Alkaline phosphatase mutants, methods of making and using the same

Abstract

The application relates to a basic phosphatase mutant and a preparation method and application thereof. The basic phosphatase mutant comprises a polypeptide obtained by replacing at least one of the following mutations of wild-type basic phosphatase, the amino acid sequence of the wild-type basic phosphatase is shown in SEQ ID No. 2, and the mutations include that lysine at the 51st position is replaced by asparagine and histidine at the 113th position is replaced by glutamic acid. By replacing lysine at the 51st position by asparagine or replacing histidine at the 113th position by glutamic acid, the hydrogen bond strength at the dimer interface of the wild-type basic phosphatase protein molecule can be weakened, so that the thermal stability of the enzyme is weakened and the thermal sensitivity of the enzyme is improved. It is verified through experiments that compared with the wild-type basic phosphatase, the stability and specific activity of the basic phosphatase mutant in the research are greatly improved, the specific enzyme activity of the protein is greater than 1200 U / mg, and the protein can be completely inactivated at 65 DEG C for 15 min.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an alkaline phosphatase mutant, a preparation method and application thereof. BACKGROUND

[0002] Alkaline phosphatases (EC 3.1.3.1) are enzymes that are widely present in prokaryotic cells and mammals, and are mainly used for removing phosphate groups of nucleotides, proteins and alkaloids. At present, there are many types of alkaline phosphatases that are applied in biology and medicine. As a commonly used enzyme in laboratories, alkaline phosphatase is often used for non-specific dephosphorylation in molecular cloning to weaken the environmental influence from empty vectors or recombinant vectors. Fragments treated by phosphatase lack 5' phosphoryl markers, so as to avoid self-circularization and reduce the influence of environmental directionality during synthesis. Most alkaline phosphatases are homodimers composed of two identical subunits, and have 10 groups of conserved sequences of beta folds in the middle part of the enzyme.

[0003] Commercially available alkaline phosphatases (AP), such as calf intestinal alkaline phosphatase (CIAP) and Escherichia coli alkaline phosphatase (ECAP), have poor heat sensitivity and are difficult to inactivate by heating after use. SUMMARY

[0004] Therefore, it is necessary to provide an alkaline phosphatase mutant with high heat sensitivity, a preparation method and application thereof.

[0005] An alkaline phosphatase mutant, which comprises a polypeptide obtained by substituting at least one of the following mutations to a wild-type alkaline phosphatase, the amino acid sequence of the wild-type alkaline phosphatase is shown as SEQ ID No. 2, the mutations include: lysine at position 51 is replaced by asparagine (i.e. K51N), histidine at position 113 is replaced by glutamic acid (i.e. H113E).

[0006] The wild-type alkaline phosphatase with the amino acid sequence shown in SEQ ID No. 2 is an alkaline phosphatase of Antarctic bacteria TAB5. It is found that the lysine at position 51 and the histidine at position 113 of the wild-type alkaline phosphatase are amino acids at the dimer interface of the enzyme. By replacing the lysine at position 51 with asparagine or replacing the histidine at position 113 with glutamic acid, the hydrogen bond strength at the dimer interface of the wild-type alkaline phosphatase protein molecule is weakened, thereby reducing the thermal stability of the enzyme and improving the heat sensitivity of the enzyme. It is verified by experiments that, compared with the wild-type alkaline phosphatase, the stability and specific activity of the alkaline phosphatase mutant of the present research are greatly improved, the specific enzyme activity of the protein is greater than 1200 U / mg, and the protein can be completely inactivated at 65℃ for 15 min.

[0007] In one embodiment, the amino acid sequence of the alkaline phosphatase mutant contains one of the polypeptides shown in SEQ ID No. 3-SEQ ID No. 4.

[0008] A polynucleotide encoding the alkaline phosphatase mutant.

[0009] A recombinant vector containing the polynucleotide.

[0010] A recombinant engineering bacterium containing the recombinant vector.

[0011] A preparation method of the alkaline phosphatase mutant, comprising the following steps: expanding culture of the recombinant engineering bacterium, adding an inducer for continuous culture, solid-liquid separation after the culture is completed, collecting cells and crushing, and extracting the alkaline phosphatase mutant from the crushed cells.

[0012] In one embodiment, in the step of adding an inducer for continuous culture, the inducer is IPTG with a concentration of 0.05mM-0.5mM;

[0013] In one embodiment, in the step of adding an inducer for continuous culture, the induction temperature is 16℃-30℃.

[0014] In one embodiment, in the step of adding an inducer for continuous culture, the induction time is 4h-16h.

[0015] In one embodiment, the step of extracting the alkaline phosphatase mutant from the crushed cells comprises: performing ammonium sulfate fractionation on the crushed cells, and then performing chromatographic purification to obtain the alkaline phosphatase mutant.

[0016] In one embodiment, the step of performing ammonium sulfate fractionation on the crushed cells comprises:

[0017] The crushed cells are treated with ammonium sulfate with a final concentration of 0.4M, solid-liquid separation is performed, and the first supernatant is collected.

[0018] The supernatant is treated with ammonium sulfate to a final concentration of 0.7 M, and solid-liquid separation is performed to collect a second supernatant.

[0019] In one embodiment, the step of chromatographically purifying the second supernatant comprises:

[0020] The second supernatant is subjected to Ni column affinity purification to obtain an affinity eluate;

[0021] The affinity eluate is subjected to anion exchange chromatographic purification to obtain the alkaline phosphatase mutant.

[0022] In one embodiment, the step of chromatographically purifying the affinity eluate comprises:

[0023] The affinity eluate is loaded using a loading buffer, wherein the loading buffer comprises 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% (v / v) Triton X-100, and 10% (v / v) glycerol;

[0024] Gradient elution is performed using a first elution buffer and a second elution buffer, wherein the first elution buffer comprises 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% (v / v) Triton X-100, and 10% (v / v) glycerol, the second elution buffer comprises 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% (v / v) Triton X-100, 1 M NaCl, and 10% (v / v) glycerol, and the gradient elution is from 100% (v / v) of the first elution buffer and 0% (v / v) of the second elution buffer to 0% (v / v) of the first elution buffer and 100% (v / v) of the second elution buffer within 10 column volumes.

[0025] The alkaline phosphatase mutant, the recombinant vector, or the recombinant engineering bacteria are used for preparing molecular cloning reagents. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 a three-dimensional spatial structure of alkaline phosphatase TAP;

[0027] Figure 2 a catalytic mechanism diagram of alkaline phosphatase;

[0028] Figure 3SDS-PAGE figure in the purification process of alkaline phosphatase TAP mutant, in which lane 1 is the supernatant of bacterial slurry crushing, lane 2 is the sample after ammonium sulfate precipitation and redissolving, lane 3 is the sample for affinity chromatography, lane 4 is the flow-through of affinity chromatography, lane 5 is the elution with 50 mM imidazole, lane 6 is the elution with 500 mM, lane 7 is the elution with 500 mM again, lane 8 is Marker, lane 9 is the sample for Q FF, lane 10 is the flow-through of Q FF, lane 11 is the elution with 100 mM NaCl of Q FF, lane 12 is the elution with 500 mM NaCl of Q FF, lane 13 is the elution with 1 M NaCl of Q FF;

[0029] Figure 4 pH range comparison figure of TAP-WT, TAP-K51N, TAP-H113E and other mutants. DETAILED DESCRIPTION

[0030] In order to make the above objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the specific embodiments and drawings. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the scope of the present application, so the present application is not limited to the specific implementation disclosed below.

[0031] One embodiment of the present application provides an alkaline phosphatase mutant which can be used for preparing a molecular cloning reagent. The alkaline phosphatase mutant comprises a polypeptide obtained by substituting at least one of the following mutations to a wild-type alkaline phosphatase, the amino acid sequence of which is shown as SEQ ID No. 2, the mutations comprising: substituting lysine at position 51 with asparagine, substituting histidine at position 113 with glutamic acid.

[0032] The wild-type alkaline phosphatase with the amino acid sequence shown as SEQ ID No. 2 is a kind of alkaline phosphatase of Antarctic bacteria TAB5, named TAP. The mature TAP protein consists of 353 residues. The sequence identity of TAP with Bacillus subtilis AP is 38%. The glycine cluster on TAP plays an important role in cold adaptation. Each monomer of TAP dimer contains a β-sheet surrounded by α-helices. The crystal structure shows that TAP has fewer salt bridges and lacks disulfide bonds which are important for TAP stability. The interface of TAP is less hydrophilic than ECAP. However, compared with ECAP, TAP has two additional metal binding sites named M4 and M5. 2+ The M4 site shows a stabilizing contribution, while the Mg 2+ The M5 site plays a specific role in the dephosphorylation function of AP.

[0033] The catalytic mechanism of alkaline phosphatase is shown in Figure 2 Specifically, first, the free cysteine nucleophile on the enzyme active site attacks the phosphorus atom in the phosphate group and bonds to it; then, the P-O bond connecting the phosphate group to the tyrosine accepts a proton from a suitably acidic amino acid (such as aspartate) or a water molecule, protonates, and forms a phospho-cysteine intermediate; this intermediate is hydrolyzed by another water molecule, and the enzyme active site is freed up to continue the next dephosphorylation reaction. The active site of metallophosphatases binds two metal ions that are essential for catalysis. The nature of these two metal ions has been the subject of much study, and no consensus has been reached. The best evidence suggests that the two metals can be any combination of magnesium, manganese, iron, and zinc, and that the two metal ions are connected by a hydroxide ion. This hydroxide ion is thought to participate in the nucleophilic attack on phosphorus.

[0034] It was found that the 51st lysine and the 113th histidine of the wild-type alkaline phosphatase are amino acids at the dimer interface of the enzyme protein, and by replacing the 51st lysine with asparagine or replacing the 113th histidine with glutamic acid, the hydrogen bond strength at the dimer interface of the wild-type alkaline phosphatase protein molecule can be weakened, thereby reducing the thermal stability of the enzyme and improving the thermal sensitivity of the enzyme. It was verified by experiments that compared with the wild-type alkaline phosphatase, the stability and specific activity of the alkaline phosphatase mutant of the present research were greatly improved, the specific enzyme activity of the protein was greater than 1200 U / mg, and it could be completely inactivated at 65°C for 15 min.

[0035] The wild-type alkaline phosphatase (named TAP-WT) has a nucleotide sequence as shown in SEQ ID No. 1.

[0036] Specifically, the sequence as shown in SEQ ID No. 1 is GTGCTGGTGA AAAACGAACC GCAGCTGAAAACCCCGAAAA ACGTGATTCT GCTGATTAGC GATGGCGCGG GCCTGAGTCA GATTAGCAGC ACCTTTTATTTTAAAGAAGG CACCCCGAAC TATACGCAGT TTAAAAACAT TGGCCTGATT AAAACGAGCA GTAGCCGCGAAGATGTGACC GATAGCGCGA GCGGCGCGAC CGCGTTTAGC TGCGGCATTA AAACCTATAA CGCGGCGATTGGCGTGGCGG ATGATAGCAC CGCGGTGAAA AGCATTGTGG AAATTGCGGC GCTGAACAAC ATTAAAACCGGCGTGGTGGC GACGAGCAGC ATTACCCATG CGACCCCGGC GAGCTTTTAT GCGCATGCGC TGAACCGCGGCCTGGAAGAG GAAATTGCGA TGGATATGAC CGAAAGCGAT CTGGATTTTT TTGCGGGCGG TGGCCTGAACTATTTTACCA AACGCAAAGA TAAAAAAGAT GTGCTGGCGA TTCTGAAAGG CAATCAGTTT ACCATTAACACCACCGGCCT GACCGATTTT AGCAGCATTG CGAGCAACCG CAAAATGGGC TTTCTGCTGG CGGATGAAGCGATGCCGACG ATGGAAAAAG GCCGCGGCAA CTTTCTGAGC GCGGCGACCG ATCTGGCGAT TCAGTTTCTGAGCAAAGATA ACAGCGCGTT TTTTATTATG AGCGAAGGCA GTCAGATTGA TTGGGGCGGC CATGCGAACAACGCGAGCTA TCTGATTAGC GAAATTAACG ATTTTGATGA TGCGATTGGC ACCGCGCTGG CGTTTGCGAAAAAAGATGGC AACACCCTGG TGATTGTGAC GAGCGATCAT GAAACCGGCG GCTTTACCCT GGCGGCGAAAAAAAACAAAC GCGAAGATGG CAGCGAATATAGCGATTATA CCGAAATTGG CCCGACCTTT AGCACCGGCGGCCATAGCGC GACCCTGATT CCGGTGTTTG CGTATGGCCC GGGCAGCGAA GAATTTATTG GCATTTATGAAAACAACGAA ATTTTTCATA AAATTCTGAA AGTGACCAAA TGGAATCAGT AA.

[0037] Specifically, the sequence as shown in SEQ ID No. 2 is VLVKNEPQLK TPKNVILLIS DGAGLSQISSTFYFKEGTPN YTQFKNIGLI KTSSSREDVT DSASGATAFS CGIKTYNAAI GVADDSTAVK SIVEIAALNNIKTGVVATSS ITHATPASFY AHALNRGLEE EIAMDMTESD LDFFAGGGLN YFTKRKDKKD VLAILKGNQFTINTTGLTDF SSIASNRKMG FLLADEAMPT MEKGRGNFLS AATDLAIQFL SKDNSAFFIM SEGSQIDWGGHANNASYLIS EINDFDDAIG TALAFAKKDG NTLVIVTSDH ETGGFTLAAK KNKREDGSEY SDYTEIGPTFSTGGHSATLI PVFAYGPGSE EFIGIYENNE IFHKILKVTK WNQ.

[0038] In some embodiments, the amino acid sequence of the alkaline phosphatase mutant contains one of the polypeptides as shown in SEQ ID No. 3-SEQ ID No. 4.

[0039] Specifically, the sequence as shown in SEQ ID No. 3 is VLVKNEPQLK TPKNVILLIS DGAGLSQISSTFYFKEGTPNYTQFKNIGLI NTSSSREDVT DSASGATAFSCG IKTYNAAI GVADDSTAVK SIVEIAALNNIKTGVVATSS ITHATPASFY AHALNRGLEE EIAMDMTESD LDFFAGGGLN YFTKRKDKKD VLAILKGNQFTINTTGLTDF SSIASNRKMG FLLADEAMPT MEKGRGNFLS AATDLAIQFL SKDNSAFFIM SEGSQIDWGGHANNASYLISEINDFDDAIG TALAFAKKDG NTLVIVTSDH ETGGFTLAAK KNKREDGSEY SDYTEIGPTFSTGGHSATLIPVFAYGPGSE EFIGIYENNE IFHKILKVTK WNQ.

[0040] The sequence as shown in SEQ ID No. 4 is VLVKNEPQLK TPKNVILLIS DGAGLSQISS TFYFKEGTPNYTQFKNIGLI KTSSSREDVT DSASGATAFSCG IKTYNAAI GVADDSTAVK SIVEIAALNNIKTGVVATSSITEATPASFY AHALNRGLEE EIAMDMTESD LDFFAGGGLN YFTKRKDKKD VLAILKGNQFTINTTGLTDFSSIASNRKMG FLLADEAMPT MEKGRGNFLS AATDLAIQFL SKDNSAFFIM SEGSQIDWGGHANNASYLISEINDFDDAIG TALAFAKKDG NTLVIVTSDH ETGGFTLAAK KNKREDGSEY SDYTEIGPTFSTGGHSATLIPVFAYGPGSE EFIGIYENNE IFHKILKVTK WNQ.

[0041] In one specific example, the alkaline phosphatase mutant is named TAP-K51N, and its amino acid sequence is shown in SEQ ID No. 3.

[0042] In one specific example, the alkaline phosphatase mutant is named TAP-H113E, and its amino acid sequence is shown in SEQ ID No. 4.

[0043] The two mutants have different degrees of hydrogen bond weakening at the dimer interface relative to the wild-type Antarctic bacterial TAB5 alkaline phosphatase (TAP-WT). The wild-type alkaline phosphatase (TAP-WT) has lysine at position 51 and histidine at position 113, both of which are basic amino acids. The K51N mutation changes the lysine (basic) at the Antarctic phosphodiester dimer interface to asparagine (acidic), and the H113E mutation changes the histidine (basic) at the Antarctic phosphatase dimer interface to glutamic acid (acidic). Both mutations weaken the hydrogen bond strength at the dimer interface of the Antarctic phosphatase protein molecule, thereby weakening the thermal stability of the Antarctic phosphatase. In addition, the H113E mutation also enhances the activity of the alkaline phosphatase to some extent.

[0044] An embodiment of the present research provides a polynucleotide encoding the alkaline phosphatase mutant. The provision of the polynucleotide facilitates the production of the alkaline phosphatase by genetic engineering means.

[0045] An embodiment of the present research provides a recombinant vector containing the polynucleotide.

[0046] The recombinant vector is a cloning vector or an expression vector.

[0047] Specifically, the recombinant vector is a pET-28a(+) plasmid containing the coding sequence of the alkaline phosphatase mutant. It should be noted that the recombinant vector is not limited to the above-mentioned vector, and the alkaline phosphatase mutant gene can also be integrated into other vectors, such as pET21b, pET22b, pET32a, pQE30, etc.

[0048] Psychrophilic APs such as shrimp alkaline phosphatases (SAP) can be completely and irreversibly inactivated at 65℃. However, the recombinant expression of SAP is difficult, and the extraction cost of naturally expressed products is high, which hinders its large-scale production. The recombinant vector provided in the present research contains the coding sequence of the alkaline phosphatase mutant, and can be used for large-scale industrial production of alkaline phosphatase mutants with higher heat sensitivity, and can be used for the preparation of molecular cloning reagents.

[0049] An embodiment of the present research provides a recombinant engineering bacterium containing the recombinant vector.

[0050] The recombinant engineering bacterium can express alkaline phosphatase mutants with higher heat sensitivity, and can be used for the preparation of molecular cloning reagents. In addition, the constructed recombinant engineering bacterium with high expression has the advantages of short cultivation period, simple cultivation conditions, high yield of target protein, and simple purification.

[0051] Further, the recombinant engineering bacteria is E. coli containing the recombinant vector of the above embodiments. Alternatively, the recombinant engineering bacteria is E. coli BL21 Gold (DE3) plysS containing the recombinant vector of the above embodiments. It should be noted that the recombinant engineering bacteria is not limited to E. coli containing the recombinant vector of the above embodiments, and other microbial hosts such as gram-positive bacteria, gram-negative bacteria, yeast and fungi can also be used for expression of the target protein.

[0052] An embodiment of the present research provides a method for preparing an alkaline phosphatase mutant, characterized by comprising the following steps: expanding culture of the above-mentioned recombinant engineering bacteria, adding an inducer for continued culture, solid-liquid separation after the culture is completed, collecting cells and breaking, and extracting the alkaline phosphatase mutant in the broken cells.

[0053] In some embodiments, the inoculation amount in the step of expanding culture of the above-mentioned recombinant engineering bacteria is 1% (v / v)-3% (v / v).

[0054] In some embodiments, the inducer in the step of adding an inducer for continued culture is 0.05 mM-0.5 mM IPTG, the induction temperature is 16°C-30°C, and the induction time is 4 h-16 h.

[0055] The method for breaking the cells is ultrasonic lysis. It should be noted that the method for breaking the cells is not limited to the above-mentioned method, and other breaking methods such as chemical lysis reagents can also be used for breaking.

[0056] The step of extracting the alkaline phosphatase mutant in the broken cells comprises: ammonium sulfate fractionation of the broken cells, and then chromatographic purification to obtain the alkaline phosphatase mutant.

[0057] The step of ammonium sulfate fractionation of the broken cells comprises S110-S120.

[0058] S110, the broken cells are treated with 0.4 M ammonium sulfate to a final concentration, solid-liquid separation is performed, and a first supernatant is collected.

[0059] Specifically, the method for solid-liquid separation is centrifugation. It should be noted that the method for solid-liquid separation is not limited to centrifugation, and other separation methods such as filtration can also be used.

[0060] S120, the supernatant is treated with 0.4 M ammonium sulfate to a final concentration, solid-liquid separation is performed, and a second supernatant is collected.

[0061] Specifically, the method for solid-liquid separation is centrifugation. It should be noted that the method for solid-liquid separation is not limited to centrifugation, and other separation methods such as filtration can also be used.

[0062] The step of chromatographically purifying the second supernatant comprises S130-S140:

[0063] S130, subjecting the second supernatant to Ni column affinity purification to obtain an affinity eluate.

[0064] S140, subjecting the affinity eluate to anion exchange chromatographic purification to obtain the alkaline phosphatase mutant.

[0065] Specifically, in the step of subjecting the affinity eluate to anion exchange chromatographic purification:

[0066] The affinity eluate is loaded using a loading buffer, the loading buffer comprising 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% (v / v) Triton X-100 and 10% (v / v) glycerol;

[0067] Gradient elution is performed using a first elution buffer and a second elution buffer, the first elution buffer comprising 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% (v / v) Triton X-100 and 10% (v / v) glycerol, the second elution buffer comprising 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% (v / v) Triton X-100, 1 M NaCl and 10% (v / v) glycerol, the gradient elution being from 100% (v / v) of the first elution buffer and 0% (v / v) of the second elution buffer to 0% (v / v) of the first elution buffer and 100% (v / v) of the second elution buffer within 10 column volumes.

[0068] Specifically, the chromatographic column used for anion exchange chromatography is a HiTrap Q FF column.

[0069] The alkaline phosphatase mutant of the present study has a significantly improved stability and specific activity compared to wild-type alkaline phosphatase, with a specific enzyme activity of more than 1200 U / mg, and can be completely inactivated at 65°C for 15 min.

[0070] The following is the specific examples section.

[0071] In the examples, reagents and instruments are used as commonly selected in the art unless otherwise specified. The experimental methods not specified in the examples are usually carried out according to conventional conditions, such as the conditions described in the literature, books or the methods recommended by the kit manufacturers. The reagents used in the examples are commercially available.

[0072] Example 1: Construction of recombinant expression vector and recombinant strain

[0073] Antarctic bacteria TAB5 alkaline phosphatase (TAP-WT) cDNA was synthesized according to the reported protein sequence (GenBank No: CAB82508.1). After optimization of E. coli codons, the nucleotide sequence was translated, the nucleotide sequence is shown in SEQ ID No. 1, and the corresponding amino acid sequence of the artificially synthesized Antarctic bacteria TAB5 alkaline phosphatase wild type (TAP-WT) is shown in SEQ ID No. 2. The three-dimensional structure of the wild type alkaline phosphatase is shown in Figure 1 The target gene fragment was cloned into the expression vector pET-28a(+) to construct the recombinant vector pET-28a(+)-TAP. The successfully constructed recombinant expression vector was transformed into E. coli BL21 Gold (DE3) plysS to obtain a recombinant strain expressing Antarctic bacteria TAB5 alkaline phosphatase wild type (TAP-WT). Based on the same inventive concept, the gene fragment of Antarctic bacteria TAB5 alkaline phosphatase mutant was cloned into the expression vector pET-28a(+) to construct the recombinant vectors pET-28a(+)-TAP-K51N and pET-28a(+)-TAP-H113E, and transformed into E. coli BL21 Gold (DE3) plysS to obtain recombinant strains of Antarctic bacteria TAB5 alkaline phosphatase mutants. The amino acid sequence of mutant TAP-K51N is shown in SEQ ID No. 3; the amino acid sequence of TAP-H113E is shown in SEQ ID No. 4. Based on the same inventive concept and method, mutant TAP-K51N / H113E, i.e. a double mutant combination mutant with mutations at mutant sites K51N and H113E, can be obtained.

[0074] Example 2: Expression of alkaline phosphatase TAP

[0075] Single colonies were screened on plates containing 100 μg / mL kanamycin and enriched in LB liquid medium at 37°C overnight. The culture was inoculated in a conical flask containing 1000 mL of LB liquid medium at a ratio of 1:100, and cultured at 37°C with shaking until the OD 600 When the OD of the bacterial solution was about 0.5-0.8, IPTG solution was added to the shake flask to a final concentration of 0.5 mM, and the culture was incubated at 20°C with shaking for 16 h. The bacterial cells were collected by centrifugation at 4°C, 5000 rpm for 20 min. The collected bacterial slurry was frozen at -80°C.

[0076] Example 3: Purification of alkaline phosphatase TAP

[0077] Ni affinity chromatography and anion exchange chromatography were used to purify alkaline phosphatase TAP.

[0078] The affinity chromatography step was as follows:

[0079] ① Take the slurry collected in Example 2, resuspend in 400 mL buffer A (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl2, 10% glycerol, 0.1% Triton X-100 and 10 mM imidazole) and lyse by sonication. Centrifuge the lysate at 12000 r / min for 20 min at 4°C. Slowly add 9.7 g of ground ammonium sulfate powder to the lysate at 4°C while stirring, and let stand for 2 h at 4°C. Centrifuge at 12000 r / min for 15 min to collect the supernatant. Continue to slowly add 10.9 g of ground ammonium sulfate powder to the supernatant at 4°C while stirring, and let stand for 2 h at 4°C. Centrifuge at 12000 r / min for 15 min to collect the precipitate, and resuspend the precipitate in buffer A.

[0080] ② Equilibrate the Ni-NTA column with buffer A;

[0081] ③ Load the protein at a flow rate of 1 ml / min, and collect the flow-through;

[0082] ④ Wash the column with wash buffer B (20 mM Tris-HCl, pH 8.0, 1 M NaCl, 1 mM MgCl2, 10% glycerol and 50 mM imidazole) for 10 column volumes, and collect the wash;

[0083] ⑤ Elute with elution buffer C (20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 500 mM imidazole and 10% glycerol).

[0084] The entire procedure is analyzed by SDS-PAGE, and the results are shown in Figure 3 .

[0085] The anion exchange chromatography step is as follows:

[0086] ① Equilibrate the HiTrap Q FF column with buffer D (20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% Triton X-100 and 10% glycerol), load the one-step chromatography eluate into the column, and elute the alkaline phosphatase TAP by changing the gradient ratio of buffer D and buffer E (20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% Triton X-100, 1 M NaCl and 10% glycerol), and determine the protein content by ultraviolet spectrophotometry.

[0087] Example 4: Activity determination of alkaline phosphatase TAP

[0088] (1) Enzyme activity definition

[0089] The amount of enzyme required to hydrolyze 1 μmol of pNPP to generate p-nitrophenol per minute is defined as one unit of activity.

[0090] (2) Reagent preparation

[0091] Reagent I (1 M diethanolamine): Weigh 10.514 g of diethanolamine reagent, add 60 ml of UP water and 5 ml of 0.1 M MgCl2, adjust the pH to 9.8 with 2 M HCl and then dilute to 100 ml.

[0092] Reagent II (pNPP solution): 0.674 M pNPP solution: weigh 2.5 g of p-nitrophenylphosphatedisodium salt, dissolve in 10 ml of reagent I.

[0093] Enzyme dilution: 30 mM pH 7.6 triethanolamine containing 1 mM MgCl2, 0.1 mM ZnCl2, 0.1% Triton X-100.

[0094] Reaction mixture: 30 mL of reagent I + 0.5 mL of reagent II.

[0095] (3) Enzyme activity determination

[0096] In a 1 mL cuvette, add 900 μL of the reaction mixture and zero the spectrophotometer, add 30 μL of the enzyme solution diluted to the appropriate concentration, mix by blowing, and measure the absorbance change (ΔAs) at 37°C within 1 min.

[0097] Enzyme activity calculation formula:

[0098] Specific activity of the enzyme solution (U / ml) = OD value * dilution factor * 1.676.

[0099] Example 5: Comparison of the specific activity of alkaline phosphatase mutants and wild-type alkaline phosphatase

[0100] Alkaline phosphatase TAP, in which the 51st amino acid lysine is mutated to asparagine, is denoted as TAP-K51N; alkaline phosphatase TAP, in which the 51st amino acid lysine is mutated to arginine, is denoted as TAP-K51R.

[0101] Alkaline phosphatase TAP, in which the 113th amino acid histidine is mutated to glutamic acid, is denoted as TAP-H113E; Antarctic bacteria TAB5 alkaline phosphatase, in which the 113th amino acid histidine is mutated to cysteine, is denoted as TAP-H113C.

[0102] The enzyme activity and protein specific activity of wild-type alkaline phosphatase and mutants TAP-K51N, TAP-K51R, TAP-H113E and TAP-H113C were measured according to the methods of Examples 3, 4 and 5. The results are shown in Table 1.

[0103] Table 1 Comparison of enzyme activity and specific protein activity between wild-type and mutant strains

[0104]

[0105] As shown in Table 1, compared to the wild-type TAP-WT, the mutants TAP-K51N and TAP-H113E exhibit a significantly higher specific activity of protein. Those skilled in the art will recognize that the mutant TAP-K51N / H113E demonstrates a greater increase in specific activity than the mutants TAP-K51N and TAP-H113E, an effect that can be verified using the experimental methods described in Examples 1-5. Therefore, the alkaline phosphatase mutants provided in this application can effectively enhance alkaline phosphatase activity.

[0106] Example 6: Comparison of pH range of action between alkaline phosphatase mutant and wild-type alkaline phosphatase

[0107] The activities of TAP-WT, TAP-K51N, TAP-K51R, TAP-H113E, and TAP-H113C from Example 5 were measured under different pH conditions. The results are shown in [Figure 1]. Figure 4 .

[0108] from Figure 4 It can be seen that all alkaline phosphatases have certain activities when the pH range is 7.0-11.0.

[0109] Example 7: Comparison of heat resistance between Antarctic bacterial TAB5 alkaline phosphatase mutant and Antarctic bacterial TAB5 alkaline phosphatase wild type

[0110] The residual activity of TAP-WT, TAP-K51N, TAP-K51R, TAP-H113E, and TAP-H113C from Example 5 was measured after treatment at 25℃, 30℃, 40℃, 45℃, 50℃, 55℃, 60℃, and 65℃ for 5 min and 15 min, respectively. The results are shown in Table 2 below.

[0111] Table 2 Residual activity of wild-type and different mutants at different treatment temperatures and times.

[0112]

[0113]

[0114] As can be seen from Table 2, the mutants TAP-K51N and TAP-H113E are more sensitive to high temperature than the wild type, and are more likely to be inactivated at high temperature. Based on this, one skilled in the art can know that the mutant TAP-K51N / H113E has stronger high-temperature sensitivity than the mutants TAP-K51N and TAP-H113E, and can be verified by the experimental method described in Example 7.

[0115] Example 8: Reversibility of Antarctic bacteria TAB5 alkaline phosphatase mutants and Antarctic bacteria TAB5 alkaline phosphatase wild type after heat treatment inactivation

[0116] Take the samples of the TAB5 alkaline phosphatase wild type and mutants in Example 7 after 65°C treatment, cool to room temperature (25°C) and place for 20 min, and re-determine their activity. The determination results are shown in Table 3.

[0117] Table 3 Activity of wild type and different mutants after recovery

[0118] Sample TAP-WT TAP-K51N TAP-K51R TAP-H113E TAP-H113C Residual activity 7.65％ 0％ 19.33％ 0％ 11.24％

[0119] As can be seen from Table 3, the mutants TAP-K51N and TAP-H113E are not reversible in activity after heat treatment inactivation and recovery at room temperature. Based on this, one skilled in the art can know that the mutant TAP-K51N / H113E is also not reversible in activity after heat treatment inactivation and recovery at room temperature, and can be verified by the experimental method described in Example 8.

[0120] Compared with the wild type Antarctic bacteria TAB5 alkaline phosphatase (TAP-WT), the above mutants have different degrees of hydrogen bond weakening at the dimer interface. The 51st lysine and 113th histidine of the wild type alkaline phosphatase (TAP-WT) are both basic amino acids, K51N is mutated to Antarctic phosphodiester dimer interface lysine (basic) to asparagine (acidic), H113E is mutated to Antarctic phosphatase dimer interface histidine (basic) to glutamic acid (acidic), both of these two mutations can weaken the hydrogen bond strength at the dimer interface of Antarctic phosphatase protein molecules, thereby weakening the thermal stability of Antarctic phosphatase, in addition, H113E mutation also enhances the activity of alkaline phosphatase to some extent. The alkaline phosphatase mutants of the present study can be used for preparing molecular cloning reagents.

[0121] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. An alkaline phosphatase mutant, characterized in that, The amino acid sequence of the alkaline phosphatase mutant is shown in SEQ ID No. 3 or SEQ ID No.

4.

2. A polynucleotide, comprising, The polynucleotide encodes the alkaline phosphatase mutant of claim 1.

3. A recombinant vector, characterized in that, The polynucleotide of claim 2.

4. A recombinant engineered bacterium, characterized in that, The recombinant vector of claim 3.

5. A method for producing an alkaline phosphatase mutant, characterized by, The method comprises the following steps: expanding the culture of the recombinant engineering bacteria of claim 4, adding an inducer for continued culture, solid-liquid separation after the culture is completed, collecting cells and breaking, and extracting the alkaline phosphatase mutant from the broken cells.

6. The method for preparing the alkaline phosphatase mutant according to claim 5, characterized in that, In the step of adding an inducer for continued culture, the inducer is 0.05 mM-0.5 mM IPTG; In the step of adding an inducer for continued culture, the induction temperature is 16℃-30℃; In the step of adding an inducer for continued culture, the induction time is 4h-16h.

7. The method for preparing the alkaline phosphatase mutant according to claim 5, characterized in that, The step of extracting the alkaline phosphatase mutant from the broken cells comprises: performing ammonium sulfate fractionation on the broken cells, and then performing chromatographic purification to obtain the alkaline phosphatase mutant.

8. The method for preparing an alkaline phosphatase mutant according to claim 7, wherein The step of performing ammonium sulfate fractionation on the broken cells comprises: The broken cells are treated with ammonium sulfate at a final concentration of 0.4 M, solid-liquid separation is performed, and a first supernatant is collected; The supernatant is treated with ammonium sulfate at a final concentration of 0.7 M, solid-liquid separation is performed, and a second supernatant is collected.

9. The method for preparing the alkaline phosphatase mutant according to claim 8, characterized in that, The step of performing chromatographic purification on the second supernatant comprises: The second supernatant is subjected to Ni column affinity purification to obtain an affinity eluate; The affinity eluate is subjected to anion exchange chromatographic purification to obtain the alkaline phosphatase mutant.

10. The method for preparing the alkaline phosphatase mutant according to claim 9, characterized in that, In the step of performing anion exchange chromatographic purification on the affinity eluate: The affinity eluate is loaded with a loading buffer, and the loading buffer comprises: 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% v / v Triton X-100, and 10% v / v glycerol; Gradient elution is performed with a first elution buffer and a second elution buffer, the first elution buffer comprises: 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% v / v Triton X-100, and 10% v / v glycerol, the second elution buffer comprises: 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 0.1% v / v Triton X-100, 1 M NaCl, and 10% v / v glycerol, and the gradient elution is gradient elution from 100% v / v of the first elution buffer and 0% v / v of the second elution buffer to 0% v / v of the first elution buffer and 100% v / v of the second elution buffer within 10 column volumes.

11. Use of the alkaline phosphatase mutant of claim 1, the polynucleotide of claim 2, the recombinant vector of claim 3, or the recombinant engineering bacteria of claim 4 in the preparation of a molecular cloning reagent.

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