Alkaline phosphatase mutant as well as preparation method and application thereof

Abstract

The invention relates to an alkaline phosphatase mutant as well as a preparation method and application thereof. The alkaline phosphatase mutant comprises polypeptide obtained by mutating an amino acid sequence of parent alkaline phosphatase; the mutation comprises the following steps: replacing threonine at the 124 site on the amino acid sequence of the parent alkaline phosphatase with leucine; the nucleotide sequence of the parent alkaline phosphatase is as shown in SEQ ID NO.1, and / or the amino acid sequence of the parent alkaline phosphatase is as shown in SEQ ID NO.3. The alkaline phosphatase mutant has relatively high expression quantity, can be applied to dephosphorylation of an enzymolysis phosphoric acid substrate, and particularly can be used for labeling an antibody and / or catalyzing chemiluminescence of a luminescent substrate.

Description

Technical Field

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

[0002] Alkaline phosphatase (EC 3.1.3.1) is a zinc-containing glycoprotein that has maximum activity in an alkaline environment (optimum pH is around 10). For ALP derived from bacteria, the optimal pH is 8.0, while for ALP derived from cattle, it is 8.5. It can hydrolyze various natural and synthetic monophosphate compound substrates, remove the phosphate groups on the substrate molecules, and generate phosphate ions and free hydroxyl groups. Such substrates include nucleic acids, proteins, alkaloids, etc.

[0003] Alkaline phosphatase is a homodimeric protein with a molecular weight of 56KDa. Each monomer is composed of 449 amino acids. The complete ALP molecule presents a typical α / β topological structure. At the same time, each monomer has an active center, which is composed of Asp101-Ser102-Ala103 triplet, Arg166, water molecules, three metal ions and their ligand amino acids. ALP is encoded by the phoA gene. Like many secretory proteins, it synthesizes a monomer precursor with a signal peptide at the amino terminus in the cytoplasm. The signal peptide guides the precursor to be transported across the inner membrane and then removed, forming a homodimer.

[0004] In scientific research, the enzyme is used to catalyze the removal of 5' phosphate groups from nucleic acid molecules, thereby converting the 5'-P end of the DNA or RNA fragment into a 5'-OH end. At the same time, alkaline phosphatase is also one of the most commonly used marker enzymes in current immunodiagnostic reagent products. Compared with horseradish peroxidase (HRP), the advantages of ALP as a marker enzyme are high stability and high sensitivity, and the disadvantages are high cost and difficulty in labeling. Clinically, the determination of ALP is mainly used for the diagnosis and differential diagnosis of bone and hepatobiliary system diseases, especially the differential diagnosis of jaundice. For unexplained high serum levels of alkaline phosphatase, isoenzymes can be measured to help clarify their organ sources.

[0005] Early commercial supply of alkaline phosphatase was mainly extracted from bovine small intestine, which was relatively expensive. In subsequent research and development, the secretory expression of alkaline phosphatase was achieved using the Pichia pastoris expression system, which reduced the purification cost and increased the yield of alkaline phosphatase, but the yield still could not meet market demand. Summary of the invention

[0006] Based on this, it is necessary to provide an alkaline phosphatase mutant with a higher expression level and a preparation method and application thereof.

[0007] An alkaline phosphatase mutant, the alkaline phosphatase mutant comprising: a polypeptide obtained by a mutation in the amino acid sequence of a parent alkaline phosphatase; the mutation comprising: threonine at position 124 in the amino acid sequence of the parent alkaline phosphatase is replaced by leucine; the nucleotide sequence of the parent alkaline phosphatase is as shown in SEQ ID NO.1, and / or the amino acid sequence of the parent alkaline phosphatase is as shown in SEQ ID NO.3.

[0008] This study found that the above alkaline phosphatase mutant includes: a polypeptide obtained by replacing the threonine at position 124 in the amino acid sequence of the parent alkaline phosphatase with leucine, so that the alkaline phosphatase mutant has a higher expression level. It has been verified by experiments that the extracellular expression level of the above alkaline phosphatase mutant fermentation broth after culturing for 264 hours is 484.296 mg / L, which is 39% higher than the extracellular expression level of the parent alkaline phosphatase (346.903 mg / L), and the intracellular expression level is 650.506 mg / L, which is 132% higher than the intracellular expression level of the parent alkaline phosphatase (279.493 mg / L). The overall expression level is 81% higher than that of the parent alkaline phosphatase.

[0009] In some embodiments, the alkaline phosphatase mutant contains a polypeptide having a nucleotide sequence as shown in SEQ ID NO.2;

[0010] Alternatively, the alkaline phosphatase mutant contains a polypeptide having an amino acid sequence as shown in SEQ ID NO.4.

[0011] A polynucleotide encoding the alkaline phosphatase mutant as described above.

[0012] In some embodiments, the polynucleotide sequence contains the nucleotide sequence shown in SEQ ID NO.4.

[0013] A recombinant vector, comprising the polynucleotide described above.

[0014] A recombinant engineered bacterium, wherein the recombinant engineered bacterium contains the recombinant vector described above.

[0015] In some embodiments, the recombinant engineered bacteria is Pichia pastoris containing the recombinant vector.

[0016] A method for preparing an alkaline phosphatase mutant comprises the following steps: expanding the culture of the above-mentioned recombinant engineering bacteria, adding an inducer to continue the culture, collecting the culture after the culture is completed, and obtaining the alkaline phosphatase mutant.

[0017] Use of the alkaline phosphatase mutant described above, or the alkaline phosphatase mutant obtained by the method for preparing the alkaline phosphatase mutant described above, in enzymatic dephosphorylation of phosphate substrates;

[0018] Furthermore, the alkaline phosphatase mutant is used to label antibodies and / or catalyze luminescent substrates to produce chemiluminescence.

[0019] A method for dephosphorylating a phosphate substrate by enzymatic hydrolysis, comprising the following steps: mixing a phosphate substrate with an alkaline phosphatase mutant, and allowing the alkaline phosphatase mutant to catalyze the enzymatic hydrolysis of the phosphate substrate, wherein the alkaline phosphatase mutant is selected from at least one of the alkaline phosphatase mutants described above and the alkaline phosphatase mutants obtained by the preparation method of the alkaline phosphatase mutants described above. DETAILED DESCRIPTION

[0020] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described in the application, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0021] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used in this application and in the specification of this application are only for the purpose of describing the implementation methods and embodiments and are not intended to limit this application.

[0022] Unless otherwise specified or there is a contradiction, the terms and phrases used in this application have the following meanings:

[0023] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0024] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0025] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0026] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0027] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0028] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0029] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0030] In this application, the nomenclature of amino acids and DNA sequences is as follows: Amino acid residues use the recognized IUPAC nomenclature, either in the form of three-letter abbreviations or single-letter symbols. DNA sequences use the recognized IUPAC nomenclature.

[0031] In the present application, the identifier of the alkaline phosphatase mutant is "the amino acid replaced by the original amino acid position" to indicate the mutated amino acid in the alkaline phosphatase mutant. For example, T124L (or Thr124Leu) means that the amino acid at position 124 is replaced by leucine (L) from the threonine (T) of the parent alkaline phosphatase.

[0032] In the present application, the definition of parent alkaline phosphatase: The term "parent" or "parent sequence" refers to the sequence that existed before the mutation of the present invention, on the basis of which the present invention replaces the amino acids at specific positions to produce new mutants.

[0033] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0034] One embodiment of the present study provides an alkaline phosphatase mutant, which includes: a polypeptide obtained by a mutation in the amino acid sequence of a parent alkaline phosphatase; the mutation includes: the threonine at position 124 in the amino acid sequence of the parent alkaline phosphatase is replaced by leucine; the nucleotide sequence of the parent alkaline phosphatase is as shown in SEQ ID NO.1, and / or the amino acid sequence of the parent alkaline phosphatase is as shown in SEQ ID NO.3.

[0035] This study found that the above-mentioned alkaline phosphatase mutant includes: a polypeptide obtained by replacing the threonine at position 124 of the amino acid sequence of the parent alkaline phosphatase with leucine, so that the alkaline phosphatase mutant has a higher expression level, and can be applied to the application of the alkaline phosphatase mutant in the dephosphorylation of the enzymatic hydrolysis phosphate substrate, and optionally, can be used to label antibodies and / or catalyze the luminescent substrate to produce chemiluminescence. It has been verified by experiments that the extracellular expression level of the above-mentioned alkaline phosphatase mutant fermentation broth after 264h of culture is 484.296mg / L, which is 39% higher than the extracellular expression level of the parent alkaline phosphatase (346.903mg / L), and the intracellular expression level is 650.506mg / L, which is 132% higher than the intracellular expression level of the parent alkaline phosphatase (279.493mg / L), and the overall expression level is 81% higher than that of the parent alkaline phosphatase.

[0036] Wherein, the sequence shown in SEQ ID NO.1 is:

[0037]

[0038] The sequence shown in SEQ ID NO.3 is:

[0039] LIPAEEENPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQ CNTTRGNEVTSVINRAKKAGKAVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQKNGCQDIAAQLVYNMDIDVILGGGRMYMFPEGTPDPEYPDDASVNGVRKDKQNLVQEWQAK HQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLAEMTEAALQVLSRNPRGFYLFVEGGRIDHGHHDGKAYMALTEAIMFDNAIAKANELTSELDTLILVTADHSH VFSFGGYTLRGTSIFGLAPGKALDSKSYTSILYGNGPGYALGGGSRPDVNGSTSEEPSYRQQAAVPLASETHGGEDVAVFARGPQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPA.

[0040] In some embodiments, the alkaline phosphatase mutant contains a polypeptide having a nucleotide sequence as shown in SEQ ID NO.2;

[0041] Alternatively, the alkaline phosphatase mutant contains a polypeptide having an amino acid sequence as shown in SEQ ID NO.4.

[0042] Wherein, the sequence shown in SEQ ID NO.2 is:

[0043]

[0044] The sequence shown in SEQ ID NO.4 is:

[0045] LIPAEEENPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQ CNTLRGNEVTSVINRAKKAGKAVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQKNGCQDIAAQLVYNMDIDVILGGGRMYMFPEGTPDPEYPDDASVNGVRKDKQNLVQEWQAK HQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLAEMTEAALQVLSRNPRGFYLFVEGGRIDHGHHDGKAYMALTEAIMFDNAIAKANELTSELDTLILVTADHSH VFSFGGYTLRGTSIFGLAPGKALDSKSYTSILYGNGPGYALGGGSRPDVNGSTSEEPSYRQQAAVPLASETHGGEDVAVFARGPQAHLVHGVQEETFVAHIMAFAGCVEPYTDCNLPAPA.

[0046] One embodiment of the present study provides a polynucleotide encoding the above alkaline phosphatase mutant. Providing the above polynucleotide is conducive to producing the above alkaline phosphatase by genetic engineering.

[0047] In some embodiments, the polynucleotide sequence contains the nucleotide sequence shown in SEQ ID NO.4.

[0048] One embodiment of the present study provides a recombinant vector, which contains the above-mentioned polynucleotide.

[0049] Among them, the recombinant vector is a cloning vector or an expression vector.

[0050] Specifically, the recombinant vector is a pPICZαA plasmid containing the coding sequence of the alkaline phosphatase mutant. It should be noted that the recombinant vector is not limited to the vectors mentioned above, and the alkaline phosphatase mutant gene can also be integrated into other vectors, such as pET21b, pET22b, pET32a, pQE30 and other vectors.

[0051] One embodiment of the present study provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium contains the above-mentioned recombinant vector.

[0052] The above recombinant engineering bacteria can express the above alkaline phosphatase mutant and can be used to prepare molecular cloning reagents. In addition, the constructed highly efficient expression recombinant engineering bacteria has the advantages of short culture cycle, simple culture conditions, high yield of target protein and simple purification.

[0053] Further, the recombinant engineered bacteria is a Pichia yeast containing the recombinant vector. Optionally, the recombinant engineered bacteria is a Pichia yeast X33 containing the recombinant vector of the above embodiment. It should be noted that the recombinant engineered bacteria is not limited to the Pichia yeast containing the recombinant vector of the above embodiment, and microbial hosts such as Gram-positive bacteria, Gram-negative bacteria, yeast and fungi can also be used to express the target protein.

[0054] One embodiment of the present study provides a method for preparing an alkaline phosphatase mutant, comprising the following steps: expanding the culture of the above-mentioned recombinant engineered bacteria, adding an inducer and culturing, and collecting the culture after the culture is completed to obtain the alkaline phosphatase mutant.

[0055] The inducer is methanol. The step of adding the inducer and continuing the culture comprises: adding 1% (V / V) methanol every 24 hours.

[0056] Specifically, the inducer is formaldehyde. The steps of expanding the culture of the recombinant engineered bacteria, adding the inducer and culturing include: inoculating the seed liquid of the recombinant engineered bacteria into a fermentation tank filled with a fermentation medium for fermentation, and controlling the dissolved oxygen content in the fermentation medium to be above 30%; starting glycerol feeding when the dissolved oxygen suddenly rises to above 90%, stopping glycerol feeding when the wet weight of the bacteria reaches 200g / L during the glycerol feeding stage, and starting starvation for 1 hour when the dissolved oxygen rises to above 90%; and then starting the methanol induction stage, that is, adding 1% (V / V) methanol every 24 hours.

[0057] The fermentation medium is BMGY medium, which includes the following components: 10 g yeast powder, 20 g peptone dissolved in 700 mL water, 100 mL pH 6.0 1 mol / L potassium phosphate buffer, 100 mL 10×YNB, 2 mL 500×biotin, and 10 mL glycerol are added when used.

[0058] In the methanol induction stage, the culture medium used for methanol induction is BMMY culture medium, which includes the following components: 10 g yeast powder and 20 g peptone dissolved in 700 mL water, then sterilized at 121°C for 20 min, and when used, 100 mL 1 mol / L potassium phosphate buffer pH 6.0, 100 mL 10×YNB, 2 mL 500×biotin, and 2.5 mL methanol are added at 60°C.

[0059] Among them, the fermentation time (i.e. the fermentation time in the fermentation tank) is 192h-264h.

[0060] The step of collecting the culture after the culture is completed includes: after the culture is completed, the solid-liquid separation is performed to obtain the supernatant and the solid; the solid is crushed and the alkaline phosphatase mutant in the crushed solid is extracted. The cell crushing method is ultrasonic lysis. It should be noted that the cell crushing method is not limited to the above-mentioned method, and can also be other crushing methods, such as crushing with a chemical lysis reagent.

[0061] One embodiment of the present study provides the use of the above-mentioned alkaline phosphatase mutant, or the alkaline phosphatase mutant obtained by the above-mentioned preparation method of the alkaline phosphatase mutant, in enzymatic dephosphorylation of phosphate substrates.

[0062] Furthermore, the alkaline phosphatase mutant is used to label antibodies and / or catalyze luminescent substrates to produce chemiluminescence.

[0063] One embodiment of the present study provides a method for enzymatic dephosphorylation of a phosphate substrate, comprising the following steps: mixing the phosphate substrate with an alkaline phosphatase mutant, and allowing the alkaline phosphatase mutant to catalyze the enzymatic hydrolysis of the phosphate substrate, wherein the alkaline phosphatase mutant is selected from at least one of the above-mentioned alkaline phosphatase mutants and the alkaline phosphatase mutants obtained by the above-mentioned preparation method of the alkaline phosphatase mutant.

[0064] The present application also provides the above-mentioned alkaline phosphatase mutant and its preparation method and application. The above-mentioned alkaline phosphatase mutant has a significantly improved expression level compared with its parent alkaline phosphatase. Under suitable conditions, the extracellular expression level of the above-mentioned alkaline phosphatase mutant after culturing the fermentation broth for 264 hours is 484.296 mg / L, which is 39% higher than the extracellular expression level of the parent alkaline phosphatase (346.903 mg / L). The intracellular expression level is 650.506 mg / L, which is 132% higher than the intracellular expression level of the parent alkaline phosphatase (279.493 mg / L). The overall expression level is 81% higher than that of the parent alkaline phosphatase, and can be applied to the application of alkaline phosphatase mutants in enzymatic dephosphorylation of phosphate substrates. Optionally, it can be used to label antibodies and / or catalyze luminescent substrates to produce chemiluminescence.

[0065] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0066] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0067] The culture medium used in the following examples (configured according to the Pichia yeast fermentation manual) is as follows:

[0068] YPD medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L glucose, and 18 g / L agar for solid culture medium.

[0069] BMGY medium: 10 g yeast powder and 20 g peptone were dissolved in 700 mL water. When used, 100 mL pH 6.01 mol / L potassium phosphate buffer, 100 mL 10×YNB, 2 mL 500×biotin, and 10 mL glycerol were added.

[0070] BMMY medium: 10 g yeast powder and 20 g peptone were dissolved in 700 mL water and sterilized at 121°C for 20 min. When used, 100 mL 1 mol / L potassium phosphate buffer (pH 6.0), 100 mL 10×YNB, 2 mL 500×biotin, and 2.5 mL methanol were added at 60°C.

[0071] PTM1 basic salts ( / L) for glycerol feed: CuSO 4 -5H 2 O 6g; sodium iodide 0.08g; MnSO 4 -H 2 O 3.0g; Sodium molybdate-2H 2 O 0.2g; Boric acid 0.02g; Cobalt chloride 0.5g; ZnCl 2 20.0g; FeSO 4 65.0g; Biotin 0.2g; H 2 SO 4 5ml.

[0072] The activity determination method of alkaline phosphatase used in the following examples is:

[0073] (1) Reaction principle and definition of enzyme activity

[0074] Reaction principle: Alkaline phosphatase can catalyze the hydrolysis of the phosphate substrate PNPP (4-nitrophenyl phosphate disodium salt hexahydrate) to produce PNP (p-nitrophenol). PNP appears yellow under alkaline conditions, and its content can be detected at a wavelength of 405nm. The higher the PNP content, the higher the enzyme activity. Source

[0075] Definition of enzyme activity: Under the conditions of pH 9.6 and 25°C, the amount of enzyme used to hydrolyze 1 μmoL PNP per minute is 1 enzyme activity unit, i.e. 1U.

[0076] (2) Reagent configuration

[0077] Working solution: Store in a brown bottle away from light, stable at 2-8℃ for 6 hours. It is best to prepare and use it immediately. The prepared liquid should be colorless and transparent. If it turns yellow, it cannot be used.

[0078] Add 120 mL 3.9 mM phosphatase substrate, 1.25 mL 1 M magnesium chloride, and 15 mL water. Adjust to pH 9.6 with 2 M sodium hydroxide at 25°C, make up to 145 mL with water, recheck pH at 25°C and readjust if necessary. Recheck pH at 25°C before use and adjust to pH 9.6 with 2 M sodium hydroxide. Discard if pH < 9.50 or solution is very yellow.

[0079] 2M sodium hydroxide: weigh 8g of sodium hydroxide and dissolve it in 100mL of pure water.

[0080] 1M Magnesium Chloride: Weigh 9.521 g of anhydrous magnesium chloride and dissolve in water to adjust the final volume to 100 mL. Stable at 2-8°C for 1 month.

[0081] 3.9mM phosphatase substrate: Weigh 14.5mg phosphatase substrate (pNPP) and dissolve it in 10mL 0.025M glycine / NaOH solution, pH 9.6. Store away from light. It is best to prepare it before use. The prepared liquid should be colorless and transparent. If it turns yellow, it cannot be used.

[0082] 1M Glycine / NaOH pH 9.6: Dissolve 37.5g of glycine in approximately 450mL of water and adjust the pH to 9.6 with 2M sodium hydroxide at 25°C. Add water to a final volume of 500mL. Filter through a 0.22μm filter and store at room temperature for 1 year.

[0083] 0.025M Glycine / NaOH pH 9.6: Dilute 1M Glycine / NaOH pH 9.6 40-fold, stable for 1 week at 2-8°C.

[0084] Enzyme diluent: Add 1 mL of 1 M magnesium chloride and 1 mL of 0.1 M zinc chloride to 1 L of 0.025 M glycine / NaOH solution at pH 9.6, add 100 mL of glycerol and stir well. If necessary, adjust the pH to 9.6 with 2 M sodium hydroxide at 25 °C. Stable at 2-8 °C for 2 weeks.

[0085] 0.1M Zinc Chloride: Dissolve 272 mg of zinc chloride in water and adjust to a final volume of 20 mL. Stable for 1 month at 2-8°C.

[0086] Stop solution: 2M Na 2 CO 3 , weigh 10.6g Na 2 CO 3 Dissolve in 50 mL of pure water.

[0087] (3) Measurement method

[0088] Make a PNP standard curve:

[0089] Since PNP has low solubility in water, DMOS can be used as solvent to prepare 100 mM PNP, and then Na 2 CO 3 Diluted to 10 mM, then diluted 14 times in 2-fold ratios to 2 CO 3 As 0mM PNP, the OD corresponding to the gradient of PNP from 10mM to 0mM was detected by the microplate reader at a wavelength of 405nm 405 The OD value corresponding to each concentration 405 Subtract the OD corresponding to 0 mM 405 Post-production PNP concentration and OD 405 The standard curve of the values.

[0090] Test tube:

[0091] First, add 5 μL of enzyme diluent to each well, add 145 μL of enzyme diluent with a pipette, mix well, and then measure the OD. 405 The value was taken as the value of 0min, and after reacting at 25℃ in the dark for 4min, 100μL of stop solution was added with a spray gun, and the OD was measured. 405 As a value of 4 min, the reaction is best carried out on a constant temperature incubator shaker, protected from light.

[0092] Control tube: Use 5 μL enzyme diluent instead of enzyme diluent, and the rest is the same as the test tube.

[0093] Enzyme activity calculation:

[0094] Substitute OD according to the standard curve 405 (4min-0min) Calculate the concentration of PNP (mM), the volume of each well is 0.25mL, and the amount of PNP substance (μmoL) can be calculated. According to the definition of enzyme activity, the amount of PNP substance / reaction time (min) is 1U, and then divide it by the volume of added enzyme (mL) to get the volumetric enzyme activity of alkaline phosphatase (U / mL), and then divide it by the protein concentration (mg / mL) to get the specific activity of the enzyme (U / mg).

[0095] Volume enzyme activity (U / mL) = OD 405Substitute the PNP concentration obtained by the standard curve into 0.25 / 4 / 5*1000

[0096] Specific enzyme activity (U / mg) = volumetric enzyme activity (U / mL) / protein concentration (mg / mL)

[0097] The plasmids involved in the examples are shown in Table 1:

[0098] Table 1 Plasmids used in the examples

[0099] Plasmids use Resistance pPICZαA Expression vector Zeocin (fungus)

[0100] Note: pPICZαA plasmid is a commercial plasmid.

[0101] The primer information involved in the embodiment is shown in Table 2:

[0102] Table 2 Primer information used in the examples

[0103]

[0104] Example 1: Obtaining alkaline phosphatase mutants

[0105] (1) Acquisition of mutant genes

[0106] The gene sequence of the parent alkaline phosphatase was synthesized, and the gene was used as a template to construct a plasmid of the parent alkaline phosphatase on pPICZαA with primers CIAPII-F and CIAPII-R, and amplified by homologous recombination. The amplification reaction system is shown in Table 3. Among them, the gene sequence of the parent alkaline phosphatase is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.3.

[0107] Table 3 Amplification reaction system

[0108] template 1μL 2X Taq Enzyme Mix 25μL Primer CIAPII-F 2μL Primer CIAPII-R 2μL Sterile water 20uL

[0109] The PCR product was subjected to agarose gel electrophoresis, and the size of the alkaline phosphatase electrophoresis band was about 1500 bp. The PCR product was then recovered using a small amount of DNA recovery kit to obtain the parent alkaline phosphatase DNA fragment with EcoRI and Not I restriction sites.

[0110] The recovered parent alkaline phosphatase DNA fragment was used as a template, T124L-F and T124L-R were used as primers, and PCR was performed according to the above reaction system to obtain the mutant alkaline phosphatase DNA fragment.

[0111] (2) Construction of recombinant vector

[0112] 1) Extract pPICZαA plasmid, perform agarose gel electrophoresis after double enzyme digestion with EcoRI and Not I, and then recover the product with a DNA gel recovery kit to obtain a linearized vector sequence. The double enzyme digestion system is shown in Table 4.

[0113] Table 4 Double enzyme digestion system

[0114]

[0115]

[0116] 2) The linear vector fragment obtained by enzyme digestion and the mixed target gene fragments were connected by seamless cloning to form recombinant plasmids pPICZαA-CIAPII (parent) and pPICZαA-T124L (mutant). The seamless cloning enzyme reaction system is shown in Table 5.

[0117] Table 5 Seamless cloning enzyme reaction system

[0118] Seamless cloning enzyme 5μL Linear vector xμL Insert yμL

[0119] After mixing evenly, react in a 50°C water bath for 15 min.

[0120] PS: First, use NanoDrop 2000C ultra-micro spectrophotometer to measure the DNA concentration of linear vector and target gene according to the formula:

[0121] pmols = mass ng / (fragment length bp×0.65 kDa),

[0122] x:y=1:2;x+y=5,

[0123] In addition, the amount of x and y added should be between 0.01 and 0.25 pmols, and the amount of linear vector and insert added can be calculated accordingly.

[0124] 3) Recombinant pPICZαA-CIAPII and pPICZαA-T124L plasmids were extracted respectively, and the extraction process was referred to the operating manual of the kit. After single enzyme digestion with Sal I, agarose gel electrophoresis was performed, and the product was recovered by a DNA gel recovery kit to obtain a linearized recombinant plasmid. The single enzyme digestion system is shown in Table 6.

[0125] Table 6 Single enzyme digestion system

[0126] <![CDATA[ddH 2 The]]> 27μL Plasmid template 15μL Q.cut Buffer 5μL Restriction endonuclease Sal I 3μL

[0127] After mixing, the enzyme digestion was carried out in a water bath at 37°C for 2 h. After the reaction was completed, the digestion products were subjected to agarose gel electrophoresis and then recovered using a small amount of DNA recovery kit: linear recombinant plasmids pPICZαA-CIAPII and pPICZαA-T124L.

[0128] Example 2: Transformation and screening of recombinant alkaline phosphatase parent and mutant plasmids in Pichia pastoris

[0129] Take 15 μL of linear fragment and 100 μL of X33 yeast competent cells and mix them, transfer them to an ice-precooled electroporation cup (2 mm gap between the two electrodes), and put them into an electroporator for electroporation. The electroporation parameters are: 1500V, 25 μF, 200Ω; then quickly add 200 μL, 1 mL of 1M sorbitol solution to the electroporation cup, and then add 800 μL of YPD medium, mix well and transfer to a 1.5 mL EP tube, and activate it on a shaker at 30°C for 2 hours; centrifuge the bacterial suspension to remove the supernatant, add 80 μL of YPD medium to resuspend, spread on a YPD plate containing 100 ug / mL Zeocin, invert it in a 30°C incubator, and culture until a single colony appears.

[0130] Pick the transformed yeast single colony and inoculate it in a 5mL YPD liquid medium sterile tube containing 100ug / mL Zeocin, and then inoculate it in a 1L YPD liquid medium triangular shake flask containing 100ug / mL Zeocin at 30℃250r / min for 24h. After continuing to culture at 30℃250r / min for 24h, add 1% methanol every 24h for induction and sampling, and detect the alkaline phosphatase activity in the sample. The screening result is to obtain a single colony with alkaline phosphatase activity in the expression product, extract the plasmid, send it to a biological company for sequencing, and name the mutant T124L / X33, and the parent original strain CIAPII / X33. After sequencing, it can be seen that the obtained alkaline phosphatase mutant is a mutation of the 124th threonine (Thr) in the amino acid sequence of the parent alkaline phosphatase to leucine (Leu). The amino acid sequence of the alkaline phosphatase mutant is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.2.

[0131] Example 3: Expression of alkaline phosphatase and its mutants in Pichia pastoris and determination of enzymatic properties

[0132] Scrape an appropriate amount of bacterial liquid from the glycerol tube and inoculate it on a non-resistant YPD solid medium with dense streaks. After about two days of bacterial growth, pick a small amount of bacteria and transfer it to three zones on a YPD solid medium containing 100μg / mL Zeocin resistance and grow for 4 days. Pick two single colonies from the plate containing Zeocin resistance and inoculate them into 2 bottles of 50mL BMGY first-level seed liquid respectively, and culture them in a shaking incubator at 30℃ and 250rpm for 20-24h. Inoculate 4 100mL BMGY second-level seed bottles from the BMGY first-level seed liquid at a 5% inoculation rate, and culture them in a shaking incubator at 30℃ and 250rpm for 16-20h until the bacterial concentration reaches a 20-fold diluted OD 600=15-20. A 10% inoculation amount was transferred to a fermentation tank (the total volume of the fermentation tank was 7L) with a liquid volume of 2.5L of fermentation medium (i.e., BMGY medium) for scale-up experiments. Among them, during the growth stage and the methanol induction stage of the fermentation process, the dissolved oxygen content in the fermentation tank was controlled to be above 30%, and 50% w / v of 12mlPTM1 per liter of glycerol was added for feeding during the glycerol feeding stage. The dissolved oxygen content in the fermentation tank was controlled to be above 20%, the stirring speed was 800-900rpm, and the temperature was 30°C. When the dissolved oxygen suddenly rose to above 90% during the growth stage, glycerol feeding was started; when the wet weight of the bacteria reached 200g / L during the glycerol feeding stage, glycerol feeding was stopped, and when the dissolved oxygen rose to above 90%, starvation for 1h was started, and then the methanol induction stage was started. In the early stage of fermentation, defoaming agent was appropriately added to control the generation of foam during the fermentation process. (Note: The operating steps of this experiment were carried out in accordance with the Pichia yeast fermentation manual)

[0133] The test results are shown in Table 7. In Table 7, expression level / OD 600 is the expression level and OD 600 ratio.

[0134] As can be seen from Table 7, the extracellular expression level of the alkaline phosphatase mutant fermentation broth of the present application was 484.296 mg / L after 264 h of culture, which was 39% higher than the extracellular expression level of the parent alkaline phosphatase (346.903 mg / L), and the intracellular expression level was 650.506 mg / L, which was 132% higher than the intracellular expression level of the parent alkaline phosphatase (279.493 mg / L). The overall expression level was increased by 81% compared with the parent alkaline phosphatase.

[0135] Table 7 Fermentation culture and expression level of Example 3

[0136]

[0137]

[0138] The alkaline phosphatase mutant of the present application has a significantly higher expression level than its parent alkaline phosphatase, and can be applied to the application of alkaline phosphatase mutants in enzymatic dephosphorylation of phosphate substrates, especially for labeling antibodies and / or catalyzing luminescent substrates to produce chemiluminescence.

[0139] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0140] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An alkaline phosphatase mutant, characterized in that: The alkaline phosphatase mutant includes: a polypeptide obtained by a mutation in the amino acid sequence of the parent alkaline phosphatase; the mutation includes: the threonine at position 124 in the amino acid sequence of the parent alkaline phosphatase is replaced by leucine; the nucleotide sequence of the parent alkaline phosphatase is as shown in SEQ ID NO.1, and / or the amino acid sequence of the parent alkaline phosphatase is as shown in SEQ ID NO.

3.

2. The alkaline phosphatase mutant according to claim 1, characterized in that The alkaline phosphatase mutant contains a polypeptide having a nucleotide sequence as shown in SEQ ID NO.2; Alternatively, the alkaline phosphatase mutant contains a polypeptide having an amino acid sequence as shown in SEQ ID NO.

4.

3. A polynucleotide, characterized in that The polynucleotide encodes the alkaline phosphatase mutant according to claim 1 or 2.

4. The polynucleotide according to claim 3, characterized in that The polynucleotide sequence contains the nucleotide sequence shown in SEQ ID NO.

4.

5. A recombinant vector, characterized in that: The recombinant vector contains the polynucleotide according to any one of claims 3-4.

6. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contains the recombinant vector according to claim 4.

7. The recombinant engineered bacterium according to claim 6, characterized in that The recombinant engineering bacteria is Pichia pastoris containing the recombinant vector.

8. A method for preparing an alkaline phosphatase mutant, characterized in that: The method comprises the following steps: expanding the culture of the recombinant engineering bacteria according to any one of claims 6 to 7, adding an inducer to continue the culture, collecting the culture after the culture is completed, and obtaining an alkaline phosphatase mutant.

9. Use of the alkaline phosphatase mutant according to any one of claims 1 to 2, or the alkaline phosphatase mutant obtained by the preparation method of the alkaline phosphatase mutant according to claim 8 in enzymatic dephosphorylation of phosphate substrates; Furthermore, the alkaline phosphatase mutant is used to label antibodies and / or catalyze luminescent substrates to produce chemiluminescence.

10. A method for dephosphorylating a phosphate substrate by enzymatic hydrolysis, characterized in that: The method comprises the following steps: mixing a phosphate substrate with an alkaline phosphatase mutant, and allowing the alkaline phosphatase mutant to catalyze the enzymatic hydrolysis of the phosphate substrate, wherein the alkaline phosphatase mutant is selected from at least one of the alkaline phosphatase mutants described in claims 1-2 and the alkaline phosphatase mutant obtained by the preparation method of the alkaline phosphatase mutant described in claim 8.

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