CN117660517A

CN117660517A - Combined expression system of non-specific peroxidase and application thereof

Info

Publication number: CN117660517A
Application number: CN202311695889.5A
Authority: CN
Inventors: 薛亚平; 赵理想; 沈其; 邹树平; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2023-12-12
Filing date: 2023-12-12
Publication date: 2024-03-08

Abstract

The invention relates to the technical field of bioengineering, in particular to a combined expression system of non-specific peroxidase and application thereof. The combined expression system of the non-specific peroxidase comprises a host cell and an expression vector transformed into the host cell, wherein the expression vector comprises a first expression vector inserted with a gene for encoding a non-specific peroxidase protein UPO and a second expression vector inserted with a gene for encoding disulfide isomerase PDI, and the host cell is Pichia pastoris (Komagataelaphaffii) GS115. The invention constructs a disulfide bond isomerase and non-specific peroxidase co-expression combined expression system, so that the expression quantity of the non-specific peroxidase is obviously improved, and the concentration of the non-specific peroxidase in the collected culture medium supernatant is increased without generating other proteins because the disulfide bond isomerase is expressed in a host cell and the non-specific peroxidase can be secreted and expressed outside the host cell, and finally the concentration of the non-specific peroxidase protein in a 5 liter bioreactor can reach 333.3mg/L.

Description

Combined expression system of non-specific peroxidase and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a combined expression system of non-specific peroxidase and application thereof.

Background

In 2004, nonspecific peroxidase (UPO, E.C.1.11.2.1), a novel heme sulfate peroxidase with mono (per) oxidase activity, was found in agrocybe cylobeguerita (previously known as agrocybe aegerita). To date, there have been reports of over 300 non-specific peroxidase substrates, and the number is increasing. Notably, the non-specific peroxidases and cytochrome P450 enzymes have similar properties when they do not activate C-H bonds in specific oxygenated organic molecules. Cytochrome P450 enzymes not only catalyze monooxygenation reactions, but also dealkylation, S-oxidation, N-oxidation, alcohol and aldehyde oxidation, dehalogenation and denitrification. However, commercial use of cytochrome P450 enzymes is still limited to whole cell oxidation procedures due to the instability and complexity of the isolation preparation. In contrast, a non-specific peroxidase is a relatively stable extracellular enzyme, and has the advantages of high activity, no need for expensive or complex redox partners, and the like. In addition, the nonspecific peroxidase operates in a self-sufficient manner, being "driven" by an appropriate amount of hydrogen peroxide. Hydrogen peroxide is both the primary electron acceptor and the source of oxygen. Thus, a non-specific peroxidase is considered to be a self-sufficient peroxidase.

At present, the expression level of the non-specific peroxidase is not high, and in order to improve the production efficiency of the non-specific peroxidase, a yeast host is the key point of research. Pichia pastoris (original Pichia pastoris) has a high-efficiency heterologous expression system, and the culture condition is simple, so that the method is suitable for high-density fermentation. Accordingly, pichia has been widely used for the production of recombinant proteins. There are several strategies reported to increase the efficient secretory expression of heterologous proteins in pichia pastoris, but most literature focuses on single-or two-factor optimization, which still has less than ideal overall improvement of heterologous protein secretion levels.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides a combined expression system of non-specific peroxidase, and is applied to the production of the non-specific peroxidase so as to solve the problem of non-ideal expression yield of the non-specific peroxidase in the prior art.

In order to achieve the above purpose, the present invention is realized by the following technical scheme:

in a first aspect, the invention provides a combination expression system of a non-specific peroxidase, comprising a host cell and an expression vector transformed into the host cell, the expression vector comprising a first expression vector inserted with a gene encoding a non-specific peroxidase protein UPO and a second expression vector inserted with a gene encoding a disulfide isomerase PDI, the host cell being Pichia pastoris (Komagataelaphaffii) GS115.

The invention constructs a combined expression system for co-expression of disulfide isomerase and non-specific peroxidase, so that the expression quantity of the non-specific peroxidase is obviously improved, and the concentration of the non-specific peroxidase in the culture medium supernatant is increased and other proteins are not newly produced at the same time because the disulfide isomerase is expressed in a host cell and the non-specific peroxidase can be secreted and expressed outside the host cell. Finally, the concentration of the nonspecific peroxidase protein in the 5 liter bioreactor can reach 333.3mg/L.

Disulfide isomerase is a member of the family of sulphur oxidoreductases, whose main function is to catalyze the formation of correct disulfide bonds in new and formed proteins on the endoplasmic reticulum. Accumulation of misfolded proteins to some extent can cause stress in the endoplasmic reticulum of the cell, thereby placing a burden on the metabolism of the cell. The nonspecific peroxidase contains a pair of disulfide bonds, and more disulfide bond isomerase can help to form correct disulfide bonds, so that the expression level is improved. The host cell in the invention is Pichia pastoris (Komagataella phaffii) GS115, and the foreign protein secreted by the Pichia pastoris is little, so that the high-secretion foreign protein is easy to separate from the culture medium; in addition, compared with Saccharomyces cerevisiae, the glycosylation degree of Pichia pastoris is lower, and the produced nonspecific peroxidase is closer to the natural protein.

Preferably, the gene encoding the non-specific peroxidase protein UPO is derived from agrocybe cylindracea (Cyclocybe aegerita) TM-A1.

Preferably, the nucleotide sequence of the gene for encoding the non-specific peroxidase protein UPO is shown as SEQ ID NO. 1.

Preferably, the nucleotide sequence of the gene encoding disulfide isomerase protein PDI is shown as SEQ ID NO. 2.

Preferably, the original vector of the first expression vector is a pPIC series vector, and the original vector of the second expression vector is a pPIC series vector.

Preferably, the original vector of the first expression vector is a vector pPICZ alpha A.

Preferably, the original vector of the second expression vector is a vector pPIC3.5K.

Preferably, the construction method comprises the following steps:

(1) Synthesizing a codon optimized gene encoding a non-specific peroxidase UPO;

(2) Inserting the gene for encoding the non-specific peroxidase UPO obtained in the step (1) into a vector pPICZ alpha A to construct a first expression vector pPICZ alpha A-UPO;

(3) Linearizing the first expression vector pPICZ alpha A-UPO obtained in the step (2) and then electrically introducing the linearized first expression vector pPICZ alpha A-UPO into pichia pastoris (Komagataella phaffii) GS115 to obtain an engineering strain;

(4) Inserting a gene encoding disulfide isomerase PDI into the vector pPIC3.5K to construct a second expression vector pPIC3.5K-PDI;

(5) And (3) linearizing the second expression vector pPIC3.5K-PDI obtained in the step (4) and then electrically introducing the linearized second expression vector pPIC3.5K-PDI into the engineering strain obtained in the step (3), thereby obtaining the combined expression system of the non-specific peroxidase.

Further preferably, the gene encoding the non-specific peroxidase UPO is inserted downstream of the promoter and the signal peptide in the vector pPICZαA, and the gene encoding the disulfide isomerase PDI is inserted downstream of the promoter in the vector pPIC3.5K.

The first expression vector was constructed to contain a signal peptide downstream of the promoter and the second expression vector was constructed to have no signal peptide downstream of the promoter. Thus, the nonspecific peroxidase protein is secreted outside the cell, while the disulfide isomerase protein remains inside the cell.

In the first expression vector, an XhoI enzyme cutting site is added to the upstream sequence of a gene for encoding non-specific peroxidase UPO, and a NotI enzyme cutting site is added to the downstream sequence of the gene; the first expression vector pPICZαA-UPO was constructed by first cutting the vector pPICZαA with the linearized enzyme BspE I and then ligating with the UPO gene.

In the second expression vector, the vector pPICC 3.5K is firstly cut by XhoI enzyme and NotI enzyme, and then is connected with the PDI gene, so that a second expression vector pPICC 3.5K-PDI is constructed.

Preferably, the gene encoding the non-specific peroxidase UPO and the gene encoding the disulfide isomerase PDI are overexpressed.

Further preferably, the copy number of the first expression vector pPICZ alpha A-UPO is 2-3, and the copy number of the second expression vector pPIC3.5K-PDI is at least 2.

Pichia pastoris GS115, plasmid pPICZ alpha A and plasmid pPIC3.5K are commercial products, and are purchased from Invitrogen Corp.

In a second aspect, the invention provides the use of a combination expression system of said non-specific peroxidase in the production of a non-specific peroxidase.

The BSM culture medium is adopted for batch fed-batch culture, the whole process is divided into a batch culture stage and a fed-batch culture stage, and the carbon source used in the batch culture stage is glycerol, so that the purpose of the glycerol is to accumulate thalli; the carbon source used in the feed culture stage is methanol, and the purpose of the carbon source is protein expression. The whole culture medium section requires Dissolved Oxygen (DO) >25%.

Compared with the prior art, the invention has the following beneficial effects:

(1) According to the invention, the host cell is selected as the pichia pastoris, and the self-secreted foreign protein is little, so that the high-secreted foreign protein, namely the non-specific peroxidase, is easy to separate from the culture medium, the glycosylation degree of the pichia pastoris is lower, and the produced non-specific peroxidase is more similar to the natural protein;

(2) According to the invention, a host for co-expression of the non-specific peroxidase and the disulfide bond isomerase is constructed, so that the expression of the non-specific peroxidase is obviously improved, and the concentration of the non-specific peroxidase in the collected culture medium supernatant is increased and other proteins are not produced at the same time because the disulfide bond isomerase is expressed in cells but the secretion table of the non-specific peroxidase reaches the outside of the cells;

(3) The combined expression system of the non-specific peroxidase further improves the yield of the non-specific peroxidase, and finally the protein concentration of the non-specific peroxidase in a 5 liter bioreactor can reach 333.3mg/L, thus providing a foundation for industrial mass production.

Drawings

FIG. 1 is a schematic construction diagram of a first expression vector pPICZ alpha A-UPO.

FIG. 2 shows Western-blot analysis of cloned strains of non-specific peroxidases selected at different bleomycin concentrations.

FIG. 3 is a schematic diagram showing the construction of a second expression vector pPIC3.5K-PDI.

FIG. 4 shows Western-blot analysis of the expression of a multicopy UPO gene into which a PDI gene is introduced in a host.

FIG. 5 is a graph showing the results of RT-PCR to determine whether the PDI gene is induced.

FIG. 6 is a schematic diagram of SDS-PAGE analysis and Western-blot analysis of fermentation supernatants from 5 liter bioreactors at different culture times.

FIG. 7 shows UPO gene expression levels and enzyme activities at different fermentation times.

FIG. 8 shows the result of non-specific peroxidase Ni purification.

Detailed Description

The invention is further described below with reference to the drawings and specific examples. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.

Example 1

Construction of recombinant plasmid pPICZ alpha A-UPO

The UPO gene was synthesized in Beijing qingke biotechnology Co.Ltd according to the nucleotide sequence of UPO. Primers were designed based on UPO nucleotide sequence to PCR amplify UPO up (SEQ ID NO. 3) and UPO dn (SEQ ID NO. 4). The UPO gene amplification product was then digested with Xho I and Not I and recovered. The empty pPICZ alpha A vector was digested with Xho I and Not I, and the vector fragment was recovered by electrophoresis. The digested pPICZ alpha A vector and the digested UPO expression sequence were mixed in a ratio of 2:1, and ligated with T4 ligase in a water bath at 16℃for 12 hours to form pPICZ alpha A-UPO (construction principle is shown in FIG. 1). After transformation of pPICZαA-UPO into DH 5. Alpha. The cells were plated on LB agar plates containing 25. Mu.g/mL bleomycin and incubated overnight at 37 ℃. Several clones on the plates were selected and inoculated into 5mL of LB liquid medium containing 25. Mu.g/mL bleomycin, and incubated overnight at 37 ℃. The obtained positive clone was sent to Beijing qingke biotechnology Co.Ltd for sequencing verification to be correct.

Example 2

Recombinant plasmid pPICZ alpha A UPO for converting Pichia pastoris GS115

The pPICZ alpha A-UPO plasmid obtained in example 1, which was verified to be correct, was linearized with SacI and transformed into Pichia pastoris GS115 in a conductometric manner, and the transformed cells were incubated in YPD liquid medium at 30℃for 2 hours and then plated onto YPD agar plates (1%yeast extract,2%peptone,2% glucose, 1M sorbitol, 2% agarose) containing 100, or 800. Mu.g/mL bleomycin. After 3 days of incubation at 30℃approximately 100, 4 single colonies were grown on YPDS agar plates of 100, or 800. Mu.g/mL bleomycin, respectively. 4 (designated as L1-4) and 3 (designated as H1-3) single colonies were picked on YPDS agar plates of 100 and 800. Mu.g/mL bleomycin, respectively.

Example 3

Fusion protein IGH for enhanced expression in multicopy hosts

Two pairs of RT-PCR primers are designed to identify the UPO gene copy number of each strain. Wherein, two primers of RTGAP up (SEQ ID NO. 5) and RTGAP dn (SEQ ID NO. 6) are used for amplifying the housekeeping genes of the strain. RTUPO up (SEQ ID NO. 7), RTUPO dn (SEQ ID NO. 8) two primers were used to amplify the UPO gene. RT-PCR amplification procedure was 95℃and 5minutes; then 95 ℃ for 30s;55 ℃ for 30s;72 ℃,30s; cycling was performed 35 times, at a final 72℃and 5minutes.

The copy number of the 8 clones UPO selected in example 2 was determined by RT-PCR and the results are shown in Table 1.

TABLE 1

Strain	UPO Gene copy number	Strain	UPO Gene copy number
				L1	0.8±0.2	H1	2.7±0.3
L2	0.7±0.2	H2	2.3±0.2
				L3	0.8±0.3	H3	3.8±0.4
L4	0.9±0.1

8 clones selected in example 2 were inoculated into a cell culture medium containing 30mL of BMGY medium (1%yeast extract,2%peptone,100mmol/L potassium phosphate, pH 6.0,4X 10) ^-5 % biotin, 1.34% amino acid-free yeast nitrogen source, 1% glycerol), at 200rpm/min at 30℃to OD=2-6, and changing to 30mL MMY medium (1%yeast extract,2%peptone,100mmol/L potassium phosphate, pH 6.0,4 ×10) ^-5 % biotin, 1.34% amino acid free yeast nitrogen source, 1% methanol). Then methanol accounting for 1% of the total culture medium volume is added every 24 hours, supernatant is centrifugally taken after 96 hours of induction, and the expression products are analyzed by Western-blot, and each strain is simultaneously expressed in triplicate, wherein the Western-blot results are shown in figure 2, and a is a monoclonal selected from bleomycin plates of 100 mug/mL (L1-4) and 800 mug/mL (H1-3); b is the corresponding strain, and the gray value quantitative software Image J is used for quantification and then the unit thallus expression quantity comparison is calculated (variance is from three parallels). It can be seen by combining Table 1 and FIG. 2 that hosts containing 2 to 3 copies of the UPO gene are expressed in significantly higher amounts than single copy hosts.

Example 4

Amplification of PDI Gene the primers were designed to amplify the PDI gene fragments PDI up (SEQ ID NO. 9) and PDI dn (SEQ ID NO. 10) using the yeast genome as template. An EcoRI cleavage site and a protecting base are introduced into both the upstream and downstream primers used for amplification. PCR reaction conditions: mu.L of yeast genome, 1. Mu.L of 10. Mu. Mol/L of each of the upstream and downstream primers, 2.5mmol/L of dNTP mix 4. Mu.L, 5 XBuffer (Mg) ²⁺ plus) 10. Mu.L, primeSTAR DNA polymerase 0.5. Mu.L, the remainder ddH ₂ And O is complemented. The PCR reaction condition is 98 ℃ and 5minutes; then 98 ℃ for 20s;55 ℃ for 30s;72 ℃,2minutes; cycling was performed 30 times, at 72℃again, for 10minutes. The PCR product expected to be 1573bp was obtained by 0.8% gel electrophoresis analysis.

Example 5

Construction of recombinant vector pPIC3.5K-PDI

The amplified product of PDI obtained in example 4 was recovered and purified by using a DNA fragment recovery kit. The purified PCR product is digested with EcoRI; the empty plasmid pPIC3.5K was digested likewise with EcoRI, and the linearized fragment was recovered by electrophoresis. The digested PDI fragment was mixed with vector pPIC3.5K at a ratio of 3:1, ligated with T4 ligase in a water bath at 16℃for 12h, and DH 5. Alpha. Competent cells were transformed. Transformed DH 5. Alpha. Cells were plated on LB agar plates containing ampicillin (50. Mu.g/mL) and incubated at 37℃for 16h. Several clones on the plates were selected and submitted to gene sequencing by Beijing qingke biotechnology Co., ltd, and the sequencing result determined that DH 5. Alpha. Strain (pPICC 3.5K-PDI) with correct PDI insertion direction and gene sequence was used for the subsequent experiments.

Example 6

Transformation of pPICC 3.5K-PDI GS115 expression Strain containing multiple copies of UPO Gene

The pPICC 3.5K-PDI plasmid obtained in example 5 was linearized with BspE I and transformed into the GS115 expression strain H1 containing multicopy UPO (see Table 1). The transformed cells were first plated on RDB agar plates (1 mol/L sorbitol, 1% glucose, 4X 10) ^-5 % biotin, 1.34% yeast nitrogen source without amino acid, 0.005% amino acid mixture (0.005% each of glutamic acid, methionine, lysine, leucine, isoleucine) and culturing at 28℃for 3-5 days. Positive clones on RDB were then plated on YPD plates containing 0.2 and 1.5mg/mL geneticin and transformants were picked on a further selection.

Example 7

Co-expression of PDI gene to increase UPO gene expression

Three transformants obtained in example 6 were selected for comparison of UPO gene expression, one strain selected from YPD plates of 0.2mg/mL geneticin and two strains selected from YPD plates of 1.5mg/mL geneticin. Pichia pastoris GS115 expression strain with single copy UPO gene and Pichia pastoris GS115 expression strain with multi-copy UPO gene (the copy number of UPO gene is 2.7 determined by RT-PCR method) are selected as the expression strainsAnd (3) controlling. Each strain was inoculated into a culture medium (1% yeastex, 2% peptone,100mmol/L potassium phosphate, pH 6.0,4X 10) containing 30mL of BMGY ^-5 % biotin, 1.34% amino acid-free yeast nitrogen source, 1% glycerol), at 200rpm/min, at 30℃until OD600 = 2-6, and 30mL BMMY medium (1%yeast extract,2%peptone,100mmol/L potassium phosphate, pH 6.0,4 ×10) ^-5 % biotin, 1.34% amino acid-free yeast nitrogen source, 1% methanol), 1% methanol/24 h thereafter, induction for 3d followed by centrifugation to collect the supernatant, western-blot analysis of the expression product. The results are shown in FIG. 4, wherein lane 1 in FIG. 4.A is a 1.5mg/mL geneticin YPD plate of the coexpression PDI strain, lane 2 is a 0.2mg/mL geneticin YPD plate of the coexpression PDI gene strain, lane 3 is a multi-copy UPO gene strain, lane 4 is a single-copy UPO gene strain, and each strain is expressed in triplicate. FIG. 4.B shows the quantitative result of Western-blot banding using software Image J, and shows that when PDI gene and UPO gene are co-expressed, the UPO gene expression level in the supernatant increases significantly.

Example 8

Identification of PDI Gene mRNA level in Co-expressed PDI Gene Strain

RT-PCR was used to detect mRNA level changes in the co-expression hosts, with Pichia pastoris GS115 expression strains containing multiple copies of the UPO gene as controls. The induced yeast cells were broken with lysozyme and then mRNA was extracted from the cells using the RNeasy Mini Kit. Reverse transcription of the extracted mRNA was performed using PrimeScript RTReagent Kit kit to cDNA for RT-PCR analysis. Primers used for analysis of the transcriptional level of PDI gene were RTPDI up (SEQ ID NO. 11) and RT PDI dn (SEQ ID NO. 12), respectively, and internal reference sequences were RTGAP up (SEQ ID NO. 5) and RTGAP dn (SEQ ID NO. 6). PCR reaction system: into a 20. Mu.L reaction system, 2. Mu.L of cDNA sample, 10. Mu. Mol/L of upstream and downstream primers each 0.8. Mu.L, 10. Mu. LSYBR Premix Ex Taq II, 0.4. Mu.L LROX Reference, 6. Mu.L ddH were added ₂ O. The PCR reaction condition is 95 ℃ for 30s; then 95 ℃ for 5s;55 ℃ for 30s;72 ℃,30s; the sample was cycled 30 times and the solubility curve analysis was used to determine that the specificity of the amplified product was good, and three wells were made per sample. RT-PCR results obtained with 2 ^-ΔΔCt Method meterThe result of calculation of mRNA level change of PDI is shown in FIG. 5, wherein the sample No.1 is a strain with multiple copies of UPO gene, the sample No.2 is a strain with coexpression of PDI gene selected on YPD plate with 1.5mg/mL geneticin, and the result shows that the mRNA amount of PDI gene in the strain with coexpression of PDI gene is improved by about 20 times, which indicates that the transcription level of PDI gene is obviously improved.

Example 9

The co-expression strain was cultured in an amplified manner using the strain H1 having the highest expression level as the starting strain, and inoculated into a 3mLYPD test tube, followed by shaking culture at 30℃and 220rpm/min for 20 hours. These cultures were then inoculated into 50ml lypd shake flasks at an inoculum size of 10.0% and incubated at 30 ℃ and 220rpm/min to od600=16-20. Thereafter, the seed culture was transferred to a 5 liter fermenter with an inoculum size of 10.0%. BSM medium is used in the whole fermentation process, and the medium consists of 85.0% of H ₃ PO ₄ (26.7mL/L)、CaSO ₄ ·2H ₂ O(0.93g/L)、K ₂ SO ₄ (18.2g/L)、MgSO ₄ ·2H ₂ O (14.9 g/L), KOH (4.13 g/L), glycerin (40.0 g/L) and PTM1 (4.0 mL/L). PTM1 is composed of CuSO ₄ ·5H ₂ O(6.0g/L)、KI(0.088g/L)、MnSO ₄ ·H ₂ O(3.0g/L)、H ₃ BO ₃ (0.02g/L)、CoCl ₂ ·6H ₂ O(0.5g/L)、ZnCl ₂ (20.0g/L)、FeSO ₄ ·7H ₂ O (65.0 g/L), biotin (0.2 g/L) and H ₂ SO ₄ (5 mL/L). In the glycerol incubation stage, a 50.0% glycerol solution was used and 12mL/L PTM1 was added; in the methanol induction phase, pure methanol containing 12mL/LPTM1 was used as an inducer. The fermentation conditions were as follows: the initial working volume was 3 liters, the stirring speed was set at 200rpm/min and the aeration was 1vvm. The temperature in the growth stage was maintained at 30℃and the pH was adjusted to 5.5 with ammonia. The dissolved oxygen level is maintained above 25.0% by the dissolved oxygen and velocity coupling. After glycerol depletion, the strain was starved for 30 minutes and the incubation temperature was reduced to 20 ℃. And starting methanol feeding to induce expression, wherein the flow rate is coupled with dissolved oxygen. When the dissolved oxygen is reduced to below 25.0%, the methanol feeding is stopped, and when the dissolved oxygen is increased to above 25.0%, the methanol flow is restored. Sampling every 12 hours, SDS-PAGE andwestern-blot analysis of the expression products and determination of UPO gene expression and enzyme activity during fermentation. The results are shown in fig. 6 and 7. FIG. 6 a is a schematic diagram of SDS-PAGE analysis, and b is a schematic diagram of Western-blot analysis; FIG. 7 shows UPO gene expression levels and enzyme activities at different fermentation times. As can be seen from FIG. 7, the concentration of the unspecific peroxidase protein in the 5 liter bioreactor can reach 333.3mg/L.

Example 10

Non-specific peroxidase Ni purification

The culture in the 5 liter bioreactor was centrifuged at 8000rpm/min for 10minutes to pellet the cells. The HIS-select nickel affinity gel column (Sigma-Aldrich) was washed three times with 10mM potassium dihydrogen phosphate buffer (pH 7.0). Then, three column volumes of supernatant were added. When the supernatant was completely drained, 10mM imidazole buffer was added again in twice the column volume. Then 1mL 200mM imidazole buffer was added and the eluate was collected. This procedure was repeated five times. Finally, 2mL of the obtained high-concentration protein eluent is added into a desalting column, and the high-concentration protein eluent is drained. Then 1mL of potassium dihydrogen phosphate buffer without imidazole was added and the eluate was collected. This procedure was repeated five times. The protein concentration was measured by the nanorange protein quantification kit, and the results of protein purification are shown in FIG. 8.

Example 11

Non-specific peroxidase enzyme activity detection

The peroxidation activity of the nonspecific peroxidase was determined by the oxidation of 2,2' -azobis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS). In a solution containing 100mM sodium phosphate/citrate buffer (pH 5.5), 0.3mM AMBTS and 1mM H ₂ O ₂ To the solution of (2) was added 1. Mu.L of a purified enzyme solution obtained by passing through a nickel column to a total volume of 200. Mu.L, and an enzymatic reaction was carried out at room temperature, and the change in absorbance at 418nm was recorded by using a microplate reader. The results of the enzyme activity assay are shown in FIG. 7. Wherein the unit enzyme activity is defined as the amount of enzyme required to oxidize 1. Mu. Mol of ABTS per minute.

Claims

1. A combined expression system for a non-specific peroxidase comprising a host cell and an expression vector transformed into said host cell, wherein said expression vector comprises a first expression vector inserted with a gene encoding a non-specific peroxidase protein UPO and a second expression vector inserted with a gene encoding a disulfide isomerase PDI, and said host cell is pichia pastoris GS115.

2. The combination expression system of non-specific peroxidase according to claim 1, wherein said gene encoding a non-specific peroxidase protein UPO is derived from Agrocybe cylindracea (Cyclocybe aegerita) TM-A1.

3. The combination expression system of non-specific peroxidase according to claim 1 or 2, wherein a nucleotide sequence of said gene encoding non-specific peroxidase protein UPO is shown in SEQ ID NO. 1.

4. The combination expression system of non-specific peroxidases according to claim 1, wherein the nucleotide sequence of the gene encoding disulfide isomerase protein PDI is shown in SEQ ID No. 2.

5. The combination expression system of a non-specific peroxidase according to claim 1, wherein a primary vector of said first expression vector is a pPIC series vector and a primary vector of said second expression vector is a pPIC series vector.

6. The combination expression system of a non-specific peroxidase according to claim 5, wherein a primary vector of said first expression vector is a vector pPICZ αA.

7. The combination expression system of a non-specific peroxidase according to claim 5 or 6, wherein a primary vector of said second expression vector is a vector ppic3.5k.

8. The combination expression system of non-specific peroxidases as claimed in claim 1, wherein the construction method comprises the steps of:

(1) Synthesizing a codon optimized gene encoding a non-specific peroxidase UPO;

9. The combination expression system of a non-specific peroxidase according to claim 8, wherein said gene encoding a non-specific peroxidase UPO and said gene encoding a disulfide isomerase PDI are overexpressed.

10. Use of a combination expression system of a non-specific peroxidase according to any one of claims 1 to 9 for the production of a non-specific peroxidase.