AU2019385785B2 - Glucose oxidase M5GOD and coding genes and applications thereof - Google Patents
Glucose oxidase M5GOD and coding genes and applications thereof Download PDFInfo
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- AU2019385785B2 AU2019385785B2 AU2019385785A AU2019385785A AU2019385785B2 AU 2019385785 B2 AU2019385785 B2 AU 2019385785B2 AU 2019385785 A AU2019385785 A AU 2019385785A AU 2019385785 A AU2019385785 A AU 2019385785A AU 2019385785 B2 AU2019385785 B2 AU 2019385785B2
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- 230000031700 light absorption Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- AZQWKYJCGOJGHM-UHFFFAOYSA-N para-benzoquinone Natural products O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
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- 239000000843 powder Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 101150116497 sacm1l gene Proteins 0.000 description 1
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- 238000002798 spectrophotometry method Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
- A23B4/00—Preservation of meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
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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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Abstract
The present invention discloses a glucose oxidase M5GOD and coding genes
and applications thereof, and relates to the field of genetic engineering and
fermentation engineering. An amino acid sequence of the glucose oxidase provided
by the present invention is shown as SEQ ID NO.2. Moreover, the present
invention further provides coding genes coding the glucose oxidase, wherein a
nucleotide sequence of the coding genes is shown as SEQ ID NO.4. The present
invention also provides recombinant vectors and recombinant strains including the
genes and applications of the recombinant vectors and recombinant strains. The
glucose oxidase in the present invention has excellent properties, has an optimum
pH value of 5.5 and an optimum temperature of 30°C, and also has excellent
hypothermophile property. The glucose oxidase serves as a novel enzyme
preparation and can be widely applied to industries of feed, foods and medicines.
1
Description
Description
GLUCOSE OXIDASE M5GOD AND CODING GENES AND APPLICATIONS THEREOF
Technical Field
The present invention relates to the technical field of genetic engineering and fermentation engineering, and particularly relates to a glucose oxidase M5GOD and coding genes and applications thereof.
Background
Glucose oxidase (ECI.1.3.4, GOD) is an aerobic dehydrogenase, is mainly distributed in multiple animals, plants and microbes, takes molecular oxygen as an electron acceptor, and is capable of specifically catalytically oxidizing p-D-glucose into gluconic acid and producing hydrogen peroxide. The glucose oxidase has wide applications in many fields such as chemistry, pharmacy, foods, beverages, clinical diagnosis and biotechnologies, and includes glucose biosensors, food preservatives and the like used in diabetes detection. The glucose oxidase serves as one of the natural biological preservatives, is capable of consuming oxygen to produce hydrogen peroxide, and has huge potential application value. Xu et al. (LWT 92 (2018): 339-346) preserve litopenaeus vannamei by utilizing the glucose oxidase, and discover that the glucose oxidase not only effectively prevents browning of prawns, but also has excellent effects of maintaining quality indexes such as color, smell, hardness, elasticity and chewiness. At present, the major producing strains of the glucose oxidase include Aspergillus niger and penicillium. However, the Aspergillus niger and penicillium are low in production output and complicated in purification process, are low in enzyme activity in a low-temperature environment, and have a poor effect in the preservation application aspect of aquatic products.
Description
Therefore, how to provide the glucose oxidase, optimize physicochemical properties of the glucose oxidase and provide a more excellent producing strain constructed by a genetic engineering method is a problem that urgently needs to be solved by those skilled in the art.
Summary In view of this, the present invention provides a glucose oxidase M5GOD and coding genes and applications thereof. In the present invention, a new glucose oxidase gene is obtained from penicillium, and coded glucose oxidase has high activity and stability in acidic and neutral ranges and has excellent hypothermophile property. The characteristics mean that the new glucose oxidase in the present invention will have higher application value in preservation of aquatic products. In order to realize the above purpose, the present invention adopts the following technical solution: An amino acid sequence of the glucose oxidase M5GOD is shown as SEQ ID NO.2. SEQ ID NO.1 is as follows: MKSIILASALASLAAAQGFTPAEQIDVQASLISDPNKVAGQTFDYIIAG GGLTGLTVAAKLSENPNITVLVIEKGFYESNNGPIIENPNDYGLIFGSSVDQ NYLTVPMAINNRTLEVKSGKGLGGSTLINGDSWTGPDKVQIDSWETVLG NTGWNYDALKGYMKEAELARYPTASQIAAGIYFNETCHGFNGTVNAGPR DDGTPYSPLMKALMNTTSAKGVPTQLDFLCGRPRGVSMIYNNLLPDQTRA DAAREWLLPNYKRPNLSILTGQVVGKVLFTQTATGPKATGVNFGTNKAIN FNVLAKHEVLLAAGSAISPLILEHSGIGLKSVLDQFNITQLVELPVGLNMQ DQTTTTVRARAKASSAGQGQAVYFANFTEVFGDYSARATDLLNTKLSQW ANETVARGGFNNATALLIQYENYRNWLLNEDVAYVELFLDTNGKMNFDL WDLIPFTRGSTHIAHADPYLQSFSNNPMFLLNELDLLGQAAGSMLAREIQN SGELANYFDGEDIPGANLLPYNATLDGWVGYVKQNFRANWHAVGTCSM
Description
MSRELGGVVDPTAKVYGTQGLRVIDGSIPPTQVSSHVMTVFYGMALKIAD AVLADYKP. The full length of the glucose oxidase includes 605 amino acids, and first 16 amino acids on terminal N refer to a signal peptide sequence 'MKSIILASALASLAAA', SEQ ID NO.5. Therefore, a theoretical molecular weight of mature glucose oxidase M5GOD is 63.860 KDa, and an amino acid sequence of the glucose oxidase is shown as SEQ ID NO.2 as follows: QGFTPAEQIDVQASLISDPNKVAGQTFDYIIAGGGLTGLTVAAKLSENP NITVLVIEKGFYESNNGPIIENPNDYGLIFGSSVDQNYLTVPMAINNRTLEV KSGKGLGGSTLINGDSWTGPDKVQIDSWETVLGNTGWNYDALKGYMKE AELARYPTASQIAAGIYFNETCHGFNGTVNAGPRDDGTPYSPLMKALMNT TSAKGVPTQLDFLCGRPRGVSMIYNNLLPDQTRADAAREWLLPNYKRPNL SILTGQVVGKVLFTQTATGPKATGVNFGTNKAINFNVLAKHEVLLAAGSAI SPLILEHSGIGLKSVLDQFNITQLVELPVGLNMQDQTTTTVRARAKASSAG QGQAVYFANFTEVFGDYSARATDLLNTKLSQWANETVARGGFNNATALL IQYENYRNWLLNEDVAYVELFLDTNGKMNFDLWDLIPFTRGSTHIAHADP YLQSFSNNPMFLLNELDLLGQAAGSMLAREIQNSGELANYFDGEDIPGANL LPYNATLDGWVGYVKQNFRANWHAVGTCSMMSRELGGVVDPTAKVYG TQGLRVIDGSIPPTQVSSHVMTVFYGMALKIADAVLADYKP. The glucose oxidase is wide in pH operation range. In a pH range of 3-7, the glucose oxidase may maintain enzyme activity of 50% or higher and has an optimum pH value of 5.5. An optimum operation temperature of the glucose oxidase is 30°C. Further, a glucose oxidase gene can code genes of the glucose oxidase. A nucleotide sequence of the genes is shown as SEQ ID NO.4. SEQ ID NO.3 is as follows: ATGAAGTCCATCATTCTTGCCTCTGCCCTCGCCTCTCTAGCTGCAGC CCAGGGCTTCACTCCAGCCGAGCAGATTGATGTCCAGGCCAGCCTGAT CTCCGACCCTAACAAGGTCGCCGGCCAGACATTCGACTACATCATCGCT
Description
Description
CCAACTGGCACGCTGTCGGGACTTGTTCTATGATGTCCCGGGAGCTTGG TGGTGTTGTTGATCCTACGGCCAAGGTCTACGGTACCCAGGGTCTTCGT GTCATTGATGGTTCTATTCCACCCACCCAGGTGTCATCTCACGTTATGA CCGTTTTCTACGGTATGGCTTTGAAGATTGCCGATGCTGTTCTGGCTGA CTACAAGCCC. In the present invention, by virtue of a PCR method, the glucose oxidase gene M5GOD is separated and cloned. cDNA complete sequence analysis results show that, the full length of the glucose oxidase gene is 1815 bp, wherein a base sequence of the signal peptide is ATGAAGTCCATCATTCTTGCCTCTGCCCTCGCCTCTCTAGCTGCAGCC', SEQ ID NO.6. In addition, the nucleotide sequence takes TAA as a termination codon. Therefore, a nucleotide sequence coding mature glucose oxidase proteins has a full length of 1767 bp, as shown in SEQ ID NO.4 as follows: CAGGGCTTCACTCCAGCCGAGCAGATTGATGTCCAGGCCAGCCTG ATCTCCGACCCTAACAAGGTCGCCGGCCAGACATTCGACTACATCATCG CTGGAGGTGGTCTGACAGGTCTTACCGTTGCGGCCAAGCTGTCTGAGA ACCCTAACATCACCGTCCTTGTCATCGAAAAGGGCTTCTACGAGTCCAA TAATGGGCCCATCATCGAAAACCCCAACGACTATGGCTTGATCTTCGGT AGCTCTGTTGACCAAAACTACCTCACCGTTCCCATGGCCATCAACAACC GTACCCTGGAAGTCAAGTCTGGCAAGGGTCTCGGTGGTTCCACGTTGAT TAACGGTGACTCCTGGACCGGTCCCGACAAGGTCCAGATTGACTCCTG GGAGACTGTCTTGGGAAATACCGGTTGGAACTATGACGCCCTCAAGGG GTACATGAAGGAAGCCGAGCTTGCTCGTTACCCAACCGCCAGTCAGAT TGCCGCCGGTATCTACTTCAACGAAACCTGCCATGGATTCAACGGCACC GTTAACGCCGGACCCCGTGATGATGGTACCCCTTACTCTCCCCTTATGA AAGCCCTCATGAACACCACCTCTGCCAAGGGTGTTCCCACTCAGCTTGA CTTCCTCTGCGGTCGCCCTCGTGGTGTCTCCATGATCTACAACAACTTG CTGCCTGACCAGACCCGTGCGGATGCTGCTCGCGAGTGGCTTCTTCCCA ACTATAAGCGCCCCAACTTGAGCATTCTTACCGGCCAGGTTGTTGGAAA GGTTCTCTTCACTCAAACCGCGACTGGCCCCAAGGCTACTGGTGTTAAT
Description
TTTGGCACCAACAAGGCCATCAATTTCAACGTCTTGGCCAAGCACGAG GTCCTTTTGGCTGCTGGCTCTGCCATCTCGCCCCTAATCCTCGAGCACTC TGGTATTGGTCTAAAGTCTGTCCTCGACCAGTTCAATATCACCCAGCTC GTCGAGCTTCCCGTCGGTCTCAATATGCAGGACCAGACCACCACCACTG TCCGCGCCCGAGCCAAGGCGTCTTCTGCTGGTCAGGGCCAGGCCGTCTA CTTTGCCAACTTCACTGAGGTCTTTGGTGACTACTCCGCTCGAGCTACC GATTTACTCAACACCAAGCTCTCCCAGTGGGCCAACGAGACTGTTGCAC GCGGAGGCTTCAACAACGCCACCGCTCTCTTAATCCAGTATGAGAACT ACCGTAACTGGCTCCTGAACGAGGACGTTGCCTATGTCGAGCTTTTCCT CGACACCAACGGAAAGATGAACTTTGACTTGTGGGATCTCATCCCCTTC ACGCGCGGCTCTACGCACATAGCACACGCCGACCCTTATCTGCAGTCCT TCTCCAACAATCCCATGTTCCTGTTGAACGAGCTTGACCTTCTTGGCCA GGCTGCTGGCTCGATGCTGGCTCGTGAGATCCAGAACTCGGGTGAGCT GGCCAACTACTTTGACGGCGAGGATATCCCCGGGGCAAATCTCTTGCCC TACAACGCTACCCTCGATGGCTGGGTTGGATATGTCAAGCAGAACTTTC GTGCCAACTGGCACGCTGTCGGGACTTGTTCTATGATGTCCCGGGAGCT TGGTGGTGTTGTTGATCCTACGGCCAAGGTCTACGGTACCCAGGGTCTT CGTGTCATTGATGGTTCTATTCCACCCACCCAGGTGTCATCTCACGTTAT GACCGTTTTCTACGGTATGGCTTTGAAGATTGCCGATGCTGTTCTGGCT GACTACAAGCCC. By performing blast aligning on the sequence of the glucose oxidase M5GOD gene and a derived amino acid sequence in NCBI, amino acid sequence identity of the gene and the glucose oxidase derived from the penicillium is 74%, which indicates that the M5GOD is a new glucose oxidase. Further, the present invention provides a recombinant vector including genes coding the glucose oxidase. Preferably, the recombinant vector is pPIC ZaA-M5GOD. The glucose oxidase gene in the present invention is inserted into appropriate restriction sites of an expression vector, so that the nucleotide sequence of the glucose oxidase gene is operably connected with an expression regulation
Description
sequence. As a most preferred embodiment of the present invention, preferably the glucose oxidase gene is inserted between EcoR 1 and Not 1 restriction sites on plasmid pPIC ZaA. The nucleotide sequence is located at the downstream of a promoter and regulated by the promoter, thereby obtaining a recombinant yeast expression plasmid pPIC ZaA-M5GOD. Further, the present invention provides a recombinant strain comprising the gene coding the glucose oxidase or the recombinant vector. Preferably, the recombinant strain is pichia pastoris X33/M5GOD. Preferably, the host cells are pichic pastoris. Preferably, the recombinant yeast expression plasmid is used to transform the pichic pastoris, thereby obtaining the recombinant strain X33/M5GOD. Further, the present invention provides an application of the glucose oxidase M5GOD in food preservation. Further, the present invention provides an application of the gene, the recombinant vector or the recombinant strain in industrial production of the glucose oxidase. Further, a method for preparing the glucose oxidase M5GOD includes the following steps: 1) transforming host cells by using the recombinant vector so as to obtain a recombinant strain; 2) culturing the recombinant strain, inducing expression of glucose oxidase, and collecting supernatant; 3) recovering and purifying the supernatant, thereby obtaining the glucose oxidase M5GOD. In the present invention, the new glucose oxidase gene is cloned from the penicillium, and the coded glucose oxidase has higher enzyme activity under acidic and neutral conditions and has excellent hypothermophile property. Through the above technical solutions, compared with the prior art, the present invention discloses the glucose oxidase M5GOD as well as the coding genes and applications thereof. Technical effects are achieved as follows: the
Description
hypothermophile glucose oxidase, the gene coding the glucose oxidase, the recombinant vector including the glucose oxidase, the recombinant strain including the glucose oxidase gene, the method for preparing the glucose oxidase and the application of the glucose oxidase are provided.
Description of Drawings
To more clearly illustrate technical solutions in embodiments of the present invention or in the prior art, drawings to be used in the description of the embodiments or the prior art are simply introduced below. Apparently, the drawings in the descriptions below are the embodiments of the present invention only. Other drawings may be obtained by those ordinary skilled in the art according to the provided drawings on premise of not contributing creative work. Fig. 1 is a schematic diagram of an optimum pH value of recombinant glucose oxidase. Fig. 2 is a schematic diagram of pH stability of recombinant glucose oxidase. Fig. 3 is a schematic diagram of an optimal reaction temperature of recombinant glucose oxidase. Fig. 4 is a schematic diagram of heat stability of recombinant glucose oxidase. Fig. 5 is a schematic diagram of volatile basic nitrogen content of recombinant glucose oxidase applied to preservation. Fig. 6 is a schematic diagram of total bacterial count of recombinant glucose oxidase applied to preservation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and fully described below in combination with drawings in the embodiments of the present invention. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other
Description
embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention. The embodiments of the present invention disclose a glucose oxidase M5GOD as well as coding genes and applications thereof. Experimental materials and reagents: Strains and vectors: Escherichia coli DH5a, pichia pastoris X33 and a vector pPIC ZaA are all purchased from Invitrogen Corporation. Enzymes and other biochemical reagents: Restriction enzymes and ligase are purchased from the corporation, and the rest are domestic biochemical reagents. Culture media: Seed medium (/L): 2 g of NaNO3, 1 g of K2 HPO4 , 0.5 g of KCl, 0.01 g of MgSO4 and 30 g of sucrose; Escherichia coli culture medium LB: 1% of peptone, 0.5% of yeast extract and 1% of NaCl, having a pH value of 7.0; Yeast medium: 2% of peptone, 1% of yeast powder and 2% of glucose; BMGY medium: 1% of yeast extract, 2% of peptone, 1.34% of YNB, 0.00004% of Biotin and 1% of glycerin (V/V); BMMY medium: in addition to glycerin replaced with 0.5% of methanol, other components are the same as those of the BMGY medium, and a pH value of the BMMY medium is 4.0. Notes: Molecular biology experimental methods that are not described in detail in embodiments below are performed by referring to specific methods listed in the third edition of Molecular Cloning Manual written by J. Sambrook, or performed according to kit and product specifications. Embodiment 1 Cloning of glucose oxidase coding gene M5GOD in penicillium Extraction of genome DNA of penicillium:
Description
The penicillium was cultured in a seed medium for 5 days, filtered by sterile filter paper and placed in a mortar; 2 ml of an extracting solution was added, and grinding was performed for 5 min; and grinding fluid was placed in a centrifuge tube, and the DNA was extracted by a genome extraction kit. Synthetic primers F1 and RI were designed according to a sequence of the glucose oxidase gene as follows: Fl: 5'-AGAGAGGCTGAAGCTGAATTCCAGGGCTTCACTCCAGCCG-3', SEQIDNO. 7; According to a primer design principle of recombinant connection, the designed primer sequence is a fusion primer, and a sequence 'AGAGAGGCTGAAGCTGAATTC' at the front end is a gene sequence of a vector pPIC ZaA and a target gene on the vector pPIC ZaA at the junction of EcoR 1 restriction sites. RI: 5'-TGTTCTAGAAAGCTGGCGGCCGCTTAGGGCTTGTAGTCAGC CAGAA-3', SEQIDNO. 8, wherein a sequence 'TGTTCTAGAAAGCTGGCGGCCGC' at the front end is a gene sequence of the vector pPIC ZaA and the target gene on the vector pPIC ZaA at the junction of Not 1 restriction sites. Amplification was performed by taking total penicillium DNA as a template. PCR parameters are as follows: pre-degeneration at 94°C for 5 min, degeneration at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 2 min, and heat preservation at 72°C for 10 min after 30 cycles. The recovered segment was linked with the vector pPIC ZaA for transformation and then transferred to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. Embodiment 2 Construction of engineered strains of glucose oxidase (1) Construction of expression vector and expression in pichia pastoris An expression vector pPIC ZaA was subjected to double enzyme digestion (EcoR 1 and Not 1), and M5GOD coding the glucose oxidase was subjected to
Description
double enzyme digestion (EcoR 1 and Not 1); digested gene segments coding mature glucose oxidase were linked with the expression vector pPIC ZaA so as to construct a yeast expression vector pPIC ZaA- M5GOD; the yeast expression vector was transferred into Escherichia coli competent cells DH5a; positive transformants were selected for performing DNA sequencing, wherein sequencing showed that transformants with accurate sequences were used for large-scale preparation of recombinant plasmids; the expression plasmid vector DNA was linearized by a restriction enzyme Sac1; yeast X33 competent cells were subjected to electric shock transformation; transformed cells were coated on a YPD plate and cultured at 30°C for 2-3 days; and transformants growing on the plate were selected for conducting further expression experiments. Specific operations should refer to pichia pastoris expression operation manual. Recombinant expression including signal peptide sequences was constructed in the same way as follows: Synthetic primers F2 and RI were designed according to a sequence of the glucose oxidase gene as follows: F2: 5'-AGAGAGGCTGAAGCTGAATTCATGAAGTCCATCATTCTTGCC TC-3', SEQIDNO. 9; RI: 5'-TGTTCTAGAAAGCTGGCGGCCGCTTAGGGCTTGTAGTCAGC CAGAA-3', SEQIDNO. 8. Amplification was performed by taking total penicillium DNA as a template. PCR parameters are as follows: pre-degeneration at 94°C for 5 min, degeneration at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 2 min, and heat preservation at 72°C for 10 min after 30 cycles. The recovered segment was linked with the vector pPIC ZaA for transformation and then transferred to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing. (2) Screening of transformants with high glucose oxidase activity
Description
Single colonies were selected from a plate on which transformants grow by using sterilized toothpicks; the colonies were placed on the plate according to numbers; the plate was cultured in an incubator at 30°C for 2 days until bacterial colonies grow; the transformants were picked from the plate according to numbers and inoculated in a 3 ml of BMGY medium so as to perform shaking culture at °C for 48 h; bacterial bodies were centrifugally collected; 1 ml of BMMY induction medium including 1% of methanol was added; induction culture was continuously performed at 30°C; enzyme activity of supernatant of each strain was subjected to sampling detection within 48 h; and the transformants with high glucose oxidase activity were screened from the strains. Embodiment 3 Fermentation of recombinant glucose oxidase in pichia pastoris (1) Large-scale expression of recombinant glucose oxidase in shake flask The screened transformants with high enzyme activity were inoculated in a 300 ml of BMGY fluid medium, shaking culture was performed at 30°C at 200 rpm for 48 h, and cell enrichment was performed; 4000 x g centrifugation was performed for 5 min, and the supernatant was gently removed; the cells were transferred into 100 ml of a BMMY fluid medium including 1% of methanol; and induction culture was performed at 30°C and 200 rpm for 72 h. During induction culture, the methanol solution was added once every 24 h, so that the methanol concentration was maintained at about 1%. 10000 x g centrifugation was performed for 10 min, and the supernatant was collected. The activity of the glucose oxidase was determined, and an expression quantity of the recombinant glucose oxidase was 15 U/ml. (2) Purification of recombinant glucose oxidase Supernatant of the recombinant glucose oxidase subjected to shake flask expression was collected, subjected to desalination and concentration by utilizing a kDa membrane bag first, and purified by anion exchange column
Description
chromatography; the collected electrophoretically pure fluid served as a sample for expressing enzymatic properties; and protein content of purified enzyme liquid was determined by utilizing a coomassie brilliant blue method, and specific activity of zymoprotein was calculated. Embodiment 4 Analysis of partial properties of recombinant glucose oxidase (1) Optimum pH and pH stability of glucose oxidase M5GOD Enzyme activity of a purified glucose oxidase sample in Embodiment 3 was determined at different pH values so as to determine the optimum pH value of the sample. Buffer solutions used at different pH values were prepared as follows: a glycine-hydrochloric acid buffer solution having a pH value of 1.0-3.0; acetic acid-sodium acetate series buffer solutions having a pH value of 4.0-6.0 and Tris-hydrochloric acid series buffer solutions having a pH value of 7.0-9.0. The purified glucose oxidase existed in different buffer systems. Enzyme activity detection method of recombinant glucose oxidase The glucose oxidase in the present invention is subjected to enzyme activity determination by adopting 4-aminoantipyrine spectrophotometry. Under an aerobic condition, hydrogen peroxide produced by glucose dehydrogenation was catalyzed to produce red quinone imide with colorless reduced 4-aminoantipyrine and phenol in the presence of horse radish peroxidase, wherein the maximum light absorption existed at 500 nm; crude enzyme fluid was directly diluted to about 10 U/ml with a buffer solution; 4 test tubes of 150*15 were taken, 2 ml of buffer, 0.3 ml of glucose, 0.4 ml of phenol, 0.1 ml of 4-aminoantipyrine and 0.1 ml of horse radish peroxidase were added into the test tubes, and preheating was performed at 30°C for 5 min; 0.1 ml of distilled water was added into one tube, and the tube served as blank for zero setting; a water bath kettle was placed beside a spectrophotometer so as to conveniently operate; 0.1 ml of sample solution was added into a sample tube, then timing was started, and after vortex blending, colorimetric assay was
Description
immediately performed at a wavelength of 500 nm by using a lcm cuvette; an absorbance value at 30 sec was read as Ao, and the absorbance value was read as A1 within 1 min after reaction, thereby obtaining AAoo=A1 -Ao. A calculation formula of the enzyme activity is as follows: Enzyme activity X1 (U/mL or U/g) in the sample is calculated according to a formula as follows: X1 =AA50 0 xfxBx1000/(887xtxAxd)=33.82xAA 50oxf
In the formula: f...................... a dilution ratio of enzyme fluid B.....................a volume of reaction solution (3 ml) 1000.................a unit conversion factor of an extinction coefficient 887................... an extinction coefficient (L-mol-1 -cm-1
) t.......................reaction time (min), that is, a time difference of 1 min between reading A1 and Ao. A......................a volume of added sample (0.1 ml) d.........................a thickness (cm) of the cuvette. Definition of an enzyme activity unit: under conditions of a pH value of 6.0 and a temperature of 30°C, 1 umol of p-D-glucose may be oxidized to produce an enzyme amount of D-gluconic acid and hydrogen peroxide every minute, and the enzyme amount is defined as one enzyme activity unit (IU). The optimum pH result is determined at 30°C (See Fig. 1). It shows that, the optimum pH value is 5.5, and in a pH value range of 4.0-7.0, the enzyme activity may be maintained at 60% or higher. The enzyme fluid is treated in the buffer solutions at different pH values at °C for 2 h, and the enzyme activity is determined at the optimum pH value so as to research pH stability of the enzyme. Analysis results show (see Fig. 2) that the enzyme is basically stable at a pH value between 2.0 and 5.0, and the enzyme activity may be maintained at 80% or higher. At pH values of 6.0 and 7.0 and after
Description
treatment, the enzyme activity can also be maintained at about 60% respectively, which indicates that the enzyme has excellent pH stability. (2) Optimum temperature and heat stability of glucose oxidase M5GOD Under a condition of the pH value of 6.0, the enzyme activity of the purified glucose oxidase sample is determined at different temperatures of 5-70°C. Analyzed experimental results show that, an optimum reaction temperature of the enzyme is 30°C, and the enzyme still has enzyme activity of 60% or higher at -50°C (see Fig. 3). Heat stability determination is as follows: after treated at different temperatures such as 40°C, 45°C, 50°C and 55°C for 5 min, the glucose oxidase sample is subjected to enzyme activity determination at 30°C. Heat stability experiments show (see Fig. 4) that the glucose oxidase has basically lost the enzyme activity after treated at 55°C. Embodiment 5 Preservation application experiment of recombinant glucose oxidase Step 1: a fresh and alive grass carp was killed, the head, tail, internal organs, bone, scale and skin of the grass carp were removed, and the grass carp was cleaned with sterile water; Step 2: the grass carp meat was cut by a cutter disinfected by boiling, cut to have almost uniform length, width and thickness, and randomly divided into 4 groups; Step 3: the clean grass carp meat was impregnated in a mixed solution of glucose oxidase prepared in Embodiment 3 and glucose for a period of time, wherein enzyme activity of the glucose oxidase solution was 1 U/ml, content of the glucose is 4%, a ratio of the fish to a preservative is 1:1 (kg/L), and impregnation time is 10 min; Step 4: the grass carp meat was taken out and placed on boulting cloth and drained at a room temperature;
Description
Step 5: the grass carp meat was separately charged in sterile polyethylene preservation bags and subjected to refrigerated preservation at 4°C. Embodiment 7 Influences of the recombinant glucose oxidase in the present invention on grass carp quality in refrigeration and preservation of grass carp are as follows: According to Embodiment 6, the grass carp was preserved and taken out on Day 0, 2, 4, 6, 8 and 10; total bacterial count and total volatile basic nitrogen content were determined, wherein results (see Fig. 5 and Fig. 6) showed that, during refrigeration, the total bacterial count and total volatile basic nitrogen content of the grass carp treated with the glucose oxidase in the present invention were obviously lower than those in a blank control group; and a preservation effect was subjected to sensory evaluation. Results are as shown in Table 1. Table 1
Refrigeration time/d Blank control group Experimental group
0 Fresh
Surface glossiness starts to lose, the fishThe fish is normal in glossiness 2 meat still has elasticity, and slightand clear in texture, and has
unpleasant odor exists. elasticity and no odor.
Color of the fish becomes dark, fishy Surface glossiness is lost, fish
4 smell comes out, and original fish flesh flesh elasticity still exists, and
elasticity is lost. delicate flavor lacks.
Color of the fish becomes yellow and Color of the fish meat becomes
6 dark, delicate flavor lacks,fishy smell is dark, and slight unpleasant odor
serious, and viscous liquid exists belowexists.
Description
Color of the fish meat becomes Serious fishy smell exists, the fish meat delicate flavor 8 presents dark red, and fish flesh textureyellow, lacks,fishy smell comes out, and is unclear. pressed fish meat may restore.
Fishy smell and ammonia smell are Color of the fish becomes serious, the fish meat presents brown, yellow and dark, fishy smell is 10 viscous liquid is increased, elasticity serious, and viscous liquid exists lacks, and the pressed fish meat cannot below the fish meat. restore.
In conclusion, the glucose oxidase in the present invention has high catalytic
efficiency under the low-temperature condition, and can be effectively applied to
low-temperature preservation of aquatic products. During application of the
glucose oxidase in preservation of the aquatic products, the aquatic products are
impregnated in the low-temperature glucose oxidase solution, oxygen may be
removed in subsequent preservation, H 2 0 2 is produced to inhibit reproduction of
microorganisms, deterioration is effectively inhibited, and tissue elasticity is
maintained. Therefore, it is indicated that, the low-temperature glucose oxidase in
the present invention has potential application values in preservation of the aquatic
products.
Each embodiment in the description is described in a progressive way. The
difference of each embodiment from each other is the focus of explanation. The
same and similar parts among all of the embodiments can be referred to each other.
The above description of the disclosed embodiments enables those skilled in
the art to realize or use the present invention. Many modifications to these
embodiments will be apparent to those skilled in the art. The general principle
defined herein can be realized in other embodiments without departing from the
spirit or scope of the present invention. Therefore, the present invention will not be
Description
limited to these embodiments shown herein, but will conform to the widest scope consistent with the principle and novel features disclosed herein.
Description
Sequence Table
<110> Ocean University of China Shanghao Science and Technology Co., Ltd.
<120> Glucose oxidase M5GOD and coding genes and applications thereof
<160> 9
<170> SIPO Sequence Listing 1.0
<210> 1 <211> 605 <212> PRT <213> Glucose oxidase M5GOD (Penicillium)
<400> 1 Met Lys Ser Ile Ile Leu Ala Ser Ala Leu Ala Ser Leu Ala Ala Ala 1 5 10 15 Gln Gly Phe Thr Pro Ala Glu Gln Ile Asp Val Gln Ala Ser Leu Ile 20 25 30 Ser Asp Pro Asn Lys Val Ala Gly Gln Thr Phe Asp Tyr Ile Ile Ala 35 40 45 Gly Gly Gly Leu Thr Gly Leu Thr Val Ala Ala Lys Leu Ser Glu Asn 50 55 60 Pro Asn Ile Thr Val Leu Val Ile Glu Lys Gly Phe Tyr Glu Ser Asn 70 75 80
Description
Asn Gly Pro Ile Ile Glu Asn Pro Asn Asp Tyr Gly Leu Ile Phe Gly 85 90 95 Ser Ser Val Asp Gln Asn Tyr Leu Thr Val Pro Met Ala Ile Asn Asn 100 105 110 Arg Thr Leu Glu Val Lys Ser Gly Lys Gly Leu Gly Gly Ser Thr Leu 115 120 125 Ile Asn Gly Asp Ser Trp Thr Gly Pro Asp Lys Val Gln Ile Asp Ser 130 135 140 Trp Glu Thr Val Leu Gly Asn Thr Gly Trp Asn Tyr Asp Ala Leu Lys 145 150 155 160 Gly Tyr Met Lys Glu Ala Glu Leu Ala Arg Tyr Pro Thr Ala Ser Gln 165 170 175 Ile Ala Ala Gly Ile Tyr Phe Asn Glu Thr Cys His Gly Phe Asn Gly 180 185 190 Thr Val Asn Ala Gly Pro Arg Asp Asp Gly Thr Pro Tyr Ser Pro Leu 195 200 205 Met Lys Ala Leu Met Asn Thr Thr Ser Ala Lys Gly Val Pro Thr Gln 210 215 220 Leu Asp Phe Leu Cys Gly Arg Pro Arg Gly Val Ser Met Ile Tyr Asn 225 230 235 240 Asn Leu Leu Pro Asp Gln Thr Arg Ala Asp Ala Ala Arg Glu Trp Leu 245 250 255 Leu Pro Asn Tyr Lys Arg Pro Asn Leu Ser Ile Leu Thr Gly Gln Val 260 265 270 Val Gly Lys Val Leu Phe Thr Gln Thr Ala Thr Gly Pro Lys Ala Thr 275 280 285 Gly Val Asn Phe Gly Thr Asn Lys Ala Ile Asn Phe Asn Val Leu Ala 290 295 300
Description
Lys His Glu Val Leu Leu Ala Ala Gly Ser Ala Ile Ser Pro Leu Ile 305 310 315 320 Leu Glu His Ser Gly Ile Gly Leu Lys Ser Val Leu Asp Gln Phe Asn 325 330 335 Ile Thr Gln Leu Val Glu Leu Pro Val Gly Leu Asn Met Gln Asp Gln 340 345 350 Thr Thr Thr Thr Val Arg Ala Arg Ala Lys Ala Ser Ser Ala Gly Gln 355 360 365 Gly Gln Ala Val Tyr Phe Ala Asn Phe Thr Glu Val Phe Gly Asp Tyr 370 375 380 Ser Ala Arg Ala Thr Asp Leu Leu Asn Thr Lys Leu Ser Gln Trp Ala 385 390 395 400 Asn Glu Thr Val Ala Arg Gly Gly Phe Asn Asn Ala Thr Ala Leu Leu 405 410 415 Ile Gln Tyr Glu Asn Tyr Arg Asn Trp Leu Leu Asn Glu Asp Val Ala 420 425 430 Tyr Val Glu Leu Phe Leu Asp Thr Asn Gly Lys Met Asn Phe Asp Leu 435 440 445 Trp Asp Leu Ile Pro Phe Thr Arg Gly Ser Thr His Ile Ala His Ala 450 455 460 Asp Pro Tyr Leu Gln Ser Phe Ser Asn Asn Pro Met Phe Leu Leu Asn 465 470 475 480 Glu Leu Asp Leu Leu Gly Gln Ala Ala Gly Ser Met Leu Ala Arg Glu 485 490 495 Ile Gln Asn Ser Gly Glu Leu Ala Asn Tyr Phe Asp Gly Glu Asp Ile 500 505 510 Pro Gly Ala Asn Leu Leu Pro Tyr Asn Ala Thr Leu Asp Gly Trp Val 515 520 525
Description
Gly Tyr Val Lys Gln Asn Phe Arg Ala Asn Trp His Ala Val Gly Thr 530 535 540 Cys Ser Met Met Ser Arg Glu Leu Gly Gly Val Val Asp Pro Thr Ala 545 550 555 560 Lys Val Tyr Gly Thr Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro 565 570 575 Pro Thr Gln Val Ser Ser His Val Met Thr Val Phe Tyr Gly Met Ala 580 585 590 Leu Lys Ile Ala Asp Ala Val Leu Ala Asp Tyr Lys Pro 595 600 605
<210> 2 <211> 589 <212> PRT <213> Glucose oxidaseM5GOD(Penicillium)
<400> 2 Gln Gly Phe Thr Pro Ala Glu Gln Ile Asp Val Gln Ala Ser Leu Ile 1 5 10 15 Ser Asp Pro Asn Lys Val Ala Gly Gln Thr Phe Asp Tyr Ile Ile Ala 20 25 30 Gly Gly Gly Leu Thr Gly Leu Thr Val Ala Ala Lys Leu Ser Glu Asn 35 40 45 Pro Asn Ile Thr Val Leu Val Ile Glu Lys Gly Phe Tyr Glu Ser Asn 50 55 60 Asn Gly Pro Ile Ile Glu Asn Pro Asn Asp Tyr Gly Leu Ile Phe Gly 70 75 80 Ser Ser Val Asp Gln Asn Tyr Leu Thr Val Pro Met Ala Ile Asn Asn
Description
85 90 95 Arg Thr Leu Glu Val Lys Ser Gly Lys Gly Leu Gly Gly Ser Thr Leu 100 105 110 Ile Asn Gly Asp Ser Trp Thr Gly Pro Asp Lys Val Gln Ile Asp Ser 115 120 125 Trp Glu Thr Val Leu Gly Asn Thr Gly Trp Asn Tyr Asp Ala Leu Lys 130 135 140 Gly Tyr Met Lys Glu Ala Glu Leu Ala Arg Tyr Pro Thr Ala Ser Gln 145 150 155 160 Ile Ala Ala Gly Ile Tyr Phe Asn Glu Thr Cys His Gly Phe Asn Gly 165 170 175 Thr Val Asn Ala Gly Pro Arg Asp Asp Gly Thr Pro Tyr Ser Pro Leu 180 185 190 Met Lys Ala Leu Met Asn Thr Thr Ser Ala Lys Gly Val Pro Thr Gln 195 200 205 Leu Asp Phe Leu Cys Gly Arg Pro Arg Gly Val Ser Met Ile Tyr Asn 210 215 220 Asn Leu Leu Pro Asp Gln Thr Arg Ala Asp Ala Ala Arg Glu Trp Leu 225 230 235 240 Leu Pro Asn Tyr Lys Arg Pro Asn Leu Ser Ile Leu Thr Gly Gln Val 245 250 255 Val Gly Lys Val Leu Phe Thr Gln Thr Ala Thr Gly Pro Lys Ala Thr 260 265 270 Gly Val Asn Phe Gly Thr Asn Lys Ala Ile Asn Phe Asn Val Leu Ala 275 280 285 Lys His Glu Val Leu Leu Ala Ala Gly Ser Ala Ile Ser Pro Leu Ile 290 295 300 Leu Glu His Ser Gly Ile Gly Leu Lys Ser Val Leu Asp Gln Phe Asn
Description
305 310 315 320 Ile Thr Gln Leu Val Glu Leu Pro Val Gly Leu Asn Met Gln Asp Gln 325 330 335 Thr Thr Thr Thr Val Arg Ala Arg Ala Lys Ala Ser Ser Ala Gly Gln 340 345 350 Gly Gln Ala Val Tyr Phe Ala Asn Phe Thr Glu Val Phe Gly Asp Tyr 355 360 365 Ser Ala Arg Ala Thr Asp Leu Leu Asn Thr Lys Leu Ser Gln Trp Ala 370 375 380 Asn Glu Thr Val Ala Arg Gly Gly Phe Asn Asn Ala Thr Ala Leu Leu 385 390 395 400 Ile Gln Tyr Glu Asn Tyr Arg Asn Trp Leu Leu Asn Glu Asp Val Ala 405 410 415 Tyr Val Glu Leu Phe Leu Asp Thr Asn Gly Lys Met Asn Phe Asp Leu 420 425 430 Trp Asp Leu Ile Pro Phe Thr Arg Gly Ser Thr His Ile Ala His Ala 435 440 445 Asp Pro Tyr Leu Gln Ser Phe Ser Asn Asn Pro Met Phe Leu Leu Asn 450 455 460 Glu Leu Asp Leu Leu Gly Gln Ala Ala Gly Ser Met Leu Ala Arg Glu 465 470 475 480 Ile Gln Asn Ser Gly Glu Leu Ala Asn Tyr Phe Asp Gly Glu Asp Ile 485 490 495 Pro Gly Ala Asn Leu Leu Pro Tyr Asn Ala Thr Leu Asp Gly Trp Val 500 505 510 Gly Tyr Val Lys Gln Asn Phe Arg Ala Asn Trp His Ala Val Gly Thr 515 520 525 Cys Ser Met Met Ser Arg Glu Leu Gly Gly Val Val Asp Pro Thr Ala
Description
530 535 540 Lys Val Tyr Gly Thr Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro 545 550 555 560 Pro Thr Gln Val Ser Ser His Val Met Thr Val Phe Tyr Gly Met Ala 565 570 575 Leu Lys Ile Ala Asp Ala Val Leu Ala Asp Tyr Lys Pro 580 585
<210> 3 <211> 1815 <212> DNA <213> Artificial Sequence
<400> 3 atgaagtcca tcattcttgc ctctgccctc gcctctctag ctgcagccca gggcttcact 60 ccagccgagc agattgatgt ccaggccagc ctgatctccg accctaacaa ggtcgccggc 120 cagacattcg actacatcat cgctggaggt ggtctgacag gtcttaccgt tgcggccaag 180 ctgtctgaga accctaacat caccgtcctt gtcatcgaaa agggcttcta cgagtccaat 240 aatgggccca tcatcgaaaa ccccaacgac tatggcttga tcttcggtag ctctgttgac 300 caaaactacc tcaccgttec catggccatc aacaaccgta ccctggaagt caagtctggc 360 aagggtctcg gtggttccac gttgattaac ggtgactcct ggaccggteccgacaaggtc 420 cagattgact cctgggagac tgtcttggga aataccggtt ggaactatga cgccctcaag 480 gggtacatga aggaagccga gcttgctcgt tacccaaccg ccagtcagat tgccgccggt 540 atctacttca acgaaacctg ccatggattc aacggcaccg ttaacgccgg accccgtgat 600 gatggtaccc cttactctcc ccttatgaaa gccctcatga acaccacctc tgccaagggt 660 gttcccactc agcttgactt cctctgcggt cgccctcgtg gtgtctccat gatctacaac 720 aacttgctgc ctgaccagac ccgtgcggat gctgctcgcg agtggcttct tcccaactat 780 aagcgcccca acttgagcat tcttaccggc caggttgttg gaaaggttct cttcactcaa 840
Description
accgcgactg gccccaaggc tactggtgtt aattttggca ccaacaaggc catcaatttc 900 aacgtcttgg ccaagcacga ggtccttttg gctgctggct ctgccatctc gcccctaatc 960 ctcgagcact ctggtattgg tctaaagtct gtcctcgacc agttcaatat cacccagctc 1020 gtcgagcttc ccgtcggtct caatatgcag gaccagacca ccaccactgt ccgcgcccga 1080 gccaaggcgt cttctgctgg tcagggccag gccgtctact ttgccaactt cactgaggtc 1140 tttggtgact actccgctcg agctaccgat ttactcaaca ccaagctctc ccagtgggcc 1200 aacgagactg ttgcacgcgg aggcttcaac aacgccaccg ctctcttaat ccagtatgag 1260 aactaccgta actggctcct gaacgaggac gttgcctatg tcgagctttt cctcgacacc 1320 aacggaaaga tgaactttga cttgtgggat ctcatcccct tcacgcgcgg ctctacgcac 1380 atagcacacg ccgaccctta tctgcagtec ttctccaaca atcccatgtt cctgttgaac 1440 gagcttgacc ttcttggcca ggctgctggc tcgatgctgg ctcgtgagat ccagaactcg 1500 ggtgagctgg ccaactactt tgacggcgag gatatccccg gggcaaatct cttgccctac 1560 aacgctaccc tcgatggctg ggttggatat gtcaagcaga actttcgtgc caactggcac 1620 gctgtcggga cttgttctat gatgtcccgg gagcttggtg gtgttgttga tctacggcc 1680 aaggtctacg gtacccaggg tcttcgtgtc attgatggtt ctattccacc cacccaggtg 1740 tcatctcacg ttatgaccgt tttctacggt atggctttga agattgccga tgctgttctg 1800 gctgactaca agccc 1815
<210> 4 <211> 1767 <212> DNA <213> Artificial Sequence
<400> 4 cagggcttca ctccagccga gcagattgat gtccaggcca gcctgatctc cgaccctaac 60 aaggtcgccg gccagacatt cgactacatc atcgctggag gtggtctgac aggtcttacc 120 gttgcggcca agctgtctga gaaccctaac atcaccgtec ttgtcatcga aaagggcttc 180 tacgagtcca ataatgggcc catcatcgaa aaccccaacg actatggctt gatcttcggt 240
Description
agctctgttg accaaaacta cctcaccgtt cccatggcca tcaacaaccg taccctggaa 300 gtcaagtctg gcaagggtct cggtggttec acgttgatta acggtgactc ctggaccggt 360 cccgacaagg tccagattga ctcctgggag actgtcttgg gaaataccgg ttggaactat 420 gacgccctca aggggtacat gaaggaagcc gagcttgctc gttacccaaccgccagtcag 480 attgccgccg gtatctactt caacgaaacc tgccatggat tcaacggcac cgttaacgcc 540 ggaccccgtg atgatggtac cccttactct ccccttatga aagccctcat gaacaccacc 600 tctgccaagg gtgttcccac tcagcttgac ttcctctgcg gtcgccctcg tggtgtctcc 660 atgatctaca acaacttgct gcctgaccag acccgtgcgg atgctgctcg cgagtggctt 720 ctteccaact ataagcgccc caacttgagc attcttaccg gccaggttgt tggaaaggtt 780 ctcttcactc aaaccgcgac tggccccaag gctactggtg ttaattttgg caccaacaag 840 gccatcaatt tcaacgtctt ggccaagcac gaggtccttt tggctgctgg ctctgccatc 900 tcgcccctaa tcctcgagca ctctggtatt ggtctaaagt ctgtcctcga ccagttcaat 960 atcacccagc tcgtcgagct tcccgtcggt ctcaatatgc aggaccagac caccaccact 1020 gtccgcgccc gagccaaggc gtcttctgct ggtcagggcc aggccgtcta ctttgccaac 1080 ttcactgagg tctttggtga ctactccgct cgagctaccg atttactcaa caccaagctc 1140 tcccagtggg ccaacgagac tgttgcacgc ggaggcttca acaacgccac cgctctctta 1200 atccagtatg agaactaccg taactggctc ctgaacgagg acgttgccta tgtcgagctt 1260 ttcctcgaca ccaacggaaa gatgaacttt gacttgtggg atctcatcccc ttcacgcgc 1320 ggctctacgc acatagcaca cgccgaccct tatctgcagt ccttctccaa caatcccatg 1380 ttcctgttga acgagcttga ccttcttggc caggctgctg gctcgatgct ggctcgtgag 1440 atccagaact cgggtgagct ggccaactac tttgacggcg aggatatcccc ggggcaaat 1500 ctcttgccct acaacgctac cctcgatggc tgggttggat atgtcaagca gaactttcgt 1560 gccaactggc acgctgtcgg gacttgttct atgatgtccc gggagcttgg tggtgttgtt 1620 gatcctacgg ccaaggtcta cggtacccag ggtcttcgtg tcattgatgg ttctattcca 1680 cccacccagg tgtcatctca cgttatgacc gttttctacg gtatggcttt gaagattgcc 1740 gatgctgttc tggctgacta caagccc 1767
<210> 5
Description
<211> 16 <212> PRT <213> Artificial Sequence
<400> 5 Met Lys Ser Ile Ile Leu Ala Ser Ala Leu Ala Ser Leu Ala Ala Ala 1 5 10 15
<210> 6 <211> 48 <212> DNA <213> Artificial Sequence
<400> 6 atgaagtcca tcattcttgc ctctgccctc gcctctctag ctgcagcc 48
<210> 7 <211> 40 <212> DNA <213> Artificial Sequence
<400> 7 agagaggctg aagctgaatt ccagggcttc actccagccg 40
<210> 8 <211> 46 <212> DNA <213> Artificial Sequence
Description
<400> 8 tgttctagaa agctggcggc cgcttagggc ttgtagtcag ccagaa 46
<210> 9 <211> 44 <212> DNA <213> Artificial Sequence
<400> 9 agagaggctg aagctgaatt catgaagtec atcattcttg cctc 44
Claims (9)
1. A glucose oxidase M5GOD, wherein an amino acid sequence of the glucose oxidase M5GOD is shown as SEQ ID NO.2.
2. A recombinant vector comprising genes coding the glucose oxidase of claim 1, wherein a nucleotide sequence of the genes is shown as SEQ ID NO.4.
3. The recombinant vector according to claim 2, wherein the recombinant vector is pPIC ZaA-M5GOD.
4. The recombinant vector according to claim 3, wherein a preparation method of the recombinant vector pPIC ZaA-M5GOD comprises: the glucose oxidase gene is inserted between EcoR 1 and Not 1 restriction sites on plasmid pPIC ZaA; the nucleotide sequence is located at the downstream of a promoter and regulated by the promoter, thereby obtaining a recombinant yeast expression plasmid pPIC ZaA-M5GOD.
5. A recombinant strain comprising the recombinant vector of claim 2 or 3.
6. The recombinant strain according to claim 5, wherein the recombinant strain is pichia pastoris X33/M5GOD; and the recombinant yeast expression plasmid pPIC ZaA-M5GOD is used to transform pichic pastoris, thereby obtaining the recombinant strain.
7. An application of the glucose oxidase M5GOD of claim 1 in food preservation.
8. An application of the recombinant vector of claim 2 or 3 or the recombinant strain of claim 5 or 6 in industrial production of the glucose oxidase.
9. A method for preparing the glucose oxidase M5GOD of claim 1, comprising the following steps: 1) transforming host cells by using the recombinant vector of claim 2 or 3 so as to obtain a recombinant strain; 2) culturing the recombinant strain, inducing expression of glucose oxidase, and collecting supernatant; 3) recovering and purifying the supernatant, thereby obtaining the glucose oxidase M5GOD.
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| PCT/CN2019/123895 WO2021103123A1 (en) | 2019-11-25 | 2019-12-09 | Glucose oxidase m5god, coding gene thereof and application thereof |
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| CN101481680B (en) * | 2009-01-19 | 2011-06-22 | 崔增学 | Production method of glucose oxidase |
| CN103981158A (en) * | 2009-12-05 | 2014-08-13 | 天野酶株式会社 | Mutant enzyme and application thereof |
| CN101955953A (en) * | 2010-09-09 | 2011-01-26 | 中国农业科学院生物技术研究所 | Glucose oxidase mutant gene, expression and application thereof |
| FR2979918B1 (en) * | 2011-09-08 | 2013-12-27 | Centre Nat Rech Scient | AMAGASAKIENSE PENICILLIUM GLUCOSE OXIDASE MUTANTS |
| CN103275942B (en) * | 2013-04-03 | 2014-09-24 | 北京挑战生物技术有限公司 | Glucose oxidase GODJ4A, and gene and application thereof |
| CN103981159B (en) * | 2014-06-05 | 2016-04-13 | 青岛蔚蓝生物集团有限公司 | A kind of glucose oxidase enzyme mutant and application thereof |
| CN104711274B (en) * | 2015-03-17 | 2019-01-29 | 中国科学院微生物研究所 | A kind of preparation method and applications recombinating penicillium amagasakiense glucose oxidase |
| CN105420252A (en) * | 2015-12-07 | 2016-03-23 | 河北省微生物研究所 | Glucose oxidase gene GOD, protein coded by GOD, pichia pastoris transformed by GOD and preparation method of pichia pastoris |
| CN108220262A (en) * | 2018-02-26 | 2018-06-29 | 大连大学 | A kind of marine low temperature glucose oxidase and its application in seafood freshing |
| CN109652390A (en) * | 2019-02-25 | 2019-04-19 | 大连大学 | A kind of marine low temperature glucose oxidase and its application |
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Non-Patent Citations (3)
| Title |
|---|
| Simpson, C. et al., 2007. Isolation, purification and characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally active at neutral pH. Protein expression and purification, 51(2), pp.260-266. * |
| Sukhacheva, M.V., Davydova, M.E. and Netrusov, A.I., 2004. Production of Penicillium funiculosum 433 glucose oxidase and its properties. Applied Biochemistry and Microbiology, 40(1), pp.25-29. * |
| Yuivar, Y. et al., 2017. Biochemical and thermodynamical characterization of glucose oxidase, invertase, and alkaline phosphatase secreted by Antarctic yeasts. Frontiers in molecular biosciences, 4, p.86. * |
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