CN114836402B - 一种gh16家族热稳定性增强的葡聚糖酶突变体及其构建方法与应用 - Google Patents
一种gh16家族热稳定性增强的葡聚糖酶突变体及其构建方法与应用 Download PDFInfo
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- CN114836402B CN114836402B CN202210446191.9A CN202210446191A CN114836402B CN 114836402 B CN114836402 B CN 114836402B CN 202210446191 A CN202210446191 A CN 202210446191A CN 114836402 B CN114836402 B CN 114836402B
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Abstract
一种GH16家族热稳定性增强的葡聚糖酶突变体及其构建方法与应用。包括以葡聚糖酶AtGlu16D为母本将Asp219分别突变为Arg219和Ser219后得到突变体AtGlu16D_D219R和AtGlu16D_D219S,突变体AtGlu16D_D219R能够耐受61℃的温度处理,比野生型提高12.5℃。突变体AtGlu16D_D219S能够耐受54.5℃的温度处理,比野生型提高6℃,且均能在偏酸性条件下具有80%以上的酶活力,对GH16家族葡聚糖酶,以及β‑sandwich jelly‑roll结构酶的热稳定性改良,具有重要的指导意义。
Description
技术领域
本发明涉及基因工程、蛋白质工程领域,具体涉及一种GH16家族热稳定性增强的葡聚糖酶突变体及其构建方法与应用。
背景技术
葡聚糖根据糖苷键的类型分为α-葡聚糖和β-葡聚糖。相比α-葡聚糖,β-葡聚糖具有优良的生化活性和特点。β-葡聚糖又可以根据葡萄糖单体之间的糖苷键的类型细分为谷类和非谷类。水稻、小麦、玉米等农作物中富含β-葡聚糖,并且葡聚糖是我们日常生活中重要的膳食纤维,它通过影响代谢活动来降低胆固醇和血糖。在燕麦和大麦中有一种天然存在的碳水化合物,可溶性 β-1,3-葡聚糖,其具有免疫激活剂的功能,从而可以促进哺乳动物的免疫系统的反应。然而,β-葡聚糖通会增加食糜粘度和影响饲料的消化率,因此β-葡聚糖酶被广泛应用于工业生产中以提高经济效益。
β-葡聚糖酶在我们日常生活中应用广泛,它具有降低人体的胆固醇和血糖的功效,从而促进人体的代谢活动来维持优良的健康水平。由于β-葡聚糖酶可以提高饲料的消化和转化效率,且可以消除饲料中的抗营养因子,其还可被用作补充剂并在饲料行业被使用广泛。β-葡聚糖酶可降低食糜粘度的功效使其在食品制造业也被广泛使用。在目前的工业应用中,影响β-葡聚糖酶普及率的关键因素在于其热环境下的耐受性。目前,基因挖掘和理性设计等方法被应用于筛选获得性质优良的β-葡聚糖酶,且对β-葡聚糖酶的酶学性质的改造也在不断深入。然而,影响β-葡聚糖酶热稳定性的氨基酸位点等关键因素仍不清晰,因此,探究影响β-葡聚糖酶热稳定性的关键因素对拓展其在工业生产中的应用价值具有重要意义。
发明内容
本发明提供了一种GH16家族热稳定性增强的葡聚糖酶突变体及其构建方法与应用,该突变体为定点突变后筛选得到,突变体AtGlu16D_D219R能够耐受61 ℃的温度处理,比野生型(48.5℃)提高12.5 ℃。突变体AtGlu16D_D219S能够耐受54.5 ℃的温度处理,比野生型(48.5 ℃)提高6℃,突变体AtGlu16D_D219R和AtGlu16D_D219S在偏酸性条件下具有80%以上的酶活力。
一种GH16家族热稳定性增强的葡聚糖酶突变体,以AtGlu16D为模板,对其D219位点进行定点突变,获得突变株,所述AtGlu16D的核苷酸序列如SEQ ID NO.5所示,
SEQ ID NO.5:
gcttatactttgcaagatgattactctggtgattctttttttgatggttttactttcttcaccggtgctgatccaactaacggttttgttgattacgttgatgaggctactgctcagtctaacggttacatctctacttctggtgatgctcctgtttacatgggtgttgatcatactaacattgctggttcttctggtagacagtctgttagaatttcttctgatgctacttataaccatggtttgtttattttggacttgggtcatatgccaggtggagtttgtggtacttggcctgctttttggttggttggtgctgattggccaaacaacggtgagattgatattattgaaggtgttaatcagcagtctggtaatgatatgactttgcatacttctgatggttgttctgtttcttcttctgctgattttactggttctatgaccacatctaattgttacgtttccgctgctggtcagtctaacaacgctggttgtggtattactgatcctgatggtacttcttacggtactgcttttaatgctaacaacggtggtgtttttgctactgaatggacttctgatgctatttctatttggtttttcgaaagaggttctattccagatgatattgattcaggtaaccctgatccagattcctggggttctccagttgctagatttcaaggtgattgtgatattgatgctcattttgataacttgcagattattttcgacactactttttgtggtgattgggctggtaatgtttggggttctggttcttgtgcttctgttgcttcttcttgttctgattacgttgctaataacccagaagcttttgctgatgcttattggattattaattccttgaaggtttaccaggatgatggtgatgatgatgatactgttgctggtttggttgattatggtgatgatgaagatgatgaagacgatcaggatgatgataacgatgatgatgactctgatgatgatgatgattctgatgataacgacgatgattctgactctgatgacgattctgatgacgaagatgatgatgatgatgatgatgatgatgattccgacgatgatgatgatgatgaatctagaccatggagaggaggtggtggaggtagaaacagacatggttcaaagttgtacgcttctacttctacttcttcttttccatctccatcttttatgagaaaggctaagtgggaaagaagaaacccaatggctggtatgattttttaa。
作为改进的时,当D219位点突变成R219时,获得葡聚糖酶突变体AtGlu16D_D219R,编码葡聚糖酶突变体AtGlu16D_D219R的核苷酸序列如SEQ ID NO.1;当D219位点突变成S219获得葡聚糖酶突变体AtGlu16D_D219S,编码葡聚糖酶突变体AtGlu16D_D219S的核苷酸序列如SEQ ID NO.2;
SEQ ID NO.1:
gcttatactttgcaagatgattactctggtgattctttttttgatggttttactttcttcaccggtgctgatccaactaacggttttgttgattacgttgatgaggctactgctcagtctaacggttacatctctacttctggtgatgctcctgtttacatgggtgttgatcatactaacattgctggttcttctggtagacagtctgttagaatttcttctgatgctacttataaccatggtttgtttattttggacttgggtcatatgccaggtggagtttgtggtacttggcctgctttttggttggttggtgctgattggccaaacaacggtgagattgatattattgaaggtgttaatcagcagtctggtaatgatatgactttgcatacttctgatggttgttctgtttcttcttctgctgattttactggttctatgaccacatctaattgttacgtttccgctgctggtcagtctaacaacgctggttgtggtattactgatcctgatggtacttcttacggtactgcttttaatgctaacaacggtggtgtttttgctactgaatggacttctgatgctatttctatttggtttttcgaaagaggttctattccagatgatattgattcaggtaaccctgatccacgttcctggggttctccagttgctagatttcaaggtgattgtgatattgatgctcattttgataacttgcagattattttcgacactactttttgtggtgattgggctggtaatgtttggggttctggttcttgtgcttctgttgcttcttcttgttctgattacgttgctaataacccagaagcttttgctgatgcttattggattattaattccttgaaggtttaccaggatgatggtgatgatgatgatactgttgctggtttggttgattatggtgatgatgaagatgatgaagacgatcaggatgatgataacgatgatgatgactctgatgatgatgatgattctgatgataacgacgatgattctgactctgatgacgattctgatgacgaagatgatgatgatgatgatgatgatgatgattccgacgatgatgatgatgatgaatctagaccatggagaggaggtggtggaggtagaaacagacatggttcaaagttgtacgcttctacttctacttcttcttttccatctccatcttttatgagaaaggctaagtgggaaagaagaaacccaatggctggtatgattttttaa;
SEQ ID NO.2:
gcttatactttgcaagatgattactctggtgattctttttttgatggttttactttcttcaccggtgctgatccaactaacggttttgttgattacgttgatgaggctactgctcagtctaacggttacatctctacttctggtgatgctcctgtttacatgggtgttgatcatactaacattgctggttcttctggtagacagtctgttagaatttcttctgatgctacttataaccatggtttgtttattttggacttgggtcatatgccaggtggagtttgtggtacttggcctgctttttggttggttggtgctgattggccaaacaacggtgagattgatattattgaaggtgttaatcagcagtctggtaatgatatgactttgcatacttctgatggttgttctgtttcttcttctgctgattttactggttctatgaccacatctaattgttacgtttccgctgctggtcagtctaacaacgctggttgtggtattactgatcctgatggtacttcttacggtactgcttttaatgctaacaacggtggtgtttttgctactgaatggacttctgatgctatttctatttggtttttcgaaagaggttctattccagatgatattgattcaggtaaccctgatccatcttcctggggttctccagttgctagatttcaaggtgattgtgatattgatgctcattttgataacttgcagattattttcgacactactttttgtggtgattgggctggtaatgtttggggttctggttcttgtgcttctgttgcttcttcttgttctgattacgttgctaataacccagaagcttttgctgatgcttattggattattaattccttgaaggtttaccaggatgatggtgatgatgatgatactgttgctggtttggttgattatggtgatgatgaagatgatgaagacgatcaggatgatgataacgatgatgatgactctgatgatgatgatgattctgatgataacgacgatgattctgactctgatgacgattctgatgacgaagatgatgatgatgatgatgatgatgatgattccgacgatgatgatgatgatgaatctagaccatggagaggaggtggtggaggtagaaacagacatggttcaaagttgtacgcttctacttctacttcttcttttccatctccatcttttatgagaaaggctaagtgggaaagaagaaacccaatggctggtatgattttttaa。
进一步改进的是,所述葡聚糖酶突变体AtGlu16D_D219R的氨基酸序列如SEQ IDNO.3;葡聚糖酶突变体AtGlu16D_D219S的氨基酸序列如SEQ ID NO.4,
SEQ ID NO.3:
aytlqddysgdsffdgftfftgadptngfvdyvdeataqsngyistsgdapvymgvdhtniagssgrqsvrissdatynhglfildlghmpggvcgtwpafwlvgadwpnngeidiiegvnqqsgndmtlhtsdgcsvsssadftgsmttsncyvsaagqsnnagcgitdpdgtsygtafnannggvfatewtsdaisiwffergsipddidsgnpdpRswgspvarfqgdcdidahfdnlqiifdttfcgdwagnvwgsgscasvasscsdyvannpeafadaywiinslkvyqddgddddtvaglvdygddeddeddqdddnddddsdddddsddndddsdsdddsddeddddddddddsddddddesrpwrgggggrnrhgsklyaststssfpspsfmrkakwerrnpmagmif;
SEQ ID NO.4:
aytlqddysgdsffdgftfftgadptngfvdyvdeataqsngyistsgdapvymgvdhtniagssgrqsvrissdatynhglfildlghmpggvcgtwpafwlvgadwpnngeidiiegvnqqsgndmtlhtsdgcsvsssadftgsmttsncyvsaagqsnnagcgitdpdgtsygtafnannggvfatewtsdaisiwffergsipddidsgnpdpSswgspvarfqgdcdidahfdnlqiifdttfcgdwagnvwgsgscasvasscsdyvannpeafadaywiinslkvyqddgddddtvaglvdygddeddeddqdddnddddsdddddsddndddsdsdddsddeddddddddddsddddddesrpwrgggggrnrhgsklyaststssfpspsfmrkakwerrnpmagmif。
一种重组载体,含有编码葡聚糖酶突变体AtGlu16D_D219R或 葡聚糖酶突变体AtGlu16D_D219S的质粒。
一种重组菌株,表达上述含有编码葡聚糖酶突变体AtGlu16D_D219R或 葡聚糖酶突变体AtGlu16D_D219S的重组载体。
上述重组菌株的构建方法,包括以下步骤:
步骤1,将葡聚糖酶AtGlu16D作为模板,通过定点突变的方法构建重组质粒;
步骤2,将葡聚糖酶突变体AtGlu16D_D219R或AtGlu16D_D219S的重组质粒转入DMT感受态,得重组载体;
步骤3,将突变重组载体进行线性化处理,通过电击法转入GS115毕赤酵母感受态,诱导表达后筛选有催化活力的突变菌株;
步骤4,培养重组菌株,大量表达葡聚糖酶突变体;
步骤5,回收并且纯化所表达的有催化活力的重组菌株。
上述葡聚糖酶突变体、葡聚糖酶突变体AtGlu16D_D219R或葡聚糖酶突变体AtGlu16D_D219S,重组载体,或重组载体在降解β-葡聚糖上的应用。
有益效果:
与现有技术相比,本发明一种GH16家族热稳定性增强的葡聚糖酶突变体及其构建方法与应用,在热稳定性方面,突变体AtGlu16D_D219R在50℃下的半衰期(t 1/2)为71 min,是野生酶AtGlu16D(4 min)的18倍;突变体AtGlu16D_D219R的T 50值为61℃,相较野生酶AtGlu16D(48.5℃)提高12.5℃;突变体AtGlu16D_D219S在50 ℃下的半衰期(t 1/2)为42min,是野生酶AtGlu16D(4 min)的10倍;突变体AtGlu16D_D219S的T 50值为54.5℃,相较野生酶AtGlu16D(48.5 ℃)提高6℃。突变体AtGlu16D_D219R和AtGlu16D_D219S的催化效率和比活力也得到提高。突变体AtGlu16D_D219R和AtGlu16D_D219S的最适pH和最适温度和野生型基本保持一致,符合食品以及饲料的工业应用要求。
附图说明
图1为葡聚糖酶AtGlu16D及其突变体的最适pH;
图2为葡聚糖酶AtGlu16D及其突变体的pH稳定性;
图3为葡聚糖酶AtGlu16D及其突变体的最适温度;
图4为50℃下耐热葡聚糖酶突变体与野生型的半衰期(t 1/2);
图5为葡聚糖酶AtGlu16D及其突变体的T 50。
具体实施方式
将结合附图和实施例详细描述本发明的内容。
1、菌株及载体: Pichia pastoris GS115作为宿主, pPIC9γ作为质粒载体,均为市售材料;
2、酶类及其它生化试剂:Taq酶和Pfu酶均购自全式金生物科技公司,大麦葡聚糖和昆布多糖均购自源叶公司;分析纯试剂均从国药集团购买;
3、培养基:
(1)LB培养基:0.5%酵母提取物,1%蛋白胨,1% NaCl, pH 7.0;
(2)YPD培养基:,2%蛋白胨,1%酵母提取物,2%葡萄糖;
(3)MD固体培养基:2%葡萄糖,1.5%琼脂糖,1.34% YNB,0.00004% Biotin;
(4)BMGY培养基:1%酵母提取物,2%蛋白胨,1%甘油(V/V),1.34% YNB,0.00004%Biotin;
(5)BMMY培养基:1% 酵母提取物,2%蛋白胨,0.5%甲醇(V/V),1.34% YNB,0.00004%Biotin。
实施例1 葡聚糖酶AtGlu16D及其突变体的构建
合成基因序列Atglu16d(NCBI参考序列:XP_035356354.1)并首次表达获得GH16家族葡聚糖酶AtGlu16D,以葡聚糖酶AtGlu16D为出发材料,采用定点突变的方法将Asp219位点分别突变为Arg219和Ser219,所用引物序列如表1所示,定点突变方法和克隆方法参考文献(You, et al., 2019)。
表1 引物合成清单
实施例2 葡聚糖酶AtGlu16D及其突变体的制备
将PCR获得的线性重组表达载体直接转化DMT感受态,菌落PCR验证,获得突变体AtGlu16D_D219R和AtGlu16D_D219S的核苷酸序列,将重组质粒线性化后转化毕赤酵母GS115,获得重组酵母菌株AtGlu16D_D219R和AtGlu16D_D219S。
用含有2 mL BMGY 培养基的10 mL 试管培养含有重组质粒的酵母菌株,将试管置于30℃摇床以220 rpm转速培养48 h。以3000G的转速离心培养液,离心5 min后弃去上清液。配置含有0.5%甲醇的2 mL BMMY 培养基,使用BMMY 培养基重悬沉淀。将菌体与培养基混合均匀,之后将试管放置于30℃的摇床以220 rpm的转速培养48 h。取上清离心后用于酶活性检测,筛选到具有催化活性的突变体AtGlu16D_D219R和AtGlu16D_D219S。
将重组酵母菌株AtGlu16D、AtGlu16D_D219R和AtGlu16D_D219S放大发酵体系,首先接种于YPD培养基中获得种子培养液。从种子培养液中取1 %接种于l L三角瓶中,其中含有200 mL BMGY 培养基。将其放置于30℃的摇床中, 以220 rpm的转速培养48 h;以3000G的转速离心培养液,离心5 min后弃去上清液。配置含有0.5%甲醇的200 mL BMMY 培养基,使用BMMY 培养基重悬沉淀。将菌体与培养基混合均匀,之后将试管放置于30℃的摇床以220 rpm的转速培养48 h。以开始培养菌体为准,每隔12 h向培养基中加一次1 mL的甲醇。每次加甲醇前,取一部分上清液离心后进行酶活性检测。纯化蛋白使用的是阴离子交换法。所表达的葡聚糖酶经过纯化之后,其蛋白质的含量达到总蛋白的90%以上。
实施例3 葡聚糖酶AtGlu16D及其突变体的酶学性质分析
一、葡聚糖酶的酶活力测定
以1%(w/v)的大麦葡聚糖作为底物测定葡聚糖酶AtGlu16D及其突变体的活力。方法根据Yang等人所报道的 (Yang 10.1021/jf800303b)。
以下为具体的方法:1 mL的反应体系中含有100μL酶液和900μL葡聚糖底物。在给定的pH和温度条件下,将葡聚糖酶与底物混合后水浴反应10 min。反应结束后加入1.5 mLDNS终止反应。在沸水中水浴5 min以促进其显色。试管冷却后在酶标仪中测定其540 nm吸光度时的OD值。上述条件下,每分钟生成1 μmol 葡萄糖当量的还原糖所需的酶量定义为葡聚糖酶活力单位(U)。
二、葡聚糖酶AtGlu16D及其突变体的性质测定
1、葡聚糖酶AtGlu16D及其突变体的最适pH测定如下:
将实施例2中纯化的AtGlu16D、AtGlu16D_D219R和AtGlu16D_D219S在不同的pH下进行酶促反应,测定完成后分析其最适pH。用0.1 mol/L柠檬酸-磷酸氢二钠配置成不同pH的缓冲液。用缓冲液稀释底物(大麦葡聚糖)后配置成可以使用的底物溶液。在最适温度下对葡聚糖酶AtGlu16D及其突变体进行酶活力测定。
结果(图1)表明,葡聚糖酶AtGlu16D及其突变体的最适pH值均为4.0。突变体AtGlu16D_D219R和AtGlu16D_D219S在pH 3.0-9.0范围具有较高的酶催化活性(图2)。
2、葡聚糖酶AtGlu16D及其突变体的最适温度测定如下:
将实施例2纯化的AtGlu16D、AtGlu16D_D219R和AtGlu16D_D219S在不同的温度下进行酶促反应,测定完成后分析其最适温度。突变体AtGlu16D_D219R和AtGlu16D_D219S酶促反应最适温度测定结果(图3)表明, 野生型葡聚糖酶AtGlu16D和突变体AtGlu16D_D219R、AtGlu16D_D219S的最适温度都是45℃。
3、葡聚糖酶AtGlu16D及其突变体的热稳定性测定如下:
将葡聚糖酶突变体AtGlu16D_D219R、AtGlu16D_D219S和野生型AtGlu16D在50 ℃处理一定时间后热稳定性逐渐降低,但突变体AtGlu16D_D219R和AtGlu16D_D219S的热稳定性均优于野生型。通过拟合曲线(图4)可以发现,突变体AtGlu16D_D219R的t 1/2值为71 min,较野生型酶t 1/2值(4 min)提高18倍;突变体AtGlu16D_D219S的t 1/2值为42 min,较野生型酶t 1/2值提高10倍。通过拟合曲线(图5)可以发现,突变体AtGlu16D_D219R的T 50值为61 ℃,较野生型酶T 50值(48.5℃)提高12.5℃;突变体AtGlu16D_D219S的T 50值为54.5℃,较野生型酶T 50值提高6℃。
4、葡聚糖酶AtGlu16D及其突变体的动力学测定如下:
在最适条件下,分别以大麦葡聚糖和昆布多糖为底物测定重组葡聚糖酶AtGlu16D_D219R、AtGlu16D_D219S和AtGlu16D的动力学参数和比活力,结果如表2所示。
表2 AtGlu16D及其突变体在最适条件下的动力学和比活
当以大麦葡聚糖为底物时,AtGlu16D_D219R的k cat/K m和specific activity 分别为928 mL/s·mg和1318 U/mg。AtGlu16D_D219S的k cat/K m和specific activity 分别为816mL/s·mg和1338 U/mg。野生型的k cat/K m和specific activity分别为696 mL/s·mg和1177U/mg。突变体AtGlu16D_D219R和AtGlu16D_D219S较野生型的催化效率和比活均有提高。
当以昆布多糖为底物时,AtGlu16D_D219R的k cat/K m和specific activity 分别为508 mL/s·mg和455 U/mg。AtGlu16D_D219S的k cat/K m和specific activity 分别为526mL/s·mg和381 U/mg。野生型的k cat/K m和specific activity分别为299 mL/s·mg和295U/mg。突变体AtGlu16D_D219R和AtGlu16D_D219S较野生型的催化效率和比活均有提高。
序列表
<110> 江苏科技大学
<120> 一种GH16家族热稳定性增强的葡聚糖酶突变体及其构建方法与应用
<160> 9
<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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gcttatactt tgcaagatga ttactctggt gattcttttt ttgatggttt tactttcttc 60
accggtgctg atccaactaa cggttttgtt gattacgttg atgaggctac tgctcagtct 120
aacggttaca tctctacttc tggtgatgct cctgtttaca tgggtgttga tcatactaac 180
attgctggtt cttctggtag acagtctgtt agaatttctt ctgatgctac ttataaccat 240
ggtttgttta ttttggactt gggtcatatg ccaggtggag tttgtggtac ttggcctgct 300
ttttggttgg ttggtgctga ttggccaaac aacggtgaga ttgatattat tgaaggtgtt 360
aatcagcagt ctggtaatga tatgactttg catacttctg atggttgttc tgtttcttct 420
tctgctgatt ttactggttc tatgaccaca tctaattgtt acgtttccgc tgctggtcag 480
tctaacaacg ctggttgtgg tattactgat cctgatggta cttcttacgg tactgctttt 540
aatgctaaca acggtggtgt ttttgctact gaatggactt ctgatgctat ttctatttgg 600
tttttcgaaa gaggttctat tccagatgat attgattcag gtaaccctga tccagattcc 660
tggggttctc cagttgctag atttcaaggt gattgtgata ttgatgctca ttttgataac 720
ttgcagatta ttttcgacac tactttttgt ggtgattggg ctggtaatgt ttggggttct 780
ggttcttgtg cttctgttgc ttcttcttgt tctgattacg ttgctaataa cccagaagct 840
tttgctgatg cttattggat tattaattcc ttgaaggttt accaggatga tggtgatgat 900
gatgatactg ttgctggttt ggttgattat ggtgatgatg aagatgatga agacgatcag 960
gatgatgata acgatgatga tgactctgat gatgatgatg attctgatga taacgacgat 1020
gattctgact ctgatgacga ttctgatgac gaagatgatg atgatgatga tgatgatgat 1080
gattccgacg atgatgatga tgatgaatct agaccatgga gaggaggtgg tggaggtaga 1140
aacagacatg gttcaaagtt gtacgcttct acttctactt cttcttttcc atctccatct 1200
tttatgagaa aggctaagtg ggaaagaaga aacccaatgg ctggtatgat tttttaa 1257
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<212> DNA
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gcttatactt tgcaagatga ttactctggt gattcttttt ttgatggttt tactttcttc 60
accggtgctg atccaactaa cggttttgtt gattacgttg atgaggctac tgctcagtct 120
aacggttaca tctctacttc tggtgatgct cctgtttaca tgggtgttga tcatactaac 180
attgctggtt cttctggtag acagtctgtt agaatttctt ctgatgctac ttataaccat 240
ggtttgttta ttttggactt gggtcatatg ccaggtggag tttgtggtac ttggcctgct 300
ttttggttgg ttggtgctga ttggccaaac aacggtgaga ttgatattat tgaaggtgtt 360
aatcagcagt ctggtaatga tatgactttg catacttctg atggttgttc tgtttcttct 420
tctgctgatt ttactggttc tatgaccaca tctaattgtt acgtttccgc tgctggtcag 480
tctaacaacg ctggttgtgg tattactgat cctgatggta cttcttacgg tactgctttt 540
aatgctaaca acggtggtgt ttttgctact gaatggactt ctgatgctat ttctatttgg 600
tttttcgaaa gaggttctat tccagatgat attgattcag gtaaccctga tccacgttcc 660
tggggttctc cagttgctag atttcaaggt gattgtgata ttgatgctca ttttgataac 720
ttgcagatta ttttcgacac tactttttgt ggtgattggg ctggtaatgt ttggggttct 780
ggttcttgtg cttctgttgc ttcttcttgt tctgattacg ttgctaataa cccagaagct 840
tttgctgatg cttattggat tattaattcc ttgaaggttt accaggatga tggtgatgat 900
gatgatactg ttgctggttt ggttgattat ggtgatgatg aagatgatga agacgatcag 960
gatgatgata acgatgatga tgactctgat gatgatgatg attctgatga taacgacgat 1020
gattctgact ctgatgacga ttctgatgac gaagatgatg atgatgatga tgatgatgat 1080
gattccgacg atgatgatga tgatgaatct agaccatgga gaggaggtgg tggaggtaga 1140
aacagacatg gttcaaagtt gtacgcttct acttctactt cttcttttcc atctccatct 1200
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<210> 3
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gcttatactt tgcaagatga ttactctggt gattcttttt ttgatggttt tactttcttc 60
accggtgctg atccaactaa cggttttgtt gattacgttg atgaggctac tgctcagtct 120
aacggttaca tctctacttc tggtgatgct cctgtttaca tgggtgttga tcatactaac 180
attgctggtt cttctggtag acagtctgtt agaatttctt ctgatgctac ttataaccat 240
ggtttgttta ttttggactt gggtcatatg ccaggtggag tttgtggtac ttggcctgct 300
ttttggttgg ttggtgctga ttggccaaac aacggtgaga ttgatattat tgaaggtgtt 360
aatcagcagt ctggtaatga tatgactttg catacttctg atggttgttc tgtttcttct 420
tctgctgatt ttactggttc tatgaccaca tctaattgtt acgtttccgc tgctggtcag 480
tctaacaacg ctggttgtgg tattactgat cctgatggta cttcttacgg tactgctttt 540
aatgctaaca acggtggtgt ttttgctact gaatggactt ctgatgctat ttctatttgg 600
tttttcgaaa gaggttctat tccagatgat attgattcag gtaaccctga tccatcttcc 660
tggggttctc cagttgctag atttcaaggt gattgtgata ttgatgctca ttttgataac 720
ttgcagatta ttttcgacac tactttttgt ggtgattggg ctggtaatgt ttggggttct 780
ggttcttgtg cttctgttgc ttcttcttgt tctgattacg ttgctaataa cccagaagct 840
tttgctgatg cttattggat tattaattcc ttgaaggttt accaggatga tggtgatgat 900
gatgatactg ttgctggttt ggttgattat ggtgatgatg aagatgatga agacgatcag 960
gatgatgata acgatgatga tgactctgat gatgatgatg attctgatga taacgacgat 1020
gattctgact ctgatgacga ttctgatgac gaagatgatg atgatgatga tgatgatgat 1080
gattccgacg atgatgatga tgatgaatct agaccatgga gaggaggtgg tggaggtaga 1140
aacagacatg gttcaaagtt gtacgcttct acttctactt cttcttttcc atctccatct 1200
tttatgagaa aggctaagtg ggaaagaaga aacccaatgg ctggtatgat tttttaa 1257
<210> 4
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aaccctgatc caagatcctg gggttctcca gt 32
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tcttggatca gggttacctg aatcaatatc atctggaa 38
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aaccctgatc catcttcctg gggttctcca gt 32
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<213> 人工序列(Artificial Sequence)
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agatggatca gggttacctg aatcaatatc atctggaa 38
<210> 8
<211> 418
<212> PRT
<213> 氨基酸序列(Aminoacid Sequence)
<400> 8
Ala Tyr Thr Leu Gln Asp Asp Tyr Ser Gly Asp Ser Phe Phe Asp Gly
1 5 10 15
Phe Thr Phe Phe Thr Gly Ala Asp Pro Thr Asn Gly Phe Val Asp Tyr
20 25 30
Val Asp Glu Ala Thr Ala Gln Ser Asn Gly Tyr Ile Ser Thr Ser Gly
35 40 45
Asp Ala Pro Val Tyr Met Gly Val Asp His Thr Asn Ile Ala Gly Ser
50 55 60
Ser Gly Arg Gln Ser Val Arg Ile Ser Ser Asp Ala Thr Tyr Asn His
65 70 75 80
Gly Leu Phe Ile Leu Asp Leu Gly His Met Pro Gly Gly Val Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Leu Val Gly Ala Asp Trp Pro Asn Asn Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Ser Gly Asn Asp Met
115 120 125
Thr Leu His Thr Ser Asp Gly Cys Ser Val Ser Ser Ser Ala Asp Phe
130 135 140
Thr Gly Ser Met Thr Thr Ser Asn Cys Tyr Val Ser Ala Ala Gly Gln
145 150 155 160
Ser Asn Asn Ala Gly Cys Gly Ile Thr Asp Pro Asp Gly Thr Ser Tyr
165 170 175
Gly Thr Ala Phe Asn Ala Asn Asn Gly Gly Val Phe Ala Thr Glu Trp
180 185 190
Thr Ser Asp Ala Ile Ser Ile Trp Phe Phe Glu Arg Gly Ser Ile Pro
195 200 205
Asp Asp Ile Asp Ser Gly Asn Pro Asp Pro Arg Ser Trp Gly Ser Pro
210 215 220
Val Ala Arg Phe Gln Gly Asp Cys Asp Ile Asp Ala His Phe Asp Asn
225 230 235 240
Leu Gln Ile Ile Phe Asp Thr Thr Phe Cys Gly Asp Trp Ala Gly Asn
245 250 255
Val Trp Gly Ser Gly Ser Cys Ala Ser Val Ala Ser Ser Cys Ser Asp
260 265 270
Tyr Val Ala Asn Asn Pro Glu Ala Phe Ala Asp Ala Tyr Trp Ile Ile
275 280 285
Asn Ser Leu Lys Val Tyr Gln Asp Asp Gly Asp Asp Asp Asp Thr Val
290 295 300
Ala Gly Leu Val Asp Tyr Gly Asp Asp Glu Asp Asp Glu Asp Asp Gln
305 310 315 320
Asp Asp Asp Asn Asp Asp Asp Asp Ser Asp Asp Asp Asp Asp Ser Asp
325 330 335
Asp Asn Asp Asp Asp Ser Asp Ser Asp Asp Asp Ser Asp Asp Glu Asp
340 345 350
Asp Asp Asp Asp Asp Asp Asp Asp Asp Ser Asp Asp Asp Asp Asp Asp
355 360 365
Glu Ser Arg Pro Trp Arg Gly Gly Gly Gly Gly Arg Asn Arg His Gly
370 375 380
Ser Lys Leu Tyr Ala Ser Thr Ser Thr Ser Ser Phe Pro Ser Pro Ser
385 390 395 400
Phe Met Arg Lys Ala Lys Trp Glu Arg Arg Asn Pro Met Ala Gly Met
405 410 415
Ile Phe
<210> 9
<211> 418
<212> PRT
<213> 氨基酸序列(Aminoacid Sequence)
<400> 9
Ala Tyr Thr Leu Gln Asp Asp Tyr Ser Gly Asp Ser Phe Phe Asp Gly
1 5 10 15
Phe Thr Phe Phe Thr Gly Ala Asp Pro Thr Asn Gly Phe Val Asp Tyr
20 25 30
Val Asp Glu Ala Thr Ala Gln Ser Asn Gly Tyr Ile Ser Thr Ser Gly
35 40 45
Asp Ala Pro Val Tyr Met Gly Val Asp His Thr Asn Ile Ala Gly Ser
50 55 60
Ser Gly Arg Gln Ser Val Arg Ile Ser Ser Asp Ala Thr Tyr Asn His
65 70 75 80
Gly Leu Phe Ile Leu Asp Leu Gly His Met Pro Gly Gly Val Cys Gly
85 90 95
Thr Trp Pro Ala Phe Trp Leu Val Gly Ala Asp Trp Pro Asn Asn Gly
100 105 110
Glu Ile Asp Ile Ile Glu Gly Val Asn Gln Gln Ser Gly Asn Asp Met
115 120 125
Thr Leu His Thr Ser Asp Gly Cys Ser Val Ser Ser Ser Ala Asp Phe
130 135 140
Thr Gly Ser Met Thr Thr Ser Asn Cys Tyr Val Ser Ala Ala Gly Gln
145 150 155 160
Ser Asn Asn Ala Gly Cys Gly Ile Thr Asp Pro Asp Gly Thr Ser Tyr
165 170 175
Gly Thr Ala Phe Asn Ala Asn Asn Gly Gly Val Phe Ala Thr Glu Trp
180 185 190
Thr Ser Asp Ala Ile Ser Ile Trp Phe Phe Glu Arg Gly Ser Ile Pro
195 200 205
Asp Asp Ile Asp Ser Gly Asn Pro Asp Pro Ser Ser Trp Gly Ser Pro
210 215 220
Val Ala Arg Phe Gln Gly Asp Cys Asp Ile Asp Ala His Phe Asp Asn
225 230 235 240
Leu Gln Ile Ile Phe Asp Thr Thr Phe Cys Gly Asp Trp Ala Gly Asn
245 250 255
Val Trp Gly Ser Gly Ser Cys Ala Ser Val Ala Ser Ser Cys Ser Asp
260 265 270
Tyr Val Ala Asn Asn Pro Glu Ala Phe Ala Asp Ala Tyr Trp Ile Ile
275 280 285
Asn Ser Leu Lys Val Tyr Gln Asp Asp Gly Asp Asp Asp Asp Thr Val
290 295 300
Ala Gly Leu Val Asp Tyr Gly Asp Asp Glu Asp Asp Glu Asp Asp Gln
305 310 315 320
Asp Asp Asp Asn Asp Asp Asp Asp Ser Asp Asp Asp Asp Asp Ser Asp
325 330 335
Asp Asn Asp Asp Asp Ser Asp Ser Asp Asp Asp Ser Asp Asp Glu Asp
340 345 350
Asp Asp Asp Asp Asp Asp Asp Asp Asp Ser Asp Asp Asp Asp Asp Asp
355 360 365
Glu Ser Arg Pro Trp Arg Gly Gly Gly Gly Gly Arg Asn Arg His Gly
370 375 380
Ser Lys Leu Tyr Ala Ser Thr Ser Thr Ser Ser Phe Pro Ser Pro Ser
385 390 395 400
Phe Met Arg Lys Ala Lys Trp Glu Arg Arg Asn Pro Met Ala Gly Met
405 410 415
Ile Phe
Claims (6)
1.一种GH16家族热稳定性增强的葡聚糖酶突变体,其特征在于,所述葡聚糖酶突变体为葡聚糖酶突变体AtGlu16D_D219R或葡聚糖酶突变体AtGlu16D_D219S,编码所述葡聚糖酶突变体AtGlu16D_D219R的核苷酸序列为SEQ ID NO.1,编码所述葡聚糖酶突变体AtGlu16D_D219S的核苷酸序列为SEQ ID NO.2。
2.根据权利要求1所述的一种GH16家族热稳定性增强的葡聚糖酶突变体,其特征在于,所述葡聚糖酶突变体AtGlu16D_D219R的氨基酸序列如SEQ ID NO.3;葡聚糖酶突变体AtGlu16D_D219S的氨基酸序列如SEQ ID NO.4。
3.一种重组载体,其特征在于,其为含有编码权利要求1中所述葡聚糖酶突变体AtGlu16D_D219R或葡聚糖酶突变体AtGlu16D_D219S的核苷酸的质粒。
4.一种重组菌株,其特征在于,表达权利要求3所述的重组载体。
5.权利要求4所述的重组菌株的构建方法,其特征在于,包括以下步骤:
步骤1,将NCBI序列 XP_035356354.1葡聚糖酶AtGlu16D作为模板,通过定点突变的方法构建重组质粒;
步骤2,将葡聚糖酶突变体AtGlu16D_D219R或AtGlu16D_D219S的重组质粒转入DMT感受态,得重组载体;
步骤3,将突变重组载体进行线性化处理,通过电击法转入GS115毕赤酵母感受态,诱导表达后筛选有催化活力的突变菌株;
步骤4,培养重组菌株,大量表达葡聚糖酶突变体;
步骤5,回收并且纯化所表达的有催化活力的重组菌株。
6.权利要求1所述的葡聚糖酶突变体、权利要求3所述的重组载体,或权利要求4所述的重组菌株在降解β-葡聚糖上的应用。
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