Preparation method and application of phospholipase C crystal and phospholipase C mutant

Abstract

The present invention discloses a preparation method and application of a phospholipase C crystal and a phospholipase C mutant. The crystal has a tetrahedral morphology, and its spatial structure is a fine three-dimensional structure. The structure contains 433 amino acids, and contains 2 molecules in each asymmetric unit. Each molecule contains 15 α-helical structures and 14 β-folded structures. The protein space group of the crystal is P212121. Based on the wild-type phospholipase RePLC shown in SEQ ID NO.1, a site-directed mutation occurs: the mutant R205K formed by the R mutation at the 205th position has a significant improvement in its enzyme activity. The non-specific phospholipase C (RePLC) crystal provided by the present invention provides the necessary conditions for analyzing the structure of non-specific phospholipase C from fungi, and provides a basis for further improving the performance of phospholipase RePLC through protein engineering in the future.

Description

Technical Field

[0001] The present invention relates to, in particular to, a preparation method and application of a phospholipase C crystal and a phospholipase C mutant. Background Art

[0002] Phospholipase C (PLC, EC 3.1.4.3) is an important industrial enzyme that can catalyze the hydrolysis of the Sn-3 ester bond of phospholipids to generate diacylglycerol (DAG) and phosphorylated head groups. It is widely used in food, feed, daily chemicals and other industrial fields. According to the substrate specificity of PLC, it can be mainly divided into two categories: phosphatidylinositol-specific phospholipase C (PI-PLC) and non-specific phospholipase C (NPC), among which NPC has received more attention due to its wider substrate selectivity.

[0003] The biological function of enzyme proteins depends on the molecular structure of proteins. Understanding structural information is helpful for better application and modification of enzymes. X-ray crystallography is one of the most important research methods for obtaining the three-dimensional crystal structure of proteins. Obtaining high-quality protein crystals is still a bottleneck problem in protein structure analysis. Currently, only a few NPC structures have been analyzed. After expression, purification and concentration, in order to obtain crystals that can be used for X-ray diffraction from high-purity and high-uniformity protein solutions, it is necessary to transform the proteins in the solution from a random state to a certain ordered solid state, that is, protein crystals. Protein crystallization is related to conditions such as the concentration of the protein solution, pH value, ionic strength, buffer system, growth temperature, precipitant concentration, and additive composition. Because it is impossible to predict the optimal conditions for crystal growth under existing conditions, protein crystallization is still a "trial and error" process of continuous exploration, and protein crystals with very regular internal structures are still difficult to obtain.

[0004] At present, the main industrial application of phospholipase C is oil degumming. Phospholipase C degumming can not only reduce the generation of wastewater and the use of acid and alkali, but also the product can further increase the oil yield, so it is more favored by the industry. The product diacylglycerol (DAG) also plays an important role in information transmission and cell metabolism, and can activate protein kinase K (PKC), thereby triggering a series of intracellular functions, such as cell proliferation, signal spatial distribution, etc. Therefore, phospholipase C has important application prospects in the edible oil industry. In previous reports, NPC was only found in bacteria and plants, and the protein structure database (PDB) only had NPC structures from bacteria B.cereus and C.perfringens, lacking structural information of fungal NPC. Summary of the invention

[0005] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a phospholipase C crystal.

[0006] Another object of the present invention is to provide a method for preparing the above crystal.

[0007] Another object of the present invention is to provide a phospholipase C mutant.

[0008] Another object of the present invention is to provide the application of the above-mentioned phospholipase C mutant.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A phospholipase C crystal is formed by crystallization of phospholipase C (RePLC) protein. The crystal has a tetrahedral morphology and a fine three-dimensional structure. The structure contains 433 amino acids, and each asymmetric unit contains 2 molecules. Each molecule contains 15 α-helical structures and 14 β-folded structures. The unit cell is α=β=γ=90.0°, and the protein space group of the crystal is P212121.

[0011] The structural characteristics of the active region of the crystal are as follows: its catalytic active center is mainly composed of conservative structures α9, α10, α14 and β1, β5, β7, β8, and its catalytic residues are mainly composed of glutamic acid (E61, E342), threonine (T201), arginine (R205) and aspartic acid (D257, D341). The volume of its catalytic pocket is Depth

[0012] The method for preparing the crystal comprises the following steps:

[0013] Take the purified phospholipase C protein solution, adjust the concentration to 10-20 mg / mL, inoculate it into an equal volume of crystal buffer, place the obtained crystal solution on a plastic coverslip, then invert the crystal drop on a crystallization plate, seal the middle gap with vaseline, place it in a biological incubator at 16°C for crystal culture, and collect the crystals after they grow.

[0014] The crystal buffer consists of polyethylene glycol 1500 and pH buffer, wherein the mass percentage of polyethylene glycol 1500 is 20-30%.

[0015] The pH buffer solution is a Tris solution with a molar concentration of 0 to 0.3 M and a pH value of 6.5 to 7.5.

[0016] The amino acid sequence of the phospholipase C is shown in SEQ ID NO.1.

[0017] The phospholipase C, the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2.

[0018] The phospholipase C is derived from the fungus Rasamsonia emersonii.

[0019] A phospholipase C mutant, whose amino acid sequence is shown in SEQ ID NO.3.

[0020] The nucleotide sequence of the gene encoding the phospholipase C mutant is shown in SEQ ID NO.4.

[0021] The preparation method of the phospholipase C and phospholipase C mutant comprises the following steps:

[0022] The corresponding coding gene is integrated into the pPICZαA plasmid, transferred into Escherichia coli, cultured and verified to obtain the recombinant plasmid, and the recombinant plasmid is transferred into Pichia pastoris for culture and induction. The obtained crude enzyme solution is purified to obtain the corresponding enzyme.

[0023] A genetically engineered bacterium is obtained by transferring a plasmid integrating the above-mentioned phospholipase C or phospholipase C mutant coding gene into Pichia pastoris X-33.

[0024] Application of the above-mentioned phospholipase C crystal and phospholipase C mutant in oil degumming.

[0025] Compared with the prior art, the present invention has the following advantages and effects:

[0026] (1) The genetically engineered bacterium P. pastoris X-33 / pPICZαA-replc provided by the present invention is a high-yield strain with good commercial application prospects. It has high enzyme production, low impurity protein, and simple purification, which is conducive to further expanding the application field of phospholipase C.

[0027] (2) The present invention performs molecular design based on the crystal structure of RePLC and also provides a non-specific phospholipase C (RePLC) mutant with improved activity, which is one of the mutants obtained after the following mutations occur in the amino acids in the wild-type RePLC shown in SEQ ID NO.1: RePLC mutant R205K in which the arginine R at position 205 is mutated to lysine K; RePLC mutant R205Q in which the arginine R at position 205 is mutated to glutamine Q; RePLC mutant T201C in which the threonine T at position 201 is mutated to cysteine ​​C; RePLC mutant T201S in which the threonine T at position 201 is mutated to serine S; RePLC mutant C101S in which the cysteine ​​C at position 101 is mutated to serine S; RePLC mutant N310D in which the asparagine N at position 310 is mutated to aspartic acid D.

[0028] (3) The present invention obtains non-specific phospholipase C (RePLC) mutants RePLC-R205K / R205Q, RePLC-T201C / T201S, RePLC-C101S, and RePLC-N310D by introducing a single point mutation into the wild-type non-specific phospholipase C (RePLC). Experiments have shown that the activity of RePLC-R205K in the mutant obtained by the present invention is greatly improved, reaching 1.5 times that of the wild type. Therefore, compared with the wild type, the non-specific phospholipase C (RePLC) mutant RePLC-R205K is more suitable for industrial application.

[0029] (4) In summary, the present invention provides the first fungal non-specific phospholipase C (RePLC) crystal, which provides the necessary conditions for analyzing the structure of fungal non-specific phospholipase C. The RePLC crystal structure contains detailed information related to the active center of the enzyme. The enzyme molecule is modified through protein engineering technology to improve the catalytic activity of the enzyme, making it more able to meet the needs of industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Polarized light microscope photo of non-specific phospholipase C (RePLC) crystals.

[0031] Figure 2 X-ray diffraction pattern of non-specific phospholipase C (RePLC) crystal.

[0032] Figure 3 Schematic diagram of the asymmetric unit structure of non-specific phospholipase C (RePLC).

[0033] Figure 4 Schematic representation of the monomer structure of nonspecific phospholipase C (RePLC).

[0034] Figure 5 SDS-PAGE images of wild-type and mutant non-specific phospholipase C (RePLC).

[0035] Figure 6 Results of enzyme activity determination of wild-type and mutant non-specific phospholipase C (RePLC). DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0037] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0038] Example 1 Construction of non-specific phospholipase C (RePLC) recombinant genetic engineering bacteria P. pastoris X-33 / pPICZαA-replc

[0039] (1) obtaining the complete amino acid sequence of phospholipase C from Rasamsonia emersonii, the amino acid sequence of which is shown in SEQ ID NO.1;

[0040] (2) Based on the amino acid sequence obtained in step (1), the gene coding sequence was designed according to the codon preference of Pichia pastoris, and its base sequence is shown in SEQ ID NO. 2. An EcoR I restriction site was introduced upstream, and a Sal I restriction site was introduced downstream. The resulting non-specific phospholipase C (RePLC) gene sequence was synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.;

[0041] (3) Double digest the purified gene fragment of the non-specific phospholipase C (RePLC) gene synthesized in step (2) and the plasmid pPICZαA with restriction endonucleases EcoR I and Sal I, connect, and transform into E. coli TOP10 competent cells; spread on LB (containing 25ug / ml bleomycin) plates. After the colonies grow, pick the positive clones, use the kit to extract the plasmid for sequencing verification, and obtain the pPICZαA-replc recombinant plasmid;

[0042] (4) The pPICZαA-replc recombinant plasmid obtained in step (3) was electroporated into Pichia pastoris X-33 competent cells, spread on YPD (containing 100 ug / ml bleomycin) plates, and after colonies grew, positive clones were picked to obtain the recombinant genetically engineered bacteria P. pastoris X-33 / pPICZαA-replc expression strain.

[0043] Example 2 Expression of non-specific phospholipase C (RePLC)

[0044] The genetically engineered bacteria P. pastoris X-33 / pPICZαA-replc prepared in Example 1 was added to 4LYPG liquid culture medium (containing 100 ug / ml bleomycin), cultured on a shaker at 30°C at 240 rpm, induced by adding 1% methanol every 24 h, induced at 30°C for 4 days, centrifuged at 20,000 rpm for 15 minutes, and the supernatant was collected to obtain the crude enzyme solution.

[0045] Example 3 Purification of non-specific phospholipase C (RePLC)

[0046] (1) Load the crude enzyme solution onto a Ni-NTA agarose gel column produced by Newprobe; then rinse the column with 20 ml of solution A (20 mM Tris-HCl, 300 mM sodium chloride, 20 mM imidazole, pH 7.0) to remove most of the impurities; then elute with solution B (20 mM Tris-HCl, 300 mM sodium chloride, 200 mM imidazole, pH 7.0) and collect the eluate (protein concentration is 1.0 mg / ml, total volume is about 50 ml).

[0047] (2) Desalting the eluate using a fast protein liquid chromatography system: using a HiPrep 26 / 10 desalting column (Cytiva), the equilibration and elution buffer was 20 mM Tris-HCl, 300 mM sodium chloride, pH 7.0, and the flow rate was 2 mL / min to collect the eluate with the protein peak, i.e., the desalted eluate.

[0048] (3) The desalted eluate was further passed through a gel exclusion chromatography column HiLoad 16 / 600 Superdex 200 pg (Cytiva), with an equilibration and elution buffer of 20 mM Tris-HCl, 300 mM sodium chloride, pH 7.0, and a flow rate of 1 mL / min. The peak eluate was collected to obtain a higher purity and more uniform protein (protein concentration was 2.0 mg / ml, and the total volume was about 9 ml).

[0049] (4) The peak eluate was concentrated to 1 ml using an Amicon Ultra-15 (Millipore) ultrafiltration tube with a cutoff of 10 kDa. This was the RePLC protein solution (protein concentration was 16 mg / mL). The solution was then divided into 100 μL portions using EP tubes and stored at -80°C for later use.

[0050] Example 4 Preparation of non-specific phospholipase C (RePLC) crystals

[0051] (1) Preparation of crystallization (growth) buffer

[0052] The crystallization buffer consists of precipitant polyethylene glycol 1500 and pH buffer, wherein the mass percentage of the precipitant polyethylene glycol is 30%; the pH buffer is a Tris solution, the molar concentration of the buffer is 0.1M, and the pH value is 7.0.

[0053] (2) Preparation of non-specific phospholipase C (RePLC) aqueous solution

[0054] The non-specific phospholipase C (RePLC) protein obtained in Example 3 was prepared into an aqueous solution with a concentration of 13 mg / mL.

[0055] (3) Preparation of crystallization solution

[0056] Take 0.5 μL of the non-specific phospholipase C (RePLC) aqueous solution in step (2) and inoculate it into 0.5 μL of the crystal buffer solution in step (1); use the hanging drop method to place the above crystal solution on a plastic cover glass, then invert the crystal drop on a 96-well crystallization plate, seal the middle gap with vaseline, place it in a biological incubator at 16°C for crystal culture, and collect crystal photos under a polarizing microscope after 2 weeks, as shown in Figure 2. Figure 1 As shown, the obtained crystal has a tetrahedral morphology.

[0057] Example 5 Structural determination of non-specific phospholipase C (RePLC) crystals

[0058] First, the non-specific phospholipase C (RePLC) crystals obtained in Example 4 were immersed in antifreeze solutions containing 5%, 10%, 15% and 25% (v / v) glycerol, respectively, and then quickly frozen in liquid nitrogen. Diffraction data collection, X-ray diffraction patterns such as Figure 2 The diffraction data of the crystals were processed by HKL3000 and PHNEIX software packages. 5% of the data were randomly selected for R-free calculations. The initial structure was modeled using Alphafold2 as a template, and phase information was obtained by molecular replacement. The crystal structure model of non-specific phospholipase C (RePLC) was finally optimized using PHNEIX. The quality of the model was tested using MOLPROBITY. The parameters for data collection and structure modification are as follows:

[0059] Crystal space group: P212121;

[0060] Unit cell parameters: α=β=γ=90.0°;

[0061] Resolution range :25.91-2.50;

[0062] Data integrity (%): 100.

[0063] Example 6 Spatial structural characteristics of non-specific phospholipase C (RePLC) crystals

[0064] like Figure 3As shown, there are 2 molecules in each asymmetric unit, each molecule contains 15 α-helical structures and 14 β-folded structures. Residues 33-464 and 2 N-acetylglucosamine ligands can be seen in chain A, and residues 32-464 and 3 N-acetylglucosamine ligands can be seen in chain B; the crystal structure contains a phosphatase domain.

[0065] like Figure 4 As shown, the crystal of non-specific phospholipase C (RePLC) contains three pairs of disulfide bonds, namely C87-C101, C196-C207, and C297-C298. The structural characteristics of the active region of the crystal are: its catalytic activity center is mainly composed of conservative structures α9, α10, α14 and β1, β5, β7, β8, and its catalytic residues are mainly composed of glutamic acid (E61, E342), threonine (T201), arginine (R205) and aspartic acid (D257, D341). The volume of its catalytic pocket is Depth Among them, the arginine (R205) side chain at the bottom of the RePLC catalytic pocket carries a positive charge, which can electrostatically interact with the negatively charged group at the head of the substrate, thereby affecting the enzyme activity.

[0066] The RePLC crystal structure is the first structure of fungal NPC resolved, covering detailed information related to the enzyme active center. Subsequent experiments will transform the enzyme molecule through protein engineering technology to improve the enzyme's catalytic activity, making it better meet the needs of industrial applications.

[0067] Example 7 Construction of non-specific phospholipase C (RePLC) mutant

[0068] (1) The recombinant genetically engineered bacteria E. coli TOP10 / pPICZαA-replc clone strain obtained in Example 1 was inoculated into a test tube and cultured for 10 to 12 hours, and its plasmid was extracted as a template for subsequent PCR.

[0069] (2) The mutants were constructed using the Site-directed Mutagenesis Kit (BBI). The specific steps were as follows:

[0070] 1. According to the product instructions, primers were designed using the target sequence as a template. A total of 6 mutation primer pairs were designed. The specific primer sequences are shown in Table 1;

[0071] 2. Use high-fidelity Primer STAR enzyme to introduce point mutations through PCR;

[0072] 3. Digest the template plasmid with Dpn I;

[0073] 4. Purify the digestion product;

[0074] 5. Introduce the purified product into E. coli TOP10 competent cells;

[0075] 6. Pick a single colony and inoculate it into 5 mL LB medium. After culturing at 37°C overnight, extract the plasmid for sequencing;

[0076] 7. After ensuring the sequence is correct, the mutant was induced to express and the protein was extracted according to Examples 2 and 3. After purification, it was analyzed by SDS-PAGE gel electrophoresis according to the Laemmli method to obtain a single protein band. The results are as follows: Figure 5 shown.

[0077] For any steps not covered in the above experimental method, please refer to the kit instructions.

[0078] Table 1 Nucleotide sequences of primers

[0079]

[0080] Example 8 Enzyme activity determination

[0081] The enzyme activity was determined using p-nitrophenylphosphocholine (p-NPPC) as a substrate to determine the enzyme activity of the purified enzymes prepared in Example 3 and Example 7. The specific determination method is:

[0082] Prepare 50mM Tris-HCl buffer (pH 7.0) containing 10mM substrate as substrate solution, the reaction system is 200μL, take 20μL enzyme solution (2mg / mL) and add 180μL substrate solution, react at the optimal temperature for 10min, inactivate and terminate the reaction, measure the absorbance at 410nm, and calculate the enzyme activity by determining the content of the product p-nitrophenol (pNP) through the standard curve. The enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1μM pNP per minute of reaction under the optimal reaction conditions.

[0083] The results of enzyme activity determination of all the above non-specific phospholipase C (RePLC) mutants are as follows Figure 6 As shown, WT is a non-specific phospholipase C that has not been mutated. The experimental results show that the activity of the mutant RePLC-R205K is greatly improved, reaching 1.5 times that of the wild type, while other mutation sites do not show obvious activity improvement and may even cause enzyme inactivation.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A phospholipase C mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. The gene encoding the phospholipase C mutant according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A phospholipase C crystal, characterized in that: It is formed by crystallization of phospholipase C (RePLC) protein. The crystal has a tetrahedral morphology and a fine three-dimensional spatial structure. It contains 433 amino acids, 2 molecules in each asymmetric unit, and each molecule contains 15 α-helical structures and 14 β-folded structures. The structure has a unit cell of a = 62.81 Å, b = 134.528 Å, c = 137.51 Å, α= β = γ = 90.0°, and the protein space group of the crystal is P212121. The amino acid sequence of the phospholipase C is shown in SEQ ID NO.

1.

4. The method for preparing the crystal according to claim 3, characterized in that The steps include: Take the purified phospholipase C protein solution, adjust the concentration to 13 mg / mL, inoculate it into an equal volume of crystal buffer, place the obtained crystal droplet on a plastic cover glass, then invert the crystal droplet on a crystallization plate, seal the middle gap with vaseline, place it in a biological incubator at 16°C for crystal culture, and collect the crystals after they grow out; The crystal buffer is composed of polyethylene glycol 1500 and pH buffer, wherein the mass percentage of polyethylene glycol 1500 is 30%; The pH buffer solution is a Tris solution with a molar concentration of 0.1 M and a pH value of 7.

0.

5. The crystal according to claim 3, characterized in that: The phospholipase C, the nucleotide sequence of its encoding gene is shown in SEQ ID NO.

2.

6. The method for preparing the phospholipase C mutant according to claim 1, characterized in that The steps include: The corresponding coding gene is integrated into the pPICZαA plasmid, transferred into Escherichia coli, cultured and verified to obtain the recombinant plasmid, and the recombinant plasmid is transferred into Pichia pastoris for culture and induction. The obtained crude enzyme solution is purified to obtain the corresponding enzyme.

7. A genetically engineered bacterium, characterized in that: The plasmid integrating the phospholipase C mutant encoding gene of claim 1 is transferred into Pichia pastoris P. pastoris Obtained after X-33.

8. Use of the phospholipase C mutant according to claim 1 in oil degumming.

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