CN114395541A - 一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用 - Google Patents
一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用 Download PDFInfo
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- CN114395541A CN114395541A CN202210161778.5A CN202210161778A CN114395541A CN 114395541 A CN114395541 A CN 114395541A CN 202210161778 A CN202210161778 A CN 202210161778A CN 114395541 A CN114395541 A CN 114395541A
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Abstract
本发明涉及基因工程技术领域，特别是涉及一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1‑MUT、其编码基因和应用。本发明的经过多次突变和高通量筛选获得的热稳定性和比活提高的葡萄糖氧化酶突变体，其氨基酸序列如SEQ ID NO：2所示。与SEQ ID NO：1所示的亲本葡萄糖氧化酶相比，其热稳定性和比活有了显著的提升，有利于该酶在工业化生产中应用。
Description
技术领域
本发明涉及基因工程技术领域，特别是涉及一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用。
背景技术
葡萄糖氧化酶(GOD，EC 1.1.3.4)是一种氧化还原酶，由两个亚基组成二聚体，含有2个黄素腺嘌呤二核苷酸(FAD)结合位点。每个单体含有2个不同的区域：一个与部分FAD非共价但紧密结合，主要为β折叠；另一个与底物β-D-葡萄糖结合，由4个α-螺旋支撑1个反平行的β-折叠。不同来源的GOD理化性质存在差异，分子量在130～175kDa范围内，在有氧条件下，能专一性地催化β-D-葡糖生成葡萄糖酸和过氧化氢。
GOD被广泛应用于食品、化工、生物医疗等领域。在食品行业，GOD可被用于催化葡萄糖耗尽真空袋中氧气，抑制微生物生长和繁殖，延长食品保质期，还可提升面制品的口感。在化工领域，GOD不仅常被应用于漂白脱色工艺，还是葡萄糖酸及其衍生物生产中的关键酶。医疗领域中，GOD是葡萄糖检测试剂盒的关键原料，也被加入牙膏中用于降低口腔疾病发生率，还能作为生物电池的电极，为生物传感器和人造器官提供持续的能源。此外，GOD的用途在转基因植物及其他生物的生产中提出，所述转基因植物及其他生物对于害虫或疾病具有降低的敏感性或增加的抗性。因此，大规模高效生产GOD具有重要的经济价值。然而，GOD在进一步的加工生产过程中，往往需要经历高温的过程，从而不可避免地使得GOD的酶活下降甚至失活。例如，在饲料加工过程中的短暂高温过程会造成葡萄糖氧化酶的失活，影响葡萄糖氧化酶的应用效果，在葡萄糖氧化酶产量得到保证的前提下，葡萄糖氧化酶的耐温性能越来越受到关注。
GOD广泛分布于动植物和微生物体内。目前工业水平的GOD生产，主要以黑曲霉和青霉作为生产菌株，相对青霉GOD而言，黑曲霉表达的GOD热稳定性较好，但都存在酶活水平低、分离纯化复杂的问题。毕赤酵母(Pichiapastoris)遗传背景清楚，易于进行遗传操作，分泌的蛋白糖基化适中，是一种进行外源蛋白表达的常用宿主，尤其是利用P.pastoris进行蛋白分泌表达，可大大简化分离纯化的过程，具有重要的应用价值。
发明内容
本发明的目的是提供一种热稳定性和比活提高的葡萄糖氧化酶突变体。
本发明的再一目的是提供编码上述热稳定性和比活提高的葡萄糖氧化酶突变体的基因。
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。
本发明的再一目的是提供一种提高葡萄糖氧化酶的热稳定和比活的方法。
本发明的再一目的是提供上述热稳定性和比活提高的葡萄糖氧化酶突变体的应用。
本发明热稳定性和比活提高的葡萄糖氧化酶突变体是基于亲本黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶(GOx1)基因(Genebank:FJ979866.1)进行突变的，相对于氨基酸序列SEQ ID NO:1所示的亲本葡萄糖氧化酶进行以下取代：
T10K、E14L、V20Y、T30A、T34V、A36E、R37K、D43N、D70E、S74T、Q90R、V106I、S163A、I167L、A173V、I185L、V186M、K187R、S226A、Q243R、G274S、A288S、A292T、D360K、A362T、A418G、D492N、Y509E、N510H、M556L、I575V。
根据本发明，经过多次突变和高通量筛选获得的热稳定性和比活提高的葡萄糖氧化酶突变体，其氨基酸序列如SEQ ID NO：2所示。
SEQ ID NO：2：
SNGIEASLLKDPKLVAGRTYDYIIAGGGLAGLTVAEKLTENPNITVLVIESGSYESDRGPIIEDLNAYGEIFGTSVDHAYETVELATNNRTALIRSGNGLGGSTLINGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNAACHGLNGTVHVGPRDTGDDYSPLMRALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRADAAREWLLPNYQRPNLRVLTGQYVGKVLLSQNATTPRAVGVEFGTHKSNTHNVYAKHEVLLSAGSTVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGKYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTGGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGNNLAYDADLSAWVEYIPEHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQLSSHVMTVFYAMALKIADAVLADYASMQ。
根据本发明的葡萄糖氧化酶基因编码上述葡萄糖氧化酶突变体，其核苷酸序列如SEQ ID NO：3所示。
SEQ ID NO：3
tctaatggtattgaggcttccttgttgaaagacccaaaacttgtcgccggtagaacctacgactacatcattgccggtggtggtttggctggtttgaccgttgctgagaagttgaccgagaatcctaacatcactgttttggttattgagtccggttcctacgagtctgaccgtggtccaattattgaggatttgaatgcctacggtgaaatcttcggaacttctgtcgaccacgcctatgagaccgttgagttggctactaacaatagaactgctttgatccgttccggtaacggtttgggaggatccactttgattaacggtggaacctggactagaccacataaagcccaagtcgactcctgggagactgtcttcggaaacgaaggttggaactgggactctgttgctgcttactcccttcaggctgaaagagctcgtgccccaaatgctaagcagatcgccgctggtcactactttaacgccgcatgccacggtttgaacggtactgttcacgttggaccacgtgatactggtgatgactactctccattgatgagagccttgatgtctgctgtcgaagatcgtggagtccctaccaagaaggacttgggttgcggagaccctcatggtgtctccatgttcccaaacaccttgcacgaggaccaagttcgtgctgacgctgccagagaatggttgcttcctaactaccagagaccaaacttgagggtcttgactggtcagtacgtcggtaaggtcttgttgtctcagaacgctaccaccccaagagctgttggtgtcgagttcggtactcacaagtctaacacccacaacgtctacgctaagcatgaggtccttttgtccgccggttctactgtttccccaaccatcttggagtattctggaattggtatgaaatctattttggagcctttgggaatcgacaccgttgttgaccttccagttggtttgaacttgcaggaccagaccacctccactgtccgttctcgtattacttccgctggtgctggacaaggtcaagctgcctggttcgctaccttcaatgagacctttggtaagtacaccgagaaggcccacgagttgttgaacaccaagttggagcaatgggctgaagaggctgtcgctagaggtggattccataataccaccgccttgttgatccaatacgaaaattatagagattggattgttaaggacaatgttgcttactccgagttgtttttggataccggtggagtcgcttcctttgacgtctgggacttgttgcctttcacccgtggttacgttcacattttggacaaagatccttacttgcgtcacttcgcctacgacccacagtacttcttgaacgagttggacttgttgggtcaagctgctgctactcagttggcccgtaacatttctaactctggtgccatgcaaacctacttcgctggagagaccattccaggaaacaacttggcctacgatgccgacttgtctgcctgggtcgagtacatccctgaacatttccgtccaaactatcacggtgtcggaacctgctccatgatgccaaaggaaatgggtggagtcgtcgacaatgccgctcgtgtttacggagtccagggtttgagagtcatcgacggttctatcccaccaacccaattgtcctcccacgtcatgactgtcttctacgctatggccttgaagatcgctgacgctgttcttgctgactacgcttctatgcagtaa。
根据本发明的提高葡萄糖氧化酶的热稳定和比活的方法，包括将氨基酸序列SEQID NO：1所示的亲本葡萄糖氧化酶进行以下取代的步骤：
T10K、E14L、V20Y、T30A、T34V、A36E、R37K、D43N、D70E、S74T、Q90R、V106I、S163A、I167L、A173V、I185L、V186M、K187R、S226A、Q243R、G274S、A288S、A292T、D360K、A362T、A418G、D492N、Y509E、N510H、M556L、I575V。
SEQ ID NO：1
SNGIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYAEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLSAWVEYIPYNFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAILADYASMQ。
本发明提供了包含所述葡萄糖氧化酶突变体编码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。
本发明提供包含所述葡萄糖氧化酶突变体编码基因的重组菌株，所述宿主细胞是细菌或真菌细胞，优选地，酵母细胞或丝状真菌细胞，更优选地，为毕赤酵母。
本发明还提供一种制备热稳定性和比活提高的葡萄糖氧化酶的方法，包括以下步骤：
构建包含编码所述葡萄糖氧化酶突变体的基因的重组表达载体；
将所述重组表达载体导入宿主细胞中；
诱导所述宿主细胞表达葡萄糖氧化酶。
本发明提供的热稳定性和比活提高的葡萄糖氧化酶突变体可在食品、化工、医药、农业或饲料领域中应用，可应用于饲料添加剂、食品添加剂、葡萄糖酸钠或葡萄糖酸钙的生产。
本发明提供的葡萄糖氧化酶突变体，与SEQ ID NO：1所示的亲本葡萄糖氧化酶相比，其热稳定性和比活有了显著的提升，有利于该酶在工业化生产中应用。
附图说明
图1显示本发明获得的突变体GOx1-MUT与亲本葡萄糖氧化酶的最适反应pH；
图2显示亲本葡萄糖氧化酶GOx1及突变体GOx1-MUT的最适反应温度；
图3显示亲本葡萄糖氧化酶GOx1及突变体GOx1-MUT的及热稳定性。
具体实施方式
下述实施方法是为了更好的解释本发明，而不应该被解释为限制本发明的目的。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。
将包含编码葡萄糖氧化酶突变体的多核苷酸序列的载体引入宿主细胞，并诱导宿主细胞表达出葡萄糖氧化酶突变体，以用于医药、饲料添加剂、食品添加剂、葡萄糖酸钠或葡萄糖酸钙的生产应用。
本发明还提供一种制备热稳定性和比活提高的葡萄糖氧化酶的方法，包括以下步骤：
a)构建包含编码本发明的葡萄糖氧化酶突变体的基因的重组表达载体；
b)将所述重组表达载体导入宿主细胞中；
c)诱导所述宿主细胞表达葡萄糖氧化酶。
本发明还提供一种热稳定性和比活提高的葡萄糖氧化酶的生产方法，其包括以下步骤：
在适合的条件下培养所述的重组菌株，以产生葡萄糖氧化酶；
纯化得到所述产生的葡萄糖氧化酶；
以及任选地，加工所述产生的葡萄糖氧化酶。
重组葡萄糖氧化酶被分泌到营养培养基中，则可直接从培养基中回收。如果表达的葡萄糖氧化酶突没有分泌，则可从细胞裂解液中回收。
葡萄糖氧化酶蛋白质可在多种表达体系中表达，且相应地必须选择适当的下游加工和纯化步骤。在本发明的一些实施方案中，葡萄糖氧化酶可以在细菌宿主中表达，且蛋白质分泌至周质或细胞外间隙。表达生物体的培养根据标准发酵法以适当体积制备。在优选的实施方案中，细胞在发酵罐中生长，且任选地控制生长条件如pH、温度、氧和/或营养素供给。纯化的第一步包括使用几种技术如沉降、微滤、离心或絮凝中的一种或多种从上清液分离细胞。在优选的实施方案中，适用的方法为微滤。如果在细胞内表达，对细胞进行处理，从细胞内间隙释放蛋白质。这些处理可包括例如加压、酶促、渗透压休克、冷冻、超声或其它处理，从而产生细胞提取物，其可以进行进一步的纯化，或可以不进行。
在本发明的一些实施方案中，葡萄糖氧化酶通过诱导培养后，分泌至上清液，进一步由上清液或浓缩的上清液的蛋白纯化可利用包括下述的几种方法中的一种或多种进行：提取或分级法例如硫酸铵或乙醇或酸沉淀，或层析法，包括但不限于离子交换、疏水相互作用、羟磷灰石、通过凝胶过滤的粒径分级、磷酸纤维素或凝集素层析和亲和层析、或其任意组合。在一些优选的方法中，亲和性标记蛋白通过金属螯合剂亲和层析纯化，从而获得高纯度目标蛋白。在另一些优选的实施例中，通过HPLC纯化获得高纯度的目标蛋白。
在本发明进一步的实施方案中，使用例如但不限于流化床干燥、输送器干燥、喷雾干燥或转鼓干燥或其任意组合等方法将包含表达的葡萄糖氧化酶的发酵细胞悬浮液作为整体干燥。
实验材料和试剂：
1、菌株与载体：含有葡萄糖氧化酶GOx1基因和表达质粒的菌株，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：
超保真2×Master Mix PCR聚合酶、限制性内切酶等购自NEB公司，质粒提取试剂盒、纯化试剂盒购于上海生工公司；
3、培养基
大肠杆菌培养基为LB培养基(1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0)。LB+Amp培养基为LB培养基加入终浓度为100ug/mL的氨苄青霉素，LB+Zeo培养基为LB培养基加入终浓度为25ug/mL的Zeocin；
酵母培养基为YPD培养基(1％酵母提取物，2％蛋白胨，2％葡萄糖)。酵母筛选培养基为YPD+Zeo培养基(YPD+Zeo培养基为YPD培养基加入终浓度为100ug/mL的Zeocin)；
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4.35mL PTM1，其中，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自Sigma公司，葡萄糖购自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，然后在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、葡萄糖氧化酶GOx1基因合成及载体构建
黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶(GOx1)基因(Genebank:FJ979866.1)，其氨基酸序列如SEQ ID NO:1所示。
在葡萄糖氧化酶GOx1基因的5’端和3’端引入EcoRI和XbaI酶切位点，并连接至Puc57-amp载体上，将GOx1-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-GOx1。
实施例2、易错PCR随机突变
以上述pPICzαA-GOx1为模板，使用GeneMorph II随机突变PCR试剂盒(Stratagene)随机引入突变。
以下的引物进行PCR扩增：
GOx1-F 5’-tctaatggtattgaggcttccttg-3’，
GOx1-R 5’-ttactgcatagaagcgtagtcagc-3’。
琼脂糖电泳检测PCR扩增结果，纯化回收PCR扩增的目的产物。用限制性内切酶DpnI消解模板，采用化学转化热激法将分解完的产物转入大肠杆菌Top10感受态细胞中，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。用PmeI将测序正确的突变体质粒线性化，纯化线性质粒片段，采用电转化法转入毕赤酵母X33感受态细胞中，采用YPD+Zeo培养基筛选，获得酵母重组转化子。
实施例3、高通量筛选高比活突变菌株
用牙签逐个将实施例2得到的酵母重组转化子挑至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，计算比活。经过高通量筛选得到比活提升的酵母重组转化子。
实施例4、高通量筛选耐热提升的葡萄糖氧化酶突变体
将实施例3得到的与亲本葡萄糖氧化酶GOx1酶活接近或提升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，筛选出耐热性相对于GOx1显著提高的葡萄糖氧化酶突变体。
实施例5、组合突变与筛选
在实施例3和实施例4中，比活或耐热正向突变位点的基础上，进行双位点或者多位点组合突变，采用镍亲和层析纯化法分别将亲本葡萄糖氧化酶GOx1和葡萄糖氧化酶突变体进行纯化，继续采用实施案例3和实施案例4中的高通量方法进行筛选，如表1和表2所示，分别测定相应的酶活和耐热性，并计算出比活。如以下表1所示，以突变体比活除以亲本葡萄糖氧化酶GOx1比活，来计算突变体的相对比活。通过多轮组合突变和筛选，实验最终筛选到最优的组合突变，命名为GOx1-MUT。
表1亲本葡萄糖氧化酶GOx1和组合突变体GOx1-MUT比活力比较
由表1可以看出，GOx1-MUT相对于亲本葡萄糖氧化酶GOx1的比活明显提高。
表2亲本葡萄糖氧化酶GOx1和组合突变体GOx1-MUT耐热比较
同样地，由表2可以看出，GOx1-MUT相对于亲本葡萄糖氧化酶GOx1的热稳定性明显提高。
实施例6亲本葡萄糖氧化酶GOx1及突变体GOx1-MUT的最适反应pH
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1所示。其中，以pH5.5条件下测得葡萄糖氧化酶的酶活力为对照，计算酶各自在不同pH条件下的相对酶活。从图1可知，GOx1-MUT在不同pH条件下的相对酶活与亲本葡萄糖氧化酶GOx1基本一致，最适反应pH范围是pH5.0-pH6.0，当pH为5.5时，测得亲本葡萄糖氧化酶GOx1及突变体GOx1-MUT的酶活力为最高值。
实施例7亲本葡萄糖氧化酶GOx1及突变体GOx1-MUT的最适反应温度范围及热稳定性
在pH5.5的条件下，分别在25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃，测定葡萄糖氧化酶的酶活力，其中，以37℃条件下测得葡萄糖氧化酶的酶活力为对照，计算酶各自在不同温度条件下的相对酶活。结果如图2显示，葡萄糖氧化酶的最适反应温度范围是25℃-50℃。
为了研究葡萄糖氧化酶GOx1和突变体GOx1-MUT在不同温度下的稳定性，分别将上清液在35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃、85℃，静置5min。37℃、pH5.5的条件下作为未热处理的样品，以该未热处理的样品的相对酶活性为100％。结果如图3所示，亲本葡萄糖氧化酶GOx1及突变体GOx1-MUT在50℃以下静置5min后，仍然保留有100％的相对酶活，而GOx1-MUT在85℃下静置5min后，残余酶活还能保持大于60％；在75℃下静置5min后，残余酶活还能保持大于70％，显著优于亲本GOx1。
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> 一种热稳定性和比活提高的葡萄糖氧化酶突变体GOx1-MUT、其编码基因和应用
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Claims (9)
1.一种热稳定性和比活提高的葡萄糖氧化酶突变体，其特征在于，其氨基酸序列如SEQID NO：2所示。
2.一种葡萄糖氧化酶基因，其特征在于，编码权利要求1所述的热稳定性和比活提高的葡萄糖氧化酶突变体。
3.根据权利要求2所述的葡萄糖氧化酶基因，其特征在于，其核苷酸序列如SEQ ID NO：3所示。
4.一种提高葡萄糖氧化酶的热稳定和比活的方法，其特征在于，所述方法包括相对于氨基酸序列SEQ ID NO：1所示的亲本葡萄糖氧化酶进行以下取代的步骤：T10K、E14L、V20Y、T30A、T34V、A36E、R37K、D43N、D70E、S74T、Q90R、V106I、S163A、I167L、A173V、I185L、V186M、K187R、S226A、Q243R、G274S、A288S、A292T、D360K、A362T、A418G、D492N、Y509E、N510H、M556L、I575V。
5.包含权利要求2所述的葡萄糖氧化酶基因的重组表达载体。
6.包含权利要求2所述的葡萄糖氧化酶基因的重组菌株。
7.根据权利要求6所述的重组菌株，其特征在于，所述重组菌株为重组毕赤酵母菌。
8.一种制备热稳定性和比活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：
构建包含编码权利要求1所述热稳定性和比活提高的葡萄糖氧化酶突变体的基因的重组表达载体；
将所述重组表达载体导入宿主细胞中；
诱导所述宿主细胞表达葡萄糖氧化酶。
9.权利要求1所述的热稳定性和比活提高的葡萄糖氧化酶突变体用于制备饲料添加剂、食品添加剂或药物的应用。
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