Potato patatin mutant, preparation method and application thereof

Abstract

The application provides a potato Patatin mutant and a preparation method and application thereof, adopts a Pichia pastoris expression system to perform heterologous expression on Patatin, and explores enzymatic properties. High cell density fermentation is performed under optimal fermentation conditions, and the expression amount can reach 121 mg / L; p-NPP is used as a substrate to verify the enzymatic properties and substrate specificity of Patatin, and it is found that the optimal substrate is a short-chain fatty acid; the Patatin D286A mutant obtained through directional design and modification significantly improves the denaturation temperature of the protein, improves the selectivity to long-chain substrates, and does not change the folding of the protein; molecular dynamics simulation shows that the D286A mutant destroys the helical configuration of the domain 280-286, and converts it into a flexible loop, so that the long-chain substrate pNP-C16 can be better accommodated in the active pocket. The application lays a foundation for efficiently synthesizing and mass-producing Patatin, improving the catalytic efficiency, stability and specificity of Patatin lipase in hydrolyzing long-chain fatty acids, and applying Patatin in medium-chain fatty acids and other functional lipids.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a potato Patatin mutant, a preparation method and application thereof. BACKGROUND

[0002] Potato (Solanum tuberosum) is an important tuber crop, with a global annual production of 350 billion kilograms. In recent years, people have begun to focus on sustainable protein sources such as potato protein. Patatin (GenBank accession number: AY033231.1) is a potato storage protein, accounting for about 40% of the soluble tuber protein of potato. It is a high-quality plant glycoprotein with multiple physiological functions, containing up to 76% essential amino acids, of which the monosaccharide residues include mannose, rhamnose, glucose, galactose, xylose and arabinose. Due to its significant solubility, foaming and emulsifying properties, it can be used as a food additive. At the same time, Patatin is a suitable animal protein substitute, with antioxidant properties, cholesterol and blood pressure and blood lipid regulation, anti-proliferation and other effects. Compared with other potato storage proteins, Patatin also has non-specific ester acylhydrolase activity and acyltransferase activity, which can hydrolyze glycolipids, phospholipids, monoacylglycerol and diacylglycerol, and pNP fatty acid esters. Therefore, a full study of the esterase activity and environmental tolerance of Patatin can provide a theoretical basis for its use in food processing and utilization.

[0003] At present, the preparation methods of Patatin at home and abroad mainly include obtaining from potato tubers and using recombinant genetically engineered bacteria for heterologous expression, and then isolating and purifying the pure protein. Heterologous expression is based on the source of protein synthesis by organisms, by cloning the DNA fragment encoding the target protein into a designated expression plasmid, and then introducing the constructed recombinant plasmid into a suitable expression host for expression, thereby obtaining the target protein. Common expression systems include eukaryotic expression systems mainly based on Pichia pastoris and prokaryotic expression systems mainly based on Escherichia coli.

[0004] The Pichia pastoris expression system has many advantages, such as: the expression level of Pichia pastoris is high, with a maximum expression amount exceeding 20 g / L; as a single-cell organism, Pichia pastoris is as easy to operate as Escherichia coli and Saccharomyces cerevisiae; the foreign gene is integrated into the yeast chromosome, so it can be replicated with the replication of the chromosome, is relatively stable and is not easy to lose; the Pichia pastoris process is relatively mature and easy to scale up. Pichia pastoris expression can directly secrete the target protein into the fermentation broth; Pichia pastoris belongs to eukaryotes, and as a eukaryotic expression system, it can help protein to undergo post-translational modifications such as glycosylation, fatty acylation and protein phosphorylation.

[0005] Patatin is a non-specific lipase with the ability to activate lipolysis, which can specifically hydrolyze sulfatide, phospholipid and glycerol diester and regulate lipid metabolism in vivo. However, the substrate specificity mechanism of Patatin protein hydrolyzing different chain length fatty acids is still unclear. In addition, if Patatin can be expressed through eukaryotic and prokaryotic expression systems to improve protein yield and activity, it will be beneficial to realize its industrial production; and there is very little research on its enzymatic properties and applications, and deepening the research on its enzymatic properties can make it more widely used in food, medicine and other fields. The present application provides a new type of Patatin fat hydrolytic enzyme which can hydrolyze long chain fatty acids through directional design and modification, providing a theoretical application basis for its application in medium chain fatty acids and other functional lipids. SUMMARY

[0006] The technical problem to be solved is to provide a potato Patatin mutant, a preparation method and application thereof, which aims at the low content of natural potato Patatin source limiting the processing scale application, and the technical deficiency that the natural lipase can only hydrolyze short chain fatty acids.

[0007] The technical scheme is a potato Patatin mutant, wherein the Patatin mutant is Patatin F108A, Patatin D286A or Patatin M325A, and the amino acid sequences thereof correspond to SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 respectively.

[0008] The preparation method of the above-mentioned potato Patatin mutant comprises the following steps: cloning the coding sequence of the potato Patatin mutant in claim 1 into an expression vector to obtain a recombinant vector; transforming the recombinant vector into a host cell for expression and purification, so as to obtain the potato Patatin mutant.

[0009] The expression vector is pET51b(+).

[0010] The host cell is Pichia pastoris.

[0011] The method for preparing the potato Patatin mutant comprises the following steps:

[0012] S1. Constructing a pET51-Patatin recombinant vector: linearizing the target gene Patatin and the vector pET51b(+) using XhoI and NotI restriction enzymes, combining the target gene with the vector to obtain a pET51-Patatin recombinant vector;

[0013] S2. Constructing a mutant recombinant plasmid: using the pET51-Patatin recombinant vector as a template, performing PCR site-directed mutation, digesting the PCR product, and transforming the digested product into a DH5α competent cell to obtain a Patatin mutant recombinant plasmid;

[0014] S3. Patatin recombinant expression: transforming the Patatin mutant recombinant plasmid into Pichia pastoris to obtain an expression strain; culturing the expression strain in a YPD liquid medium, and when OD600 = 2, centrifuging to discard the supernatant and collect the bacterial cells; 600

[0015] S4. Purification: resuspending the bacterial cells in a BMGY medium, culturing and then standing overnight; discarding the upper culture medium, adding a BMMY medium to perform induction, adding methanol for induction every 24 hours, centrifuging to collect the bacterial cells, resuspending the bacterial cells in a buffer to crush the bacterial cells, centrifuging to collect the supernatant, and purifying the Patatin mutant through a nickel affinity column, further purifying through gel filtration chromatography, and obtaining the purified Patatin mutant.

[0016] Further, the method for preparing further comprises high-density fermentation of the recombinant Pichia pastoris.

[0017] Further, the PCR reaction program in the step S2 is as follows: 30 cycles of 95℃ denaturation for 10s, 55℃ annealing for 5s, and 72℃ extension for 15s.

[0018] Further, the high-density fermentation step is as follows: transforming the Patatin mutant recombinant plasmid into Pichia pastoris to obtain an expression strain; culturing the expression strain in a YPD liquid medium, and when OD600 = 2, centrifuging to discard the supernatant and collect the bacterial cells; resuspending the bacterial cells in a BMGY medium, culturing and then standing overnight; discarding the upper culture medium, adding a BMMY medium containing YNB at a content of 9.68% to perform induction for 72 hours, adding methanol for induction every 24 hours, so that the final concentration of methanol is 0.44%, centrifuging to collect the bacterial cells, resuspending the bacterial cells in a buffer to crush the bacterial cells, centrifuging to collect the supernatant, and purifying the Patatin mutant through a nickel affinity column, further purifying through gel filtration chromatography, and obtaining the purified Patatin mutant.​

[0019] The application of the potato Patatin mutant prepared by the preparation method in lipase hydrolysis of fatty acids.

[0020] Beneficial effects:

[0021] 1. The expression amount of the lipase Patatin mutant prepared by the application can reach 121 mg / L after high cell density fermentation under the optimal fermentation conditions, the Patatin mutant belongs to alkaline lipase, the optimal reaction temperature is 35 DEG C, 1 mmol / L of Ca 2+ , Zn 2+ and Mg 2+ can significantly improve the lipase activity of Patatin, and 1 mmol / L and above of Fe 2+ , Fe 3+ and Cu 2+ have inhibitory effect on the lipase activity of Patatin, Triton X-100 and SDS have certain improving effect on the enzyme activity, but Tween-20 can significantly reduce the enzyme activity, and the optimal substrate of Patatin is short-chain fatty acid.

[0022] 2. The Patatin D286A mutant designed and modified by site-directed mutation of the application significantly improves the denaturation temperature of the protein, improves the selectivity of the protein to long-chain substrates, and does not change the folding of the protein; molecular dynamics simulation shows that the D286A mutant destroys the helical configuration of the domain 280-286 and converts it into a flexible loop, and the loop can better accommodate the long-chain substrate pNP-C16 in the active pocket.

[0023] 3. The application lays a foundation for efficient synthesis and large-scale production of Patatin, and improves the catalytic efficiency, stability and specificity of lipase hydrolysis of long-chain fatty acids. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a graph of the influence of four factors on the expression of Patatin, wherein A is the methanol addition amount, B is the YNB addition amount, C is the inoculation amount, and D is the culture time;

[0025] Figure 2 It is a response surface graph and contour graph of the interactive influence of enzyme activity, wherein A is the methanol concentration and YNB concentration, B is the methanol concentration and inoculation amount, and C is the YNB concentration and inoculation amount;

[0026] Figure 3 It is a graph of the influence of pH, temperature, organic solvent and surfactant on the lipase activity of Patatin, wherein A is pH, B and C are temperature, and D is organic solvent and surfactant;

[0027] Figure 4 Figure 6 is a graph of the substrate specificity and Patatin and its mutant related properties of lipase Patatin, wherein A is the residual relative enzyme activity of wild type Patatin enzyme to different chain length of p-nitrophenyl ester; B is the relative enzyme activity of Patatin wild type, M325A, F108A and D286A variants to different chain length of p-nitrophenyl ester; C is the fluorescence intensity of Patatin wild type, M325A, F108A and D286A variants; D is the circular dichroism of Patatin wild type, M325A, F108A and D286A variants;

[0028] Figure 5 Figure 7 is a graph of the flexibility change of dynamic loop 258-302 in lipase Patatin D286A mutant, wherein A is the ΔRMSF value of Loop 258-302 of lipase Patatin complexed with pNP-C4; B is the ΔRMSF value of Loop 258-302 of lipase Patatin complexed with pNP-C16;

[0029] Figure 6 Figure 8 is a graph of the affinity of long chain substrate pNP-C16 to lipase Patatin D286A mutant, wherein A is the RMSD value of lipase Patatin complexed with p-nitrophenyl ester (pNP-C4 and pNP-C16); B is the number of hydrogen bonds between lipase Patatin and long chain substrate pNP-C16; C is the helix structure of domain 280-286 destroyed by D286A mutation. DETAILED DESCRIPTION

[0030] The present application will be further described below in conjunction with the accompanying drawings and examples, which are illustrative of the present application and the present application is not limited to the following examples:

[0031] The main materials involved in the embodiments of the present application are as follows: the carrier pET51b(+) is purchased from Shanghai Yage Biological Technology Co., Ltd.; the Pichia pastoris is purchased from Shanghai Blue Sky Bio-Technology Co., Ltd.

[0032] The amino acid sequence of wild type Patatin is as follows:

[0033] MHHHHHHAMAQLGEMVTVLSIDGGGIRGIIPATILEFLEGQLQEMDNNADARLADYFDVIG

[0034] GTSTGGLLTAMISTPNENNRPFAAAKEIVPFYFEHGPQIFNPSGQILGPKYDGKYLMQVLQE

[0035] KLGETRVHQALTEVVISSFDIKTNKPVIFTKSNLANSPELDAKMYDISYSTAAAPTYFPPHYF

[0036] VTNTSNGDEYEFNLVDGAVATVADPALLSISVATRLAQKDPAFASIRSLNYKKMLLLSLGTGT

[0037] TSEFDKTYTAKEAATWTAVHWMLVIQKMTDAASSYMTDYYLSTAFQALDSKNNYLRVQE

[0038] NALTGTTTEMDDASEANMELLVQVGENLLKKPVSEDNPETYEEALKRFAKLLSDRKKLRANKASY;

[0039] The amino acid sequence of Patatin F108A is shown in SEQ ID NO. 1 :

[0040] MHHHHHHAMAQLGEMVTVLSIDGGGIRGIIPATILEFLEGQLQEMDNNADARLADYFDVIG

[0041] GTSTGGLLTAMISTPNENNRPFAAAKEIVPFYFEHGPQIANPSGQILGPKYDGKYLMQVLQE

[0042] KLGETRVHQALTEVVISSFDIKTNKPVIFTKSNLANSPELDAKMYDISYSTAAAPTYFPPHYF

[0043] VTNTSNGDEYEFNLVDGAVATVADPALLSISVATRLAQKDPAFASIRSLNYKKMLLLSLGTGT

[0044] TSEFDKTYTAKEAATWTAVHWMLVIQKMTDAASSYMTDYYLSTAFQALDSKNNYLRVQE

[0045] NALTGTTTEMDDASEANMELLVQVGENLLKKPVSEDNPETYEEALKRFAKLLSDRKKLRANKASY;

[0046] The amino acid sequence of Patatin D286A is shown in SEQ ID NO. 2:

[0047] MHHHHHHAMAQLGEMVTVLSIDGGGIRGIIPATILEFLEGQLQEMDNNADARLADYFDVIG

[0048] GTSTGGLLTAMISTPNENNRPFAAAKEIVPFYFEHGPQIFNPSGQILGPKYDGKYLMQVLQE

[0049] KLGETRVHQALTEVVISSFDIKTNKPVIFTKSNLANSPELDAKMYDISYSTAAAPTYFPPHYF

[0050] VTNTSNGDEYEFNLVDGAVATVADPALLSISVATRLAQKDPAFASIRSLNYKKMLLLSLGTGT

[0051] TSEFDKTYTAKEAATWTAVHWMLVIQKMTAAASSYMTDYYLSTAFQALDSKNNYLRVQE

[0052] NALTGTTTEMDDASEANMELLVQVGENLLKKPVSEDNPETYEEALKRFAKLLSDRKKLRANKASY;

[0053] The amino acid sequence of Patatin M325A is shown in SEQ ID NO. 3:

[0054] MHHHHHHAMAQLGEMVTVLSIDGGGIRGIIPATILEFLEGQLQEMDNNADARLADYFDVIG

[0055] GTSTGGLLTAMISTPNENNRPFAAAKEIVPFYFEHGPQIFNPSGQILGPKYDGKYLMQVLQE

[0056] KLGETRVHQALTEVVISSFDIKTNKPVIFTKSNLANSPELDAKMYDISYSTAAAPTYFPPHYF

[0057] VTNTSNGDEYEFNLVDGAVATVADPALLSISVATRLAQKDPAFASIRSLNYKKMLLLSLGTGT

[0058] TSEFDKTYTAKEAATWTAVHWMLVIQKMTDAASSYMTDYYLSTAFQALDSKNNYLRVQE

[0059] NALTGTTTEADDASEANMELLVQVGENLLKKPVSEDNPETYEEALKRFAKLLSDRKKLRANKASY.

[0060] Example 1

[0061] A method for preparing a potato Patatin mutant, comprising the following steps:

[0062] S1. Preparation of Pichia pastoris competence: Pichia pastoris monoclonal strain is taken from YPD solid medium and cultured at 30°C, and the culture solution is transferred to 50 mL YPD medium and cultured until OD 600 = 1.3; centrifuged at 1500 r / min for 5 min at 4°C, resuspended the cells with ice double-distilled water; centrifuged, resuspended with 25 mL ice double-distilled water; centrifuged again, resuspended the cells with 2 mL 1 mol / L ice sorbitol solution; centrifuged again, resuspended with sorbitol solution to make the total volume 100 μL, and obtain Pichia pastoris competence;

[0063] S2. Pichia pastoris electroporation: the pET51-Patatin expression vector is treated with endonuclease SacI to obtain linearized pET51-Patatin expression vector, 5 μL linearized expression vector is mixed with Pichia pastoris competence, poured into ice electroporation cup, and placed for 5 min, the electroporation parameters are voltage 2 kV, capacitance 25 μF, resistance 200 Ω, and pulse time 5.0 ms; 1 mL ice sorbitol solution is immediately added after the end of electroporation, mixed, transferred to 1.5 mL centrifuge tube, and cultured at 30°C for 2 h; the transformation solution in the centrifuge tube is diluted to 100 μL, and spread on YPD medium, cultured at 30°C overnight, single colonies are selected, and verified using 5-AOX and 3-AOX amplification technology;

[0064] S3. Construction of pET51-Patatin recombinant vector: the target gene Patatin and the vector pET51b(+) are linearized using XhoI and NotI restriction endonucleases, respectively, the target gene is collected and purified using gel recovery kit, and the target gene is combined with the vector to obtain pET51-Patatin recombinant vector;

[0065] S4. Mutant recombinant plasmid construction: PCR site-directed mutagenesis was performed using pET51-Patatin expression vector as template. The mutants of Patatin were F108A, D286A and M325A. The PCR reaction program was as follows: 30 cycles of amplification, 95℃ denaturation for 10 s, 55℃ annealing for 5 s, and 72℃ extension for 15 s. The PCR product was verified by 1% agarose gel electrophoresis, digested with Dpn I for 1 h, and then transformed into DH5α competent cells to obtain Patatin mutant recombinant plasmid.

[0066] S5. Patatin recombinant expression: The Patatin mutant recombinant plasmid was transformed into Pichia pastoris to obtain an expression strain. The expression strain was cultured in YPD liquid medium, and when OD600 = 2, the supernatant was discarded after centrifugation, and the bacterial cells were collected. 600

[0067] S6. Purification: The bacterial cells were resuspended in BMGY medium and cultured for 24 h, then stood overnight. The upper BMGY medium was discarded, and BMMY medium was added for induction for 72 h. Methanol was added every 24 h for induction. The bacteria were collected by centrifugation, resuspended in buffer, and the bacterial cells were broken. The supernatant was collected and the Patatin mutant was purified by nickel affinity column. Gel filtration chromatography was used for further purification to obtain purified Patatin mutant.

[0068] S7. High-density fermentation of recombinant Pichia pastoris: The Patatin mutant recombinant plasmid was transformed into Pichia pastoris to obtain an expression strain. The expression strain was cultured in YPD liquid medium, and the inoculation amount was 10.42%. When OD600 = 2, the supernatant was discarded after centrifugation, and the bacterial cells were collected. The bacterial cells were resuspended in BMGY medium and cultured, then stood overnight. The upper medium was discarded, and BMMY medium containing YNB at a content of 9.68% was added for induction for 72 h. Methanol was added every 24 h for induction, and the final concentration of methanol was 0.44%. The bacteria were collected by centrifugation, resuspended in buffer, and the bacterial cells were broken. The supernatant was collected and the Patatin mutant was purified by nickel affinity column. Gel filtration chromatography was used for further purification to obtain purified Patatin mutant.

[0069] The high-cell-density fermentation process was divided into a basic culture stage (glycerol as carbon source, temperature 28℃, rotation speed 500 r / min, pH 5.0), a fed-batch culture stage and an induction stage (maintaining dissolved oxygen at 20-30%, temperature 28℃, pH 5.0 to the end of fermentation).

[0070] The Patatin mutant prepared according to the above high-cell-density fermentation method had an expression amount of 121 mg / L.

[0071] ​The effects of methanol addition (0.0, 0.5, 1.0, 1.5, 2.0%), YNB content (5.0, 7.5, 10.0, 12.5, 15.0%), inoculum size (1.0, 5.0, 10.0, 15.0, 20%), and culture time (48, 60, 72, 84, 96 h) on the production of Patatin by Pichia pastoris were investigated as single factors. Figure 1 The effects of methanol addition, YNB addition, inoculum size, and culture time on Patatin expression were shown. When Patatin production reached its peak, the methanol addition was only 0.5% (…). Figure 1 A), but when other factors were taken into account, Patatin production began to decline, most likely due to excessive methanol intake, which had a harmful effect. Research indicates that when methanol concentration exceeds 3.65%, the growth and reproduction of Pichia pastoris are significantly hindered; therefore, precise control of Pichia pastoris fermentation is essential. Enzyme activity reached its peak at 10% YNB content, at 131.37 U / mg, but gradually decreased with increasing YNB content. Figure 1 B); Figure 1 In C, enzyme yield gradually increased with increasing inoculum size, reaching its maximum at an inoculum size of 10%; different culture times had little effect on Patatin yield, with yields consistently reaching 110 U / mg. Figure 1 D). Based on single-factor experiments, a Box-Behnken model was used, with methanol concentration, YNB concentration, and inoculum size as influencing factors, and these were divided into three levels. Response surface methodology was then conducted (e.g., Figure 2 The optimal fermentation conditions were found to be: 0.44% methanol, 9.68% YNB, and 10.42% inoculum. The verification experiment showed that the optimal conditions for cultivation using response surface methodology resulted in a final Patatin enzyme activity of 130.21 U / mg.

[0072] Effects of metal ions on patatin enzyme activity

[0073] Table 1 Effects of metal ions on patatin enzyme activity

[0074]

[0075] Note: Different lowercase letters in each column represent significant differences (P < 0.05).

[0076] As shown in Table 1, 1 mmol / L Ca 2+ Zn 2+ and Mg 2+It can significantly enhance the lipase activity of Patatin, while Fe at 1 mmol / L and above 2+ Fe 3+ and Cu 2+ It has an inhibitory effect on the lipase activity of Patatin.

[0077] Application of potato Patatin mutants in the hydrolysis of substrates with different chain lengths:

[0078] I. Effects of pH, temperature, organic solvents and surfactants on the activity of Patatin lipase

[0079] Figure 3 In solution A, the activity of lipase increases significantly with increasing buffer pH. When the pH reaches 10.0, the lipase activity reaches 100%, indicating that the optimal reaction pH for Patatin is 10.0. When the pH is between 4.0 and 6.0, the lipase activity decreases significantly, with a relative enzyme activity of approximately 30%. This may be due to the presence of an acidic environment, which causes some groups to dissociate, thus hindering the binding of lipase to the substrate and reducing the reaction rate, resulting in a decrease in enzyme activity. Patatin has good alkali resistance and can be effectively used in alkaline environments to enhance its function. Figure 3 In sample B, as the temperature increased from 20℃ to 60℃, the activity of lipase first rose and then fell. When the temperature reached 35℃, the catalytic activity of lipase reached its maximum of 100%, indicating that the optimal reaction temperature for Patatin is approximately 35℃. At 40-50℃, the relative activity of lipase remained above 40%, but when the temperature rose to 60℃, their relative activity decreased significantly, with only about 20% remaining. After treating the enzyme at 70℃ for 30 minutes, the relative enzyme activity was 0%, and Patatin was completely inactivated. Figure 3 C); by Figure 3 As shown in Figure D, hydrophilic organic solvents such as methanol, ethanol, and isopropanol significantly enhance the activity of lipase, with 30% methanol increasing the activity by over 60%. Due to the high adsorption capacity of the lipase surface, it is more easily adsorbed by organic solutes with high adsorption capacity, thus accelerating its binding to the substrate. Although Triton X-100 and SDS both have a certain effect on enhancing enzyme activity, SDS has a more significant promoting effect, with a relative enzyme activity increase of 124.52 ± 4.14%. Tween-80 and EDTA did not significantly enhance enzyme activity, but Tween-20 significantly reduced enzyme activity. II. Substrate specificity of lipase Patatin and related properties of Patatin and its mutants.

[0080] Figure 4In A, the wild-type Patatin showed very strong hydrolytic ability when treating nitrophenyl butyrate (pNP-C4), but relatively low activity when treating nitrophenyl myristate (pNP-C14) and nitrophenyl palmitate (pNP-C16). It was found through research that the effect of the lipase Patatin on short-chain nitrophenyl esters was significantly better than that on other types of substrates, which indicated that pNP-C4 was the ideal substrate for the lipase Patatin, and the lipase Patatin could more effectively treat C4-C8 type nitrophenyl esters.

[0081] In order to improve the substrate preference of Patatin and explore the influence of structural changes on catalytic activity, mutants F108A, D286A and M325A were constructed, and the substrate specificity of the mutants was studied by using p-NPP (p-nitrophenyl phosphate) with different chain lengths as substrates. Figure 4 B), it was found that, compared with other mutants, the mutant D286A had stronger specificity for long-chain substrates. Figure 4 In C, the Tm values of Patatin and its mutants were determined by fluorescence quantitative PCR, and the Tm value of Patatin was 66.18℃. The Tm values of the mutants were relatively lower than that of the wild type, and the Tm value of F108A was 60.57℃, the Tm value of D286A was 60.82℃, and the Tm value of M325A was 62.34℃. Figure 4 The circular dichroism spectrum of D shows that the structures of the three mutants after mutation are basically similar to those of the wild type, so it can be inferred that the mutation does not affect the folding of the protein.

[0082] III. Changes in flexibility of dynamic loop 258-302 in the lipase Patatin D286A mutant

[0083] As shown in Figure 5 Compared with the wild-type lipase (WT), the D286A mutant resulted in a decrease in the ΔRMSF value of the dynamic loop 258-302, indicating that the rigidity of this domain was significantly increased and the flexibility was significantly reduced; in addition, the RMSD of the D286A mutant with the pNP-C4 / pNP-C16 complex was significantly lower than the RMSD of the D286A mutant with the pNP-C4 / pNP-C16 complex (as shown in Figure 6 A, B), indicating that the D286A mutant stabilized the binding of Patatin to the substrate.

[0084] IV. Affinity of the lipase Patatin D286A mutant for long-chain substrate pNP-C16

[0085] Figure 6A shows the RMSD values of the complex of lipase Patatin and p-nitrophenyl ester (short-chain substrate: pNP-C4, long-chain substrate: pNP-C16). Compared with wild-type Patatin, the number of hydrogen bonds formed by the D286A mutant and pNP-C16 is significantly increased (B), indicating that the affinity between the D286A mutant Patatin and pNP-C16 is enhanced, and the increase in the specific activity of the D286A mutant for pNP-C16 determined by experiments also supports this mechanism (B). Figure 6 Figure 4 B). Figure 6 As can be seen in C, the D286A mutation destroys the helical structure of the domain 280-286 and converts it into a flexible loop, which can better regulate the long-chain substrate pNP-C16, which may be the key molecular mechanism for the high specificity of the D286A mutant for long-chain substrates.

[0086] The above only describes the preferred embodiments of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solutions of the present application, still falls within the scope of protection of the technical solutions of the present application.​

Claims

1. A potato Patatin mutant, characterized by: The Patatin mutant is Patatin D286A, and its amino acid sequence is SEQ ID NO.

2.

2. The method for preparing a potato Patatin mutant according to claim 1, characterized in that: The coding sequence of the potato Patatin mutant described in claim 1 is cloned into an expression vector to obtain a recombinant vector; the recombinant vector is transformed into host cells for expression and purification to obtain the potato Patatin mutant. The expression vector is pET51b(+); The host cell is Pichia pastoris.

3. The method for preparing a potato Patatin mutant according to claim 1, characterized in that, Includes the following steps: S1. Construction of pET51-Patatin recombinant vector: After linearizing the target gene Patatin and the vector pET51b(+) with XhoI and NotI restriction endonucleases respectively, the target gene was combined with the vector to obtain the pET51-Patatin recombinant vector. S2. Construction of mutant recombinant plasmid: Using pET51-Patatin recombinant vector as template, PCR site-directed mutagenesis was performed, the PCR product was digested, and the digested product was transferred into DH5α competent cells to obtain the Patatin mutant recombinant plasmid. S3. Patatin Recombinant Expression: The Patatin mutant recombinant plasmid was transformed into Pichia pastoris to obtain the expression strain; the expression strain was cultured in YPD liquid medium, and when OD... 600 Centrifuge at 2 hours, discard the supernatant, and collect the bacterial cells; S4. Purification: The bacterial cells were resuspended in BMGY medium and cultured overnight. The supernatant was discarded, and BMMY medium was added for induction. Methanol was added every 24 hours for induction. The cells were collected by centrifugation, and the bacterial cells were resuspended in buffer to disrupt them. The supernatant was collected by centrifugation and purified by nickel affinity column purification. The purified Patatin mutant was further purified by gel filtration chromatography to obtain the purified Patatin mutant.

4. The method for preparing a potato Patatin mutant according to claim 3, characterized in that: The preparation method also includes high-density fermentation of recombinant Pichia pastoris.

5. The method for preparing a potato Patatin mutant according to claim 3, characterized in that, The PCR reaction program in step S2 is as follows: 30 cycles of amplification, 95°C denaturation for 10 seconds, 55°C annealing for 5 seconds, and 72°C extension for 15 seconds.

6. The method for preparing a potato Patatin mutant according to claim 4, characterized in that, The high-density fermentation steps are as follows: The recombinant plasmid of the Patatin mutant is transformed into Pichia pastoris to obtain the expression strain; the expression strain is cultured in YPD liquid medium with an inoculum of 10.42%; when OD600 = 2, the supernatant is discarded by centrifugation, and the cells are collected; the cells are resuspended in BMGY medium and cultured overnight; the upper medium is discarded, and BMMY medium with YNB content of 9.68% is added for induction for 72 h, with methanol added every 24 h to induce induction until the final methanol concentration is 0.44%; the cells are collected by centrifugation, and the cells are resuspended in buffer to disrupt the cells; the supernatant is collected by centrifugation and purified by nickel affinity column purification; the purified Patatin mutant is further purified by gel filtration chromatography.

7. The application of the potato Patatin mutant prepared by the preparation method according to any one of claims 2-6 in the hydrolysis of fatty acids by lipase.

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