Glycerol phosphate oxidase and mutants, methods of making and uses thereof

Abstract

This application discloses a glycerol phosphate oxidase, its mutant, preparation method, and application. In this application, the glycerol phosphate oxidase mutant, obtained by mutating Enterococcus spp. glycerol phosphate oxidase, exhibits better stability than Enterococcus spp. glycerol phosphate oxidase or Streptococcus agalactiae spp. glycerol phosphate oxidase expressed in Escherichia coli through genetic engineering, after screening of bacterial strains and optimization of synonymous codon bias, yields high production, and the obtained product exhibits good activity and stability.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to glycerol phosphate oxidase and its mutants, preparation methods and applications. Background Technology

[0002] Triglycerides (TG), also known as neutral fats, are an important routine indicator of blood lipids in clinical practice. Measuring serum TG levels can reveal the body's TG metabolism, and can also be used for the clinical diagnosis of hypertriglyceridemia and metabolic syndrome, as well as to assess the risk of coronary heart disease. Currently, the most convenient method for measuring serum TG is enzymatic analysis. Glycerol phosphate oxidase, as one of the key enzymes in the coupled enzyme method for measuring triglyceride levels, is now widely used in the clinical diagnosis of heart disease and hyperlipidemia due to its unique high specificity and sensitivity.

[0003] Glycerol phosphate oxidase (GPO, EC1.1.3.21) is an enzyme that oxidizes 3-glycerol phosphate to dihydroxyacetone phosphate and H2O2 using O2. Based on the electron transfer pathway during the enzyme reaction, enzymes that can oxidize glycerol phosphate are classified into three categories: those via NAD+, those via O2, and those via O2-. 十 Enzymes that transfer electrons include those that transfer electrons via the cytochrome chain and those that are directly linked to O2. The first two types involve dehydrogenation during the reaction and are called glycerol phosphate dehydrogenases. The third type does not involve dehydrogenation and is called glycerol phosphate oxidase (GPO). Glycerol phosphate oxidase is the last enzyme in the reaction measured using a TG kit, requiring higher specificity, enzyme activity, and specific activity.

[0004] Glyceryl phosphate oxidase (GPO oxidase) has a wide range of sources, including Streptomyces, Enterococcus faecalis, and Lactobacillus, whose GPO oxidase genes have been identified. Currently, domestic research is relatively limited, with most studies focusing on direct extraction from primitive microorganisms. For example, Zhang Simin et al. cloned and enzymatically identified GPO oxidase from Mycoplasma; Bao Lingsheng et al. extracted GPO oxidase from Enterococcus faecalis; and Yu Caixia et al. screened and cultivated Enterococcus faecalis and optimized fermentation production conditions. Despite the considerable amount of research, the complex culture conditions and production processes of the relevant production strains result in low enzyme yields using existing methods, and most methods produce inclusion bodies, leading to high costs and hindering large-scale fermentation production. Furthermore, the resulting GPO oxidase exhibits poor stability, limiting its application. Therefore, there is a need in this field to develop low-cost methods for preparing highly stable GPO oxidase. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing recombinant glycerol phosphate oxidase.

[0006] Another object of the present invention is to provide a polynucleotide sequence encoding glycerol phosphate oxidase.

[0007] Another object of the present invention is to provide a vector adapted to a polynucleotide sequence encoding glycerol phosphate oxidase.

[0008] Another object of the present invention is to provide a kit containing a polynucleotide sequence encoding glycerol phosphate oxidase.

[0009] To address the aforementioned technical problems, in a first aspect, the present invention provides a glycerol phosphate oxidase mutant, wherein the glycerol phosphate oxidase mutant is selected from any of the following:

[0010] (i) It has the amino acid sequence shown in SEQ ID NO.4;

[0011] (ii) Having an amino acid sequence that is more than 95% homologous to, for example, SEQ ID NO.4.

[0012] In a second aspect, the present invention provides a polynucleotide encoding glycerol phosphate oxidase or a mutant thereof, said polynucleotide being codon-optimized and selected from any of the following:

[0013] (i) Polynucleotides having the sequence shown in SEQ ID NO.1-3;

[0014] (ii) Polynucleotides having greater than 95% homology to sequences shown in SEQ ID NO. 1-3; and

[0015] (iii) A polynucleotide having a sequence complementary to the polynucleotide sequence described in (i) or (ii).

[0016] In some preferred embodiments, the glycerol phosphate oxidase is Enterococcus glycerol phosphate oxidase or Streptococcus agalactiae glycerol phosphate oxidase.

[0017] In a third aspect, the present invention provides an expression vector comprising the polynucleotides provided in the second aspect of the present invention.

[0018] In some preferred embodiments, the expression vector includes a polynucleotide sequence expressing a His×6 tag; more preferably, the 3' end of the polynucleotide in the expression vector is linked to a polynucleotide sequence expressing a His×6 tag.

[0019] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

[0020] In a fourth aspect, the present invention provides a host cell comprising the expression vector provided in the third aspect of the present invention; or

[0021] The host cell genome contains polynucleotides as provided in the second aspect of the present invention.

[0022] In some preferred embodiments, the host cell is *Escherichia coli*.

[0023] In some preferred embodiments, the host cell is Escherichia coli Rosetta (DE3) strain.

[0024] The fifth aspect of the present invention provides a method for preparing glycerol phosphate oxidase, the method comprising the step of: transforming a host cell with a polynucleotide vector as described in the second aspect of the present invention;

[0025] The host cells are cultured to express the glycerol phosphate oxidase; and

[0026] The target protein is isolated to obtain the glycerol phosphate oxidase.

[0027] In some preferred embodiments, the host cell is obtained by transforming Escherichia coli with a plasmid containing the polynucleotide described in the first aspect of the invention.

[0028] In some preferred embodiments, the host cells are cultured in an oscillating environment.

[0029] In some preferred embodiments, the host cells are cultured at temperatures ranging from 16 to 39°C, such as 18°C ​​or 37°C.

[0030] In some preferred embodiments, the culture medium used to culture the host cells contains a kanamycin resistance gene.

[0031] In some preferred embodiments, IPTG is used to induce the expression of the target protein when culturing the host cells.

[0032] In some preferred embodiments, the host cells are cultured until the OD600 is between 0.6 and 0.8, and then induced with IPTG to express the target protein.

[0033] In some preferred embodiments, the step of isolating the target protein includes:

[0034] The supernatant of the lysed target protein is passed through a chromatography column for elution, and the eluent is collected.

[0035] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column, such as HisTrapTMFF.

[0036] A sixth aspect of the present invention provides a kit comprising: a polynucleotide as provided in a second aspect of the present invention; or

[0037] Such as the expression vector provided in the third aspect of the present invention; or

[0038] Host cells as described in the fourth aspect of the present invention; or

[0039] Or glycerol phosphate oxidase prepared according to the method described in the fifth aspect of the present invention; or

[0040] The glycerol phosphate oxidase mutant as described in the first aspect of the present invention.

[0041] Compared with the prior art, the present invention has at least the following advantages:

[0042] (1) The glycerol phosphate oxidase mutant provided in this invention is obtained by mutation of Enterococcus spp. glycerol phosphate oxidase and has better stability than Enterococcus spp. glycerol phosphate oxidase or Streptococcus agalactiae spp. glycerol phosphate oxidase.

[0043] (2) The glycerol phosphate oxidase Escherichia coli expression system provided in this invention has high yield, good activity and good stability of the obtained product after screening of strain sources and optimization of synonymous codon preference.

[0044] 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

[0045] 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.

[0046] Figure 1 This is a diagram showing the SDS-PAGE identification results of the target protein according to an embodiment of the present invention;

[0047] Figure 2 This is another SDS-PAGE identification result of the target protein according to an embodiment of the present invention;

[0048] Figure 3 This is an electrophoresis image of the target protein according to an embodiment of the present invention;

[0049] Figure 4 This is another electrophoresis image of the target protein according to an embodiment of the present invention;

[0050] Figure 5 This is a standard curve of OD changing with enzyme concentration according to an embodiment of the present invention. Detailed Implementation

[0051] Through extensive and in-depth research, the inventors have developed a method for preparing glycerol phosphate oxidase based on a prokaryotic expression system. This method, through screening of several bacterial strains and optimization using synonymous codon bias, yielded optimized codons suitable for the *E. coli* system, which, when introduced into *E. coli*, can express a large amount of soluble protein.

[0052] Furthermore, several mutants were obtained by mutating the enterococcal glycerol oxidase with a high soluble expression level. By optimizing the gene sequence encoding the mutants using synonymous codon preference, an optimized codon encoding the mutants was obtained. This codon can express glycerol oxidase in large quantities in the E. coli expression system. The resulting product maintains enzyme activity while improving the soluble expression yield and stability of the expressed glycerol oxidase.

[0053] Obtain the nucleic acid sequence related to the target gene / protein.

[0054] In this invention, the full-length nucleotide sequence or fragments of the target protein or its elements can typically be obtained using PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. 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.

[0055] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0056] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0057] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0058] In one embodiment of the present invention, the amino acid sequence of Enterococcus phosphoglycerate oxidase is obtained through the NCBI database, and analyzed to obtain the gene sequence information encoding the target protein.

[0059] In another embodiment of the present invention, the amino acid sequence of Streptococcus agalactiae glycerol phosphate oxidase is obtained from the NCBI database, and analyzed to obtain the gene sequence information encoding the target protein.

[0060] Target gene mutant

[0061] This invention also relates to glycerol phosphate oxidase mutants, which are obtained by substituting, adding or deleting any one or more amino acids in the original glycerol phosphate oxidase amino acid sequence. The resulting glycerol phosphate oxidase mutants do not change the function and activity of the original glycerol phosphate oxidase.

[0062] In one embodiment of the present invention, the amino acid sequence of Enterococcus phosphoglycerate oxidase is mutated to obtain a phosphoglycerate oxidase mutant. The obtained phosphoglycerate oxidase mutant maintains the same function and activity as the original phosphoglycerate oxidase, but improves its stability.

[0063] Synonymous codon preference optimization

[0064] This invention also relates to polynucleotide sequences optimized by synonymous codon preference. Synonymous codon preference optimization is performed on the obtained gene sequence, and the optimized target gene sequence can express the same amino acid sequence as the target protein (or a mutant of the target protein).

[0065] The present invention also relates to polynucleotides with greater than 95% homology to the sequences shown in SEQ ID NO:1-3; and polynucleotides complementary to the sequences shown in SEQ ID NO:1-3.

[0066] Vector of the target gene

[0067] This invention also relates to vectors comprising the polynucleotides of this invention. 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 one embodiment of this invention, pET-28a(+) is selected as the vector to obtain more efficient expression.

[0068] 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.

[0069] In addition to essential replication and selection elements, and elements assisting expression and translation, expression vectors may also include various tags, such as lysis-promoting tags. In one embodiment of the present invention, the vector further includes a polynucleotide sequence expressing a His×6 tag, preferably, the polynucleotide sequence expressing the His×6 tag is linked to the 3' end (C-terminus) of the target gene sequence.

[0070] In this invention, the "His×6 tag" refers to a fusion tag composed of six histidine residues, which can be purified in the presence of nonionic surfactants or under denaturing conditions. When inclusion bodies are present, after dissolving with a high concentration of denaturant, impurities are removed by metal chelate affinity chromatography, so that the refolding is not interfered with by other proteins and the purity is higher.

[0071] Transformation of host cells using a vector containing the target gene

[0072] This invention also relates to host cells genetically engineered using the vector or fusion protein coding sequence of this invention. A vector containing a codon-optimized target gene can be inserted, transfected, or otherwise transformed into a host cell by known methods to obtain a transformant containing the codon-optimized target gene of this invention and capable of expressing the target protein. In this invention, "host cell" refers to a cell incorporating exogenous polynucleotides and / or a vector. The host cell can be a eukaryotic or prokaryotic host cell, preferably a bacterium, and more preferably *Escherichia coli* Rosetta(DE3) strain.

[0073] Methods for preparing target proteins

[0074] This invention also relates to a method for preparing a truncated form of a target protein, which can be used to express or produce recombinant proteins using the polynucleotide sequence of this invention. Generally, the method involves the following steps:

[0075] (1) Transform or transduce suitable host cells using the polynucleotide (or variant) encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0076] (2) Host cells cultured in a suitable culture medium;

[0077] (3) Isolate and purify proteins from culture media or cells.

[0078] In step (1), the recombinant expression vector containing the polynucleotide is transformed or transduced into a suitable host cell by conventional techniques known to those skilled in the art. When the host is Escherichia coli, heat shock and electroconversion methods can be used.

[0079] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media, preferably SB, TB, or SOC media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature change or chemical induction), and the cells are cultured for a further period. In a preferred embodiment of this invention, to promote the expression of the target protein and increase the expression level of soluble proteins, host cells are cultured in TB media containing a kanamycin resistance gene.

[0080] The proteins described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods 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, as well as combinations of these methods. In one embodiment of the invention, affinity chromatography is used to molecularly target the protein.

[0081] In this invention, any exemplary or illustrative terminology (e.g., “”) used with respect to certain embodiments herein is merely for the purpose of better presenting the invention and does not limit the scope of the invention as otherwise claimed. No terminology herein should be construed as indicating an element not described in the claims that is indispensable to the implementation of this invention.

[0082] If the definition or use of a term in a cited reference is inconsistent with or inconsistent with the definition of a term described herein, the definition of the term described herein shall be used instead of the definition of the term in the cited reference.

[0083] The various terms used herein are as follows. If a term used in the claims is not defined below, the broadest definition of that term given by a person skilled in the art should be given, as reflected in the publication printed at the time of application or in the published patent.

[0084] As used herein, the term "isolated" refers to a nucleic acid or polypeptide isolated from at least one other component (e.g., a nucleic acid or polypeptide) present in its natural source. In one embodiment, the nucleic acid or polypeptide is found to be present only (if any) in a solvent, buffer, ion, or other component normally present in its solution. The terms "isolated" and "purified" do not include nucleic acids or polypeptides present in their natural source.

[0085] As used herein, the terms "polynucleotide" and "polynucleotide sequence" can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding.

[0086] This invention also relates to variants of the aforementioned polynucleotides that encode protein fragments, analogs, and derivatives having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the encoded polypeptide.

[0087] As used in this article, the term "codon optimization" refers to the 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 performing protein expression or production (including E. coli, yeast, mammalian blood cells, plant cells, insect cells, etc.).

[0088] As used herein, 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 in the arranged polynucleotide or polypeptide containing the same nucleotide or amino acid residues, and comparing this to the number of positions in the arranged polynucleotide or polypeptide containing different nucleotide or amino acid residues. 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., the 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., the insertion of one or two amino acids in the polypeptide or the deletion of amino acids). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residues 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 residues by the total number of nucleotide or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0089] As used herein, the terms “sequence complement” and “reverse sequence complement” are used interchangeably and refer to a sequence that is in the opposite direction to the original polynucleotide sequence and is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, then its reverse complementary sequence is GTTCAT.

[0090] As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to transcription, translation, post-translational modification, and secretion. Post-expression can be harvested, i.e., the host cell or the expressed product can be recovered.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] Example 1

[0096] (1) Synthesis of recombinant plasmids

[0097] The gene sequence of Enterococcus phosphoglycerate oxidase provided by NCBI was obtained. After codon preference optimization for Escherichia coli, optimized codon I (SEQ ID NO:1) was obtained. The optimized codon was ligated into the vector pet28a to synthesize recombinant plasmid A-1.

[0098] The gene sequence of glycerol phosphate oxidase from *Streptococcus agalactiae* provided by NCBI was obtained. After codon preference optimization for *Escherichia coli*, the optimized codon II (SEQ ID NO:2) was obtained. The optimized codon was then linked to the vector pet28a to synthesize recombinant plasmid A-2.

[0099] Several sets of mutations were performed on the amino acid sequence of glycerol phosphate oxidase derived from Enterococcus spp., and the codons of the mutated amino acid sequences were optimized to obtain several sets of optimized mutant codons. For example, the optimized codon III (SEQ ID NO:3, the corresponding mutant amino acid sequence is SEQ ID NO:4) was used to synthesize recombinant plasmid A-3 in the same manner.

[0100] Table 1

[0101]

[0102]

[0103]

[0104]

[0105] (2) Introduction of recombinant plasmids into host Escherichia coli

[0106] Take 1 μL of the expression plasmid A-1 prepared in step (1), add it to 30 μL of E. coli competent cells BL21(DE3) under ice bath conditions, incubate on ice for 30 min, incubate in water at 42℃ for 45 s, immediately place on ice for 2 min, add 400 μL of antibiotic-free SOC medium, and incubate at 37℃ and 230 rpm for 45 min with shaking. Take 100 μL of bacterial culture and spread it evenly on LB agar plates containing 100 μg / mL kanamycin resistance, and incubate overnight at 37℃.

[0107] Expression plasmids A-2 and A-3 were treated in the same way and introduced into E. coli for culture.

[0108] Example 2

[0109] In this embodiment, soluble glycerol phosphate oxidase was successfully expressed by culturing the monoclonal strain prepared in Example 1. The specific steps are as follows:

[0110] Single clones from Example (1) were aseptically inoculated into SB and SOC media containing 100 μg / mL kanamycin resistance, respectively, and cultured at 37°C with shaking at 220 rpm until the OD600 was between 0.6 and 0.8. Induction with IPTG was then performed, and the clones were incubated overnight at 37°C and 18°C ​​with shaking, respectively. Samples were then sonicated and analyzed by SDS-PAGE. The results are shown in [Figure number missing]. Figure 1 and Figure 2 .

[0111] Figure 1In the table, columns 1-4 show the identification results of the E. coli expression product with optimized codon I, which was expressed in the supernatant at both 18℃ and 37℃ (target band 70kDa). Columns 6-9 show the identification results of the E. coli expression product with optimized codon III, which was expressed in the supernatant at both 18℃ and 37℃.

[0112] Figure 2 To optimize the identification results of E. coli expression products with codon II, by Figure 1 It can be seen that TB medium at 18℃ showed expression in the supernatant.

[0113] Example 3

[0114] In this embodiment, the recombinant expression products of optimized codon I and optimized codon II from Example 2 were purified.

[0115] The specific steps are as follows:

[0116] Weigh 4g of recombinant enterococcal cells and 4g of recombinant streptococcal cells, respectively, and resuspend them in 20mL Lysis Buffer on ice. Cell disruption was performed by sonication: 10# probe, 10% power, 5.5s sonication, 9.9s pause, for 30min. Centrifuge at 20000rpm, 4℃ for 30min, collect the supernatant, filter through a 0.22μm membrane, and purify with 1mL Ni-NTA. The flow rate was 0.5mL / min. After loading, rinse with 20mL Lysis Buffer to remove UV light and adjust conductivity to baseline. The elution program was as follows: Step 1: 0% B, 8CV, 1.5mL / min; Step 2: 0-60% B, 20CV, 1.5mL / min; Step 3: 100% B, 15CV, 1.5mL / min. Refer to Table 2 for mobile phase preparation.

[0117] Table 2

[0118] Tris 50mM 50mM 50mM 50mM NaCl 50mM 50mM 1M 300mM Glycerol - - - - Imidazole - 500mM - - pH 8.0 8.0 8.0 8.0

[0119] The codon I recombination purification product was optimized, and the electrophoresis results were as follows: Figure 3 As shown.

[0120] according to Figure 3 The target protein bound well to the column and eluted at a concentration of 150 mM imidazole. Based on the SDS results, 10.5 mL of eluent 2A11-2B7 was used for dialysis, yielding 10.5 mL of sample. The concentration was determined using BCA assay. 2 =0.996, sample concentration was 2.885 mg / mL, yield was 30.29 mg, and bacterial count was 7.57 mg / g.

[0121] The same method was used to process the optimized codon II recombination purified expression product, and the electrophoresis results were as follows: Figure 4 As shown.

[0122] according to Figure 4 The target protein bound well to the column and eluted at a concentration of 200 mM imidazole. Based on the SDS results, 10 mL of eluent (14-26) was used for dialysis, yielding 10.5 mL of sample. The concentration was determined using the BCA method. The dialyzed sample was diluted 5-fold, and the concentration was measured using the BCA method. 2 =0.996, sample concentration was 2.68 mg / mL, yield was 26.8 mg, and bacterial count was 6.7 mg / g.

[0123] The experimental results above show that the soluble expression level of Enterococcus phosphoglycerate oxidase in Escherichia coli is higher than that of Streptococcus agalactiae.

[0124] Example 4

[0125] In this embodiment, expression plasmids A-1 and A-3 were introduced into *E. coli*, the bacterial cells were cultured and collected, and the obtained bacterial cells were purified according to the method described in Example 3, followed by activity assay. The specific steps for the activity assay are as follows:

[0126] (1) Solution preparation

[0127] 0.2M DL-α-glycerophosphate: Take 6.25g DL-α-glycerophosphate, 125μL Triton-100, and 1M Tris-HCl pH 8.0-6.25mL, and bring the volume to 100mL with deionized water.

[0128] 0.1% 4-Aminoantipyrine: Weigh 0.05g of 4-aminoantipyrine powder and dissolve it in 50mL of deionized water. Store in the dark.

[0129] 0.1% Phenol: Weigh 0.05g of phenol powder and dissolve it in 50mL of deionized water. Store in the dark.

[0130] Enzyme dilution solution: 2 mL of 1M Tris-HCl, 5 mL of 4% BSA, and deionized water to a final volume of 100 mL.

[0131] Working solution (10 mL): Prepare according to Table 3 below.

[0132] Table 3

[0133] 0.2M DL-α-glycerophosphate 5mL 0.1% 4-Aminoantipyrine 1mL 0.1% phenol 2mL Enzyme dilution 2mL peroxidase 50U

[0134] (2) Solution preparation

[0135] Preparation of positive control enzyme (glycerol phosphate oxidase control): Dissolve 500U of positive control enzyme in 0.5mL of PBS pH 7.4 buffer (containing 50% glycerol) to prepare an enzyme solution of 1U / μL. Then, further dilute the solution stepwise according to the gradient. The diluent is PBS pH 7.4 buffer.

[0136] Absorbance measurement method: Preheat the microplate reader for 30 min, add 100 μL of working solution, and measure the absorbance value A1 at 500 nm. Then add 1 μL of enzyme solution of each concentration, react for 5 min, and measure the absorbance value A2 at 500 nm. Calculate the OD difference between the sample and the blank, A2-A1, and plot the glycerol phosphate oxidase standard curve as follows. Figure 5 The sample concentration was calculated.

[0137] The absorbance of the recombinant purified expression products from Enterococcus (optimized codon I and optimized codon III) was measured using the method described above, and the results are shown in Table 4 below.

[0138] Table 4

[0139]

[0140] The concentration of the glycerol phosphate oxidase stock solution purified and expressed by recombinant codon I from Enterococcus was 2.885 mg / mL, and the average activity was 0.25 U / μL. Therefore, the specific activity was (0.25*1000) / 2.885=86.66 U / mg.

[0141] The concentration of the glycerol phosphate oxidase stock solution purified and expressed by recombinant codon III optimized from Enterococcus was 2.68 mg / ml, with an average activity of 0.3802 U / μL and a specific activity of (0.3802*1000) / 2.68=141.86 U / mg. The specific activity of the enzyme after mutation was significantly improved compared with that of wild type.

[0142] Example 5

[0143] Stability determination procedure: (1) Dilute the sample to be tested to 400 μL with enzyme stock solution (0.5 mg / ml); (2) Add to the sample plate and push into the differential scanning calorimeter (DSC) for 3 water scans, 1 buffer scan, and 1 sample scan; (3) Detect at a temperature of 20-100℃ and a scanning rate of 100℃ / h to obtain the Tm value of the sample.

[0144] Table 5

[0145] Optimize codon I recombinase samples 49.83 Optimize codon II recombinase samples 46.65 Optimize codon III mutant enzyme samples 53.51 Commercially available glycerol phosphate oxidase 52.13

[0146] The test results are shown in Table 5. The higher the Tm value, the better the thermal stability of the enzyme. The Tm of the mutant enzyme sample is about 3.5℃ higher than that of the wild type, and the value is slightly higher than that of commercially available enzymes, indicating that the thermal stability of the enzyme is slightly improved after mutation.

[0147] 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. A mutant of a glycerol phosphate oxidase, characterized in that, The amino acid sequence of the glycerol phosphate oxidase mutant is shown in SEQ ID NO.

4.

2. An isolated polynucleotide encoding a glycerol phosphate oxidase mutant, characterized in that, The polynucleotide is codon-optimized, and the sequence of the polynucleotide is shown in SEQ ID NO.

3.

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

4. The expression vector according to claim 3, characterized in that, The expression vector is pET-28a.

5. A host cell, characterized in that, The host cell comprises the expression vector as described in any one of claims 3 or 4; or The host cell genome contains the polynucleotides as described in claim 2.

6. A method for preparing a glycerol phosphate oxidase mutant, characterized in that, The method includes the following steps: Transform host cells using a vector containing the polynucleotide as described in claim 2; The host cells were cultured to express the target protein; and The target protein is isolated to obtain the glycerol phosphate oxidase mutant.

7. The method according to claim 6, characterized in that, When culturing the host cells, the host cells are cultured at a temperature of 16 to 39°C; And / or, when culturing the host cells, culture them until the OD600 is between 0.6 and 0.8, and then induce expression of the target protein using IPTG.

8. The method according to claim 6, characterized in that, The step of separating the target protein includes: The supernatant of the lysed target protein is passed through a chromatography column for elution, and the eluent is collected.

9. A reagent kit, characterized in that, The kit comprises: the glycerol phosphate oxidase mutant as described in claim 1, or The polynucleotide as described in claim 2; or The expression vector as described in any one of claims 3 or 4; or The host cell as described in claim 5.

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