Phospholipase B from aspergillus fumigatus and preparation method thereof

Abstract

The invention provides phospholipase B from aspergillus fumigatus and a preparation method thereof. The amino acid sequence of the enzyme is as shown in SEQ ID NO: 1, and a catalytic triad of the enzyme is composed of Ser-122, Asp-178 and His-211. The invention provides a coding gene (SEQ ID NO: 2) of the gene, an expression vector pGEX-6P-1 containing the gene, a host cell escherichia coli BL21 (DE3), and a corresponding recombinant expression and purification method. The enzyme can specifically hydrolyze sn-1 and sn-2 fatty acid ester bonds of a GPI anchor, so that GPI-APs on a membrane can be efficiently released. Compared with common PI-PLC, the PlbH has the remarkable advantages that the PlbH is more excellent in enzymatic property, more stable in effect, efficient in release capacity for various GPI-APs and the like, and a brand new tool enzyme which can replace PI-PLC and is complementary in action mechanism is provided for GPI-APs research.

Description

Technical Field

[0001] This invention relates to the field of enzyme gene engineering technology, specifically to a phospholipase B derived from Aspergillus fumigatus and its preparation method. Background Technology

[0002] Phospholipases are a class of enzymes that hydrolyze phospholipid bonds and are widely involved in key biological processes such as membrane construction, signal transduction, and metabolic regulation. Based on their hydrolysis sites, phospholipases can be classified into several types, including A1, A2, B, C, and D. Among them, phospholipase B can simultaneously hydrolyze the acyl bonds at both the Sn-1 and Sn-2 positions of phospholipid molecules, exhibiting dual activity as a lysophospholipase and a hydrolase.

[0003] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a class of important functional proteins that are anchored to the cell membrane surface through the glycosylphosphatidylinositol structure. They participate in various physiological and pathological processes such as cell recognition, immune response, and signal transduction. The isolation and functional study of GPI-APs usually require their specific release from the cell membrane.

[0004] Currently, in scientific research, phosphatidylinositol-specific phospholipase C (PI-PLC) is commonly used to act on phosphatidylinositol, releasing GPI-APs from the cell membrane. Therefore, PI-PLC is widely used in the study and identification of cell surface GPI-APs. However, PI-PLC, as a commonly used, especially commercially available, technology, has clear limitations: its catalytic activity is usually highly dependent on calcium ions, making it easily inhibited by metal ion chelating agents (such as EDTA). Furthermore, most PI-PLCs can only cleave unacylated phosphatidylinositol and cannot function on GPI structures with acylated inositol rings. These characteristics limit the application of PI-PLC in complex, low-calcium, or metal ion chelation systems.

[0005] Therefore, there is an urgent need in this field for a novel tool enzyme with superior enzymatic properties, more stable function, and efficient release capability for a variety of GPI-APs, in order to overcome the many limitations of existing PI-PLCs in practical applications.

[0006] To address the aforementioned issues, this invention has for the first time identified and developed a phospholipase B with unique functions from Aspergillus fumigatus. Unlike PI-PLC, which is commonly used to release GPI-APs, this enzyme, as phospholipase B, can efficiently and specifically act on the fatty acyl chain of GPI anchors. By cleaving fatty acids at the sn-1 and sn-2 positions, it achieves the release of GPI-APs. This not only provides a novel tool for GPI-AP research that can replace PI-PLC and has a complementary mechanism of action, but also expands the application boundaries of phospholipase B. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a phospholipase B derived from Aspergillus fumigatus and its preparation method, thereby solving the technical problem of "many limitations of existing phosphatidylinositol-specific phospholipase C in practical applications".

[0008] To achieve the above objectives, the present invention is implemented using the following technical solution: In this invention, the phospholipase B derived from Aspergillus fumigatus is named PlbH; the PlbH gene refers to the nucleotide sequence encoding the phospholipase B; the PlbH protein refers to the protein encoded by the PlbH gene. This abbreviation will be used in the following invention description and specific embodiments.

[0009] In a first aspect, the present invention provides a phospholipase B derived from Aspergillus fumigatus, comprising the amino acid sequence shown in SEQ ID NO:1.

[0010] Specifically, the catalytic triplet of phospholipase B is composed of serine at position 122, aspartic acid at position 178, and histidine at position 211.

[0011] In a second aspect, the present invention provides a gene encoding the above-mentioned phospholipase B, comprising the nucleotide sequence shown in SEQ ID NO:2.

[0012] Thirdly, the present invention provides an expression vector containing the above-mentioned coding gene.

[0013] Fourthly, the present invention provides a recombinant bacterium comprising the above-described expression vector.

[0014] Preferably, the expression vector is pGEX-6P-1.

[0015] Preferably, the recombinant bacteria is constructed using Escherichia coli BL21(DE3) as the host.

[0016] Fourthly, the present invention provides a method for preparing the above-mentioned phospholipase B, comprising the following steps: (1) Insert the nucleic acid molecule shown in SEQ ID NO:2 into the pGEX-6P-1 expression vector to construct a recombinant plasmid; (2) The recombinant plasmid was introduced into Escherichia coli BL21(DE3) to obtain recombinant bacteria; (3) Cultivate the recombinant bacteria and induce the expression of the phospholipase B; (4) The phospholipase B was purified from the culture.

[0017] Fifthly, the present invention provides the application of the above-mentioned phospholipase B in the release of glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell membrane.

[0018] Compared with the prior art, the beneficial effects achieved by the present invention are: (1) This invention successfully cloned and heterologously expressed phospholipase B from Aspergillus fumigatus for the first time. It releases protein by hydrolyzing the fatty acid ester bonds at the sn-1 and sn-2 positions of the glycerol backbone in the GPI anchor. Its mechanism of action is completely different from that of traditional PI-PLC. The enzyme is stable in activity over a wide pH range and retains >90% of its activity under EDTA treatment, overcoming the limitation of PI-PLC's strict dependence on the reaction environment.

[0019] (2) The phospholipase B of the present invention can effectively release at least four different GPI-anchored proteins from the cell membrane, and can achieve broad-spectrum and efficient release to multiple targets.

[0020] (3) The phospholipase B of the present invention is not only highly efficient and stable, but its unique mechanism of action enables it to release GPI-APs that PI-PLC cannot act on, solving the application blind spots of existing tools due to their mechanism of action, and providing a brand-new key tool for basic research in life sciences and the development of related technologies. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the homology analysis of Aspergillus fumigatus PlbH.

[0022] Figure 2 This is a schematic diagram illustrating the structural characteristics of Aspergillus fumigatus PlbH. Figure 2 A is a schematic diagram of the α / β-hydrolase domain in the PlbH hydrolase superfamily. Figure 2 B represents the homology modeling of PlbH, resulting in a 3D visualization of the three-level structure.

[0023] Figure 3 For prokaryotic expression of PlbH, Figure 3 A represents the results of purifying GST-PlbH recombinant protein using GST tag purification resin. Figure 3 B is a schematic diagram illustrating the use of the PGEX-6P1-PlbH vector to express the GST-PlbH recombinant protein. Figure 3 C is the LC-MS / MS image of the purified GST-PlbH recombinant protein.

[0024] Figure 4 Thin-layer chromatography analysis of phospholipase B activity in PlbH.

[0025] Figure 5 SDS-PAGE analysis of GPI-APs components released by PlbH.

[0026] Figure 6 Western blot detection of Mp1 protein release from PlbH.

[0027] Figure 7 Analysis of hydrolysis sites of GPI-anchor using PlbH. Figure 7 A is a schematic diagram showing the release of glycerol from GPI-APs after treatment with pyridine hydrofluoric acid. Figure 7 B represents the model for the release of GPI-APs from the plasma membrane by PlbH. Figure 7 Comparison of high performance liquid chromatography results for C, where C represents glycerol. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Based on the above-mentioned invention, the present invention provides a method for efficiently preparing Aspergillus fumigatus phospholipase B (PlbH) and applying it to release glycosylphosphatidylinositol anchored proteins (GPI-APs) through genetic engineering. Those skilled in the art will understand that the following specific embodiments are merely descriptions of preferred embodiments of the present invention without departing from the essence of the present invention, and are not intended to limit the present invention.

[0030] To obtain the recombinant PlbH protein, the gene can be constructed in a suitable expression vector and transferred into a host cell for expression. The expression system can be a prokaryotic expression system (such as the Escherichia coli system) or a eukaryotic expression system (such as yeast, insect cell or mammalian cell system).

[0031] Preferably, a prokaryotic expression system is used to achieve rapid and low-cost production. The expression vector may contain tag sequences that are easy to purify or detect, such as glutathione S-transferase tags, histidine tags, etc.

[0032] Purification of recombinant proteins can be performed based on the selected expression system and tag. It can be a fusion protein with a GST tag, which can be specifically purified using glutathione affinity resin; or a protein with a His tag, which can be purified using nickel ion affinity resin. The purified protein can be transferred to a suitable storage buffer by dialysis or chromatography.

[0033] The purified phospholipase B can be used to hydrolyze GPI anchors on biofilms (especially fungal cell membranes), thereby releasing the GPI-anchored proteins attached thereto. The biofilms are not limited to Aspergillus fumigatus, but can also be derived from other fungi, protists or higher eukaryotes. The reaction can be carried out in a buffer system close to physiological conditions (Hank's balanced salt solution at pH 7.4), or it can be optimized in other pH or ionic strength buffers as needed.

[0034] In a preferred embodiment of the present invention, the host cell of the phospholipase B derived from Aspergillus fumigatus is Escherichia coli BL21(DE3), and the expression vector is pGEX-6P-1.

[0035] In a preferred embodiment of the present invention, the expression vector contains a GST tag coding sequence for expressing the GST-PlbH recombinant protein.

[0036] In the following embodiments of the present invention, the culture medium and reagent formulations used are as follows: LB liquid medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 15g / L, deionized water to 1L; Sevage reagent: a solution of chloroform and n-butanol in a volume ratio of 3:1.

[0037] Buffer II solution: 20 mM Tris-HCl, 2 mM EDTA.

[0038] The liquid CM medium used in this invention is a complete culture medium for culturing Aspergillus fumigatus, and its composition can refer to conventional formulations in the art, such as containing a carbon source, a nitrogen source, and inorganic salts. Specifically, the CM medium used in the embodiments of this invention contains 10 g / L glucose, 5 g / L yeast extract, and 5 g / L peptone.

[0039] According to the product manual, the unit U of PI-PLC is defined as the amount of enzyme required to release 1.0 μmol of water-soluble phosphorus from the substrate within 1 minute under conditions of 37°C and pH 7.5.

[0040] Example 1: Cloning, Expression, Purification, and Identification of the PlbH Gene (1) The cDNA sequence of the PlbH gene synthesized by Beijing Sangon Biotech Co., Ltd. was cloned into the pGEX-6P-1 vector carrying the GST protein coding sequence to construct the recombinant plasmid pGEX-PlbH, see below. Figure 3 B; (2) pGEX-PlbH was introduced into Escherichia coli BL21(DE3) to induce the expression of recombinant protein. The cells were inoculated into LB liquid medium containing 50 µg / mL ampicillin and cultured at 37°C with shaking until OD.600 The value reached 0.6; then, isopropyl-β-D-thiogalactoside (IPTG) at a concentration of 0.4 mmol / L was added, and the cells were induced and cultured at 16℃ for another 12 h to prepare cell extracts; (3) Collect cell extracts and separate and purify recombinant proteins using a GST tag purification kit to obtain purified GST-PlbH recombinant protein.

[0041] Homology analysis of PlbH, such as Figure 1 As shown in the figure, multiple sequence alignments were performed on the PlbH protein and its homologous proteins from mice (CAJ18409.1), rats (XP_032771444.1), Saccharomyces cerevisiae (NP_013219.1), Caenorhabditis elegans (NP_001293296.1), and Aspergillus niger (EHA25481.1). Conserved residues in all proteins are highlighted in red. Based on the principle of high conservation of the sequence characteristics (nucleophilic group, acidic group, and basic group in sequence) and topological position characteristics (all three active sites are located on the ring structure formed between the β-sheet and the α-helix) of the hydrolase family catalytic triad, it is inferred that its catalytic triad consists of serine at position 122 (Ser-122), aspartic acid at position 178 (Asp-178), and histidine at position 211 (His-211), which are highlighted in green in the figure.

[0042] Above the protein sequence of Aspergillus fumigatus PlbH (accession number: XP_747799.1), the predicted secondary structural elements are labeled using two symbols: α-helix and β-sheet, where the β-sheet is marked and numbered with arrows 1-9 and the α-helix is ​​marked with helix symbols 1-5.

[0043] Homology comparison results showed that the PlbH gene shared 35%, 27%, and 78% sequence homology with phospholipase B genes from mice, rats, and Aspergillus niger, respectively. The PlbH genomic sequence is 1048 bp long, containing 4 introns and 5 exons; its cDNA sequence is 726 bp long, encoding a protein of 241 amino acids. This protein (PlbH) contains the conserved lipase characteristic motif GXSXG, further supporting the inference of the aforementioned catalytic triplet.

[0044] Figure 2 A schematic diagram illustrating the structural characteristics of Aspergillus fumigatus PlbH, as shown below. Figure 2 As shown in Figure A, PlbH possesses an α / β-hydrolase domain.

[0045] like Figure 2As shown in Figure B, multiple alignments of the amino acid sequences of PlbH and its orthologs from different species were performed, followed by homology modeling and visualization of the tertiary structure. The results indicate that the catalytic triplet is located in the active pocket center of the PlbH protein, suggesting that PlbH is a new member of the α / β-hydrolase family.

[0046] like Figure 3 As shown in Figure A, the band size of the GST-PlbH recombinant protein is between 40-50 kDa. The band is clear and well-defined, indicating that the GST tagging system is effective during the purification process, the protein expression is successful, and the purification effect is good.

[0047] like Figure 3 As shown in C, the purified GST-PlbH recombinant protein was confirmed by LC-MS / MS. The matching peptides are shown in bold red. The mass spectrometry sequence coverage reached 89% with the theoretical sequence, proving that PlbH was successfully expressed.

[0048] Example 2; Phospholipase B activity of GST-PlbH recombinant protein This example is used to verify whether the GST-PlbH recombinant protein prepared in Example 1 has the expected broad-spectrum lysophospholipase (phospholipase B) activity.

[0049] Four lysophosphatidylinosides, namely lysophosphatidylcholine (LPC), lysophosphatidylserine (LPS), lysophosphatidylglycerol (LPG), and lysophosphatidylethanolamine (LPE), were used as substrates.

[0050] The reaction mixture contains: 50 mM Tris-HCl buffer (pH 8.0), 1 mM dithiothreitol (DTT) and 100 µg of any of the above lysophospholipid substrates, with a total volume of 200 µL; Add 50 µg of the GST-PlbH recombinant protein purified in Example 1 to each reaction system, and place the reaction system at 30 °C. o Incubate in a water bath at C for 1 hour. Add 800 µL of chloroform-methanol mixture (chloroform to methanol volume ratio 2:1) and 20 µL of 2% phosphoric acid to each system to terminate the reaction. Allow to stand and separate into layers. Take the lower organic phase, dry it with nitrogen, and redissolve the residue with 10 µL of chloroform. Spot the solution onto a thin-layer chromatography plate and develop it with a mixture of chloroform / methanol / 28% ammonia (volume ratio 65:25:10) as the developing solvent.

[0051] Figure 4Thin-layer chromatography (TLC) analysis of the phospholipase B activity of purified GST-PlbH recombinant protein shows that PlbH can hydrolyze LPC, LPS, LPG, and LPE, releasing free fatty acids. The chemical reaction equation above illustrates the principle of lysophospholipid hydrolysis by phospholipase B, indicating that PlbH has broad-spectrum phospholipase B activity.

[0052] Example 3: GST-PlbH recombinant protein releases GPI-APs components from the cell membrane This embodiment provides a method for releasing glycosylphosphatidylinositol-anchored proteins (GPI-APs) on the cell membrane using the GST-PlbH recombinant protein prepared in Example 1, specifically including the following steps: (1) Take 1×10 8 Fresh Aspergillus fumigatus conidia were inoculated into liquid CM medium and cultured at 37°C for 48 h. The mycelium was collected, rapidly ground in liquid nitrogen, and resuspended in 0.05 M Tris-HCl buffer at pH 7.8. The mixture was centrifuged at 13000 r / min for 15 min, and the supernatant was collected. The supernatant was ultracentrifuged at 50000 g for 10 min, and the precipitate was collected to obtain the cell membrane component. The cell membrane component was resuspended in buffer II solution at pH 8, and then ultracentrifuged at 50000 g for 1 h. The resuspension and centrifugation were repeated three times to obtain the purified cell membrane component. (2) Add 50 µg of the purified GST-PlbH recombinant protein prepared in Example 1 to the purified cell membrane fraction above, incubate at 30 °C for 2 h, and determine the activity using Hank's balanced salt solution at pH 7.4. After the reaction is complete, collect the supernatant by ultracentrifugation at 50000 g for 40 min, and then... o Add an equal volume of pyridine hydrofluoric acid to the supernatant at C and incubate for 3 h to release glycerol. Remove the protein and organic solvent with 50 μL of Sevage reagent to obtain the GPI-APs component.

[0053] Figure 7 A is a schematic diagram of the structure of GPI-APs releasing glycerol after treatment with pyridine hydrofluoric acid.

[0054] Figure 7 Model B represents the release of GPI-APs from the plasma membrane by PlbH, indicating that PlbH hydrolyzes the ester bonds at the sn-1 and sn-2 positions of the GPI-anchor glycerol ester in GPI-APs, cleaving the saturated fatty acid chain and releasing GPI-APs from the cell membrane.

[0055] Figure 7C represents the high-performance liquid chromatography (HPLC) comparison results of glycerol. Blank represents the cell membrane treatment with an equal volume of buffer instead of PlbH as a blank control. Enzyme digestion is the product obtained by treating the supernatant of cell membranes treated with PlbH for 1 hour with HF after ultracentrifugation. Standard substance is the glycerol standard control group. Figure 7 C verified that PlbH performs the function of phospholipase B. After the fatty acids at the sn-1 and sn-2 positions are removed, the glycerol backbone is exposed, and then free glycerol is released by HF. If PlbH only cuts one fatty acid like phospholipase A, or cuts at other positions like phospholipase C, complete free glycerol will not be produced.

[0056] Example 4; Identification and analysis of GPI-APs released by GST-PlbH recombinant protein This embodiment is used to specifically identify and analyze the types of GPI-APs released from the cell membrane after treatment by the method in Example 3.

[0057] Following the method described in Example 3, the cell membrane components of Aspergillus fumigatus were treated with GST-PlbH recombinant protein, and a supernatant containing GPI-APs was obtained. The supernatant was then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0058] like Figure 5 As shown, multiple protein bands were visible after electrophoresis. Target bands corresponding to different molecular weights were cut off, and the proteins within were subjected to enzymatic digestion and peptide extraction, followed by LC-MS / MS analysis. The obtained mass spectrometry data were compared with protein databases.

[0059] The mass spectrometry identification results are shown in Table 1. Four GPI-APs specifically released by the GST-PlbH recombinant protein were successfully identified from the cell membrane, including: EAL84472.1 (cell wall galactomannose protein Mp1), EAL87212.1 (specified GPI-anchored cell wall protein Dan4), EAL87073.1 (functionally conserved putative protein), and EAL92273.1 (extracellular serine-threonine enriched protein).

[0060] Table 1

[0061] All identified proteins were bioinformatically predicted to be glycosylphosphatidylinositol-anchored proteins (GPI-APs), and their subcellular localization was directed towards the cell wall or extracellular space. This result directly confirms that the GST-PlbH recombinant protein prepared in Example 1 can effectively release a variety of endogenous GPI-APs from the Aspergillus fumigatus cell membrane, indicating that phospholipase B (PlbH) derived from Aspergillus fumigatus is a novel tool enzyme capable of efficiently and specifically releasing GPI-APs from the cell membrane.

[0062] To further verify this, the GST-PlbH recombinant protein prepared in Example 1 and the GPI-APs component obtained in Example 3 were co-incubated at 30°C for 1 h. The supernatant after ultracentrifugation was then used for Western blotting. The release of galactomannose protein (Mp1), specifically localized to the cell wall, by PlbH was detected using an anti-Mp1 monoclonal antibody.

[0063] like Figure 6 As shown, no Mp1 protein band was detected in the control group without PlbH, while an Mp1 band consistent with the theoretical molecular weight was detected in the experimental group with PlbH. This indicates that PlbH can release the GPI-anchored cell wall protein Mp1 from the membrane, further confirming the function of PlbH in releasing GPI-APs.

[0064] Comparative Example 1: Efficiency of GPI-APs release between GST-PlbH recombinant protein and commercial PI-PLC This comparative example is used to compare the efficiency of the GST-PlbH recombinant protein prepared in this invention and the commonly used phosphatidylinositol-specific phospholipase C (PI-PLC) in releasing GPI-APs from the cell membrane of Aspergillus fumigatus, using four proteins: EAL84472.1, EAL87212.1, EAL87073.1, and EAL92273.1 as examples.

[0065] The purified GST-PlbH recombinant protein prepared in Example 1 was set as the experimental group, the commercial phosphatidylinositol-specific phospholipase C (derived from Bacillus cereus, catalog number P5542) was set as the control group, and an equal volume of buffer was set as the blank control group.

[0066] The substrate was a cell membrane component of Aspergillus fumigatus prepared according to the method described in Example 3.

[0067] The reaction system consisted of a cell membrane fraction containing 200 µg of protein from each group, resuspended in 200 µL of Hank's balanced salt solution at pH 7.4.

[0068] To fairly compare the efficiency of the two enzymes in releasing GPI-APs under the same reaction conditions, the saturation doses of the two enzymes in the reaction system were first determined through preliminary experiments. For PlbH, saturation was achieved at 50 µg; for PI-PLC, saturation was achieved at 50 U. Therefore, these saturation doses were used in subsequent comparative experiments.

[0069] Experimental group: 50 µg of purified GST-PlbH recombinant protein prepared in Example 1 was added.

[0070] Comparative group: Add a 50U PI-PLC.

[0071] Blank control group: Add an equal volume of buffer solution.

[0072] All groups were incubated at 30°C for 60 min. After the reaction was completed, the reaction tubes were immediately placed on ice, and the supernatant was then centrifuged at 50,000 g for 40 min.

[0073] 20 µL of the supernatant was taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis. The primary antibody was a mouse monoclonal antibody against four proteins, and the secondary antibody was HRP-labeled goat anti-mouse IgG. Chemiluminescence was used for development, and the target bands were analyzed for grayscale values ​​using ImageJ software. The net signal value was calculated based on the grayscale value. The relative release efficiency was calculated as (net signal value of the control group / net signal value of the experimental group) × 100%. The results are shown in Table 2.

[0074] Table 2

[0075] As shown in Table 2, the phospholipase B derived from Aspergillus fumigatus provided by this invention has a higher release efficiency for various GPI-APs than the currently commonly used tool enzyme PI-PLC.

[0076] Comparative Example 2: Environmental stability comparison between GST-PlbH recombinant protein and commercial PI-PLC This comparative example compares the environmental stability of GST-PlbH recombinant protein and commercial PI-PLC by comparing the effects of different pH environments and metal ion chelating agents on enzyme activity.

[0077] 50 mM Tris-HCl buffer solutions with pH values ​​of 6.0, 7.4, and 8.5 were prepared as reaction systems. Two parallel reactions were set up at each pH condition: the experimental group was treated with 50 µg of purified GST-PlbH recombinant protein, and the control group was treated with 50 U of commercially available PI-PLC. Using Aspergillus fumigatus cell membrane components as substrates, the Mp1 protein release assay was performed according to the method described in Example 4, and Western blotting was used for quantitative detection. The relative activities of each enzyme at each pH condition were calculated with the activity measured at pH 7.4 as 100%, and the results are shown in Table 3.

[0078] Based on Hank's balanced salt solution at pH 7.4, three treatment conditions were set up: the control group was without any additional components; experimental group A was with 5 mM EDTA added; and experimental group B was with 2 mM CaCl2 added. Under each condition, the Mp1 protein release experiment was carried out in the experimental group (GST-PlbH) and the control group (PI-PLC). It should be noted that the preparation method of the cell membrane component is the same as step (1) in Example 3, but the final washing step with buffer II solution is omitted to obtain the untreated membrane component as the substrate. With the enzyme activity of the control group as 100% as the baseline, the change in enzyme activity after adding EDTA or CaCl2 was calculated, and the results are shown in Table 3.

[0079] Table 3

[0080] As shown in Table 3, the GST-PlbH of this invention maintains a high activity of over 80% within a pH range of 6.0 to 8.5, exhibiting broad pH adaptability. In contrast, the activity of commercially available PI-PLC is more sensitive to pH changes; at pH 6.0 and 8.5, its activity decreases to 30% and 50%, respectively, and its stability is significantly lower than that of GST-PlbH. After adding the metal ion chelating agent EDTA, the activity of GST-PlbH was not significantly affected, while the activity of PI-PLC was strongly inhibited. Supplementing Ca²⁺... + It has no effect on the activity of GST-PlbH, but it can maintain the normal activity of PI-PLC.

[0081] The above results demonstrate that the phospholipase B (GST-PlbH) provided by this invention has superior enzymatic properties compared to commercially available PI-PLC, exhibiting stable activity over a wide pH range and being independent of Ca²⁺. + As divalent metal ions, they possess higher reliability and broader application potential in various complex biochemical and cell experimental environments.

[0082] SEQ ID NO:1: Amino acid sequence of phospholipase B (PlbH) from Aspergillus fumigatus The sequence consists of 241 amino acid residues, as shown below: MAPPRAPYIVPALKKHTATVIMAHGLGDRMSLAQNWRRRGMFDEVAFIFPNAPMIPITVNFGMTMPGWHDLTKLGRELDYESAIRHQDEPGVLRSRDYFNTLIKEQIDKGIKPSRIVLGG FSQGAAISVFTGITCKEKLGGVFGLSSYLVLSDKLKNYIPENWPNKKTPFFLAHGLEDEIVLFDFGDLSAKKMKEIGLEDVTFKSYPNLGHSADPVEIEDLARFLQKVIPPEDDGQASAGL SEQ ID NO:2: Nucleotide sequence encoding phospholipase B (PlbH) from Aspergillus fumigatus. This sequence is the cDNA sequence of the PlbH gene, with a full length of 726 bp, as shown below: ATGGCTCTCCTCGCGCACCGTACATTGTGCCGGCGCTTAAAAAACACACGGCGACGGTCATCATGGCCCATGGACTGGGCGACAGGATGTCCCTTGCTCAGAACTGGCGTCGCCGGGGCATGTTCGATGAGGTGGCTTTCATCTTCCCAAATGCGCCTATGATCCCGATCACGGTGAACT TCGGAATGACCATGCCCGGATGGCACGACTTGACGAAGCTTGGTCGCGAGCTCGATTATGAATCAGCCATTCGGCACCAGGACGAGCCGGGTGTCCTTCGATCTCGCGACTACTTCAACACTTTGATCAAGGAACAGATTGATAAGGGCATCAAGCCCTCACGGATTGTTCTGGGTGGCTTC TCCCAAGGAGCTGCCATATCTGTCTTTACTGGTATTACCTGCAAAGAGAAGCTCGGCGGTGTCTTCGGTTTGTCCAGCTATCTTGTTCTCAGTGACAAGCTCAAGAATTACTACCGGAGAATTGGCCGAATAAGAAGACGCCTTTCTTCCTCGCTCATGGCTTGGAAGATGAAATCGTGC TGTTCGACTTCGGTGATCTGTCGGCGAAGAAGATGAAAGAGATCGGCTTGGAGGATGTCACTTTCAAATCTTATCCTAACTTGGGCCACTCCGCCGATCCAGTAGAGATTGAGGATTTGGCGCGATTCCTTCAGAAAAGTCATTCCTCCAGAGGACGACGGGCAGGCTTCTGCCGGATTATGA It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

Claims

1. A phospholipase B derived from Aspergillus fumigatus, characterized in that, It contains the amino acid sequence shown in SEQ ID NO:

1.

2. The phospholipase B according to claim 1, characterized in that, Its catalytic triad consists of serine at position 122, aspartic acid at position 178, and histidine at position 211.

3. A gene encoding the phospholipase B of claim 1, characterized in that, Includes the nucleotide sequence shown in SEQ ID NO:

2.

4. An expression vector comprising the encoding gene of claim 3.

5. A recombinant bacterium comprising the expression vector of claim 4.

6. The expression vector according to claim 4, characterized in that, The expression vector is pGEX-6P-1.

7. The recombinant bacteria according to claim 5, characterized in that, The recombinant bacteria were constructed using Escherichia coli BL21(DE3) as the host.

8. A method for preparing phospholipase B as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Insert the nucleic acid molecule shown in SEQ ID NO:2 into the pGEX-6P-1 expression vector to construct a recombinant plasmid; (2) The recombinant plasmid was introduced into Escherichia coli BL21(DE3) to obtain recombinant bacteria; (3) Cultivate the recombinant bacteria and induce the expression of the phospholipase B; (4) The phospholipase B was purified from the culture.

9. The use of phospholipase B as described in any one of claims 1-3 in the release of glycosylphosphatidylinositol-anchored proteins on the cell membrane.

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