CN115786289B - Ascorbate oxidase - Google Patents
Ascorbate oxidase Download PDFInfo
- Publication number
- CN115786289B CN115786289B CN202211180850.5A CN202211180850A CN115786289B CN 115786289 B CN115786289 B CN 115786289B CN 202211180850 A CN202211180850 A CN 202211180850A CN 115786289 B CN115786289 B CN 115786289B
- Authority
- CN
- China
- Prior art keywords
- ascorbate oxidase
- amino acid
- seq
- acid sequence
- aso
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an ascorbate oxidase, a preparation method and application, wherein the amino acid sequence of the ascorbate oxidase is shown as SEQ ID NO.3 or SEQ ID NO.4, and compared with wild ascorbate oxidase, the amino acid sequence SEQ ID NO.3 and the amino acid sequence SEQ ID NO.4 have higher thermal stability and/or activity; the application of the ascorbate oxidase in enhancing the anti-interference aspect of the clinical diagnosis kit can eliminate the interference of the ascorbate in the detection of the galactosamine, the total cholesterol, the triacylglycerol, the creatinine and the like.
Description
Technical Field
The invention relates to the field of biotechnology, and in particular relates to ascorbic acid oxidase.
Background
Ascorbate oxidase (ASO) is a copper-containing enzyme that is found in melons, seeds, grains, fruits and vegetables. It oxidizes ascorbic acid to produce water and dehydroascorbic acid. Compared to non-enzymatic oxidation, ascorbate oxidase produces water after action, while the former produces hydrogen peroxide.
Yeast expression systems include Saccharomyces cerevisiae expression systems, methanol yeast expression systems, and other yeast expression systems. Pichia pastoris is a yeast variety capable of efficiently expressing recombinant proteins, on one hand, the expressed proteins can be subjected to glycosylation modification because the Pichia pastoris belongs to eukaryotes, on the other hand, the Pichia pastoris is high in growth speed, and the expressed proteins can be secreted into a culture medium, so that the protein purification is facilitated. Pichia pastoris belongs to methanol nutritional yeast and can take methanol as the only carbon source.
The ascorbic acid oxidase used at home and abroad at present is mainly obtained from pumpkin. Because of the single source and high production cost, the content and quality of the ascorbate oxidase are affected by batches, and the application of the ascorbate oxidase is greatly limited. The thermal stability of the corresponding products of the companies at home and abroad is poor, and the problem of high temperature generated in reagent transportation cannot be satisfied. Thus, a new ascorbate oxidase gene and yeast expression system are needed.
Disclosure of Invention
It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.
It is still another object of the present invention to provide an ascorbate oxidase, the amino acid sequence of which is shown as SEQ ID NO.3 or SEQ ID NO.4, wherein the amino acid sequence SEQ ID NO.3 is obtained by mutating the amino acid sequence SEQ ID NO.1, and the amino acid sequence SEQ ID NO.4 is obtained by mutating the amino acid sequence SEQ ID NO.2, and the amino acid sequences SEQ ID NO.3 and SEQ ID NO.4 have higher thermal stability and/or activity than the wild type ascorbate oxidase.
The application of the ascorbate oxidase in enhancing the anti-interference of a clinical diagnosis kit is also provided, and the protein expressed by the ascorbate oxidase can eliminate the interference of the kit in detecting the ascorbate in urine galactose, total cholesterol, triacylglycerol, creatinine and the like.
To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided an ascorbate oxidase having an amino acid sequence as shown in SEQ ID NO.3 or SEQ ID NO.4.
Preferably, the amino acid sequence SEQ ID NO.3 of the ascorbate oxidase is obtained by mutating the amino acid sequence SEQ ID NO.1 of the ascorbate oxidase, and the specific mutation is as follows:
error-prone PCR random mutation is carried out on the amino acid sequence SEQ ID NO.1 of the ascorbate oxidase, the 275 th amino acid of the amino acid sequence SEQ ID NO.1 is mutated from K to Q, and the 429 th amino acid is mutated from D to N, so that the amino acid sequence SEQ ID NO.3 of the ascorbate oxidase is obtained.
Preferably, the amino acid sequence SEQ ID NO.4 of the ascorbate oxidase is obtained by mutating the amino acid sequence SEQ ID NO.2 of the ascorbate oxidase;
error-prone PCR random mutation is carried out on the amino acid sequence SEQ ID NO.2 of the ascorbate oxidase, the 149 th amino acid of the amino acid sequence SEQ ID NO.2 is mutated from V to D, and the 317 th amino acid is mutated from P to R, thus obtaining the amino acid sequence SEQ ID NO.4 of the ascorbate oxidase.
Preferably, the nucleotide sequence of the ascorbic acid oxidase with the amino acid sequence shown as SEQ ID NO.3 is shown as SEQ ID NO. 5.
Preferably, the nucleotide sequence of the ascorbic acid oxidase with the amino acid sequence shown as SEQ ID NO.4 is shown as SEQ ID NO. 6.
In another aspect, the present invention provides a recombinant vector comprising the ascorbate oxidase gene.
The invention also provides engineering bacteria containing the recombinant vector.
The invention also provides a preparation method of the engineering bacteria, which comprises the following specific steps:
s1, constructing a vector for expressing ascorbate oxidase protein;
s2, transfecting the vector which is constructed in the step S1 and expresses the ascorbate oxidase protein into cells;
s3, obtaining expressed ascorbate oxidase protein from the transfected cells in the step S2;
s4, purifying the ascorbate oxidase protein obtained in the step S3.
In another aspect, the invention provides the use of the ascorbate oxidase in enhancing the anti-interference properties of a clinical diagnostic kit.
The invention at least comprises the following beneficial effects:
firstly, the invention carries out error-prone PCR random mutation on an amino acid sequence SEQ ID NO.1, the 275 th amino acid of the amino acid sequence SEQ ID NO.1 is mutated from K to Q, the 429 th amino acid is mutated from D to N, the amino acid sequence SEQ ID NO.3 of the ascorbic acid oxidase is obtained, the error-prone PCR random mutation is carried out on an amino acid sequence SEQ ID NO.2 of the ascorbic acid oxidase, the 149 th amino acid of the amino acid sequence SEQ ID NO.4 is mutated from V to D, and the 317 th amino acid is mutated from P to R, thus obtaining the amino acid sequence SEQ ID NO.4 of the ascorbic acid oxidase, wherein the amino acid sequences SEQ ID NO.3 and SEQ ID NO.4 have higher thermal stability and/or activity compared with wild ascorbic acid oxidase.
And secondly, the application of the ascorbate oxidase in enhancing the anti-interference aspect of a clinical diagnosis kit can eliminate the interference of the kit on the detection of the ascorbate in the urine galactose, the total cholesterol, the triacylglycerol, the creatinine and the like.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a map of the pPICZ alpha A-ASO vector according to one embodiment of the present invention;
FIG. 2 is a pH-ASO activity curve;
FIG. 3 is a graph showing the enzymatic activity of ASO after 17h at various pH;
FIG. 4 is a graph of activity at temperature-ASO;
FIG. 5 shows the enzymatic activity curves of ASO after 30min of retention at different temperatures.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
The embodiment of the application provides an ascorbate oxidase, the amino acid sequence of which is shown as SEQ ID NO.3 or SEQ ID NO.4, wherein the amino acid sequence is shown as SEQ ID NO.3: AKTRHFKWEVEYMYWSPDCIEHVVMGINGQFPGPTIRAKAGDTVVVELTNKLPTEGVVIHWHGIRQLGTPWADGTAFISQCAINSGETFHYRFKVDRAGTYFYHGHLGMQRSAGLYGSLLVDVAEGEKEPFHYDGEFNLLLSDWWHKSVHEQEVGLSSNPFRWIGEPQSLLINGRGQYNCSLAAQYSDTNSSQCKLRGNEQCAPQILHVRSNKTYRLRVASSTALASLNLAIGNHKMVVVEADGNYLQPFKVNDLDIYSGESYSVLITTNQDPSQNYWLSIGVRGRLPATPPGLTILNYQPTSASKFPTSPPPVTPPWNDYHHSKMFSKSIFALMGSPKPPTSYDRRISLLNTQNKIDGFTKWAINNVSLALPPTPYLGSIKYGLRNAFDQKSPPENFPDNYDVMRPPINPNSTTGSGVYMFGLNTTVNVILQNANALSDNVSEIHPWHLHGHDFWVLGYGEGKFSAKDEKKLNFKNPPLRNTAVIFPYGWTALRFVADNPGVWAFHCHIEPHLHMGMGVVFAEGVHHVKKIPNEALTCGLTAKLLYKNRGR; amino acid sequence SEQ ID No.4: AKARHFNWEVEYMYRSPDCLEHIVMGINGQFPGPTIRAKAGDTLVIELSNKLHTEGVVIHWHGIRQLGTPWADGTASISQCAINPGETFKYRFKVDRRGTYFYHGHYGMQRSAGLYGSLIVDVADGEKEPFHYDGELNLLLSDWWHKGDHEQEVGLSSNPFRWIGEPQSVLINGRGQYNCSMAAKFSNPPIGQCKFRGNEQCAPQILKVQPNKTYRLRIASTTALASLNLAIQGHKMVVVEADGNHVQPFAMNDLDIYSGESYSVLLTTDQNPSRNYWISIGVRAREPKTPQALTILNYSPTSASRIPMSQPPVTPRWNDYNHSKAFTKSIYALMGSPKPPKTSNRRIVLLNTQNRVNGFIKWSINNVSLVLPSTPYLGSLKFGLNNSFDQKSPPDNYDSSYDIMKPAVNQNSTQGSGIYTIGLNTTVDVILQNANTLAKDVSEIHPWHLHGHDFWVLGYGESKFKEGDEKTFNLKNPPLRNTAVIFPYGWTALRFVADNPGVWAFHCHIEPHLHMGMGVVFAQGVHRVGQIPREALACGLTGNKHN.
In another technical scheme, the amino acid sequence SEQ ID NO.3 of the ascorbate oxidase is obtained by mutating the amino acid sequence SEQ ID NO.1 of the ascorbate oxidase, and the specific mutation is as follows:
error-prone PCR random mutation is carried out on the amino acid sequence SEQ ID NO.1 of the ascorbate oxidase, the 275 th amino acid of the amino acid sequence SEQ ID NO.1 is mutated from K to Q, the 429 th amino acid is mutated from D to N, and the amino acid sequence SEQ ID NO.3 and the amino acid sequence SEQ ID NO.1 of the ascorbate oxidase are obtained: AKTRHFKWEVEYMYWSPDCIEHVVMGINGQFPGPTIRAKAGDTVVVELTNKLPTEGVVIHWHGIRQLGTPWADGTAFISQCAINSGETFHYRFKVDRAGTYFYHGHLGMQRSAGLYGSLLVDVAEGEKEPFHYDGEFNLLLSDWWHKSVHEQEVGLSSNPFRWIGEPQSLLINGRGQYNCSLAAQYSDTNSSQCKLRGNEQCAPQILHVRSNKTYRLRVASSTALASLNLAIGNHKMVVVEADGNYLQPFKVNDLDIYSGESYSVLITTNQDPSKNYWLSIGVRGRLPATPPGLTILNYQPTSASKFPTSPPPVTPPWNDYHHSKMFSKSIFALMGSPKPPTSYDRRISLLNTQNKIDGFTKWAINNVSLALPPTPYLGSIKYGLRNAFDQKSPPENFPDNYDVMRPPINPNSTTGSGVYMFGLNTTVDVILQNANALSDNVSEIHPWHLHGHDFWVLGYGEGKFSAKDEKKLNFKNPPLRNTAVIFPYGWTALRFVADNPGVWAFHCHIEPHLHMGMGVVFAEGVHHVKKIPNEALTCGLTAKLLYKNRGR the nucleotide sequence of the edited amino acid sequence SEQ ID NO.1 is: GCAAAAACTAGACATTTTAAGTGGGAGGTTGAATACATGTACTGGTCTCCAGATTGTATTGAACACGTCGTAATGGGAATCAATGGACAATTTCCTGGACCAACCATCAGAGCTAAAGCTGGTGACACAGTTGTGGTCGAACTAACCAATAAGCTGCCAACTGAAGGTGTTGTGATTCATTGGCACGGTATTAGACAACTTGGAACGCCTTGGGCCGATGGTACCGCTTTTATTAGTCAATGTGCTATCAACTCTGGCGAAACTTTTCATTATCGATTTAAAGTTGACAGAGCAGGTACGTACTTTTACCATGGACACTTGGGTATGCAAAGAAGTGCTGGTCTGTATGGTTCTCTATTGGTTGATGTGGCAGAAGGCGAAAAGGAACCTTTCCATTACGACGGAGAGTTTAACTTGCTATTGTCAGACTGGTGGCACAAGTCAGTACATGAACAAGAGGTCGGATTGTCATCAAATCCTTTCAGATGGATTGGAGAGCCCCAATCTCTGCTAATCAACGGTAGAGGACAATATAATTGTTCTTTGGCTGCCCAATATTCTGATACTAACTCTTCCCAATGTAAATTGAGAGGTAATGAACAATGTGCACCCCAAATCCTACATGTTAGATCCAATAAAACCTACAGATTGAGAGTTGCCTCTTCTACCGCATTAGCATCTTTAAATTTGGCAATTGGCAATCATAAGATGGTTGTTGTGGAAGCAGATGGCAACTATTTGCAGCCATTTAAGGTCAATGATCTAGATATTTACTCTGGAGAATCATATAGTGTATTGATTACTACAAACCAAGATCCATCAAAGAACTACTGGTTATCAATTGGTGTTAGAGGCAGGTTACCTGCAACTCCTCCTGGACTTACAATTTTGAACTATCAACCAACATCCGCCTCCAAATTTCCTACTTCACCTCCACCTGTCACCCCACCATGGAACGATTATCATCATTCTAAGATGTTTAGTAAGAGTATTTTTGCACTAATGGGATCACCCAAACCACCAACAAGTTACGATCGTAGAATTAGTCTGCTGAATACACAGAATAAGATTGATGGATTTACCAAATGGGCTATAAACAATGTCTCCTTGGCACTTCCACCAACACCCTATTTGGGCTCTATTAAATACGGATTGAGAAACGCCTTCGACCAGAAGTCACCTCCAGAAAATTTTCCAGATAATTATGATGTGATGAGGCCACCAATTAACCCCAATTCTACTACGGGCTCCGGTGTCTACATGTTCGGTCTGAACACAACTGTAGATGTCATCTTGCAAAATGCTAATGCCTTGTCCGATAACGTGTCTGAAATTCATCCTTGGCACTTGCATGGTCATGATTTCTGGGTTCTTGGATATGGAGAAGGTAAATTTAGTGCTAAAGACGAAAAAAAGCTTAACTTCAAAAACCCACCTTTGAGAAATACCGCAGTGATTTTCCCTTACGGATGGACGGCTCTGAGATTCGTTGCTGACAATCCTGGTGTGTGGGCCTTCCATTGTCATATTGAACCACATCTGCACATGGGTATGGGAGTTGTTTTTGCCGAAGGTGTTCATCATGTTAAAAAGATCCCTAATGAAGCATTGACATGCGGATTGACTGCAAAGTTGTTGTATAAGAACCGTGGTAGA.
In the above-described embodiments, one skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids from the sequences of the invention to obtain variants of the ascorbate oxidase amino acid sequence without substantially affecting ascorbate oxidase activity. They are all considered to be included within the scope of the present invention.
In another technical scheme, the amino acid sequence SEQ ID NO.4 of the ascorbyl oxidase is obtained by mutating the amino acid sequence SEQ ID NO.2 of the ascorbyl oxidase;
error-prone PCR random mutation is carried out on the amino acid sequence SEQ ID NO.2 of the ascorbate oxidase, the 149 th amino acid of the amino acid sequence SEQ ID NO.2 is mutated from V to D, and the 317 th amino acid is mutated from P to R, so that the amino acid sequence SEQ ID NO.4 and the amino acid sequence SEQ ID NO.2 of the ascorbate oxidase are obtained: AKARHFNWEVEYMYRSPDCLEHIVMGINGQFPGPTIRAKAGDTLVIELSNKLHTEGVVIHWHGIRQLGTPWADGTASISQCAINPGETFKYRFKVDRRGTYFYHGHYGMQRSAGLYGSLIVDVADGEKEPFHYDGELNLLLSDWWHKGVHEQEVGLSSNPFRWIGEPQSVLINGRGQYNCSMAAKFSNPPIGQCKFRGNEQCAPQILKVQPNKTYRLRIASTTALASLNLAIQGHKMVVVEADGNHVQPFAMNDLDIYSGESYSVLLTTDQNPSRNYWISIGVRAREPKTPQALTILNYSPTSASRIPMSQPPVTPPWNDYNHSKAFTKSIYALMGSPKPPKTSNRRIVLLNTQNRVNGFIKWSINNVSLVLPSTPYLGSLKFGLNNSFDQKSPPDNYDSSYDIMKPAVNQNSTQGSGIYTIGLNTTVDVILQNANTLAKDVSEIHPWHLHGHDFWVLGYGESKFKEGDEKTFNLKNPPLRNTAVIFPYGWTALRFVADNPGVWAFHCHIEPHLHMGMGVVFAQGVHRVGQIPREALACGLTGNKHN the nucleotide sequence of the edited amino acid sequence SEQ ID NO.2 is: GCAAAAGCCAGACACTTTAATTGGGAGGTAGAGTACATGTATAGATCTCCAGATTGTTTGGAACACATTGTCATGGGAATCAACGGCCAATTCCCTGGTCCCACAATCCGTGCAAAGGCTGGTGACACACTTGTTATTGAATTGTCTAACAAACTTCACACAGAGGGAGTTGTTATCCACTGGCATGGTATTCGACAACTGGGTACTCCTTGGGCAGATGGTACTGCCTCCATTTCTCAATGTGCCATAAATCCAGGCGAAACATTTAAATACAGATTCAAAGTCGATAGGAGAGGTACATACTTTTATCACGGTCATTACGGCATGCAACGTTCTGCTGGTTTGTATGGAAGTCTTATCGTGGATGTGGCTGACGGTGAGAAAGAACCTTTTCACTACGATGGTGAGTTGAACTTGCTGTTGTCTGACTGGTGGCATAAGGGAGTCCATGAACAAGAAGTAGGACTTTCCTCAAACCCCTTCAGGTGGATTGGAGAACCACAATCAGTCTTGATTAACGGAAGAGGTCAGTATAACTGCAGTATGGCCGCCAAATTTTCCAATCCCCCAATTGGCCAGTGTAAGTTTCGAGGTAACGAACAATGTGCTCCCCAAATTTTGAAGGTGCAGCCCAACAAAACCTACAGACTAAGAATAGCATCCACTACCGCTCTAGCTTCATTGAACCTGGCTATACAAGGTCACAAGATGGTTGTTGTTGAAGCCGACGGAAATCATGTCCAACCATTTGCTATGAATGACCTTGACATTTATTCTGGAGAATCTTACTCCGTTCTTTTGACCACAGATCAGAACCCATCCAGAAACTATTGGATTAGTATTGGTGTTAGAGCTAGAGAACCTAAAACACCTCAGGCTCTAACTATTCTGAACTATTCCCCAACATCTGCTTCCCGAATACCTATGAGTCAACCCCCAGTCACACCACCTTGGAACGATTACAACCATTCTAAAGCATTTACCAAGTCTATTTATGCATTAATGGGTTCTCCAAAGCCTCCTAAGACTTCCAACAGAAGAATTGTTCTGTTAAACACTCAAAACAGAGTTAATGGTTTTATTAAATGGTCCATCAACAATGTCTCTTTGGTCCTACCTTCTACGCCTTACTTAGGATCCCTAAAATTCGGTTTGAATAATTCTTTCGATCAAAAGTCACCACCTGACAACTATGATTCTTCTTACGATATTATGAAGCCTGCTGTTAATCAAAATTCAACACAGGGTTCCGGTATATACACAATTGGTCTGAACACTACAGTGGATGTTATATTGCAGAACGCCAACACTCTAGCTAAGGACGTGTCCGAAATTCATCCTTGGCACCTTCACGGTCACGATTTCTGGGTTCTGGGTTACGGTGAGAGTAAGTTTAAGGAGGGTGACGAAAAGACCTTCAACCTAAAGAATCCACCTTTGAGAAACACGGCAGTTATATTCCCTTACGGATGGACGGCCCTTAGGTTTGTCGCTGACAATCCTGGTGTTTGGGCTTTTCATTGTCATATAGAGCCCCACTTGCATATGGGTATGGGAGTTGTTTTTGCTCAGGGTGTGCATAGAGTTGGTCAGATTCCTAGAGAAGCCCTAGCTTGTGGTCTGACTGGAAATAAACATAAT.
In the above-described embodiments, one skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids from the sequences of the invention to obtain variants of the ascorbate oxidase amino acid sequence without substantially affecting ascorbate oxidase activity. They are all considered to be included within the scope of the present invention.
In the above technical scheme, if the same group of amino acid residues are substituted in the amino acid sequence, for example, R is substituted by K or I is substituted by L, the functions of the residues in the protein structure domain are not changed, so that the three-dimensional structure of the protein is not influenced, and the functions of the protein can still be realized. For example, D and E, S and T, A and G, Q and N, P and G, F and W, A and V, C and M, etc., are substituted for each other without affecting the steric structure and functional activity of the protein. Amino acid residue substitutions of the same genus may occur at any one amino acid residue position on the ascorbate oxidase. In contrast, amino acid residues of different classes are substituted, or amino acid substitutions do not meet the substitution rules listed above, and are likely to change the structure of the protein and cause differences in function.
The ascorbate oxidase provided by the invention can be modified or mutated to obtain derivative protein. The term "derivative protein" as used herein means a protein having an amino acid sequence different from that of an ascorbate oxidase having the above amino acid sequence, or having a modified form which does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants may be obtained by various techniques such as irradiation or random mutagenesis by mutagens, or by techniques such as site-directed mutagenesis or other known molecular biology. "derivatized proteins" also include analogs having residues of natural amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids.
The modified forms include: chemical forms of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylating acid or deglycosylating enzyme). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).
In another technical scheme, the nucleotide sequence of the ascorbic acid oxidase with the amino acid sequence shown as SEQ ID NO.3 is shown as SEQ ID NO. 5: GCAAAAACTAGACATTTTAAGTGGGAGGTTGAATACATGTACTGGTCTCCAGATTGTATTGAACACGTCGTAATGGGAATCAATGGACAATTTCCTGGACCAACCATCAGAGCTAAAGCTGGTGACACAGTTGTGGTCGAACTAACCAATAAGCTGCCAACTGAAGGTGTTGTGATTCATTGGCACGGTATTAGACAACTTGGAACGCCTTGGGCCGATGGTACCGCTTTTATTAGTCAATGTGCTATCAACTCTGGCGAAACTTTTCATTATCGATTTAAAGTTGACAGAGCAGGTACGTACTTTTACCATGGACACTTGGGTATGCAAAGAAGTGCTGGTCTGTATGGTTCTCTATTGGTTGATGTGGCAGAAGGCGAAAAGGAACCTTTCCATTACGACGGAGAGTTTAACTTGCTATTGTCAGACTGGTGGCACAAGTCAGTACATGAACAAGAGGTCGGATTGTCATCAAATCCTTTCAGATGGATTGGAGAGCCCCAATCTCTGCTAATCAACGGTAGAGGACAATATAATTGTTCTTTGGCTGCCCAATATTCTGATACTAACTCTTCCCAATGTAAATTGAGAGGTAATGAACAATGTGCACCCCAAATCCTACATGTTAGATCCAATAAAACCTACAGATTGAGAGTTGCCTCTTCTACCGCATTAGCATCTTTAAATTTGGCAATTGGCAATCATAAGATGGTTGTTGTGGAAGCAGATGGCAACTATTTGCAGCCATTTAAGGTCAATGATCTAGATATTTACTCTGGAGAATCATATAGTGTATTGATTACTACAAACCAAGATCCATCACAGAACTACTGGTTATCAATTGGTGTTAGAGGCAGGTTACCTGCAACTCCTCCTGGACTTACAATTTTGAACTATCAACCAACATCCGCCTCCAAATTTCCTACTTCACCTCCACCTGTCACCCCACCATGGAACGATTATCATCATTCTAAGATGTTTAGTAAGAGTATTTTTGCACTAATGGGATCACCCAAACCACCAACAAGTTACGATCGTAGAATTAGTCTGCTGAATACACAGAATAAGATTGATGGATTTACCAAATGGGCTATAAACAATGTCTCCTTGGCACTTCCACCAACACCCTATTTGGGCTCTATTAAATACGGATTGAGAAACGCCTTCGACCAGAAGTCACCTCCAGAAAATTTTCCAGATAATTATGATGTGATGAGGCCACCAATTAACCCCAATTCTACTACGGGCTCCGGTGTCTACATGTTCGGTCTGAACACAACTGTAAATGTCATCTTGCAAAATGCTAATGCCTTGTCCGATAACGTGTCTGAAATTCATCCTTGGCACTTGCATGGTCATGATTTCTGGGTTCTTGGATATGGAGAAGGTAAATTTAGTGCTAAAGACGAAAAAAAGCTTAACTTCAAAAACCCACCTTTGAGAAATACCGCAGTGATTTTCCCTTACGGATGGACGGCTCTGAGATTCGTTGCTGACAATCCTGGTGTGTGGGCCTTCCATTGTCATATTGAACCACATCTGCACATGGGTATGGGAGTTGTTTTTGCCGAAGGTGTTCATCATGTTAAAAAGATCCCTAATGAAGCATTGACATGCGGATTGACTGCAAAGTTGTTGTATAAGAACCGTGGTAGA.
In another technical scheme, the nucleotide sequence of the ascorbic acid oxidase with the amino acid sequence shown as SEQ ID NO.4 is shown as SEQ ID NO. 6: GCAAAAGCCAGACACTTTAATTGGGAGGTAGAGTACATGTATAGATCTCCAGATTGTTTGGAACACATTGTCATGGGAATCAACGGCCAATTCCCTGGTCCCACAATCCGTGCAAAGGCTGGTGACACACTTGTTATTGAATTGTCTAACAAACTTCACACAGAGGGAGTTGTTATCCACTGGCATGGTATTCGACAACTGGGTACTCCTTGGGCAGATGGTACTGCCTCCATTTCTCAATGTGCCATAAATCCAGGCGAAACATTTAAATACAGATTCAAAGTCGATAGGAGAGGTACATACTTTTATCACGGTCATTACGGCATGCAACGTTCTGCTGGTTTGTATGGAAGTCTTATCGTGGATGTGGCTGACGGTGAGAAAGAACCTTTTCACTACGATGGTGAGTTGAACTTGCTGTTGTCTGACTGGTGGCATAAGGGAGACCATGAACAAGAAGTAGGACTTTCCTCAAACCCCTTCAGGTGGATTGGAGAACCACAATCAGTCTTGATTAACGGAAGAGGTCAGTATAACTGCAGTATGGCCGCCAAATTTTCCAATCCCCCAATTGGCCAGTGTAAGTTTCGAGGTAACGAACAATGTGCTCCCCAAATTTTGAAGGTGCAGCCCAACAAAACCTACAGACTAAGAATAGCATCCACTACCGCTCTAGCTTCATTGAACCTGGCTATACAAGGTCACAAGATGGTTGTTGTTGAAGCCGACGGAAATCATGTCCAACCATTTGCTATGAATGACCTTGACATTTATTCTGGAGAATCTTACTCCGTTCTTTTGACCACAGATCAGAACCCATCCAGAAACTATTGGATTAGTATTGGTGTTAGAGCTAGAGAACCTAAAACACCTCAGGCTCTAACTATTCTGAACTATTCCCCAACATCTGCTTCCCGAATACCTATGAGTCAACCCCCAGTCACACCACGTTGGAACGATTACAACCATTCTAAAGCATTTACCAAGTCTATTTATGCATTAATGGGTTCTCCAAAGCCTCCTAAGACTTCCAACAGAAGAATTGTTCTGTTAAACACTCAAAACAGAGTTAATGGTTTTATTAAATGGTCCATCAACAATGTCTCTTTGGTCCTACCTTCTACGCCTTACTTAGGATCCCTAAAATTCGGTTTGAATAATTCTTTCGATCAAAAGTCACCACCTGACAACTATGATTCTTCTTACGATATTATGAAGCCTGCTGTTAATCAAAATTCAACACAGGGTTCCGGTATATACACAATTGGTCTGAACACTACAGTGGATGTTATATTGCAGAACGCCAACACTCTAGCTAAGGACGTGTCCGAAATTCATCCTTGGCACCTTCACGGTCACGATTTCTGGGTTCTGGGTTACGGTGAGAGTAAGTTTAAGGAGGGTGACGAAAAGACCTTCAACCTAAAGAATCCACCTTTGAGAAACACGGCAGTTATATTCCCTTACGGATGGACGGCCCTTAGGTTTGTCGCTGACAATCCTGGTGTTTGGGCTTTTCATTGTCATATAGAGCCCCACTTGCATATGGGTATGGGAGTTGTTTTTGCTCAGGGTGTGCATAGAGTTGGTCAGATTCCTAGAGAAGCCCTAGCTTGTGGTCTGACTGGAAATAAACATAAT.
The embodiment of the application provides a recombinant vector which contains the ascorbate oxidase gene.
The embodiment of the application provides engineering bacteria containing the recombinant vector of claim 6.
The embodiment of the application provides a preparation method of engineering bacteria, which comprises the following specific steps:
s1, constructing a vector for expressing ascorbate oxidase protein;
s2, transfecting the vector which is constructed in the step S1 and expresses the ascorbate oxidase protein into cells;
s3, obtaining expressed ascorbate oxidase protein from the transfected cells in the step S2;
s4, purifying the ascorbate oxidase protein obtained in the step S3.
The embodiment of the application provides an application of the ascorbate oxidase in enhancing the anti-interference aspect of a clinical diagnosis kit.
Example 1 vector construction of ASO and shake flask fermentation
1. Experimental materials
pPICZ alpha A vector plasmid, the X-33 strain was purchased from Invitrogen corporation; YNB was purchased from Beijing Tiangen Biotech Co., ltd; peptone and yeast powder were purchased from OXOID; biotin was purchased from Sigma; ni and SP columns were purchased from GE company; sacI enzyme and cleavage Buffer were purchased from NEB (Beijing) Inc.
2. Construction of ASO Yeast expression
The sequences of ascorbate oxidase are searched and compared in NCBI database, 18 suspected sequences are selected, codons are optimized, and then the sequences are synthesized by the Optimago, restriction sites EcoR I and Sal I are reserved at the N end and the C end respectively, the target fragment and pPICZ alpha A vector are connected by T4 ligase after double restriction of EcoR I and Sal I, the DNA sequence C section of ascorbate oxidase contains a 6 XHis tag, DH5 alpha competence is transformed, LLB (Low Salt LB medium) solid plates (Zeocin 25 mug/mL) are coated, 12 transformants are selected by colony PCR of each gene, the recombinant is identified by double restriction, the strains with the successful vector named pPICZ alpha A-ASO1 and 18 sequences are respectively numbered, and the partial map is shown in figure 1.
3. Ascorbate oxidase expression plasmid miniprep
Ascorbate oxidase positive clone DH 5. Alpha. Strain was inoculated into 10mL of LLB medium containing 25. Mu.g/mL zeocin and cultured to mid-log growth (OD 600 =0.5-0.7), 0.85mL of the bacterial solution was removed, sterilized glycerol was added to preserve the strain at-80 ℃, and the remaining bacteria were continued to be cultured overnight at 37 ℃. Extracting plasmid with plasmid miniprep kit, and packaging for preservation.
LLB (Low Salt LB) medium: trypton l%, yeast Extract 0.5%, naCL 0.5%, pH7.5. 2% agar powder is added for autoclaving at 121 ℃ for 20min when the flat plate is manufactured. Can be stored at room temperature for several months, and when used for culturing pPICZ alpha A prokaryotic host bacteria DH5 alpha, the culture medium is cooled to at least 55 ℃, then Zeocin is added to the final concentration of 25 mug/mL, and can be stored for 1-2 weeks at 4 ℃.
4. Ascorbate oxidase expression plasmid linearization
The expression plasmid pPICZαA-ASO 1-18 was digested with SacI (209 bp) endonuclease, which was digested at the 5' AOX1 site of the expression vector pPICZαA. The cleavage system was 100. Mu.L (plasmid more than 10. Mu.g), and after cleavage, the plasmid was checked for cleavage by electrophoresis. The linearized yeast run before and after the run versus electrophoresis, the cut strip runs slowly, the whole plasmid runs before, if the cut is incomplete, then there are two strips in the linearized lane. After the enzyme digestion is completed, EDTA is added to stop the reaction, or the heat inactivation condition is 65 ℃ for 20min.
5. Phenol chloroform extraction of plasmids
(1) About 100 mu L of the system after enzyme digestion is supplemented to 400 mu L;
(2) Adding equal volume of phenol chloroform (lower layer of phenol chloroform), mixing, standing at 4deg.C for 10min;
(3) Taking an upper water sample, adding 1/10 volume of 3M sodium acetate and 2.5 volumes of pre-cooled 100% ethanol, placing at-20 ℃, centrifuging for 20min at 1h and 4 ℃, and removing the supernatant;
(4) Adding 250 mu L of 80% ethanol to clean DNA, centrifuging for 20min at 4 ℃, and removing supernatant;
(5) Blow-drying, adding 10. Mu.L of sterile ddH 2 O, preserving at-20 ℃ for standby.
6. Preparation of yeast competence and electrotransformation of methanol-philic yeast
(1) The saccharomycetes are streaked and then are selected and cloned in a 5mL YPD 50mL centrifuge tube, and cultured overnight at 30 ℃ and 220 rpm; 0.25mL of seed solution was re-inoculated into 2L Erlenmeyer flasks containing 500mL of fresh medium and cultured overnight.
(2)OD 600 =1.3-1.5, generally shaking for 12-18 h without longer, 1500g, centrifuging at 4deg.C for 5min;
(3) 500mL of pre-chilled (0deg.C) sterile ddH was added 2 O,1500g, centrifuging at 4 ℃ for 5min; 250mL of pre-chilled (0 ℃) sterile ddH was added 2 O,1500g, centrifuging at 4 ℃ for 5min;
(4) The pellet was resuspended in 20mL ice-cold (0deg.C) 1M sorbitol, 1500g, and centrifuged at 4deg.C for 5min; the pellet was resuspended in 1mL ice-cold (0deg.C) 1M sorbitol to a final volume of about 1.5mL and placed in an ice bath for use without preserving the cells.
(5) Mixing 80 μL of the above cell+linear DNA (5-10 μg), transferring into 0.2cm electric rotating cup, placing on ice for 5min, wiping off water of metal sheet of the electric rotating cup, and applying voltage: 1500V, resistance: 400 Ω, capacitance: 25 μF, pulse time: 10mS, starting electric shock;
(6) Immediately add 1mL ice-cold (0 ℃) 1M sorbitol, transfer to 15mL sterile centrifuge tube;
(7) Standing and incubating for 1-2h at 30 ℃;
(8) Taking 50-200 mu L, coating YPDS flat plate containing zeocin100 mu g/mL;
(9) Placing the strain in a 30 ℃ incubator for 2-3 days, and growing transformants on the plates;
note that: YPD or YPDs medium configuration (Yeast Extract Peptone Dextrose Medium, yeast extract/tryptone/dextrose medium): the liquid YPD medium with the concentration of 1% of Yeast extract, 2% of peptone, dextrose (glucose)%, 1Msorbitol, 2%agar,Zeocin 100 mug/mL can be stored at normal temperature, is the most basic medium of pichia pastoris, and can be stored for several months at 4 ℃. Zeocin 100. Mu.g/mL was added to the medium to prepare YPDZ medium, which was stored at 4℃for 1-2 weeks.
7. Identification and trial expression of transformants
(1) And (5) after single colony grows on the YPDS plate, 10-20 single clones are selected, and the back of the plate is marked. Inoculating to fresh YPD medium, culturing, collecting fungus, adding glycerol, storing at-80deg.C, transferring into small triangular flask containing 50mL BMGY, culturing at 30deg.C and 200rpm until OD 600 ＝1-1.5；
(2) Transferring the culture system in the small triangular flask into a 50mL centrifuge tube, 3000g, and collecting bacteria in 5min;
(3) The pellet was resuspended to OD with BMMY 600 =0.3 (about 100-200 mL);
(4) Transferring to 500mL big triangular flask, inducing and culturing at 30deg.C and 200rpm, taking out 1mL of the culture medium at intervals, quick-freezing, storing at-80deg.C, supplementing 0.5% methanol, and sampling at time points: 0h, 24h, 48h, 72h, 96h;
(5) Centrifuging the samples taken at each time point for 1min to obtain supernatant, adding 10 mu L L loading buffe to 30 mu L of the supernatant, boiling for 10min, centrifuging (13000 rpm,5 min), and taking 15 mu L of SDS-PAGE;
note that: BMGY medium: yeast extract 1%, peptone 2%, potassium phosphate buffer (pH 6.0) 100mmol/L, YNB 1.34%, biotin (4X 10) -5 )％，Glycerol 1％
Pichia pastoris induction expression pre-culture medium, YNB and Biotin filtration sterilization. After 24 hours of incubation, typically left overnight, the BMGY medium was removed after yeast precipitation, and the BMMY medium was changed to enter the induction expression stage.
BMMY medium: yeast extract 1%, peptone 2%, potassium phosphate buffer (pH 6.0) 100mmol/L, YNB 1.34%, biotin (4X 10) -5 )％，methanol 3％
Pichia pastoris induced expression medium, YNB and Biotion filtration sterilization. Shaking flask culture is followed by induction with 3% methanol every 24 hours, typically for 72 hours.
8. Shaking flask fermentation of pPICZ alpha A-ASO strain
Inoculating the constructed yeast strain expressing the protein of the ascorbate oxidase into 10mL YPD culture medium, and shake culturing at 150rpm and 29 ℃ for 24 hours to reach the logarithmic growth phase, and taking the yeast strain as a first-stage seed; first seed was prepared by mixing 1:100 is inoculated to 100mL of BMGY culture medium, after shake culture at 150rpm and 29 ℃ for 4 days, 1.8v/v% methanol is added every day to induce expression, and fermentation is finished on the 7 th day; transferring the fermentation liquor into a centrifugal barrel, centrifuging for 15min at 12000g, and collecting the supernatant to obtain the crude enzyme solution of the cells.
9. Purification
And (5) keeping a small sample of the fermentation broth for activity measurement on the collected supernatant. The remainder was purified, and the ascorbate oxidase was purified by nickel column affinity chromatography.
EXAMPLE 2 determination of ascorbate oxidase Activity
One unit of ascorbate oxidase activity is defined as the amount of enzyme that oxidizes 1.0 mu moL of ascorbate per minute at 37℃and pH 5.5.
The reaction formula:
the disappearance of ascorbic acid was measured spectrophotometrically at 245 nm.
Reagent configuration
ASO enzyme activity detection step
1. The following reaction mixture was prepared in a reaction tube (UV quartz cup) and equilibrated at 30℃for 5minutes.
| Composition of the components | Volume of | |
| A | Substrate solution | 0.5mL |
| B | Na 2 HPO 4 Solution | 0.5mL |
(the pH of the reaction mixture was adjusted to 5.5)
2. Add 0.1mL of ASO enzyme solution to be tested, mix upside down.
3. After 5minutes of reaction at 30 ℃, 3.0mL of HCL solution (C) was added to terminate the reaction, and the mixture was inverted and mixed.
4. The spectrophotometer was zeroed with distilled water and the OD blanc was measured at 245nm (OD test) while the enzyme reagent (D) was used instead of ASO enzyme solution.
ASO enzyme solution pre-diluted to 0.15-0.25U/mL with pre-chilled enzyme reagent (D); if the ASO enzyme activity exceeds 60U/mL, the ASO enzyme can be diluted by pre-chilled sterile water, and the ASO enzyme can be diluted before activity measurement.
ASO enzyme Activity calculation
The ASO enzyme activity unit calculating method comprises the following steps:
weight ASO enzyme Activity (U/mg) = (U/mL) ×1/C
Wherein the meaning of each symbol or number is as follows: vt, total volume (4.1 mL); vs, sample volume (0.1 mL); 10.0, millimole extinction coefficient (cm) of ascorbic acid at pH1.0 2 /mM); 1.0, light passing path (cm); t, reaction time (5 minutes); df, dilution factor; c, enzyme concentration (C mg/mL)
The crude enzyme solution of the cells was taken to measure the enzyme activity, high-yield transformants were selected, ASO was purified after fermentation and lyophilized, and the activity was measured again, and the activity of the mutant strain ASO3m4 was increased by 112% compared with ASO3, as shown in Table 1.
TABLE 1ASO Activity statistics
| Numbering device | Activity (U/mg-solid) |
| ASO1 | 230 |
| ASO1m2 | 225 |
| ASO3 | 268 |
| ASO3m4 | 570 |
EXAMPLE 3 thermal stability random mutagenesis of ascorbate oxidase Gene
Randomly mutating the expression vectors pPICZ alpha A-ASO1 and pPICZ alpha A-ASO3 of the ascorbate oxidase sequences SEQ ID NO 1 and SEQ ID NO 2 by error-prone PCR, cutting gel, recovering to obtain a mutated DNA fragment, and connecting the mutated DNA fragment with the pPICZ alpha A plasmid to construct a recombinant vector. The plasmid is firstly linearized, then transferred into the competence of X-33 Pichia pastoris expression bacteria by electrotransformation, the plating plate is picked and cloned, and the plating plate is subjected to expansion culture to obtain a transformation product culture solution.
The conditions for random mutation by the error-prone PCR method are as follows:
25. Mu.L of 2 Xerror-prone PCR buffer (100mM KCL,15mM MgCL) 2 50mM Tris-HCl pH8.3,0.02% gelatin; 5. Mu.M dATP, 5. Mu.M dGTP, 5. Mu.M dTTP and 5. Mu.M dCTP;0.1mM MnCL 2 0.5U Taq DNA polymerase), 500nM primer ASO-For, 500nM primer ASO-Rev, 25ng plasmid DNA, sterile water to 50. Mu.L reaction volume;
the amplification procedure was: the reaction system is carried out for 8min at 95 ℃;31 cycles of "95℃30s,56℃30s,72℃1min50s";72 ℃ for 10min; the resulting PCR amplified gene product was separated by 1% agarose gel electrophoresis, and a DNA fragment was recovered.
Further, in the PCR amplification reaction of pPICZαA-ASO1 and pPICZαA-ASO3, the following primer pairs were used, respectively:
ASO-For1:5’-GAATTCGCAAAAACTAGACA-3’；
ASO-Rev1:5’-GTCGACTCTACCACGGTTCT-3’；
ASO-For3:5’-GAATTCGCAAAAGCCAGACA-3’；
ASO-Rev3:5’-GTCGACATTATGTTTATTTC-3’。
mutant screening
Cloning after random mutation, culturing bacterial liquid, performing ultrasonic cell disruption after expanding culture and induction, heating in water bath at a plurality of different incremental temperatures, and then measuring the activity of the cell disruption liquid heated at different temperatures.
In the random mutation experiment of pPICZαA-ASO1, clone which is not lost by ascorbic acid oxidase under the heating of 60 ℃ is obtained, the clone is the mutant ascorbic acid oxidase genetic engineering strain with good heat stability, the amino acid sequence of the strain is shown as SEQ ID NO.3, and the carrier of the strain is pPICZαA-ASO1m2.
In a random mutation experiment on pPICZαA-ASO3, a clone which does not lose the ascorbate oxidase under the heating of 60 ℃ is obtained, the activity of the ascorbate oxidase is increased by 112 percent by the mutation, the clone is a mutant ascorbate oxidase genetic engineering strain with good heat stability and high activity, the amino acid sequence of the clone is shown as SEQ ID NO.4, and the vector of the clone is pPICZαA-ASO3m4.
Example 4 influence of pH and temperature on ASO enzyme Activity and stability
1. Influence of pH on ASO enzyme Activity and stability
Preparing enzyme detection solution (pH 3.0-6.0, acetate buffer, pH5.0-9.0, phosphate buffer) with buffers of different pH (pH 3-9), measuring enzyme activity according to the above method, and determining optimal reaction pH value of ASO. ASO activity was measured at 30℃in a buffer at pH3-9, and as shown in FIG. 2, the enzyme activity was 0 at pH3.0, and as pH increased, the ascorbate oxidase activity was gradually increased to a maximum at pH 5.5. And then starts to decrease. Thus, the enzyme has activity between pH5 and 8, and the optimal reaction pH for the enzyme is 5.5.
The enzyme solution was diluted with Britton-Robinson buffer having a different pH (pH 3-12), and the residual enzyme activity was measured after incubation at 25℃for 17 hours. The pH stability of ASO was examined by preparing a curve of pH and enzyme activity with the enzyme activity of ASO at the optimum pH. As a result, as shown in FIG. 3, the activity of ascorbate oxidase was gradually increased in the treatment at pH6-10, and the activity of ascorbate oxidase was not affected in the treatment at pH6-10, and then the activity was decreased with the increase in pH.
2) Influence of temperature on ASO enzyme Activity and stability
The enzyme activities at different temperatures were determined as described above in50 mM phosphate buffer pH5.5 to determine the optimal reaction temperature for ASO. ASO activity data were obtained at 25deg.C, 30deg.C, 35deg.C, 40deg.C, 45deg.C and 50deg.C, the highest activity temperature was 30deg.C, and a temperature-ASO activity curve was prepared, and the result showed in FIG. 4 that ASO activity increased at 25-30deg.C and ASO activity gradually decreased at 30-50deg.C.
The heat stability of ASO is studied, the enzyme solution with the enzyme activity of 12U/mL is respectively kept at 20 ℃,30 ℃, 40 ℃,50 ℃, 60 ℃ and 70 ℃ for 30min in50 mM phosphate buffer with the pH value of 8.0, the temperature is quickly cooled to room temperature, the residual enzyme activity is measured, and the enzyme activity curve of ASO after being kept at different temperatures for 30min is prepared, and as a result, the ASO is treated for 30min at 20-50 ℃ without losing the enzyme activity basically, as shown in figure 5; the enzyme is basically lost to activity after 30min of treatment at 70 ℃; after 30min of treatment at 60℃the mutants ASO1m2 and ASO3m4 showed substantially no loss of viability, whereas ASO1 and ASO3 showed substantially no loss of viability.
Example 5 Yeast fermenter Process for ASO
Shake flask culture of ASO fermentation seed liquid:
the shake flask contains BMGY culture medium 5-10% of the initial fermentation broth volume, and a colony is selected from MGY plate or bacterial liquid is taken from frozen glycerol stock and inoculated into shake flask, and cultured on shake flask at 30deg.C and 250rpm for 16-24 hr until OD 600 ＝2-6。
Batch fermentation of ASO glycerol:
1. the fermentor and fermentation basal salt medium containing 4% glycerol were sterilized.
2. After sterilization and cooling, when the temperature was reduced to 30 ℃, stirring and aeration were turned on and the pH of the medium was adjusted to 5.0 with 28% ammonia. 4.35mL of sterile PTM1 base salt was added per liter of medium.
3. Inoculating seed liquid with initial fermentation volume of 5-10% into the fermentation tank. DO values were close to 100% before the start of the culture. Oxygen is consumed after the start of culture, resulting in a decrease in DO value. Oxygen is added as needed to ensure that the DO value exceeds 20%.
4. Batch fermentation was performed until glycerol was completely consumed (18-24 h), marked by an increase in DO to 100%. The time for complete consumption of glycerol will vary with the initial broth density.
5. The completion of each fermentation stage requires sampling and at least twice daily. 10mL of sample was taken at each time point, and another 1mL of sample was taken from 10 mL. Samples were used to analyze cell growth (OD 600 And wet cell weight), pH, microscopic observation, protein concentration or activity. The centrifuged cells and supernatant were stored at-80℃and used for the subsequent analysis.
The cell yield achieved at this stage was 90-150g/L wet cells. Recombinant ASO enzymes are not produced due to the lack of induction of methanol.
ASO glycerol feed culture:
introduction: once all glycerol is consumed in the batch fermentation culture, glycerol feed needs to begin to increase cellular biomass under limiting conditions immediately. When preparing for methanol induction, it is first necessary to determine that glycerol is depleted by DO values.
1. The feed was added with 50% w/v glycerin containing 12mL of PTM1 per liter. The feed rate was set at 18.15mL per hour per liter of initial broth volume.
2. Glycerol feed will be conducted for about 4 hours or more. The cell yield should reach 180-220g/L wet cells after this stage is completed but no recombinant egg mass is produced.
Note that: the expression level of the protein depends on the amount of cells produced in glycerol feed culture. The feed duration will be staggered to optimize protein production, typically in the approximate range of 50-300g/L wet cells. The maximum concentration of glycerol in the feed is 4%, and higher glycerol concentrations can cause poisoning problems.
If the dissolved oxygen is less than 20%, the feed of glycerol or methanol should be stopped and nothing to raise the dissolved oxygen should be done until the dissolved oxygen is stable. At this point stirring, aeration, agitation or oxygen replenishment is initiated.
ASO methanol feed culture:
glycerol must be consumed cleanly before methanol feed can be used to fully induce the AOX1 promoter. Methanol is slowly fed to adapt the culture to growth in methanol, if methanol is fed too fast, the cells will be killed.
Once the culture was conditioned with methanol, the status of the culture was analyzed with DO values and the time point of methanol addition was determined to optimize protein expression. The growth liquid generates a lot of heat on methanol. The control of the temperature at this stage is very important.
Methanol feed culture of ASO strain (Mut +):
1. the glycerol feed was terminated and induction with 100% methanol per liter containing 12ml of PTM1 base salt was started. The feed rate was set at 3.5mL/h per liter of initial broth volume.
2. Methanol will accumulate in the broth during the first 2-3 hours and the DO value will change during the reaction of the culture with methanol.
3. If the DO value cannot be maintained above 20%, stopping feeding methanol, and continuing feeding methanol at the current speed after the DO value is stable. Stirring, aeration, pressure or oxygen supplementation was added to maintain DO above 20%.
4. When the culture is fully adapted to methanol (2-4 h) and methanol is the limiting growth factor, there is a stable DO reading and a faster DO stabilization time point (typically less than 1 min). This lower feed rate was maintained under limiting conditions for a minimum of 1h after the culture was acclimatized to methanol and before doubling the feed rate. The addition rate was then doubled to about 7mL/h per liter of initial broth volume.
5. After 2h of feeding at a rate of 7mL// L, the methanol feeding rate was increased to 10.5mL/h per liter of initial broth volume. This flow rate runs through the rest of the fermentation process.
6. The whole methanol feed culture was added together to approximately 700mL per liter of initial volume of broth for approximately 72 hours.
Harvesting ASO fermentation supernatant:
the culture was transferred to a centrifuge tube (1000 mL) and centrifuged to separate the cells from the supernatant.
PTM1 basic salt: cuSO 4 -5H 2 O,5g; sodium iodide, 0.08g; mnSO 4 -H 2 O,3g; sodium molybdate dihydrate, 0.2g; boric acid, 0.02g; cobalt chloride, 0.5g; znCL (ZnCL) 2 ，20g；FeSO 4 65g; biotin, 0.2g; h 2 SO 4 5mL of water was added to 1L and the mixture was sterilized by filtration.
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.
Claims (7)
1. The ascorbate oxidase is characterized in that the amino acid sequence of the ascorbate oxidase is shown as SEQ ID NO.3 or SEQ ID NO.4.
2. The gene for encoding the ascorbate oxidase of claim 1, wherein the nucleotide sequence is shown as SEQ ID NO.5, and the amino acid sequence of the encoded ascorbate oxidase is shown as SEQ ID NO.3.
3. The gene for encoding the ascorbate oxidase of claim 1, wherein the nucleotide sequence is shown in SEQ ID NO.6, and the amino acid sequence of the encoded ascorbate oxidase is shown in SEQ ID NO.4.
4. A recombinant vector comprising the ascorbate oxidase gene of claim 2 or 3.
5. An engineering bacterium comprising the recombinant vector of claim 4.
6. The method for preparing engineering bacteria according to claim 5, comprising the following specific steps:
s1, constructing a vector for expressing ascorbate oxidase protein;
s2, transfecting the vector which is constructed in the step S1 and expresses the ascorbate oxidase protein into cells;
s3, obtaining expressed ascorbate oxidase protein from the transfected cells in the step S2;
s4, purifying the ascorbate oxidase protein obtained in the step S3.
7. Use of the ascorbate oxidase of claim 1 for enhancing the anti-interference properties of a clinical diagnostic kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211180850.5A CN115786289B (en) | 2022-09-27 | 2022-09-27 | Ascorbate oxidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211180850.5A CN115786289B (en) | 2022-09-27 | 2022-09-27 | Ascorbate oxidase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115786289A CN115786289A (en) | 2023-03-14 |
| CN115786289B true CN115786289B (en) | 2023-06-06 |
Family
ID=85432238
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211180850.5A Active CN115786289B (en) | 2022-09-27 | 2022-09-27 | Ascorbate oxidase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115786289B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997009431A1 (en) * | 1995-09-01 | 1997-03-13 | Novo Nordisk Biotech, Inc. | Blue copper oxidase mutants with enhanced activity |
| JPH1175848A (en) * | 1997-09-03 | 1999-03-23 | Ichibiki Kk | Variant ascorbic acid oxidase |
| JP2006068003A (en) * | 2004-08-04 | 2006-03-16 | Asahi Kasei Pharma Kk | Oxidase and oxidation reagent containing the same |
| JP2008178301A (en) * | 2007-01-23 | 2008-08-07 | Asahi Kasei Pharma Kk | Ascorbate oxidase |
| JP2015171359A (en) * | 2014-02-24 | 2015-10-01 | 池田食研株式会社 | Modified pyranose oxidases |
| CN111662888A (en) * | 2020-06-15 | 2020-09-15 | 北京达成生物科技有限公司 | Diaphorase mutant with high thermal stability, gene and preparation method thereof |
| CN111876394A (en) * | 2020-07-01 | 2020-11-03 | 华南农业大学 | Application of ascorbic acid oxidase RIP5 in regulation and control of drought resistance of rice |
| CN112608938A (en) * | 2020-12-22 | 2021-04-06 | 华中农业大学 | Application of OsAO2 gene in controlling drought resistance of rice |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8993255B2 (en) * | 2008-10-10 | 2015-03-31 | Toyo Boseki Kabushiki Kaisha | Protein having fructosyl valyl histidine oxidase activity, modified protein, and use of the protein or the modified protein |
-
2022
- 2022-09-27 CN CN202211180850.5A patent/CN115786289B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997009431A1 (en) * | 1995-09-01 | 1997-03-13 | Novo Nordisk Biotech, Inc. | Blue copper oxidase mutants with enhanced activity |
| JPH1175848A (en) * | 1997-09-03 | 1999-03-23 | Ichibiki Kk | Variant ascorbic acid oxidase |
| JP2006068003A (en) * | 2004-08-04 | 2006-03-16 | Asahi Kasei Pharma Kk | Oxidase and oxidation reagent containing the same |
| JP2008178301A (en) * | 2007-01-23 | 2008-08-07 | Asahi Kasei Pharma Kk | Ascorbate oxidase |
| JP2015171359A (en) * | 2014-02-24 | 2015-10-01 | 池田食研株式会社 | Modified pyranose oxidases |
| CN111662888A (en) * | 2020-06-15 | 2020-09-15 | 北京达成生物科技有限公司 | Diaphorase mutant with high thermal stability, gene and preparation method thereof |
| CN111876394A (en) * | 2020-07-01 | 2020-11-03 | 华南农业大学 | Application of ascorbic acid oxidase RIP5 in regulation and control of drought resistance of rice |
| CN112608938A (en) * | 2020-12-22 | 2021-04-06 | 华中农业大学 | Application of OsAO2 gene in controlling drought resistance of rice |
Non-Patent Citations (5)
| Title |
|---|
| L-ascorbate oxidase-like [Carya illinoinensis];NCBI;Genbank;Accession No. XP_042959338.1 * |
| L-ascorbate oxidase-like [Hibiscus syriacus];NCBI;Genbank;Accession No. XP_039015061.1 * |
| The role of quaternary interactions on the stability and activity of ascorbate peroxidase;Protein Science;第7卷(第10期);2089–2098 * |
| 植物体内自由基清除剂抗坏血酸研究进展;闫慧芳;夏方山;王明亚;毛培胜;;草业科学(08) * |
| 芥蓝抗坏血酸过氧化物酶结构稳定性及分子改造研究;席加富;中国优秀硕士学位论文全文数据库基础科学辑（电子期刊）(第2期);A006-362 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115786289A (en) | 2023-03-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ingram et al. | Expression of different levels of ethanologenic enzymes from Zymomonas mobilis in recombinant strains of Escherichia coli | |
| US7056717B2 (en) | Genes involved in isoprenoid compound production | |
| CN101454449B (en) | Glucose dehydrogenase | |
| CN113832083B (en) | Bacillus beleisi and application thereof in vinegar brewing | |
| CN108103081B (en) | Acetaldehyde dehydrogenase and application thereof in anti-alcohol health-care product | |
| CN113430147B (en) | Bacillus villagens QH-20011 with low pH tolerance and application thereof | |
| CN115786289B (en) | Ascorbate oxidase | |
| CN112852859B (en) | Method for improving synthesis capacity of filamentous fungi organic acid | |
| CN115948373B (en) | Algin lyase mutant Pl7AaM and application thereof | |
| JP4903129B2 (en) | Thermostable yeast strain | |
| CN109957538A (en) | A kind of genetic engineering bacterium and its preparation method and application preparing sarcosine oxidase | |
| US9290782B2 (en) | Modified algae for improved biofuel productivity | |
| CN111808836B (en) | Heat-resistant mutant enzyme of pullulanase I and preparation method and application thereof | |
| CN105779446B (en) | Glycerol 3-phosphate kinase promoter and terminator and its application | |
| CN111378583B (en) | Trichoderma reesei and application thereof | |
| CN106754769A (en) | A kind of tomato Inappropriate ADH syndrome gene and application | |
| CN104342409B (en) | Recombinate the preparation method of ginseng superoxide dismutase | |
| CN113913410B (en) | Construction and application of yak rumen anaerobic fungus xylanase gene engineering bacteria | |
| KR100578539B1 (en) | Phytase produced from Citrobacter braakii | |
| CN106318941B (en) | A kind of glucose derepresses evoked promoter and terminator and its application | |
| CN105779447B (en) | Glyceraldehyde 3-phosphate dehydro-genase promoter and terminator and its application | |
| CN106148338B (en) | Na+/ Pi cotransporter promoter and terminator and its application | |
| CN116004398B (en) | Aspergillus niger strain capable of expressing lactase and protease and application thereof | |
| CN111826368B (en) | Mutant enzyme of type I pullulanase and preparation method and application thereof | |
| CN114752588B (en) | Heparinase II |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |