WO2023236638A1 - 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 - Google Patents

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 Download PDF

Info

Publication number
WO2023236638A1
WO2023236638A1 PCT/CN2023/086142 CN2023086142W WO2023236638A1 WO 2023236638 A1 WO2023236638 A1 WO 2023236638A1 CN 2023086142 W CN2023086142 W CN 2023086142W WO 2023236638 A1 WO2023236638 A1 WO 2023236638A1
Authority
WO
WIPO (PCT)
Prior art keywords
goxm10
glucose oxidase
mutant
seq
mutation
Prior art date
Application number
PCT/CN2023/086142
Other languages
English (en)
French (fr)
Inventor
涂涛
黄火清
闫亚茹
张伟
罗会颖
姚斌
Original Assignee
中国农业科学院北京畜牧兽医研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国农业科学院北京畜牧兽医研究所 filed Critical 中国农业科学院北京畜牧兽医研究所
Publication of WO2023236638A1 publication Critical patent/WO2023236638A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)

Definitions

  • the invention belongs to the field of feed biotechnology, and specifically relates to glucose oxidase GoxM10 mutant E361P with improved thermal stability and derivative mutants and applications thereof.
  • Glucose oxidase (Gox; EC1.1.3.4) is an aerobic dehydrogenase with good characteristics. It uses oxygen molecules as electron acceptors under aerobic conditions to catalyze ⁇ -D-glucose to generate D-gluconolactone. and hydrogen peroxide, followed by hydrolysis of D-gluconolactone into gluconic acid and water.
  • Gox has received widespread attention from the feed industry as a new antibiotic alternative additive.
  • Gox as an alternative to antibiotics and growth promoters, is critical to ensuring animal health and performance without compromising human health. A large number of application studies have shown that Gox, as a green feed additive, can be used to prevent gastrointestinal infections and diarrhea in livestock and promote animal growth.
  • Gox must have good thermal stability and acid stability.
  • the existing technology has improved the stability of Gox derived from Aspergillus niger. For example, a mutant GoxM4 with a T 50 increased by 7.5°C and a mutant GoxM10 with a T m increased by 9°C compared to GoxM4 were obtained.
  • the object of the present invention is to provide a mutant of glucose oxidase GoxM10 with improved thermostability.
  • Another object of the present invention is to provide genes encoding the above mutants.
  • Another object of the present invention is to provide a recombinant vector containing the above mutant gene.
  • Another object of the present invention is to provide a recombinant strain containing the above mutant gene.
  • Another object of the present invention is to provide applications of the above mutants.
  • Yet another object of the present invention is to provide a method for preparing glucose oxidase with improved stability.
  • Another object of the present invention is to provide a method for improving the thermal stability of glucose oxidase GoxM10. Law.
  • site-directed mutation is performed on the glucose oxidase whose amino acid sequence is shown as GoxM10ID NO:1.
  • the glucose oxidase with the amino acid sequence shown in GoxM10 ID NO:1 is subjected to 203, 219, 338, 361, 419 for single point mutation, and then 361/203, 361/219, 361/338 , 361/419 double-point combination mutation, then 361/419/203, 361/419/338 three-point combination mutation, and further 361/419/193/497, 361/419/4/471 four-point combination mutation, and 361/419/471/193/497 six-point combined mutation to obtain a glucose oxidase mutant with improved acid and thermal stability.
  • the glucose oxidase with an amino acid sequence as shown in SEQ ID NO:1 is subjected to G203C mutation to obtain a mutant with an amino acid sequence with improved thermal stability as shown in SEQ ID NO:2.
  • a gene encoding the above-mentioned glucose oxidase mutant G203C with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 3.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO:1 is subjected to E219P mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO:4.
  • a gene encoding the above-mentioned glucose oxidase mutant E219P with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 5.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1 is subjected to S338P mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO: 6.
  • a gene encoding the above-mentioned glucose oxidase mutant S338P with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 7.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1 is subjected to E361P mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO: 8.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 9.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO:1 is subjected to A419I mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO:10.
  • a glucose oxidation method encoding the above-mentioned thermal stability improvement.
  • the nucleotide sequence of the gene of enzyme mutant A419I is shown in SEQ ID NO: 11.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO:1 is subjected to E361P/G203C mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO:12.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/G203C with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 13.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO:1 is subjected to E361P/E219P mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO:14.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/E219P with improved stability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 15.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO:1 is subjected to E361P/S338P mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO:16.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/S338P with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 17.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO:1 is subjected to E361P/A419I mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO:18.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/A419I with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 19.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1 is subjected to E361P/A419I/G203C mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO: 20.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/A419I/G203C with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 21.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1 is subjected to E361P/A419I/S338P mutation, and the resulting amino acid sequence is a mutant shown in SEQ ID NO: 22.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/A419I/S338P with improved thermostability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 23.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO: 1 is subjected to E361P/A419I/D193N/D497N mutation, and the obtained amino acid sequence is as SEQ ID NO.
  • the mutant shown in NO:24 is subjected to E361P/A419I/D193N/D497N mutation, and the obtained amino acid sequence is as SEQ ID NO.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/A419I/D193N/D497N with improved acid and thermal stability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 25.
  • the glucose oxidase with the amino acid sequence shown in SEQ ID NO:1 is subjected to E361P/A419I/A4D/N471E mutation to obtain a mutant with the amino acid sequence shown in SEQ ID NO:26.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/A419I/A4D/N471E with improved acid and thermal stability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 27.
  • the glucose oxidase with an amino acid sequence as shown in SEQ ID NO:1 is subjected to E361P/A419I/D193N/D497N/A4D/N471E mutation to obtain a mutation with an amino acid sequence as shown in SEQ ID NO:28 body.
  • a gene encoding the above-mentioned glucose oxidase mutant E361P/A419I/D193N/D497N/A4D/N471E with improved acid and thermal stability is also provided, and its nucleotide sequence is as shown in SEQ ID NO: 29 Show.
  • a recombinant vector containing the above-mentioned glucose oxidase mutant gene is also provided.
  • the starting vector of the recombinant expression vector is pPIC9, and the recombinant expression vector is specifically pPIC9-goxm10-G203C, pPIC9-goxm10 -E219P, pPIC9-goxm10-S338P, pPIC9-goxm10-E361P, pPIC9-goxm10-A419I, pPIC9-goxm10-E361P/G203C, pPIC9-goxm10-E361P/E219P, pPIC9-goxm10-E361P/S338P, p PIC9-goxm10-E361P /A419I, pPIC9-goxm10-E361P/A419I/
  • a recombinant strain containing the above glucose oxidase mutant gene is also provided.
  • the method for preparing glucose oxidase with improved thermostability according to the present invention includes the following steps:
  • the present invention provides the application of the above-mentioned glucose oxidase mutant with acid stability or thermal stability, which can be specifically applied in the fields of food and feed.
  • the mutant of the present invention Compared with glucose oxidase GoxM10, the mutant of the present invention has the same optimal reaction temperature, both at 40°C, and its thermal stability is significantly improved. After incubation at 65°C for 180 minutes, the remaining enzyme activity of GoxM10 was 61%.
  • the remaining enzyme activities of /A419I/S338P, E361P/A419I/A4D/N471E, E361P/A419I/D193N/D497N and E361P/A419I/A4D/N471E/D193N/D497N are 65%, 63%, 69%, 74%, respectively.
  • the half-lives of the four double-point mutants E361P/G203C, E361P/E219P, E361P/S338P and E361P/A419I were 495min, 433min, 462min and 578min respectively, which were 1.3, 1.1, 1.2 and 1.5 times that of GoxM10 (385min) respectively.
  • the glucose enzyme mutant with improved acid stability or thermal stability provided by the present invention is suitable for application in the fields of food and feed, and has very broad application prospects.
  • Figure 1 shows the thermal stability comparison of GoxM10 and mutants treated at 65°C for 180 min
  • Figure 2 shows the thermal stability comparison of GoxM10 and mutants treated at 75°C for 12 min
  • Figure 3 shows a comparison of the optimal temperatures of GoxM10 and mutants
  • Figure 4 shows the acid stability comparison of GoxM10 and mutants treated at pH 2.5 for 30 minutes
  • Figure 5 shows the acid stability comparison of GoxM10 and mutants treated at pH 2.75 for 60 minutes
  • Figure 6 shows a comparison of the optimal pH of GoxM10 and mutants.
  • the expression host is Pichiapastoris GS115, and the expression plasmid vector is pPIC9.
  • Enzymes and other biochemical reagents endonuclease/high-fidelity DNA polymerase and recombinase.
  • Escherichia coli LB medium 1% peptone, 0.5% yeast extract, 1% NaCl, natural pH.
  • Pichia pastoris YPD medium 1% yeast extract, 2% peptone, 2% glucose, natural pH
  • BMGY 1% yeast extract, 2% peptone, 10% amino-free yeast nitrogen source, 0.1% biotin, pH natural
  • BMMY 1% yeast extract, 2% peptone, 10% amino-free yeast nitrogen source, 0.1% biotin, 1% methanol, pH natural).
  • the glucose oxidase with the amino acid sequence shown in GoxM10 ID NO:1 is subjected to single point mutations 203, 219, 338, 361, and 419, and then 361/203, 361/219, 361/338 , 361/419 double-point combination mutation, and then carry out the three-point combination mutation of 361/419/203, 361/419/338, and then proceed to the four-point combination mutation of 361/419/193/497, 361/419/4/471. and 361/419/471/193/497 six-point combination mutations to obtain glucose oxidation with improved acid and thermal stability Enzyme mutants.
  • glucose oxidase whose amino acid sequence is shown as GoxM10ID NO:1 is mutated as follows:
  • E361P/G203C E361P/E219P, E361P/S338P, E361P/A419I;
  • the goxm10 gene fragment was amplified by PCR, and the vector pPIC9 nucleic acid fragment was obtained by double enzyme digestion. The two were connected by recombinase to obtain the recombinant plasmid pPIC9-goxm10, and transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115. (pPIC9-goxm10).
  • the primers used in PCR are as follows:
  • GoxM10-F (SEQ ID No:30,30bp), GoxM10-R (SEQ ID No:31,30bp).
  • GoxM10-F and GoxM10-R are used to amplify the gene coding sequence of GoxM10; the vector pPIC9 is obtained by inserting the preserved bacterial strain into liquid LB medium and culturing it overnight, and then extracting the plasmid.
  • the PCR product and the extracted plasmid were tested by nucleic acid electrophoresis.
  • the band sizes of goxm10 and vector pPIC9 were 1746bp and 8088bp respectively.
  • the vector was digested with EcoRI and NotI, the PCR product and the digested product were recovered and purified respectively. .
  • the recovered goxM10 and pPIC9 gene fragments were recombinantly ligated using recombinase, and then the recombinant products were transformed into E. coli JM109 competent cells and spread on LB (containing 100 ⁇ g/mL Amp) for screening.
  • the recombinant plasmid pPIC9-goxm10 is digested with BglII restriction endonuclease.
  • the digested product is recovered and transformed into Pichia pastoris GS115 competent cells by electroporation to obtain the recombinant expression strain GS115 (pPIC9-goxm10).
  • Design mutation primers based on the mutants use plasmid pPIC9-goxm10 as a template, and use point mutation reagents
  • the cassette introduces mutated amino acids, and the PCR product containing the mutated amino acid is digested with DpnI to remove the template.
  • the digested PCR product is transformed into E. coli JM109 competent cells, and is sequenced and verified to obtain E. coli containing the glucose oxidase mutant plasmid.
  • the primers used are as follows: Gox-G203C-F (SEQ ID No: 32), Gox-G203C-R (SEQ ID No: 33); Gox-E219P-F (SEQ ID No: 34), Gox-E219P-R (SEQ ID No:35); Gox-S338P-F (SEQ ID No:36), Gox-S338P-R (SEQ ID No:37); Gox-E361P-F (SEQ ID No:38), Gox-E361P -R (SEQ ID No: 39); Gox-A419I-F (SEQ ID No: 40), Gox-A419I-R (SEQ ID No: 41).
  • the correctly sequenced mutant plasmid was digested with BglII, and the digested product was recovered and transformed into Pichia pastoris GS115 competent cells by electroporation to obtain recombinant expression strains GS115 (pPIC9-goxm10-G203C), GS115 (pPIC9-goxm10-E219P), and GS115 GS115 ( pPIC9-goxm10-E361P/S338P), GS115 (pPIC9-goxm10-E361P/A419I), GS115 (pPIC9-goxm10-E361P/A419I/G203C), GS115 (pPIC9-goxm10-E361P/A419I/S338P), GS11 5(pPIC9- goxm10-E361P/A419I/A4D/N471E), GS115 (pPIC9-goxm10-E361
  • the liquid was purified with an anion column.
  • the purification liquid A was 10mM disodium hydrogen phosphate-citrate buffer (pH 6.5).
  • Liquid B was liquid A plus 1M NaCl. Purify the protein and collect the eluate.
  • the purified enzyme solution was diluted to the same protein concentration with 0.1M disodium hydrogen phosphate-citrate buffer (pH 6.0), incubated in a water bath at 65°C and 75°C for different times, and finally measured according to standard methods. Relative to the remaining enzyme activity, and calculate t 1/2 . As shown in Figure 1, the remaining enzyme activity of GoxM10 after incubation at 65°C for 180 minutes was 61%.
  • the half-lives of the four double-point mutants E361P/G203C, E361P/E219P, E361P/S338P and E361P/A419I were 495min, 433min, 462min and 578min respectively, which were 1.3, 1.1, 1.2 and 1.5 times that of GoxM10 (385min).
  • the purified enzyme solution was diluted to an appropriate multiple, and the enzyme activity was measured at pH 6.0 and different temperature gradients (20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70°C).
  • the enzyme activity at the optimal reaction temperature is set to 100%, and then the relative enzyme activity at other temperatures is calculated to obtain the optimal reaction temperature of GoxM10 and its mutants.
  • Optimum reaction temperature and G oxM10 is also 40 °C.
  • Method 1 Place GoxM10 and mutant enzyme solutions of the same concentration in accurately configured 0.1M disodium hydrogen phosphate-citrate buffer at pH 2.5, 2.75, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0, and then Incubate at 37°C for 1 hour. After the incubation, the remaining enzyme activity was measured under standard conditions, and the enzyme activity of the untreated sample was recorded as 100%.
  • Method 2 Place GoxM10 and mutant enzyme solutions of the same concentration in 0.1M disodium hydrogen phosphate-citrate buffer at pH 2.5, and then incubate at 37°C for 10min, 20min, 30min, 40min, 50min and 60min.
  • the substrate mixtures for Gox enzyme activity measurement with different pH gradients (3.0, 4.0, 5.0, 6.0, 7.0, 8.0) were prepared respectively.
  • the enzyme activities of GoxM10 and its mutants were measured at 30°C, and the optimal The enzyme activity under reaction pH conditions is set to 100%, and then the relative enzyme activities under other pH conditions are calculated to obtain the optimal reaction pH of GoxM10 and its mutants.
  • the optimal reaction pH of mutants E361P/A419I/A4D/N471E, E361P/A419I/D193N/D497N and E361P/A419I/A4D/N471E/D193N/D497N is all 6.0 compared with GoxM10.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Polymers & Plastics (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Husbandry (AREA)
  • Food Science & Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种热稳定性改善的葡萄糖氧化酶GoxM10突变体及其衍生突变体和在饲料工业中的应用。

Description

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 技术领域
本发明属于饲料生物技术领域，具体涉及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。
背景技术
葡萄糖氧化酶(Gox；EC1.1.3.4)是一种特性良好的需氧脱氢酶，在有氧条件下利用氧分子作为电子受体，催化β-D-葡萄糖生成D-葡萄糖酸内酯和过氧化氢，随后D-葡萄糖酸内酯水解成葡萄糖酸和水。近年来Gox作为一种新型抗生素替代添加剂受到饲料行业的普遍关注。Gox作为抗生素和生长促进剂的替代品，在不损害人类健康的前提下对保证动物健康和生产性能至关重要。大量应用研究表明，Gox作为一种绿色饲料添加剂，可用于预防牲畜胃肠道感染和腹泻，并有促进动物生长的作用。
当前大部分动物饲料均需要经过高温制粒，所以提高Gox的热稳定性对于其在饲料中的广泛应用至关重要。此外，能够在酸性环境中维持较高的活性，以便在动物胃肠道发挥最大作用也至关重要。所以为了满足Gox在饲料工业中的应用，Gox要具备良好的热稳定性和酸稳定性。现有技术已对黑曲霉(Aspergillus niger)来源Gox稳定性方面做了改进，例如获得了T50增加7.5℃的突变体GoxM4以及比GoxM4的Tm提高了9℃的突变体GoxM10。
发明内容
本发明的目的是提供一种热稳定性改善的葡萄糖氧化酶GoxM10的突变体。
本发明的再一目的是提供编码上述突变体的基因。
本发明的再一目的是提供包含上述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突变体基因的重组菌株。
本发明的再一目的是提供上述突变体的应用。
本发明的再一目的是提供制备稳定性提高的葡萄糖氧化酶的方法。
本发明的再一目的是提供一种改善葡萄糖氧化酶GoxM10的热稳定性的方 法。
根据本发明的具体实施方式，对氨基酸序列如GoxM10ID NO:1所示的葡萄糖氧化酶进行定点突变。
根据本发明的具体实施方式，将氨基酸序列如GoxM10ID NO:1所示的葡萄糖氧化酶进行203、219、338、361、419进行单点突变，然后进行361/203、361/219、361/338、361/419双点组合突变，然后进行361/419/203、361/419/338三点组合突变，进一步进行361/419/193/497、361/419/4/471四点组合突变，以及361/419/4/471/193/497六点组合突变，从而获得酸和热稳定性改进的葡萄糖氧化酶突变体。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行G203C突变，得到热稳定性提高的氨基酸序列如SEQ ID NO:2所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体G203C的基因，其核苷酸序列如SEQ ID NO:3所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E219P突变，得到氨基酸序列如SEQ ID NO:4所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体E219P的基因，其核苷酸序列如SEQ ID NO:5所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行S338P突变，得到氨基酸序列如SEQ ID NO:6所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体S338P的基因，其核苷酸序列如SEQ ID NO:7所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P突变，得到氨基酸序列如SEQ ID NO:8所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体E361P的基因，其核苷酸序列如SEQ ID NO:9所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行A419I突变，得到氨基酸序列如SEQ ID NO:10所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化 酶突变体A419I的基因，其核苷酸序列如SEQ ID NO:11所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/G203C突变，得到氨基酸序列如SEQ ID NO:12所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体E361P/G203C的基因，其核苷酸序列如SEQ ID NO:13所示。
根据本发明的具体方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/E219P突变，得到氨基酸序列如SEQ ID NO:14所示的突变体。
根据本发明的具体实施方式，还提供了编码上述具有稳定性提高的葡萄糖氧化酶突变体E361P/E219P的基因，其核苷酸序列如SEQ ID NO:15所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/S338P突变，得到氨基酸序列如SEQ ID NO:16所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体E361P/S338P的基因，其核苷酸序列如SEQ ID NO:17所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/A419I突变，得到氨基酸序列如SEQ ID NO:18所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体E361P/A419I的基因，其核苷酸序列如SEQ ID NO:19所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/A419I/G203C突变，得到氨基酸序列如SEQ ID NO:20所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体E361P/A419I/G203C的基因，其核苷酸序列如SEQID NO:21所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/A419I/S338P突变，得到的氨基酸序列如SEQ ID NO:22所示的突变体。
根据本发明的具体实施方式，还提供了编码上述热稳定性提高的葡萄糖氧化酶突变体E361P/A419I/S338P的基因，其核苷酸序列如SEQ ID NO:23所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/A419I/D193N/D497N突变，得到的氨基酸序列如SEQ ID  NO:24所示的突变体。
根据本发明的具体实施方式，还提供了编码上述酸和热稳定性提高的葡萄糖氧化酶突变体E361P/A419I/D193N/D497N的基因，其核苷酸序列如SEQ ID NO:25所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/A419I/A4D/N471E突变，得到氨基酸序列如SEQ ID NO:26所示的突变体。
根据本发明的具体实施方式，还提供了编码上述酸和热稳定性提高的葡萄糖氧化酶突变体E361P/A419I/A4D/N471E的基因，其核苷酸序列如SEQ ID NO:27所示。
根据本发明的具体实施方式，将氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶进行E361P/A419I/D193N/D497N/A4D/N471E突变，得到氨基酸序列如SEQ ID NO:28所示的突变体。
根据本发明的具体实施方式，还提供了编码上述酸和热稳定性提高的葡萄糖氧化酶突变体E361P/A419I/D193N/D497N/A4D/N471E的基因，其核苷酸序列如SEQID NO:29所示。
根据本发明的具体实施方式，还提供了包含上述葡萄糖氧化酶突变体基因的重组载体，所述重组表达载体的出发载体为pPIC9，所述重组表达载体具体为pPIC9-goxm10-G203C，pPIC9-goxm10-E219P，pPIC9-goxm10-S338P，pPIC9-goxm10-E361P，pPIC9-goxm10-A419I，pPIC9-goxm10-E361P/G203C，pPIC9-goxm10-E361P/E219P，pPIC9-goxm10-E361P/S338P，pPIC9-goxm10-E361P/A419I，pPIC9-goxm10-E361P/A419I/G203C，pPIC9-goxm10-E361P/A419I/S338P，pPIC9-goxm10-E361P/A419I/D193N/D497N，pPIC9-goxm10-E361P/A419I/A4D/N471E和pPIC9-goxm10-E361P/A419I/A4D/N471E/D193N/D497N。
根据本发明的具体实施方式，还提供了包含上述葡萄糖氧化酶突变体基因的重组菌株。
根据本发明的制备热稳定提高的葡萄糖氧化酶的方法，包括以下步骤：
1)制备包含上述突变体基因的重组载体；
2)所述重组载体转化毕赤酵母GS115表达宿主；
3)发酵培养所述宿主，并分离葡萄糖氧化酶。
本发明提供了上述具有酸稳定性或热稳定性的葡萄糖氧化酶突变体的应用，具体可以应用于食品和饲料领域中。
本发明的突变体与葡萄糖氧化酶GoxM10相比，最适反应温度一致，均为40℃，其热稳定性均明显提高。在65℃保温180min后GoxM10的剩余酶活力为61％，突变体G203C、E219P、S338P、E361P、A419I、E361P/G203C、E361P/E219P、E361P/S338P、E361P/A419I、E361P/A419I/G203C和E361P/A419I/S338P、E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的剩余酶活力分别为65％、63％、69％、74％、66％、75％、68％、76％、81％、72％、62％、67％、75％和71％。在75℃保温12min后，突变体G203C、E219P、S338P、E361P、A419I、E361P/G203C、E361P/E219P、E361P/S338P、E361P/A419I、E361P/A419I/G203C、E361P/A419I/S338P、E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N纯化后的酶液保留酶活力分别为42％、37％、42％、44％、43％、47％、37％、48％、50％、38％、39％、42％、45％和44％左右，GoxM10的保留酶活力为36％。
在65℃保温，GoxM10的半衰期为385min，突变体E219P和A419I的半衰期均为433min，是GoxM10的1.1倍，突变体G203C和E361P的半衰期分别为462min和495min，是GoxM10的1.2和1.3倍，突变体S338P的半衰期和GoxM10相当。4个双点突变体E361P/G203C、E361P/E219P、E361P/S338P和E361P/A419I的半衰期分别为495min、433min、462min和578min，分别是GoxM10(385min)的1.3、1.1、1.2和1.5倍。突变体E361P/A419I/G203C、E361P/A419I/S338P、E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的半衰期分别为433、450、495、553和462min。
在pH 2.5条件下处理30min时，突变体E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的相对剩余酶活力分别为31％、29％和33％，GoxM10的剩余酶活为21％。在pH 2.75和37℃条件下处理60min，突变体E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的相对剩 余酶活力分别为42％、46％和47％左右，GoxM10的剩余酶活为24％。突变体E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的最适pH和GoxM10一致均为6.0。
本发明提供了的酸稳定性或热稳定性的改善的葡萄糖酶突变体适合于在食品和饲料领域中应用，有着非常广阔的应用前景。
附图说明
图1显示GoxM10和突变体在65℃下处理180min的热稳定性对比；
图2显示GoxM10和突变体在75℃下处理12min的热稳定性对比；
图3显示GoxM10和突变体的最适温度对比；
图4显示GoxM10和突变体在pH 2.5条件下处理30min的酸稳定性对比；
图5显示GoxM10和突变体在pH 2.75条件下处理60min的酸稳定性对比；
图6显示GoxM10和突变体的最适pH对比。
具体实施方式
试验材料和试剂
1、菌株及载体：表达宿主为Pichiapastoris GS115，表达质粒载体为pPIC9。
2、酶类及其它生化试剂：内切酶/高保真DNA聚合酶和重组酶。
3、大肠杆菌LB培养基(1％蛋白胨、0.5％酵母提取物、1％NaCl，pH自然)。毕赤酵母YPD培养基(1％酵母提取物，2％蛋白胨，2％葡萄糖，pH自然)；BMGY(1％酵母提取物，2％蛋白胨，10％无氨基酵母氮源，0.1％生物素，pH自然)；BMMY(1％酵母提取物，2％蛋白胨，10％无氨基酵母氮源，0.1％生物素，1％甲醇，pH自然)。
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。
根据本发明的具体实施方式，将氨基酸序列如GoxM10ID NO:1所示的葡萄糖氧化酶进行203、219、338、361、419进行单点突变，然后进行361/203、361/219、361/338、361/419双点组合突变，然后进行361/419/203、361/419/338三点组合突变，进步一进行361/419/193/497、361/419/4/471四点组合突变，以及361/419/4/471/193/497六点组合突变，从而获得酸和热稳定性改进的葡萄糖氧化 酶突变体。
具体地，将氨基酸序列如GoxM10ID NO:1所示的葡萄糖氧化酶进行如下突变：
G203C、E219P、S338P、E361P、A419I；
E361P/G203C、E361P/E219P、E361P/S338P、E361P/A419I；
E361P/A419I/G203C、E361P/A419I/S338；
E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N；
E361P/A419I/A4D/N471E/D193N/D497N)。
实施例1、重组菌株GS115(pPIC9-goxm10)的制备
1.扩增高热稳定性葡萄糖氧化酶GoxM10的核酸序列goxm10
采用PCR的方法扩增goxm10基因片段，采用双酶切方法获得载体pPIC9核酸片段，通过重组酶将两者连接，获得重组质粒pPIC9-goxm10，并转化毕赤酵母GS115，获得重组毕赤酵母菌株GS115(pPIC9-goxm10)。PCR所用引物如下：
GoxM10-F(SEQ ID No:30,30bp)，GoxM10-R(SEQ ID No:31,30bp)。
其中，GoxM10-F及GoxM10-R用于扩增GoxM10的基因编码序列；载体pPIC9通过将保存的菌种接到液体LB培养基过夜培养后，质粒提取得到。
扩增结束后，将PCR产物以及提取质粒进行核酸电泳检测，goxm10与载体pPIC9条带大小分别为1746bp、8088bp，将载体用EcoRI和NotI进行酶切后，将PCR产物与酶切产物分别回收纯化。
2.构建重组菌株GS115(pPIC9-goxm10)
将回收的goxM10与pPIC9基因片段通过重组酶进行重组连接，然后将重组产物转化大肠杆菌JM109感受态，并涂布于LB(含100μg/mLAmp)进行筛选。待测序正确后，利用BglⅡ限制性内切酶将重组质粒pPIC9-goxm10进行酶切，回收酶切产物后电击转化毕赤酵母GS115感受态细胞，得到重组表达菌株GS115(pPIC9-goxm10)。
实施例2、重组突变菌株GS115的制备
1.稳定性提高的重组质粒的构建
根据突变体设计突变引物，以质粒pPIC9-goxm10为模板，通过点突变试剂 盒引入突变氨基酸，含突变氨基酸的PCR产物用DpnⅠ消化处理以去除模板，消化后的PCR产物转化进入大肠杆菌JM109感受态细胞，并对其进行测序验证，获得含葡萄糖氧化酶突变质粒的大肠杆菌pPIC9-goxm10-G203C，pPIC9-goxm10-E219P，pPIC9-goxm10-S338P，pPIC9-goxm10-E361P，pPIC9-goxm10-A419I，pPIC9-goxm10-E361P/G203C，pPIC9-goxm10-E361P/E219P，pPIC9-goxm10-E361P/S338P，pPIC9-goxm10-E361P/A419I，pPIC9-goxm10-E361P/A419I/G203C，pPIC9-goxm10-E361P/A419I/S338P，pPIC9-goxm10-E361P/A419I/A4D/N471E、pPIC9-goxm10-E361P/A419I/D193N/D497N和pPIC9-goxm10-E361P/A419I/A4D/N471E/D193N/D497N。所用引物如下所示：Gox-G203C-F(SEQ ID No:32)，Gox-G203C-R(SEQ ID No:33)；Gox-E219P-F(SEQ ID No:34)，Gox-E219P-R(SEQ ID No:35)；Gox-S338P-F(SEQ ID No:36)，Gox-S338P-R(SEQ ID No:37)；Gox-E361P-F(SEQ ID No:38)，Gox-E361P-R(SEQ ID No:39)；Gox-A419I-F(SEQ ID No:40)，Gox-A419I-R(SEQ ID No:41)。
2.酸稳定性和热稳定重组菌株GS115构建
利用BglⅡ将测序正确的突变质粒进行酶切，回收酶切产物后电击转化毕赤酵母GS115感受态细胞，得到重组表达菌株GS115(pPIC9-goxm10-G203C)，GS115(pPIC9-goxm10-E219P)，GS115(pPIC9-goxm10-S338P)，GS115(pPIC9-goxm10-E361P)，GS115(pPIC9-goxm10-A419I)，GS115(pPIC9-goxm10-E361P/G203C)，GS115(pPIC9-goxm10-E361P/E219P)，GS115(pPIC9-goxm10-E361P/S338P)，GS115(pPIC9-goxm10-E361P/A419I)，GS115(pPIC9-goxm10-E361P/A419I/G203C)，GS115(pPIC9-goxm10-E361P/A419I/S338P)，GS115(pPIC9-goxm10-E361P/A419I/A4D/N471E),GS115(pPIC9-goxm10-E361P/A419I/D193N/D497N)和GS115(pPIC9-goxm10-E361P/A419I/A4D/N471E/D193N/D497N)。
实施例3、葡萄糖氧化酶GoxM10及高酸稳定性和热稳定性突变体酶蛋白的获得
1.GoxM10及突变体的诱导表达
以1％的接种量将上述得到的重组酵母表达菌株接种至50mL YPD培养基中进行种子液培养，200rpm，30℃培养48h后，以1％接种量转接至400mL BMGY培养基中，200rpm，30℃培养48h，离心收集菌体转接到200mL BMMY培养基中，每24h补加1％的甲醇进行诱导表达，共培养72h。
2.GoxM10及突变体的纯化
将诱导表达后的菌液12000rpm,10min离心，收集上清后用10kDa的膜包进行浓缩，再用10mM磷酸氢二钠-柠檬酸缓冲液(pH 6.5)进行透析，然后将透析后的粗酶液用阴离子柱纯化，纯化用A液为10mM磷酸氢二钠-柠檬酸缓冲液(pH 6.5)，B液为A液加1M NaCl，纯化蛋白，收集洗脱液。
3.GoxM10和突变体的酶学性质测定
(1)GoxM10和突变体的热稳定性测定
用0.1M磷酸氢二钠-柠檬酸缓冲液(pH 6.0)把纯化后的酶液稀释到相同的蛋白浓度，分别在65℃和75℃水浴锅中保温处理不同的时间，最后按标准方法测定相对剩余酶活力，并计算t1/2。如图1所示，65℃保温180min后GoxM10的剩余酶活力为61％，突变体G203C、E219P、S338P、E361P、A419I、E361P/G203C、E361P/E219P、E361P/S338P、E361P/A419I、E361P/A419I/G203C和E361P/A419I/S338P、E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的剩余酶活力分别为65％、63％、69％、74％、66％、75％、68％、76％、81％、72％、62％、67％、75％和71％，与GoxM10(61％)相比分别提高了约7％、3％、13％、21％、8％、23％、11％、25％、33％、18％、2％、10％、23％和16％。如图2所示，75℃保温12min后突变体G203C、E219P、S338P、E361P、A419I、E361P/G203C、E361P/E219P、E361P/S338P、E361P/A419I、E361P/A419I/G203C和E361P/A419I/S338P、E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的剩余酶活力分别为42％、37％、42％、44％、43％、47％、37％、48％、50％、38％、39％、42％、45％和44％，与GoxM10(36％)相比分别提高了约17％、3％、17％、22％、、19％、31％、3％、33％、38％、5％、8％、17％、25％和22％。如表1所示，65℃保温GoxM10的半衰期 为385min，突变体E219P和A419I的半衰期均为433min，是GoxM10的1.1倍，突变体G203C和E361P的半衰期分别为462min和495min，是GoxM10的1.2和1.3倍，突变体S338P的半衰期和GoxM10相当。4个双点突变体E361P/G203C、E361P/E219P、E361P/S338P和E361P/A419I的半衰期分别为495min、433min、462min和578min，分别是GoxM10(385min)的1.3、1.1、1.2和1.5倍。突变体E361P/A419I/G203C、E361P/A419I/S338P、E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的半衰期分别为433、450、495、553和462min。
表1
(2)GoxM10和突变体的最适温度测定
将纯化后的酶液稀释适当的倍数，在pH 6.0和不同的温度梯度(20、25、30、35、40、45、50、55、60、65、70℃)下测定酶活力，把最适反应温度下的酶活力设定为100％，然后计算其余温度下的相对酶活力，即可得到GoxM10及其突变体的最适反应温度。如图3所示，突变体G203C、E219P、S338P、E361P、A419I、E361P/G203C、E361P/E219P、E361P/S338P、E361P/A419I、 E361P/A419I/G203C、E361P/A419I/S338P、E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的最适反应温度和GoxM10一样均为40℃。
(3)GoxM10和突变体的酸稳定性测定
采用两种测定方法评估GoxM10和突变体的pH稳定性。方法一：把相同浓度的GoxM10和突变体酶液置于准确配置的pH 2.5、2.75、3.0、4.0、5.0、6.0、7.0和8.0的0.1M的磷酸氢二钠-柠檬酸缓冲液，然后于37℃保温处理1h。保温结束后在标准条件下测定其剩余酶活力，以未处理样品的酶活力记为100％。方法二：把相同浓度的GoxM10和突变体酶液置于pH 2.5的0.1M的磷酸氢二钠-柠檬酸缓冲液，然后于37℃保温处理10min、20min、30min、40min、50min和60min。保温结束后在标准条件下测定GoxM10及其突变体酶液的相对剩余酶活。如图4所示，pH 2.5条件下处理30min时，突变体E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的相对剩余酶活力分别为31％、29％和33％，与GoxM10(21％)相比分别提高了约48％、38％和57％。如图5所示，在pH 2.75和37℃条件下处理60min，突变体E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的相对剩余酶活力分别为42％、46％和47％左右，与GoxM10(24％)相比分别提高了约75％、92％和96％。
(4)GoxM10和突变体的最适pH测定
首先，分别配置不同pH梯度(3.0、4.0、5.0、6.0、7.0、8.0)的Gox酶活力测定的底物混合液，然后，在30℃下测定GoxM10及其突变体的酶活力，把最适反应pH条件下的酶活力设定为100％，然后计算其余pH条件下的相对酶活力，即可得到GoxM10及其突变体的最适反应pH。如图6，突变体E361P/A419I/A4D/N471E、E361P/A419I/D193N/D497N和E361P/A419I/A4D/N471E/D193N/D497N的最适反应pH与GoxM10相比均为6.0。
以上实施例仅用于解释本申请的技术方案，不限定本申请的保护范围。

Claims (10)

  1. 热稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，具有葡萄糖氧化酶GoxM10进行以下突变后的氨基酸序列，其中，
    所述葡萄糖氧化酶GoxM10的氨基酸序列如SEQ ID NO:1所示；
    所述葡萄糖氧化酶GoxM10进行G203C、或E219P、或S338P或、E361P、或A419I突变。
  2. 根据权利要求1所述的热稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，所述葡萄糖氧化酶GoxM10进一步进行E361P/G203C、或E361P/E219P、或E361P/S338P或E361P/A419I突变。
  3. 根据权利要求2所述的热稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，所述葡萄糖氧化酶GoxM10进一步进行E361P/A419I/G203C或E361P/A419I/S338突变。
  4. 根据权利要求2所述的热稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，所述葡萄糖氧化酶GoxM10进一步进行E361P/A419I/A4D/N471E或E361P/A419I/D193N/D497N突变。
  5. 根据权利要求3所述的热稳定性提高的葡萄糖氧化酶GoxM10突变体，其特征在于，所述葡萄糖氧化酶GoxM10进一步进行E361P/A419I/A4D/N471E/D193N/D497N突变。
  6. 热稳定性提高的葡萄糖氧化酶基因，其特征在于，所述基因编码权利要求1～5任意一项所述的热稳定性提高的葡萄糖氧化酶GoxM10突变体。
  7. 一种提高葡萄糖氧化酶突变体的热稳定性的方法，其特征在于，所述方法包括对氨基酸序列如SEQ ID NO:1所示的葡萄糖氧化酶GoxM10进行以下突变：
    所述葡萄糖氧化酶GoxM10进行G203C、或E219P、或S338P、或E361P或A419I突变；或者；
    所述葡萄糖氧化酶GoxM10进行E361P/G203C、或E361P/E219P、或E361P/S338P或E361P/A419I突变；或者
    所述葡萄糖氧化酶GoxM10进行E361P/A419I/G203C或E361P/A419I/S338突变；或者
    所述葡萄糖氧化酶GoxM10进行E361P/A419I/A4D/N471E或 E361P/A419I/D193N/D497N突变；或者
    所述葡萄糖氧化酶GoxM10进行E361P/A419I/A4D/N471E/D193N/D497N突变。
  8. 包含权利要求6所述热稳定性提高的葡萄糖氧化酶基因的重组载体。
  9. 包含权利要求6所述热稳定性提高的葡萄糖氧化酶基因的重组菌株。
  10. 权利要求1～5任意一项所述的热稳定性提高的葡萄糖氧化酶GoxM10突变体的应用。
PCT/CN2023/086142 2022-06-09 2023-04-04 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 WO2023236638A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210648703.XA CN114736879B (zh) 2022-06-09 2022-06-09 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
CN202210648703.X 2022-06-09

Publications (1)

Publication Number Publication Date
WO2023236638A1 true WO2023236638A1 (zh) 2023-12-14

Family

ID=82286786

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/086142 WO2023236638A1 (zh) 2022-06-09 2023-04-04 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

Country Status (2)

Country Link
CN (1) CN114736879B (zh)
WO (1) WO2023236638A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
US20210230561A1 (en) * 2018-06-04 2021-07-29 Institute Of Animal Science Of Chinese Academy Of Agricultural Sciences Glucose oxidase god mutant and gene and application thereof
CN114736879A (zh) * 2022-06-09 2022-07-12 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

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110577939B (zh) * 2018-06-07 2021-01-26 青岛红樱桃生物技术有限公司 耐热性提高的葡萄糖氧化酶突变体及其编码基因和应用
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
EP3978602A4 (en) * 2019-05-31 2023-07-05 Nanjing Bestzyme Bio-engineering Co., Ltd. THERMOSTABLE GLUCOSE OXIDASE
CN113528476B (zh) * 2021-08-20 2023-01-31 福建福大百特生物科技有限公司 一种葡萄糖氧化酶突变体及其编码基因和高效重组表达
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

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US20210230561A1 (en) * 2018-06-04 2021-07-29 Institute Of Animal Science Of Chinese Academy Of Agricultural Sciences Glucose oxidase god mutant and gene and application thereof
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
CN114736879A (zh) * 2022-06-09 2022-07-12 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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ITTISOPONPISAN SIRAWIT, JEERAPAN ITTHIPON: "In Silico Analysis of Glucose Oxidase from Aspergillus niger: Potential Cysteine Mutation Sites for Enhancing Protein Stability", BIOENGINEERING, vol. 8, no. 11, pages 188, XP093113638, ISSN: 2306-5354, DOI: 10.3390/bioengineering8110188 *

Also Published As

Publication number Publication date
CN114736879A (zh) 2022-07-12
CN114736879B (zh) 2022-09-27

Similar Documents

Publication Publication Date Title
WO2023236638A1 (zh) 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
WO2019233083A1 (zh) 葡萄糖氧化酶god突变体及其基因和应用
WO2019227512A1 (zh) 一种可高效表达角蛋白酶的重组枯草芽孢杆菌工程菌
CN110218708B (zh) 一种细菌漆酶及其基因、制备方法与应用
EP4006149A1 (en) Mutant glucose oxidase (god) having improved thermal stability and gene and application thereof
WO2010053161A1 (ja) 改変型フラビンアデニンジヌクレオチド依存性グルコースデヒドロゲナーゼ
CN113862233B (zh) 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
CN110066777B (zh) 一种内切菊粉酶及其在生产低聚果糖中的应用
CN107012130A (zh) 一种葡萄糖氧化酶突变体及其编码基因和应用
CN117625581B (zh) 一种N-乙酰氨基葡萄糖苷酶突变体Ea2F及其应用
CN114736880B (zh) 酸稳定性提高葡萄糖氧化酶GoxM10的突变体D497N及其衍生突变体和应用
CN114736881B (zh) 酸稳定性提高的葡萄糖氧化酶GoxM10突变体A4D及其衍生突变体和应用
CN110283797B (zh) 一种酪氨酸酶及其基因、工程菌和制备方法
CN108998403B (zh) 一种枯草芽孢杆菌重组菌及其应用
CN115960879A (zh) 一种d-阿洛酮糖3-差向异构酶突变体文库的高通量筛选方法及获得的突变体
CN108795891A (zh) 一种葡萄糖氧化酶CnGODA及其基因与应用
CN116790538B (zh) 一种具有耐碱性的氧化硫化酶突变体及其应用
CN109749948A (zh) 一种利用tef1启动子调控黏玉米谷氨酰胺转氨酶表达的重组毕赤酵母菌株及构建方法
CN114686409B (zh) 增强超氧化物歧化酶基因的表达提高谷氨酰胺转氨酶产量的方法
CN116731989B (zh) 漆酶突变体、基因工程菌及其应用
CN117737039B (zh) 一种N-乙酰氨基葡萄糖苷酶突变体De259AΔ7及其制备与应用
CN114621944B (zh) 酶活提高的精氨酸脱亚胺酶突变体
CN117402858B (zh) 一种耐热性提高的β-葡萄糖苷酶突变体
AU2021100409A4 (en) Recombinant low-temperature catalase, recombinant vector and engineered strain thereof
CN114958789B (zh) 一种葡萄糖脱氢酶突变体、其制备方法及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23818806

Country of ref document: EP

Kind code of ref document: A1