Process for the preparation of recombinant ascorate oxidase

Abstract

The application discloses a preparation method of a recombinant ascorbate oxidase. The preparation method of the ascorbate oxidase provided in the application depends on Pichia pastoris as a heterologous expression fungal host system, and methanol as a sole carbon source, and has the advantages of stable heredity, low cost, easy cultivation, scale-up, post-translational modification and secretion expression, and the like; and the ascorbate oxidase protein with low cost, large-scale production and high enzyme activity can be obtained.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to a method for preparing recombinant ascorbic acid oxidase. Background Technology

[0002] Ascorbic acid oxidase (AAO) is a polycopper oxidase that catalyzes the conversion of ascorbic acid to dehydroascorbic acid, accompanied by the reduction of molecular oxygen to water. AAO is a homodimeric copper protein, with each subunit containing four copper atoms. Three of these atoms form a trinuclear copper cluster at the junction of domain 1 (Ser31-Tyr163) and domain 3 (Asn374-Pro579), while the remaining copper atom exists as a mononuclear atom in domain 1. Copper atoms are crucial components of the enzymatic reaction of ascorbic acid oxidase; it is hypothesized that the trinuclear copper cluster is used for binding oxygen and storing electrons, while the mononuclear copper atom binds to ascorbic acid. Furthermore, phenolic compounds can also serve as competitive inhibitory substrates for ascorbic acid.

[0003] Ascorbic acid oxidase is widely found in plants, regulating physiological processes such as germination, growth and development, and abiotic stress. In practical applications, ascorbic acid oxidase is mainly used for enzymatic determination of L-ascorbic acid content, trace determination of dissolved oxygen, and elimination of interference from L-ascorbic acid in peroxidase-coupled enzyme assays. Ascorbic acid oxidase has been purified from plants such as cucumber, zucchini, pumpkin, melon, and tobacco, and its physicochemical properties, enzymatic characteristics, and catalytic mechanism have been studied in depth. However, ascorbic acid oxidase extracted directly from plants is not only costly and limited by raw materials, but the purified product also contains many impurities, which can easily interfere with the detection results. Therefore, this greatly limits the application of ascorbic acid oxidase in detection.

[0004] With the development of molecular biology, recombinant expression of target proteins by microorganisms has become a cost-effective and efficient method for obtaining large quantities of high-purity target proteins. However, there are few reports on the use of recombinant expression of ascorbic acid oxidase by microorganisms in the existing technology. Patent JP1997168389A discloses a method for expressing ascorbic acid oxidase using Escherichia coli, but this method not only has low yield but also produces enzymes with very low activity, which cannot meet the needs of commercialization and industrialization. Therefore, there is a need to develop a method for preparing highly active ascorbic acid oxidase suitable for industrial production. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing ascorbic acid oxidase.

[0006] Another object of the present invention is to provide a kit containing recombinant ascorbic acid oxidase prepared by the above method.

[0007] To address the aforementioned technical problems, the first aspect of this invention provides a polynucleotide encoding ascorbic acid oxidase protein, wherein the polynucleotide is codon-optimized and selected from any of the following:

[0008] (i) Polynucleotides with the sequence shown in SEQ ID NO.2;

[0009] (ii) polynucleotides with greater than 95% homology to the sequence shown in SEQ ID NO.2; and

[0010] (iii) A polynucleotide complementary to the polynucleotide sequence described in (i) or (ii).

[0011] In a second aspect, the present invention provides an expression vector comprising the polynucleotide provided in the first aspect of the present invention.

[0012] In some preferred embodiments, the expression vector is a Pichia pastoris expression vector, more preferably pPIC9, pPic9k, pHIL-S1, pPICZαA, pPinkα-HC or pYAM75P, and most preferably pPic9k.

[0013] In a third aspect, the present invention provides a host cell comprising the expression vector provided in the second aspect of the present invention; or

[0014] The host cell genome integrates polynucleotides as provided in the first aspect of the present invention.

[0015] In some preferred embodiments, the host cell is Pichia pastoris.

[0016] A fourth aspect of the present invention provides a method for preparing ascorbic acid oxidase protein, the method comprising the steps of: culturing the host cells described in the third aspect of the present invention to express the target protein; and

[0017] The target protein is isolated to obtain the ascorbic acid oxidase protein.

[0018] In some preferred embodiments, the host cells are cultured in BSM medium.

[0019] In some preferred embodiments, the host cells are cultured in YPD medium.

[0020] In some preferred embodiments, the host cells are first cultured in YPD medium, and the resulting single colony fallouts are inoculated into BSM medium for further culture of the host cells.

[0021] In some preferred embodiments, the host cells are cultured at 25-30°C, for example, 28°C.

[0022] In some preferred embodiments, the host cells are cultured in a medium with a pH of 4.8-5.5.

[0023] In some preferred embodiments, the host cells are cultured at a stirring speed of 200-1000 rpm, for example 250 rpm.

[0024] In some preferred embodiments, the host cells are first cultured in a medium containing glycerol, and then methanol is added for further culture to express the target protein.

[0025] In some preferred embodiments, the host cells are first cultured in a glycerol-containing medium, and after the OD value reaches 180-240, they are cultured for at least 0.8-1.2 hours, followed by the addition of methanol to continue culturing in order to express the target protein.

[0026] In some preferred embodiments, the methanol addition rate is 2-7 g / (L*h).

[0027] In some preferred embodiments, the host cells are cultured for at least 60 hours after the addition of methanol, preferably 60-120 hours.

[0028] In some preferred embodiments, the step of isolating the target protein includes:

[0029] The supernatant of the lysed target protein was eluted by passing it through a chromatography column while the flow was constant, and the eluent was collected.

[0030] A fifth aspect of the present invention provides a kit comprising: a polynucleotide as provided in the first aspect of the present invention; or

[0031] Such as the expression vector provided in the second aspect of the present invention; or

[0032] The host cell as described in the third aspect of the present invention; or

[0033] Or ascorbic acid oxidase protein prepared by the method according to the fourth aspect of the present invention.

[0034] Compared with the prior art, the present invention has at least the following advantages:

[0035] (1) This invention relies on Pichia pastoris as a fungal host system for heterologous expression, with methanol as the sole carbon source, and has advantages such as stable inheritance, low cost and easy culture, scalability, post-translational modification and secretory expression.

[0036] (2) The method in this invention utilizes Pichia pastoris to heterologously express ascorbic acid oxidase protein, which can obtain low-cost, large-scale production of ascorbic acid oxidase protein with high enzyme activity.

[0037] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0038] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0039] Figure 1 These are photographs of electro-sieving plates according to embodiments of the present invention;

[0040] Figure 2 This is an electrophoresis image of the purified expression product (target protein: 61.7 kDa) according to an embodiment of the present invention;

[0041] Figure 3 This is a standard curve diagram for ascorbic acid oxidase activity determination according to an embodiment of the present invention. Detailed Implementation

[0042] Through extensive and in-depth research, the inventors have developed a method for culturing ascorbic acid oxidase in a Pichia pastoris expression system. This method yields high output, produces enzymes with high activity, and has low fermentation production costs, making it valuable for industrial applications.

[0043] Target protein and its coding sequence

[0044] In this invention, the target protein is "ascorbic acid oxidase," and its amino acid sequence is shown in SEQ ID NO.1. The gene sequence encoding the target protein can be obtained from the amino acid sequence of the target protein according to this invention.

[0045] Synonymous codon-optimized polynucleotide sequences

[0046] To improve the expression efficiency of the target gene in a heterologous host, this invention also involves codon-optimized polynucleotide sequences, obtained by synonymous codon optimization of the gene sequence encoding the target protein. In this invention, the term "synonymous codon optimization" refers to a method of improving gene synthesis efficiency by avoiding the use of low-utilization or rare codons, based on the differences in codon utilization exhibited by the actual organisms used for protein expression or production (including *E. coli*, yeast, mammalian blood cells, plant cells, insect cells, etc.). The method of synonymous codon optimization for the target gene is not unique. Optionally, synonymous codon preference optimization can be performed on *E. coli* to improve the expression efficiency of the target gene in *E. coli*; synonymous codon preference optimization can be performed on *Pichia pastoris* to improve the expression efficiency of the target gene in *Pichia pastoris*. Even if the organism used for production is the same, the method of synonymous codon preference optimization is not limited to one. For example, in the embodiments of this invention, Pichia pastoris synonymous codon preference optimization is performed on the gene sequence encoding the target protein SEQ ID NO.1 to obtain multiple different optimized codons such as SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4. The expression yields of codons obtained through different optimization methods vary considerably.

[0047] The present invention also relates to homologous sequences of the aforementioned polynucleotides that encode protein fragments, analogs, and derivatives having the same amino acid sequence as those of the present invention. As is known in the art, a homologous sequence of a polynucleotide is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0048] In one embodiment, the aforementioned homologous sequence has a homology of greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 91%, more preferably greater than 92%, more preferably greater than 93%, more preferably greater than 94%, and more preferably greater than 95% with the polynucleotide sequence. As used in this invention, the terms "homology" and "identity" are used interchangeably and refer to the percentage of identical (i.e., same) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by arranging the nucleotide or amino acid sequences of the polynucleotide or polypeptide, scoring the number of positions containing identical nucleotide or amino acid residues in the arranged polynucleotide or polypeptide, and comparing this to the number of positions containing different nucleotide or amino acid residues in the arranged polynucleotide or polypeptide. Polynucleotides can differ at one position, for example, by containing different nucleotides (i.e., substitutions or variations) or by the deletion of nucleotides (i.e., insertion or deletion of one or two nucleotides in the polynucleotide). Polypeptides can differ at one position, for example, by containing amino acids (i.e., substitutions or variations) or by the deletion of amino acids (i.e., insertion or deletion of one or two amino acids in the polypeptide). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percentage identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0049] This invention also relates to sequences complementary to the aforementioned polynucleotide sequences. In this invention, the terms "sequence complement" and "reverse sequence complement" are used interchangeably, referring to sequences that are in the opposite direction to and complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complementary sequence is GTTCAT.

[0050] After obtaining the polynucleotide sequence information of this invention, the polynucleotide sequence can be prepared solublely using methods well known to those skilled in the art. For example, PCR amplification or chemical synthesis.

[0051] (1) PCR amplification method

[0052] Primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences. A commercially available cDNA library or a cDNA library prepared using conventional methods known to those skilled in the art can be used as a template. The template is paired with the complementary sequence of the designed primers. Then, under the action of Taq DNA polymerase, using dNTPs as reaction raw materials and the target sequence as a template, a new semi-conservative replicating strand complementary to the template DNA strand is synthesized according to the principles of base pairing and semi-conservative replication. Repeated cycles of denaturation-annealing-extension amplification are performed to obtain the relevant sequence. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order to obtain the full-length cDNA sequence.

[0053] (2) Chemical synthesis method

[0054] Chemical synthesis of target genes can be performed using methods such as superphosphate, phosphate-triester, and phosphite-triester synthesis. This process can be automated using a nucleic acid synthesizer. When the sequence length is short (typically 60-80 bp), chemical synthesis is considered a more preferred method for obtaining nucleotide sequences. Generally, longer sequences can be obtained by first synthesizing multiple small fragments and then ligating them.

[0055] Vectors containing polynucleotide sequences

[0056] This invention also relates to vectors containing polynucleotide sequences. In this invention, "vector" refers to a linear or circular DNA molecule containing a fragment encoding a target protein, said target protein being operatively linked to other fragments that provide for its transcription. Such additional fragments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more optional markers, enhancers, polyadenylation signals, vectors, etc. The vector fragment may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector may be any expression vector, either synthetic or readily manipulated with recombinant DNA, and the choice of vector generally depends on the host cell to which the vector is to be introduced. Thus, the vector may be a self-replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be a vector that integrates into the host cell genome upon introduction into a host cell and replicates along with the chromosome into which it is integrated. In one embodiment, the vector of this invention is an expression vector. In some embodiments, pPIC9, pPic9k, pHIL-S1, pPICZαA, and pYAM75P are used as vectors. In one embodiment, pPic9k was selected as the vector to obtain more efficient expression.

[0057] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Exemplarily, the vector DNA molecule is cleaved into linear molecules that can be linked to a foreign gene using a DNA endonuclease, and then a codon-optimized target gene fragment is ligated into the vector. The insertion of the foreign DNA fragment can be achieved by using sticky-end ligation at a single restriction enzyme site, directed cloning of double-restricted fragments, sticky-end ligation at different restriction enzyme sites, blunt-end ligation, artificial adapter ligation, or ligation to oligonucleotide ends.

[0058] Host cells containing polynucleotides

[0059] This invention also relates to host cells generated by genetic engineering using the vector of this invention. In this invention, "host cell" is a cell in which exogenous polynucleotides and / or a vector have been introduced. The host cell can be a eukaryotic or prokaryotic host cell, preferably a fungus, and more preferably a yeast, more preferably Pichia pastoris, such as GS115, KM71, and SMD1168 strains.

[0060] Methods well known to those skilled in the art can be used to insert, transfect, or otherwise transform vectors containing polynucleotide sequences into host cells, thereby obtaining transformants containing the polynucleotide sequences of the present invention and capable of expressing the target protein. When the host is Pichia pastoris, heat shock or electroporation methods can be used.

[0061] (1) Heat shock method

[0062] The heat shock method adheres plasmid DNA to the surface of competent E. coli cells, and then subjectes them to a short heat shock treatment at around 42°C to promote plasmid DNA uptake.

[0063] (2) Electroconversion method

[0064] The competent E. coli and plasmid mixture were added to a pre-cooled electroporation cuvette and then electroporated in an electroporator to induce plasmid DNA to enter the host cell.

[0065] After obtaining the transformants, transformants (single colonies grown on screening plates) can be selected using conventional methods in the field, such as genetic testing, sequencing, and colony PCR.

[0066] Methods for preparing target proteins

[0067] The present invention also relates to a method for preparing a target protein, comprising: (1) transforming or transducing a suitable host cell with a polynucleotide (or homologous sequence) encoding the target protein of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) culturing the host cell in a suitable culture medium; and (3) isolating and purifying the protein from the culture medium or the cell.

[0068] Transformants containing the polynucleotide sequence of this invention can be obtained and cultured using conventional methods to express the target protein encoded by the gene of this invention. Depending on the host cells used, conventional culture media are selected and cultured under conditions suitable for host cell growth. The culture medium can be optimized based on expression results to improve expression stability and efficiency. In some examples, selectable culture media include LB, LLB, YPD, BMGY, BMMY, YPDS+Zeocin, MGY, MGYH, RD, and SOC media, with BSM medium being preferred. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0069] Generally, host cells are cultured in shake flasks until single colonies grow, which are then picked and inoculated into fermenters for large-scale production. During production, fermentation conditions typically differ from those in shake flasks. Yield and stability can usually be improved by controlling the stirring speed, culture temperature, pH, and carbon supplementation. Optimal culture conditions vary depending on the microbial strain and the target protein being produced. In some embodiments of this invention, preferred culture conditions during fermentation are a pH of 4.8-5.5, a culture temperature of 25-30°C, and a stirring speed of 200-1000 rpm.

[0070] In some embodiments of the present invention, host cells are first cultured in a medium containing glycerol, and then methanol is added to the medium for further culture to express the target protein. To further increase the yield, host cells are first cultured in a medium containing glycerol, and when the OD value reaches 180-240, they are cultured for at least 0.8-1.2 hours. Then methanol is added and culture is continued. The methanol addition rate is preferably 2-7 g / (L*h). After adding methanol, the cells are cultured for at least 60 hours, preferably 60-120 hours, and then the fermentation broth of the target protein is collected for separation and purification.

[0071] In this invention, after successfully culturing the target protein, the steps of separating and purifying it are also involved, such as separating and purifying the protein from the culture medium to obtain the target protein with high purity. Although the methods for purifying the target protein are conventional techniques well known to those skilled in the art, they include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0072] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0073] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0074] Example 1

[0075] (1) Construct a gene sequence recombination expression vector containing AAO

[0076] The gene sequence of AAO was obtained and optimized for Pichia pastoris synonymous codon bias to obtain the optimized codon sequence SEQ ID NO.2. The ligation vector was pPic9k, which was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0077] (2) Plasmid linearization

[0078] Escherichia coli glycerol containing the plasmid was streaked onto LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37°C. Single colonies were then inoculated onto LB agar containing 100 μg / mL ampicillin and incubated overnight at 37°C with shaking at 230 rpm to obtain a high concentration of bacterial culture. The plasmid was extracted using a high-purity plasmid mini-prep kit from Tiangen Biotech (Beijing) Co., Ltd., following the manufacturer's instructions. Sal I and the corresponding buffer were added to the plasmid, and the mixture was digested at 37°C for 2 hours. The linearized plasmid was then precipitated with anhydrous ethanol and sodium acetate, recovered, and stored at -20°C.

[0079] (3) Preparation of Pichia pastoris competent cells

[0080] GS115 was streaked onto YPD plates and incubated at 28°C for 2-3 days to obtain single colonies. These single colonies were then inoculated onto YPD medium and incubated overnight at 28°C with shaking at 250 rpm to obtain primary bacterial culture. 0.5%-1.5% of the primary bacterial culture was then inoculated onto YPD medium and incubated at 28°C with shaking at 250 rpm until OD reached [value missing]. 600 The concentration was between 1.3 and 1.5. The cells were collected by centrifugation at 1500g at 4℃ for 5 min. The cells were washed twice with sterile water, resuspended in 1M sorbitol solution, incubated on ice for 5 min, centrifuged at 1500g at 4℃ for 5 min, and finally reconstituted with 1M sorbitol solution to obtain competent cells.

[0081] (4) Introduction of recombinant plasmids

[0082] Take 10 μL of linearized plasmid and 80 μL of GS115 competent cells into an electroporation cuvette, mix well by pipetting, and place in an electroporator after incubating on ice. After successful electroporation, add 1 mL of YPDS, mix well by pipetting, and incubate at 28℃ for at least 2 hours. Take 50-100 μL of bacterial culture and spread it evenly on a YPDS plate containing G418, and incubate at 28℃ for 5-10 days to obtain 20-50 transformants. Figure 1 As shown.

[0083] (5) Expression of recombinant proteins

[0084] Fifteen single colonies from the above plates were picked and inoculated into BMGY medium. After incubation at 28℃ and 250 rpm for 24-36 h, methanol was added to a final concentration of 0.5% for induction, and then methanol was added every 24 h to a final concentration of 0.5%. The protein concentration of the samples before induction and at 72 h and 144 h of induction was determined using the Bradford method, and samples with high protein concentrations were selected for qualitative activity testing (using the Solarbio ascorbic acid oxidase (AAO) activity assay kit). The results are shown in Table 1. The protein content of the 15 transformants increased continuously with the induction time, with the final protein concentration of GS115-3 / 6 / 7 / 10 / 12 exceeding 450 μg / mL. Qualitative activity testing was performed on the above samples, and the results are shown in Table 2. The fermentation broth of the electroporated strains GS115-7 / 12 showed enzyme activity, and GS115-12 showed the largest change in absorbance. Therefore, GS115-12 was selected as the strain for subsequent expansion culture.

[0085] Table 1. Protein concentrations in the fermentation broth before induction, at 72 h, and at 144 h after induction.

[0086]

[0087]

[0088] Table 2 Qualitative activity assay of fermentation broth after 144 hours of induction.

[0089] GS115-3 (10μL) -0.0615 GS115-6 (10μL) 0.0077 GS115-7 (10μL) 0.0939 GS115-10 (10μL) -0.0249 GS115-12 (10μL) 0.1403 Unloaded 0.012 Blank 1 0.0252 Positive enzyme (1 μL) 0.1552

[0090] The amino acid sequence of AAO in SEQ ID NO. 1

[0091] SQIRHYKWEVEYMFWAPDCNENIVMGINGQFPGPTIRANAGDTVVVELINKLHTEGVVIHWHGILQRGTPWADGTASISQ

[0092] CAINPGETFFYNFTVDNPGTFFYHGHLGMQRSAGLYGSLIVDPPQGKKEPFHYDGEINLLLSDWWHQSIHKQEVGLSSKP

[0093] IRWIGEPQTILLNGRGQFDCSIAAKYDSNLEPCKLKGSEPCAPYIFHVMPKKTYRIRIASTTALAALNFAIGNHPLLVVE

[0094] ADGNYVQPFYTSDIDIYSGESYSVLITTDQNPSENYWVSVGTRGRHPNTPPGLTLLNYLPNSVSKLPTSPPPETPAWDDF

[0095] DRSKNFTYRITAAMGSPKPPVKSNRRIFLLNTQNVINGYVKWAINDVSLALPPTPYLGAMKFNLLHAFDQNPPPEVFPED

[0096] YDIDTPPTNEKTKIGNGVYQFKIGEIVDVILQNANMMKENLSEIHPWHLHGHDFWVLGYGDGKFTAEEESSLNLKNPPLR

[0097] NTVVIFPYGWTAIRFVADNPGVWAFHCHIEPHLHMGMGVVFAEGVEKVGRIPTKALACGGTAKSLINNP

[0098] Optimized codon of SEQ ID NO. 2

[0099] AGTCAAATCAGACACTACAAATGGGAGGTTGAGTACATGTTCTGGGCACCTGATTGTAATGAGAACATTGTGATGGGTAT

[0100] TAATGGACAATTCCCTGGACCTACCATTAGAGCCAATGCTGGTGATACTGTCGTTGTCGAGCTGATTAACAAATTGCACA

[0101] CCGAAGGTGTCGTCATTCATTGGCATGGAATCTTGCAAAGAGGTACGCCTTGGGCTGATGGTACTGCTTCCATTTCTCAA

[0102] TGTGCTATTAACCCTGGAGAAACCTTCTTCTACAACTTCACTGTTGACAATCCAGGTACCTTCTTCTACCACGGTCATTT

[0103] GGGTATGCAAAGATCCGCTGGTTTGTACGGTAGTCTTATTGTTGACCCTCCACAAGGTAAGAAAGAGCCATTTCACTACG

[0104] ATGGTGAGATCAACTTGTTGCTGTCCGATTGGTGGCATCAATCCATTCACAAGCAAGAAGTCGGTCTTTCTTCCAAACCA

[0105] ATCAGATGGATTGGTGAACCACAGACAATCTTGCTTAACGGAAGAGGTCAATTCGACTGTTCCATCGCTGCTAAGTACGA

[0106] CTCTAATTTGGAGCCATGTAAGCTGAAAGGTAGTGAGCCATGTGCACCATACATCTTTCATGTGATGCCAAAGAAGACTT

[0107] ACAGAATTAGAATTGCCAGTACTACCGCTTTGGCTGCTTTGAATTTCGCTATCGGTAATCATCCATTGCTGGTTGTCGAA

[0108] GCAGATGGAAACTACGTTCAACCATTCTACACTTCCGACATTGACATCTATAGTGGTGAGTCTTACAGTGTTCTGATCAC

[0109] AACTGATCAGAATCCATCAGAGAATTACTGGGTCAGTGTTGGAACAAGAGGTAGACATCCAAATACTCCACCAGGTTTAA

[0110] CCTTGTTGAACTACCTGCCTAACTCTGTTTCAAAGCTGCCTACTAGTCCACCACCAGAGACTCCAGCTTGGGATGACTTC

[0111] GATAGATCCAAGAACTTTACCTACAGAATTACCGCTGCTATGGGAAGTCCAAAGCCACCTGTCAAGTCCAACAGAAGAAT

[0112] CTTTCTGTTGAATACTCAGAACGTTATCAATGGATACGTGAAATGGGCTATTAATGACGTTAGTTTGGCTTTGCCTCCAA

[0113] CACCTTATTTGGGTGCTATGAAATTTAACCTGTTGCATGCTTTCGACCAGAATCCACCTCCAGAAGTCTTTCCAGAAGAC

[0114] TACGATATCGATACACCTCCAACTAATGAGAAGACCAAGATCGGTAATGGTGTTTATCAGTTCAAGATTGGTGAGATTGT

[0115] TGACGTTATTCTTCAGAATGCTAACATGATGAAAGAGAACCTGTCCGAAATTCACCCTTGGCATCTGCATGGTCACGATT

[0116] TCTGGGTTCTGGGTTACGGTGATGGTAAGTTTACTGCTGAAGAAGAATCCAGTCTGAACCTTAAGAATCCACCTTTGAGA

[0117] AATACCGTTGTGATCTTCCCTTATGGTTGGACCGCCATCAGATTTGTTGCTGATAATCCAGGTGTTTGGGCATTCCATTG

[0118] TCATATTGAACCTCATTTGCACATGGGTATGGGTGTTGTGTTCGCTGAAGGTGTAGAGAAGGTCGGAAGAATACCTACTA

[0119] AGGCTCTGGCTTGTGGTGGTACTGCTAAGAGTCTGATTAACAATCCA

[0120] Example 2

[0121] Select the positive transformant GS115-12 prepared in Example 1 above, streak it onto YPD plates, and incubate at 28℃ for 3-5 days until single colonies grow. Pick a single colony and incubate it in 5 mL of YPD medium at 28℃ and 250 rpm for 18-24 hours. Transfer 1% of the culture to 100 mL of YPG medium, incubate at 28℃ and 250 rpm for 18-24 hours, and then inoculate 5% of the culture into a 5L fermenter containing 2L of inorganic salt medium (BSM medium). Fermentation conditions: stirring: 200-1000 rpm; temperature: 25-30℃; DO: 20-50% (pure oxygen is added appropriately if DO is insufficient). pH: pH is automatically controlled using ammonia water to 4.8-5.5.

[0122] After DO or pH rebound, begin adding glucose at 5-12 g / (L*h), OD 600 Once the concentration reached 180-240, glucose supplementation was stopped. Methanol induction was initiated 1 hour later at a rate of 2-7 g / (L*h), with samples taken every 24 hours. After 60-120 hours of methanol induction, the mixture was discharged from the fermentation broth and centrifuged at 4500 rpm for 5 minutes to obtain the supernatant. Protein concentration was determined using the Bradford method, as shown in Table 3. The protein content of the fermentation broth increased continuously with induction time, reaching a final concentration of 364.2 μg / mL upon discharge.

[0123] Table 3 Protein concentration during fermentation process

[0124]

[0125]

[0126] Example 3

[0127] (1) Sample processing

[0128] Concentrate the supernatant 10-15 times using an ultrafiltration system, then perform ultrafiltration with 5-15 times the volume of Buffer A. Centrifuge at 12000 rpm, 4°C for 30 min, collect the supernatant, and filter through a 0.22 μm membrane.

[0129] (2) Anion column purification

[0130] Purification was performed using a 20 mL Q-HP column. The flow rate was 3.0 mL / min. After loading, the UV filter was washed with 20 mL Buffer A and the electrolyte was charged to baseline. The elution program was as follows: Step 1: 0% C, 1.5 CV, 3.0 mL / min; Step 3: 5% C, 2 CV, 3.0 mL / min; Step 3: 100% C, 2 CV, 3.0 mL / min. The electrophoresis results after purification are shown below. Figure 2 As shown, tubes 3C7-3C10 contain the corresponding target bands and a small amount of extraneous proteins.

[0131] Buffer solutions used for ultrafiltration concentration and purification:

[0132] Buffer A: 50mM Tris, 50mM NaCl, pH8.0

[0133] Buffer C: 100mM Tris, 1M NaCl, pH 8.0

[0134] Comparative Example 1

[0135] Different codon optimization methods resulted in significant differences in the product yield of ascorbic acid oxidase in the Pichia pastoris expression system. The inventors obtained a series of codons through extensive experiments (as shown in Table 4). Some optimized codons had low expression efficiency and low yield under the same fermentation conditions. Other expression products had no enzyme activity or even no expression product.

[0136] Table 4

[0137] SEQ ID NO.3 225.1 μg / mL SEQ ID NO.4 41.2 μg / mL

[0138] In Table 4 above, the optimized codon of SEQ ID NO.3 can express a certain amount of ascorbic acid oxidase product in the Pichia pastoris expression system, but the yield is not as high as that of SEQ ID NO.2. The expression yield of the optimized codon of SEQ ID NO.3 is too low, and its application value is small.

[0139] SEQ ID NO.3

[0140] TCACAAATACGTCCATTACAAATGGGAAGTAGAATACATGTTTTGGGCTCCTGATTGTAACGAGAATATTGTGATGGGAAT

[0141] TAATGGCCAATTCCCTGGTCCTACCATTAGAGCAAATGCTGGAGACACTGTTGTCGTGGAACTTATTAATAAGCTGCACA

[0142] CTGAAGGAGTTGTAATTCATTGGCATGGCATTTTGCAAAGGGGTACCCCATGGGCTGATGGTACTGCTAGTATCTCACAG

[0143] TGTGCAATCAATCCTGGGGAGACCTTTTTTTATAATTTTACTGTTGATAACCCCGGTACCTTCTTTTATCATGGACATTT

[0144] GGGTATGCAAAGGAGTGCCGGTCTTTATGGATCTCTAATAGTGGATCCACCACAGGGAAAAAAGGAGCCATTTCATTATG

[0145] ACGGTGAGATTAACTTGTTATTATCAGACTGGTGGCATCAAAGCATACATAAACAGGAAGTGGGCTTATCTTCAAAACCT

[0146] ATTCGTTGGATCGGTGAACCGCAAACTATTCTGCTAAACGGCAGAGGCCAATTTGATTGTTCAATTGCAGCCAAATATGA

[0147] TTCCAATCTGGAACCTTGTAAGTTAAAAGGTTCTGAACCATGCGCACCTTACATCTTCCATGTCATGCCTAAAAAGACAT

[0148] ACAGAATTAGAATTGCTAGTACTACCGCCCTTGCAGCCTTAAATTTTGCGATAGGAAATCACCCTTTACTAGTGGTTGAG

[0149] GCAGACGGCAATTACGTTCAGCCATTCTACACTTCTGACATAGATATATATTCAGGTGAGTCATATTCAGTATTAATAAC

[0150] AACTGATCAAAACCCTTCCGAGAATTATTGGGTTTCTGTCGGTACCCGTGGCCGTCATCCAAACACTCCCCCTGGTCTAA

[0151] CTTTGCTAAACTATCTTCCTAATTCTGTGAGCAAGTTGCCCACAAGTCCTCCACCTGAAACACCAGCATGGGACGACTTT

[0152] GATAGATCTAAAAATTTCACATATAGAATAACAGCAGCTATGGGCTCTCCAAAGCCACCAGTAAAGAGTAACAGACGTAT

[0153] TTTTCTTCTGAATACGCAGAACGTTATAAACGGATACGTCAAGTGGGCTATTAATGACGTCTCTTTAGCTCTTCCACCAA

[0154] CTCCATACTTAGGTGCTATGAAGTTCAATCTATTGCACGCTTTTGATCAAAACCCACCACCAGAGGTTTTCCCAGAAGAC

[0155] TATGATATTGATACACCACCCACTAATGAGAAAACTAAGATCGGTAACGGCGTGTACCAGTTTAAAATTGGTGAAATTGT

[0156] GGACGTCATTTTGCAAAACGCTAATATGATGAAAGAGAATCTAAGCGAGATCCATCCTTGGCATCTGCATGGACATGACT

[0157] TCTGGGTTTTGGGATACGGCGATGGAAAGTTCACTGCTGAGGAAGAATCATCTCTAAATTTGAAAAATCCGCCTTTGAGA

[0158] AACACTGTTGTTATTTTTCCTTATGGATGGACAGCTATCCGTTTTGTAGCGGATAATCCAGGTGTGTGGGCATTTCACTG

[0159] CCATATTGAGCCACATTTACACATGGGTATGGGTGTTGTTTTCGCTGAGGGTGTCGAAAAAGTTGGGAGGATTCCCACTA

[0160] AGGCATTAGCCTGTGGTGGTACAGCCAAGTCTTTGATTAATAACCCT

[0161] SEQ ID NO.4

[0162] TCTCAAATCAGACACTACAAATGGGAAGTTGAATATATGTTCTGGGCACCAGACTGTAATGAAAATATAGTTATGGGTAT

[0163] CAATGGTCAGTTTCCAGGTCCAACCATAAGAGCTAATGCTGGTGATACCGTAGTCGTTGAATTGATTAATAAGTTACATA

[0164] CTGAAGGTGTTGTTATTCACTGGCATGGTATTTTACAAAGAGGCACACCATGGGCTGACGGTACTGCATCTATTTTCACAA

[0165] TGTGCAATCAATCCAGGTGAAACATTTCTATAACTTCACCGTGGATAATCCTGGTACTTTTTTCTATCATGGTCATTT

[0166] AGGTATGCAAAGATCTGCAGGTTTGTATGGTTCTTTGATTGTTGATCCACCACAAGGCAAGAAAGAGCCTTTCCATTATG

[0167] ATGGTGAAATTATTTATTGTCAGATTGGTGGCACCAATCTATTCATAAACAAGAAGTTGGTTTATCGTCGAAGCCA

[0168] ATTAGATGGATTGGTGAACCTCAAACAATTCTTTTGAATGGCAGAGGTCAATTCGATTGTTCTATTGCTGCTAAATACGA

[0169] TTCTAACTTGGAACCCTGTAAATTAAAAGGTTCTGAACCATGTGCTCCATACATCTTTCATGTTATGCCCAAAAAGACTT

[0170] ATAGAATCAGGATTGCTTCTACAACTGCTTTGGCAGCCTTGAATTTTGCTATTGGTAATCATCCTCTGTTGGTTGTTGAA

[0171] GCTGATGGTAACTACGTTCAACCATTCTATACTTCTGACATTGATATATACTCTGGTGAATCTTATAGCGTCTTAATAAC

[0172] GACAGATCAAAACCCATCTGAAAATTATTGGGTTTCTGTGGGTACTAGAGGTAGACATCCAAATACTCCACCAGGTTTAA

[0173] CTTTATTGAATTATTTGCCCAATTCCGTTTCTAAGTTGCCAACTTCTCCTCCACCAGAAACCCCAGCCTGGGACGACTTT

[0174] GATAGATCCAAAAATTTCACTTACAGAATCACGGCCGCAATGGGTTCTCCAAAACCACCAGTCAAGAGTAATCGTAGAAT

[0175] TTTTTTGTTGAATACCCAAAATGTAATTAACGGATATGTTAAGTGGGCTATTAATGATGTTTCTTTGGCCTTGCCTCCAA

[0176] CCCCATACTTAGGTGCAATGAAGTTCAATTTGTTGCATGCTTTTGACCAAAATCCACCACCAGAAGTTTTCCCAGAAGAC

[0177] TACGACATTGATACTCCACCAACAAACGAAAAAACAAAAATTGGTAATGGCGTTTATCAATTCAAAATAGGTGAAATCGT

[0178] TGACGTTATTTTGCAAAATGCAAATATGATGAAAGAAAACTTATCTGAAATTCACCCATGGCATTTGCATGGTCATGATT

[0179] TCTGGGTTTTAGGTTACGGTGATGGTAAATTTACTGCTGAAGAGGAATCATCTCTGAACTTGAAAAATCCACCTTTAAGA

[0180] AACACCGTGGTTATTTTTCCATACGGTTGGACTGCAATAAGATTTGTTGCTGATAATCCAGGTGTTTGGGCTTTCCATTG

[0181] TCATATAGAACCACATTTACATATGGGTATGGGTGTCGTATTTGCAGAAGGCGTTGAAAAAGTTGGTAGAATTCCAACAA

[0182] AAGCATTGGCTTGTGGTGGTACAGCTAAGTCTTTGATTAACAACCCA

[0183] [Enzyme activity detection]

[0184] AAO can directly oxidize ascorbic acid; therefore, the activity of AAO can be detected by measuring the amount of ascorbic acid oxidized, with a detection wavelength of 265 nm. The specific experimental steps are as follows:

[0185] 1) Preheat the microplate reader for 30 minutes and set the incubation temperature to 25℃.

[0186] 2) The reaction solution was prepared according to the instructions of Solarbio's ascorbic acid oxidase (AAO) kit.

[0187] 3) Add 100 μL of working solution to a 96-well microplate, and measure the OD value A1 at 265 nm. After reacting at 25 °C for 2 min, measure the OD value A2 at 265 nm. Calculate the OD difference A1-A2 before and after the reaction.

[0188] 4) The AAO-positive enzyme (512 U / μL) was serially diluted to different concentrations to establish a standard curve. Enzyme activity was calculated based on the standard curve. The results of the ascorbic acid oxidase standard curve are shown below. Figure 3 .

[0189] The absorbance of the recombinases prepared in the examples and comparative examples (optimized codon SEQ ID NO.3) was measured according to the above method, and the results are shown in Table 5 below:

[0190] Table 5. Enzyme activity assay results of the purified product.

[0191]

[0192] The concentration of the purified sample was 5.893 mg / mL, and the average activity was 82.1 U / mL. Therefore, the specific activity was 82.1 / 5.893 = 13.93 U / mg.

[0193] The purified sample concentration in the example was 6.577 mg / mL, and the average activity was 217.6 U / mL. Therefore, the specific activity was 217.6 / 6.577 = 33.08 U / mg, which was significantly higher than that of the comparative example.

[0194] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. An isolated polynucleotide encoding ascorbic acid oxidase protein, characterized in that, The polynucleotide is codon-optimized and is a polynucleotide as shown in SEQ ID NO.

2.

2. An expression carrier, characterized in that, The expression vector comprises the polynucleotide as described in claim 1.

3. The expression vector according to claim 2, characterized in that, The expression vector is a Pichia pastoris expression vector.

4. The expression vector according to claim 2, characterized in that, The expression vector is pPic9k.

5. A host cell, characterized in that, The host cell comprises the expression vector as described in any one of claims 2-4; or The host cell genome contains the polynucleotides as described in claim 1.

6. A method for preparing ascorbic acid oxidase protein, characterized in that, The method includes: Transform host cells using a vector containing the polynucleotide as described in claim 1; The host cells were cultured to express ascorbic acid oxidase protein.

7. The method according to claim 6, characterized in that, The host cells were cultured in BSM medium.

8. The method according to claim 6, characterized in that, First, the host cells were cultured in YPD medium, and single colonies were then inoculated into BSM medium for further culture.

9. The method according to claim 6, characterized in that, The host cells were first cultured in a medium containing glycerol, and then methanol was added to continue culturing in order to express the target protein.

10. The method according to claim 6, characterized in that, The host cells were cultured at 25-30°C. And / or, the host cells are cultured in a medium with a pH of 4.8-5.5; And / or, the host cells are cultured at a stirring speed of 200-1000 rpm.

11. A reagent kit, characterized in that, The kit comprises: the polynucleotide as described in claim 1; or The expression vector as described in any one of claims 2-4; or The host cell as described in claim 5.

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