WO2021103123A1 - 一种葡萄糖氧化酶m5god及其编码基因和应用 - Google Patents

一种葡萄糖氧化酶m5god及其编码基因和应用 Download PDF

Info

Publication number
WO2021103123A1
WO2021103123A1 PCT/CN2019/123895 CN2019123895W WO2021103123A1 WO 2021103123 A1 WO2021103123 A1 WO 2021103123A1 CN 2019123895 W CN2019123895 W CN 2019123895W WO 2021103123 A1 WO2021103123 A1 WO 2021103123A1
Authority
WO
WIPO (PCT)
Prior art keywords
glucose oxidase
m5god
recombinant
gene
zaa
Prior art date
Application number
PCT/CN2019/123895
Other languages
English (en)
French (fr)
Inventor
牟海津
刘哲民
苑明雪
郁东兴
郁万帅
Original Assignee
中国海洋大学
尚好科技有限公司
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 中国海洋大学, 尚好科技有限公司 filed Critical 中国海洋大学
Priority to AU2019385785A priority Critical patent/AU2019385785B2/en
Publication of WO2021103123A1 publication Critical patent/WO2021103123A1/zh

Links

Images

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
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A23B4/22—Microorganisms; Enzymes; Antibiotics
    • 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 relates to the technical field of genetic engineering and fermentation engineering, and more specifically to a glucose oxidase M5GOD and its coding gene and application.
  • Glucose oxidase (EC 1.1.3.4, GOD) is a kind of aerobic dehydrogenase, which is mainly distributed in a variety of animals, plants and microorganisms. It uses molecular oxygen as the electron acceptor and specifically reduces ⁇ -D -The catalytic oxidation of glucose to gluconic acid and the production of hydrogen peroxide.
  • Glucose oxidase has a wide range of applications in many fields such as chemistry, pharmacy, food, beverage, clinical diagnosis, and biotechnology, including glucose biosensors for diabetes detection, food preservatives, etc.
  • glucose oxidase can consume oxygen to produce hydrogen peroxide, which has great potential application value.
  • Xu et al. LWT 92(2018):339-346) used glucose oxidase to preserve the freshness of Penaeus vannamei and found that it not only effectively prevents the browning of the shrimp, but also has quality indicators such as color, odor, hardness, elasticity, and chewiness. Good retention.
  • the main production strains of glucose oxidase are Aspergillus niger and Penicillium, but the production yield of Aspergillus niger and Penicillium is low, the purification process is complicated, and the enzyme activity is low in low temperature environment, and the effect in the application of aquatic products is not good.
  • the present invention provides a glucose oxidase M5GOD and its coding gene and application.
  • the present invention obtains a new glucose oxidase gene from Penicillium.
  • the encoded glucose oxidase has high activity and stability in the acid and neutral range, and has good low temperature performance. These characteristics mean that the present invention
  • the new glucose oxidase will have more application value in the preservation of aquatic products.
  • a glucose oxidase M5GOD whose amino acid sequence is shown in SEQ ID NO.2.
  • SEQ ID NO.1 SEQ ID NO.1:
  • the enzyme has a full length of 605 amino acids, and the first 16 amino acids at the N-terminus are the signal peptide sequence "MKSIILASALASLAAA”, SEQ ID NO.5.
  • the theoretical molecular weight of the mature glucose oxidase M5GOD is 63.860KDa, and its amino acid sequence is as SEQ ID NO. 2:
  • the glucose oxidase has a wide pH action range, within the pH range of 3-7, the enzyme can maintain more than 50% of its enzyme activity, and the optimum pH is 5.5.
  • the optimum temperature of the enzyme is 30°C.
  • glucose oxidase gene which encodes the above-mentioned glucose oxidase gene, and the nucleotide sequence of the gene is shown in SEQ ID NO.4.
  • SEQ ID NO. 3 SEQ ID NO. 3:
  • the present invention isolates and clones the glucose oxidase gene M5GOD by the PCR method, and the cDNA full sequence analysis results show that the glucose oxidase gene has a full length of 1815 bp, and the base sequence of the signal peptide is: "ATGAAGTCCATCATTCTTGCCTCTGCCCTCGCCTCTCTAGCTGCAGCC", SEQ ID NO.6,
  • the nucleotide sequence uses TAA as the stop codon. Therefore, the nucleotide sequence encoding the mature glucose oxidase protein is 1767 bp in length, as shown in SEQ ID NO. 4:
  • the glucose oxidase M5GOD gene sequence and the deduced amino acid sequence were BLAST aligned in NCBI, and the gene was 74% identical to the amino acid sequence of glucose oxidase derived from Penicillium. It shows that M5GOD is a new kind of glucose oxidase.
  • the recombinant vector is pPIC ZaA-M5GOD.
  • the glucose oxidase gene of the present invention is inserted between suitable restriction enzyme cutting sites of the expression vector, so that its nucleotide sequence is operably connected with the expression control sequence.
  • a recombinant strain containing the aforementioned gene encoding glucose oxidase or the aforementioned recombinant vector containing the aforementioned gene encoding glucose oxidase or the aforementioned recombinant vector.
  • the recombinant strain is Pichia X33/M5GOD.
  • the host cell is preferably a Pichia pastoris cell
  • the recombinant yeast expression plasmid is preferably transformed into a Pichia pastoris cell (Pichic pastoris) to obtain a recombinant strain X33/M5GOD.
  • a method for preparing glucose oxidase M5GOD including the following steps:
  • the present invention clones a new glucose oxidase gene from Penicillium, and the encoded glucose oxidase has higher enzyme activity under acidic and neutral conditions, and it has better performance in adapting to low temperatures.
  • the present disclosure provides a glucose oxidase M5GOD and its coding gene and application.
  • the technical effect achieved is to provide a low temperature glucose oxidase encoding the above The glucose oxidase gene, the recombinant vector containing the above-mentioned glucose oxidase, the recombinant strain containing the above-mentioned glucose oxidase gene, the method for preparing the above-mentioned glucose oxidase, and the application of the glucose oxidase.
  • Figure 1 is a schematic diagram of the optimal pH value of recombinant glucose oxidase.
  • Figure 2 is a schematic diagram of the pH stability of recombinant glucose oxidase.
  • Figure 3 is a schematic diagram of the optimal reaction temperature for recombinant glucose oxidase.
  • Figure 4 is a schematic diagram of the thermal stability of recombinant glucose oxidase.
  • Figure 5 is a schematic diagram of the volatile base nitrogen content of recombinant glucose oxidase used in fresh-keeping applications.
  • Figure 6 is a schematic diagram of the total number of colonies used for fresh-keeping applications of recombinant glucose oxidation.
  • the embodiment of the invention discloses a glucose oxidase M5GOD and its coding gene and application.
  • Escherichia coli DH5a, Pichia pastoris X33, and vector pPIC ZaA were all purchased from Invitrogen.
  • Restriction endonucleases and ligases were purchased from the company; the others are chemical reagents produced in China.
  • Seed medium NaNO 3 2g, K 2 HPO 4 1g, KCl 0.5g, MgSO 4 0.01g, sucrose 30g;
  • E. coli culture medium LB 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0;
  • Yeast culture medium YPD 2% tryptone, 1% yeast powder, 2% glucose;
  • BMGY medium 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V);
  • BMMY medium Divided by 0.5% methanol instead of glycerol, the remaining components are the same as BMGY, pH 4.0.
  • F1 5'-AGAGAGGCTGAAGCTGAATTCCAGGGCTTCACTCCAGCCG-3', SEQ ID NO.7;
  • the designed primer sequence is a fusion primer
  • the “AGAGAGGCTGAAGCTGAATTC” sequence at the front end is the gene sequence on the pPIC ZaA vector at the junction of the pPIC ZaA vector and the target gene at the EcoR 1 restriction site.
  • R1 5'-TGTTCTAGAAAGCTGGCGGCCGCTTAGGGCTTGTAGTCAGCCAGAA-3', SEQ ID NO.8.
  • the "TGTTCTAGAAAGCTGGCGGCCGC” sequence at the front end is the gene sequence on the pPIC ZaA vector at the junction of the pPIC ZaA vector and the target gene at the Not 1 restriction site.
  • the total DNA of Penicillium was used as a template for amplification.
  • the PCR reaction parameters were: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 2 minutes, and incubation at 72°C for 10 minutes after 30 cycles.
  • the fragment was recovered and ligated with the vector pPIC ZaA for transformation and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.
  • the expression vector pPIC ZaA was double digested (EcoR 1 and Not 1), and the M5GOD encoding glucose oxidase was double digested (EcoR 1 and Not 1), and the gene fragment encoding mature glucose oxidase and the expression vector pPIC were cut out ZaA was connected to construct a yeast expression vector pPIC ZaA-M5GOD and transferred to E. coli competent cells DH5a, and positive transformants were selected for DNA sequencing. The sequencing showed that the transformants with the correct sequence were used for mass preparation of recombinant plasmids.
  • Use restriction enzyme Sac1 to linearize expression plasmid vector DNA, and transform yeast X33 competent cells by electroporation. The transformed cells are spread on YPD plates and incubated at 30°C for 2-3 days. Pick the transformants that grow on the plates.
  • Pichia pastoris expression manual for specific operations.
  • F2 5'-AGAGAGGCTGAAGCTGAATTCATGAAGTCCATCATTCTTGCCTC-3', SEQ ID NO.9;
  • R1 5'-TGTTCTAGAAAGCTGGCGGCCGCTTAGGGCTTGTAGTCAGCCAGAA-3', SEQ ID NO.8.
  • the total DNA of Penicillium was used as a template for amplification.
  • the PCR reaction parameters were: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 2 minutes, and incubation at 72°C for 10 minutes after 30 cycles.
  • the fragment was recovered and ligated with the vector pPIC ZaA for transformation and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.
  • the selected transformants with higher enzyme activity were inoculated into 300ml BMGY liquid medium and cultured at 30°C with 200rpm shaking for 48 hours for cell enrichment; centrifuged at 4000 ⁇ g for 5 min, gently discard the supernatant, and transfer the cells to In 100ml of BMMY liquid medium containing 1% methanol, the culture was induced at 30°C and 200rpm for 72h. During the induction culture, methanol solution was added every 24 hours to keep the final concentration of methanol at about 1%, and the supernatant was collected by centrifugation at 10,000 ⁇ g for 10 min. The activity of glucose oxidase was determined, and the expression level of recombinant glucose oxidase was 15 U/ml.
  • the supernatant of the recombinant glucose oxidase expressed in the shake flask was collected, and the 10kDa membrane package was first used for desalting and concentration, and then purified by anion exchange column chromatography.
  • the collected solution with electrophoresis purity is used as the sample for the study of the expression of the enzyme properties.
  • Coomassie brilliant blue method was used to determine the protein content of the purified enzyme solution, and the specific activity of the enzyme protein was calculated.
  • the purified glucose oxidase samples of Example 3 were tested for enzyme activity at different pH values to determine its optimal pH.
  • Buffer solutions for different pH values glycine-hydrochloric acid buffer with pH 1.0-3.0; acetic acid-sodium acetate buffer with pH 4.0-6.0 and Tris-hydrochloric acid buffer with pH 7.0-9.0, purified glucose oxidation Enzymes are in different buffer systems.
  • the enzyme activity of the glucose oxidase of the present invention is measured by 4-aminoantipyrine spectrophotometry.
  • the hydrogen peroxide produced by catalyzing the dehydrogenation of glucose and the colorless reduced 4-aminoantipyrine and phenol under the action of horseradish peroxidase produce red quinoneimine at 500nm There is maximum light absorption below.
  • the crude enzyme solution is directly diluted with buffer to about 10U/ml. Take 4 test tubes of 150*15, add 2ml buffer, 0.3ml glucose, 0.4ml phenol, 0.1ml 4-aminoantipyrine, 0.1ml horseradish peroxidase, preheat at 30°C for 5min.
  • the enzyme activity X1 (U/mL or U/g) in the sample is calculated according to the following formula:
  • Enzyme activity unit definition Under the condition of pH 6.0 and temperature 30 °C, the amount of enzyme that can oxidize 1 umol of ⁇ -D-glucose to produce D-gluconic acid and hydrogen peroxide per minute is defined as 1 enzyme activity unit (IU ).
  • the enzyme solution was treated in buffers with different pH values at 25°C for 2 hours, and the enzyme activity was measured at the optimum pH to study the pH stability of the enzyme.
  • the analysis results show (see Figure 2) that the pH value is basically stable between 2.0-5.0, and it can maintain more than 80% of the enzyme activity. After pH 6.0 and 7.0 and treatment, the enzyme activity can also maintain about 60% respectively, indicating that the enzyme has good pH stability.
  • thermostability determination is that the glucose oxidase samples are treated at different temperatures of 40°C, 45°C, 50°C, and 55°C for 5 minutes, and then the enzyme activity is measured at 30°C. Thermal stability experiments show (see Figure 4) that the glucose oxidase has basically lost its enzyme activity after being treated at 55°C.
  • Step 1 After selecting fresh grass carp to death, remove the head, tail, internal organs, bones, scales, and skin, and wash with sterile water;
  • Step 2 Use boiling-sterilized knives to divide the fish.
  • the length, width, and thickness of the segmentation are almost uniform, and they are randomly divided into 4 groups.
  • Step 4 Take it out on the sieve and drain at room temperature
  • Step 5 Pack them into polyethylene aseptic fresh-keeping bags and store them in a refrigerator at 4°C.
  • Example 6 the grass carp was preserved and taken out at 0, 2, 4, 6, 8, and 10 days, and the total number of colonies and the content of volatile base nitrogen were determined.
  • the results showed that during the cold storage period, The total number of colonies and the content of volatile base nitrogen of the grass carp treated with the glucose oxidase of the present invention are significantly lower than that of the blank control group. And sensory evaluation of the preservation effect. The results are shown in Table 1.
  • the glucose oxidase of the present invention has high catalytic efficiency under low temperature conditions, and can be effectively used for low-temperature preservation of aquatic products.
  • the aquatic products can be immersed in a low-temperature glucose oxidase solution to remove oxygen in the subsequent preservation process, and generate H 2 O 2 to inhibit the reproduction of microorganisms, effectively inhibit deterioration, and maintain tissue elasticity. It shows that the low-temperature glucose oxidase of the present invention has potential application value in the preservation of aquatic products.

Landscapes

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

Abstract

提供了一种葡萄糖氧化酶M5GOD及其编码基因和应用，涉及基因工程及发酵工程领域。提供了一种葡萄糖氧化酶的氨基酸序列如SEQ ID NO.2所示，且还提供了编码上述葡萄糖氧化酶的编码基因的核苷酸序列如SEQ ID NO.4所示。包含该基因的重组载体和重组菌株及其应用。所述葡萄糖氧化酶具有良好的性质，最适pH 5.5，最适温度30℃，具有较好的适低温性。所述葡萄糖氧化酶作为一种酶制剂，可广泛应用于饲料、食品、医药等行业。

Description

一种葡萄糖氧化酶M5GOD及其编码基因和应用 技术领域
本发明涉及基因工程及发酵工程技术领域，更具体的说是涉及一种葡萄糖氧化酶M5GOD及其编码基因和应用。
背景技术
葡萄糖氧化酶(Glucose oxidase,EC 1.1.3.4,GOD)是一种需氧脱氢酶，主要分布于多种动物、植物和微生物中，以分子氧为电子受体，专一性将β-D-葡萄糖催化氧化成葡萄糖酸并产生过氧化氢。葡萄糖氧化酶在化学、制药、食品、饮料、临床诊断、生物技术等众多领域有广泛的应用，包括用于糖尿病检测的葡萄糖生物传感器、食品防腐剂等。
葡萄糖氧化酶作为天然生物保鲜剂之一，能够消耗氧气产生过氧化氢，具有巨大的潜在应用价值。Xu等(LWT 92(2018):339-346)利用葡萄糖氧化酶保鲜南美白对虾，发现其不仅能有效防止对虾褐变，而且对其色泽、气味、硬度、弹性、咀嚼性等品质指标都有良好的保持作用。
目前葡萄糖氧化酶最主要的生产菌株是黑曲霉和青霉，但黑曲霉和青霉生产产量低、纯化工艺繁杂，且在低温环境下酶活性较低，在水产品保鲜应用方面效果不佳。
因此，如何提供一种葡萄糖氧化酶，优化其理化性质，并提供一种用基因工程方法构建的更优良的生产菌株是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种葡萄糖氧化酶M5GOD及其编码基因和应用。
本发明从青霉中得到了一个新的葡萄糖氧化酶基因，编码的葡萄糖氧化酶在酸性及中性的范围有高活性及稳定性，且具有较好的适低温性能，这些 特点意味着本发明的新的葡萄糖氧化酶在水产品保鲜方面将会更有应用价值。
为了实现上述目的，本发明采用如下技术方案：
一种葡萄糖氧化酶M5GOD，其氨基酸序列如SEQ ID NO.2所示。
其中，SEQ ID NO.1：
Figure PCTCN2019123895-appb-000001
该酶全长605个氨基酸，N端前16个氨基酸为信号肽序列“MKSIILASALASLAAA”，SEQ ID NO.5。
因此，成熟的葡萄糖氧化酶M5GOD的理论分子量为63.860KDa，其氨基酸序列如SEQ ID NO.2：
Figure PCTCN2019123895-appb-000002
Figure PCTCN2019123895-appb-000003
该葡萄糖氧化酶pH作用范围广，在pH值为3～7范围内，该酶能够维持其50％以上的酶活力，最适pH为5.5。该酶最适作用温度30℃。
进一步的：一种葡萄糖氧化酶基因，编码上述的葡萄糖氧化酶的基因，基因的核苷酸序列如SEQ ID NO.4所示。
其中，SEQ ID NO.3：
Figure PCTCN2019123895-appb-000004
Figure PCTCN2019123895-appb-000005
本发明通过PCR的方法分离克隆了葡萄糖氧化酶基因M5GOD，cDNA全序列分析结果表明，葡萄糖氧化酶基因全长1815bp，其中，信号肽的碱基序列为：“ATGAAGTCCATCATTCTTGCCTCTGCCCTCGCCTCTCTAGCTGCAGCC”，SEQ ID NO.6，此外，核苷酸序列以TAA为终止密码子。所以编码成熟葡萄糖氧化酶蛋白的的核苷酸序列全长1767bp，如SEQ ID NO.4所示：
Figure PCTCN2019123895-appb-000006
Figure PCTCN2019123895-appb-000007
Figure PCTCN2019123895-appb-000008
将葡萄糖氧化酶M5GOD基因序列及推导出的氨基酸序列在NCBI中进行BLAST比对，该基因与来源于青霉的葡萄糖氧化酶氨基酸序列一致性为74％。说明M5GOD是一种新的葡萄糖氧化酶。
进一步的，包含上述编码葡萄糖氧化酶的基因的重组载体。
优选的：重组载体为pPIC ZaA-M5GOD。
将本发明的葡萄糖氧化酶基因插入到表达载体合适的限制性酶切位点之间，使其核苷酸序列可操作的与表达调控序列相连接。作为本发明的一个最优选的实施方案，优选为将葡萄糖氧化酶基因插入到质粒pPIC ZaA上的EcoR1和Not 1限制性酶切位点之间，使该核苷酸序列位于启动子的下游并受其调控，得到重组酵母表达质粒pPIC ZaA-M5GOD。
进一步的：包含上述编码葡萄糖氧化酶的基因或上述重组载体的重组菌株。
优选的：重组菌株为毕赤酵母X33/M5GOD。
其中，优选所述宿主细胞为毕赤酵母细胞，优选将重组酵母表达质粒转化毕赤酵母细胞(Pichic pastoris)，得到重组菌株X33/M5GOD。
进一步的：上述葡萄糖氧化酶M5GOD在食品保鲜中的应用。
进一步的：上述基因、上述的重组载体或上述重组菌株在产业化生产葡萄糖氧化酶中的应用。
进一步的：一种制备葡萄糖氧化酶M5GOD的方法，包括以下步骤：
1)用上述的重组载体转化宿主细胞，获得重组菌株；
2)培养重组菌株，诱导葡萄糖氧化酶的表达，收集上清液；
3)回收并纯化上清液，得到葡萄糖氧化酶M5GOD。
本发明从青霉中克隆得到一个新的葡萄糖氧化酶基因，其编码的葡萄糖氧化酶在酸性和中性条件下具有较高的酶活力，并且其具有较好的适低温性能。
经由上述的技术方案可知，与现有技术相比，本发明公开提供了一种葡萄糖氧化酶M5GOD及其编码基因和应用，取得的技术效果为提供了一种适低温的葡萄糖氧化酶、编码上述葡萄糖氧化酶的基因、包含上述葡萄糖氧化酶的重组载体、包含上述葡萄糖氧化酶基因的重组菌株、制备上述葡萄糖氧化酶的方法及葡萄糖氧化酶的应用。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1为重组葡萄糖氧化酶的最适pH值示意图。
图2为重组葡萄糖氧化酶的pH稳定性示意图。
图3为重组葡萄糖氧化酶最适反应温度示意图。
图4为重组葡萄糖氧化酶热稳定性示意图。
图5为重组葡萄糖氧化酶用于保鲜应用的挥发性盐基氮含量示意图。
图6为重组葡萄糖氧化用于保鲜应用的菌落总数示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明实施例公开了一种葡萄糖氧化酶M5GOD及其编码基因和应用。
实验材料和试剂：
菌株和载体：
大肠杆菌DH5a、毕赤酵母X33、载体pPIC ZaA均购自Invitrogen公司。
酶类及其它生化试剂：
限制性内切酶和连接酶均购自公司；其它均为国产生化试剂。
培养基：
种子培养基(/L)：NaNO 3 2g，K 2HPO 4 1g，KCl 0.5g，MgSO 4 0.01g，蔗糖30g；
大肠杆菌培养基LB：1％蛋白胨，0.5％酵母提取物，1％NaCl，pH7.0；
酵母培养基YPD：2％胰蛋白胨、1％酵母粉、2％葡萄糖；
BMGY培养基：1％酵母提取物，2％蛋白胨，1.34％YNB，0.00004％Biotin，1％甘油(V/V)；
BMMY培养基：除以0.5％甲醇代替甘油，其余成份均与BMGY相同，pH4.0。
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
青霉中葡萄糖氧化酶编码基因M5GOD的克隆
提取青霉基因组DNA：
将青霉经种子培养基培养5天后，用无菌滤纸过滤放入研钵中，加入2ml提取液，研磨5min，然后将研磨液置于离心管中，采用基因组提取试剂盒提取。
根据葡萄糖氧化酶基因序列设计合成引物F1和R1：
F1：5'-AGAGAGGCTGAAGCTGAATTCCAGGGCTTCACTCCAGCCG-3'，SEQ ID NO.7；
根据重组连接的引物设计原则，设计的引物序列为融合引物，前端的“AGAGAGGCTGAAGCTGAATTC”序列是pPIC ZaA载体与目的基因在EcoR 1酶切位点连接处的pPIC ZaA载体上的基因序列。
R1：5'-TGTTCTAGAAAGCTGGCGGCCGCTTAGGGCTTGTAGTCAGCCAGAA-3'，SEQ ID NO.8。
其中，前端的“TGTTCTAGAAAGCTGGCGGCCGC”序列是pPIC ZaA载体与目的基因在Not 1酶切位点连接处的pPIC ZaA载体上的基因序列。
以青霉总DNA为模板进行扩增。PCR反应参数为：94℃预变性5min，然后94℃变性30sec，56℃退火30sec，72℃延伸2min，30个循环后72℃保温10min。将该片段回收后与载体pPIC ZaA连接转化后送北京睿博兴科生物技术有限公司测序。
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毕赤酵母中的表达
将表达载体pPIC ZaA进行双酶切(EcoR 1和Not 1)，同时将编码葡萄糖氧化酶的M5GOD双酶切(EcoR 1和Not 1)，切出编码成熟葡萄糖氧化酶的基因片段与表达载体pPIC ZaA连接，构建成酵母表达载体pPIC ZaA-M5GOD转入到大肠杆菌感受态细胞DH5a，挑选阳性转化子进行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Sac1进行线性化表达质粒载体DNA，电击转化酵母X33感受态细胞，转化细胞涂布于YPD平板上，30℃培养2-3天，挑取在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。
以同样的方式构建包含信号肽序列的重组表达，具体为：
根据葡萄糖氧化酶基因序列设计合成引物F2和R1：
F2：5'-AGAGAGGCTGAAGCTGAATTCATGAAGTCCATCATTCTTGCCTC-3'，SEQ ID NO.9；
R1：5'-TGTTCTAGAAAGCTGGCGGCCGCTTAGGGCTTGTAGTCAGCCAGAA-3'，SEQ ID NO.8。
以青霉总DNA为模板进行扩增。PCR反应参数为：94℃预变性5min，然后94℃变性30sec，56℃退火30sec，72℃延伸2min，30个循环后72℃保温10min。将该片段回收后与载体pPIC ZaA连接转化后送北京睿博兴科生物技术有限公司测序。
(2)高葡萄糖氧化酶活性转化子的筛选
用灭过菌的牙签从长有转化子的板上挑取单菌落，按照编号先点到平板上，将平板置于30℃培养箱中培养2天，至菌落长出。按编号从平板上挑取转化子接种于3ml BMGY培养基中于30℃摇床培养48h，离心收集菌体，加入1ml含有1％甲醇的BMMY诱导培养基，继续30℃诱导培养，48h后取样检测各菌株上清液的酶活性，从中筛选出高葡萄糖氧化酶活性的转化子。
实施例3
重组葡萄糖氧化酶在毕赤酵母中的发酵
(1)重组葡萄糖氧化酶在摇瓶中大量表达
将筛选出的酶活较高的转化子接种于300ml BMGY液体培养基中，30℃200rpm振荡培养48h，进行菌体富集；4000×g离心5min，轻柔弃上清，将菌体转接到100ml含有1％甲醇的BMMY液体培养基中，30℃200rpm诱导培养72h。诱导培养期间，每间隔24h补加一次甲醇溶液，使甲醇终浓度保持在1％左右，10000×g离心10min收集上清。测定葡萄糖氧化酶的活力，重组葡萄糖氧化酶的表达量为15U/ml。
(2)重组葡萄糖氧化酶的纯化
收集摇瓶表达的重组葡萄糖氧化酶上清液，首先利用10kDa膜包进行脱盐和浓缩，再经阴离子交换柱层析纯化。以达到电泳纯的收集液作为表达酶学性质研究的样品。利用考马斯亮蓝法测定纯化后酶液的蛋白质含量,计算得到酶蛋白的比活力。
实施例4
重组葡萄糖氧化酶的部分性质分析
(1)葡萄糖氧化酶M5GOD的最适pH及pH稳定性
经纯化的实施例3葡萄糖氧化酶样品在不同pH值下测定酶活以确定其最适pH。配制不同pH值所用缓冲液：pH 1.0-3.0的甘氨酸-盐酸缓冲液；pH4.0-6.0的乙酸-乙酸钠系列缓冲液及pH7.0-9.0的Tris-盐酸系列缓冲液，纯化的葡萄糖氧化酶在不同的缓冲体系。
重组葡萄糖氧化酶的酶活力检测方法
采用4-氨基安替吡啉分光光度法对本发明的葡萄糖氧化酶进行酶活测定。在有氧条件下，催化葡萄糖脱氢产生的过氧化氢与无色的还原型4-氨基安替吡啉和苯酚在辣根过氧化物酶的作用下，生成红色的醌亚胺，在500nm 下有最大的光吸收。将粗酶液直接用缓冲液稀释至约10U/ml。取4支150*15的试管，加入2ml缓冲液、0.3ml葡萄糖、0.4ml苯酚、0.1ml 4-氨基安替吡啉、0.1ml辣根过氧化物酶，30℃预热5min。向其中一管加入0.1ml蒸馏水，作为空白调零。水浴锅放在分光光度计旁以方便操作，向样品管中加入0.1ml样品溶液，此时开始计时，涡旋混匀后立即在500nm波长处用1cm比色杯比色。读取30sec时吸光度值为A 0，再反应1min后，读取吸光度值A 1，得出ΔA 500＝A 1-A 0。
酶活计算公式：
试样中酶活力X1(U/mL或U/g)按照如下公式计算：
X1＝ΔA 500×f×B×1000/(887×t×A×d)＝33.82×ΔA 500×f
式中：
f---------------------酶液稀释倍数
B--------------------反应液体积(3ml)
1000----------------消光系数单位转换系数
887-----------------消光系数(L·mol -1·cm -1)
t---------------------反应时间(min)，即读数A1与A0之间的时间差值1min。
A--------------------加入样品体积(0.1ml)
d--------------------比色皿的厚度(cm)
酶活单位定义：在pH6.0，温度30℃条件下，每分钟能将1umol的β-D-葡萄糖氧化生成D-葡萄糖酸和过氧化氢的酶量，定义为1个酶活性单位(IU)。
30℃下测定的最适pH结果(参见图1)。
表明最适pH值为5.5，在pH值4.0-7.0范围内，酶活力能够维持在60％以上。
将酶液在不同pH值的缓冲液中于25℃下处理2h，在最适pH下测定酶活性以研究该酶的pH稳定性。分析结果表明(参见图2)pH值2.0-5.0之间基本稳定，能够维持80％以上的酶活力。经pH6.0和7.0和处理后酶活力也能分别保持60％左右，说明该酶具有较良好的pH稳定性。
(2)葡萄糖氧化酶M5GOD的最适温度及热稳定性
在pH值6.0条件下，测定不同温度5-70℃下已纯化葡萄糖氧化酶样品的酶活力。分析实验结果表明，该酶的最适反应温度为30℃，在20-50℃之间依然具有60％以上的酶活力(参见图3)。
热稳定性测定为葡萄糖氧化酶样品在不同温度40℃、45℃、50℃、55℃下处理5min后，在30℃下进行酶活性测定。热稳定性实验表明(参见图4)该葡萄糖氧化酶在55℃下处理后已基本失去酶活力。
实施例5
重组葡萄糖氧化酶的保鲜应用实验
步骤1，选用鲜活草鱼致死后，去头去尾去内脏去骨去鱼鳞去皮，用无菌水清洗；
步骤2，用经过煮沸消毒的刀具进行鱼肉分割，分割成长、宽、厚几乎均匀一致，随机分为4组。
步骤3，将干净鱼肉在实施例3制备的葡萄糖氧化酶和葡萄糖的混合液中浸渍一段时间；葡萄糖氧化酶溶液酶活为1U/ml，葡萄糖为4％，鱼∶保鲜剂＝1∶1(kg/L)，浸渍时间为10min。
步骤4，取出于筛绢上室温沥干；
步骤5，分装到聚乙烯无菌保鲜袋中，4℃冷藏保存。
实施例7
本发明重组葡萄糖氧化酶在草鱼冷藏保鲜中，对草鱼品质的影响：
按照实施例6，对草鱼进行保藏，第0，2，4，6，8，10d取出，测定菌落总数，挥发性盐基氮含量，结果(参见图5和图6)表明在冷藏时间内，经本发明葡萄糖氧化酶处理的草鱼的菌落总数和挥发性盐基氮含量都明显低于空白对照组。并对保鲜效果进行感官评价。结果见表1。
表1
Figure PCTCN2019123895-appb-000009
Figure PCTCN2019123895-appb-000010
综上所述，本发明葡萄糖氧化酶在低温条件下催化效率高，可有效用于水产品低温保鲜。在水产品保鲜中应用时，水产品通过在低温葡萄糖氧化酶溶液中浸渍，能够在后续保鲜过程中除氧，产生H 2O 2抑制微生物的繁殖，有效抑制变质，保持组织弹性。说明本发明低温葡萄糖氧化酶在水产品保鲜中有潜在应用价值。
本说明书中各个实施例采用递进的方式描述，每个实施例重点说明的都是与其他实施例的不同之处，各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (10)

  1. 一种葡萄糖氧化酶M5GOD，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
  2. 一种编码权利要求1所述葡萄糖氧化酶的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.4所示。
  3. 包含权利要求2所述编码葡萄糖氧化酶的基因的重组载体。
  4. 根据权利要求3所述的重组载体，其特征在于，所述重组载体为pPIC ZaA-M5GOD。
  5. 根据权利要求4所述的重组载体，其特征在于，所述重组载体pPIC ZaA-M5GOD的制备方法为将葡萄糖氧化酶的基因插入到质粒pPIC ZaA上的EcoR 1和Not 1限制性酶切位点之间，使该核苷酸序列位于启动子的下游并受其调控，得到重组酵母表达质粒pPIC ZaA-M5GOD。
  6. 包含权利要求2所述编码葡萄糖氧化酶的基因或权利要求3或4所述重组载体的重组菌株。
  7. 根据权利要求6所述的重组菌株，其特征在于，所述重组菌株为毕赤酵母X33/M5GOD：用重组酵母表达质粒pPIC ZaA-M5GOD转化毕赤酵母细胞即得。
  8. 权利要求1所述葡萄糖氧化酶M5GOD在食品保鲜中的应用。
  9. 权利要求2所述基因、权利要求3或4所述的重组载体或权利要求5或6所述重组菌株在产业化生产葡萄糖氧化酶中的应用。
  10. 权利要求1所述葡萄糖氧化酶M5GOD的制备方法，其特征在于，包括以下步骤：
    1)用权利要求3或4所述的重组载体转化宿主细胞，获得重组菌株；
    2)培养所述重组菌株，诱导葡萄糖氧化酶的表达，收集上清液；
    3)回收并纯化所述上清液，得到葡萄糖氧化酶M5GOD。
PCT/CN2019/123895 2019-11-25 2019-12-09 一种葡萄糖氧化酶m5god及其编码基因和应用 WO2021103123A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2019385785A AU2019385785B2 (en) 2019-11-25 2019-12-09 Glucose oxidase M5GOD and coding genes and applications thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911168042.5A CN110885801B (zh) 2019-11-25 2019-11-25 一种葡萄糖氧化酶m5god及其编码基因和应用
CN201911168042.5 2019-11-25

Publications (1)

Publication Number Publication Date
WO2021103123A1 true WO2021103123A1 (zh) 2021-06-03

Family

ID=69748720

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/123895 WO2021103123A1 (zh) 2019-11-25 2019-12-09 一种葡萄糖氧化酶m5god及其编码基因和应用

Country Status (3)

Country Link
CN (1) CN110885801B (zh)
AU (1) AU2019385785B2 (zh)
WO (1) WO2021103123A1 (zh)

Families Citing this family (1)

* 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

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101481680A (zh) * 2009-01-19 2009-07-15 崔增学 葡萄糖氧化酶生产方法
CN101955953A (zh) * 2010-09-09 2011-01-26 中国农业科学院生物技术研究所 葡萄糖氧化酶突变基因及其表达和应用
CN103981158A (zh) * 2009-12-05 2014-08-13 天野酶株式会社 突变酶及其用途
CN103981159A (zh) * 2014-06-05 2014-08-13 青岛蔚蓝生物集团有限公司 一种葡萄糖氧化酶突变体及其应用
US20140305809A1 (en) * 2011-09-08 2014-10-16 Centre National De La Recherche Scientique Penicillium amagasakiense glucose oxidase mutants
CN104711274A (zh) * 2015-03-17 2015-06-17 中国科学院微生物研究所 一种重组尼崎青霉葡萄糖氧化酶的制备方法及其应用
CN105420252A (zh) * 2015-12-07 2016-03-23 河北省微生物研究所 葡萄糖氧化酶基因god、利用其编码的蛋白、转化的巴斯德毕赤酵母及其制备
CN108220262A (zh) * 2018-02-26 2018-06-29 大连大学 一种海洋低温葡萄糖氧化酶及其在海产品保鲜中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103275942B (zh) * 2013-04-03 2014-09-24 北京挑战生物技术有限公司 一种葡萄糖氧化酶godj4a及其基因和应用
CN109652390A (zh) * 2019-02-25 2019-04-19 大连大学 一种海洋低温葡萄糖氧化酶及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101481680A (zh) * 2009-01-19 2009-07-15 崔增学 葡萄糖氧化酶生产方法
CN103981158A (zh) * 2009-12-05 2014-08-13 天野酶株式会社 突变酶及其用途
CN101955953A (zh) * 2010-09-09 2011-01-26 中国农业科学院生物技术研究所 葡萄糖氧化酶突变基因及其表达和应用
US20140305809A1 (en) * 2011-09-08 2014-10-16 Centre National De La Recherche Scientique Penicillium amagasakiense glucose oxidase mutants
CN103981159A (zh) * 2014-06-05 2014-08-13 青岛蔚蓝生物集团有限公司 一种葡萄糖氧化酶突变体及其应用
CN104711274A (zh) * 2015-03-17 2015-06-17 中国科学院微生物研究所 一种重组尼崎青霉葡萄糖氧化酶的制备方法及其应用
CN105420252A (zh) * 2015-12-07 2016-03-23 河北省微生物研究所 葡萄糖氧化酶基因god、利用其编码的蛋白、转化的巴斯德毕赤酵母及其制备
CN108220262A (zh) * 2018-02-26 2018-06-29 大连大学 一种海洋低温葡萄糖氧化酶及其在海产品保鲜中的应用

Also Published As

Publication number Publication date
CN110885801B (zh) 2021-04-30
AU2019385785A1 (en) 2021-06-10
AU2019385785B2 (en) 2022-11-03
CN110885801A (zh) 2020-03-17

Similar Documents

Publication Publication Date Title
CN112813052B (zh) 一种低温活性提高的外切菊粉酶突变体MutDP121ET6
CN112646794B (zh) 低温活性提高的外切菊粉酶突变体MutY119V
CN112831485B (zh) 一种低温活性改良的外切菊粉酶突变体MutDR121EH9
CN112725306B (zh) 热盐性改变的菊粉酶突变体MutY119T及其应用
CN112980813B (zh) 低温改良的外切菊粉酶突变体MutS117G
CN112646792A (zh) 一种热稳定性降低的低温外切菊粉酶突变体MutA122Δ5及应用
CN112725304A (zh) 一种高活性的低温外切菊粉酶突变体MutAP122EK5及应用
CN101348794B (zh) 一种高活力葡萄糖氧化酶的编码基因及制备方法和应用
CN112646793A (zh) 低温适应性和盐适应性改良的菊粉酶突变体MutS120D及其应用
US20230193215A1 (en) Mutant glucose oxidase (god) having improved thermal stability and gene and application thereof
CN107012130A (zh) 一种葡萄糖氧化酶突变体及其编码基因和应用
CN112852782A (zh) 一种低温适应性改良的低温外切菊粉酶突变体MutDL121EK5及应用
CN112725307A (zh) 一种耐热性降低的低温外切菊粉酶突变体MutG169Δ4及应用
CN114835783B (zh) NCgl2747基因突变体及其在制备L-赖氨酸中的应用
EP3978602A1 (en) Thermostable glucose oxidase
CN108251389A (zh) 一种耐热性提高的葡萄糖氧化酶突变体
CN109468288A (zh) 一种高效降解组胺的新多铜氧化酶
CN101348795B (zh) 一种葡萄糖氧化酶的编码基因及制备方法和应用
CN108118037A (zh) 一种耐热性提高的葡萄糖氧化酶突变体
WO2021103123A1 (zh) 一种葡萄糖氧化酶m5god及其编码基因和应用
CN107475222A (zh) 基因工程改造的耐热人溶菌酶
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
WO2018196881A1 (zh) 一种葡萄糖氧化酶CnGODA及其基因与应用
CN112143717A (zh) 比活力提高的葡萄糖氧化酶突变体
CN113403292B (zh) 一种披发糖多孢菌来源的可降解生物胺的含铜胺氧化酶及其应用

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref document number: 2019385785

Country of ref document: AU

Date of ref document: 20191209

Kind code of ref document: A

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

Ref document number: 19954177

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19954177

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 19954177

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 05/06/2023)