CN115786289A

CN115786289A - Ascorbic acid oxidase

Info

Publication number: CN115786289A
Application number: CN202211180850.5A
Authority: CN
Inventors: 肖秀孝; 刘鹏飞; 郑长龙; 赵占勇
Original assignee: Beijing Dacheng Biotechnology Co ltd
Current assignee: Beijing Dacheng Biotechnology Co ltd
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-03-14
Anticipated expiration: 2042-09-27
Also published as: CN115786289B

Abstract

The invention discloses an ascorbic acid oxidase, a preparation method and application thereof, wherein the amino acid sequence of the ascorbic acid oxidase is shown as SEQ ID NO.3 or SEQ ID NO.4, and compared with the wild type ascorbic acid 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 ascorbic acid oxidase in enhancing the anti-interference of the clinical diagnosis kit can eliminate the interference of the ascorbic acid in the detection of the kit on the urogalactose, the total cholesterol, the triacylglycerol, the creatinine and the like.

Description

Ascorbic acid oxidase

Technical Field

The invention relates to the field of biotechnology, and more particularly relates to ascorbic acid oxidase.

Background

Ascorbic acid oxidase (ASO) is a copper-containing enzyme found in melons, seeds, grains, fruits and vegetables. It oxidizes ascorbic acid to water and dehydroascorbic acid. In contrast to non-enzymatic oxidation, ascorbic acid oxidase produces water after its action, whereas the former produces hydrogen peroxide.

The yeast expression system comprises a saccharomyces cerevisiae expression system, a methanol yeast expression system and other yeast expression systems. The pichia pastoris is a yeast variety capable of efficiently expressing recombinant proteins, on one hand, because the pichia pastoris belongs to eukaryotes, the expressed proteins can be subjected to glycosylation modification, and on the other hand, the pichia pastoris has high growth speed, can secrete the expressed proteins into a culture medium, and is convenient for protein purification. Pichia pastoris belongs to methylotrophic yeast, and can use methanol as a sole carbon source.

At present, the ascorbic acid oxidase used at home and abroad is mainly obtained from pumpkin. The source of the ascorbic acid is single, the production cost is high, and the content and the quality of the ascorbic acid oxidase are influenced by batches, so that the application of the ascorbic acid oxidase is greatly limited. The corresponding products of domestic and foreign companies have poor thermal stability and cannot meet the high temperature problem generated in reagent transportation. Therefore, a new ascorbate oxidase gene and yeast expression system are needed.

Disclosure of Invention

It is an object of the present invention to address at least the above problems and to provide at least the advantages described hereinafter.

Still another object of the present invention is to provide an ascorbic acid oxidase, the amino acid sequence of the enzyme is shown in SEQ ID No.3 or SEQ ID No.4, the amino acid sequence of SEQ ID No.3 is formed by mutation of the amino acid sequence of SEQ ID No.1, the amino acid sequence of SEQ ID No.4 is formed by mutation of the amino acid sequence of SEQ ID No.2, and the amino acid sequence of SEQ ID No.3 and the amino acid sequence of SEQ ID No.4 have higher thermal stability and/or activity than the wild-type ascorbic acid oxidase.

The protein expressed by the ascorbic acid oxidase can eliminate the interference of the kit in detecting ascorbic acid in urine galactose, total cholesterol, triacylglycerol, creatinine and the like.

To achieve these objects and other advantages in accordance with the purpose of the invention, there is provided an ascorbic acid oxidase having an amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

Preferably, the amino acid sequence SEQ ID NO.3 of the ascorbic acid oxidase is obtained by mutation of the amino acid sequence SEQ ID NO.1 of the ascorbic acid oxidase, and the specific mutation is as follows:

carrying out error-prone PCR random mutation on an ascorbic acid oxidase amino acid sequence SEQ ID NO.1, wherein the 275 th amino acid of the amino acid sequence SEQ ID NO.1 is mutated into Q from K, and the 429 th amino acid is mutated into N from D, so as to obtain the ascorbic acid oxidase amino acid sequence SEQ ID NO.3.

Preferably, the amino acid sequence SEQ ID NO.4 of the ascorbic acid oxidase is obtained by mutation of the ascorbic acid oxidase amino acid sequence SEQ ID NO. 2;

carrying out error-prone PCR random mutation on an ascorbic acid oxidase amino acid sequence SEQ ID NO.2, wherein 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 as to obtain the ascorbic acid oxidase amino acid sequence SEQ ID NO.4.

Preferably, the amino acid sequence of the coded ascorbic acid oxidase is shown in SEQ ID NO. 5as the nucleotide sequence of SEQ ID NO.3.

Preferably, the amino acid sequence of the ascorbic acid oxidase is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 6.

In another aspect of the present invention, there is provided a recombinant vector comprising said ascorbate oxidase gene.

The invention also provides an engineering bacterium containing the recombinant vector.

The invention also provides a preparation method of the engineering bacteria, which comprises the following specific steps:

s1, constructing a carrier for expressing ascorbic acid oxidase protein;

s2, transfecting the vector expressing the ascorbic acid oxidase protein constructed in the step S1 into a cell;

s3, obtaining the expressed ascorbic acid oxidase protein from the transfected cells in the step S2;

and S4, purifying the ascorbic acid oxidase protein obtained in the step S3.

The invention also provides an application of the ascorbic acid oxidase in enhancing the anti-interference of a clinical diagnosis kit.

The invention at least comprises the following beneficial effects:

firstly, the invention carries out error-prone PCR random mutation on an ascorbic acid oxidase amino acid sequence SEQ ID NO.1, the 275 th amino acid of the amino acid sequence SEQ ID NO.1 is mutated into Q from K, the 429 th amino acid is mutated into N from D, thus obtaining the amino acid sequence SEQ ID NO.3 of the ascorbic acid oxidase, carries out error-prone PCR random mutation on an ascorbic acid oxidase amino acid sequence SEQ ID NO.2, the 149 th amino acid of the amino acid sequence SEQ ID NO.4 is mutated into D from V, and the 317 th amino acid is mutated into R from P, thus obtaining the amino acid sequence SEQ ID NO.4 of the ascorbic acid oxidase, wherein compared with a wild type ascorbic acid 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.

And secondly, the application of the ascorbic acid oxidase in enhancing the anti-interference of the clinical diagnosis kit, wherein the ascorbic acid oxidase can eliminate the interference of the kit in detecting ascorbic acid in urine galactose, total cholesterol, triacylglycerol, 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 pPICZ alpha A-ASO vector map according to one embodiment of the present invention;

FIG. 2 is a pH-ASO activity curve;

FIG. 3 is a graph showing the enzyme activity of ASO after 17h at various pH;

FIG. 4 is a graph of activity at temperature versus ASO;

FIG. 5 is a graph showing the enzyme activity curves of ASO after 30min holding at different temperatures.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

The embodiment of the application provides an ascorbic acid oxidase, the amino acid sequence of the ascorbic acid oxidase is shown as SEQ ID NO.3 or SEQ ID NO.4, wherein the amino acid sequence of SEQ ID NO.3: <xnotran> AKTRHFKWEVEYMYWSPDCIEHVVMGINGQFPGPTIRAKAGDTVVVELTNKLPTEGVVIHWHGIRQLGTPWADGTAFISQCAINSGETFHYRFKVDRAGTYFYHGHLGMQRSAGLYGSLLVDVAEGEKEPFHYDGEFNLLLSDWWHKSVHEQEVGLSSNPFRWIGEPQSLLINGRGQYNCSLAAQYSDTNSSQCKLRGNEQCAPQILHVRSNKTYRLRVASSTALASLNLAIGNHKMVVVEADGNYLQPFKVNDLDIYSGESYSVLITTNQDPSQNYWLSIGVRGRLPATPPGLTILNYQPTSASKFPTSPPPVTPPWNDYHHSKMFSKSIFALMGSPKPPTSYDRRISLLNTQNKIDGFTKWAINNVSLALPPTPYLGSIKYGLRNAFDQKSPPENFPDNYDVMRPPINPNSTTGSGVYMFGLNTTVNVILQNANALSDNVSEIHPWHLHGHDFWVLGYGEGKFSAKDEKKLNFKNPPLRNTAVIFPYGWTALRFVADNPGVWAFHCHIEPHLHMGMGVVFAEGVHHVKKIPNEALTCGLTAKLLYKNRGR; </xnotran> Amino acid sequence SEQ ID No.4: <xnotran> AKARHFNWEVEYMYRSPDCLEHIVMGINGQFPGPTIRAKAGDTLVIELSNKLHTEGVVIHWHGIRQLGTPWADGTASISQCAINPGETFKYRFKVDRRGTYFYHGHYGMQRSAGLYGSLIVDVADGEKEPFHYDGELNLLLSDWWHKGDHEQEVGLSSNPFRWIGEPQSVLINGRGQYNCSMAAKFSNPPIGQCKFRGNEQCAPQILKVQPNKTYRLRIASTTALASLNLAIQGHKMVVVEADGNHVQPFAMNDLDIYSGESYSVLLTTDQNPSRNYWISIGVRAREPKTPQALTILNYSPTSASRIPMSQPPVTPRWNDYNHSKAFTKSIYALMGSPKPPKTSNRRIVLLNTQNRVNGFIKWSINNVSLVLPSTPYLGSLKFGLNNSFDQKSPPDNYDSSYDIMKPAVNQNSTQGSGIYTIGLNTTVDVILQNANTLAKDVSEIHPWHLHGHDFWVLGYGESKFKEGDEKTFNLKNPPLRNTAVIFPYGWTALRFVADNPGVWAFHCHIEPHLHMGMGVVFAQGVHRVGQIPREALACGLTGNKHN. </xnotran>

In another technical scheme, the amino acid sequence SEQ ID NO.3 of the ascorbic acid oxidase is obtained by mutating the amino acid sequence SEQ ID NO.1 of the ascorbic acid oxidase, and the specific mutation is as follows:

carrying out error-prone PCR random mutation on an ascorbic acid oxidase amino acid sequence SEQ ID NO.1, wherein 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 ascorbic acid oxidase amino acid sequence SEQ ID NO.3, the amino acid sequence SEQ ID NO.1: <xnotran> AKTRHFKWEVEYMYWSPDCIEHVVMGINGQFPGPTIRAKAGDTVVVELTNKLPTEGVVIHWHGIRQLGTPWADGTAFISQCAINSGETFHYRFKVDRAGTYFYHGHLGMQRSAGLYGSLLVDVAEGEKEPFHYDGEFNLLLSDWWHKSVHEQEVGLSSNPFRWIGEPQSLLINGRGQYNCSLAAQYSDTNSSQCKLRGNEQCAPQILHVRSNKTYRLRVASSTALASLNLAIGNHKMVVVEADGNYLQPFKVNDLDIYSGESYSVLITTNQDPSKNYWLSIGVRGRLPATPPGLTILNYQPTSASKFPTSPPPVTPPWNDYHHSKMFSKSIFALMGSPKPPTSYDRRISLLNTQNKIDGFTKWAINNVSLALPPTPYLGSIKYGLRNAFDQKSPPENFPDNYDVMRPPINPNSTTGSGVYMFGLNTTVDVILQNANALSDNVSEIHPWHLHGHDFWVLGYGEGKFSAKDEKKLNFKNPPLRNTAVIFPYGWTALRFVADNPGVWAFHCHIEPHLHMGMGVVFAEGVHHVKKIPNEALTCGLTAKLLYKNRGR, SEQ ID NO.1 : </xnotran> <xnotran> GCAAAAACTAGACATTTTAAGTGGGAGGTTGAATACATGTACTGGTCTCCAGATTGTATTGAACACGTCGTAATGGGAATCAATGGACAATTTCCTGGACCAACCATCAGAGCTAAAGCTGGTGACACAGTTGTGGTCGAACTAACCAATAAGCTGCCAACTGAAGGTGTTGTGATTCATTGGCACGGTATTAGACAACTTGGAACGCCTTGGGCCGATGGTACCGCTTTTATTAGTCAATGTGCTATCAACTCTGGCGAAACTTTTCATTATCGATTTAAAGTTGACAGAGCAGGTACGTACTTTTACCATGGACACTTGGGTATGCAAAGAAGTGCTGGTCTGTATGGTTCTCTATTGGTTGATGTGGCAGAAGGCGAAAAGGAACCTTTCCATTACGACGGAGAGTTTAACTTGCTATTGTCAGACTGGTGGCACAAGTCAGTACATGAACAAGAGGTCGGATTGTCATCAAATCCTTTCAGATGGATTGGAGAGCCCCAATCTCTGCTAATCAACGGTAGAGGACAATATAATTGTTCTTTGGCTGCCCAATATTCTGATACTAACTCTTCCCAATGTAAATTGAGAGGTAATGAACAATGTGCACCCCAAATCCTACATGTTAGATCCAATAAAACCTACAGATTGAGAGTTGCCTCTTCTACCGCATTAGCATCTTTAAATTTGGCAATTGGCAATCATAAGATGGTTGTTGTGGAAGCAGATGGCAACTATTTGCAGCCATTTAAGGTCAATGATCTAGATATTTACTCTGGAGAATCATATAGTGTATTGATTACTACAAACCAAGATCCATCAAAGAACTACTGGTTATCAATTGGTGTTAGAGGCAGGTTACCTGCAACTCCTCCTGGACTTACAATTTTGAACTATCAACCAACATCCGCCTCCAAATTTCCTACTTCACCTCCACCTGTCACCCCACCATGGAACGATTATCATCATTCTAAGATGTTTAGTAAGAGTATTTTTGCACTAATGGGATCACCCAAACCACCAACAAGTTACGATCGTAGAATTAGTCTGCTGAATACACAGAATAAGATTGATGGATTTACCAAATGGGCTATAAACAATGTCTCCTTGGCACTTCCACCAACACCCTATTTGGGCTCTATTAAATACGGATTGAGAAACGCCTTCGACCAGAAGTCACCTCCAGAAAATTTTCCAGATAATTATGATGTGATGAGGCCACCAATTAACCCCAATTCTACTACGGGCTCCGGTGTCTACATGTTCGGTCTGAACACAACTGTAGATGTCATCTTGCAAAATGCTAATGCCTTGTCCGATAACGTGTCTGAAATTCATCCTTGGCACTTGCATGGTCATGATTTCTGGGTTCTTGGATATGGAGAAGGTAAATTTAGTGCTAAAGACGAAAAAAAGCTTAACTTCAAAAACCCACCTTTGAGAAATACCGCAGTGATTTTCCCTTACGGATGGACGGCTCTGAGATTCGTTGCTGACAATCCTGGTGTGTGGGCCTTCCATTGTCATATTGAACCACATCTGCACATGGGTATGGGAGTTGTTTTTGCCGAAGGTGTTCATCATGTTAAAAAGATCCCTAATGAAGCATTGACATGCGGATTGACTGCAAAGTTGTTGTATAAGAACCGTGGTAGA. </xnotran>

In the above technical solutions, researchers in the field can substitute, add and/or delete one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids to the sequence of the present invention to obtain the variant of the amino acid sequence of the ascorbate oxidase without substantially affecting the activity of the ascorbate oxidase. All of which are considered to be included within the scope of protection of the present invention.

In another technical scheme, the amino acid sequence SEQ ID NO.4 of the ascorbic acid oxidase is obtained by mutating the amino acid sequence SEQ ID NO.2 of the ascorbic acid oxidase;

carrying out error-prone PCR random mutation on an ascorbic acid oxidase amino acid sequence SEQ ID NO.2, wherein 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 ascorbic acid oxidase amino acid sequence SEQ ID NO.4, the amino acid sequence SEQ ID NO.2: <xnotran> AKARHFNWEVEYMYRSPDCLEHIVMGINGQFPGPTIRAKAGDTLVIELSNKLHTEGVVIHWHGIRQLGTPWADGTASISQCAINPGETFKYRFKVDRRGTYFYHGHYGMQRSAGLYGSLIVDVADGEKEPFHYDGELNLLLSDWWHKGVHEQEVGLSSNPFRWIGEPQSVLINGRGQYNCSMAAKFSNPPIGQCKFRGNEQCAPQILKVQPNKTYRLRIASTTALASLNLAIQGHKMVVVEADGNHVQPFAMNDLDIYSGESYSVLLTTDQNPSRNYWISIGVRAREPKTPQALTILNYSPTSASRIPMSQPPVTPPWNDYNHSKAFTKSIYALMGSPKPPKTSNRRIVLLNTQNRVNGFIKWSINNVSLVLPSTPYLGSLKFGLNNSFDQKSPPDNYDSSYDIMKPAVNQNSTQGSGIYTIGLNTTVDVILQNANTLAKDVSEIHPWHLHGHDFWVLGYGESKFKEGDEKTFNLKNPPLRNTAVIFPYGWTALRFVADNPGVWAFHCHIEPHLHMGMGVVFAQGVHRVGQIPREALACGLTGNKHN, SEQ ID NO.2 : </xnotran> <xnotran> GCAAAAGCCAGACACTTTAATTGGGAGGTAGAGTACATGTATAGATCTCCAGATTGTTTGGAACACATTGTCATGGGAATCAACGGCCAATTCCCTGGTCCCACAATCCGTGCAAAGGCTGGTGACACACTTGTTATTGAATTGTCTAACAAACTTCACACAGAGGGAGTTGTTATCCACTGGCATGGTATTCGACAACTGGGTACTCCTTGGGCAGATGGTACTGCCTCCATTTCTCAATGTGCCATAAATCCAGGCGAAACATTTAAATACAGATTCAAAGTCGATAGGAGAGGTACATACTTTTATCACGGTCATTACGGCATGCAACGTTCTGCTGGTTTGTATGGAAGTCTTATCGTGGATGTGGCTGACGGTGAGAAAGAACCTTTTCACTACGATGGTGAGTTGAACTTGCTGTTGTCTGACTGGTGGCATAAGGGAGTCCATGAACAAGAAGTAGGACTTTCCTCAAACCCCTTCAGGTGGATTGGAGAACCACAATCAGTCTTGATTAACGGAAGAGGTCAGTATAACTGCAGTATGGCCGCCAAATTTTCCAATCCCCCAATTGGCCAGTGTAAGTTTCGAGGTAACGAACAATGTGCTCCCCAAATTTTGAAGGTGCAGCCCAACAAAACCTACAGACTAAGAATAGCATCCACTACCGCTCTAGCTTCATTGAACCTGGCTATACAAGGTCACAAGATGGTTGTTGTTGAAGCCGACGGAAATCATGTCCAACCATTTGCTATGAATGACCTTGACATTTATTCTGGAGAATCTTACTCCGTTCTTTTGACCACAGATCAGAACCCATCCAGAAACTATTGGATTAGTATTGGTGTTAGAGCTAGAGAACCTAAAACACCTCAGGCTCTAACTATTCTGAACTATTCCCCAACATCTGCTTCCCGAATACCTATGAGTCAACCCCCAGTCACACCACCTTGGAACGATTACAACCATTCTAAAGCATTTACCAAGTCTATTTATGCATTAATGGGTTCTCCAAAGCCTCCTAAGACTTCCAACAGAAGAATTGTTCTGTTAAACACTCAAAACAGAGTTAATGGTTTTATTAAATGGTCCATCAACAATGTCTCTTTGGTCCTACCTTCTACGCCTTACTTAGGATCCCTAAAATTCGGTTTGAATAATTCTTTCGATCAAAAGTCACCACCTGACAACTATGATTCTTCTTACGATATTATGAAGCCTGCTGTTAATCAAAATTCAACACAGGGTTCCGGTATATACACAATTGGTCTGAACACTACAGTGGATGTTATATTGCAGAACGCCAACACTCTAGCTAAGGACGTGTCCGAAATTCATCCTTGGCACCTTCACGGTCACGATTTCTGGGTTCTGGGTTACGGTGAGAGTAAGTTTAAGGAGGGTGACGAAAAGACCTTCAACCTAAAGAATCCACCTTTGAGAAACACGGCAGTTATATTCCCTTACGGATGGACGGCCCTTAGGTTTGTCGCTGACAATCCTGGTGTTTGGGCTTTTCATTGTCATATAGAGCCCCACTTGCATATGGGTATGGGAGTTGTTTTTGCTCAGGGTGTGCATAGAGTTGGTCAGATTCCTAGAGAAGCCCTAGCTTGTGGTCTGACTGGAAATAAACATAAT. </xnotran>

In the above technical solutions, researchers in the field can substitute, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids to the sequences of the present invention to obtain variants of the amino acid sequences of the ascorbate oxidase without substantially affecting the activity of the ascorbate oxidase. All of which are considered to be included within the scope of the present invention.

In the above technical scheme, if the same group of amino acid residue substitutions, such as R substitution by K or I substitution by L, occur in the amino acid sequence, the role of the residue in the protein domain is not changed, and therefore the steric structure of the protein is not affected, and the function 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. Substitution of an amino acid residue belonging to the same class may occur at any amino acid residue position on the ascorbate oxidase. In contrast, substitution of amino acid residues of different classes, or substitution of amino acids that do not conform to the above-listed substitution rules, is likely to result in changes in the structure and differences in function of the protein.

The ascorbic acid oxidase provided by the invention can also be modified or mutated to obtain a derivative protein. The "derived protein" of the present invention means a protein which differs in amino acid sequence from the ascorbic acid oxidase having the above-mentioned amino acid sequence, has a difference in modified form which does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or mutagenic agents, or by techniques such as site-directed mutagenesis or other known molecular biology techniques. "derived 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.

Modified forms include: chemical forms of the protein, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the protein or in further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a 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 coded ascorbic acid oxidase is shown as SEQ ID NO.3 and the nucleotide sequence is shown as SEQ ID NO. 5: <xnotran> GCAAAAACTAGACATTTTAAGTGGGAGGTTGAATACATGTACTGGTCTCCAGATTGTATTGAACACGTCGTAATGGGAATCAATGGACAATTTCCTGGACCAACCATCAGAGCTAAAGCTGGTGACACAGTTGTGGTCGAACTAACCAATAAGCTGCCAACTGAAGGTGTTGTGATTCATTGGCACGGTATTAGACAACTTGGAACGCCTTGGGCCGATGGTACCGCTTTTATTAGTCAATGTGCTATCAACTCTGGCGAAACTTTTCATTATCGATTTAAAGTTGACAGAGCAGGTACGTACTTTTACCATGGACACTTGGGTATGCAAAGAAGTGCTGGTCTGTATGGTTCTCTATTGGTTGATGTGGCAGAAGGCGAAAAGGAACCTTTCCATTACGACGGAGAGTTTAACTTGCTATTGTCAGACTGGTGGCACAAGTCAGTACATGAACAAGAGGTCGGATTGTCATCAAATCCTTTCAGATGGATTGGAGAGCCCCAATCTCTGCTAATCAACGGTAGAGGACAATATAATTGTTCTTTGGCTGCCCAATATTCTGATACTAACTCTTCCCAATGTAAATTGAGAGGTAATGAACAATGTGCACCCCAAATCCTACATGTTAGATCCAATAAAACCTACAGATTGAGAGTTGCCTCTTCTACCGCATTAGCATCTTTAAATTTGGCAATTGGCAATCATAAGATGGTTGTTGTGGAAGCAGATGGCAACTATTTGCAGCCATTTAAGGTCAATGATCTAGATATTTACTCTGGAGAATCATATAGTGTATTGATTACTACAAACCAAGATCCATCACAGAACTACTGGTTATCAATTGGTGTTAGAGGCAGGTTACCTGCAACTCCTCCTGGACTTACAATTTTGAACTATCAACCAACATCCGCCTCCAAATTTCCTACTTCACCTCCACCTGTCACCCCACCATGGAACGATTATCATCATTCTAAGATGTTTAGTAAGAGTATTTTTGCACTAATGGGATCACCCAAACCACCAACAAGTTACGATCGTAGAATTAGTCTGCTGAATACACAGAATAAGATTGATGGATTTACCAAATGGGCTATAAACAATGTCTCCTTGGCACTTCCACCAACACCCTATTTGGGCTCTATTAAATACGGATTGAGAAACGCCTTCGACCAGAAGTCACCTCCAGAAAATTTTCCAGATAATTATGATGTGATGAGGCCACCAATTAACCCCAATTCTACTACGGGCTCCGGTGTCTACATGTTCGGTCTGAACACAACTGTAAATGTCATCTTGCAAAATGCTAATGCCTTGTCCGATAACGTGTCTGAAATTCATCCTTGGCACTTGCATGGTCATGATTTCTGGGTTCTTGGATATGGAGAAGGTAAATTTAGTGCTAAAGACGAAAAAAAGCTTAACTTCAAAAACCCACCTTTGAGAAATACCGCAGTGATTTTCCCTTACGGATGGACGGCTCTGAGATTCGTTGCTGACAATCCTGGTGTGTGGGCCTTCCATTGTCATATTGAACCACATCTGCACATGGGTATGGGAGTTGTTTTTGCCGAAGGTGTTCATCATGTTAAAAAGATCCCTAATGAAGCATTGACATGCGGATTGACTGCAAAGTTGTTGTATAAGAACCGTGGTAGA. </xnotran>

In another technical scheme, the nucleotide sequence of the coded ascorbic acid oxidase is shown in SEQ ID NO.6 as SEQ ID NO.4: <xnotran> GCAAAAGCCAGACACTTTAATTGGGAGGTAGAGTACATGTATAGATCTCCAGATTGTTTGGAACACATTGTCATGGGAATCAACGGCCAATTCCCTGGTCCCACAATCCGTGCAAAGGCTGGTGACACACTTGTTATTGAATTGTCTAACAAACTTCACACAGAGGGAGTTGTTATCCACTGGCATGGTATTCGACAACTGGGTACTCCTTGGGCAGATGGTACTGCCTCCATTTCTCAATGTGCCATAAATCCAGGCGAAACATTTAAATACAGATTCAAAGTCGATAGGAGAGGTACATACTTTTATCACGGTCATTACGGCATGCAACGTTCTGCTGGTTTGTATGGAAGTCTTATCGTGGATGTGGCTGACGGTGAGAAAGAACCTTTTCACTACGATGGTGAGTTGAACTTGCTGTTGTCTGACTGGTGGCATAAGGGAGACCATGAACAAGAAGTAGGACTTTCCTCAAACCCCTTCAGGTGGATTGGAGAACCACAATCAGTCTTGATTAACGGAAGAGGTCAGTATAACTGCAGTATGGCCGCCAAATTTTCCAATCCCCCAATTGGCCAGTGTAAGTTTCGAGGTAACGAACAATGTGCTCCCCAAATTTTGAAGGTGCAGCCCAACAAAACCTACAGACTAAGAATAGCATCCACTACCGCTCTAGCTTCATTGAACCTGGCTATACAAGGTCACAAGATGGTTGTTGTTGAAGCCGACGGAAATCATGTCCAACCATTTGCTATGAATGACCTTGACATTTATTCTGGAGAATCTTACTCCGTTCTTTTGACCACAGATCAGAACCCATCCAGAAACTATTGGATTAGTATTGGTGTTAGAGCTAGAGAACCTAAAACACCTCAGGCTCTAACTATTCTGAACTATTCCCCAACATCTGCTTCCCGAATACCTATGAGTCAACCCCCAGTCACACCACGTTGGAACGATTACAACCATTCTAAAGCATTTACCAAGTCTATTTATGCATTAATGGGTTCTCCAAAGCCTCCTAAGACTTCCAACAGAAGAATTGTTCTGTTAAACACTCAAAACAGAGTTAATGGTTTTATTAAATGGTCCATCAACAATGTCTCTTTGGTCCTACCTTCTACGCCTTACTTAGGATCCCTAAAATTCGGTTTGAATAATTCTTTCGATCAAAAGTCACCACCTGACAACTATGATTCTTCTTACGATATTATGAAGCCTGCTGTTAATCAAAATTCAACACAGGGTTCCGGTATATACACAATTGGTCTGAACACTACAGTGGATGTTATATTGCAGAACGCCAACACTCTAGCTAAGGACGTGTCCGAAATTCATCCTTGGCACCTTCACGGTCACGATTTCTGGGTTCTGGGTTACGGTGAGAGTAAGTTTAAGGAGGGTGACGAAAAGACCTTCAACCTAAAGAATCCACCTTTGAGAAACACGGCAGTTATATTCCCTTACGGATGGACGGCCCTTAGGTTTGTCGCTGACAATCCTGGTGTTTGGGCTTTTCATTGTCATATAGAGCCCCACTTGCATATGGGTATGGGAGTTGTTTTTGCTCAGGGTGTGCATAGAGTTGGTCAGATTCCTAGAGAAGCCCTAGCTTGTGGTCTGACTGGAAATAAACATAAT. </xnotran>

The examples of the present application provide a recombinant vector containing the ascorbate oxidase gene.

The embodiment of the application provides an engineering bacterium containing the recombinant vector of claim 6.

The embodiment of the application provides a preparation method of the engineering bacteria, which comprises the following specific steps:

s1, constructing a carrier for expressing ascorbic acid oxidase protein;

s3, obtaining the expressed ascorbate oxidase protein from the transfected cells in the step S2;

and S4, purifying the ascorbic acid oxidase protein obtained in the step S3.

The embodiment of the application provides an application of the ascorbic acid oxidase in enhancing the anti-interference of a clinical diagnosis kit.

Example 1 vector construction and Shake flask fermentation of ASO

1. Experimental materials

pPICZ α A vector plasmid, X-33 strain was purchased from Invitrogen; YNB was purchased from Beijing Tiangen Biotech, inc.; peptone and yeast powder were purchased from OXOID; biotin was purchased from Sigma; ni and SP columns were purchased from GE; sac I enzyme and digestion Buffer were purchased from NEB (Beijing) Inc.

2. Construction of Yeast expression of ASO

The method comprises the steps of searching sequences against ascorbate oxidase in an NCBI database, selecting 18 suspected sequences, performing codon optimization, performing biosynthesis by Pomaceae, reserving enzyme cutting sites EcoR I and Sal I at an N end and a C end respectively, performing double enzyme cutting on a target fragment and a pPICZ alpha A vector by using EcoR I and Sal I, connecting the target fragment and the pPICZ alpha A vector by using T4 ligase, wherein a DNA sequence C section of the ascorbate oxidase contains 6 XHis labels, converting DH5 alpha competence, coating a LLB (Low Salt LB medium) solid plate (Zeocin 25 mu g/mL), performing colony PCR on each gene to screen 12 transformants, identifying recombinants by using double enzyme cutting, constructing successful vectors named as pPICZ alpha A-ASO1, wherein strains of the 18 sequences are respectively numbered from 1 to 18, and part of maps are shown in figure 1.

3. Ascorbic acid oxidase expression plasmid miniextract

The DH 5. Alpha. Strain was cloned as an ascorbate oxidase positive clone, inoculated into 10mL of LLB medium containing zeocin at a concentration of 25. Mu.g/mL, and cultured to the middle logarithmic growth phase (OD) ₆₀₀ = 0.5-0.7), 0.85mL of the bacterial liquid is taken out and added with sterile glycerol to preserve the strains at-80 ℃, and the rest bacteria are continuously cultured at 37 ℃ overnight. Extracting plasmid with a plasmid miniprep kit, and subpackaging and storing.

LLB (Low Salt LB) medium: trypton l%, yeast Extract 0.5%, naCL 0.5%, pH7.5%. When preparing the plate, 2% agar powder is added to sterilize for 20min at 121 ℃. Can be stored for several months at room temperature, 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 mu g/mL, and can be stored for 1-2 weeks at the temperature of 4 ℃.

4. Linearization of ascorbate oxidase expression plasmids

The expression plasmids pPICZ α A-ASO1 to 18 were digested linearly with Sac I (209 bp) endonuclease, which digested at 5' AOX1 site of the expression vector pPICZ α A. The enzyme cutting system is 100 mu L (the plasmid is more than 10 mu g), and electrophoresis is carried out to check whether the plasmid is cut after enzyme cutting. Before and after the linearization of yeast, the electrophoresis is compared, the cut band runs slowly, the whole plasmid runs in front, if the enzyme cutting is incomplete, the linearization of the lane has two bands. EDTA is added to stop the reaction after the enzyme digestion is completed, or the heat inactivation condition is 65 ℃ and 20min.

5. Phenol chloroform extraction of plasmid

(1) Supplementing about 100 mu L of system to 400 mu L after the enzyme is cut;

(2) Adding phenol chloroform (lower layer of phenol chloroform), mixing, and standing at 4 deg.C for 10min;

(3) Taking the upper water sample, adding 1/10 volume of 3M sodium acetate and 2.5 volume of precooled 100% ethanol, placing at-20 ℃,1h, centrifuging at 4 ℃ for 20min, and removing the supernatant;

(4) Adding 250 μ L80% ethanol to clean DNA, centrifuging at 4 deg.C for 20min, and removing supernatant;

(5) Blow-drying, adding 10 μ L sterile ddH ₂ O, storing at-20 ℃ for later use.

6. Preparation of Yeast competence and electrotransformation of Methanotrophic Yeast

(1) Marking yeast, selecting and cloning the yeast in a 5mL YPD and 50mL centrifuge tube, culturing overnight at 30 ℃ and 220 rpm; 0.25mL of the seed solution was re-inoculated into a 2L Erlenmeyer flask containing 500mL of fresh medium for overnight culture.

(2)OD ₆₀₀ =1.3-1.5, which is not longer than 12-18 h, and is centrifuged at 1500g and 4 ℃ for 5min;

(3) 500mL of pre-cooled (0 ℃ C.) sterile ddH was added ₂ O,1500g, and centrifuging at 4 ℃ for 5min; 250mL of pre-cooled (0 ℃) sterile ddH were added ₂ O,1500g, and centrifuging at 4 ℃ for 5min;

(4) Resuspend the pellet in 20mL ice cold (0 ℃) 1M sorbitol, 1500g, centrifuge for 5min at 4 ℃; the pellet was resuspended in 1mL ice-cold (0 ℃) 1M sorbitol to a final volume of about 1.5mL and placed in an ice bath until use without preserving the cells.

(5) Mixing 80 μ L of the above cells and linear DNA (5-10 μ g), transferring into 0.2cm electric rotary cup, standing on ice for 5min, wiping off water on the metal sheet of the electric rotary cup, and measuring voltage: 1500V, resistance: 400 Ω, capacitance: 25 μ F, pulse time: 10mS, start shock;

(6) 1mL of ice-cold (0 ℃ C.) 1M sorbitol was immediately added and transferred to a 15mL sterile centrifuge tube;

(7) Standing and incubating for 1-2h at 30 ℃;

(8) Coating 50-200 μ L YPDS plate containing zeocin100 μ g/mL;

(9) Culturing in 30 deg.c incubator for 2-3 days to grow transformant;

note: YPD or YPDS Medium configuration (Yeast Extract Peptone Dextrose Medium, yeast Extract powder/tryptone/Dextrose Medium): 1% yeast extract, 2% peptone, 2% dextrase (glucose), 2%, + -1 Msbitol, + -2% agar, 100. Mu.g/mL Zeocin liquid YPD medium can be preserved at normal temperature, is the most basic medium for Pichia pastoris, and agar YPDS plates can be preserved at 4 ℃ for several months. Zeocin 100. Mu.g/mL was added to the medium to prepare YPDZ medium, which was allowed to stand at 4 ℃ for 1 to 2 weeks.

7. Transformant identification and test expression

(1) When a single colony grows on the YPDS plate, 10-20 single colonies are picked, and the back of the plate is marked. Inoculating to fresh YPD medium, culturing, adding glycerol, storing at-80 deg.C, transferring into small triangular flask containing 50mL BMGY at 30 deg.C and 200rpm, and culturing to OD ₆₀₀ ＝1-1.5；

(2) Transferring the culture system in the small triangular flask into a 50mL centrifuge tube, collecting the strain at 3000g for 5min;

(3) Resuspending the pellet to OD with BMMY ₆₀₀ =0.3 (about 100-200 mL);

(4) Transferring to 500mL large triangular flask, inducing expression culture at 30 deg.C and 200rpm, taking out 1mL of quick-frozen medium at intervals, storing at-80 deg.C, supplementing final concentration of 0.5% methanol, and sampling at time points: 0h, 24h, 48h, 72h and 96h;

(5) Centrifuging samples taken at each time point for 1min, collecting supernatant, adding 10 μ L loading buffer into 30 μ L supernatant, decocting for 10min, centrifuging (13000rpm, 5 min), collecting 15 μ L SDS-PAGE;

note: BMGY medium: yeast extract 1%, peptone 2%, potassium phosphate buffer (pH6.0) 100mmol/L, YNB 1.34%, biotin (4X 10) ^-5 )％，Glycerol 1％

Pichia pastoris pre-induction expression medium, YNB and Biotin filter sterilization. After 24 hours of culture, the cells are generally allowed to stand overnight, and after yeast precipitation, the BMGY medium is discarded and the BMMY medium is changed to an induction expression phase.

BMMY medium: yeast extract 1%, peptone 2%, potassium phosphate buffer (pH6.0) 100mmol/L, YNB 1.34%, biotin (4X 10) ^-5 )％，methanol 3％

Pichia induction expression medium, YNB and Biotion filtration sterilization. In the case of shake flask culture, 3% methanol is added after every 24 hours of induction, generally for 72 hours.

8. Shake flask fermentation of pPICZ alpha A-ASO strain

Inoculating the constructed yeast strain expressing the protein of the ascorbic acid oxidase into 10mL YPD culture medium, and performing shake culture at 150rpm and 29 ℃ for 24h to reach logarithmic phase to serve as first-level seeds; mixing the first-class seeds according to the proportion of 1:100 to 100mL of BMGY culture medium, culturing for 4 days at 150rpm and 29 ℃ with shaking, adding 1.8v/v% methanol every day to induce expression, and ending fermentation on day 7; transferring the fermentation liquor into a centrifugal barrel, centrifuging at 12000g for 15min, and collecting the supernatant to obtain the crude cell enzyme solution.

9. Purification of

The collected supernatant was left as a small sample of fermentation broth for viability measurement. And purifying the rest by nickel column affinity chromatography to purify the ascorbic acid oxidase.

EXAMPLE 2 determination of ascorbic acid oxidase Activity

One unit of ascorbic acid oxidase activity was defined as the amount of enzyme that oxidizes 1.0. Mu. MoL ascorbic acid per minute at 37 ℃ and pH 5.5.

The reaction formula is as follows:

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 (I)	Volume of
			A	Substrate solution	0.5mL
B	Na ₂ HPO ₄ Solutions of	0.5mL

(the pH of the reaction mixture was adjusted to 5.5)

2. Adding 0.1mL of ASO enzyme solution to be detected, and reversing and mixing uniformly.

3. After reacting at 30 ℃ for 5minutes, 3.0mL of HCl solution (C) was added to terminate the reaction, and the mixture was mixed by inversion.

4. The OD value (OD test) was measured at 245nm with the spectrophotometer by zeroing with distilled water, and the OD blank was measured in the same manner with enzyme reagent (D) instead of the ASO enzyme solution.

ASO enzyme solution was previously diluted to 0.15-0.25U/mL with precooled enzyme solvent (D); if the ASO enzyme activity exceeds 60U/mL, it can be diluted with pre-cooled sterile water, just prior to activity determination.

ASO enzyme activity calculation

The ASO enzyme activity unit calculation method comprises the following steps:

weight ASO enzyme activity (U/mg) = (U/mL). Times.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); millimolar extinction coefficient (cm) of ascorbic acid under measurement conditions at 10.0, pH1.0 ² in/mM); 1.0, light passes through path (cm); t, reaction time (5 minutes); df, dilution factor; c, enzyme concentration (C mg/mL)

Crude enzyme solution of the cells was taken to measure the enzyme activity, high-yield transformants were screened, ASO was purified and lyophilized after fermentation, and the activity was measured again, with the mutant strain ASO3m4 being 112% more active than ASO3, as shown in table 1.

TABLE 1ASO Activity statistics

Numbering	Activity (U/mg-solid)
		ASO1	230
ASO1m2	225
		ASO3	268
ASO3m4	570

Example 3 Heat-stable random mutagenesis of the ascorbic acid oxidase Gene

Expression vectors pPICZ alpha A-ASO1 and pPICZ alpha A-ASO3 of ascorbic acid oxidase sequences SEQ ID NO.1 and SEQ ID NO.2 are subjected to random mutation by error-prone PCR, a mutated DNA fragment is obtained by gel cutting and recovery, and the mutated DNA fragment is connected with pPICZ alpha A plasmid to construct a recombinant vector. The plasmid is firstly linearized, then transferred into the susceptible state of the Pichia pastoris X-33 expression strain in an electrotransfer mode, coated with plates, selected for cloning, and subjected to amplification culture to obtain a transformation product culture solution.

The conditions for random mutagenesis by the error-prone PCR method are as follows:

25 μ L of 2 × error 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 a reaction volume of 50 μ L;

the amplification procedure was: the reaction system is heated to 95 ℃ for 8min;31 cycles of "95 ℃ 30s,56 ℃ 30s,72 ℃ 1min50s"; 10min at 72 ℃; the resulting PCR-amplified gene product was separated by electrophoresis on a 1% agarose gel, and the DNA fragment was recovered.

Further, in the PCR amplification reaction of pPICZ alpha A-ASO1 and pPICZ alpha A-ASO3, the following primer pairs are respectively adopted:

ASO-For1:5’-GAATTCGCAAAAACTAGACA-3’；

ASO-Rev1:5’-GTCGACTCTACCACGGTTCT-3’；

ASO-For3:5’-GAATTCGCAAAAGCCAGACA-3’；

ASO-Rev3:5’-GTCGACATTATGTTTATTTC-3’。

screening of mutant strains

Cloning after random mutation, culturing bacterial liquid, carrying out amplification culture and induction, carrying out ultrasonic cell breaking, heating in water bath at a plurality of different increasing temperatures, and testing the cell breaking liquid heated at different temperatures for survival.

In random mutation experiment of pPICZ alpha A-ASO1, the clone without loss of ascorbic acid oxidase is obtained under 60 ℃ heating, the clone is a mutated ascorbic acid oxidase gene engineering strain with good thermal stability, the amino acid sequence of the clone is shown as SEQ ID NO.3, and the vector is pPICZ alpha A-ASO1m2.

In random mutation experiments of pPICZ alpha A-ASO3, a clone without loss of ascorbic acid oxidase under heating at 60 ℃ is obtained, the mutation enables the activity of the enzyme to be increased by 112%, the clone is a mutated ascorbic acid oxidase gene engineering strain with good thermal stability and high activity, the amino acid sequence of the mutant strain is shown as SEQ ID NO.4, and the carrier is pPICZ alpha A-ASO3m4.

Example 4 Effect of pH and temperature on ASO enzymatic Activity and stability

1. Effect of pH on ASO enzyme Activity and stability

Preparing enzyme detection solution (pH 3.0-6.0, acetate buffer solution, pH5.0-9.0, phosphate buffer solution) with buffer solutions of different pH (pH 3-9), measuring enzyme activity according to the above method, and determining the optimum reaction pH value of ASO. The activity of ASO was measured at 30 ℃ in each of the buffers of pH3 to 9, and as shown in FIG. 2, the enzyme activity was 0 at pH3.0, and the ascorbate oxidase activity gradually increased with increasing pH and reached a maximum at pH 5.5. And then begins to decrease. Therefore, the enzyme has activity between pH5 and 8, and the optimum reaction pH of the enzyme is 5.5.

The enzyme solution was diluted with Britton-Robinson buffer solutions of different pH (pH 3-12), incubated at 25 ℃ for 17 hours, and the residual enzyme activity was measured. The enzyme activity of the ASO under the optimum pH value is used for preparing a curve of the pH value and the enzyme activity, and the pH stability of the ASO is inspected. As shown in FIG. 3, the ascorbic acid oxidase activity was gradually increased in the treatment at pH6 to 10, and had no effect on the ascorbic acid oxidase activity in the treatment at pH6 to 10, and then the enzyme activity was decreased as the pH was increased.

2) Effect of temperature on ASO enzyme Activity and stability

In50 mM phosphate buffer solution with pH5.5, enzyme activity at different temperatures was measured according to the above method, and the optimum reaction temperature for ASO was determined. The activity data of ASO were obtained at 25 ℃,30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃, the highest activity temperature was 30 ℃, and a temperature-ASO activity curve was prepared, and the results are shown in fig. 4, in which ASO activity at 25-30 ℃ increased and ASO activity at 30-50 ℃ gradually decreased.

Studying the heat stability of ASO, in50 mM phosphate buffer solution with pH8.0, keeping the temperature of enzyme solution with ASO enzyme activity of 12U/mL at 20 ℃,30 ℃, 40 ℃,50 ℃, 60 ℃ and 70 ℃ for 30min respectively, rapidly cooling to room temperature, measuring residual enzyme activity, making an enzyme activity curve of the ASO kept at different temperatures for 30min, and processing at 20-50 ℃ for 30min with the result as shown in figure 5, wherein the enzyme activity is basically not lost; the enzyme basically loses activity after being treated for 30min at the temperature of 70 ℃; after 30min of treatment at 60 ℃, the activity of the mutant strains ASO1m2 and ASO3m4 is not basically lost, and the activity of the mutant strains ASO1 and ASO3 is basically lost.

EXAMPLE 5 Yeast fermenter Process for ASO

Shake flask culture of ASO fermentation seed liquid:

the shake flask contains BMGY medium 5-10% of the initial fermentation broth volume, and a colony is selected from MGY plate or frozen glycerol stock solution is inoculated into shake flask, cultured on shaking table at 30 deg.C and 250rpm for 16-24 hr until OD ₆₀₀ ＝2-6。

ASO glycerol batch fermentation:

1. the fermentor and fermentation base salt medium containing 4% glycerol were sterilized.

2. After sterilization and cooling, when the temperature had dropped to 30 ℃, the 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 5-10% of the initial fermentation volume of seed liquid from the seed liquid shake flask into the fermentation tank. The DO value was close to 100% at the beginning of the culture. Oxygen is consumed when the culture is started, resulting in a decrease in DO value. Oxygen was added as needed to ensure that the DO value exceeded 20%.

4. Batch fermentation was carried out until glycerol was completely consumed (18-24 h), as indicated by an increase in DO to 100%. The time to complete glycerol consumption will vary with the initial fermentation broth density.

5. Sampling is required for the completion of each fermentation stage and is performed at least twice a day. 10mL of sample was taken at each time point and another 1mL sample was taken from 10 mL. Samples for analysis of cell growth (OD) ₆₀₀ And cell wet weight), pH, microscopic observation, concentration or activity of protein. The centrifuged cells and supernatant were stored at-80 ℃ for later analysis.

The cell yield achieved at this stage is 90-150g/L wet cells. Recombinant ASO enzymes are not produced due to lack of induction by methanol.

ASO glycerol supplemented culture:

introduction: once all glycerol was consumed in the batch fermentation culture, glycerol feeding required to start increasing cell biomass under limiting conditions. When preparing for methanol induction, it is first necessary to determine that glycerol has been depleted by DO value.

1. Adding 50% w/v of 12mL of PTM1-per liter of glycerol for feeding. The feed rate was set at 18.15mL per hour per liter of initial fermentation broth volume.

2. Glycerol feeding will take about 4 hours or more. After this stage, the cell yield should reach 180-220g/L wet cells but no recombinant egg is produced.

Note that: the expression level of the protein depends on the amount of cells produced in glycerol fed culture. The duration of the feed will be alternated to optimize the protein yield, typically roughly in the range of 50-300g/L wet cells. The maximum concentration of glycerol in the feed was 4%, higher glycerol concentrations would create toxicity problems.

If the dissolved oxygen is less than 20%, the feed of glycerol or methanol should be stopped and nothing to increase the dissolved oxygen should be done until the dissolved oxygen stabilizes. At this point, stirring, aeration, pleasure or oxygen supplementation is started.

ASO methanol feeding culture:

glycerol must be consumed clean before methanol feeding to fully induce the AOX1 promoter. Methanol is slowly fed to adapt the culture to growth in methanol, which if added too quickly will kill the cells.

Once the culture was acclimated to methanol, the DO value was used to analyze the status of the culture and to determine the time point of methanol feeding to optimize protein expression. The growth liquid generates a large amount of heat on methanol. The temperature control at this stage is very important.

Methanol fed-batch culture of ASO strain (Mut + type):

1. glycerol feed was stopped and induction was started with 100% methanol per liter containing 12mL of PTM1 base salts. The feed rate was set at 3.5mL/h per liter of initial fermentation broth volume.

2. Methanol accumulates in the fermentation broth during the first 2-3h, and the DO changes during the reaction of the culture with methanol.

3. If the DO level is not maintained above 20%, the methanol feed is stopped, and the methanol feed continues at the current rate after the DO level stabilizes. The DO is maintained above 20% by adding agitation, aeration, pressure or oxygen supplementation.

4. When the culture was fully adapted to use methanol (2-4 h) and methanol became its limiting growth factor, there was a steady 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 had been acclimated to methanol but before doubling the feed rate. The flow rate was then doubled to about 7mL/h per liter of initial broth volume.

5. After 2h at a rate of 7mL// L, the methanol feed rate was increased to 10.5mL/h per liter of initial broth volume. This feed rate is throughout the remainder of the fermentation process.

6. The total methanol feed culture was added for nearly 700mL per liter of initial broth volume for almost 72 hours.

Harvesting of ASO fermentation supernatant:

the cultures were transferred to a centrifuge tube (1000 mL) and centrifuged to separate the cells from the supernatant.

PTM1 base salts: cuSO ₄ -5H ₂ O,5g; sodium iodide, 0.08g; mnSO ₄ -H ₂ O,3g; 0.2g of sodium molybdate dihydrate; boric acid, 0.02g; 0.5g of cobalt chloride; znCL ₂ ，20g；FeSO ₄ 65g; biotin, 0.2g; h ₂ SO ₄ 5mL, add water to 1L, filter and sterilize.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims

1. The ascorbic acid oxidase is characterized in that the amino acid sequence of the ascorbic acid oxidase is shown in SEQ ID NO.3 or SEQ ID NO.4.

2. The ascorbic acid oxidase of claim 1, wherein the amino acid sequence of the ascorbic acid oxidase SEQ ID No.3 is obtained by mutating the amino acid sequence of the ascorbic acid oxidase SEQ ID No.1, wherein the mutation is:

3. The ascorbic acid oxidase of claim 1, wherein the amino acid sequence of the ascorbic acid oxidase, SEQ ID No.4, is obtained by mutation of the ascorbic acid oxidase amino acid sequence, SEQ ID No. 2;

carrying out error-prone PCR random mutation on an ascorbic acid oxidase amino acid sequence SEQ ID NO.2, wherein 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, so as to obtain the ascorbic acid oxidase amino acid sequence SEQ ID NO.4.

4. The ascorbate oxidase of claim 1, wherein an amino acid sequence encoding the ascorbate oxidase is set forth in SEQ ID No.3 and a nucleotide sequence set forth in SEQ ID No. 5.

5. The ascorbate oxidase of claim 1, wherein the amino acid sequence encoding the ascorbate oxidase is set forth in SEQ ID No.4 and the nucleotide sequence is set forth in SEQ ID No. 6.

6. A recombinant vector comprising the ascorbate oxidase gene according to claim 4 or 5.

7. An engineered bacterium comprising the recombinant vector according to claim 6.

8. The preparation method of the engineering bacteria as claimed in claim 7, which comprises the following steps:

s1, constructing a carrier for expressing ascorbic acid oxidase protein;

and S4, purifying the ascorbic acid oxidase protein obtained in the step S3.

9. Use of an ascorbate oxidase of claim 1 for enhancing interference rejection in a clinical diagnostic kit.