WO2021017292A1 - 热稳定性提高的葡萄糖氧化酶突变体god及其基因和应用 - Google Patents

热稳定性提高的葡萄糖氧化酶突变体god及其基因和应用 Download PDF

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WO2021017292A1
WO2021017292A1 PCT/CN2019/117826 CN2019117826W WO2021017292A1 WO 2021017292 A1 WO2021017292 A1 WO 2021017292A1 CN 2019117826 W CN2019117826 W CN 2019117826W WO 2021017292 A1 WO2021017292 A1 WO 2021017292A1
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god
glucose oxidase
mutant
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WO2021017292A9 (zh
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姚斌
涂涛
蒋肖
罗会颖
苏小运
黄火清
王亚茹
柏映国
王苑
师霞
张�杰
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Feed Research Institute of Chinese Academy of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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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    • 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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    • C12R2001/685—Aspergillus niger
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention relates to the field of genetic engineering, in particular to a glucose oxidase mutant GOD with improved thermal stability and its gene and application.
  • Glucose oxidase (GOD; E.C.1.1.3.4.) is an important class of oxidases, belonging to the glucose/methanol/choline (GMC) oxidoreductase family. GOD specifically catalyzes ⁇ -D-glucose to produce gluconic acid and hydrogen peroxide. The catalytic process requires the participation of oxygen. The mechanism of glucose oxidation is: glucose oxidase and catalase form an oxidoreductase system. In the presence of molecular oxygen, glucose oxidase uses FAD as a cofactor to oxidize glucose to produce D-gluconolactone, while consuming oxygen to produce hydrogen peroxide.
  • GDC glucose/methanol/choline
  • Catalase can decompose hydrogen peroxide to produce water and oxygen, and then the water combines with glucose lactone to produce gluconic acid.
  • GOD is a homodimer enzyme, composed of two 80KD monomers polymerized together, each monomer contains two regions: one binds to FAD in a non-covalent form, and the other binds to the substrate ⁇ -D glucose Combine.
  • GOD is widely distributed in animals, plants and microorganisms. Because microorganisms have the characteristics of fast growth and reproduction and a wide variety of species, they are the main source of GOD production. The main production strains are Aspergillus niger and penicillin.
  • Glucose oxidase is widely used in industries such as food, medicine, feed, and biosensors. Glucose oxidase is used as a feed additive in the feed industry. Adding GOD to the feed can inhibit the growth of good nourishment microorganisms and kill harmful intestinal microorganisms, and the produced gluconic acid can improve the pH of the intestinal tract and facilitate the absorption of nutrients. GOD is also widely used in the food industry. The addition of glucose oxidase in the brewing process can resist the oxidation of beer, maintain the flavor of the beer, and keep the beer for a long time. This is because the glucose oxidase catalysis process consumes oxygen.
  • the purpose of the present invention is to provide glucose oxidase mutants GOD-M6, GOD-M7, GOD-M8, GOD-M9, GOD-M10.
  • Another object of the present invention is to provide a gene encoding the aforementioned glucose oxidase mutant.
  • Another object of the present invention is to provide a recombinant vector containing the aforementioned glucose oxidase mutant gene.
  • Another object of the present invention is to provide a recombinant strain containing the aforementioned glucose oxidase mutant.
  • Another object of the present invention is to provide a method for preparing a mutant of glucose oxidase.
  • Another object of the present invention is to provide applications of the aforementioned glucose oxidase mutants.
  • the amino acid sequence of the glucose oxidase mutant GOD-M5 is shown in SEQ ID NO.1, wherein the 68th Asp of the glucose oxidase GOD-M5 amino acid sequence is mutated to Lys to obtain glucose Oxidase mutant GOD-M6; mutant GOD-M6's Thr 274 is mutated to Phe, and Tyr 278 is mutated to Thr to obtain mutant GOD-M7; Mutant GOD-M7 is mutated to Ser 94 For Ala, obtain the mutant GOD-M8; mutate the 31th Thr of the mutant GOD-M8 to Val to obtain the mutant GOD-M9; mutate the 88th Gln of the mutant GOD-M9 to Arg to obtain the mutant GOD -M10.
  • the amino acid sequence of the glucose oxidase mutant GOD-M6 is shown in SEQ ID NO. 2, and the amino acid sequence of the glucose oxidase mutant GOD-M7 is shown in SEQ ID NO. 3.
  • the glucose oxidase is shown in SEQ ID NO.4
  • the amino acid sequence of the glucose oxidase mutant GOD-M9 is shown in SEQ ID NO.5
  • the amino acid sequence of the glucose oxidase mutant GOD-M10 is shown in SEQ ID NO.6 shown.
  • the present invention provides a gene encoding the GOD mutant of the aforementioned glucose oxidase.
  • the 202th GAC of the god-m5 sequence of the glucose oxidase gene is changed to AAA to obtain the glucose oxidase mutant gene god-m6;
  • the ACC at the 820th position of the god-m6 sequence and the ACC at the 832th position of the god-m6 sequence TTA was changed to TTT and ACC respectively, and the glucose oxidase mutant gene god-m7 was obtained;
  • the TCC at position 280 of the god-m7 sequence was changed to GCT, and the glucose oxidase mutant gene god-m8 was obtained;
  • the god-m8 sequence was changed to 90
  • the ACT was changed to GTT, and the glucose oxidase mutant gene god-m9 was obtained;
  • the AGA at position 262 of the god-m9 sequence was changed to CAA, and the glucose oxidase mutant gene god-m10 was obtained.
  • the gene sequence of the glucose oxidase mutant god-m5 is shown in SEQ ID NO. 7
  • the gene sequence of the glucose oxidase mutant god-m6 is shown in SEQ ID NO. 8
  • glucose oxidizes The gene sequence of the enzyme mutant god-m7 is shown in SEQ ID NO.9
  • the gene sequence of the glucose oxidase mutant god-m8 is shown in SEQ ID NO.10
  • the gene sequence of the glucose oxidase mutant god-m9 is shown in As shown in SEQ ID NO.11
  • the gene sequence of the glucose oxidase mutant god-m10 is shown in SEQ ID NO.12.
  • the present invention provides a recombinant vector containing the GOD mutant gene of the above-mentioned glucose oxidase.
  • the present invention also provides a recombinant strain containing the above-mentioned glucose oxidase GOD mutant gene.
  • the preferred strain is Pichia pastoris GS115.
  • the recombinant strains containing the glucose oxidase mutant gene are recombinant G. oxidase GS115/GODM5, GS115/GOD- M6, GS115/GOD-M7, GS115/GOD-M8, GS115/GOD-M9, GS115/GOD-M10.
  • the method for preparing the GOD mutant of glucose oxidase is as follows:
  • glucose oxidase GOD-M5 After being treated at 70°C for 10 minutes, the relative remaining enzyme activity of glucose oxidase GOD-M5 was 55%.
  • the modified glucose oxidase mutants GOD-M6, GOD-M7, GOD-M8, GOD-M9, GOD-M10 The remaining enzyme activities are 60%, 71%, 75%, 99%, 100%. Therefore, the thermal stability of the glucose oxidase mutant is significantly improved.
  • the relative remaining enzyme activity of glucose oxidase GOD-M5 was 35%.
  • the modified glucose oxidase mutants GOD-M6, GOD-M7, GOD-M8, GOD-M9, GOD-M10 The remaining enzyme activities are 40%, 55%, 60%, 72%, 80%. Therefore, the thermal stability of the glucose oxidase mutant was significantly improved.
  • the relative remaining enzyme activity of GOD-M10 increased by about 2.2 times compared with GOD-M5.
  • the present invention provides multiple glucose oxidase mutants, which have the excellent properties of high catalytic efficiency and good thermal stability, break the barriers of low GOD enzyme activity and poor stability, can fully meet the needs of industrial applications, and can be well applied It has broad application prospects in the food, medicine, textile and feed industries.
  • Figure 1 shows the thermal stability analysis results of glucose oxidase mutants GOD-M5 to GOD-M10 at 70°C;
  • Figure 2 shows the thermal stability analysis results of glucose oxidase mutants GOD-M5 to GOD-M10 at 80°C;
  • Figure 3 shows the analysis results of the optimum temperature of glucose oxidase mutants GOD-M5 to GOD-M10.
  • Enzymes and other biochemical reagents the ligase was purchased from Invitrogen, the site-directed mutagenesis kit was purchased from Quanshijin Company, and the others were domestic reagents (all available from ordinary biochemical reagent companies).
  • E. coli medium LB 1% peptone, 0.5% yeast powder, 1% NaCl, pH 7.0.
  • BMGY medium 1% yeast powder, 2% peptone, 1.34% YNB, 0.000049 ⁇ Biotin, 1% glycerol (v/v).
  • BMMY medium replace glycerol with 0.5% methanol, and the other components are the same as BMGY.
  • the glucose oxidase gene godm5 is a fifth-generation mutant of wild-type god cloned in Aspergillus niger.
  • the mutation process is:
  • the Glu at position 82 of the wild-type glucose oxidase GOD was mutated to Cys to obtain the mutant GOD-M1; the 418th Val of the mutant GOD-M1 was mutated To Glu to obtain mutant GOD-M2; to mutate Asn at position 508 of mutant GOD-M2 to His to obtain mutant GOD-M3; to mutate Thr at position 32 of mutant GOD-M3 to Val to obtain Mutant GOD-M4: The 313th Asp of mutant GOD-M4 was mutated to Lys to obtain mutant GOD-M5.
  • the amino acid sequence of GOD-M5 is shown in SEQ ID NO.1.
  • the 68th Asp was mutated to Lys to obtain the glucose oxidase mutant GOD-M6; then pPIC9-godm6 was used as the template, The 274th Thr of the mutant GOD-M6 was mutated to Phe, and the Tyr 278 was mutated to Thr to obtain the mutant GOD-M7; the 94th Ser of the mutant GOD-M7 was sequentially mutated to Ala to obtain the mutant GOD-M8; Mutation of Thr 31 of mutant GOD-M8 to Val to obtain mutant GOD-M9; Mutation of Gln at position 88 of mutant GOD-M9 to Arg to obtain mutant GOD-M10.
  • the primers for each round of site-directed mutation are shown in the following table:
  • the mutants were amplified using site-directed mutagenesis reagents. After verification by the nucleic acid gel, add 1 ⁇ L of DMT enzyme to the PCR product, mix well and incubate at 37°C for 1h. Take 2-5 ⁇ L of the PCR product digested with DMT enzyme and heat-shock to transform it into competent cell DMT. The positive transformants were selected for DNA sequencing. The transformants with the correct sequence were used to prepare large quantities of recombinant plasmids.
  • the transformants with good thermal stability and high enzyme activity were selected, and they were inoculated into 300mL BMGY liquid medium in a 1L Erlenmeyer flask, cultured at 30°C, 220rpm shaker shaker for 48h; centrifuged at 4500rpm for 5min, discarded the supernatant, and transferred to the bacteria. Add 200 mL of BMMY liquid medium containing 0.5% methanol and induce culture at 30°C and 220 rpm for 48 hours.
  • methanol solution was added once every 24 hours to compensate for the loss of methanol to keep the methanol concentration at about 0.5%; centrifuge at 12000 ⁇ g for 10 minutes, collect the supernatant fermentation broth, detect enzyme activity and perform SDS-PAGE protein electrophoresis analysis.
  • Collect the recombinant glucose oxidase supernatant cultured in shake flask fermentation use a 10kDa membrane package to concentrate the fermentation broth, and replace the medium with pH 6.5 10mM disodium hydrogen phosphate-citrate buffer, and then pass it through an anion exchange column. Purify.
  • the glucose oxidase GOD enzyme activity was determined by the o-dianisidine method.
  • the specific method is as follows: Under the condition of pH 6.0, the 3mL reaction system includes 2.5 mL o-dianisidine buffer (0.2 mL of 1% o-dianisidine). Add 25m 0.1M phosphate buffer solution), 300 ⁇ L 18% glucose solution, 100 ⁇ L 0.03% horseradish peroxidase, 100 ⁇ L appropriately diluted enzyme solution. After reacting at 30°C for 3 min, the reaction was terminated with 2 mL 2M H 2 SO 4 and the absorbance was measured at 540 nm.
  • One enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 ⁇ mol gluconic acid and hydrogen peroxide per unit time under given conditions.
  • the specific activity of glucose oxidase GOD-M5 is 366U/mg, and the enzyme activities of glucose oxidase mutants GOD-M6, GOD-M7, GOD-M8, GOD-M9, GOD-M10 are 301.1U/mg, 299.3U, respectively From the overall specific activity data of GOD-M5 to GOD-M10, the specific activity of GOD-M10 is 1.2 times higher than that of GOD-M5.
  • glucose oxidase GOD mutant GODM5-GODM10 was treated at 70°C for 0, 2, 5, 10, 20, and 30 minutes, respectively. Treat them at 80°C for 0, 1, 2, 5, and 10 minutes, and then measure the relative remaining enzyme activity at 30°C.
  • the relative remaining enzyme activity of glucose oxidase mutant GOD-M5 is 55% after treatment at 70°C for 10 minutes, and the modified glucose oxidase mutant GOD-M6, GOD-M7, GOD-M8
  • the remaining enzyme activities of GOD-M9, GOD-M10 are 60%, 71%, 75%, 99%, 100%, respectively.
  • the remaining enzyme activity of the glucose oxidase mutant GOD-M5 was 35% after treatment at 80°C for 2 min.
  • the remaining enzyme activities of GOD-M9 and GOD-M10 are 40%, 55%, 60%, 72%, and 80%, respectively. From GOD-M5 to GOD-M10, the relative remaining enzyme activity increased by about 2 times.
  • Glucose oxidase GOD mutants GOD-M5 to GOD-M10 were tested for their enzyme activities at 0, 20, 30, 40, 50, 60, 70, 80°C and pH 6.0. As shown in Figure 3, the optimum temperature of glucose oxidase mutants GOD-M5 to GOD-M10 is 40°C, and the relative remaining enzyme activity can be maintained at more than 50% in the range of 20°C-70°C.

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Abstract

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GOD及其基因和应用。葡萄糖氧化酶突变体经野生型定点依次突变后，得到热稳定性和催化效率明显提高的多个突变体。葡萄糖氧化酶突变体具有良好的酶学性质，可以应用于饲料、食品、医药、纺织等行业。

Description

热稳定性提高的葡萄糖氧化酶突变体GOD及其基因和应用 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GOD及其基因和应用。
背景技术
葡萄糖氧化酶(GOD；E.C.1.1.3.4.)是一类重要的氧化酶类，属于glucose/methanol/choline(GMC)氧化还原酶家族。GOD专一性的催化β-D-葡萄糖，产生葡萄糖酸和过氧化氢，催化过程需要氧气的参与。葡萄糖氧化的作用机制为：葡萄糖氧化酶与过氧化氢酶组成一个氧化还原酶系统。葡萄糖氧化酶在分子氧存在下，以FAD为辅因子能氧化葡萄糖生成D-葡萄糖酸内酯，同时消耗氧生成过氧化氢。过氧化氢酶能够将过氧化氢分解生成水和氧，然后水又与葡萄糖内酯结合产生葡萄糖酸。GOD是一种同型二聚体酶，由两个80KD的单体聚合在一起组成，每个单体含有两个区域：一个与FAD以非共价形式结合，另一个与底物β-D葡萄糖结合。GOD广泛分布于动植物和微生物体内，由于微生物具有生长繁殖速度快、种类繁多的特点，因此是生产GOD的主要来源，主要的生产菌株为黑曲霉和青酶。
葡萄糖氧化酶在食品、医学、饲料、生物传感器等行业中有广泛应用。饲料行业中葡萄糖氧化酶作为饲料添加剂，在饲料中添加GOD可以抑制好养微生物的生长，杀死有害肠道微生物，并且产生的葡萄糖酸可以改善肠道的pH，利于营养的吸收。GOD在食品行业中的应用也非常的广泛，酿酒过程中添加葡萄糖氧化酶可以抗啤酒氧化、保持啤酒的风味，使啤酒长期保存，这是因为葡萄糖氧化酶催化过程中会消耗氧。在面包生产中，面粉中添加葡萄糖氧化酶，可以对面粉品质改良达到最佳效果，面包的比容和质量均有很大改善，它对面包抗老化性能效果较好。基于GOD本身的性质，它的应用领域在不断的扩展，国内外市场需求量急剧增加，但是产量低、酶活性低等缺陷限制了它的工业化发展。专利CN108893453A中，以来源于Aspergillus niger的葡萄糖氧化酶GOD作为母本，经过多次突变后获得热稳定提高的突变体GOD-M5，但是其仍不能满足工业要 求。
发明内容
本发明的目的是提供葡萄糖氧化酶突变体GOD-M6、GOD-M7、GOD-M8、GOD-M9、GOD-M10。
本发明的再一目的是提供上述葡萄糖氧化酶突变体的编码基因。
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的重组菌株。
本发明的再一目的是提供一种制备葡萄糖氧化酶突变体的方法。
本发明的再一目的是提供上述葡萄糖氧化酶突变体的应用。
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.1所示，其中，将葡萄糖氧化酶GOD-M5氨基酸序列的第68位Asp突变为Lys，获得葡萄糖氧化酶突变体GOD-M6；将突变体GOD-M6的第274位Thr突变为Phe，278位的Tyr突变为Thr,获得突变体GOD-M7；将突变体GOD-M7的第94位Ser突变为Ala，获得突变体GOD-M8；将突变体GOD-M8的第31位Thr突变为Val，获得突变体GOD-M9；将突变体GOD-M9的第88位Gln突变为Arg，获得突变GOD-M10。
根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M6的氨基酸序列SEQ ID NO.2所示，葡萄糖氧化酶突变体GOD-M7的氨基酸序列如SEQ ID NO.3所示，葡萄糖氧化酶突变体GOD-M8的氨基酸序列如SEQ ID NO.4所示，葡萄糖氧化酶突变体GOD-M9的氨基酸序列如SEQ ID NO.5所示，葡萄糖氧化酶突变体GOD-M10的氨基酸序列如SEQ ID NO.6所示。
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，将葡萄糖氧化酶基因god-m5序列的第202位GAC变为AAA，获得葡萄糖氧化酶突变体基因god-m6；将god-m6序列820位的ACC和832位的TTA分别变为TTT和ACC，获得葡萄糖氧化酶突变体基因god-m7；将god-m7序列280位的TCC变为GCT，获得葡萄糖氧化酶突变体基因god-m8；将god-m8序列90位的ACT变为GTT，获得葡萄糖氧化酶突变体基因god-m9；将god-m9序列262位的AGA变为CAA，获得葡萄糖氧化酶突变体基因god-m10。
根据本发明的具体实施方式，葡萄糖氧化酶突变体god-m5的基因序列如SEQ ID NO.7所示，葡萄糖氧化酶突变体god-m6的基因序列如SEQ ID NO.8所示，葡萄糖氧化酶突变体god-m7的基因序列如SEQ ID NO.9所示，葡萄糖氧化酶突变体god-m8的基因序列如SEQ ID NO.10所示，葡萄糖氧化酶突变体god-m9的基因序列如SEQ ID NO.11所示，葡萄糖氧化酶突变体god-m10的基因序列如SEQ ID NO.12所示。
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，优选的菌株为毕赤酵母GS115，含葡萄糖氧化酶突变体基因的重组菌株分别为重组赤酵母GS115/GODM5，GS115/GOD-M6，GS115/GOD-M7，GS115/GOD-M8，GS115/GOD-M9,GS115/GOD-M10。
根据本发明的具体实施方式，制备葡萄糖氧化酶GOD突变体的方法如下所述：
(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，得到重组菌株；
(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。
本发明的有益效果：
在70℃下处理10min后，葡萄糖氧化酶GOD-M5的相对剩余酶活为55％，改造后的葡萄糖氧化酶突变体GOD-M6、GOD-M7、GOD-M8、GOD-M9、GOD-M10的剩余酶活分别为60％、71％、75％、99％、100％。因此，葡萄糖氧化酶突变体的热稳定获得明显提高。
在80℃下处理2min后，葡萄糖氧化酶GOD-M5的相对剩余酶活为35％，改造后的葡萄糖氧化酶突变体GOD-M6、GOD-M7、GOD-M8、GOD-M9、GOD-M10的剩余酶活分别为40％、55％、60％，72％、80％。因此，葡萄糖氧化酶突变体的热稳定获得明显提高，80℃热处理2min后，GOD-M10相较GOD-M5相对剩余酶活提高了2.2倍左右。
本发明提供了多个葡萄糖氧化酶突变体，具有催化效率高、热稳定性好的优良性质，打破了GOD酶活性低、稳定性差的壁垒，能够完全满足工业应用需求，可以很好的应用于食品、医药、纺织及饲料行业中，具有广阔的应用前景。
附图说明
图1显示葡萄糖氧化酶突变体GOD-M5至GOD-M10在70℃热稳定性分析结果；
图2显示葡萄糖氧化酶突变体GOD-M5至GOD-M10在80℃热稳定性分析结果；
图3显示葡萄糖氧化酶突变体GOD-M5至GOD-M10的最适温度的分析结果。
具体实施方式
试验材料和试剂
1、菌株及载体：毕赤酵母(Pichia pastoris GS115)、毕赤酵母表达载体pPIC9及菌株GS115。
2、酶类及其它生化试剂：连接酶购自Invitrogen公司，定点突变试剂盒购自全式金公司，其它都为国产试剂(均可从普通生化试剂公司购买得到)。
3、培养基：
(1)大肠杆菌培养基LB(1％蛋白胨、0.5％酵母粉、1％NaCl，pH7.O)。
(2)BMGY培养基；1％酵母粉，2％蛋白胨，1.34％YNB，0.000049<Biotin，1％甘油(v/v)。
(3)BMMY培养基：以0.5％甲醇代替甘油，其余成份均与BMGY相同。
说明：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。
实施例1葡萄糖氧化酶的定点突变
葡萄糖氧化酶基因godm5为克隆于黑曲霉的野生型god的第五代突变体，突变过程为：
以来源于Aspergillus niger的葡萄糖氧化酶GOD作为母本，将野生型葡萄糖氧化酶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，GOD-M5的氨基酸序列如SEQ ID NO.1所示。
以GOD-M5的重组质粒pPIC9-godm5序列为模板，根据毕赤酵母优化密码子表，将第68位Asp突变为Lys，获得葡萄糖氧化酶突变体GOD-M6；再以pPIC9-godm6为模板，将突变体GOD-M6的第274位Thr突变为Phe，将278位的Tyr突变为Thr，获得突变体GOD-M7；依次将突变体GOD-M7的第94位Ser突变为Ala,获得突变体GOD-M8；将突变体GOD-M8的第31位Thr突变为Val，获得突变体GOD-M9；将突变体GOD-M9的第88位Gln突变为Arg,获得突变体GOD-M10。每轮定点突变的引物如下表所示：
表1本发明所需的引物
Figure PCTCN2019117826-appb-000001
实施例2葡萄糖氧化酶工程菌株的构建
(1)表达载体的构建及在酵母中的表达
以葡萄糖氧化酶重组质粒pPIC9-godm5为模板，利用定点突变试剂扩增突变体。经核酸胶验证后，将PCR产物中加入1μL DMT酶，混匀后于37℃孵育1h。取2-5μL DMT酶消化的PCR产物，热击转化至感受态细胞DMT。挑取阳性转化子进行DNA测序，测序表明序列正确的转化子用于大量制备重组质粒。用限制性内切酶Bgl II进行线性化表达质粒载体DNA，电击转化酵母GS115感受态细胞，30℃培养2-3天，挑取在MD平板上生长的转化子进行进一步的表达实验，具体操作请参考毕赤酵母表达操作手册。然后通过MM平板的显色反应筛选葡萄糖氧化酶阳性克隆子菌株，依次为：GS115/GOD-M5、GOD-M6、GOD-M7、GOD-M8、GOD-M9、GOD-M10。
实施例3重组葡萄糖氧化酶的制备
(1)葡萄糖氧化酶在毕赤酵母中摇瓶水平的大量表达
筛选出热稳定性好、酶活性较高的转化子，接种于300mL BMGY液体培养基的1L三角瓶中，30℃，220rpm摇床振荡培养48h；4500rpm离心5min，弃上清，再向菌体加入200mL含有0.5％甲醇的BMMY液体培养基，30℃，220rpm诱导培养48h。诱导培养期间，间隔24h补加一次甲醇溶液以补偿甲醇的损失，使甲醇浓度保持在0.5％左右；12000×g离心10min，收集上清发酵液，检测酶活性并进行SDS-PAGE蛋白电泳分析。
(2)重组葡萄糖氧化酶的纯化
收集摇瓶发酵培养的重组葡萄糖氧化酶上清液，利用10kDa膜包进行发酵液的浓缩，同时用pH 6.5 10mM磷酸氢二钠-柠檬酸缓冲液置换其中的培养基，然后通过阴离子交换柱来进行纯化。
实施例4测定纯化的葡萄糖氧化酶突变体的酶学性质
采用邻联茴香胺法对葡萄糖氧化酶GOD酶活性进行测定，具体方法如下：在pH 6.0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25m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。
测定葡萄糖氧化酶GODM5突变体及本发明的各个突变体的酶活性和热稳定性：
1、测定葡萄糖氧化酶GOD M5突变体及本发明的各个突变体的酶活性。将实施例3纯化的葡萄糖氧化酶突变体GOD M5及本发明的各个突变体在pH 6.0、30℃下进行酶促反应以测定其酶活性。
葡萄糖氧化酶GOD-M5的比活为366U/mg，葡萄糖氧化酶突变体GOD-M6、GOD-M7、GOD-M8、GOD-M9、GOD-M10的酶活力分别为301.1U/mg、299.3U/mg、197.9U/mg、454U/mg、445.3U/mg，从GOD-M5到GOD-M10的整体比活数据来看，GOD-M10的比活较GOD-M5提高1.2倍。
2、测定葡萄糖氧化酶突变体GOD M5及本发明的各个突变体在70℃及80℃下的稳定性：
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GODM5-GODM10分别于70℃下处理0、2、5、10、20、30min，80℃下处理0、1、2、5、10min，再在30℃下测定相对剩余酶活。
如图1所示，在70℃下处理10min后，葡萄糖氧化酶突变体GOD-M5的相对剩余酶活为55％，改造后的葡萄糖氧化酶突变体GOD-M6、GOD-M7、GOD-M8、GOD-M9、GOD-M10的剩余酶活分别为60％、71％、75％、99％、100％。
如图2所示，在80℃下处理2min后，葡萄糖氧化酶突变体GOD-M5的剩余酶活为35％，改造后的葡萄糖氧化酶突变体GOD-M6、GOD-M7、GOD-M8、GOD-M9、GOD-M10的剩余酶活分别为40％、55％、60％、72％、80％。从GOD-M5到GOD-M10，相对剩余酶活提高了2倍左右。
3.测定葡萄糖氧化酶突变体GOD-M5及本发明的各个突变体的最适温度：
葡萄糖氧化酶GOD突变体GOD-M5至GOD-M10分别在0、20、30、40、50、60、70、80℃，pH 6.0的条件下测定其酶活性。如图3所示，葡萄糖氧化酶突变体GOD-M5至GOD-M10的最适温度都为40℃，在20℃-70℃范围内都可以维持50％以上的相对剩余酶活。

Claims (10)

  1. 热稳定性提高的葡萄糖氧化酶突变体GOD-M6，其特征在于，其为将葡萄糖氧化酶GOD-M5的氨基酸序列的第68位碱基由Asp突变为Lys得到，其中，葡萄糖氧化酶GOD-M5的氨基酸序列如SEQ ID NO.1所示。
  2. 热稳定性提高的葡萄糖氧化酶突变体GOD-M7，其特征在于，其为将葡萄糖氧化酶GOD-M5的氨基酸序列的第68位碱基由Asp突变为Lys、第274碱基由Thr突变为Phe、第278位碱基由Tyr突变为Thr得到，其中，葡萄糖氧化酶GOD-M5的氨基酸序列如SEQ ID NO.1所示。
  3. 热稳定性提高的葡萄糖氧化酶突变体GOD-M8，其特征在于，其为将葡萄糖氧化酶GOD-M5的氨基酸序列的第68位碱基由Asp突变为Lys、第274碱基由Thr突变为Phe、第278位碱基由Tyr突变为Thr且第94位碱基由Ser突变为Ala得到，其中，葡萄糖氧化酶GOD-M5的氨基酸序列如SEQ ID NO.1所示。
  4. 热稳定性提高的葡萄糖氧化酶突变体GOD-M9，其特征在于，其为将葡萄糖氧化酶GOD-M5的氨基酸序列的第68位碱基由Asp突变为Lys、第274碱基由Thr突变为Phe、第278位碱基由Tyr突变为Thr、第94位碱基由Ser突变为Ala、第31位碱基由Thr突变为Val得到，其中，葡萄糖氧化酶GOD-M5的氨基酸序列如SEQ ID NO.1所示。
  5. 热稳定性提高的葡萄糖氧化酶突变体GOD-M10，其特征在于，其为将葡萄糖氧化酶GOD-M5的氨基酸序列的第68位碱基由Asp突变为Lys、第274碱基由Thr突变为Phe、第278位碱基由Tyr突变为Thr、第94位碱基由Ser突变为Ala、第31位碱基由Thr突变为Val且第88位碱基由Gln突变为Arg得到，其中，葡萄糖氧化酶GOD-M5的氨基酸序列如SEQ ID NO.1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任一项所述的葡葡糖氧化酶GOD突变体的方法，其特征在于，包括以下步骤：
    (1)用包含权利要求1～5任一项所述的葡萄糖氧化酶GOD突变体的编码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，诱导表达葡萄糖氧化酶突变体GOD；
    (3)回收并纯化所表达的葡萄糖氧化酶突变体GOD。
  10. 权利要求1～5任一项所述的热稳定性提高的葡萄糖氧化酶突变体GOD的应用。
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