Marine phospholipase c mutant and use thereof

Abstract

Provided are a marine phospholipase C mutant and a use thereof. Compared with wild-type (WT) enzyme protein, at 40°C and under conditions where enzyme protein activity remains essentially unchanged, the enzyme protein half-life of the constructed mutant can be increased to 3.46 and 9.25 times that of the original enzyme protein, and the residual phosphorus content in degummed oil can be reduced from 551 ppm to 72 ppm, which shows a significant improvement over the final residual phosphorus content of 175 ppm for the blank control and the final residual phosphorus content of 131 ppm for the WT. These results demonstrate that the enzyme has improved thermal stability, enables oil degumming under various conditions, and is suitable for a broader range of industrial applications.

Description

Marine phospholipase C mutant and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a marine phospholipase C mutant and application thereof. BACKGROUND

[0002] Phospholipase C (PLC, EC 3.1.4.3) is an important industrial enzyme, which can catalyze the hydrolysis of phospholipid Sn-3 ester bond to generate diglyceride (DAG) and phosphorylated head group, and is widely used in food, feed, daily chemical and other industrial fields. According to the substrate specificity of PLC, it can be mainly divided into two types: phosphatidylinositol-specific phospholipase C (PI-PLC) and non-specific phospholipase C (NPC), wherein NPC is more concerned due to its wider substrate selectivity.

[0003] In industry, the activity and stability of the enzyme are the key indicators to determine whether it can be industrialized. The enzyme with better temperature tolerance can reduce the amount of enzyme added during the reaction process, and the enzyme with good temperature tolerance can maintain high enzyme activity in long-term storage. The strategies to improve the thermal stability of the enzyme include introducing non-covalent / covalent interaction (hydrophobic interaction, hydrogen bond, salt bridge, aromatic ring interaction, disulfide bond), ring truncation, C-terminal and N-terminal engineering, and increasing the number of proline / reducing the number of glycine. The present application adopts the proline introduction strategy. The difficulty of the proline introduction strategy lies in the confirmation of the introduction site, and the related research is less. The present application can provide a certain reference for the proline introduction strategy.

[0004] At present, the most important industrial application of phospholipase C is oil degumming. Phospholipase C degumming can not only reduce the generation of waste water and the use amount of acid and alkali, but also can further increase the oil yield, so it is more favored by industry. The product diglyceride (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 an important application prospect in the edible oil industry field.

[0005] SUMMARY

[0006] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provides a marine phospholipase C mutant.

[0007] Another object of the present application is to provide the application of the marine phospholipase C mutant.

[0008] The object of the present application is achieved by the following technical scheme:

[0009] A marine phospholipase C mutant is at least one of the following:

[0010] TiPLC-E92P, which has an E92P mutation relative to wild-type TiPLC;

[0011] TiPLC-K105P, which has a K105P mutation relative to wild-type TiPLC;

[0012] TiPLC-T150P, which has a T150P mutation relative to wild-type TiPLC;

[0013] TiPLC-A168P, which has an A168P mutation relative to wild-type TiPLC;

[0014] TiPLC-A359P, which has an A359P mutation relative to wild-type TiPLC;

[0015] TiPLC-A375P, which has an A375P mutation relative to wild-type TiPLC;

[0016] The amino acid sequence of wild-type TiPLC is shown in SEQ ID NO. 1.

[0017] The nucleotide sequence of the coding gene of the marine phospholipase C mutant is obtained according to the codon coding rule.

[0018] The marine phospholipase C mutant, wherein:

[0019] The nucleotide sequence of the coding gene of TiPLC-E92P is shown in SEQ ID NO. 3;

[0020] The nucleotide sequence of the coding gene of TiPLC-K105P is shown in SEQ ID NO. 4;

[0021] The nucleotide sequence of the coding gene of TiPLC-T150P is shown in SEQ ID NO. 5;

[0022] The nucleotide sequence of the coding gene of TiPLC-A168P is shown in SEQ ID NO. 6;

[0023] The nucleotide sequence of the coding gene of TiPLC-A359P is shown in SEQ ID NO. 7;

[0024] The nucleotide sequence of the coding gene of TiPLC-A375P is shown in SEQ ID NO. 8.

[0025] The marine phospholipase C mutant has significantly improved thermal stability relative to wild-type.

[0026] An expression vector comprising the coding gene.

[0027] The starting plasmid of the expression vector is pPICZ alpha A.

[0028] An engineering bacterium, which contains the above-mentioned coding gene in the genome; preferably, the cell contains the above-mentioned expression vector.

[0029] The starting strain of the engineering bacterium is Pichia pastoris Strain X-33.

[0030] The marine phospholipase C mutant, the expression vector or the engineering bacterium is applied to oil processing.

[0031] The oil processing is oil degumming.

[0032] The marine phospholipase C mutant, the expression vector or the engineering bacterium is applied to the production of glycerol diester.

[0033] The present application has the following advantages and effects relative to the prior art:

[0034] Compared with the wild-type enzyme protein, the mutant constructed in the present application can increase the half-life of the enzyme protein by 3.46 and 9.25 times respectively at 40 DEG C while keeping the enzyme protein activity basically unchanged, and can reduce the residual phosphorus content of the degummed oil from 551 ppm to 72 ppm, which is significantly lower than the final residual phosphorus content of 175 ppm of the blank and the final residual phosphorus content of 131 ppm of the WT, proving that the enzyme has better thermal stability and can be used for oil degumming treatment under different conditions and is suitable for more extensive industrial application fields. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a gel electrophoresis result diagram of the total bacterial protein concentrate of the TiPLC mutant constructed in Example 1; wherein, A: E92P; B: K105P; C: T150P; D: A168P; E: A359P; F: A375P; G: N461P.

[0036] Figure 2 is a curve diagram of the optimum reaction temperature of the wild type of TiPLC.

[0037] Figure 3 is a curve diagram of the optimum reaction pH of the wild type of TiPLC.

[0038] Figure 4 is a thermal stability time curve diagram of the TiPLC mutant and the wild type in Example 4.

[0039] Figure 5 is an ln(residual enzyme activity)-time curve diagram fitted out from the results of Example 4.

[0040] Figure 6 is a comparison diagram of the residual phosphorus content after degumming experiment of the mutant E92P, A375P and the wild type phospholipase C (TiPLC) in Example 5.

[0041] Figure 7 is a comparison chart of diglyceride content after degumming experiment of mutant E92P, A375P and wild type phospholipase C in Example 5. DETAILED DESCRIPTION

[0042] The application will be further described in conjunction with the following examples and drawings, but the embodiments of the application are not limited thereto.

[0043] In the following examples, if the specific test conditions are not specified, the test conditions are generally in accordance with the conventional test conditions or in accordance with the test conditions recommended by the reagent company. If not specified, the materials, reagents, etc. used are reagents and materials obtained from commercial channels.

[0044] Example 1 Construction of wild type TiPLC / mutant recombinant expression vector and expression strain

[0045] 1.1 Construction and expression of pPICZαA-tiplc recombinant plasmid

[0046] (1) The complete amino acid sequence of Talaromyces islandicus phospholipase C (GenBank: CRG91692.1) was obtained, and the amino acid sequence is shown as SEQ ID NO: 1;

[0047] (2) According to the amino acid sequence obtained in step (1), the gene encoding the protein sequence was codon optimized according to the codon bias of Pichia pastoris. EcoR I was introduced upstream of the sequence, and Sal I restriction site was introduced downstream. The optimized non-specific phospholipase C (TiPLC) gene sequence is shown as SEQ ID NO: 2. The pPICZαA plasmid carrying the corresponding gene was synthesized by GenScript Biotech (Shanghai) Co., Ltd.;

[0048] (3) The pPICZαA plasmid carrying the TiPLC gene obtained in step (2) was transformed into E. coli TOP10 competent cells, and was coated on LB (containing 25 ug / ml bleomycin) plate. Positive clones were picked and the plasmid was extracted using a kit for sequencing verification, and the pPICZαA-tiplc recombinant plasmid was obtained.

[0049] (4) The pPICZαA-tiplc recombinant plasmid obtained in step (3) was electroporated into P. pastoris X-33 competent cells, and was coated on YPD (containing 100 ug / ml bleomycin) plate. After the colonies grew, positive clones were picked, and the recombinant genetically engineered bacteria P. pastoris X-33 / pPICZαA-tiplc expression strain was obtained.

[0050] 1.2 Construction and expression of TiPLC mutant

[0051] (1) Using the pPICZαA-tiplc recombinant plasmid obtained in step (3) of 1.1 as a template, the mutant of the application was constructed by overlap extension PCR, and the reaction system was as follows:

[0052] Table 1 PCR reaction system

[0053] The sequences of the upstream primer and the downstream primer used in the mutant construction are as follows:

[0054] Table 2 List of primers used in the construction of the mutant

[0055] The PCR amplification conditions were as follows: 98℃, 3min; 98℃, 30s; 55℃, 30s; 72℃, 3min; 30 cycles; 72℃, 5min. After the completion of the PCR reaction, the amplification product was digested with Dpn I to digest the template plasmid, and the digestion system was as follows:

[0056] Table 3 Digestion system

[0057] The Dpn I digestion system was incubated at 37℃ for 1h to obtain the digestion product.

[0058] (2) The digestion product obtained in step (1) was transformed into E. coli Top10 competent cells. The positive clones were selected and verified by gene sequencing, and the corresponding pPICZαA-tiplc mutant recombinant plasmid was obtained.

[0059] (3) The mutant recombinant plasmid obtained in step (2) was electroporated into Pichia pastoris X-33 competent cells, and the positive clones were verified by colony PCR, thereby obtaining the recombinant Pichia pastoris X-33 expression strain of the mutant pPICZαA-tiplc.

[0060] Example 2 Fermentation of the pPICZαA-tiplc mutant recombinant expression strain and purification of the recombinant protein

[0061] (1) The recombinant genetic engineering bacteria P. pastoris X-33 / pPICZαA-tiplc expression strain obtained in 1.1 and the recombinant Pichia pastoris pPICZαA-tiplc mutant expression strain obtained in 1.2 were inoculated into 5mL seed culture medium (glucose 20g / L, peptone 20g / L, yeast extract 10g / L) containing 100μg / mL of bleomycin, and cultured at 30℃, 240r / min in a shaking flask for 17-20h to the logarithmic growth phase as a seed liquid;

[0062] (2) The seed solution obtained in step (1) was inoculated into 100 mL of YPD liquid fermentation medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) at an inoculum size of 3%, and cultured in a shaking flask at 30° C. and 240 rpm for 20 to 24 h;

[0063] (3) The secondary seed liquid obtained in step (2) was inoculated into 400 mL of YPD liquid fermentation medium (20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract) at an inoculum size of 3%, and cultured at 30°C on a shaker at 240 rpm. 1% methanol was added every 24 h for induction. The culture was 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. The crude enzyme solution was detected by SDS-PAGE. The experimental results are shown in Figure 1. Bands at the corresponding positions were observed in groups A to F, but no band at the corresponding position was observed for the N461P mutation in group G, indicating that the corresponding protein was not expressed.

[0064] (4) The crude enzyme solution obtained in step (3) was loaded onto a Ni-NTA agarose gel column produced by Newprobe; the column was then washed with 20 ml of solution A (20 mM Tris-HCl, 300 mM sodium chloride, 20 mM imidazole, pH 7.5) to remove most of the impurities; the column was then eluted with solution B (20 mM Tris-HCl, 300 mM sodium chloride, 200 mM imidazole, pH 7.5), and the eluate was collected (protein concentration was 0.4 mg / ml, total volume was about 10 ml). The eluate was desalted using a fast protein liquid chromatography system: a HiPrep 26 / 10 desalting column (Cytiva) was used, the equilibration and elution buffer was 20 mM Tris-HCl, 300 mM sodium chloride, pH 7.5, and the flow rate was 2 mL / min. The eluate with the protein peak was collected, i.e., the desalted eluate. The desalted eluate was passed through an Amicon Ultra-15 (Millipore) ultrafiltration tube with a cutoff of 10 kDa to concentrate the volume to 1 ml, which was the purified TiPLC protein solution. The solution was then divided into 100 μL portions using EP tubes and stored at -80°C for later use.

[0065] Example 3 Characterization experiments of wild-type phospholipase C TiPLC and its mutants

[0066] 3.1 Determination of optimal temperature

[0067] Phospholipase C TiPLC can hydrolyze the substrate O-(p-nitrophenylphosphoryl)choline (p-NPPC) to generate p-nitrophenol (pNP) through the reaction. pNP can be detected at 410nm. Based on this principle, the optimal temperature of phospholipase C TiPLC wild type and its mutants was detected with p-NPPC as the reference substrate.

[0068] 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM pNPPC substrate was prepared as the substrate solution. The reaction system was 200 μL, 20 μL of enzyme solution (0.05 mg / mL) was added to 180 μL of substrate solution, and the reaction was carried out at 30°C, 40°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, and 90°C for 2 min, and the absorbance was measured at 410 nm. The relative enzyme activity was calculated with the maximum value measured as 100%. Each group of experiments was repeated three times. The experimental results are shown in Figure 2. The optimal temperature of wild-type phospholipase C TiPLC is 60°C.

[0069] 3.2 Determination of optimal pH

[0070] A substrate solution containing 10 mM pNPPC substrate at pH 4-10 was prepared using citric acid-sodium citrate buffer, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, and Tris-HCl buffer, respectively. The reaction system was 200 μL, 20 μL of enzyme solution (0.05 mg / mL) was added to 180 μL of substrate solution, and the reaction was carried out at 60°C for 2 min. The enzyme was inactivated in a 95°C water bath for 10 min to terminate the reaction. The absorbance at 410 nm was measured, and the absorbance at 410 nm was measured. The relative enzyme activity was calculated with the maximum value measured as 100%. Each experiment was repeated three times. The experimental results are shown in Figure 3. The optimal pH of wild-type phospholipase C TiPLC is 7.5.

[0071] 3.3 Determination of enzyme activity

[0072] Prepare 50mM Tris-HCl buffer (pH 7.5) containing 10mM substrate as the substrate solution. The reaction system is 200μL. Take 20μL enzyme solution (0.05mg / mL) and add 180μL substrate solution. React at 60℃ for 2min. Place in a 95℃ water bath to inactivate the enzyme for 10min to terminate the reaction. Measure the absorbance at 410nm, and determine the content of the product 4-nitrophenol by calibration curve to calculate the enzyme activity.

[0073] The establishment of standard curve: prepare different concentrations (0.01 mg mL -1 , 0.02mg·mL -1 , 0.03mg·mL -1 , 0.04mg·mL -1 , 0.05mg·mL-1 0.06 mg / mL -1 0.07 mg / mL -1 0.08 mg / mL -1 0.09 mg / mL -1 0.1 mg / mL -1 , aqueous solution) of 4-nitrophenol (p-nitrophenyl, pNP). The absorbance value was measured at 410 nm. Three sets of parallel experiments were set, and the average value of A 410 was taken as the ordinate, and the corresponding pNP concentration was taken as the abscissa to draw a standard curve.

[0074] The enzyme activity unit (U) was defined as the amount of enzyme required to produce 1 μM of pNP per minute under the optimum reaction conditions.

[0075] Example 4 Determination of the thermal stability of phospholipase C TiPLC wild type and its mutants

[0076] The thermal stability was determined using O-(p-nitrophenyl phosphoryl) choline (p-NPPC) as the substrate, and the specific determination method was as follows:

[0077] The reaction system was prepared according to the method of Reference Example 3, and after incubation at 40°C, it was immediately cooled to 0°C in ice water to ensure that the control variable (the unincubated control group was added to the reaction system at about 0°C) was determined at 60°C. The residual enzyme activity was determined at different times, and the enzyme activity was detected according to the method of Reference Example 3.

[0078] The ln value of the residual activity was plotted against time (h), and the slope of the straight line was the inactivation constant K d , and the half-life of the lipase at this temperature was obtained from t 1 / 2 = ln2 / K d . t 1 / 2 is the time required for the loss of 50% of the activity of the protein, and is a parameter for characterizing the kinetic stability of the protein.

[0079] The results are shown in Figures 4-5 and Table 4. The thermal stability of the mutants E92P, A168P, and A375P was improved relative to the wild type, and the half-life was increased by 2.46, 1.63, and 8.25 times, respectively.

[0080] Table 4 Determination results of the half-life of the mutants and the wild type

[0081] Example 5 Verification of the effect of phospholipase C TiPLC wild type and its mutant enzyme degumming reaction

[0082] The two mutants with the best effect in Example 4 and phospholipase C TiPLC wild type were selected to verify the effect of enzymatic degumming reaction, and the specific steps were as follows:

[0083] (1) 200 g of plant oil was weighed in a beaker and heated to 40 DEG C in an oil bath, 3% of the total oil mass was added to the enzyme, and a control group without enzyme was set, and after uniform mixing, 40 DEG C, 500 r / min stirring for 4 h, degumming reaction was carried out. After the reaction was completed, the oil sample was placed in a 95 DEG C water bath to inactivate the enzyme for 10 min.

[0084] (2) The sample was centrifuged at 10,000 r / min for 10 min to obtain the upper degummed oil sample.

[0085] (3) The phosphorus content in the oil was determined according to GB / T 5537-2008 "Determination of phospholipid content in grain and oil", and the diacylglycerol content was determined by high performance liquid chromatography (evaporation light detector).

[0086] The experimental results are shown in Figures 6 and 7, after enzyme treatment, the phosphorus content in the crude oil is significantly reduced, and the diacylglycerol yield is significantly improved, compared with the wild type, the effect of mutants E29P and A375P is more prominent, which proves that the two mutants have better thermal stability and wider application range in industrial production.

[0087] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application, all should be equivalent replacement method, all are included in the protection scope of the present application.

Claims

1. A marine phospholipase C mutant, characterized in that at least one of the following: TiPLC-E92P, which has a mutation of E92P relative to wild-type TiPLC; TiPLC-K105P, which has a mutation of K105P relative to wild-type TiPLC; TiPLC-T150P, which has a mutation of T150P relative to wild-type TiPLC; TiPLC-A168P, which has a mutation of A168P relative to wild-type TiPLC; TiPLC-A359P, which has a mutation of A359P relative to wild-type TiPLC; TiPLC-A375P, which has a mutation of A375P relative to wild-type TiPLC; wherein the amino acid sequence of wild-type TiPLC is shown in SEQ ID NO.

1.

2. The marine phospholipase C mutant of claim 1, wherein the nucleotide sequence of the encoding gene of the marine phospholipase C mutant is obtained according to the codon usage.

3. The marine phospholipase C mutant of claim 1, wherein the nucleotide sequence of the encoding gene of the marine phospholipase C mutant is obtained according to the codon usage.

4. The marine phospholipase C mutant of claim 1, wherein the nucleotide sequence of the encoding gene of the marine phospholipase C mutant is obtained according to the codon usage.

5. The marine phospholipase C mutant of claim 1, wherein the nucleotide sequence of the encoding gene of the marine phospholipase C mutant is obtained according to the codon usage.

6. The marine phospholipase C mutant of claim 1, wherein the nucleotide sequence of the encoding gene of the marine phospholipase C mutant is obtained according to the codon usage.

7. The marine phospholipase C mutant of claim 1, wherein the nucleotide sequence of the encoding gene of the marine phospholipase C mutant is obtained according to the codon usage.

8. An expression vector, comprising the encoding gene of any one of claims 2-3.

9. The expression vector of claim 8, wherein the starting plasmid of the expression vector is pPICZαA.

10. An engineered bacterium, comprising the expression vector of any one of claims 4-5 in the genome.

11. The engineered bacterium of claim 10, wherein the starting strain of the engineered bacterium is Pichia pastoris Strain X-33.

12. Use of the marine phospholipase C mutant of any one of claims 1-3, the expression vector of any one of claims 4-5, or the engineered bacterium of any one of claims 6-7 in oil processing.

13. The use of claim 12, wherein the oil processing is oil degumming.

14. Use of the marine phospholipase C mutant of any one of claims 1-3, the expression vector of any one of claims 4-5, or the engineered bacterium of any one of claims 6-7 in the production of diglycerides. ​ ​ ​ ​ ​ ​ ​ ​

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