WO2019233083A1 - 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 PDF

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WO2019233083A1
WO2019233083A1 PCT/CN2018/122270 CN2018122270W WO2019233083A1 WO 2019233083 A1 WO2019233083 A1 WO 2019233083A1 CN 2018122270 W CN2018122270 W CN 2018122270W WO 2019233083 A1 WO2019233083 A1 WO 2019233083A1
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god
glucose oxidase
mutant
glucose
mutation
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姚斌
罗会颖
涂涛
黄火清
苏小运
王亚茹
柏映国
王苑
孟昆
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中国农业科学院饲料研究所
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Priority to EP18921821.7A priority Critical patent/EP3805378A4/en
Priority to US15/734,568 priority patent/US11434473B2/en
Publication of WO2019233083A1 publication Critical patent/WO2019233083A1/zh

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)

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  • the invention belongs to the field of agricultural biotechnology, and particularly relates to a glucose oxidase GOD mutant and its gene and application.
  • Glucose oxidase is a flavin protein that can use molecular oxygen as an electron acceptor to highly specifically oxidize ⁇ -D-glucose to gluconolactone and hydrogen peroxide. For aerobic dehydrogenase.
  • the catalytic reaction process is roughly divided into two parts: reduction reaction, GOD oxidizes ⁇ -D-glucose to gluconolactone, and gluconolactone can be converted into gluconic acid in a non-enzymatic reaction, and then cofactors in GOD Flavin adenine dinucleotide (FAD) is reduced to FADH2; oxidation reaction, reduced GOD-FADH2 is reoxidized to GOD-FAD by reaction with molecular oxygen, and generates H 2 O 2 .
  • GOD has a wide range of applications, spanning many fields such as chemistry, pharmaceuticals, food, clinical diagnostics, and biotechnology.
  • GOD g., GOD co-exists with five physiological functions in protecting the intestine: 1 protecting the integrity of intestinal epithelial cells; 2 improving the acidic digestive environment of the intestine; 3 controlling the intestine Growth and reproduction of pathogenic bacteria; 4 maintain the intestinal flora ecological balance; 5 relieve intestinal mycotoxin poisoning.
  • GOD strains mainly comes from Aspergillus niger and Penicillium.
  • the production of GOD by these wild-type mold strains is low, and the expression level can be significantly increased after heterologous expression. Nevertheless, in the practical application process of GOD, higher requirements are imposed on the thermal stability and catalytic activity of GOD.
  • the present invention provides glucose oxidase mutants GOD-M1, GOD-M2, GOD-M3, GOD-M4, and GOD-M5.
  • the stability and enzyme activity were improved compared with wild glucose oxidase.
  • the object of the present invention is to provide a glucose oxidase GOD mutant.
  • Another object of the present invention is to provide a genetic engineering method for preparing the above-mentioned glucosease GOD mutant.
  • Another object of the present invention is to provide the use of the above-mentioned glucosease GOD mutant.
  • amino acid sequence of the wild-type glucose oxidase GOD is shown in SEQ ID NO: 1:
  • the Glu at position 82 of the amino acid sequence of the wild-type glucose oxidase GOD is mutated to Cys to obtain a glucose oxidase mutant GOD-M1.
  • the amino acid sequence of the glucose oxidase mutant GOD-M1 is shown in SEQ ID NO: 2:
  • the mutation site of the glucose oxidase mutant GOD further includes amino acids 418, 508, 32, and 313.
  • the 418th mutation site Val of the mutant GOD-M1 is Glu to obtain the mutant GOD-M2; the 508th Asn mutation of the mutant GOD-M2 is His to obtain the mutant GOD- M3; Thr at position 32 of mutant GOD-M3 was mutated to Val to obtain mutant GOD-M4; Asp at position 313 of mutant GOD-M4 was mutated to Lys to obtain mutant GOD-M5.
  • the amino acid sequence of the glucose oxidase mutant GOD-M2 is shown in SEQ ID No. 3:
  • the amino acid sequence of the glucose oxidase mutant GOD-M3 is shown in SEQ ID NO: 4:
  • the amino acid sequence of the glucose oxidase mutant GOD-M4 is shown in SEQ ID No. 5:
  • the amino acid sequence of the glucose oxidase mutant GOD-M5 is shown in SEQ ID NO: 6:
  • the glucose oxidase GOD mutant may further be subjected to one or more (e.g., 1, 2, 3, 4) by any of the polypeptide sequences shown in SEQ ID No. 2 to SEQ ID No. 6. , 5, 6, 7, 8, 9) are obtained by substitution, deletion and / or insertion of amino acid residues, and still have the enzyme activity of the above glucose oxidase mutant.
  • a common strategy is conservative amino acid substitution, that is, replacing amino acid residues with amino acid residues having similar side chains.
  • a family of amino acid residues with similar side chains is well defined in the art.
  • the present invention provides a gene encoding the glucose oxidase GOD mutant.
  • the glucose oxidase GOD mutant gene of the present invention includes a gene that hybridizes with a nucleotide sequence encoding any of the amino acid sequences shown in SEQ ID No. 2 to SEQ ID No. 6 under stringent conditions.
  • Nucleotide sequence As used herein, the term “hybridize under stringent conditions" is used to describe hybridization and washing conditions where nucleotide sequences that are typically at least 75% homologous to each other still hybridize to each other. This stringent condition is well known to those of ordinary skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions is: hybridize at about 45 ° C in 6 ⁇ SSC, and then wash one or more times at 50-65 ° C in 0.2 ⁇ SSC, 0.1% SDS.
  • highly stringent conditions can be achieved by increasing the hybridization temperature, for example, to 50 ° C, 55 ° C, 60 ° C, or 65 ° C.
  • the present invention also includes a polypeptide gene having the amino acid sequence shown in SEQ ID No. 2 to SEQ ID No. 6. Allele and a polypeptide with glucose oxidase mutant activity encoded by this natural variant.
  • the present invention provides a recombinant expression vector containing the aforementioned glucose oxidase GOD mutant gene.
  • the recombinant expression vector of the present invention can be designed to express a glucose oxidase mutant protein in prokaryotic or eukaryotic cells.
  • the glucose oxidase mutant gene can be in a bacterial cell such as E. coli, yeast (such as Pichia pastoris, Aspergillus niger), insect cells (such as Sf9 cells or silkworm cells using a baculovirus expression vector) or plant cells (such Pseudomonas-mediated expression in Arabidopsis, tobacco, corn, etc.).
  • the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced.
  • the host cell may be any prokaryotic or eukaryotic cell, including but not limited to those host cells described above.
  • Pichia cells are preferred.
  • Pichia pastoris is a methanol yeast that can be metabolized using methanol as the sole carbon source.
  • As an effective expression system many glucose oxidase genes have been successfully expressed in Pichia pastoris.
  • the novel glucose oxidase mutant gene provided by the present invention is also expressed in Pichia pastoris and has a high expression level. Therefore, large-scale production of glucose oxidase mutants by fermentation is very easy and cost-effective.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in the Molecular Cloning Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, NY, 1989, Sambrook et al.) And other laboratory manuals.
  • the present invention also provides a recombinant strain containing the above-mentioned glucose oxidase GOD mutant gene, preferably the strain is E. coli, yeast (Pichia yeast cells, Saccharomyces cerevisiae cells, or polymorphous yeast cells, etc.), Bacillus, or lactic acid Bacillus.
  • the recombinant expression plasmid is preferably transformed into Pichia yeast cells, and the recombinant strains are respectively GS115 / GOD-M1, GS115 / GOD-M2, GS115 / GOD-M3, GS115 / GOD-M4, and GS115 / GOD-M5.
  • a method for preparing a glucose oxidase GOD mutant includes the following steps:
  • the wild-type glucose oxidase GOD had an enzyme activity of 229.6 U / mg.
  • the modified glucose oxidase mutants GOD-M1, GOD-M2, GOD-M3, GOD-M4, and GOD-M5 had respectively improved their enzyme activities to 352.5U / mg, 366.8U / mg, 379.8U / mg, 392.1U / mg, 381.2U / mg, the increase rate was 54%, 59.8%, 65.4%, 70.8%, 66%.
  • the residual enzyme activity of wild-type glucose oxidase GOD was 14.5 U / mg, and the modified glucose oxidase mutants GOD-M1, GOD-M2, GOD-M3, GOD-M4, GOD-
  • the remaining enzyme activities of M5 were 55.9 U / mg, 73.1 U / mg, 179.2 U / mg, 189.8 U / mg, and 211.2 U / mg, which were increased by 2.6, 4.0, 11.4, 12.1, and 13.6 times, respectively.
  • the residual enzyme activity of wild-type GOD was 4.5 U / mg
  • the residual glucose oxidase mutants GOD-M1, GOD-M2, GOD-M3, GOD-M4, GOD-M5 remained Enzyme activities were 23.6 U / mg, 35.5 U / mg, 98.6 U / mg, 117.2 U / mg, and 137.0 U / mg, which were increased by 4.2, 6.9, 20.9, 25.0, and 29.4 times, respectively.
  • the present invention provides a glucose oxidase GOD mutant with high enzyme activity and improved thermal stability, which can well meet the application needs in the fields of food, medicine, feed, and textile industry, and is suitable for food, medicine, and feed. And textile industry, it has a very broad application prospect.
  • Figure 1 shows the stability of the wild type and each mutant for 10 min at 70 ° C
  • Figure 2 shows the stability of wild type and each mutant for 2 min at 80 ° C.
  • Enzymes and other biochemical reagents Point mutation kits and other biochemical reagents were purchased by biochemical reagent companies.
  • LB medium 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0;
  • YPD medium 1% yeast extract, 2% peptone, 2% glucose
  • MD solid medium 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin;
  • MM solid medium 1.5% agarose, 1.34% YNB, 0.00004% Biotin, 0.5% methanol;
  • BMGY medium 1% yeast extract, 2% peptone, 1% glycerol (V / V), 1.34% YNB, 0.00004% Biotin;
  • BMMY medium 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol (V / V).
  • the 82th Glu of the glucose oxidase GOD shown in SEQ ID NO.1 was mutated to Cys to obtain mutant GOD-M1; the mutant GOD The 418th Val of -M1 is mutated to Glu to obtain mutant GOD-M2; the 508th Asn of mutant GOD-M2 is mutated to His to obtain mutant GOD-M3; the mutant GOD-M3 is Thr at position 32 was mutated to Val to obtain mutant GOD-M4; Asp at position 313 of mutant GOD-M4 was mutated to Lys to obtain mutant GOD-M5.
  • the specific method is as follows: Under the condition of pH 6.0, 3 mL of the reaction system includes 2.5 mL of o-anisidine buffer (0.2 mL of 1% o-anisidine is added to 25 mL of 0.1 M phosphate buffer), 300 ⁇ L 18% Glucose solution, 100 ⁇ L 0.03% horseradish peroxidase, 100 ⁇ L appropriate diluted enzyme solution. After reacting at 30 ° C for 3 minutes, the reaction was terminated with 2 mL of 2M H 2 SO 4 , and the absorbance was measured at 540 nm.
  • One enzyme unit (U) is defined as the amount of enzyme required to produce 1 ⁇ mol of gluconic acid and hydrogen peroxide per unit time under given conditions.
  • the purified glucose oxidase GOD mutant and wild-type glucose oxidase GOD of Example 2 were subjected to an enzymatic reaction at pH 6.0 and 30 ° C. to determine their enzymatic activities.
  • the specific activity of wild-type glucose oxidase GOD was 29.6U / mg, and the modified glucose oxidase mutants GOD-M1, GOD-M2, GOD-M3, GOD-M4, and GOD-M5 had their enzymatic activities increased to 352.5U, respectively. / mg, 366.8U / mg, 379.8U / mg, 392.1U / mg, 381.2U / mg, the increase rates were 54%, 59.8%, 65.4%, 70.8%, 66%.
  • a glucose oxidase GOD mutant and a wild-type glucose oxidase GOD were treated at 70 ° C for 10 minutes and 80 ° C for 2 minutes, respectively.
  • the remaining enzyme activity was measured at 30 ° C.

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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

葡萄糖氧化酶GOD突变体及其基因和应用 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H 2O 2。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，本发明提供了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所提高。
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 ID NO.1所示：
Figure PCTCN2018122270-appb-000001
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。
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 ID NO.2所示：
Figure PCTCN2018122270-appb-000002
Figure PCTCN2018122270-appb-000003
根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD的突变位点还包括418位氨基酸、508位氨基酸、32位氨基酸、313位氨基酸等。
根据本发明的具体实施方式，将突变体GOD-M1的第418突变位Val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-M2的氨基酸序列如SEQ ID NO.3所示：
Figure PCTCN2018122270-appb-000004
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 ID NO.4所示：
Figure PCTCN2018122270-appb-000005
Figure PCTCN2018122270-appb-000006
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 ID NO.5所示：
Figure PCTCN2018122270-appb-000007
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 ID NO.6所示：
Figure PCTCN2018122270-appb-000008
Figure PCTCN2018122270-appb-000009
根据本发明的具体实施方式，葡萄糖氧化酶GOD突变体还可由SEQ ID NO.2～SEQ ID NO.6所示的任一多肽序列经过一个或多个(例如，1、2、3、4、5、6、7、8、9)氨基酸残基的取代、缺失和/或插入获得，并仍然具有以上葡萄糖氧化酶突变体的酶活性。例如，一个常见的策略是保守氨基酸取代，即将氨基酸残基用具有相似侧链的氨基酸残基替换。具有相似侧链的氨基酸残基的家族在本领域已有明确定义。因此，本发明的葡萄糖氧化酶GOD突变体一个或几个氨基酸位点被来自同一侧链类的另一氨基酸残基替换，将不会在实质上影响突变体的酶活性。此外，本领域技术人员公知，在为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、6His或Flag的标签，或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。
本发明提供了编码上述葡萄糖氧化酶GOD突变体的基因。
根据本发明的具体实施方式，本发明葡萄糖氧化酶GOD突变体基因包括由在严谨条件下与编码SEQ ID NO.2～SEQ ID NO.6所示的任一氨基酸序列的核苷酸序列杂交的核苷酸序列。如此处所用，术语“在严谨条件下杂交”是用来描述典型地相互间至少75％同源的核苷酸序列仍可相互杂交的杂交和清洗条件。此严谨条件为本领域普通技术人员所公知，可在Current Protocols in Molecular  Biology,John Wiley&Sons,N.Y.(1989),6.3.1-6.3.6中找到。严谨杂交条件的一个优选、非限制性实例为：在6×SSC中于约45℃杂交，然后在0.2×SSC、0.1％SDS中于50-65℃洗涤一次或多次。本领域技术人员能够理解，高度严谨条件可通过提高杂交温度，例如至50℃、55℃、60℃或65℃来实现。
另外，本领域普通技术人员将会理解：由于自然变异所致的遗传多态性可在群体中的个体间存在。葡萄糖氧化酶GOD突变体基因可发生这种自然变异、并且不改变葡萄糖氧化酶突变体功能活性，因此，本发明也包括由SEQ ID NO.2～SEQ ID NO.6所示氨基酸序列的多肽基因的等位基因和此天然变体所编码的具有葡萄糖氧化酶突变体活性的多肽。
本发明提供了包含上述化葡萄糖氧化酶GOD突变体基因的重组表达载体。本发明的重组表达载体可设计用于在原核或真核细胞中表达葡萄糖氧化酶突变体蛋白。例如，葡萄糖氧化酶突变体基因可在如大肠杆菌的细菌细胞、酵母(如毕赤酵母、黑曲霉)、昆虫细胞(如使用杆状病毒表达载体的Sf9细胞或家蚕细胞)或植物细胞(如脓杆菌介导的拟南芥、烟草、玉米等)中表达。从而，本发明涉及已导入本发明的重组表达载体的宿主细胞。宿主细胞可为任何原核或真核细胞，其包括但不限于上述的那些宿主细胞。优选毕赤酵母细胞。巴斯德毕赤酵母(Pichia pastoris)是一种甲醇酵母，能够以甲醇作为唯一碳源进行代谢。作为有效的表达系统，许多葡萄糖氧化酶基因已成功地在毕赤酵母中表达。同样本发明提供的新的葡萄糖氧化酶突变体基因也在毕赤酵母中表达，并具有高表达水平。因此，通过发酵大规模生产葡萄糖氧化酶突变体非常容易，并且成本较低。
载体DNA可通过常规转化或转染技术导入原核或真核细胞中。转化或转染宿主细胞的合适方法可在《分子克隆》实验室手册第二版(冷泉港实验室出版社,NY,1989，Sambrook等人)和其它实验室手册中找到。
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。
野生型葡萄糖氧化酶GOD的酶活为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：
LB培养基：0.5％酵母提取物，1％蛋白胨，1％NaCl，pH 7.0；
YPD培养基：1％酵母提取物，2％蛋白胨，2％葡萄糖；
MD固体培养基：2％葡萄糖，1.5％琼脂糖，1.34％YNB，0.00004％Biotin；
MM固体培养基：1.5％琼脂糖，1.34％YNB，0.00004％Biotin，0.5％甲醇；
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；
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
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SEQ ID 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
Figure PCTCN2018122270-appb-000010
Figure PCTCN2018122270-appb-000011
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，100μL 0.03％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 H 2SO 4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。

Claims (15)

  1. 葡萄糖氧化酶GOD突变体，其特征在于，所述葡萄糖氧化酶GOD突变体是由具有SEQ ID NO.1所示的氨基酸序列的葡萄糖氧化酶GOD经过点突变得到，所述葡萄糖氧化酶GOD的突变位点包括第82位氨基酸。
  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，所述葡萄糖GOD第82位的突变方式为Glu突变为Cys。
  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，所述葡萄糖GOD的突变位点还包括第418位氨基酸。
  4. 根据权利要求3所述的葡萄糖氧化酶GOD突变体，其特征在于，所述葡萄糖GOD第418位的突变方式为Val突变为Glu。
  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，所述葡萄糖GOD的突变位点还包括第508位氨基酸。
  6. 根据权利要求4所述的葡萄糖氧化酶GOD突变体，其特征在于，所述葡萄糖GOD第508位的突变方式为Asn突变为His。
  7. 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，所述葡萄糖GOD的突变位点还包括第32位氨基酸。
  8. 根据权利要求7所述的葡萄糖氧化酶GOD突变体，其特征在于，所述葡萄糖GOD第32位的突变方式为Thr突变为Val。
  9. 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，所述葡萄糖GOD的突变位点还包括第313位氨基酸。
  10. 根据权利要求9所述的葡萄糖氧化酶GOD突变体，其特征在于，所述葡萄糖GOD第313位的突变方式为Asp突变为Lys。
  11. 葡萄糖氧化酶GOD突变体基因，其特征在于，编码权利要求1～10任一项所述的葡萄糖氧化酶GOD突变体。
  12. 包含权利要求11所述的葡萄糖氧化酶GOD突变体基因的重组表达载体。
  13. 包含权利要求11所述的葡萄糖氧化酶GOD突变体基因的重组菌株。
  14. 制备权利要求1～10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。
  15. 根据权利要求1～10任一项所述的葡萄糖氧化酶GOD突变体的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111500473A (zh) * 2020-06-05 2020-08-07 宏葵生物（中国）股份有限公司 微生物发酵生产低温葡萄糖氧化酶的方法

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN108893453B (zh) * 2018-06-04 2020-05-22 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
CN111349622B (zh) * 2018-12-20 2022-04-08 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
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
CN110628738B (zh) * 2019-09-27 2021-01-12 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
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
CN113862233B (zh) * 2021-12-03 2022-03-25 中国农业科学院北京畜牧兽医研究所 提高葡萄糖氧化酶的酸稳定性的方法及突变体q241e/r499e、基因和应用
CN114395541B (zh) * 2022-02-22 2024-02-06 广东溢多利生物科技股份有限公司 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
CN115029327A (zh) * 2022-04-24 2022-09-09 广东溢多利生物科技股份有限公司 葡萄糖氧化酶突变体GOx-MUT7~11及其编码基因和应用
CN115029328B (zh) * 2022-04-24 2024-03-15 广东溢多利生物科技股份有限公司 葡萄糖氧化酶突变体GOx-MUT1~6及其编码基因和应用
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
CN114736879B (zh) * 2022-06-09 2022-09-27 中国农业科学院北京畜牧兽医研究所 热稳定性改善的葡萄糖氧化酶GoxM10突变体E361P及其衍生突变体和应用

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105026558A (zh) * 2013-01-28 2015-11-04 霍夫曼-拉罗奇有限公司 衍生自黑曲霉的新型葡萄糖氧化酶
CN105209611A (zh) * 2013-04-24 2015-12-30 弗劳恩霍夫应用研究促进协会 新型葡糖氧化酶变体
WO2016031611A1 (ja) * 2014-08-29 2016-03-03 天野エンザイム株式会社 変異酵素及びその用途
CN107012130A (zh) * 2017-06-02 2017-08-04 中国农业科学院饲料研究所 一种葡萄糖氧化酶突变体及其编码基因和应用
CN107189991A (zh) * 2017-05-08 2017-09-22 中国农业科学院饲料研究所 一种葡萄糖氧化酶突变体及其编码基因和应用
CN107988177A (zh) * 2016-10-26 2018-05-04 青岛蔚蓝生物集团有限公司 葡萄糖氧化酶突变体
CN108251391A (zh) * 2017-08-18 2018-07-06 青岛蔚蓝生物集团有限公司 新型葡萄糖氧化酶突变体
CN108374001A (zh) * 2018-03-30 2018-08-07 广东溢多利生物科技股份有限公司 提高比活的葡萄糖氧化酶突变体及其编码基因和应用
CN108893453A (zh) * 2018-06-04 2018-11-27 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

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6312593B2 (ja) * 2011-08-25 2018-04-18 エフ ホフマン−ラ ロッシュ アクチェン ゲゼルシャフト グルコースオキシダーゼ

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105026558A (zh) * 2013-01-28 2015-11-04 霍夫曼-拉罗奇有限公司 衍生自黑曲霉的新型葡萄糖氧化酶
CN105209611A (zh) * 2013-04-24 2015-12-30 弗劳恩霍夫应用研究促进协会 新型葡糖氧化酶变体
WO2016031611A1 (ja) * 2014-08-29 2016-03-03 天野エンザイム株式会社 変異酵素及びその用途
CN107988177A (zh) * 2016-10-26 2018-05-04 青岛蔚蓝生物集团有限公司 葡萄糖氧化酶突变体
CN107189991A (zh) * 2017-05-08 2017-09-22 中国农业科学院饲料研究所 一种葡萄糖氧化酶突变体及其编码基因和应用
CN107012130A (zh) * 2017-06-02 2017-08-04 中国农业科学院饲料研究所 一种葡萄糖氧化酶突变体及其编码基因和应用
CN108251391A (zh) * 2017-08-18 2018-07-06 青岛蔚蓝生物集团有限公司 新型葡萄糖氧化酶突变体
CN108374001A (zh) * 2018-03-30 2018-08-07 广东溢多利生物科技股份有限公司 提高比活的葡萄糖氧化酶突变体及其编码基因和应用
CN108893453A (zh) * 2018-06-04 2018-11-27 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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"current protocols in molecular biology", 1989, JOHN WILEY & SONS
SAMBROOK ET AL.: "Molecular cloning"
See also references of EP3805378A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111500473A (zh) * 2020-06-05 2020-08-07 宏葵生物（中国）股份有限公司 微生物发酵生产低温葡萄糖氧化酶的方法

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