CN116042570A - Recombinant alkaline phosphatase mutant and preparation method thereof - Google Patents

Abstract

The application discloses a recombinant alkaline phosphatase mutant and a preparation method thereof. In this application, the alkaline phosphatase derived from Shewanellasp. is used as the transformation object for transformation, and the amino acid sequence is subjected to site-directed mutation, which improves the heat sensitivity of alkaline phosphatase and makes it suitable for the detection of flight mass spectrometry. After the reaction, it can pass Heating for a short time inactivates the enzyme; the alkaline phosphatase mutant provided by the application is easy to express in a prokaryotic expression system, and the cost is reduced and it is suitable for mass production.

Description

Recombinant alkaline phosphatase mutant and its preparation method

technical field

The invention relates to the field of gene detection, in particular to a recombinant alkaline phosphatase mutant and a preparation method thereof.

Background technique

Alkaline phosphatase (Alkaline phosphatase, AP, E 3.1.3.1) is a kind of non-specific phosphate monoesterase, which belongs to homodimeric metalloprotease; it can catalyze the hydrolysis of phosphate monoester to generate inorganic phosphate and corresponding alcohols and phenols Classes or sugars, in the presence of high-concentration phosphate group acceptors, AP catalyzes the transfer reaction of phosphate groups; alkaline phosphatase exists widely in various organisms in nature, and AP enzymes from different sources have larger molecular weights and sequences Different, but different enzyme active sites are highly conserved. Each monomer of AP enzyme has an active center, which consists of aspartic acid-serine-alanine triplet, arginine, water molecule, metal ion and its Ligand amino acids constitute the active center region of the AP enzyme. Among them, Escherichia coli AP enzyme has been studied more thoroughly. Because of its clear catalytic mechanism and wide range of substrates, it is widely used as a signal enzyme in the fields of medicine, immunology and analytical biotechnology.

In the human body, AP enzyme is an important hydrolase in the phosphate metabolic pathway, which can catalyze the hydrolysis and dephosphorylation of various phosphates in proteins, nucleic acids and small molecules; therefore, in clinical medicine, it is mostly used to detect the concentration of AP in serum Vitality is an important means of diagnosis and detection of diseases. For example, the detection of serum bone AP enzyme can be used for early diagnosis of bone metabolic diseases, detection of treatment effect and prognosis of the disease; in immunology research, enzyme-linked immunoassay (ELISA) uses AP enzyme The labeled antibody reacts, that is, the detected substance mainly interacts with the substrate that can develop color after alkaline phosphatase reacts with the chromogenic reagent or dephosphorylate, which has better stability and sensitivity; at the same time, alkaline Phosphatase can catalyze the removal of the 5' terminal phosphate group of DNA molecules, thereby preventing self-ligation of the carrier, and replacing isotope-labeled nucleotide probes for molecular hybridization, etc.

Among the common alkaline phosphatase products in the market, bovine intestinal alkaline phosphatase (MAP) has the highest activity, about 20-40 times that of Escherichia coli alkaline phosphatase (EAP), but MAP has poor thermal stability and requires Mg2+ Activation, while the cost of expression in Escherichia coli is low and the thermostability is good, but the activity of the product is low. Therefore, how to obtain high-activity EAP enzymes while maintaining good thermal stability is the current mainstream transformation direction. In the prior art, although some researchers have improved the enzyme activity through mutation modification, the modified bacteria are not suitable for large-scale production. After the modified EAP enzyme engineering bacteria are cultured in enlarged volume, the expression of the target protein is greatly reduced. The production application of AP enzyme is restricted. Therefore, how to develop an alkaline phosphatase with efficient expression, high activity and high thermostability is an urgent problem to be solved at present.

Contents of the invention

The purpose of the present invention is to provide a thermosensitive alkaline phosphatase mutant.

Another object of the present invention is to provide a nucleic acid molecule.

Another object of the present invention is to provide a vector adapted to the polynucleotide sequence encoding alkaline phosphatase and its mutants.

Another object of the present invention is to provide a method for preparing alkaline phosphatase and its mutants.

Another object of the present invention is to provide a kit containing polynucleotide sequences encoding alkaline phosphatase and its mutants.

In order to solve the above technical problems, the first aspect of the present invention provides an alkaline phosphatase mutant, said alkaline phosphatase mutant is selected from one or more positions of the following group in the wild-type alkaline phosphatase sequence Mutations: W 348, Q349, V365, S 369 and R 372; wherein, the numbering of amino acid residues adopts the numbering shown in SEQ ID NO.7.

In some preferred schemes, the amino acid sequence of the wild-type alkaline phosphatase is shown in SEQ ID NO.:7.

In some preferred schemes, the number of mutation sites is 1 to 5, such as 1, 2, 3, 4 or 5.

In some preferred schemes, the amino acid sequence of the alkaline phosphatase mutant has at least 80% homology compared with SEQ ID NO.7; more preferably, has at least 90% homology; most preferably Preferably, at least 95% homology; eg, at least 96%, 97%, 98%, 99% homology.

In some preferred schemes, the alkaline phosphatase mutant is mutated on the basis of the wild-type alkaline phosphatase shown in SEQ ID NO.: 7, and the alkaline phosphatase mutant comprises Mutation sites: W 348A, Q349A, V 365A, S 369A and R 372A.

In some preferred schemes, the alkaline phosphatase mutant is selected from the group:

In some preferred schemes, the amino acid sequence of the alkaline phosphatase is selected from the following group:

(i) amino acid sequence as shown in SEQ ID NO.1-6; With

(ii) a polynucleotide with an amino acid sequence homology greater than 95% as shown in SEQ ID NO.1-6.

The second aspect of the present invention provides a polynucleotide molecule encoding the alkaline phosphatase mutant described in the first aspect of the present invention.

The third aspect of the present invention provides a vector, characterized in that the vector contains the nucleic acid molecule described in the second aspect of the present invention.

The fourth aspect of the present invention provides a host cell, characterized in that the host cell contains the vector of the third aspect of the present invention or the nucleic acid molecule of the second aspect of the present invention is chromosomally integrated.

In some preferred solutions, the host cells are prokaryotic cells or eukaryotic cells.

In some preferred embodiments, the host cell is Escherichia coli (Escherichia coli).

In some preferred schemes, the host cell is Escherichia coli BL21 (DE3) strain.

In some preferred embodiments, the eukaryotic cells are yeast cells.

The fifth aspect of the present invention provides a method for preparing the alkaline phosphatase mutant described in the first aspect of the present invention, characterized in that it comprises the steps of:

(i) under suitable conditions, cultivate the host cell described in the fourth aspect of the present invention, thereby expressing the alkaline phosphatase mutant; and

(ii) isolating the alkaline phosphatase mutant.

In some preferred schemes, the host cells are cultivated with SB, TB, LB, SOC medium, more preferably with TB and LB medium, most preferably with TB medium. the host cells described above.

In some preferred embodiments, the host cells are cultured at a temperature of 16 to 19°C or 35 to 39°C.

The sixth aspect of the present invention provides a kit, characterized in that the kit comprises the alkaline phosphatase mutant described in the first aspect of the present invention.

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

(1) The present invention transforms the alkaline phosphatase derived from Shewanella sp. as the transformation object, carries out site-directed mutation to the amino acid sequence, improves the thermosensitivity of alkaline phosphatase, makes it applicable to the detection of flight mass spectrometry, after the reaction is completed The enzyme can be inactivated by heating for a short time;

(2) The alkaline phosphatase mutant provided by the present invention is easy to express in a prokaryotic expression system, and is suitable for mass production with reduced cost.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

One or more embodiments are exemplified by pictures in the accompanying drawings, and these exemplifications are not intended to limit the embodiments.

Fig. 1 is according to the three-dimensional structure schematic diagram of Shewanella sp source alkaline phosphatase in the embodiment of the present invention;

Fig. 2 is a schematic diagram of the structure comparison of alkaline phosphatase derived from Shewanella sp and E.coli according to the embodiment of the present invention;

Fig. 3 is according to the alkaline phosphatase structural comparison partial enlarged view of Shewanella sp source in the embodiment of the present invention;

Fig. 4 is a graph showing the results of small-scale expression and identification of alkaline phosphatase in Escherichia coli according to an embodiment of the present invention;

Fig. 5 is according to the electrophoresis diagram of nickel column purification of alkaline phosphatase in the embodiment of the present invention;

Fig. 6 is according to the alkaline phosphatase nickel column QP column purification electrophoresis figure in the embodiment of the present invention;

Fig. 7 is according to the alkaline phosphatase/mutant enzyme activity determination standard curve figure in the embodiment of the present invention;

Fig. 8 is a thermosensitivity graph of alkaline phosphatase/mutant in an example according to the present invention.

Detailed ways

In the prior art, alkaline phosphatase with good heat resistance (inactivated by treatment at 65° C. for 5 minutes) is mainly derived from bovine small intestine and shrimp. The alkaline phosphatase derived from bovine small intestine is mostly extracted and purified from natural products, the alkaline phosphatase derived from shrimp is mostly expressed by yeast, and the prokaryotic source of Escherichia coli (E.coli) and marine efficient phosphorus removal bacteria (Shewanella sp) The expression activity is high, but the heat resistance is poor, and heat inactivation at 65°C is not ideal. Through extensive and in-depth research, the present inventor unexpectedly found that the mutant of alkaline phosphatase described in the present invention has good thermosensitivity while retaining good enzyme activity.

alkaline phosphatase mutant

In the present invention, the target protein is alkaline phosphatase derived from Shewanella sp (the amino acid sequence is shown in SEQ ID NO.7), and the mutant is obtained by mutation of alkaline phosphatase through preset mutation methods, and the preset mutation methods include One or several sites selected from the following mutations: W 348, Q 349, V 365, S 369 and R 372. Preferably, the amino acid selected from the mutation site is mutated to alanine, and the preset mutation methods include any one or a combination of several: W 348A, Q 349A, V365A, S 369A and R 372A. When at least two, more preferably at least three, more preferably at least four, and more preferably all of the amino acids in these mutation sites are mutated, and are mutated into alanine (A).

In some embodiments of the present invention, the amino acid sequence of the alkaline phosphatase mutant is selected from any of the following,

(i) amino acid sequence as shown in SEQ ID NO.1-6; With

(ii) a polynucleotide with an amino acid sequence homology greater than 95% as shown in SEQ ID NO.1-6.

The target protein in the present invention can be prepared by conventional methods in the art, such as site-directed mutagenesis. Replace one or several bases in the target nucleic acid fragment or plasmid with other bases by methods such as polymerase chain reaction (PCR), and transform the plasmid containing the mutated nucleic acid fragment into Escherichia coli to obtain transformants, culture Transformants yield mutants. A commercially available site-directed mutagenesis kit was used for site-directed mutagenesis to obtain mutants.

Nucleic acid sequence encoding target protein mutant

In the present invention, the full-length nucleotide sequence or its fragments of the target protein mutant or its elements can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as Template, amplified to obtain related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order. Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Primers for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

Synonymous codon preference optimization

To overcome the potential problem of reduced yield when expressing heterologous proteins in host cells, the present invention relates to synonymous codon bias optimized polynucleotide sequences. Optimize the synonymous codon preference of the obtained target gene sequence, adjust the synonymous codon of the gene according to the codon preference of the host, thereby eliminating rare codons and improving the expression efficiency of heterologous genes. The target gene sequence optimized by synonymous codon bias can express the same amino acid sequence as the target protein. The embodiment of the present invention relates to the optimized codon sequence obtained by codon optimization of the gene sequence encoding alkaline phosphatase mutant, as shown in SEQ ID NO: 8-13, and the present invention also relates to SEQ ID NO: The homology of the sequence shown in 8-13 is greater than 80%, preferably greater than 85%, more preferably greater than 90%, more preferably greater than 91%, more preferably greater than 95% polynucleotide; and SEQ ID NO:8-13 Polynucleotides complementary to the indicated sequences.

Carrier of target gene

The present invention also relates to vectors comprising the polynucleotides of the present invention. "Vector" in the present invention means a linear or circular DNA molecule comprising a segment encoding a protein of interest operably linked to other segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, vectors, and the like. A vector segment may be derived from a host organism, another organism, plasmid or viral DNA, or may be synthetic. The vector can be any expression vector that is synthetic or convenient for recombinant DNA manipulation, and the choice of the vector usually depends on the host cell into which the vector is to be introduced. Thus, a vector may be an autonomously replicating vector, ie a vector, which exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome of the host cell and replicates with the chromosome into which it has integrated. In one embodiment, the vector of the invention is an expression vector. In one embodiment of the present invention, pET-28a(+) is selected as the vector to obtain higher expression efficiency.

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 appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Exemplarily, the DNA endonuclease is used to cut the carrier DNA molecule into a linear molecule that can be connected with the foreign gene, and then the codon-optimized target gene fragment is connected to the carrier, and the sticky end connection with a single enzyme cutting site can be selected , Directional cloning of double-digested fragments, sticky-end ligation of different restriction enzyme sites, blunt-end ligation, artificial adapter ligation or ligation with oligonucleotide ends to achieve the insertion of exogenous DNA fragments.

Transform host cells with the vector containing the gene of interest

The present invention also relates to host cells produced by genetic engineering using the vector or fusion protein coding sequence of the present invention. The vector containing the codon-optimized gene of interest can be inserted, transfected, or otherwise transformed into host cells by known methods, so as to obtain a transformant containing the codon-optimized gene of interest of the present invention and capable of expressing the protein of interest . A "host cell" in the present invention is a cell into which exogenous polynucleotides and/or vectors have been introduced. The host cell can be a eukaryotic host cell or a prokaryotic host cell, and the host cell is preferably a bacterium, and preferably Escherichia coli, more preferably Escherichia coli Rosetta (DE3) strain.

Method for preparing target protein

The present invention also relates to a method for preparing the target protein, and the polynucleotide sequence of the present invention can be used to express or produce the recombinant protein. Generally speaking, there are the following steps:

(1) Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2) host cells cultured in a suitable medium;

(3) Separation and purification of protein from culture medium or cells.

Wherein, step (1) transforming or transducing suitable host cells with the recombinant expression vector containing the polynucleotide can be carried out by conventional techniques well known to those skilled in the art. When the host is Escherichia coli, heat shock and electroporation can be selected. law etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. According to the host cells used, the medium used in the culture can be selected from various conventional mediums, preferably SB, TB, LB or SOC medium. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time. In order to promote the expression of the target protein and increase the expression level of the soluble protein, in a preferred embodiment of the present invention, host cells cultured in TB or LB medium are used, and the medium used contains a kanamycin resistance gene.

In order to further promote the soluble expression of the target protein, in a preferred embodiment of the present invention, after culturing the host cells until the OD ₆₀₀ is between 0.6-0.8, IPTG is used to induce, and at 17 to 19 ° C or 35 to 39 Cultivation was continued for about 8 to 12 hours at °C.

The protein in the above method may be expressed inside the cell, or on the cell membrane, or secreted outside the cell. Proteins can be isolated and purified by various separation methods by taking advantage of their physical, chemical and other properties, if desired. Therefore, in the present invention, after the target protein is successfully cultured, it also involves the steps of isolating and purifying it, for example, in step (3), separating and purifying the protein from the culture medium to obtain a high-purity target protein. Although the method of purifying the target protein can be a conventional means well known to those skilled in the art, including but not limited to: conventional refolding treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic destruction, supertreatment, ultracentrifugation , Molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

The eluted and purified target protein product is dialyzed, and the dialyzed sample is collected. Dialyzed samples can be measured by BCA method concentration, to calculate the yield.

In this disclosure, use of any exemplary or exemplary language (eg, "") provided with respect to certain embodiments herein is for the purpose of better presenting the invention only and does not limit the scope of the invention otherwise claimed. scope. Nothing in the text should be construed as indicating any non-recited element in the claims indispensable to the practice of the invention.

If the definition or use of a term in a cited document is inconsistent or inconsistent with the definition of the term described herein, the definition of the term described herein applies instead of the definition of the term in the cited document.

Various terms used in this article are as follows. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the art have given that term as reflected in printed publications or issued patents at the time of filing.

As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component (eg, nucleic acid or polypeptide) of the nucleic acid or polypeptide in its natural source. In one embodiment, a nucleic acid or polypeptide is found only in the presence, if any, of solvents, buffers, ions or other components normally present in solution thereof. The terms "isolated" and "purified" do not include nucleic acids or polypeptides as they exist in their natural source.

As used herein, the terms "polynucleotide" and "polynucleotide sequence" may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.

The present invention also relates to variants of the above polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide.

As used herein, the term "codon optimization" refers to avoiding codon usage differences based on the actual expression or production of proteins (including Escherichia coli, yeast, mammalian blood cells, plant cells, insect cells, etc.) A way to improve the efficiency of gene synthesis by using low-utilization or rare codons.

As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (ie identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following methods. Align the nucleotide or amino acid sequences of polynucleotides or polypeptides, score the number of positions in the aligned polynucleotides or polypeptides that contain the same nucleotide or amino acid residue, and compare this with the aligned polynucleotides or polypeptides Compare the number of positions containing different nucleotide or amino acid residues in . Polynucleotides may differ at a position, for example, by the inclusion of different nucleotides (i.e., substitutions or variations) or deletions of nucleotides (i.e., insertion or deletion of one or two nucleotides in a polynucleotide ). Polypeptides may differ at one position, for example, by the inclusion of amino acids (ie, substitutions or variations) or deletions of amino acids (ie, insertions or deletions of amino acids in one or both polypeptides). 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, percent identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

As used herein, the terms "sequence complementarity" and "reverse sequence complementarity" are used interchangeably to refer to a sequence that is oriented in the opposite direction to an original polynucleotide sequence and that is complementary to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAT.

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. Following expression, it can be harvested, ie, the host cells or the expression product are recovered.

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions or the conditions suggested by the manufacturer are usually followed. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercially available channels unless otherwise specified.

Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the application belongs. Exemplary implementation of the application.

Example 1

In this example, the modification region and modification strategy of alkaline phosphatase were determined through three-dimensional structure comparison, and several modified alkaline phosphatase mutants were obtained. The specific steps are as follows:

(1) Three-dimensional structural comparison to formulate renovation strategies

Alkaline phosphatase derived from Shewanella sp was selected as the object of transformation for three-dimensional structure modeling, and Alphafold2 was used to establish the three-dimensional structure of Shewanella sp (Figure 1). The structures of alkaline phosphatases derived from Shewanella sp and E.coli were compared, and the comparison results are shown in Figure 2.

According to Figure 2, the following conclusions can be drawn:

First, R166 and D153 are active site amino acids. As shown in Figure 2, the orange is the SAP derived from Shewanella sp, the light cyan is the SAP derived from E.coli, and the green is the substrate, including Zn, Mg and PO4 molecules. Structural comparison found that the active sites of alkaline phosphatase derived from Shewanella sp and E.coli are highly conserved, and the two amino acids in red are R166 and D153, which bind to the substrate, and these two amino acids were preliminarily identified is an important active site amino acid.

Second, the four helices of amino acids 330-390 are key areas for transformation. As shown in Figure 3, the difference between SAP derived from Shewanella sp and SAP derived from E.coli is mainly at the four helices of amino acids 330-390 (Figure 3). Multiple amino acids in the two helices participate in the formation of intramolecular interactions, and these sites are far away from the active center. The mutants may weaken or destroy the formation of intramolecular helices, thereby achieving the purpose of reducing thermal stability. Amino acid residues at positions 330-390 were mutated to construct mutants, and it was found that most of the mutants had very low expression activity, and further testing was needed to determine the mutation site.

(2) Test to determine the mutation site

Some of the mutants obtained after the test are shown in Table 1 below:

Table 1

amino acid site amino acid name mutagenic amino acid 348 Trp(W) Ala(A) 349 Gln(Q) Ala(A) 365 Val(V) Ala(A) 369 Ser(S) Ala(A) 372 Arg(R) Ala(A)

The test results show that among the mutation sites listed in Table 1, Trp348 and Arg372 are extremely important, and therefore, can be used as priority modification sites.

(3) Determine the sequence of the alkaline phosphatase mutant

As shown in Table 2, sequences 1-5 are sequentially Trp 348, Gln 349, Val 365, Ser 369, Arg 372 single-point mutants, and sequence 6 is a multi-point mutant (Trp348/Gln 349/Val365/Ser 369 /Arg372).

Table 2

serial number name SEQ ID NO.1 alkaline phosphatase mutant A-1 SEQ ID NO.2 alkaline phosphatase mutant A-2 SEQ ID NO.3 alkaline phosphatase mutant A-3 SEQ ID NO.4 alkaline phosphatase mutant A-4 SEQ ID NO.5 alkaline phosphatase mutant A-5 SEQ ID NO.6 alkaline phosphatase mutant B-1 SEQ ID NO.7 alkaline phosphatase before mutation

SEQ ID NO.1:

MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSP ELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQAQAQADLARGLGFELNADEVTQLSTARMQGLETMTEAIRK IIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

SEQ ID NO.2:

MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSP ELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWAADLARGLGFELNADEVTQLSTARMQGLETMTEAIRKII DKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

SEQ ID NO.3:

MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSP ELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWQADLARGLGFELNADEATQLSTARMQGLETMTEAIRKII DKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

SEQ ID NO.4:

MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSP ELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWQADLARGLGFELNADEVTQLATARMQGLETMTEAIRK IIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

SEQ ID NO.5:

MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSP ELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWQADLARGLGFELNADEVTQLSTAAMQGLETMTEAIRK IIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

SEQ ID NO.6:

MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSP ELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQAAADLARGLGFELNADEATQLATAAMQGLETMTEAIRKIID KRTGTGWTTSGHTGTDVQVFAAGPAAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

SEQ ID NO.7:

MSVTKTSLLLLTIGLVFSASSKAAPELENGPMKPPSKPKNIVIMVGDGMGPSYTSAYRYFKDNPDTEEVEQTVFDRLLVGMASTYPASVSGYVTDSAAAATALATGVKSYNGAISVDTQKQHLPTMLEKAKALGLSTGVAVTSQINHATPAAFLAHNESRKNYDALALSYLDTNADVLLGGGQKYFSP ELLEKFTAKGYQHISRFEDLATITQPKVIGLFAQVQLPWALDEKNANRLSTMTQKALDLLSQNEQGFVLLVEGSLIDWAGHSNDIANTGEMDEFANALEVVEQFVRQHPDTLMVATADHNTGGLSIGAGGDYRWNPEILRNMSASTDTLALAALGGDQWQADLARGLGFELNADEVTQLSTARMQGLETMTEAIRK IIDKRTGTGWTTSGHTGTDVQVFAAGPAAELFNGHQDNTDIANKIFTLLPKPKKAKTE

Example 2

In this example, according to the alkaline phosphatase mutant sequence obtained in Example 1 and the unmutated Shewanella sp alkaline phosphatase sequence, an adapted prokaryotic expression method was developed, and several alkaline phosphatases expressed by Escherichia coli were obtained mutants and alkaline phosphatase. The specific steps are as follows:

(1) Construction of alkaline phosphatase mutant plasmid

According to the amino acid sequence of Shewanella sp alkaline phosphatase, the signal peptide aa1-23 at the N-terminal was removed to obtain the sequence shown in SEQ ID NO.7, and the corresponding gene sequence was obtained by analyzing SEQ ID NO.7, and it was synonymous with Escherichia coli The codon preference was optimized, and the optimized codon I was obtained. The optimized codon I was connected to the vector pET-28a(+), and the recombinant plasmid C was entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize.

According to the sequence of several Shewanella sp alkaline phosphatase mutants obtained in Example 1, the corresponding gene sequences were analyzed and obtained, and these gene sequences were optimized for Escherichia coli synonymous codon preference, respectively obtaining the optimization shown in Table 3 a. Connect these optimized codons to the vector pET-28a(+), and entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize several recombinant plasmids.

table 3

Optimized codon Ⅰ (SEQ ID NO.8):

GCTCCAGAACTGGAAAACGGTCCAATGAAGCCTCCGTCTAAACCGAAGAACATTGTCATTATGGTCGGTGACGGTATGGGTCCGAGCTACACTTCCGCATATCGTTACTTCAAAGACAACCCAGACACCGAGGAGGTTGAACAGACTGTTTTCGACCGCCTGCTGGTGGGTATGGCAAGCACCTACCCTGCAAGCGTTTCCGCTACGTAACTGATA GCGCAGCGGCAGCTACCGCTCTGGCTACTGGTGTTAAATCTACAATGGTGCGATTTCTGTTGACACCCAAAAGCAGCATCTGCCAACTATGCTGGAAAAAGCTAAAGCTCTGGGCCTGTCTACCGGCGTAGCGGTTACCTCTCAGATCAACCACGCGACTCCGGCGGCATTTCTGGCTCACAACGAAAGCCGCAAAAACTACGACGCTCTGGCT CTGAGCTATCTGGACACTAACGCGGATGTACTGCTGGGTGGCGGTCAGAAATACTTCTCTCCGGAGCTGCTGGAAAAGTTCACTGCGAAAGGCTACCAGCACATCAGCCGTTTCGAAGATCTGGCAACCATTACGCAGCCAAAAGTCATGGTCTGTTCGCCCAAGTGCAGCTGCCGTGGGCACTGGATGAAAAAAAACGCCAATCGTCTGTCCACC ATGACTCAGAAAGCACTGGACCTGCTGTCTCAGAACGAACAGGGTTTCGTTCTGCTGGTCGAAGGCAGCCTGATTGACTGGGCAGGTCATTCCAACGACATCGCAAACACCATGGGTGAAATGGATGAATTCGCCAACGCCCTGGAGGTAGTTGAACAGTTCGTACGTCAGCACCCGGACACTCTGATGGTTGCAACCGCGGACCACAATACCGGC GGTCTGTCTATTGGTGCAGGTGGTGACTACCGTTGGTGACTACCGTTGGAATCCGGAAATCCTGCGTAACATGAGCGCCTCTACTGATACTCTGGCACTGGCTGCGCTGGGTGGTGATCAATGGCAGGCTGATCTGGCACGTGGCCTGGGTTTCGAACTGAACGCGGATGAAGTCACTCAGCTGTCTACCGCTCGTATGCAGGGTCTGGAAACCATGACCGAAGCA ATCCGTAAGATCATCGATAAACGCACCGGTACGGGTTGGACTACTAGCGGTCACACCGGTACCGATGTTCAGGTATTCGCCGCTGGTCCGGCAGCTGAACTGTTCAACGGTCACCAGGACAACCACCGACATCGCGAACAAAATCTTCACTCTGCTGCCAAAACCGAAAAAAGCGAAAACCGAG

Optimized codon Ⅱ-1 (SEQ ID NO.9):

GCTCCAGAACTGGAAAACGGCCCGATGAAACCACCATCTAAACCTAAAAACATCGTGATTATGGTAGGTGATGGTATGGGTCCATCTTACACTTCTGCGTACCGTTACTTCAAAAGACAACCCGGACACTGAAGAAGTTGAACAGACTGTTTTTGACCGTCTGCTGGTTGGTATGGCGTCCACTTATCCGGCTTCCGTTTCTGGTTACGTAACCGATTCTGC GGCTGCTGCTACTGCTCTGGCGACTGGTGTTAAATCTTATAACGGTGCAATCTCCGTCGACACCCAAAAACAGCACCTGCCGACCATGCTGGAAAAGGCTAAAGCTCTGGGTCTGTCCACCGGCGTCGCTGTTACCAGCCAGATCAACCATGCTACCCCAGCGGCCTTCCTGGCGCATAATGAAAGCCGCAAAAACTACGATGCGCTGGCACTGT CTTACCTGGACACTAACGCGGACGTACTGCTGGGTGGTGGTCAGAATACTTCTCTCCGGAACTGCTGGAAAAGTTCACCGCTAAGGGTTACCAGCACATCAGCCGTTTTGAAGATCTGGCAACCATTACCCAGCCGAAAGTGATCGGCCTGTTTGCCCAGGTTCAGCTGCCTTGGGCGCTGGATGAGAAAAACGCGAATCGTCTGTCCACTATGA CTCAGAAAGCGCTGGATCTGCTGTCTCCAGAACGAACAGGGCTTTGTCCTGCTGGTGGAAGGCAGCCTGATCGATTGGGCGGGTCACTCCAACGATATCGCTAACACCATGGGCGAAATGGATGAATTTGCTAATGCGCTGGAAGTGTTGAACAGTTTGTCCGTCAGCACCCGGACACGCTGATGGTTGCGACTGCTGATCACACACCG GTGGTCTGTCTATCGGTGCAGGTGGTGATTACCGCTGGAACCCTGAAATTCTGCGTAACATGAGCGCTTCTACGGATACTCTGGCGCTGGCTGCTCTGGGTGGCGATCAAGCACAGGCAGATCTGGCACGTGGTCTGGGTTTTGAGCTGAACGCAGATGAGGTGACTCAGCTGTCCACGGCGCGTATGCAGGGTCTGGAAACCATGACCGAAGCG ATCCGTAAAATCATTGACAAACGCACCGGCACTGGTTGGACTACCTCCGGTCACACTGGCACCGATGTTCAGGTTTTCGCTGCAGGTCCGGCAGCAGAACTGTTTAACGGTCATCAGGATAACACCGACATTGCAAACAAAATCTTCACTCTGCTGCCAAAACCAAAAAAGCGAAAACCGAA

Optimized codon Ⅱ-2 (SEQ ID NO.10):

GCTCCGGAACTGGAAAATGGCCCTATGAAACCGCCGTCTAAACCTAAAAACATCGTAATCATGGTTGGCGATGGCATGGGTCCGTCCTATACTTCTGCCTACCGCTATTTCAAAGACAACCCAGACACCGAGGAAGTTGAACAGACTGTATTCGACCGTCTGCTGGTTGGTATGGCTTCCACCTATCCGGCATCTGTTTCTGGCTATGTAACCGATT CTGCAGCTGCAGCCACTGCACTGGCAACCGGTGTGAAATCTTATAACGGTGCTATTTCTGTTGATACGCAGAAACAGCACCTGCCGACCATGCTGGAAAAAGCGAAAGCACTGGGCCTGTCCACCGGTGTTGCTGTTACCTCTCAGATCAACCATGCGACCCCGGCTGCTTTTCTGGCACATAACGAATCCCGTAAGAACTACGACGCGCTGGCC CTGTCTTATCTGGATACCAACGCGGATGTACTGCTGGGTGGCGGTCAGAAATATTTCTCCCCTGAACTGCTGGAAAAGTTTACCGCCAAAGGTTATCAGCATATCTCTCGCTTCGAAGATCTGGCGACTATCACCCAGCCGAAAGTTATTGGCCTGTTCGCACAGGTTCAACTGCCGTGGGCACTGGATGAAAAAAACGCCAACCGTCTGAGCACCAT GACTCAGAAAGCGCTGGACCTGCTGAGCCAGAACGAGCAGGGTTTCGTACTGCTGGTCGAAGGCAGCCTGATTGACTGGGCAGGTCATAGCAACGATATCGCAAACACGATGGGCGAGATGGACGAATTTGCGAACGCGCTGGAAGTGGTAGAACAGTTCGTCCGCAGCATCCTGACACCCTGATGGTTGCGACCGCTGACCACAATACCGGTG GCCTGTCTATTGGTGCTGGTGGCGACTATCGTTGGAACCCGGAAATCCTGCGTAACATGAGCGCTTCCACCGATACCCTGGCACTGGCTGCTCTGGGTGGCGATCAATGGGCAGCTGATCTGGCCCGTGGTCTGGGTTTCGAACTGAACGCTGATGAAGTTACCCAGCTGTCTACGGCGCGTATGCAGGGCCTGGAGACTATGACTGAAGCC ATCCGCAAAAATCATCGACAAGCGTACCGGCACTGGCTGGACTACCTCTGGCCACACTGGTACCGACGTACAGGTTTTCGCAGCAGGTCCAGCCGCTGAACTGTTCAACGGTCACCAGGACAACACTGACATCGCCAACAAAATCTTCACCCTGCTGCCGAAACCGAAAAAAGCAAAAACCGAA

Optimized codon Ⅱ-3 (SEQ ID NO.11):

GCACCTGAACTGGAGAACGGTCCAATGAAACCGCCGTCTAAACCTAAAAACATTGTTATTATGGTCGGTGATGGTATGGGTCCGTCTTATACGTCCGCTTACCGTTATTTTAAAGACAACCCGGACACCGAGGAAGTGGAGCAGACCGTTTTCGATCGTCTGCTGGTTGGCATGGCGTCTACTTATCCTGCGTCCGTGTCTGGCTATGTGACT GATTCTGCTGCTGCTGCAACCGCACTGGCAACTGGTGTGAAAAGCTATAACGGCGCTATCTCTGTCGACACTCAGAAACAGCACCTGCCGACGATGCTGGAAAAAGCGAAAGCTCTGGGCCTGTCTACCGGTGTCGCGGTAACCAGCCAAATCAACCACGCTACGCCGGCAGCCTTTCTGGCACATAACGAAAGCCGTAAAAACTACGATGCG CTGGCGCTGAGCTACCTGGACACCAACGCAGACGTTCTGCTGGGCGGTGGTCAGAATACTTCTCTCCGGAACTGCTGGAAAAATTTACCGCAAAGGGCTATCAGCACATTAGCCGCTTCGAAGACCTGGCCACCATCACCCAGCCGAAAGTTATCGGTCTGTTCGCTCAGGTGCAACTGCCGTGGGCGCTGGATGAGAAAAAATGCTAACCGTCTGT CTACGATGACCCAGAAAGCCCTGGATCTGCTGTCCCAGAACGAACAGGGCTTCGTTCTGCTGGTAGAAGGCTCTCTGATTGACTGGGCTGGCCACAGCAACGATATCGCAAACACCATGGGTGAGATGGACGAGTGAGATGGACGAGTTCGCTAATGCGCTGGAAGTAGTGGAACAGTTTGTACGTCAGCACCCGGATACTCTGATGGTGGCCACGGCGGACCAC AACACTGGTGGTCTGAGCATCGGTGCAGGTGGCGACTACCGTTGGAACCCGGAAATCCTGCGTAACATGAGCGCTTCTACCGATACCCTGGCACTGGCTGCACTGGGCGGTGATCAGTGGCAGGCTGATCTGGCGCGTGGTCTGGGTTTCGAGCTGAACGCTGACGAAGCGACTCAGCTGAGCACTGCCCGTATGCAGGGCCTGGAAAC GATGACCGAAGCGATCCGTAAAATCATCGATAAACGTACCGGTACCGGTTGGACTACCTCCGGTCACACTGGCACCGACGTTCAGGTTTTTTGCGGCAGGTCCGGCAGCGGAGCTGTTTAATGGTCACCAGGACAATACCGATATCGCGAACAAAATTTTCACCCTGCTGCCGAAGCCGAAGAAAGCAAAACTGAG

Optimized codon Ⅱ-4 (SEQ ID NO.12):

GCTCCTGAACTGGAAAACGGTCCTATGAAACCGCCGTCTAAACCGAAAAATATCGTTATTATGGTTGGTGACGGTATGGGTCCTTCCTATACCTCTGCGTACCGTTACTTCAAAAGACAACCCGGATACTGAAGAAGTTGAACAAACGTCTTCGATCGTCTGCTGGTCGGCATGGCTTCCACCTACCCGGCAAGCGTAAGCGGTTACGTAACTGA TTCTGCAGCTGCGGCTACTGCTCTGGCAACGGGTGTAAAAAGCTACAACGGCGCGATTTCCGTGGACACCCAGAAGCAGCACCTGCCAACTATGCTGGAAAAAGCAAAAGCTCTGGGCCTGTCTACCGGTGTGGCAGTCACTTCTCAGATCAACCACGCTACTCCAGCGGCTTTCCTGGCGCATAACGAAAGCCGTAAAAACTATGATGCGCTGGCT CTGTCTTACCTGGACACCAACGCAGACGTACTGCTGGGTGGCGGTCAAAAAGTACTTCTCCCCGGAGCTGCTGGAAAAGTTCACCGCGAAAGGCTACCAGCATATCTCCCGCTTCGAAGATCTGGCTACCATCACGCAGCCGAAAGTTATCGGTCTGTTTGCCCAAGTACAGCTGCCGTGGGCTCTGGATGAAAAAACGCAAACCGTCTGTCCACCAT GACCCAGAAAGCTCTGGATCTGCTGTCCCAGAACGAACAGGGTTTCGTTCTGCTGGTGGAAGGCTCTCTGATTGACTGGGCGGGCCATTCCAACGACATTGCTAACACGATGGGTGAAATGGACGAGTTTGCGAACGCACTGGAAGTTGTTGAACAGTTCGTGCGCCAGCATCCGGATACCCTGATGGTTGCGACCGCTGACCACAACACTGGT GGTCTGTCTATTGGTGCAGGCGGTGATTACCGTTGGAACCCGGAAATCCTGCGCAACATGTCTGCGTCTACTGACACCCTGGCACTGGCTGCTCTGGGTGGTGATCAGTGGCAAGCTGACCTGGCACGTGGTCTGGGCTTTGAACTGAATGCGGATGAAGTAACCCAGCTGGCGACTGCGCGTATGCAGGGTCTGGAAACGATGACCGAG GCGATTCGTAAAATCATCGACAAACGTACCGGCACTGGTTGGACTACTTCCGGCCACACTGGTACCGATGTTCAGGTCTTCGCGGCTGGTCCAGCAGCAGAACTGTTCAACGGTCATCAGGATAACACGGACATCGCGAATAAAATTTTCACCTGCTGCCGAAGCCGAAAAAAGCGAAAACTGAG

Optimized codon Ⅱ-5 (SEQ ID NO.13):

GCGCCAGAACTGGAAAATGGTCCAATGAAACCGCCGTCTAAACCGAAGAACATCGTAATCATGGTTGGCGACGGCATGGGTCTCGTCTTACACCTCTGCGTACCGTTATTTCAAAGACAACCCGGATACCGAGGAAGTTGAACAAACGTTTTCGATCGTCTGCTGGTGGGCATGGCATCTACTTATCCGGCTTCTGTATCCGGTTACGTTACCGA TTCTGCAGCAGCTGCTACCGCACTGGCGACTGGTGTTAAAAGCTACAACGGTGCGATCTCCGTCGATACTCAGAAACAGCATCTGCCGACTATGCTGGAAAAAGCGAAAGCGCTGGGTCTGTCTACTGGTGTAGCGGTCACGAGCCAAATCAACCACGCTACTCCGGCAGCTTTTCTGGCTCACAACGAATCTCGTAAAAACTACGACGCCCTGGCG CTGTCCTACCTGGATACCAACGCTGACGTACTGCTGGGTGGCGGCCAGAAATATTCAGCCCGGAACTGCTGGAAAAATTCACCGCTAAAGGCTATCAGCACATCTCCCGTTTTGAAGACCTGGCAACTATCACCCAGCCGAAAGTCATTGGCCTGTTTGCACAGGTTCAACTGCCGTGGGCACTGGACGAGAAAAAACGCTAATCGTCTGTCTACT ATGACCCAAAAGGCGCTGGACCTGCTGTCTCCAAAACGAACAGGGTTTCGTCCTGCTGGTTGAAGGTAGCCTGATTGACTGGGCAGGCCACTCCAACGATATTGCGAACACGATGGGCGAAATGGACGAATTCGCTAACGCACTGGAGGTGGTGGAACAGTTCGTTCGCCAGCATCCTGACACCCTGATGGTTGCTACCGCGGACCACAACACCG GTGGTCTGAGCATTGGTGCAGGTGGCGATTACCGTTGGAATCCGGAAATCCTGCGCAACATGTCTGCGTCTACTGATACCCTGGCTCTGGCTGCTCTGGGTGGTGACCAATGGCAAGCAGACCTGGCTCGTGGTCTGGGTTTCGAACTGAACGCGGATGAAGTTACGCAGCTGTCTACTGCCGCTATGCAAGGCCTGGAGACCATGACTGAAG CTATCCGCAAGATCATTGATAAGCGTACCGGCACGGGTTGGACTACTAGCGGCCACACCGGTACCGACGTACAGGTTTTGCCGCTGGTCCGGCAGCAGAACTGTTCAACGGTCACCAGGACAACACCGATATTGCTAACAAAGATCTTCACGCTGCTGCCAAAACCGAAAAAAGCCAAGACTGAA

Optimized codon Ⅲ-1 (SEQ ID NO.14):

GCGCCTGAACTGGAAAACGGCCCAATGAAACCACCGTCTAAACCAAAAAATATCGTGATTATGGTTGGCGACGGTATGGGTCTCGTCTTACACTAGCGCTTACCGCTACTTCAAAAGACAACCCGGATACTGAAGAAGTAGAACAACCGTGTTTGATCGTCTGCTGGTGGGCATGGCTTCTACCTACCCGGCATCCGTGAGCGGTTACGTTACGGA TTCTGCTGCGGCAGCTACCGCTCTGGCAACTGGTGTGAAATCTACAACGGCGCAATCTCTGTCGACACTCAGAAACAGCACCTGCCGACTATGCTGGAAAAAGCGAAAGCTCTGGGTCTGTCCACTGGTGTGGCTGTTACCCTCCCAGATCAACCATGCTACCCCTGCTGCTTTTCTGGCTCATAATGAATCTCGTAAAAACTATGATGCCCTGGC GCTGAGCTACCTGGATACGAATGCTGACGTTCTGCTGGGTGGCGGTCAGAATACTTTTAGCCCAGAGCTGCTGGAAAAATTCACGGCGAAAGGTTACCAGCACATCTCCCGTTTCGAAGACCTGGCGACTATTACTCAGCCGAAAGTTATCGGTCTGTTCGCACAGGTGCAACTGCCGTGGGCTCTGGATGAAAAGAACGCTAACCGTCTGTCCA CCATGACCCAGAAAGCGCTGGACCTGCTGTCTCAGAACGAGCAAGGTTTCGTGCTGCTGGTCGAAGGTTCCCTGATCGACTGGGCTGGTCACTCTAATGACATCGCAAACACCATGGGTGAGATGGACGAATTTGCCAACGCGCTGGAAGTTGTTGAACAGTTTGTACGTCAGCATCCGGACACTCTGATGGTGGCAACCGCAGATCAACACC GGTGGTCTGTCTATCGGTGCTGGTGGTGACTACCGCTGGAACCCGGAAATCCTGCGTAACATGTCTGCGAGCACTGATACTCTGGCGCTGGCAGCTCTGGGTGGTGACCAAGCAGCCGCAGATCTGGCTCGTGGTCTGGGTTTTGAACTGAACGCGGATGAAGCGACCCAGCTGGCAACTGCAGCGATGCAGGGCCTGGAAACCATGACCGA GGCAATCCGTAAAATCATTGATAAACGCACCGGCACTGGCTGGACTACTAGCGGTCACACCGGCACTGATGTCCAGGTTTTCGCGGCAGGTCCGGCTGCTGAACTGTTCAACGGCCACCAGGACAACACCGATATTGCGAACAAGATCTTCACCCTGCTGCCGAAGCCGAAAAAAGCAAAGACCGAA

(2) Recombinant plasmid introduced into host Escherichia coli

Take 1 μL of the expression plasmid obtained in step (1), add it to 30 μL of Escherichia coli competent BL21(DE3) under ice bath conditions, place in ice bath for 30 minutes, place in 42°C water bath for 45 seconds, immediately place on ice for 2 minutes, add 400 μL of In the SOC medium containing antibiotics, shake culture at 37°C and 230rpm for 45min. Take 100 μL of the bacterial solution and evenly spread it on the LB plate containing 100 μg/mL kana-resistant, and cultivate overnight in a 37°C incubator.

(3) Target gene expression

Pick the single clones prepared in step (2), and inoculate them in TB and LB medium containing 100 μg/mL kana-resistance respectively by aseptic operation, culture at 37°C with shaking at 220 rpm until OD600 is between 0.6-0.8, IPTG For induction, they were cultured overnight at 37°C and 18°C with shaking respectively. The samples were ultrasonically crushed for SDS-PAGE identification. Exemplarily, the identification results of some samples are shown in FIG. 4 . Figure 4 is a graph showing the identification results of unmutated alkaline phosphatase. It was identified that TB and LB medium were expressed in the supernatant at 18°C (target band 46kDa). The product identification results are listed in Table 4.

Table 4

(4) Purification of expression products (nickel column + QP column)

Weigh about 10 g of recombinant bacteria, add 50 ml Lysis buffer containing 600 mM guanidine hydrochloride, and resuspend using a vortex shaker. Ultrasonic disruption of cells: Ф10 probe, power 10%, working for 5.5s, stopping for 9.9s, ultrasonic disruption for 30min. Centrifuge at 20000rpm at 4°C for 30min, take the supernatant, and filter it with a 0.22μm membrane. Take the filtered supernatant, put it on a 5ml Ni column, and follow the steps below to wash and elute the target protein: Elution step1: 0% B, 5CV, Step2: 0-60% B, 15CV, Step3: 100% B , 6CV, flow rate: sample loading: 2.5ml/min (sample pump), elution: 5ml/ml, collection: 8ml/tube (15ml). The collected samples were directly diluted 6 times with 50mM Tris-HCl, 5% glycerol, pH7.0 solution, used for Q-HP column purification, using 5ml QP column for purification, the mobile phase flow rate was 3ml/min, and 20ml 50mM Tris-HCl Buffer washes UV and conductance to baseline, elution procedures include: Step 1: 0% B, 8CV, 3ml/min; Step 2: 0—60% B, 20CV, 3ml/min; Step3: 100% B, 15CV, 3ml/min. The eluted samples are collected for electrophoretic separation. For example, the electropherograms of some samples are shown in FIG. 5 and FIG. 6 . In Figure 5, LaneS: 0.22 μm, LaneF1-3: passing through, Lane2A1-2B3 gradient elution, Lane2C3: 100% elution, 10% SDS-PAGE, run: 2.0 μl. In Figure 6, LaneS: Ni eluted diluted samples, Lane1A1-2A1 passed through, Lane2A2-2B3 gradient eluted, 2B3: 2.36U/μl, 10% SDS-PAGE, run: 2.0μl.

The target protein was mainly eluted at 250mM NaCl. F was dialyzed overnight in the dialysate, and the volumes of collected dialyzed samples were 20ml. BCA was used to measure the concentration, and the result was: R ² =0.995, the concentration was 18.2 mg/ml, the yield was 433.2 mg, and the yield was 43.32 mg/g bacteria. The purified product yield and yield statistics are shown in Table 5.

table 5

codon number Corresponding alkaline phosphatase/mutant Yieldmg/g Optimized codon Ⅰ Unmutated alkaline phosphatase 43.32mg/g Optimized codon Ⅱ-1 alkaline phosphatase mutant A-1 35.27mg/g Optimized codon Ⅱ-2 alkaline phosphatase mutant A-2 32.46mg/g Optimized codon Ⅱ-3 alkaline phosphatase mutant A-3 34.07mg/g Optimized codon Ⅱ-4 alkaline phosphatase mutant A-4 28.53mg/g Optimized codon Ⅱ-5 alkaline phosphatase mutant A-5 26.37mg/g Optimized codon Ⅲ-1 alkaline phosphatase mutant B-1 31.47mg/g

Example 3

In this example, the activity of the alkaline phosphatase mutant prepared in Example 2 was determined. Specific steps are as follows:

(1) Solution preparation

Prepare 2 mL of working solution according to Table 6 below.

Table 6

Reagent add volume 500mM PNPP 200μL 10X rCutSmart buffer 200μL ddH2O 1.6mL

(2) Activity test

Preparation of positive alkaline phosphatase (commercially available active alkaline phosphatase): 1U/μL positive alkaline phosphatase (diluted with PBS pH 7.4 buffer solution (containing 50% glycerol), then gradually diluted according to the gradient, the diluent is PBS pH 7.4 buffer.

Instrument determination: preheat the microplate reader for 30 minutes, and set the temperature to 37°C. Microplate reader program setting: add 50 μL of working solution, detect the absorbance value A1 at 405 nm, then add 1 μL of positive enzyme solution of each concentration, react for 5 minutes, and measure the absorbance value A2 at 405 nm. Calculate the OD difference A2-A1 between the alkaline phosphatase mutant prepared in Example 2 and the blank, and calculate and obtain the enzyme activity value, and the statistics are shown in Table 7 below.

Table 7

Example 4

In this example, the thermosensitivity assay was performed on the alkaline phosphatase mutant prepared in Example 2. Specific steps are as follows:

Take the alkaline phosphatase mutants prepared in Example 2 and divide them into 5 groups on average. The 4 experimental groups were treated at 65, 75, 85, and 95°C for 15 minutes respectively; a control group was not subjected to heat treatment, and then each group of samples was tested The OD value of the reaction, the higher the OD value, the higher the enzyme activity, the OD value of the control group increases with the prolongation of the reaction time, and the OD of the experimental group has almost no change with the prolongation of the reaction time, indicating that the 65, 75, 85, 95 ℃ treatment for 15 minutes , can inactivate the mutant enzyme.

It can be seen from Figure 8 that the alkaline phosphatase mutant can be completely inactivated by high temperature treatment for a short time, and the modified alkaline phosphatase has good heat sensitivity.

Those of ordinary skill in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in practical applications, various changes can be made to it in form and details without departing from the spirit and spirit of the present invention. scope.

Claims (10)

1. an alkaline phosphatase mutant, is characterized in that, described alkaline phosphatase mutant is selected from one or more positions of lower group in wild-type alkaline phosphatase sequence to mutate: W 348, Q 349, V 365, S 369 and R 372; wherein, the amino acid residue numbering adopts the numbering shown in SEQ ID NO.7. 2. alkaline phosphatase mutant according to claim 1, is characterized in that, the aminoacid sequence of described wild-type alkaline phosphatase is as shown in SEQ ID NO.:7. 3. The alkaline phosphatase mutant according to claim 1, wherein the number of mutation sites is 1 to 5. 4. alkaline phosphatase mutant according to claim 1, is characterized in that, the aminoacid sequence of described alkaline phosphatase mutant has at least 90% homology compared with SEQ ID NO.7. 5. alkaline phosphatase mutant according to claim 1, is characterized in that, described alkaline phosphatase mutant is mutated on the basis of wild-type alkaline phosphatase shown in SEQ ID NO.:7, and The alkaline phosphatase mutant comprises a mutation site selected from the group: W 348A, Q 349A, V 365A, S 369A and R 372A. 6. A polynucleotide molecule, characterized in that the polynucleotide molecule encodes the alkaline phosphatase mutant according to any one of claims 1-5. 7. A carrier, characterized in that the carrier contains the nucleic acid molecule of claim 6. 8. A host cell, characterized in that the host cell contains the vector according to claim 7 or has the nucleic acid molecule according to claim 6 integrated into its chromosome. 9. a method for preparing the alkaline phosphatase mutant according to claim 1, is characterized in that, comprises the steps: (i) cultivating the host cell according to claim 8 under suitable conditions, thereby expressing the alkaline phosphatase mutant; and (ii) isolating the alkaline phosphatase mutant. 10. A test kit, characterized in that the test kit comprises the alkaline phosphatase mutant according to any one of claims 1 to 5.

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