CN113403290A - 热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用 - Google Patents
热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用 Download PDFInfo
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- CN113403290A CN113403290A CN202110574650.7A CN202110574650A CN113403290A CN 113403290 A CN113403290 A CN 113403290A CN 202110574650 A CN202110574650 A CN 202110574650A CN 113403290 A CN113403290 A CN 113403290A
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Abstract
本发明涉及基因工程领域，具体涉及热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用。所述葡萄糖氧化酶突变体通过将氨基酸序列如SEQIDNO：1所示的葡萄糖氧化酶的第4位、第70位、第86位、第96位、第274位、第508位中至少一个氨基酸进行取代获得。本发明提供葡萄糖氧化酶突变体，与野生型葡萄糖氧化酶相比，其热稳定性有了显著的提升，有利于该酶在工业化生产中应用。本发明对来源于黑曲霉的葡萄糖氧化酶进行分子改造，通过定点突变技术和易错PCR方法提高了葡萄糖氧化酶的热稳定性，为葡萄糖氧化酶的工业化应用奠定基础。
Description
技术领域
本发明涉及基因工程领域，具体涉及热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用。
背景技术
葡萄糖氧化酶(EC 1.1.3.4，GOD)作为一种高特异性氧化还原酶，能够催化β-D-葡萄糖转化为葡萄糖酸，并产生过氧化氢(H2O2)，在医疗检测、食品、生物燃料电池、畜牧养殖等领域发挥着极其重要的作用。葡萄糖氧化酶作为一种新型的绿色添加剂有其独特的作用方式，与普通酶制剂不同，它并不是通过改善营养素的消化利用和减轻抗营养因子的方式来改善对其动物生产性能的影响。其方式可能是一方面通过与饲料中葡萄糖作用而产生的葡萄糖酸来发挥作用，另一方面，葡萄糖氧化酶在与葡萄糖作用过程中消耗O2，使消化道更易形成厌氧环境，促使厌氧有益菌的增殖。综合起来，葡萄糖氧化酶通过在肠道中的作用而可能起到酸化剂和益生菌的作用。
GOD广泛分布于动植物和微生物体内，由于微生物生长繁殖快、来源广，是生产GOD的主要来源，主要生产菌株为黑曲霉和青霉。葡萄糖氧化酶具有较差的热稳定性，当温度超过65℃时，葡萄糖氧化酶的酶活快速下降，而饲料制粒过程的温度在75℃以上，葡萄糖氧化酶较差的热稳定性已成为阻碍其广泛应用的关键因素。因此，提供热稳定的葡萄糖氧化酶性具有重要的应用价值，将带来巨大的市场竞争力。如何更有效地提高葡萄糖氧化酶耐热性能也是今后研究的方向。随着研究的不断深入，葡萄糖氧化酶的应用前景将会更加广阔。
CN 103981159A公开了一种葡萄糖氧化酶突变体及其应用，通过将第172位氨基酸由Asn变为Arg，第525位氨基酸由Cys变为Asn，提高了突变酶的耐热性。
目前大多数酶反应需要在较温和的条件下进行以维持其正常活性，而在实际应用的逆境中(如高热、高酸、高盐等)，酶的耐受性却较差、容易失活从而导致反应效率下降，极大地限制了其推广和应用。因此，对酶分子进行抗逆改造以提高其稳定性和催化活性，是当前研究的热点也是难点。本发明的葡萄糖氧化酶突变体对于拓宽葡萄糖氧化酶的实际应用具有重要价值。
发明内容
本发明的目的是提供葡萄糖氧化酶突变体。
本发明的再一目的是提供葡萄糖氧化酶突变体编码基因。
本发明的再一目的是提供包含上述葡萄糖氧化酶突变体编码基因的重组载体。
本发明的再一目的是提供包含上述葡萄糖氧化酶突变体编码基因的重组菌株。
根据本发明提供的热稳定性提高的葡萄糖氧化酶，基于氨基酸序列如SEQ ID NO：1所示的葡萄糖氧化酶的第4位、第70位、第86位、第96位、第274位、第508位中至少一个氨基酸发生了突变。
根据本发明的具体实施方案，所述突变为I4A、I4P、I4Y、D70Q、D70K、A86F、A86*(第86位氨基酸被删除)、A86Q、S96A、S96N、S96F、G274S、G274Q、G274H、P508A、P508H或P508C中之一或多个组合。
在本发明的优选具体实施方案中，所述取代为I4A、D70K、A86*、S96A、G274Q、P508H。
在本发明的优选具体实施方案中，所述取代为I4P、D70K、A86F、S96A、G274S、P508A。
在本发明的优选具体实施方案中，所述取代为I4A、D70D、A86Q、S96F、G274Q、P508H。
在本发明的优选具体实施方案中，所述取代为I4Y、D70Q、A86Q、S96A，G274H、P508C。
在本发明的优选具体实施方案中，所述取代为I4Y、D70Q、A86Q、S96N、G274Q、P508A。
在本发明的优选具体实施方案中，所述取代为I4P、D70K、A86*、S96A、G274S、P508C。
本发明还提供了编码上述突变体的基因。
本发明还提供了包含编码上述葡萄糖氧化酶突变体基因的重组表达载体，优选所述重组表达载体为毕赤酵母重组表达载体或者黑曲霉重组表达载体。
本发明还提供了包含编码上述葡萄糖氧化酶突变体基因的重组菌株，在本发明的一些实施方案中，上述宿主细胞是真菌细胞，优选酵母细胞或丝状真菌细胞，更优选毕赤酵母或黑曲霉。
本发明再一方面提供所述葡萄糖氧化酶突变体在食品、化工、医药、农业或饲料领域中的应用。
本发明提供葡萄糖氧化酶突变体，与野生型葡萄糖氧化酶相比，其热稳定性有了显著的提升，有利于该酶在工业化生产中应用。本发明对来源于黑曲霉的葡萄糖氧化酶进行分子改造，通过定点突变技术和易错PCR方法提高了葡萄糖氧化酶的热稳定性，为葡萄糖氧化酶的工业化应用奠定基础。
附图说明
图1显示在pH5.5的条件下分别在30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃，测定葡萄糖氧化酶的酶活力；
图2显示在温度37℃的条件下，分别在pH3，pH3.5，pH4，pH4.5，pH5，pH5.5，pH6，pH6.5，pH7，pH7.5条件下测定葡萄糖氧化酶的酶活力。
具体实施方式
以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
本发明的载体构建的规程和方法，使用基因工程领域惯用的规程和方法。
定义：
“基因”指在产生多肽中涉及的DNA片段，包括在编码区之前和之后的区域，以及在单个编码片段(外显子)之间的间插序列(内含子)。
“氨基酸取代”是指：将1个或多个氨基酸残基用其它化学上类似的氨基酸残基取代。例如：将某个疏水性残基用其它疏水性残基取代，将某个极性残基用具有相同电荷的其它极性残基取代。能够进行这样的取代的功能上类似的氨基酸，按氨基酸分类是本技术领域公知的。具体的例子，作为非极性(疏水性)氨基酸可以列举出丙氨酸、缬氨酸、异亮氨酸、亮氨酸、脯氨酸、色氨酸、苯丙氨酸、甲硫氨酸等。作为极性(中性)氨基酸，可以列举出甘氨酸、丝氨酸、苏氨酸、酪氨酸、谷氨酰胺、天冬酰胺、半胱氨酸等。
在本说明书中，针对碱基序列或氨基酸序列而言的“同一性”是指：在所比较的序列之间，构成各序列的碱基或氨基酸残基的一致程度。在本说明书中示出的任何“同一性”的数值，均只要是使用本领域技术人员公知的同源性检索程序计算出的数值即可。
如本文中使用的“表达载体”指含有与一个或多个合适的控制序列有效连接的DNA编码序列的DNA构建体，所述控制序列能够实现编码序列在宿主中的表达。此类控制序列包括实现转录的启动子，控制此类转录的任选的操纵基因序列，编码合适的mRNA核糖体结合位点的序列，和控制转录和翻译终止的序列。载体可以是质粒、噬菌体颗粒，或单纯的是潜在的基因组插入序列。一旦转化到合适的宿主中，载体就可以复制并独立于宿主基因组发挥功能，或者在一些情况下，可以自身整合到基因组中。质粒是最常使用的表达载体形式。然而，本发明意在包括发挥等同功能的其它形式的表达载体，所述表达载体是或者将是本领域已知的。
“启动子”指涉及结合RNA聚合酶以起始基因转录中的调控序列。启动子可以是诱导型启动子或组成型启动子。可用于本发明的诱导型启动子的非限制性实例是糖化酶启动子，该启动子是诱导型启动子。
术语“宿主细胞”指细胞或细胞系，用于多肽生产的重组表达载体可以转染到所述细胞或细胞系中以表达多肽。
真菌细胞能以本身已知的方式的通过涉及原生质体形成、原生质体的转化、以及细胞壁的再生的过程进行转化。
根据发明，将包含编码过氧化氢酶的多核苷酸序列的载体引入宿主细胞。从包含编码多肽的多核苷酸和包含与过氧化氢酶编码序列有效连接的调控序列的表达载体表达包含过氧化氢酶活性的多肽。
术语“重组菌株”当用于指细胞、核酸、蛋白质或载体时，表示通过引入异源的核酸或蛋白质，或者改变天然的核酸或蛋白质而修饰的细胞、核酸、蛋白质或载体，或者所述细胞源自这样修饰的细胞。
术语“选择性标记”或“选择标记”指能够在宿主细胞中表达的基因，使那些含有引入的核酸或载体的宿主易于选择。
本发明的蛋白质的修饰氨基酸序列的优选可以是其氨基酸具有1或多个(优选1或数个或者1、2、3或4个)保守取代的氨基酸序列。
如本文中使用的，术语“转化”指具有非天然核酸序列的细胞，所述核酸序列整合到其基因组中，或者作为附加体质粒在多个世代中维持。可以使用任何本领域普遍已知的多种技术实施将载体引入到黑曲霉宿主细胞中，所述载体包含编码过氧化氢酶多肽的多核苷酸序列，例如，转化、电穿孔、核微注射、转导、转染、用磷酸钙DNA沉淀孵育、用DNA包被的微粒高速轰击或原生质体融合。
实验材料和试剂：
1、菌株与载体
大肠杆菌菌株Top10、毕赤酵母X33、载体pPICZαA、载体pGAPzαA、抗生素Zeocin购自Invitrogen公司。
2、酶与试剂盒
PCR酶、质粒提取试剂盒、胶纯化试剂盒购于上海生工公司，限制性内切酶购于NEB公司。
3、培养基
大肠杆菌培养基为LB(1％蛋白胨，0.5％酵母提取物，1％NaCL，pH7.0)。LB-Amp为LB培养基加100μg/ml氨苄青霉素。LB-Zeocin为LB培养基加25μg/ml Zeocin。酵母培养基为YPD(1％酵母提取物，2％蛋白胨，2％葡萄糖)。酵母筛选培养基为YPDZ(YPD+100μg/mlZeocin)。酵母诱导培养基BMGY(1％酵母提取物，2％蛋白胨，1.34％YNB，0.00004％Biotin，1％甘油(v/v))和BMMY(除以0.5％甲醇代替甘油，其余成分与BMGY相同)。重组酵母发酵基本盐培养基：磷酸氢二铵5％、磷酸二氢钾0.5％、七水硫酸镁1.5％、硫酸钾1.95％、硫酸钙0.1％、消泡剂0.03％。高压后每升加4.35mlPTM1。PTM1(微量盐溶液)：硫酸铜0.6％、碘化钾0.018％。一水硫酸锰0.3％、二水钼酸钠0.02％、硼酸0.002％、流水氯化钴0.05％、氯化锌2％、七水硫酸铁6.5％、浓硫酸0.5％、生物素0.02％
4、化学试剂：
葡萄糖氧化酶标准品、邻联茴香胺盐酸盐及辣根过氧化物购于Sigma公司，葡萄糖购于OXIOD公司，其他试剂购于广州化学试剂厂。
5、葡萄糖氧化酶测定方法
葡萄糖氧化酶活性测定采用邻—联茴香胺分光光度法。在葡萄糖氧化酶的作用下，葡萄糖和氧反应，生成葡萄糖酸和过氧化氢，过氧化氢和无色的还原型邻联茴香胺在过氧化物酶的作用下，生成水和红色的氧化型邻联茴香胺。在540nm下测定反应液吸光值，依据标准曲线计算葡萄糖氧化酶的酶活。
实施例1、黑曲霉(Aspergillus niger)葡萄糖氧化酶(GOD)基因合成
黑曲霉Aspergillus niger的葡萄糖氧化酶(GOD)基因针对毕赤酵母密码子偏好性进行优化，由苏州金维智生物科技有限公司合成，人工合成该基因(核酸序列如SEQ IDNO.1所示，氨基酸序列如SEQ ID NO.2所示)。分别在葡萄糖氧化酶优化基因(GOX)的5’端和3’端引入EcoRI和XbaI酶切位点，并连接至Puc57-amp载体上，将GOX-Puc57接种至LBA培养基，培养24小时候，提取质粒，用EcoRI和XbaI酶切，切胶回收目的基因片段，产物纯化回收，连接到表达载体pPICzαA，得到表达载体pPICzαA-GOX。
实施例2、易错PCR突变
以上述pPICzαA-GOX为模板，使用GeneMorph II随机突变PCR试剂盒(Stratagene)随机引入突变。
以表1中的引物进行PCR扩增：
表1
| 所用引物为： | |
| GOX-F | 5’-tctaatggtattgaggcttccttg-3’ |
| GOX-R | 5’-ttactgcatagaagcgtagtcagc-3’ |
反应程序如表2所示：
表2
琼脂糖电泳检测PCR扩增结果，纯化回收PCR产物。用限制性内切酶DpnI将原始质粒分解，将分解完的产物再用热激法转入大肠杆菌Top10，通过菌液PCR验证重组转化子，提取验证正确的转化子的质粒进行测序，从而确定相应的突变体。将测序正确的突变体，用PmeI线性化，转入毕赤酵母X33。
实施例3、高通量筛选耐热提升的葡萄糖氧化酶突变菌株
将实施例2得到的酵母重组转化子用牙签逐个挑至24孔板，每个孔中加入1mL含有BMGY培养基，30℃，220rpm培养24h左右，离心去上清。再分别加入1.6mL BMMY培养基进行诱导培养。培养24h后，离心取上清，将上述上清液分别取出200μL至96孔板，进行葡萄糖氧化酶酶活测定和热处理残余酶活测定。通过多轮的筛选比较。最终，申请人筛选到能显著提高葡萄糖氧化酶GOD的耐热性突变位点经过高通量筛选得到17个有效突变位点分别为I4A、I4P、I4Y、D70Q、D70K、A86F、A86*、A86Q、S96A、S96N、S96F、G274S、G274Q、G274H、P508A、P508H和P508C。这17个突变体的相对比活如表3所示。
表3原始葡萄糖氧化酶和突变体葡萄糖氧化酶耐热比较
从表1中的数据可以看出，与野生型葡萄糖氧化酶相比，本发明的葡萄糖氧化酶突变体I4A、I4P、I4Y、D70Q、D70K、A86F、A86*、A86Q、S96A、S96N、S96F、G274S、G274Q、G274H、P508A、P508H和P508C的热稳定性有提升。
实施例4、位点组合突变
将实施例2中酶活提高的有效突变I4A、I4P、I4Y、D70Q、D70K、A86F、A86*(删除第86位氨基酸)、A86Q、S96A、S96N、S96F、G274S、G274Q、G274H、P508A、P508H和P508C的进行组合突变。得到的酵母重组转化子用牙签逐个挑至24孔板，每个孔中加入1mL含有BMGY培养基，30℃，220rpm培养24h左右，离心去上清。再分别加入1.6mLBMMY培养基进行诱导培养。培养24h后，离心取上清，将上述上清液分别取出200μL至96孔板，进行葡萄糖氧化酶酶活测定和热处理残余酶活测定。通过多轮的筛选比较。通过实验最终得到6个酶活显著提高的组合突变分别命名为GOX-MUT-1、GOX-MUT-2、GOX-MUT-3、GOX-MUT-4、GOX-MUT-5、GOX-MUT-6。
其中，
GOX-MUT-1包含的突变位点为：I4A、D70K、A86*(删除第86位氨基酸)、S96A、G274Q、P508H；GOX-MUT-2包含的突变位点为：I4P、D70K、A86F、S96A、G274S、P508A；
GOX-MUT-3包含的突变位点为：I4A、D70D、A86Q、S96F、G274Q、P508H；GOX-MUT-4包含的突变位点为：I4Y、D70Q、A86Q、S96A，G274H、P508C；GOX-MUT-5包含的突变位点为：I4Y、D70Q、A86Q、S96N、G274Q、P508A；GOX-MUT-6包含的突变位点为：I4P、D70K、A86*、S96A、G274S、P508C。
表4原始葡萄糖氧化酶和组合突变体葡萄糖氧化酶相对比活
| 编号 | 相对比活(％) |
| 原始GOX对照 | 100 |
| GOX-MUT-1 | 86 |
| GOX-MUT-2 | 77 |
| GOX-MUT-3 | 95 |
| GOX-MUT-4 | 39 |
| GOX-MUT-5 | 56 |
| GOX-MUT-6 | 89 |
其中，酶的比活力是指在特定条件下，单位重量(mg)蛋白质所具有的酶活力单位数，相对比活为原始菌株与突变体的比活力的比值，如表4所述，6个突变体的比活力均高于原始菌株，酶活也均提高。
表5原始葡萄糖氧化酶和组合突变体葡萄糖氧化酶耐热比较
| 编号 | 75℃水浴3分钟残余酶活(％) |
| 原始GOX对照 | 2.6％ |
| GOX-MUT-1 | 31.2％ |
| GOX-MUT-2 | 41.5％ |
| GOX-MUT-3 | 27.3％ |
| GOX-MUT-4 | 39.8％ |
| GOX-MUT-5 | 33.7％ |
| GOX-MUT-6 | 23.6％ |
通过多点组合突变筛选到6个提高葡萄糖氧化酶耐热的突变体。相对于原始葡萄糖氧化酶的2.6％的保留率，突变后葡萄糖氧化酶75℃水浴3分钟残余酶活残余提高到23.6％-41.5％。
实施例5原始葡萄糖氧化酶及突变体GOX-MUT-1、GOX-MUT-2、GOX-MUT-3、GOX-MUT-4、GOX-MUT-5和GOX-MUT-6的最适反应温度
在pH5.5的条件下，分别在30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃，测定葡萄糖氧化酶的酶活力，结果如图1所示。从图1可知，葡萄糖氧化酶的最适反应温度范围是25℃-55℃。MUT-1,MUT-2在60℃，70℃反应酶活要高于原始葡萄糖氧化酶。
实施例6原始葡萄糖氧化酶及突变体GOX-MUT-1、GOX-MUT-2、GOX-MUT-3、GOX-MUT-4、GOX-MUT-5和GOX-MUT-6的在不同pH条件下的相对酶活。
在温度37℃的条件下，分别在pH3，pH3.5，pH4，pH4.5，pH5，pH5.5，pH6，pH6.5，pH7，pH7.5条件下测定葡萄糖氧化酶的酶活力，结果如图2所示。从图2可知，在提高葡萄糖氧化酶的耐热水平同时，GOX-MUT-1、GOX-MUT-2、GOX-MUT-3、GOX-MUT-4、GOX-MUT-5和GOX-MUT-6在不同pH条件下的相对酶活与原始葡萄糖氧化酶基本一致，另外在pH6.0和pH6.5条件下相对酶活高于原始葡萄糖氧化酶。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
序列表
<110> 广东溢多利生物科技股份有限公司
<120> 热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 583
<212> PRT
<213> 黑曲霉(Aspergillus niger)
<400> 1
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Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu
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Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val
35 40 45
Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp
50 55 60
Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr
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Glu Thr Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser
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Gly Asn Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr
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Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn
115 120 125
Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu
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Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe
145 150 155 160
Asn Ala Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg
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Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala
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Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp
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Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val
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Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro
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Asn Leu Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser
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Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His
260 265 270
Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala
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Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly
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Met Lys Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu
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Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser
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Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala
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Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ala Glu Lys Ala His Glu Leu
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Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly
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Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg
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Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg
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Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Ile Leu
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Claims (10)
1.热稳定性提高的葡萄糖氧化酶突变体，其特征在于，所述葡萄糖氧化酶突变体通过将氨基酸序列如SEQ ID NO：1所示的葡萄糖氧化酶的第4位、第70位、第86位、第96位、第274位、第508位中至少一个氨基酸进行突变获得。
2.根据权利要求1所述的热稳定性提高的葡萄糖氧化酶突变体，其特征在于，
第4位氨基酸的突变为I4A、I4P或I4Y；
第70位氨基酸的突变为D70Q或D70K；
第86位氨基酸的突变为A86F或A86Q，或者被删除；
第96位氨基酸的突变为S96A、S96N或S96F；
第274位氨基酸的突变为G274S、G274Q或G274H；
第508位氨基酸的突变为P508A、P508H或P508C。
3.根据权利要求1所述的热稳定性提高的葡萄糖氧化酶突变体，其特征在于，
所述取代为I4A、D70K、S96A、G274Q、P508H以及第86位氨基酸被删除；或
所述突变为I4P、D70K、A86F、S96A、G274S、P508A；或
所述突变为I4A、D70D、A86Q、S96F、G274Q、P508H；或
所述突变为I4Y、D70Q、A86Q、S96A，G274H、P508C；或
所述突变为I4Y、D70Q、A86Q、S96N、G274Q、P508A；或
所述突变为I4P、D70K、S96A、G274S、P508C以及第86位氨基酸被删除。
4.编码权利要求1所述热稳定性提高的葡萄糖氧化酶突变体的基因。
5.包含权利要求4所述基因的重组载体。
6.根据权利要求5所述的重组载体，其特征在于，所述重组载体为毕赤酵母重组表达载体或者黑曲霉重组表达载体。
7.包含权利要求4所述基因的重组菌株。
8.根据权利要求8所述的重组菌株，其特征在于，所述重组菌株为重组酵母细胞或丝状真菌细胞，更优选毕赤酵母或黑曲霉。
9.一种制备热稳定性提高的葡萄糖氧化酶的方法，其特征在于，所述方法包括以下步骤：
构建编码权利要求1所述的热稳定性提高的葡萄糖氧化酶突变体的基因的重组表达载体；
将获得重组表达载体导入宿主细胞中；
诱导宿主细胞表达葡萄糖氧化酶。
10.权利要求1所述热稳定性提高的葡萄糖氧化酶突变体的应用。
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| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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