CN108893453A - 葡萄糖氧化酶god突变体及其基因和应用 - Google Patents
葡萄糖氧化酶god突变体及其基因和应用 Download PDFInfo
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Abstract
本发明以来源于Aspergillus niger的葡萄糖氧化酶GOD作为母本，经点突变后得到催化效率和热稳定性提高的葡萄糖氧化酶GOD‑M5。本发明的突变体相对于野生型GOD的比活提高了66％；在70℃下处理10min后，本发明突变体的酶活力相对于野生型提高了13.6倍；在80℃下处理2min后，本发明突变体的酶活力相对于野生型提高了29.4倍。
Description
技术领域
本发明属于农业生物技术领域，具体涉及葡萄糖氧化酶GOD突变体及其基因和应用。
背景技术
葡萄糖氧化酶(glucose oxidase，GOD)是一种黄素蛋白，其能够以分子氧为电子受体，高度专一的将β-D-葡萄糖氧化成葡萄糖酸内酯和过氧化氢，因此又称为需氧脱氢酶。催化反应过程大致分为两个部分：还原反应，GOD将β-D-葡萄糖氧化成葡萄糖酸内酯，而葡萄糖酸内酯能够在非酶促反应下转化成葡萄糖酸，随后GOD中的辅因子黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)被还原成FADH2；氧化反应，还原态的GOD-FADH2通过与分子氧反应被重新氧化成GOD-FAD，并生成H2O2。GOD的应用范围十分广泛，横跨了化学、制药、食品、临床诊断、生物技术等众多领域。尤其是在畜牧业领域，GOD的出现改变了传统的畜禽预防保健单一针对病原菌的模式。基于反应过程中需要消耗氧气，产生葡萄糖酸和过氧化氢的特性，GOD在保护肠道方面并存了五项生理功效：①保护肠道上皮细胞完整；②改善肠道酸性消化环境；③控制肠道病菌生长繁殖；④保持肠道菌群生态平衡；⑤解除肠道霉菌毒素中毒。
目前，工业化生产GOD的菌株主要来源于黑曲霉和青霉。这些野生型霉菌菌株生产GOD的产量较低，异源表达后可显著提高表达量。尽管如此，在GOD的实际应用过程中，对GOD的热稳定性和催化活性提出了更高的要求。
发明内容
为了解决现有的葡萄糖氧化酶热稳定性、催化活性有待提高的问题，本发明提供葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4和GOD-M5，其热稳定性及酶活力较野生葡萄糖氧化酶均有提高。
本发明的目的是提供葡萄糖氧化酶GOD突变体。
本发明的再一目的是提供编码上述葡萄糖氧化酶GOD突变体的基因。
本发明的再一目的是提供包含上述葡萄糖酶GOD突变体基因的重组载体。
本发明的再一目的是提供包含上述葡萄糖酶GOD突变体基因的重组菌株。
本发明的再一目的是提供制备上述葡萄糖酶GOD突变体的基因工程方法。
本发明的再一目的是提供上述葡萄糖酶GOD突变体的应用。
根据本发明的具体实施方式，野生型葡萄糖氧化酶GOD的氨基酸序列如SEQ IDNO.1所示：
GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYNFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*
根据本发明的具体实施方式，将野生型葡萄糖氧化酶GOD氨基酸序列的第82位Glu突变为Cys，获得葡萄糖氧化酶突变体GOD-M1；将突变体GOD-M1的第418位Val突变为Glu，获得突变体GOD-M2；将突变体GOD-M2的第508位Asn突变为His，获得突变体GOD-M3；将突变体GOD-M3的第32位Thr突变为Val，获得突变体GOD-M4；将突变体GOD-M4的第313位Asp突变为Lys，获得突变体GOD-M5。
根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M1的氨基酸序列如SEQ IDNO.2所示：
GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYNFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*
根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M2的氨基酸序列如SEQ IDNO.3所示：
GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYNFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*
根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M3的氨基酸序列如SEQ IDNO.4所示：
GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*
根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M4的氨基酸序列如SEQ IDNO.5所示：
GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTVAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*
根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M5的氨基酸序列如SEQ IDNO.6所示：
GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTVAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIKTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*
本发明提供了编码上述葡萄糖氧化酶GOD突变体的基因。
本发明提供了包含上述化葡萄糖氧化酶GOD突变体基因的重组载体。
本发明还提供了包含上述葡萄糖氧化酶GOD突变体基因的重组菌株，优选所述菌株为大肠杆菌、酵母菌(毕赤酵母细胞、啤酒酵母细胞或多型逊酵母细胞等)、芽孢杆菌或乳酸杆菌。其中，优选将重组表达质粒转化毕赤酵母细胞，得到重组菌株分别为GS115/GOD-M1，GS115/GOD-M2，GS115/GOD-M3，GS115/GOD-M4，GS115/GOD-M5。
根据本发明的具体实施方式，制备葡萄糖氧化酶GOD突变体的方法，包括如下步骤：
(1)用含有葡萄糖氧化酶GOD突变体基因的重组载体转化宿主细胞，得重组菌株；
(2)培养重组菌株，诱导重组葡萄糖氧化酶GOD突变体表达；
(3)回收并纯化所表达的葡萄糖氧化酶GOD突变体。
相对于野生型葡萄糖氧化酶GOD的比活229.6U/mg，本发明改造后的葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的酶活力分别提高到了352.5U/mg、366.8U/mg、379.8U/mg、392.1U/mg、381.2U/mg，提高幅度分别为54％、59.8％、65.4％、70.8％、66％。
在70℃下处理10min后，野生型葡萄糖氧化酶GOD的剩余酶活为14.5U/mg，改造后的葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的剩余酶活分别为55.9U/mg、73.1U/mg、179.2U/mg、189.8U/mg、211.2U/mg，分别提高了2.6、4.0、11.4、12.1、13.6倍。
在80℃下处理2min后，野生型GOD的剩余酶活为4.5U/mg，改造后的葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的剩余酶活分别为23.6U/mg、35.5U/mg、98.6U/mg、117.2U/mg、137.0U/mg，分别提高了4.2、6.9、20.9、25.0、29.4倍。
本发明提供了一种高酶活力与热稳定性提高的葡萄糖氧化酶GOD突变体，其能很好的满足食品、医药、饲料以及纺织工业等领域中应用的需求，适合于在食品、医药、饲料以及纺织工业等领域中应用，有着非常广阔的应用前景。
附图说明
图1显示野生型和各突变体在70℃下处理10min的稳定性；
图2显示野生型和各突变体在80℃下处理2min的稳定性。
具体实施方式
试验材料和试剂
1、菌株及载体：表达宿主Pichia pastoris GS115，表达质粒载体pPIC9。
2、酶类及其它生化试剂：点突变试剂盒及其它生化试剂都为生化试剂公司购得。
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：1％酵母提取物，2％蛋白胨，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)MM固体培养基：1.5％琼脂糖，1.34％YNB，0.00004％Biotin，0.5％甲醇；
(5)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；
(6)BMMY培养基：1％酵母提取物，2％蛋白胨，1.34％YNB，0.00004％Biotin，0.5％甲醇(V/V)。
实施例1定点突变
野生型葡萄糖氧化酶基因god克隆于黑曲霉。
以来源于Aspergillus niger的葡萄糖氧化酶GOD作为母本，将氨基酸序列如SEQID NO.1所示的葡萄糖氧化酶GOD的第82位Glu突变为Cys，获得突变体GOD-M1；将突变体GOD-M1的第418位Val突变成Glu，获得突变体GOD-M2；将突变体GOD-M2的第508位Asn突变成His，获得突变体GOD-M3；将突变体GOD-M3的第32位Thr突变成Val，获得突变体GOD-M4；将突变体GOD-M4的第313位Asp突变成Lys，获得突变体GOD-M5。
通过定点突变PCR的方法引入突变位点，并对其进行测序验证。设计所用引物如表1所示：
表1.所述突变体特异性引物
实施例2制备葡萄糖氧化酶GOD突变体
将PCR产物中加入1μL DMT酶，混匀后于37℃孵育1h。取2-5μL DMT酶消化产物热击转化至感受态细胞中。测序验证正确后，将含有所述突变体基因的重组质粒分别转化毕赤酵母GS115感受态细胞，获得重组酵母菌株GS115/GOD-M1，GS115/GOD-M2，GS115/GOD-M3，GS115/GOD-M4，GS115/GOD-M5。
取含有重组质粒的GS115菌株，接种于300mL BMGY培养基的1L三角瓶中，置于30℃，220rpm摇床培养48h；后将培养液3000g离心5min，弃上清，沉淀用100mL含有0.5％甲醇的BMMY培养基重悬，并再次置于30℃，220rpm条件下诱导培养。每隔12h补加0.5mL甲醇，使菌液中的甲醇浓度保持在0.5％，同时取上清用于酶活性检测。
实施例3分析葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶的活性
一、采用邻联茴香胺法对葡萄糖氧化酶GOD酶活进行测定
具体方法如下：在pH6.0的条件下，3mL的反应体系包括2.5mL邻联茴香胺缓冲液(0.2mL的1％邻联茴香胺加入到25mL的0.1M磷酸缓冲液中)，300μL 18％葡萄糖溶液，100μL0.03％辣根过氧化物酶，100μL适当的稀释酶液。在30℃下反应3min后，用2mL 2M H2SO4终止反应，并在540nm下测定吸光值。1个酶活单位(U)定义为在给定的条件下，单位时间内生成1μmol葡萄糖酸和过氧化氢所需的酶量。
二、测定葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶的酶活及热稳定性
1、测定葡萄糖氧化酶GOD突变体和野生型的酶活性:
将实施例2纯化的葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶GOD在pH 6.0、30℃下进行酶促反应以测定其酶活性。
野生型葡萄糖氧化酶GOD的比活为29.6U/mg，改造后的葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的酶活力分别提高到352.5U/mg、366.8U/mg、379.8U/mg、392.1U/mg、381.2U/mg，提高幅度分别为54％、59.8％、65.4％、70.8％、66％。
2、测定葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶GOD在70℃或80℃下的热稳定性
在0.1mol/L柠檬酸-磷酸氢二钠缓冲液(pH 6.0)缓冲液体系中，葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶GOD分别于70℃下处理10min，80℃下处理2min，再在30℃下测定剩余酶活性。
如图1所示，70℃下处理10min后，野生型葡萄糖氧化酶GOD的剩余酶活为14.5U/mg，葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的剩余酶活分别为55.9U/mg、73.1U/mg、179.2U/mg、189.8U/mg、211.2U/mg，分别提高了2.6、4.0、11.4、12.1、13.6倍。
如图2所示，在80℃下处理2min后，野生型葡萄糖氧化酶GOD的剩余酶活为4.5U/mg，改造后的葡萄糖氧化酶GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的剩余酶活分别为23.6U/mg、35.5U/mg、98.6U/mg、117.2U/mg、137.0U/mg，分别提高了4.2、6.9、20.9、25.0、29.4倍。
序列表
<110> 中国农业科学院饲料研究所
<120> 葡萄糖氧化酶GOD突变体及其基因和应用
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Claims (10)
1.葡萄糖氧化酶GOD突变体，其特征在于，其氨基酸序列如SEQ ID NO：2所示。
2.根据权利要求1所述的葡萄糖氧化酶GOD突变体，其特征在于，其氨基酸序列如SEQID NO：3所示。
3.根据权利要求1所述的葡萄糖氧化酶GOD突变体，其特征在于，其氨基酸序列如SEQID NO：4所示。
4.根据权利要求1所述的葡萄糖氧化酶GOD突变体，其特征在于，其氨基酸序列如SEQID NO：5所示。
5.根据权利要求1所述的葡萄糖氧化酶GOD突变体，其特征在于，其氨基酸序列如SEQID NO：6所示。
6.葡萄糖氧化酶GOD突变体基因，其特征在于，编码权利要求1～5任一项所述的葡萄糖氧化酶GOD突变体。
7.包含权利要求6所述的葡萄糖氧化酶GOD突变体基因的重组载体。
8.包含权利要求6所述的葡萄糖氧化酶GOD突变体基因的重组菌株。
9.制备权利要求1～5任一项所述的葡糖糖氧化酶GOD突变体的方法，其特征在于，包括以下步骤：
(1)用含有葡萄糖氧化酶GOD突变体基因的重组载体转化宿主细胞，得重组菌株；
(2)培养重组菌株，诱导重组葡萄糖氧化酶GOD突变体表达；
(3)回收并纯化所表达的葡萄糖氧化酶GOD突变体。
10.根据权利要求1～5任一项所述的葡萄糖氧化酶GOD突变体的应用。
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810561796.6A CN108893453B (zh) | 2018-06-04 | 2018-06-04 | 葡萄糖氧化酶god突变体及其基因和应用 |
| PCT/CN2018/122270 WO2019233083A1 (zh) | 2018-06-04 | 2018-12-20 | 葡萄糖氧化酶god突变体及其基因和应用 |
| US15/734,568 US11434473B2 (en) | 2018-06-04 | 2018-12-20 | Glucose oxidase GOD mutant and gene and application thereof |
| EP18921821.7A EP3805378A4 (en) | 2018-06-04 | 2018-12-20 | GLUCOSE OXIDASE (GOD) MUTANT AND RELATED GENE AND APPLICATION |
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| CN108034642A (zh) * | 2017-11-13 | 2018-05-15 | 中国农业科学院饲料研究所 | 葡萄糖氧化酶CnGOD19及其改良酶、基因和应用 |
| WO2019233083A1 (zh) * | 2018-06-04 | 2019-12-12 | 中国农业科学院饲料研究所 | 葡萄糖氧化酶god突变体及其基因和应用 |
| CN110628738A (zh) * | 2019-09-27 | 2019-12-31 | 华东理工大学 | 提高葡萄糖氧化酶活性的方法、突变体及其应用 |
| CN111004786A (zh) * | 2019-12-25 | 2020-04-14 | 广东溢多利生物科技股份有限公司 | 一种葡萄糖氧化酶及其载体与应用 |
| CN111349622A (zh) * | 2018-12-20 | 2020-06-30 | 南京百斯杰生物工程有限公司 | 一种葡萄糖氧化酶突变体及其在工业化生产中的应用 |
| CN112301009A (zh) * | 2019-07-26 | 2021-02-02 | 中国农业科学院北京畜牧兽医研究所 | 热稳定性提高的葡萄糖氧化酶突变体god及其基因和应用 |
| 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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| CN108034642A (zh) * | 2017-11-13 | 2018-05-15 | 中国农业科学院饲料研究所 | 葡萄糖氧化酶CnGOD19及其改良酶、基因和应用 |
| CN108034642B (zh) * | 2017-11-13 | 2020-12-11 | 中国农业科学院北京畜牧兽医研究所 | 葡萄糖氧化酶CnGOD19及其改良酶、基因和应用 |
| WO2019233083A1 (zh) * | 2018-06-04 | 2019-12-12 | 中国农业科学院饲料研究所 | 葡萄糖氧化酶god突变体及其基因和应用 |
| CN111349622A (zh) * | 2018-12-20 | 2020-06-30 | 南京百斯杰生物工程有限公司 | 一种葡萄糖氧化酶突变体及其在工业化生产中的应用 |
| CN112301009A (zh) * | 2019-07-26 | 2021-02-02 | 中国农业科学院北京畜牧兽医研究所 | 热稳定性提高的葡萄糖氧化酶突变体god及其基因和应用 |
| WO2021017292A1 (zh) | 2019-07-26 | 2021-02-04 | 中国农业科学院饲料研究所 | 热稳定性提高的葡萄糖氧化酶突变体god及其基因和应用 |
| CN112301009B (zh) * | 2019-07-26 | 2022-12-09 | 中国农业科学院北京畜牧兽医研究所 | 热稳定性提高的葡萄糖氧化酶突变体god及其基因和应用 |
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| CN110628738A (zh) * | 2019-09-27 | 2019-12-31 | 华东理工大学 | 提高葡萄糖氧化酶活性的方法、突变体及其应用 |
| CN111004786A (zh) * | 2019-12-25 | 2020-04-14 | 广东溢多利生物科技股份有限公司 | 一种葡萄糖氧化酶及其载体与应用 |
| CN111004786B (zh) * | 2019-12-25 | 2021-12-07 | 广东溢多利生物科技股份有限公司 | 一种葡萄糖氧化酶及其载体与应用 |
| 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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| EP3805378A4 (en) | 2022-07-06 |
| US20210230561A1 (en) | 2021-07-29 |
| EP3805378A1 (en) | 2021-04-14 |
| CN108893453B (zh) | 2020-05-22 |
| US11434473B2 (en) | 2022-09-06 |
| WO2019233083A1 (zh) | 2019-12-12 |
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