CN114836405B - 一种热稳定性提高的琼胶酶突变体及其应用 - Google Patents
一种热稳定性提高的琼胶酶突变体及其应用 Download PDFInfo
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Abstract
本发明公开了一种热稳定性提高的琼胶酶突变体及其应用，属于基因工程技术和酶工程领域。本发明提供了一种琼胶酶突变体，所述突变体为，将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第86位、第373位、第374位、第496位、第507位、或第747位的氨基酸进行突变得到的，本发明的琼胶酶突变体提高了琼胶酶的热稳定性以及水解活力。突变体酶相比较于野生型酶显示出优越的耐热性，能够在较高温度下进行工业化生产，从而提高琼胶原料的利用率及琼胶寡糖的产率，具有良好的工业应用前景。
Description
技术领域
本发明涉及一种热稳定性提高的琼胶酶突变体及其应用，属于基因工程技术和酶工程领域。
背景技术
红藻在所有的商业藻类植物中产量最高，在海洋中存量巨大，琼脂是其细胞壁的主要成分。琼脂作为凝胶和增稠剂，是一种公认的安全的食品添加剂，也可应用于医药工业，但附加值低。琼脂糖的主要结构是由重复β-D-半乳糖和3,6-无水-α-L-半乳糖(AHG)二糖单元连接组成。据报道，从琼脂中提取的寡糖具有多种生物活性，包括高效的抗氧化、抗炎、益生元效应、抑菌和美白等功效，极大的提高了琼脂的附加值。
降解琼脂制备琼胶寡糖(琼寡糖(AOS)和新琼寡糖(NAOS))的方法包括物理、化学和酶解三种方法，各有其独特的优缺点。物理方法包括热、微波和超声波处理，但由于琼脂降解效率低、可控性差，利用物理降解的研究较少。化学降解，如利用稀释酸、温和酸，可以裂解α(1,3)键，得到具有AHG残基的AOS，具有成本低、得率高、快速、简便等优点，然而化学法存在降解产物复杂且环境污染严重等问题。近年来酶解法凭借着产物均一、工艺简单、能耗低、无污染等特点成为研究热点。因此，酶解产琼胶寡糖被认为是可持续和最有商业化生产琼胶寡糖前景的方法。但当体系温度低于40℃(溶胶-凝胶转变温度)时，琼脂形成凝胶，极大的降低了酶解效率。然而大多数琼胶酶的最适温度为30℃-40℃之间，热稳定性较差，当体系温度高于50℃时其活性受到较大损害，琼胶酶的实际工业应用有较大的限制。
现有技术当中，有通过基因工程的手段提高琼胶酶的热稳定性：
例如，Su,Xu,Yan,Xie,和Lin等人将来源于Vibrio sp.ZC-1的琼胶酶AgaA在T5010(半衰期为10分钟的温度)中的稳定性提高了1.5℃(公开于SU B-M,XU X-Q,YAN R-X,etal.Mutagenesis on the surface of a beta-agarase from Vibrio sp.ZC-1increasedits thermo-stability[J].Enzyme and Microbial Technology,2019,127:22-31.论文中)；
Jang，Lee和Kim等人通过对来源于Zobellia galactanivorans的琼胶酶AgaB进行随机诱变，得到了Tm提高了5.2℃的突变体E99K-T307I(公开于JANG M-K,LEE S W,LEE D-G,et al.Enhancement of the thermostability of a recombinant beta-agarase,AgaB,from Zobellia galactanivorans by random mutagenesis[J].BiotechnologyLetters,2010,32(7):943-9.论文中)；
Shi,Lu,Ma,Fu,和Yu等人对来源于Pseudoalteromonas sp.CY24的琼胶酶AgaB进行突变，得到突变体S2，其Tm比野生型高4.6℃(公开于CHAO S,XINZHI L,CUIPING M,etal.Enhancing the thermostability of a novel beta-agarase AgaB throughdirected evolution[J].Applied Biochemistry and Biotechnology,2008,151(1):51-9.论文中)；
Zhang等人改造来自Agarivorans gilvus WH0801的琼胶酶AgWH50C，获得的突变体K621F比野生型的熔解温度(Tm)高0.88℃，最佳温度从30℃提高到38℃(公开于ZHANG P,ZHANG J,ZHANG L,et al.Structure-based design of agarase AgWH50C fromAgarivorans gilvus WH0801 to enhance thermostability[J].Applied Microbiologyand Biotechnology,2019,103(3):1289-98.论文中)。
但是，现有的琼胶酶的热稳定性还是不能够满足工业生产的需求，因此，开发高耐热性琼胶酶将能显著提高琼胶酶的生产应用价值，更有助于工业化生产琼胶寡糖，是实现海洋资源高值化的关键技术之一。
发明内容
发明人前期通过对不同来源、水解琼脂糖得到不同产物的琼胶酶进行研究，发现来源于Saccharophagus degradans 2-40的琼胶酶Aga50D(NCBI登录号：ABD81904)与琼脂糖结合的晶体结构(PDB:4BQ2)结构完整，单一产新琼二糖。
本发明在对野生型来源于Saccharophagus degradans 2-40的琼胶酶及本发明的突变体进行表征后，发现单一突变体极大的提高了琼胶酶的耐热性，其表观熔融温度(Tm)最高提高了23℃，是现有报道中热稳定性提高最多的琼胶酶突变体，相较于之前报道来自于Agarivorans gilvus WH0801菌株的AgWH50C琼胶酶突变体K621F，其耐热性提高的效果更好，并且酶活也有一定的提升，这都有利于提高琼脂糖原料利用率，增加新琼二糖的产量，赋予琼胶酶更大的工业应用价值。
本发明提供了热稳定性提高的琼胶酶突变体，以及能够表达所述琼胶酶突变体的大肠杆菌工程菌。
本发明提供了一种琼胶酶突变体，所述突变体为，将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第86位、第373位、第374位、第496位、第507位、或第747位的氨基酸进行突变得到的。
在本发明的一种实施方式中，所述亲本酶琼胶酶来源于Saccharophagusdegradans 2-40。
在本发明的一种实施方式中，编码所述亲本酶琼胶酶的核苷酸序列如SEQ IDNO.2所示。
在本发明的一种实施方式中，所述突变体为，将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第86位的丙氨酸突变为天冬氨酸；命名为：A86D。
在本发明的一种实施方式中，所述突变体为，将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第373位的丝氨酸突变为丙氨酸；命名为：S373A。
在本发明的一种实施方式中，所述突变体为，将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第374位的苯丙氨酸突变为色氨酸；命名为：F374W。
在本发明的一种实施方式中，所述突变体为，将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第496位的丙氨酸突变为脯氨酸；命名为：A496P。
在本发明的一种实施方式中，所述突变体为，将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第507位的缬氨酸突变为赖氨酸；命名为：V507K。
在本发明的一种实施方式中，所述突变体为，将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第747位的丝氨酸突变为谷氨酰胺；命名为：S747Q。
本发明还提供了编码上述突变体的基因。
本发明提供了携带上述基因的重组载体。
在本发明的一种实施方式中，所述重组载体以pPIc9k、pHIL-S1、pPIcza、pET28a中的任意一种为表达载体。
本发明提供了表达上述突变体，或含有上述基因，或含有上述重组载体的重组细胞。
在本发明的一种实施方式中，所述重组细胞以原核细胞或真核细胞为表达宿主。
在本发明的一种实施方式中，所述重组细胞以大肠杆菌E.coli BL(21)DE3、毕赤酵母GS115、毕赤酵母KM71、毕赤酵母KM7中的任意一种为表达宿主。
本发明提供了一种制备上述突变体的方法，所述方法步骤如下：
(1)根据确定的突变位点，设计定点突变的突变引物，以携带琼胶酶编码基因的载体为模板进行定点突变；构建含编码突变体的基因的载体；
(2)将含编码突变体的基因的载体转化进微生物细胞；
(3)挑选阳性克隆进行发酵培养，离心收集细胞，细胞破壁上清即为琼胶酶突变体的粗酶液。
本发明还提供了一种提高琼胶酶的热稳定性的方法，所述方法为，
将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第86位的丙氨酸突变为天冬氨酸；或将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第373位的丝氨酸突变为丙氨酸；或将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第374位的苯丙氨酸突变为色氨酸；或将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第496位的丙氨酸突变为脯氨酸；或将氨基酸序列如SEQ IDNO.1所示的琼胶酶的第507位的缬氨酸突变为赖氨酸；或将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第747位的丝氨酸突变为谷氨酰胺。
本发明还提供了一种水解琼胶的方法，所述方法为，将上述突变体，或上述重组细胞添加至含有琼胶的反应体系中，进行分解反应。
本发明还提供了一种制备新琼二糖的方法，所述方法为，利用上述突变体，或上述重组细胞反应制备得到。
本发明还提供了上述突变体，或上述基因，或上述重组载体，或上述重组细胞在制备含有水解琼胶的产品或制备含有新琼二糖的产品中的应用。
本发明还提供了上述突变体，或上述基因，或上述重组载体，或上述重组细胞在工业、医药、生化、食品领域中的应用。
有益效果
(1)本发明公开了热稳定性提高的琼胶酶突变体，属于基因工程技术和酶工程领域。采用本发明的方法获得了热稳定性显著提高的突变体，是现有报道中热稳定性提高最多的琼胶酶突变体，且本发明的突变体在热稳定提高同时并提高了酶活。
(2)本发明提供的突变体A86D相对野生酶的酶活为132.55％，酶活略微提高；突变体A86D在50℃下孵育15min，60min及6h后仍然保留89.91％，63.68％及42.15％的原始酶活，而野生酶在50℃下孵育15min，60min后只保留17.14％，10.79％的原始酶活，孵育6h后完全丧失酶活。
(3)本发明提供的突变体A86D在60℃下孵育180min后，仍保留了72.18％的原始酶活，且在孵育6h后，仍然保留了30.83％的原始酶活，而野生酶在60℃下孵育30min后就完全失活。
(4)本发明提供的突变体A86D相比较于野生酶显示出优越的耐热性，能够在较高温度下进行工业化生产，从而提高琼胶原料的利用率及琼胶寡糖的产率，具有良好的工业应用前景。
附图说明
图1：质粒构建示意图。
图2：野生酶Aga50D及其突变体的酶活表征。
图3：野生酶Aga50D及其突变体的Tm表征。
图4：野生酶Aga50D及突变体酶纯化后的SDS-PAGE图；其中，图中M：蛋白质分子量标准，1：纯化后野生型Aga50D，2-15：纯化后的突变体，依次为PD1、A86D、V172N、K259P、S286D、S373A、F374W、N400R、A496P、V507K、P677H、S705M、Y706F、S747Q。
图5：野生酶Aga50D及其突变体在50℃下的热稳定性表征。
图6：野生酶Aga50D及其突变体在60℃下的热稳定性表征。
图7：野生酶Aga50D及其突变体最适反应温度表征。
图8：野生酶Aga50D及其突变体最适反应pH表征。
图9：野生酶Aga50D及其突变体动力学参数表征。
图10：野生酶Aga50D及其突变体产物分析。
具体实施方式
下述实施例中所涉及的pET28a-Aga50D及其突变体由苏州金唯智生物科技有限公司合成。下述实施例中所涉及的主要试剂：BCA浓度测定试剂盒购自碧云天生物技术有限公司，基因合成由金唯智生物科技有限公司完成，其他常用试剂为国产分析纯。
下述实施例中所涉及的培养基如下：
LB液体培养基：称取1g蛋白胨，0.5g酵母提取物，1g NaCl。使用去离子水溶解后定容至100mL，高压灭菌锅，121℃灭菌20min。
LB固体培养基：在LB液体培养基基础上添加1.8％的琼脂粉。
LB液体抗性培养基：在LB液体培养基基础上添加卡那霉素，终浓度为50μg·mL-1。
LB固体培养基：在LB固体培养基基础上添加卡那霉素，终浓度为50μg·mL-1。
下述实施例中所涉及的酶的纯化方法如下：
(1)平衡：用50mM Tris-HCl，500mM NaCl，pH7.5的缓冲液平衡镍柱；
(2)上样：预处理的样品以1mL/min的流速上样；
(3)冲洗：用50mM Tris-HCl，500mM NaCl，pH7.5，20mM咪唑的缓冲液冲洗杂蛋白；
(4)洗脱：用50mM Tris-HCl，500mM NaCl，300mM咪唑，pH7.5的缓冲液洗脱并收集目的蛋白，即得到纯化的酶。
下述实施例中所涉及的检测方法如下：
突变体酶活测定：
采用3,5-二硝基水杨酸法(DNS)测定酶活。琼胶酶在一定的条件下催化水解琼脂糖生成还原糖，3,5-二硝基水杨酸与还原糖在热条件下还原生成棕红色氨基络合物，在一定范围内颜色深浅与还原糖量成正比，可在520nm波长下测定，计算酶活。酶活单位定义：在30℃，pH7.0条件下，每分钟催化产生相当于1μmol D-半乳糖所需的酶量定义为一个活力单位。
酶活测定步骤：
(1)预热：取2mL 1mg·mL-1的琼脂糖溶液(pH7.0)于比色管中；
(2)反应：加入0.1mL的酶液，震荡混匀，反应20min，加入1.5mL DNS终止反应，沸水浴5min，立即冷却。
(3)测量：在520nm条件下测吸光值并计算酶活。
Tm值测定
采用示差扫描荧光定量法(DSF)。天然的蛋白质处于折叠状态，疏水部分隐藏在内部，随着温度身高，蛋白结构逐渐解体，暴露疏水部分，此时，对疏水部分亲和的染料结合到蛋白质上，呈现体系荧光信号强度上升；当温度到达某一个点时，展开的蛋白质链聚集，荧光染料不能结合便重新回到环境中或在高温下荧光猝灭，导致荧光信号下降，通过追踪检测荧光信号的变化，便可测定出蛋白质的Tm。将SYPRO Orange染料稀释100倍，取5μL染料与20μL蛋白质混合置于96孔薄壁PCR板中。并在ABI StepOnePlus实时荧光定量PCR仪系统中从25℃加热至99℃，监测荧光变化。
在50℃、60℃下的酶热稳定性测定步骤
(1)预热：取2mL琼脂糖溶液(1mg·mL-1，pH7.0)于比色管中，置于50℃或60℃的水浴分别保温30、60、90、120、180min和6h；
(2)反应：加入0.1mL的酶液，震荡混匀，反应20min，加入1.5mL DNS终止反应，沸水浴5min，立即冷却。
(3)测量：在520nm条件下测吸光值并计算酶活。
新琼二糖(NA2)的检测方法
将酶解样品及新琼寡糖标准品(购自青岛博智汇力生物科技有限公司，纯度大于98％，稀释至1mg·mL-1)，过0.22μm滤膜后进行离子色谱(ICS-5000，美国赛默飞世尔科技公司)检测。检测条件为：色谱柱：Dionex CarboPac PA-200阴离子交换柱，包括分析柱(4mm×250mm)和保护柱(4mm×50mm)；流动相：100mmol·L-1NaOH溶液和150mmol·L-1的NaAc溶液，流速为0.5mL·min-1；安培检测器采用四电位脉冲安培法检测；柱温30℃，进样体积2μL。
下面结合附图和实施例，对本发明进行具体描述。
实施例1：突变体的构建
具体步骤如下：
(1)含有野生型琼胶酶Aga50D的重组载体pET28a-Aga50D的构建
根据NCBI公开的Saccharophagus degradans 2-40中，编码所述亲本酶琼胶酶的核苷酸序列如SEQ ID NO.2所示，送至金唯智生物科技有限公司进行基因合成并重组质粒。载体为pET28a，重组质粒命名为pET28a-Aga50D。
(2)含有突变体的重组载体的构建(如图1所示)
设计定点突变引物，以步骤(1)获得的重组质粒pET28a-Aga50D为模板进行定点突变，分别获得含有突变体A86D、V172N、K259P、S286D、S373A、F374W、N400R、A496P、V507K、P677H、S705M、Y706F和S747Q的重组质粒pET28a-A86D、pET28a-V172N、pET28a-K259P、pET28a-S286D、pET28a-S373A、pET28a-F374W、pET28a-N400R、pET28a-A496P、pET28a-V507K、pET28a-P677H、pET28a-S705M、pET28a-Y706F和pET28a-S747Q。
所涉及的引物序列如下：
引入A86D突变的定点突变引物为：
A86D-F:5’-GGTTAAAGTTGGATATGCAGTC-3’；
A86D-R:5’-CTTGGACTGCATATCCAAC-3’。
引入V172N突变的定点突变引物为：
V172N-F:5’-CCCGATAGTGGAGACAACAACG-3’；
V172N-R:5’-GGCGAGGTTTAAATCGTTGTTGTC-3’。
引入K259P突变的定点突变引物为：
K259P-F:5’-CCAAAGTTGATTACCCGGGTAAAATC-3’；
K259P-R:5’-CTAAACTATGGATTTTACCCGGGTAATC-3’。
引入S286D突变的定点突变引物为：
S286D-F:5’-CAAGCCAATGCCTGATCGCTC-3’；
S286D-R:5’-CGCCAAACTTAGAGCGATCAGG-3’。
引入S373A突变的定点突变引物为：
S373A-F:5’-GCAGTGAGCGAAAAAGCTTTTG-3’；
S373A-R:5’-GCGCGTAGCAAAAGCTTTTTC-3’。
引入F374W突变的定点突变引物为：
F374W-F:5’-GAGCGAAAAATCATGGGCTAC-3’；
F374W-R:5’-GCGCGTAGCCCATGATT-3’。
引入N400R突变的定点突变引物为：
N400R-F:5’-CCCTCTCGCACGCCATTATAAC-3’；
N400R-R:5’-CGACGGTAGTTATAATGGCGTGC-3’。
引入A496P突变的定点突变引物为：
A496P-F:5’-GGATTTTTGGGGCCCAATGCC-3’；
A496P-R:5’-CGAATACATCTGGCATTGGGCC-3’。
引入V507K突变的定点突变引物为：
V507K-F:5’-CGACCCAGAATTTAAAAAGCGC-3’；
V507K-R:5’-CGTTTCCATAGCGCGCTTTTTAA-3’。
引入P677H突变的定点突变引物为：
P677H-F:5’-CTACAAAGAGGGCTTGCACAAGC-3’；
P677H-R:5’-GCCCACTTCTGCTTGTGCAAG-3’。
引入S705M突变的定点突变引物为：
S705M-F:5’-GGTGCTATGGATCACGGTATGTATC-3’；
S705M-R:5’-CCGGGGTGATACATACCGTG-3’。
引入Y706F突变的定点突变引物为：
Y706F-F:5’-CTATGGATCACGGTTCGTTTCACC-3’；
Y706F-R:5’-GTGAATTAAACCGGGGTGAAACGAAC-3’。
引入S747Q突变的定点突变引物为：
S747Q-F:5’-CTGGTTCCAGTATATGGATCAACCATTAAC-3’；
S747Q-R:5’-CTCTGCCCGTTAATGGTTGATCC-3’。
PCR反应体系如下：
表1突变PCR反应体系
PCR反应条件为：95℃预变性3min，95℃变性30s，56℃退火30s，72℃延申3min48s，共30个循环。
将目的片段胶回收后，转化至E.coli BL21(DE3)，将转化子涂布于卡那霉素(50μg/mL)LB平板，37℃静置培养过夜，待长出菌落后，挑单菌落至含有卡那霉素(50μg/mL)的液体LB培养基中，37℃，200rpm培养过夜，菌液送金唯智生物科技有限公司测定。分别得到含有正确突变体的突变体工程菌：
E.coli BL21(DE3)/pET28a-A86D、E.coli BL21(DE3)/pET28a-V172N、E.coliBL21(DE3)/pET28a-K259P、E.coli BL21(DE3)/pET28a-S286D、E.coli BL21(DE3)/pET28a-S373A、E.coli BL21(DE3)/pET28a-F374W、E.coli BL21(DE3)/pET28a-N400R、E.coli BL21(DE3)/pET28a-A496P、E.coli BL21(DE3)/-V507K、E.coli BL21(DE3)/pET28a-P677H、E.coli BL21(DE3)/pET28a-S705M、E.coli BL21(DE3)/pET28a-Y706F和E.coli BL21(DE3)/pET28a-S747Q；
按照上述方法，制备得到含有原始酶的工程菌E.coli BL21(DE3)/pET28a-Aga50D。
PD1组合突变体在pET28a-Aga50D的基础上组合叠加A86D、V172N、K259P、V274K、S286D、A351P、A355P、S373A、F374W、A386V、N400R、A496P、V507K、S619E、H635K、P677H、H703R、S705M、Y706F和S747Q 20个突变位点，引物序列同上，方法同上，得到工程菌E.coliBL21(DE3)/pET28a-A86D/V172N/K259P/V274K/S286D/A351P/A355P/S373A/F374W/A386V/N400R/A496P/V507K/S619E/H635K/P677H/H703R/S705M/Y706F/S747Q；将其命名为E.coliBL21(DE3)/PD1。
实施例2：酶的纯化与酶学性质测定
(1)摇瓶发酵生产酶
分别将实施例1得到的基因工程菌在含卡那霉素(50μg/mL)LB平板上划线，37℃培养12h后，挑取单菌落接种于卡那霉素(50μg/mL)的LB液体培养基中摇瓶发酵，37℃，转速200rpm培养12h，获得种子液。
吸取1mL种子液转移到100mL卡那霉素(50μg/mL)LB液体培养基中摇瓶发酵，37℃培养至OD600达到0.6～0.8时，加入终浓度0.5mM的IPTG，降低温度到16℃，诱导产酶，培养12h后离心收集菌体。
(2)酶的纯化
离心收集的菌体加入适量(2～3倍体积)裂解缓冲液(50mmol/L Tris-HCl，100mmol/L NaCl，pH＝7.5)重悬菌体，在冰上超声破碎15～20min，30％功率，超声2s，停3s，超声破碎后，4℃，8000rpm离心10min收集上清液，0.22μm滤膜过滤后。进行Ni2+柱亲和层析纯化获得突变体酶。经过酶活测定，SDS-PAGE电泳检测，结果如图4所示。
分别制备得到含有野生型的琼胶酶及突变体酶A86D、V172N、K259P、S286D、S373A、F374W、N400R、A496P、V507K、P677H、S705M、Y706F、S747Q和PD1的纯酶液。
(3)酶活测定
测定上述野生型琼胶酶及突变体酶A86D、V172N、K259P、S286D、S373A、F374W、N400R、A496P、V507K、P677H、S705M、Y706F、S747Q和PD1的酶活，将野生型酶活设定为100％，检测突变体酶的相对酶活，结果如表2及图2所示。
表2野生型及其突变体相对酶活
结果显示：经差示扫描荧光测定上述野生型琼胶酶及突变体酶PD1、A86D、V172N、K259P、S286D、S373A、F374W、N400R、A496P、V507K、P677H、S705M、Y706F和S747Q酶的Tm值，其中效果显著的有PD1(ΔTm＝6.828℃)、S747Q(ΔTm＝5.677℃)、S705M(ΔTm＝2.163℃)、P677H(ΔTm＝2.069℃)。而A86D的热熔融温度(Tm＝65.176℃，ΔTm＝23.202℃)，远高于野生型(Tm＝41.974℃)，耐热性极大提高，如表2及图3所示。
虽然S373A、F374W、A496P的酶活都得到了提高，但是其热熔融温度的增长均不显著。
结合相对酶活，选择综合表现优越的突变体PD1、A86D、S747Q继进行后续实验。
(4)在50℃下的酶热稳定性
经过50℃的酶热稳定性实验，结果如图5所示。
结果显示，突变体A86D耐热性显著增强：
在50℃下，实验结果表明，野生酶Aga50D在孵育30min后，仅保留原始酶活的13.65％，在孵育60min后，仅保留10.79％，到180min后，几乎丧失所有酶活；
突变体S747Q在50℃下孵育15min后，保留72.17％的原始酶活，孵育30min后，保留23.71％的原始酶活，孵育至180min时完全失活。
突变体A86D在50℃下孵育30min后，保留81.39％的原始酶活，孵育60min后，保留63.68％的原始酶活；孵育6h后仍然保留了42.15％的原始酶活。
突变体PD1在50℃下孵育30min后，保留95.51％的原始酶活，孵育60min后，保留83.37％的原始酶活，孵育6h后仍然保留了55.28％的原始酶活。
因此，追加60℃的耐热性实验，进一步探索PD1、A86D优越的耐热性能。
(5)在60℃下的酶热稳定性
经过60℃的酶热稳定性实验，结果如图6所示。
结果显示，突变体A86D耐热性显著增强：
在60℃下，实验结果表明，野生酶Aga50D耐热性差，在60℃下孵育30min已完全失活。
突变体S747Q在60℃下孵育30min后，保留12.26％的原始酶活，孵育60min后仅保留0.94％的原始酶活，在孵育90min后完全失活。
突变体PD1在60℃下孵育30min后，保留8.50％的原始酶活，孵育60min后仅保留4.50％的原始酶活，在孵育90min后完全失活。
突变体A86D在60℃下孵育30min后，保留98.75％的原始酶活，孵育60min后，保留82.46％的原始酶活，孵育180min后，保留72.18％的原始酶活，在孵育6h后，仍然保留了30.83％的原始酶活，显示出极其优越的耐热性，具备极佳的工业应用潜力。
实施例3：酶学性质测定
(1)最适反应温度
为了进一步探究突变体酶的特性，对PD1、A86D、S747Q进行进一步的酶学性质研究。用pH＝7的Tris-HCl缓冲液配制0.1％琼脂糖底物，加入终浓度为0.2mg·mL-1的酶液于不同温度下反应20min测定酶活，由图7可知野生酶与突变体的最适反应温度均为30℃。
(2)最适反应pH
用50mM柠檬酸缓冲液配制pH 5～6范围的1mg·mL-1琼脂糖底物，用50mM Tris-HCl缓冲液配制pH 7～8范围的0.1％琼脂糖底物，用甘氨酸氢氧化钠缓冲液配制pH 9～10范围的0.1％琼脂糖底物，加入终浓度为0.2mg·mL-1的酶液于不同温度下反应20min测定酶活，由图8可知野生酶与突变体的最适反应pH均为7，性质相似。
(3)动力学参数
用pH＝7的Tris-HCl缓冲液配制浓度分别为1mg·mL-1、2mg·mL-1、3mg·mL-1、4mg·mL-1、5mg·mL-1、8mg·mL-1和10mg·mL-1的琼脂糖底物，加入终浓度为0.2mg·mL-1的酶液在最适反应条件下反应，然后测定还原糖的生成量，按Lineweaver－Burk法作图(图9)。图中4条曲线的线型拟和方程分别为y＝589.92x+15.37(WT)、y＝621.46x+31.568(PD1)、y＝506.45x+22.215(A86D)及y＝436.23x+58.247(S747Q)。由此可推算出野生型与突变体的各项动力学参数(表3)。
在最适反应条件下，野生型与突变体的动力学参数列于表3中。
表3野生型Aga50D和突变体的动力学参数。
从表中数据可见，突变体的Km值较WT的降低，说明突变体与底物琼脂糖的结合能力明显上升，这一结论与前面相对酶活的结论一致。突变体的Kcat/Km都比WT的大，说明突变体的催化能力均有提高。
实施例4：酶的应用
用超纯水配制1mg·mL-1的琼脂糖底物溶液后，分别加入实施例2的步骤(2)制备得到的突变体及野生型的纯酶液：0.2mg·mL-1的酶液(9.528U·mg-1)，在50℃的水浴摇床中反应，转速为：200rpm，反应时间为：1h～3h，反应结束后采用沸水浴使反应终止。
每隔1h、2h、3h取出部分反应液，并离心除去反应液中未反应的多糖后，取上清液，即得酶解产物样品：新琼二糖(NA2)。分别检测上述得到的产物新琼二糖(NA2)的含量。
结果显示(如图10所示)，在50℃的情况下，野生型酶水解琼脂糖产新琼二糖的得率低，猜测是由于不耐热所至，且热失活快；而突变体酶PD1、A86D、S747Q具有一定的耐热性，高温条件下，仍能发挥催化作用，且由于在50℃的情况下，琼脂糖底物仍呈溶液状态，促进酶与底物的结合，进一步提高新琼二糖的得率。
其中，水解反应3h，采用野生型酶水解琼脂糖后得到的产物新琼二糖(NA2)的产量为278.678mg/mL；
采用突变体酶PD1水解琼脂糖后得到的产物新琼二糖(NA2)的产量为2036.281mg/mL；
采用突变体酶A86D水解琼脂糖后得到的产物新琼二糖(NA2)的产量为1670.887mg/mL；
采用突变体酶S747Q水解琼脂糖后得到的产物新琼二糖(NA2)的产量为1965.719mg/mL。
在50℃条件下，突变体酶由于具备优越的耐热性，从而展现出卓越的水解效率，极大的提高了新琼二糖得率，具有良好的工业应用价值。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种热稳定性提高的琼胶酶突变体及其应用
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<400> 2
ggatccatgt tattcgattt tgaaaacgat caagtccctt caaatattca ttttttaaat 60
gcgcgtgcaa gtatagaaac ctataccggt ataaatggcg agccgagtaa agggttaaag 120
ttggcgatgc agtccaagca gcacagttat actggccttg ccattgtgcc agagcagcct 180
tgggattgga gcgagtttac ctctgctagc ttgtatttcg atatagtcag tgttggcgat 240
cattccacac aattttattt agatgttacc gaccaaaatg gcgccgtgtt tacccgcagt 300
attgatattc cagtgggtaa aatgcaatcg tactacgcca agttaagcgg tcacgattta 360
gaagtgcccg atagtggaga cgttaacgat ttaaacctcg cctctggctt gcgttctaac 420
ccgcctacat ggacatctga cgataggcag tttgtttgga tgtggggagt gaaaaattta 480
gatttgtcgg gcattgctaa aatatcgcta agtgtgcaaa gcgcaatgca cgataaaaca 540
gttattatcg ataatattcg tattcaaccc aacccgccgc aagatgaaaa cttccttgtc 600
ggtttggtag acgagtttgg ccaaaacgcc aaagttgatt acaagggtaa aatccatagt 660
ttagaagaat tgcatgcagc gcgcgatgtg gaactggccg agcttgatgg caagccaatg 720
cctagtcgct ctaagtttgg cggttggttg gccggcccca agctaaaagc tacagggtac 780
tttcgcacag aaaaaattaa cggtaaatgg atgctagtag acccagaagg gtacccttac 840
tttgctacgg gtttagacat tattcgccta tctaattcat ctaccatgac tggttacgat 900
tacgatcaag ctactgttgc tcagcgctct gccgacgatg taacacctga agactcaaaa 960
ggtttaatgg cagtgagcga aaaatcattt gctacgcgcc acctagcatc gccaacacga 1020
gcggcaatgt ttaactggtt gccagattac gatcaccctc tcgcaaatca ttataactac 1080
cgtcgctctg cgcattccgg cccactgaaa cgcggtgaag cctacagctt ctacagtgcc 1140
aaccttgagc gtaaatacgg tgaaacttac cccggttctt acttggataa gtggcgcgaa 1200
gtaacggtag acagaatgct aaactggggc tttacctcgc taggcaactg gactgaccca 1260
gcatattacg acaacaatcg cataccgttt ttcgcgaatg gttgggtaat aggggatttt 1320
aaaaccgtat ctagcggtgc ggatttttgg ggcgcaatgc cagatgtatt cgacccagaa 1380
tttaaagtgc gcgctatgga aacggcacgc gtggtttcag aagaaattaa aaatagccct 1440
tggtgcgtag gggtatttat cgataacgaa aaaagcttcg gtcgccccga ttccgataag 1500
gcgcaatacg gtattcccat tcataccctc ggtcgcccaa gcgaaggtgt gcctactagg 1560
caggcgttta gtaagctgct taaagccaaa tacaaaacta tagccgcgtt aaacaatgcc 1620
tgggggttaa agcttagttc ttgggctgag tttgatttgg gcgtagatgt aaaagcgctg 1680
ccggtaaccg atactctgcg cgcagattac tcaatgttac tttcggccta tgcggaccaa 1740
tattttaagg tggtacacgg cgcggttgaa cattacatgc cgaaccactt gtatttaggc 1800
gcacgctttc ctgattgggg aatgccaatg gaggtagtga aagctgccgc aaaatacgcc 1860
gatgtggtta gctataattc ctacaaagag ggcttgccta agcagaagtg ggctttttta 1920
gcagagctag ataagccgag tataatcggt gagtttcaca taggtgctat ggatcacggt 1980
tcgtatcacc ccggtttaat tcacgctgcg tcgcaggccg atagaggtga aatgtacaaa 2040
gattatatgc aatcggtaat tgataacccc tacttcgtag gcgcgcactg gttccagtat 2100
atggattcgc cattaacggg cagagcttat gatggtgaaa actacaatgt gggttttgtg 2160
gatgttaccg acacgccgta ccaagaaatg gtggatgcag caaaagaagt aaatgcgaaa 2220
atatacaccg aaaggctagg cagcaaataa gaattc 2256
Claims (10)
1.一种琼胶酶突变体，其特征在于，所述突变体为将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第86位的丙氨酸突变为天冬氨酸得到的；或将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第747位的丝氨酸突变为谷氨酰胺。
2.编码权利要求1所述突变体的基因。
3.携带权利要求2所述基因的重组载体。
4.表达权利要求1所述突变体，或含有权利要求2所述基因，或含有权利要求3所述的重组载体的宿主细胞。
5.根据权利要求4所述的宿主细胞，其特征在于，所述宿主细胞以原核细胞或真核细胞为表达宿主。
6.一种提高琼胶酶的热稳定性的方法，其特征在于，所述方法为，
将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第86位的丙氨酸突变为天冬氨酸；或将氨基酸序列如SEQ ID NO.1所示的琼胶酶的第747位的丝氨酸突变为谷氨酰胺。
7.一种水解琼胶的方法，其特征在于，将权利要求1所述的突变体，或权利要求4或5所述的宿主细胞添加至含有琼胶的反应体系中，进行水解反应。
8.一种制备新琼二糖的方法，其特征在于，利用权利要求1所述的突变体，或权利要求4或5所述的宿主细胞反应制备得到。
9.权利要求1所述突变体，或权利要求2所述基因，或权利要求3所述重组载体，或权利要求4所述宿主细胞在制备含有水解琼胶的产品中的应用。
10.权利要求1所述突变体，或权利要求2所述基因，或权利要求3所述重组载体，或权利要求4所述宿主细胞在制备含有新琼二糖的产品中的应用。
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| PCT/CN2022/126972 WO2023193421A1 (zh) | 2022-04-08 | 2022-10-24 | 一种热稳定性提高的琼胶酶突变体及其应用 |
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