；以突变体F173C为催化剂时，产物中果糖与葡萄糖质量比为4:1，果糖质量产率为24％；以突变体S175V为催化剂时，产物中果糖与葡萄糖质量比为3:1，果糖质量产率为33％；以突变体F179T为催化剂时，产物中果糖与葡萄糖质量比为11:1，果糖质量产率为76％；以突变体F179A为催化剂时，产物中果糖与葡萄糖质量比为3:1，果糖质量产率为33％。
表4 野生型磷酸酶及其突变体对体外多酶合成果糖的影响
上述结果表明，第179位点突变效果最好。第179位苯丙氨酸Phe突变为苏氨酸Thr改变了活性口袋附近的空间结构，阻碍了葡萄糖6-磷酸和葡萄糖1-磷酸进入活性口袋，而有利于底物果糖6-磷酸进入活性中心，从而提高酶对果糖6-磷酸的偏好性，使一锅法合成果糖的副产物大幅减少，纯度和产率得到提高。
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。
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<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果糖中的应用
<160> 13
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 大西洋嗜热菌(Thermosipho atlanticus)
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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 Cys 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> 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
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 Val 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> 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 Ser Met
165 170 175
Asn Ile Thr 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 Ala 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> 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 gagatctgca 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> 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 aagtcatgaa 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> 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 catcactctg 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 catcgctctg 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> 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

Claims (9)

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所示氨基酸序列突变为下列之一：第173位苯丙氨酸突变为半胱氨酸，第175位丝氨酸突变为缬氨酸，第179位苯丙氨酸突变为苏氨酸或丙氨酸。
2.一种权利要求1所述磷酸酶突变体的编码基因。
3.一种权利要求2所述磷酸酶突变体编码基因构建的重组基因工程菌。
4.一种权利要求1所述磷酸酶突变体在催化麦芽糊精生产果糖中的应用。
5.如权利要求4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、葡萄糖磷酸变位酶和葡萄糖磷酸异构酶，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，在50～70℃条件下反应8～48h，冰浴终止反应，获得含果糖的反应液，反应液分离纯化，获得果糖。
6.如权利要求5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为50-200μg/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50-200μg/mL；所述葡萄糖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。
7.如权利要求5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，再次离心后的上清液即为催化剂。
8.如权利要求5所述的应用，其特征在于所述葡聚糖磷酸化酶、葡萄糖磷酸变位酶和葡萄糖磷酸异构酶均以含所述酶相应编码基因的重组菌发酵培养获得的湿菌体经超声破碎、热处理后的上清液形式加入。
9.如权利要求8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0.1mM，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，再次离心，取上清液。
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