CN113005104A - 一个热稳定性提高的昆布二糖磷酸化酶突变体及其应用 - Google Patents

一个热稳定性提高的昆布二糖磷酸化酶突变体及其应用 Download PDF

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CN113005104A
CN113005104A CN201911329886.3A CN201911329886A CN113005104A CN 113005104 A CN113005104 A CN 113005104A CN 201911329886 A CN201911329886 A CN 201911329886A CN 113005104 A CN113005104 A CN 113005104A
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游淳
孙尚尚
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

本发明公布了一个热稳定性提高的昆布二糖磷酸化酶突变体及其在高温合成昆布二糖中的应用，属于酶工程技术领域。本发明所公开的热稳定性明显提高的昆布二糖磷酸化酶突变体是以假单胞菌Paenibacillus sp.YM1来源的昆布二糖磷酸化酶为基础，通过定点饱和突变和随机突变的方法建立基因突变库，再通过筛选基因突变库的方法获得的。然后将获得的昆布二糖磷酸化酶突变体用于构建体外多酶体系，在高温下以淀粉为底物制备昆布二糖。该突变体热稳定性相较野生型有明显提高，能保证其在工艺生产高温环节中酶活不丧失，替代昆布二糖磷酸化酶野生型应用于在高温下合成昆布二糖，可提高整个体外多酶体系的反应速率，缩短反应时间，从而提高昆布二糖的工业化潜力。

Description

一个热稳定性提高的昆布二糖磷酸化酶突变体及其应用
技术领域
本发明属于酶工程技术领域，具体涉及一个昆布二糖磷酸化酶突变体及其在高温合成昆布二糖中的应用。
背景技术
昆布二糖(laminaribiose)是β-1,3糖苷键连接的还原性二糖，是一种功能多样的高价值寡糖。它可以作为透明质酸的合成前体应用于制药及化妆品行业，作为促发芽剂和天然防腐剂应用于农业领域，作为食品添加剂应用于食品保健品行业，还可以用于调控嗜热菌如热纤维梭菌的蛋白表达。昆布二糖的传统制备方法是酸解或者酶解β-1,3聚糖，如海带多糖，茯苓多糖，地衣多糖和凝胶多糖，但这些天然聚糖原料供应受限，水解过程难以控制，产物分离步骤繁杂，昆布二糖的生产成本较高。随着工业酶生物技术的发展，有科学家开始尝试酶法合成昆布二糖。国内学者尝试利用3个酶(淀粉磷酸化酶，葡萄糖苷酶和昆布二糖磷酸化酶)，以淀粉为底物生产昆布二糖，在50℃下反应12小时即可达到约80％转化率，然而昆布二糖磷酸化酶的热稳定性差限制了其在高于50℃温度下应用。
昆布二糖磷酸化酶(laminaribiose phosphorylase,LBP,EC 2.4.1.31)是一种磷酸化酶，属于糖苷水解酶94家族，能可逆催化昆布二糖生成葡萄糖1-磷酸和葡萄糖。昆布二糖磷酸化酶最早发现于罗马蜗牛(HelixPomatia)体内，但并未分离出纯酶。随后，4种不同来源的昆布二糖磷酸化酶相继被鉴定，分别来源于小眼虫(Euglenagracilis)，变胞藻(Astasia ocellata)，无胆甾原体(Acholeplasma laidlawii)和假单胞菌(Paenibacillussp.)。其中来源于小眼虫，变胞藻和无胆甾原体的昆布二糖磷酸化酶均为中温酶，最适温度为30-40℃；来源于假单胞菌的昆布二糖磷酸化酶最适温度为55℃，但热稳定性较差，纯酶液于60℃处理，半衰期仅为10min左右，不适用于体外生物合成体系工业化规模生产昆布二糖。
为了促进昆布二糖磷酸化酶用于昆布二糖的工业化应用，急需一个热稳定性提高的昆布二糖磷酸化酶突变体，能保证其在工艺生产高温环节中酶活不丧失，并保持其高比酶活。
发明内容
本发明所要解决的技术问题是提供一热稳定性提高的昆布二糖磷酸化酶，并将其应用于以淀粉为底物催化生产昆布二糖的体外多酶体系中，达到高温高转化率高效率合成昆布二糖的目的。
为解决上述技术问题，本发明所采取的技术方案是：
步骤1：使用计算软件分析来源于假单胞菌的昆布二糖磷酸化酶的晶体结构，确定突变位点；
步骤2：将步骤1中的突变位点分别进行饱和突变建库筛选；
步骤3：将步骤2中初筛后获得的阳性克隆进行表达纯化，测定热稳定性；
步骤4：将步骤3中复筛所得的热稳定性提高最突出的位点作为起始序列，对其他位点进行迭代饱和突变；
步骤5：将步骤4中最终筛选到的突变体序列进行随机突变建库筛选，进一步提高昆布二糖磷酸化酶的热稳定性；
步骤6：将步骤5中最终筛选到的突变体用于以淀粉为底物，经多酶催化制备昆布二糖的体系中，于60℃反应8小时，对比野生型和突变体昆布二糖的生成情况。
为了开发热稳定性高的昆布二糖磷酸化酶(本文中简称LBP)，本发明以假单胞菌Paenibacillus sp.YM1来源的昆布二糖磷酸化酶(Genebank:BAJ10826，即SEQ ID NO:1)为基础，通过定点饱和突变和易错PCR的方法建立LBP基因突变库，再通过筛选基因突变库获得热稳定性明显提高的LBP突变体。
本发明提供了一种酶突变体，所述酶的氨基酸序列为SEQ ID NO.2，其为SEQ IDNO.1第11位的E替换为I、第12位的Q替换为V的突变体。
本发明提供了一种酶突变体，所述酶的氨基酸序列为SEQ ID NO.3，其为SEQ IDNO.2第171位的D替换为N。
本发明提供了一种酶突变体，所述酶的氨基酸序列为SEQ ID NO.4，其为SEQ IDNO.3第877位的C替换为F。
本发明提供了一种酶突变体，所述酶的氨基酸序列为SEQ ID NO.5，其为SEQ IDNO.4第452位的D替换为G。
本发明提供了一种酶突变体，所述酶的氨基酸序列为SEQ ID NO.6，其为SEQ IDNO.5第26位的L替换为Q。
本发明提供了一种酶突变体，所述酶的氨基酸序列为SEQ ID NO.7，其为SEQ IDNO.6第186位的I替换为T。
本发明技术方案与现有技术相比，具有以下有益效果：
本发明得到LBP酶突变体，相较于野生型LBP，突变体在60℃的半衰期从10min提高到9.76h，最高提高了58.6倍，比酶活与野生型相当；突变体在65℃的半衰期从2min提高到3.11h，最高提高了93.3倍。
附图说明
图1为LBP半理性设计突变结果，60℃半衰期测定。
图2为LBP随机突变结果，65℃半衰期测定。
图3为LBP突变位点整理分析。
图4为淀粉转化生成昆布二糖的体外多酶催化途径的示意图；其中：IA为异淀粉酶，αGP为淀粉磷酸化酶，αG为葡萄糖苷酶，LBP为昆布二糖磷酸化酶。
图5为体外多酶催化10g/L IA处理过的淀粉高温合成昆布二糖情况。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但是应理解所述实施例仅是范例性的，不对本发明的范围构成任何限制。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。
本发明实施例中用到如下材料
可溶性淀粉，ACROS公司产品，产品编号：424490020；
pET28a载体，Novagen，Madison，WI；
大肠杆菌表达菌BL21(DE3)，Invitrogen，Carlsbad，CA；
本发明中的所有酶均能在Sigma公司购买得到，所有酶也都可以按照基因工程方法通过原核表达获得。
昆布二糖的测定方法：
高效液相色谱(HPLC)检测法：RID检测器，HPX-87H柱(Bio-Rad Hercules,CA)，流动相：5mM H2SO4，流速0.6mL/min，柱温55℃，进样体积20μL。
培养基(g/L)：胰蛋白胨10，酵母粉5，NaCl 10。
筛选方法：采用平板菌落覆盖二层板显色的方法进行筛选，将8mL含50mMHEPESbuffer(pH 7.0),2mM昆布二糖,2mM磷酸根离子,4mM镁离子,0.5mM NAD+，50μM四硝基蓝四氮唑(TNBT),10μM吩嗪硫酸甲酯(PMS),1U/mL磷酸葡萄糖变位酶(PGM)，1U/mL葡萄糖6-磷酸脱氢酶(G6PDH)的0.5％(w/v)融化的琼脂糖溶液(60℃)小心的倒在热处理后的平板菌落上。室温放置30分钟后，阳性菌落将出现蓝黑色晕圈。
实施例1理性分析，确定突变位点
假单胞菌的LBP为同源二聚体，催化口袋位于两个单体上结合区域，其晶体结构已经公布(PDB:6gh2)。纯酶的最适温度为55℃，在60℃的半衰期为10min通过来源于假单胞菌LBP的PDB晶体结构，分别采用B-FITTER和ETSS软件进行计算分析，确定E11、Q12、D171、C877为可能的提高热稳定性的位点。
利用快速PCR技术，以表达有野生型LBP的基因的质粒pET20b-lbp为模板，进行定点饱和突变。
引入E11和Q12突变的引物：
正向引物：GGTNNKNNKGGTGAATTTCGTCTGGAACAGCCGGAAC
反向引物：CTGAAATTTCCAGCCTTTCTGACCCATGGTATATC
引入D171突变的引物：
正向引物：GTCGTAGTGCCNNKGATCTGCGTGATCATC
反向引物：CATACAGCGGAATTGCGGCGGTAGG
引入C877突变的引物：
正向引物：GCCAGAGCGTGGATNNKCAGAATGATGGC
反向引物：CATTCAGACTAACGCTATCAACACGATACTGGCC
引物磷酸化体系：NEB T4 PNK缓冲液2μL，ATP(100mM)1μL，NEB T4 PNK 1μL，引物(100μM)2μL，加入双蒸水14μL。
PCR反应体系均为：缓冲液10μL，dNTPs(各2.5mM)4μL，磷酸化正向引物(10μM)1μL，磷酸化反向引物(10μM)1μL，模板DNA 1μL，NEB high-fidelity Q5 DNApolymerase(2.5U/μL)0.5μL，加入双蒸水32.5μL。
PCR反应扩增条件均为：98℃预变性2min；随后98℃ 10s，55℃ 15s，72℃ 2min进行30个循环，最后72℃保温5min。
将PCR产物经过Dpn I消化0.5h后，经TIAN quickMidipurification kit(Tiangen,Beijing,China)纯化后，NEB Quick Ligation kit将线性的质粒连接成环状，分别转入大肠杆菌E.coli TOP10的感受态细胞中，涂布到含有琼脂的LB固体培养基中培养过夜，平板60℃热处理1h，覆盖显色二层平板，挑取阳性克隆，提取质粒，转入大肠杆菌E.coliBL21(DE3)的感受态细胞中，进行酶的表达纯化，测定酶的热稳定性。
经筛选，得到E11I Q12V突变体(即突变体M1)，其热稳定性相对野生型提高的最明显，将LBP纯酶在60℃的半衰期从10min提高到1.56h(图2)。
利用快速PCR技术，以表达有突变体LBP-M1的基因的质粒pET20b-lbpm1为模板，进行D177和C877位点的饱和突变，经筛选，得到的突变体M2(E11I Q12VD171N)，将LBP纯酶在60℃的半衰期继续提高到2.59h(图2)。
利用快速PCR技术，以表达有突变体LBP-M2的基因的质粒pET20b-lbpm2为模板，进行C877位点的饱和突变，经筛选，得到的突变体M3(E11I Q12V D171N C877F)，将LBP纯酶在60℃的半衰期继续提高到3.46h(图2)。
实施例2随机突变进一步提高热稳定性
通过计算分析和迭代饱和突变，来源于假单胞菌的LBP在60℃的热稳定性有了明显提高，采用随机突变进行建库筛选以期得到热稳定性更优的突变体。
利用易错PCR技术，以表达有突变体LBP-M3的基因的质粒pET20b-lbpm3为模板，进行随机突变。
建库方法：
lbp基因片段采用易错PCR技术以pET20b-lbpm3为模板进行扩增，引物IF：CTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGTCAGAAAGGCTGGAAATTTCAGGGTGAACAGGGTG，IR：ACTAATATTACGGCCCAGGGTCACAATC。
引物磷酸化体系：NEB T4 PNK缓冲液2μL，ATP(100mM)1μL，NEB T4 PNK 1μL，IF(100μM)2μL，加入双蒸水14μL。
PCR反应体系(50μL)：1ng/μL模板，0.2mM dATP，0.2mM dGTP，1mM dCTP，1mM dTTP，5mM MgCl2，0.4μM引物(磷酸化的IF和IR)，0.05U/LNEB Taqpolymerase。
易错PCR反应扩增条件均为：94℃预变性4min；随后94℃ 30s，56℃ 30s，68℃2min进行18个循环，最后72℃保温5min。
质粒骨架采用快速PCR技术以pET20b为模板进行扩增，引物VF：CTCGAGCACCACCACCACCACCACTGAGATCC，VR：CACCCTGTTCACCCTGAAATTTCCAGCCTTTCTGACCCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAG。
引物磷酸化体系：NEB T4 PNK缓冲液2μL，ATP(100mM)1μL，NEB T4 PNK 1μL，VR(100μM)2μL，加入双蒸水14μL。
PCR反应体系为：缓冲液10μL，dNTPs(各2.5mM)4μL，VF(10μM)1μL，磷酸化的VR(10μM)1μL，模板DNA 1μL，NEB high-fidelity Q5 DNApolymerase(2.5U/μL)0.5μL，加入双蒸水32.5μL。
PCR反应扩增条件为：98℃预变性2min；随后98℃ 10s，55℃ 15s，72℃ 2min进行30个循环，最后72℃保温5min。
PCR反应体系均为：缓冲液10μL，dNTPs(各2.5mM)4μL，磷酸化正向引物(10μM)1μL，磷酸化反向引物(10μM)1μL，模板DNA 1μL，NEB high-fidelity Q5 DNApolymerase(2.5U/μL)0.5μL，加入双蒸水32.5μL。
PCR反应扩增条件均为：98℃预变性2min；随后98℃ 10s，55℃ 15s，72℃ 2min进行30个循环，最后72℃保温5min。
将片段和质粒骨架的PCR产物经过Dpn I消化0.5h后，经TIAN quick Midipurification kit(Tiangen,Beijing,China)纯化后，进行重叠延伸PCR(OE-PCR)。
PCR反应体系均为：缓冲液10μL，dNTPs(各2.5mM)4μL，磷酸化引物IF/VR(10μM)1μL，片段2ng/μL，载体骨架4ng/μL(与片段等摩尔浓度)，NEB high-fidelity Q5DNApolymerase(2.5U/μL)0.5μL，加入双蒸水至50μL。
PCR反应扩增条件均为：98℃预变性2min；随后98℃ 10s，60℃ 15s，72℃ 4min进行30个循环，最后72℃保温10min。
NEB Quick Ligation kit将线性的质粒连接成环状，转入大肠杆菌E.coli TOP10的感受态细胞中，涂布到含有琼脂的LB固体培养基中培养过夜，平板70℃热处理1h，覆盖显色二层平板，挑取阳性克隆，提取质粒，转入大肠杆菌E.coli BL21(DE3)的感受态细胞中，进行酶的表达纯化，测定酶的热稳定性。
在65℃进行突变体的筛选，得到D452G突变体(即突变体M4)，其热稳定性相对突变体LBP-M3提高的最明显，将LBP纯酶在65℃的半衰期从2min提高到15min(图3)，在60℃的半衰期从3.46h提高到5.13h(图2)。
利用易错PCR技术，以表达有突变体LBP-M4的基因的质粒pET20b-lbpm4为模板，继续进行建库筛选，得到的突变体M5(E11I Q12V D171N C877F D452G L26Q)，将LBP纯酶在65℃的半衰期继续提高到1.21h(图3)，在60℃的半衰期从5.13h提高到7.70h(图2)。
利用易错PCR技术，以表达有突变体LBP-M5的基因的质粒pET20b-lbpm5为模板，继续进行建库筛选，得到的突变体M6(E11I Q12V D171N C877F D452G L26Q I186T)，将LBP纯酶在65℃的半衰期继续提高到3.11h(图3)，在60℃的半衰期从7.7h提高到9.76h(图2)。
实施例3提高反应温度至60℃时昆布二糖的生成情况
通过体外多酶催化体系将淀粉转化为昆布二糖的催化途径见图4。其中涉及的关键酶与关键步骤包括：(1)淀粉磷酸化酶(αGP，EC 2.4.1.1)，用于从淀粉上释放出葡萄糖-1-磷酸；(2)葡萄糖苷酶(αG，EC 3.2.1.20)，用于从淀粉上释放出葡萄糖；(3)昆布二糖磷酸化酶(LBP，EC 2.4.1.31)突变体M6，用于催化葡萄糖-1-磷酸和葡萄糖生成昆布二糖。异淀粉酶可辅助水解淀粉，即在反应体系中增加能够帮助淀粉水解的异淀粉酶(IA，EC3.2.1.68)能够提高昆布二糖的得率。
在本实施例中，淀粉磷酸化酶来源于海栖热袍菌(Thermotoga maritima)，其基因在KEGG上的编号为TM1168；葡萄糖苷酶来源于淡紫色拟青霉(Paecilomyces lilacinus)，其基因在KEGG上的编号为QAQ81244；异淀粉酶来源于硫化叶菌(Sulfolobus tokodaii)，其基因在KEGG上的编号为ST0928。这些基因组DNA都可从ATCC的官方网站(www.atcc.org)上获得。这三个基因分别用F1/R1、F2/R2和F3/R3从相应的基因组DNA中通过PCR获取，其中，F1：GTTTAACTTTAAGAAGGAGATATAGTGCTGGAGAAACTTCCCGAG，R1：GTGGTGGTGGTGGTGCTCGAGTCAGAGAACCTTCTTCCAGAC，F2:CATCATCATCATCATCACAGCAGCGGCTTGAAAAAAACATGGTGGAAAGAAG，R2:GTGGTGGTGGTGGTGGTGCTCGAGTTCTTTCCAGATGTATACGCGCGCC，F3：GTTTAACTTTAAGAAGGAGATATACCATGGGTCAGAAAGGCTGGAAATTTC，R3：CAGTGGTGGTGGTGGTGGTGCTCGAGACTAATATTACGGCCCAGGGTCAC，并通过Simple Cloning(You C,Zhang XZ,ZhangY-HP.2012.Simple cloningvia direct transformation ofPCR product(DNA Multimer)to Escherichia coli andBacillus subtilis.Appl.Environ.Microbiol.78(5):1593-5.)的方法克隆至pET20b载体(Novagen,Madison,WI)中，获得相应的表达载体pET20b-TmαGP,pET20b-PlαG和pET20b-StIA。昆布二糖磷酸化酶来源于假单胞菌(Paenibacillus sp.YM1)，使用实施例2中的突变体M6，序列为SEQ ID NO.7，表达载体为pET20b-lbpm6。然后，将这四个质粒分别转化至大肠杆菌表达菌BL21(DE3)(Invitrogen,Carlsbad,CA)中，进行蛋白质表达与纯化，蛋白质纯化的结果如图2所示。
反应体系中含有5mM乙酸钠缓冲液(pH 5.5)，0.5mM二价锌离子，5U/mL的异淀粉酶，200g/L的淀粉，在85℃进行催化反应，反应时间为3个小时。
随后反应体系中含有100mM HEPES缓冲液(pH 6.5)，5mM的二价锌离子，20mM的磷酸钾(pH 6.5)，3U/mL淀粉磷酸化酶，1U/mL葡萄糖苷酶，2U/mL昆布二糖磷酸化酶，10g/LIA处理过的淀粉，在60℃进行催化反应，反应时间为8个小时。
高效液相色谱检测昆布二糖的浓度。取反应样品94.5μL，加入5.5μL 10％的硫酸终止反应。离心取上清，采用HPLC检测昆布二糖出峰面积和峰高计算昆布二糖的浓度。
经检测，反应结束后，经由LBP突变体M6催化生成昆布二糖的终浓度(图5)是22.4mM，相对淀粉(10g/L，约55.5mM葡萄糖当量，2分子葡萄糖当量合成1分子昆布二糖)转化率为80.7％，而经由LBP野生型催化生成昆布二糖的终浓度只有5.4mM，相对淀粉转化率为19.4％。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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<110> 中国科学院天津工业生物技术研究所
<120> 一个热稳定性提高的昆布二糖磷酸化酶突变体及其应用
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His Gly Ala Trp Pro Leu Met Thr Thr Leu Leu Tyr Leu His Gln Ser
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Gly Asp Leu Asp Leu Leu Phe Gln Pro Gln Ser Tyr Phe Arg Asp Val
450 455 460
Phe Val Lys Arg Cys Arg Glu Arg Asp Ala Ser Trp Thr Pro Glu Gln
465 470 475 480
Gly Asn Lys Leu Leu Thr Ala Asp Gly Gln Ile Tyr Glu Gly Thr Ile
485 490 495
Leu Glu His Ile Leu Leu Gln Asn Ile Val Pro Phe Phe Asn Val Gly
500 505 510
Glu His Gly Asn Ile Lys Leu Glu Gly Ala Asp Trp Asn Asp Gly Leu
515 520 525
Asp Leu Ala Pro Glu Arg Gly Glu Ser Val Ala Phe Thr Ala Phe Tyr
530 535 540
Ala Ser Asn Leu Met Glu Leu Ser Glu Leu Leu Leu Glu Leu Gln Lys
545 550 555 560
Arg Thr Gly Lys Asp Ser Leu Asp Ile Ala Glu Glu Met Ala Leu Leu
565 570 575
Leu Asp Thr Leu Gly Lys Pro Ile Ser Tyr Asp Ser Ile Gln Glu Lys
580 585 590
Arg Ser Leu Leu Asp Arg Tyr Tyr Asp Ala Val Thr Pro Arg Val Ser
595 600 605
Gly Lys Lys Leu Leu Leu Asp Ile Arg Lys Val Ala Glu Asp Leu Lys
610 615 620
Arg Lys Ala Asp Trp Ala Val Ala His Leu Arg Gly Ser Glu Trp Ile
625 630 635 640
Gln Ser Lys Glu Gly Tyr Ala Trp Phe Asn Gly Tyr Tyr Asn Asn Asp
645 650 655
Gly Glu Arg Val Glu Gly Asp His Pro Asp Gly Val Arg Met Thr Leu
660 665 670
Thr Gly Gln Val Phe Ala Ile Met Gly Gly Val Ala Thr Asp Glu Gln
675 680 685
Thr Glu Lys Ile Ser Gln Ala Val Asn Arg Tyr Leu Lys Asp Glu Arg
690 695 700
Ile Gly Tyr Arg Leu Asn Ser Arg Phe Gly Gly Ile Gln Gln Asn Leu
705 710 715 720
Gly Arg Ala Phe Gly Phe Ala Phe Gly His Lys Glu Asn Gly Ala Met
725 730 735
Phe Ser His Met Thr Val Met Tyr Ala Asn Ala Leu Tyr Lys Arg Gly
740 745 750
Phe Val Gln Glu Gly Phe Glu Val Leu Asp Ser Ile Tyr Arg Leu Ser
755 760 765
Ala Asp Phe Glu Asn Ser Arg Ile Tyr Pro Gly Val Pro Glu Tyr Ile
770 775 780
Asn Glu Arg Gly Arg Gly Met Tyr Thr Tyr Leu Thr Gly Ser Ala Ser
785 790 795 800
Trp Leu Leu Leu Thr Gln Leu Thr Glu Val Tyr Gly Val Lys Gly Arg
805 810 815
Phe Gly Asp Leu Arg Leu Glu Pro Lys Leu Val Gln Ala Gln Phe Asp
820 825 830
Gly Ser Gly Glu Ala Ala Val Glu Thr Leu Phe Ala Gly Arg Met Leu
835 840 845
Arg Val Val Tyr Arg Asn Pro Gln Ala Ala Glu His Gly Gln Tyr Arg
850 855 860
Val Asp Ser Val Ser Leu Asn Gly Gln Ser Val Asp Phe Gln Asn Asp
865 870 875 880
Gly Ala Gly Cys Leu Ile Gly Arg Ser Leu Ile Glu Ala Leu Pro Ala
885 890 895
Asp Gly Val His Glu Leu Ile Val Thr Leu Gly Arg Asn Ile Ser
900 905 910
<210> 5
<211> 911
<212> PRT
<213> 人工序列()
<400> 5
Met Gly Gln Lys Gly Trp Lys Phe Gln Gly Ile Val Gly Glu Phe Arg
1 5 10 15
Leu Glu Gln Pro Glu His Asn Ser Tyr Leu Tyr Phe Pro Leu Val Asn
20 25 30
Glu Ala Gly Met Met Ser Ala Val Thr Pro Asn Leu His Gly Glu Ile
35 40 45
Thr Ser Gly His Asn Thr Phe Leu Met Glu Pro Val Ser Ala Glu Ser
50 55 60
Leu His Asn Ser Lys Ala Ser Arg Asn Phe Trp Val Phe Ile Glu Gly
65 70 75 80
Tyr Gly Ala Trp Ser Val Ser Gly Asn Ser Ala Arg Gln Asn Ala Ala
85 90 95
Arg Phe Thr Gly Glu Glu Glu Arg Ser Ala Val Glu Ala Gly Phe Leu
100 105 110
Trp His Ala Val Thr Arg Glu Asn Glu Lys Ala Gly Leu Lys Ala Arg
115 120 125
Thr Val Ser Phe Val Pro Val Thr Asp Asp Lys Ile Glu Leu Met Arg
130 135 140
Val Thr Leu Thr Asn Thr Gly Asn Ala Pro Leu Lys Leu Thr Pro Thr
145 150 155 160
Ala Ala Ile Pro Leu Tyr Gly Arg Ser Ala Asn Asp Leu Arg Asp His
165 170 175
Arg His Val Thr Ser Leu Leu His Arg Ile Phe Thr Ser Glu Tyr Gly
180 185 190
Ile Glu Val Gln Pro Ala Leu Ser Phe Asp Glu Arg Gly His Arg Val
195 200 205
Asn Lys Val Thr Tyr Gly Val Phe Gly Ala Glu Ala Gly Gly Thr Ala
210 215 220
Pro Ala Gly Phe Phe Pro Val Thr Glu Asp Phe Ile Gly Glu Gly Gly
225 230 235 240
Ala Leu Asp Trp Pro Glu Ala Val Val Ala Asn Arg Glu Pro Asp Ala
245 250 255
Gln Ala Gly Thr Ala Val Glu Gly Tyr Glu Ala Val Gly Ala Leu Arg
260 265 270
Phe Ala Pro Val Glu Leu Ala Pro Gly Lys Ser Val Ser Tyr Val Val
275 280 285
Ala Met Val Ile Ser Gly Asp Arg Ile Asp Val Gly Arg Tyr Ala Ala
290 295 300
Asp Tyr Leu Ala Ala Gly Arg Phe Asp Ala Leu Leu Glu Gln Asn Arg
305 310 315 320
Ala Tyr Trp Arg Asp Lys Leu Asp Thr Val Arg Phe Ser Ser Gly Asp
325 330 335
Gly Glu Gln Asp Leu Trp Met Lys Trp Val Thr Leu Gln Pro Ile Leu
340 345 350
Arg Arg Leu Tyr Gly Asn Ser Phe Leu Pro Tyr His Asp Tyr Gly Arg
355 360 365
Gly Gly Arg Gly Trp Arg Asp Leu Trp Gln Asp Cys Leu Ala Leu Met
370 375 380
Val Met Glu Pro Ala Glu Val Arg His Leu Leu Leu Asn Asn Tyr Ala
385 390 395 400
Gly Val Arg Met Asp Gly Ser Asn Ala Thr Ile Ile Gly Ala Gly Pro
405 410 415
Gly Glu Phe Val Ala Asp Arg Asn Asn Ile Pro Arg Val Trp Met Asp
420 425 430
His Gly Ala Trp Pro Leu Met Thr Thr Leu Leu Tyr Leu His Gln Ser
435 440 445
Gly Asp Leu Gly Leu Leu Phe Gln Pro Gln Ser Tyr Phe Arg Asp Val
450 455 460
Phe Val Lys Arg Cys Arg Glu Arg Asp Ala Ser Trp Thr Pro Glu Gln
465 470 475 480
Gly Asn Lys Leu Leu Thr Ala Asp Gly Gln Ile Tyr Glu Gly Thr Ile
485 490 495
Leu Glu His Ile Leu Leu Gln Asn Ile Val Pro Phe Phe Asn Val Gly
500 505 510
Glu His Gly Asn Ile Lys Leu Glu Gly Ala Asp Trp Asn Asp Gly Leu
515 520 525
Asp Leu Ala Pro Glu Arg Gly Glu Ser Val Ala Phe Thr Ala Phe Tyr
530 535 540
Ala Ser Asn Leu Met Glu Leu Ser Glu Leu Leu Leu Glu Leu Gln Lys
545 550 555 560
Arg Thr Gly Lys Asp Ser Leu Asp Ile Ala Glu Glu Met Ala Leu Leu
565 570 575
Leu Asp Thr Leu Gly Lys Pro Ile Ser Tyr Asp Ser Ile Gln Glu Lys
580 585 590
Arg Ser Leu Leu Asp Arg Tyr Tyr Asp Ala Val Thr Pro Arg Val Ser
595 600 605
Gly Lys Lys Leu Leu Leu Asp Ile Arg Lys Val Ala Glu Asp Leu Lys
610 615 620
Arg Lys Ala Asp Trp Ala Val Ala His Leu Arg Gly Ser Glu Trp Ile
625 630 635 640
Gln Ser Lys Glu Gly Tyr Ala Trp Phe Asn Gly Tyr Tyr Asn Asn Asp
645 650 655
Gly Glu Arg Val Glu Gly Asp His Pro Asp Gly Val Arg Met Thr Leu
660 665 670
Thr Gly Gln Val Phe Ala Ile Met Gly Gly Val Ala Thr Asp Glu Gln
675 680 685
Thr Glu Lys Ile Ser Gln Ala Val Asn Arg Tyr Leu Lys Asp Glu Arg
690 695 700
Ile Gly Tyr Arg Leu Asn Ser Arg Phe Gly Gly Ile Gln Gln Asn Leu
705 710 715 720
Gly Arg Ala Phe Gly Phe Ala Phe Gly His Lys Glu Asn Gly Ala Met
725 730 735
Phe Ser His Met Thr Val Met Tyr Ala Asn Ala Leu Tyr Lys Arg Gly
740 745 750
Phe Val Gln Glu Gly Phe Glu Val Leu Asp Ser Ile Tyr Arg Leu Ser
755 760 765
Ala Asp Phe Glu Asn Ser Arg Ile Tyr Pro Gly Val Pro Glu Tyr Ile
770 775 780
Asn Glu Arg Gly Arg Gly Met Tyr Thr Tyr Leu Thr Gly Ser Ala Ser
785 790 795 800
Trp Leu Leu Leu Thr Gln Leu Thr Glu Val Tyr Gly Val Lys Gly Arg
805 810 815
Phe Gly Asp Leu Arg Leu Glu Pro Lys Leu Val Gln Ala Gln Phe Asp
820 825 830
Gly Ser Gly Glu Ala Ala Val Glu Thr Leu Phe Ala Gly Arg Met Leu
835 840 845
Arg Val Val Tyr Arg Asn Pro Gln Ala Ala Glu His Gly Gln Tyr Arg
850 855 860
Val Asp Ser Val Ser Leu Asn Gly Gln Ser Val Asp Phe Gln Asn Asp
865 870 875 880
Gly Ala Gly Cys Leu Ile Gly Arg Ser Leu Ile Glu Ala Leu Pro Ala
885 890 895
Asp Gly Val His Glu Leu Ile Val Thr Leu Gly Arg Asn Ile Ser
900 905 910
<210> 6
<211> 911
<212> PRT
<213> 人工序列()
<400> 6
Met Gly Gln Lys Gly Trp Lys Phe Gln Gly Ile Val Gly Glu Phe Arg
1 5 10 15
Leu Glu Gln Pro Glu His Asn Ser Tyr Gln Tyr Phe Pro Leu Val Asn
20 25 30
Glu Ala Gly Met Met Ser Ala Val Thr Pro Asn Leu His Gly Glu Ile
35 40 45
Thr Ser Gly His Asn Thr Phe Leu Met Glu Pro Val Ser Ala Glu Ser
50 55 60
Leu His Asn Ser Lys Ala Ser Arg Asn Phe Trp Val Phe Ile Glu Gly
65 70 75 80
Tyr Gly Ala Trp Ser Val Ser Gly Asn Ser Ala Arg Gln Asn Ala Ala
85 90 95
Arg Phe Thr Gly Glu Glu Glu Arg Ser Ala Val Glu Ala Gly Phe Leu
100 105 110
Trp His Ala Val Thr Arg Glu Asn Glu Lys Ala Gly Leu Lys Ala Arg
115 120 125
Thr Val Ser Phe Val Pro Val Thr Asp Asp Lys Ile Glu Leu Met Arg
130 135 140
Val Thr Leu Thr Asn Thr Gly Asn Ala Pro Leu Lys Leu Thr Pro Thr
145 150 155 160
Ala Ala Ile Pro Leu Tyr Gly Arg Ser Ala Asn Asp Leu Arg Asp His
165 170 175
Arg His Val Thr Ser Leu Leu His Arg Ile Phe Thr Ser Glu Tyr Gly
180 185 190
Ile Glu Val Gln Pro Ala Leu Ser Phe Asp Glu Arg Gly His Arg Val
195 200 205
Asn Lys Val Thr Tyr Gly Val Phe Gly Ala Glu Ala Gly Gly Thr Ala
210 215 220
Pro Ala Gly Phe Phe Pro Val Thr Glu Asp Phe Ile Gly Glu Gly Gly
225 230 235 240
Ala Leu Asp Trp Pro Glu Ala Val Val Ala Asn Arg Glu Pro Asp Ala
245 250 255
Gln Ala Gly Thr Ala Val Glu Gly Tyr Glu Ala Val Gly Ala Leu Arg
260 265 270
Phe Ala Pro Val Glu Leu Ala Pro Gly Lys Ser Val Ser Tyr Val Val
275 280 285
Ala Met Val Ile Ser Gly Asp Arg Ile Asp Val Gly Arg Tyr Ala Ala
290 295 300
Asp Tyr Leu Ala Ala Gly Arg Phe Asp Ala Leu Leu Glu Gln Asn Arg
305 310 315 320
Ala Tyr Trp Arg Asp Lys Leu Asp Thr Val Arg Phe Ser Ser Gly Asp
325 330 335
Gly Glu Gln Asp Leu Trp Met Lys Trp Val Thr Leu Gln Pro Ile Leu
340 345 350
Arg Arg Leu Tyr Gly Asn Ser Phe Leu Pro Tyr His Asp Tyr Gly Arg
355 360 365
Gly Gly Arg Gly Trp Arg Asp Leu Trp Gln Asp Cys Leu Ala Leu Met
370 375 380
Val Met Glu Pro Ala Glu Val Arg His Leu Leu Leu Asn Asn Tyr Ala
385 390 395 400
Gly Val Arg Met Asp Gly Ser Asn Ala Thr Ile Ile Gly Ala Gly Pro
405 410 415
Gly Glu Phe Val Ala Asp Arg Asn Asn Ile Pro Arg Val Trp Met Asp
420 425 430
His Gly Ala Trp Pro Leu Met Thr Thr Leu Leu Tyr Leu His Gln Ser
435 440 445
Gly Asp Leu Gly Leu Leu Phe Gln Pro Gln Ser Tyr Phe Arg Asp Val
450 455 460
Phe Val Lys Arg Cys Arg Glu Arg Asp Ala Ser Trp Thr Pro Glu Gln
465 470 475 480
Gly Asn Lys Leu Leu Thr Ala Asp Gly Gln Ile Tyr Glu Gly Thr Ile
485 490 495
Leu Glu His Ile Leu Leu Gln Asn Ile Val Pro Phe Phe Asn Val Gly
500 505 510
Glu His Gly Asn Ile Lys Leu Glu Gly Ala Asp Trp Asn Asp Gly Leu
515 520 525
Asp Leu Ala Pro Glu Arg Gly Glu Ser Val Ala Phe Thr Ala Phe Tyr
530 535 540
Ala Ser Asn Leu Met Glu Leu Ser Glu Leu Leu Leu Glu Leu Gln Lys
545 550 555 560
Arg Thr Gly Lys Asp Ser Leu Asp Ile Ala Glu Glu Met Ala Leu Leu
565 570 575
Leu Asp Thr Leu Gly Lys Pro Ile Ser Tyr Asp Ser Ile Gln Glu Lys
580 585 590
Arg Ser Leu Leu Asp Arg Tyr Tyr Asp Ala Val Thr Pro Arg Val Ser
595 600 605
Gly Lys Lys Leu Leu Leu Asp Ile Arg Lys Val Ala Glu Asp Leu Lys
610 615 620
Arg Lys Ala Asp Trp Ala Val Ala His Leu Arg Gly Ser Glu Trp Ile
625 630 635 640
Gln Ser Lys Glu Gly Tyr Ala Trp Phe Asn Gly Tyr Tyr Asn Asn Asp
645 650 655
Gly Glu Arg Val Glu Gly Asp His Pro Asp Gly Val Arg Met Thr Leu
660 665 670
Thr Gly Gln Val Phe Ala Ile Met Gly Gly Val Ala Thr Asp Glu Gln
675 680 685
Thr Glu Lys Ile Ser Gln Ala Val Asn Arg Tyr Leu Lys Asp Glu Arg
690 695 700
Ile Gly Tyr Arg Leu Asn Ser Arg Phe Gly Gly Ile Gln Gln Asn Leu
705 710 715 720
Gly Arg Ala Phe Gly Phe Ala Phe Gly His Lys Glu Asn Gly Ala Met
725 730 735
Phe Ser His Met Thr Val Met Tyr Ala Asn Ala Leu Tyr Lys Arg Gly
740 745 750
Phe Val Gln Glu Gly Phe Glu Val Leu Asp Ser Ile Tyr Arg Leu Ser
755 760 765
Ala Asp Phe Glu Asn Ser Arg Ile Tyr Pro Gly Val Pro Glu Tyr Ile
770 775 780
Asn Glu Arg Gly Arg Gly Met Tyr Thr Tyr Leu Thr Gly Ser Ala Ser
785 790 795 800
Trp Leu Leu Leu Thr Gln Leu Thr Glu Val Tyr Gly Val Lys Gly Arg
805 810 815
Phe Gly Asp Leu Arg Leu Glu Pro Lys Leu Val Gln Ala Gln Phe Asp
820 825 830
Gly Ser Gly Glu Ala Ala Val Glu Thr Leu Phe Ala Gly Arg Met Leu
835 840 845
Arg Val Val Tyr Arg Asn Pro Gln Ala Ala Glu His Gly Gln Tyr Arg
850 855 860
Val Asp Ser Val Ser Leu Asn Gly Gln Ser Val Asp Phe Gln Asn Asp
865 870 875 880
Gly Ala Gly Cys Leu Ile Gly Arg Ser Leu Ile Glu Ala Leu Pro Ala
885 890 895
Asp Gly Val His Glu Leu Ile Val Thr Leu Gly Arg Asn Ile Ser
900 905 910
<210> 7
<211> 911
<212> PRT
<213> 人工序列()
<400> 7
Met Gly Gln Lys Gly Trp Lys Phe Gln Gly Ile Val Gly Glu Phe Arg
1 5 10 15
Leu Glu Gln Pro Glu His Asn Ser Tyr Gln Tyr Phe Pro Leu Val Asn
20 25 30
Glu Ala Gly Met Met Ser Ala Val Thr Pro Asn Leu His Gly Glu Ile
35 40 45
Thr Ser Gly His Asn Thr Phe Leu Met Glu Pro Val Ser Ala Glu Ser
50 55 60
Leu His Asn Ser Lys Ala Ser Arg Asn Phe Trp Val Phe Ile Glu Gly
65 70 75 80
Tyr Gly Ala Trp Ser Val Ser Gly Asn Ser Ala Arg Gln Asn Ala Ala
85 90 95
Arg Phe Thr Gly Glu Glu Glu Arg Ser Ala Val Glu Ala Gly Phe Leu
100 105 110
Trp His Ala Val Thr Arg Glu Asn Glu Lys Ala Gly Leu Lys Ala Arg
115 120 125
Thr Val Ser Phe Val Pro Val Thr Asp Asp Lys Ile Glu Leu Met Arg
130 135 140
Val Thr Leu Thr Asn Thr Gly Asn Ala Pro Leu Lys Leu Thr Pro Thr
145 150 155 160
Ala Ala Ile Pro Leu Tyr Gly Arg Ser Ala Asn Asp Leu Arg Asp His
165 170 175
Arg His Val Thr Ser Leu Leu His Arg Thr Phe Thr Ser Glu Tyr Gly
180 185 190
Ile Glu Val Gln Pro Ala Leu Ser Phe Asp Glu Arg Gly His Arg Val
195 200 205
Asn Lys Val Thr Tyr Gly Val Phe Gly Ala Glu Ala Gly Gly Thr Ala
210 215 220
Pro Ala Gly Phe Phe Pro Val Thr Glu Asp Phe Ile Gly Glu Gly Gly
225 230 235 240
Ala Leu Asp Trp Pro Glu Ala Val Val Ala Asn Arg Glu Pro Asp Ala
245 250 255
Gln Ala Gly Thr Ala Val Glu Gly Tyr Glu Ala Val Gly Ala Leu Arg
260 265 270
Phe Ala Pro Val Glu Leu Ala Pro Gly Lys Ser Val Ser Tyr Val Val
275 280 285
Ala Met Val Ile Ser Gly Asp Arg Ile Asp Val Gly Arg Tyr Ala Ala
290 295 300
Asp Tyr Leu Ala Ala Gly Arg Phe Asp Ala Leu Leu Glu Gln Asn Arg
305 310 315 320
Ala Tyr Trp Arg Asp Lys Leu Asp Thr Val Arg Phe Ser Ser Gly Asp
325 330 335
Gly Glu Gln Asp Leu Trp Met Lys Trp Val Thr Leu Gln Pro Ile Leu
340 345 350
Arg Arg Leu Tyr Gly Asn Ser Phe Leu Pro Tyr His Asp Tyr Gly Arg
355 360 365
Gly Gly Arg Gly Trp Arg Asp Leu Trp Gln Asp Cys Leu Ala Leu Met
370 375 380
Val Met Glu Pro Ala Glu Val Arg His Leu Leu Leu Asn Asn Tyr Ala
385 390 395 400
Gly Val Arg Met Asp Gly Ser Asn Ala Thr Ile Ile Gly Ala Gly Pro
405 410 415
Gly Glu Phe Val Ala Asp Arg Asn Asn Ile Pro Arg Val Trp Met Asp
420 425 430
His Gly Ala Trp Pro Leu Met Thr Thr Leu Leu Tyr Leu His Gln Ser
435 440 445
Gly Asp Leu Gly Leu Leu Phe Gln Pro Gln Ser Tyr Phe Arg Asp Val
450 455 460
Phe Val Lys Arg Cys Arg Glu Arg Asp Ala Ser Trp Thr Pro Glu Gln
465 470 475 480
Gly Asn Lys Leu Leu Thr Ala Asp Gly Gln Ile Tyr Glu Gly Thr Ile
485 490 495
Leu Glu His Ile Leu Leu Gln Asn Ile Val Pro Phe Phe Asn Val Gly
500 505 510
Glu His Gly Asn Ile Lys Leu Glu Gly Ala Asp Trp Asn Asp Gly Leu
515 520 525
Asp Leu Ala Pro Glu Arg Gly Glu Ser Val Ala Phe Thr Ala Phe Tyr
530 535 540
Ala Ser Asn Leu Met Glu Leu Ser Glu Leu Leu Leu Glu Leu Gln Lys
545 550 555 560
Arg Thr Gly Lys Asp Ser Leu Asp Ile Ala Glu Glu Met Ala Leu Leu
565 570 575
Leu Asp Thr Leu Gly Lys Pro Ile Ser Tyr Asp Ser Ile Gln Glu Lys
580 585 590
Arg Ser Leu Leu Asp Arg Tyr Tyr Asp Ala Val Thr Pro Arg Val Ser
595 600 605
Gly Lys Lys Leu Leu Leu Asp Ile Arg Lys Val Ala Glu Asp Leu Lys
610 615 620
Arg Lys Ala Asp Trp Ala Val Ala His Leu Arg Gly Ser Glu Trp Ile
625 630 635 640
Gln Ser Lys Glu Gly Tyr Ala Trp Phe Asn Gly Tyr Tyr Asn Asn Asp
645 650 655
Gly Glu Arg Val Glu Gly Asp His Pro Asp Gly Val Arg Met Thr Leu
660 665 670
Thr Gly Gln Val Phe Ala Ile Met Gly Gly Val Ala Thr Asp Glu Gln
675 680 685
Thr Glu Lys Ile Ser Gln Ala Val Asn Arg Tyr Leu Lys Asp Glu Arg
690 695 700
Ile Gly Tyr Arg Leu Asn Ser Arg Phe Gly Gly Ile Gln Gln Asn Leu
705 710 715 720
Gly Arg Ala Phe Gly Phe Ala Phe Gly His Lys Glu Asn Gly Ala Met
725 730 735
Phe Ser His Met Thr Val Met Tyr Ala Asn Ala Leu Tyr Lys Arg Gly
740 745 750
Phe Val Gln Glu Gly Phe Glu Val Leu Asp Ser Ile Tyr Arg Leu Ser
755 760 765
Ala Asp Phe Glu Asn Ser Arg Ile Tyr Pro Gly Val Pro Glu Tyr Ile
770 775 780
Asn Glu Arg Gly Arg Gly Met Tyr Thr Tyr Leu Thr Gly Ser Ala Ser
785 790 795 800
Trp Leu Leu Leu Thr Gln Leu Thr Glu Val Tyr Gly Val Lys Gly Arg
805 810 815
Phe Gly Asp Leu Arg Leu Glu Pro Lys Leu Val Gln Ala Gln Phe Asp
820 825 830
Gly Ser Gly Glu Ala Ala Val Glu Thr Leu Phe Ala Gly Arg Met Leu
835 840 845
Arg Val Val Tyr Arg Asn Pro Gln Ala Ala Glu His Gly Gln Tyr Arg
850 855 860
Val Asp Ser Val Ser Leu Asn Gly Gln Ser Val Asp Phe Gln Asn Asp
865 870 875 880
Gly Ala Gly Cys Leu Ile Gly Arg Ser Leu Ile Glu Ala Leu Pro Ala
885 890 895
Asp Gly Val His Glu Leu Ile Val Thr Leu Gly Arg Asn Ile Ser
900 905 910

Claims (10)

1.一种昆布二糖磷酸化酶(LBP)突变体，其特征在于，是将氨基酸序列如SEQ ID NO.1所示的昆布二糖磷酸化酶进行迭代饱和突变和/或随机突变，获得热稳定性提高的昆布二糖磷酸化酶突变体。突变体氨基酸序列如SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、SEQ ID NO.6和SEQ ID NO.7所示。所述突变体的氨基酸序列包含突变位点E11I、Q12V、D171N、C877F、D452G、L26Q、I186T其中的一个或多个或任何位点的组合。
2.编码权利要求1所述昆布二糖磷酸化酶突变体的基因。
3.携带权利要求2所述基因的载体或细胞。
4.一种获得权利要求1所述昆布二糖磷酸化酶突变体的方法，其特征在于，以含有野生型或突变体LBP的编码基因的pET20b-lbp为模版，将如序列SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示的基因进行定点饱和突变，将如序列SEQ ID NO.4、SEQ ID NO.5和SEQ IDNO.6所示的基因进行随机突变，将携带编码突变体的基因的载体转入大肠杆菌BL21(DE3)，培养重组菌得到昆布二糖磷酸化酶突变体。
5.权利要求1-4所述昆布二糖磷酸化酶初始来源于假单胞菌(Paenibacillussp.YM1)。
6.权利要求1-5所述昆布二糖磷酸化酶经多轮定点饱和突变及随机突变，将LBP纯酶在60℃的半衰期从10min提高到9.76h，在65℃的半衰期从2min提高到3.11h。
7.一种高温下催化淀粉制备昆布二糖的方法，其特征在于，以大肠杆菌BL21(DE3)为宿主，分别重组表达海栖热袍菌(Thermotoga maritima)来源的淀粉磷酸化酶；淡紫色拟青霉(Paecilomyces lilacinus)来源的葡萄糖苷酶和硫化叶菌(Sulfolobus tokodaii)来源的异淀粉酶，纯化获得重组酶，构建体外多酶体系，在高温下催化淀粉制备昆布二糖。
8.权利要求7所述体外多酶体系还包括二价锌离子、磷酸钾以及乙酸钠和HEPES缓冲液。
9.权利要求7-8所述体外多酶体系中的酶组分可以是任何来源的具有相同功能的酶资源。
10.应用权利要求1-6所述昆布二糖磷酸化酶突变体应用于体外多酶催化淀粉制备昆布二糖的方法。其特征在于，所述体外多酶体系以耐高温的昆布二糖磷酸化酶突变体为催化剂，结合其他耐高温酶，在高温下催化淀粉生产昆布二糖的应用。
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CN113621666A (zh) * 2021-08-24 2021-11-09 山东大学 一种生物合成昆布二糖的方法

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JPH10304882A (ja) * 1996-11-08 1998-11-17 Hayashibara Biochem Lab Inc コージビオースホスホリラーゼとその製造方法並びに用途
CN109706200A (zh) * 2017-10-26 2019-05-03 中国科学院天津工业生物技术研究所 一种制备昆布二糖的方法
US20190322990A1 (en) * 2018-04-23 2019-10-24 Danisco Us Inc Synthesis of glucan comprising beta-1,3 glycosidic linkages with phosphorylase enzymes

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Publication number Priority date Publication date Assignee Title
JPH10304882A (ja) * 1996-11-08 1998-11-17 Hayashibara Biochem Lab Inc コージビオースホスホリラーゼとその製造方法並びに用途
CN109706200A (zh) * 2017-10-26 2019-05-03 中国科学院天津工业生物技术研究所 一种制备昆布二糖的方法
US20190322990A1 (en) * 2018-04-23 2019-10-24 Danisco Us Inc Synthesis of glucan comprising beta-1,3 glycosidic linkages with phosphorylase enzymes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113621666A (zh) * 2021-08-24 2021-11-09 山东大学 一种生物合成昆布二糖的方法

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