Potato Patatin mutant as well as preparation method and application thereof

Abstract

The invention provides a potato Patatin mutant as well as a preparation method and application thereof, Patatin is subjected to heterologous expression by adopting a pichia pastoris expression system, and enzymatic properties are explored. High-cell-density fermentation is carried out under the optimal fermentation condition, and the expression quantity can reach 121 mg / L; the enzymatic property and substrate specificity of Patatin are verified by taking p-NPP as a substrate, and the optimal substrate of Patatin is found to be short-chain fatty acid; according to the Patatin D286A mutant modified by directional design, the denaturation temperature of the protein is obviously increased, the selectivity to a long-chain substrate is improved, and the folding of the protein is not changed; molecular dynamics simulation shows that a D286A mutant can destroy the helical configuration of a structural domain 280-286 and convert the structural domain 280-286 into a flexible ring, so that a long-chain substrate pNP-C16 can be better accommodated in an active pocket. The invention lays a foundation for efficient synthesis and large-scale production of Patatin, improvement of catalytic efficiency, stability and specificity of Patatin lipase hydrolysis of long-chain fatty acid, and application of Patatin lipase in functional lipids such as medium-chain fatty acid.

Description

Technical Field

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

[0002] Potato (Solanum tuberosum) is an important tuber crop with an annual global production of 350 billion kilograms. In recent years, people have begun to pay attention to 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 various physiological functions, containing up to 76% essential amino acids, and its monosaccharide residues include mannose, rhamnose, glucose, galactose, xylose and arabinose. Due to its remarkable solubility, foaming property and emulsifying property, it can be used as a food additive. At the same time, Patatin is a suitable alternative to animal protein, with antioxidant properties, regulating cholesterol, blood pressure and blood lipids, and anti-proliferation effects. Compared with other potato storage proteins, Patatin also has non-specific acyl hydrolase activity and acyl transferase activity, and can hydrolyze glycolipids, phospholipids, monoacylglycerols and diacylglycerols, pNP fatty acid esters. Therefore, a full study of the esterase activity and environmental tolerance of Patatin can provide a theoretical basis for its food processing and utilization. At present, the preparation methods of Patatin at home and abroad are mainly divided into two ways: obtaining from potato tubers and heterologous expression using recombinant genetic engineering bacteria, and then separating and purifying to obtain pure protein. Heterologous expression is based on the source of protein synthesis in organisms. By cloning the DNA fragment encoding the target protein into a specified expression plasmid, and then introducing the constructed recombinant plasmid into a suitable expression host for expression, the target protein can be obtained. The common expression systems are two types: the eukaryotic expression system mainly based on Pichia pastoris and the prokaryotic expression system mainly based on Escherichia coli. The Pichia pastoris expression system has many advantages. For example, the expression level of Pichia pastoris is high, and the highest expression level has exceeded 20 g / L; as a single-celled organism, Pichia pastoris is as easy to operate as Escherichia coli and Saccharomyces cerevisiae; since the foreign gene is integrated into the yeast chromosome, it can be replicated along with the replication of the chromosome, is relatively stable and not easily lost; the process of Pichia pastoris is relatively mature and easy to scale up. The expression of Pichia pastoris can directly secrete the target protein into the fermentation broth; Pichia pastoris belongs to eukaryotes, and as a eukaryotic expression system, it can help the protein to carry out post-translational modifications such as glycosylation, fatty acylation, and protein phosphorylation. Patatin is a non-specific lipase with the ability to activate lipolysis, which can specifically hydrolyze sulfur lipids, phospholipids, diglycerides, etc. and regulate lipid metabolism in the body. However, the substrate specificity mechanism of Patatin protein in hydrolyzing fatty acids with different chain lengths is still unclear. In addition, if Patatin can be expressed through eukaryotic and prokaryotic expression systems to increase protein yield and activity, it will be beneficial to realize its industrial production; and there are very few studies on its enzymatic properties and applications. Deepening the research on its enzymatic properties can enable it to be more widely used in the fields of food, medicine, etc. Through directed design and modification, the present invention provides a novel Patatin lipase that can hydrolyze long-chain fatty acids, providing a theoretical application basis for its application in functional lipids such as medium-chain fatty acids. Summary of the Invention

[0003] Technical problems to be solved: The purpose of the present invention is to provide a potato Patatin mutant, its preparation method and application in view of the technical deficiencies such as the low content of natural sources of existing potato Patatin restricting the application of processing scale and the natural lipase can only hydrolyze short-chain fatty acids. The Patatin is heterologously expressed using the Pichia pastoris expression system, the optimal conditions of the enzyme expression system are studied, and the enzymatic properties are explored. The present invention also uses site-directed mutagenesis enzyme engineering technology in order to obtain better results, explore its effect on natural substrates, and conduct a preliminary exploration of its decomposition products, in order to provide a theoretical basis for the high-efficiency expression and industrial production of Patatin and open up a new path for its application; through protein molecular recombination technology, the present invention excavates and develops a novel lipase that specifically hydrolyzes long-chain fatty acids, which can be used in the future for the application of converting long-chain fatty acids into medium-chain fatty acids in food.

[0004] Technical solution: A potato Patatin mutant, wherein the Patatin mutant is Patatin F108A, Patatin D286A or Patatin M325A, and their amino acid sequences correspond to SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3 respectively. The preparation method of the above-mentioned potato Patatin mutant, cloning the coding sequence of the potato Patatin mutant described in claim 1 into an expression vector to obtain a recombinant vector; transforming the recombinant vector into a host cell for expression and purification, and the above-mentioned potato Patatin mutant can be obtained; The expression vector is pET51b(+); The host cell is Pichia pastoris. The preparation method of the above-mentioned potato Patatin mutant 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 the pET51-Patatin recombinant vector as a template, PCR site-directed mutagenesis was carried out. The PCR product was digested and the digested product was transferred into DH5α competent cells to obtain the Patatin mutant recombinant plasmid; S3. Recombinant expression of Patatin: The Patatin mutant recombinant plasmid was transferred into Pichia pastoris to obtain an expression strain; The expression strain was cultured in YPD liquid medium. When OD 600 = 2, the supernatant was discarded by centrifugation and the cells were collected; S4. Purification: The cells were resuspended in BMGY medium, cultured and then left standing overnight; The upper layer of the medium was discarded, and BMMY medium was added for induction. Methanol was added for induction every 24 h. The cells were collected by centrifugation. The cell pellet was resuspended in buffer to lyse the cells, and the supernatant was collected by centrifugation and the Patatin mutant was purified through a nickel affinity column, and further purified by gel filtration chromatography to obtain the purified Patatin mutant. Furthermore, the preparation method further includes high-density fermentation of recombinant Pichia pastoris. Furthermore, the PCR reaction program in step S2 is: 30 cycles of amplification, denaturation at 95 °C for 10 s, annealing at 55 °C for 5 s, and extension at 72 °C for 15 s. Furthermore, the steps of high-density fermentation are as follows: The Patatin mutant recombinant plasmid was transferred into Pichia pastoris to obtain an expression strain; The expression strain was cultured in YPD liquid medium with an inoculation amount of 10.42%. When OD600 = 2, the supernatant was discarded by centrifugation and the cells were collected; The cells were resuspended in BMGY medium, cultured and then left standing overnight; The upper layer of the medium was discarded, and BMMY medium with a YNB content of 9.68% was added for induction for 72 h. Methanol was added for induction every 24 h to make the final concentration of methanol 0.44%. The cells were collected by centrifugation. The cell pellet was resuspended in buffer to lyse the cells, and the supernatant was collected by centrifugation and the Patatin mutant was purified through a nickel affinity column, and further purified by gel filtration chromatography to obtain the purified Patatin mutant. Application of the potato Patatin mutant prepared by the above preparation method in the hydrolysis of fatty acids by lipase. Beneficial effects: 1. After high cell density fermentation of the lipase Patatin mutant prepared by the present invention under the optimal fermentation conditions, the expression level can reach 121 mg / L. It belongs to an alkaline lipase, and its optimal reaction temperature is 35 °C; 1 mmol / L of Ca 2+ , Zn 2+ and Mg 2+ can significantly enhance the lipase activity of Patatin, while Fe 2+ , Fe 3+ and Cu 2+ at 1 mmol / L and above have an inhibitory effect on the lipase activity of Patatin; Triton X-100 and SDS both have a certain promoting effect on the enzyme activity, but Tween-20 can significantly reduce the enzyme activity; the optimal substrate of Patatin is short-chain fatty acid; 2. The Patatin D286A mutant designed and modified by site-directed mutagenesis of the present invention significantly increases the denaturation temperature of the protein, improves its selectivity for long-chain substrates, and does not change the protein folding; molecular dynamics simulation shows that the D286A mutant will disrupt the helical configuration of domain 280-286 and transform it into a flexible loop, which can better accommodate the long-chain substrate pNP-C16 in the active pocket; 3. The present invention lays a foundation for the efficient synthesis and large-scale production of Patatin, and improving the catalytic efficiency, stability and specificity of lipase in hydrolyzing long-chain fatty acids. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 It is a diagram showing the influence of four factors on the expression of Patatin. Among them, A is the methanol addition amount; B is the YNB addition amount; C is the inoculation amount; D is the culture time; Figure 2 It is a response surface diagram and contour diagram of the interactive influence on enzyme activity. Among them, A is the methanol concentration and YNB concentration; B is the methanol concentration and inoculation amount; C is the YNB concentration and inoculation amount; Figure 3 It is a diagram showing the influence of pH, temperature, organic solvents and surfactants on the lipase activity of Patatin. Among them, A is the pH; B and C are the temperature; D is the organic solvents and surfactants; Figure 4It is a graph of the substrate specificity of lipase Patatin and related properties of Patatin and its mutants. Among them, A is the residual relative enzyme activity of wild-type Patatin enzyme towards p-nitrophenyl esters with different chain lengths; B is the relative enzyme activity of Patatin wild-type, M325A, F108A and D286A variants towards p-nitrobenzene with different chain lengths; C is the fluorescence intensity of Patatin wild-type, M325A, F108A and D286A variants; D is the circular dichroism spectrum of Patatin wild-type, M325A, F108A and D286A variants. Figure 5 It is the flexibility change of the dynamic loop 258 - 302 in the lipase Patatin D286A mutant. Among them, A is the ΔRMSF value of Loop258 - 302 in the complex of lipase Patatin and pNP-C4; B is the ΔRMSF value of Loop258 - 302 in the complex of lipase Patatin and pNP-C16. Figure 6 It is the affinity of the lipase Patatin D286A mutant for the long-chain substrate pNP-C16. Among them, A is the RMSD value of the complex of lipase Patatin and p-nitrophenyl esters (pNP-C4 and pNP-C16); B is the number of hydrogen bonds between lipase Patatin and the long-chain substrate pNP-C16; C is that the D286A mutation disrupts the helical structure of domain 280 - 286. Detailed implementation manners The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: The main materials involved in the embodiments of the present invention are as follows: The vector pET51b(+) was purchased from Shanghai Yaji Biotechnology Co., Ltd.; Pichia pastoris was purchased from Shanghai Beyotime Biotechnology Co., Ltd. The amino acid sequence of wild-type Patatin is: MHHHHHHAMAQLGEMVTVLSIDGGGIRGIIPATILEFLEGQLQEMDNNADARLADYFDVIG GTSTGGLLTAMISTPNENNRPFAAAKEIVPFYFEHGPQIFNPSGQILGPKYDGKYLMQVLQE KLGETRVHQALTEVVISSFDIKTNKPVIFTKSNLANSPELDAKMYDISYSTAAAPTYFPPHYF VTNTSNGDEYEFNLVDGAVATVADPALLSISVATRLAQKDPAFASIRSLNYKKMLLLSLGTGT TSEFDKTYTAKEAATWTAVHWMLVIQKMTDAASSYMTDYYLSTAFQALDSKNNYLRVQE NALTGTTTEMDDASEANMELLVQVGENLLKKPVSEDNPETYEEALKRFAKLLSDRKKLRANKASY; The amino acid sequence of Patatin F108A is shown in SEQ ID NO.1: MHHHHHHAMAQLGEMVTVLSIDGGGIRGIIPATILEFLEGQLQEMDNNADARLADYFDVIG GTSTGGLLTAMISTPNENNRPFAAAKEIVPFYFEHGPQIANPSGQILGPKYDGKYLMQVLQE KLGETRVHQALTEVVISSFDIKTNKPVIFTKSNLANSPELDAKMYDISYSTAAAPTYFPPHYF VTNTSNGDEYEFNLVDGAVATVADPALLSISVATRLAQKDPAFASIRSLNYKKMLLLSLGTGT TSEFDKTYTAKEAATWTAVHWMLVIQKMTDAASSYMTDYYLSTAFQALDSKNNYLRVQE NALTGTTTEMDDASEANMELLVQVGENLLKKPVSEDNPETYEEALKRFAKLLSDRKKLRANKASY; The amino acid sequence of Patatin D286A is shown in SEQ ID NO.2: MHHHHHHAMAQLGEMVTVLSIDGGGIRGIIPATILEFLEGQLQEMDNNADARLADYFDVIG GTSTGGLLTAMISTPNENNRPFAAAKEIVPFYFEHGPQIFNPSGQILGPKYDGKYLMQVLQE KLGETRVHQALTEVVISSFDIKTNKPVIFTKSNLANSPELDAKMYDISYSTAAAPTYFPPHYF VTNTSNGDEYEFNLVDGAVATVADPALLSISVATRLAQKDPAFASIRSLNYKKMLLLSLGTGT TSEFDKTYTAKEAATWTAVHWMLVIQKMTAAASSYMTDYYLSTAFQALDSKNNYLRVQE NALTGTTTEMDDASEANMELLVQVGENLLKKPVSEDNPETYEEALKRFAKLLSDRKKLRANKASY; The amino acid sequence of Patatin M325A is shown in SEQ ID NO.3: MHHHHHHAMAQLGEMVTVLSIDGGGIRGIIPATILEFLEGQLQEMDNNADARLADYFDVIG GTSTGGLLTAMISTPNENNRPFAAAKEIVPFYFEHGPQIFNPSGQILGPKYDGKYLMQVLQE KLGETRVHQALTEVVISSFDIKTNKPVIFTKSNLANSPELDAKMYDISYSTAAAPTYFPPHYF VTNTSNGDEYEFNLVDGAVATVADPALLSISVATRLAQKDPAFASIRSLNYKKMLLLSLGTGT TSEFDKTYTAKEAATWTAVHWMLVIQKMTDAASSYMTDYYLSTAFQALDSKNNYLRVQE NALTGTTTEADDASEANMELLVQVGENLLKKPVSEDNPETYEEALKRFAKLLSDRKKLRANKASY。 Example 1 A method for preparing a potato Patatin mutant, comprising the following steps: S1. Preparation of Pichia pastoris competent cells: Take a Pichia pastoris monoclonal strain from the YPD solid medium, culture it at 30 °C, transfer the culture solution to 50 mL of YPD medium, and continue to culture until OD 600 = 1.3; Centrifuge at 1500 r / min for 5 min at 4 °C, then resuspend the cells with ice-cold double-distilled water; Centrifuge, resuspend with 25 mL of ice-cold double-distilled water; Centrifuge again, resuspend the cells with 2 mL of 1 mol / L sorbitol solution; After centrifugation again, resuspend with sorbitol solution to make the total volume 100 μL to obtain Pichia pastoris competent cells; S2. Pichia pastoris electrotransformation: The pET51-Patatin expression vector was treated with the endonuclease SacI to obtain the linearized pET51-Patatin expression vector. 5 μL of the linearized expression vector was mixed with Pichia pastoris competent cells, poured into an ice-cold electroporation cuvette, and allowed to stand for 5 min. The electroporation parameters were a voltage of 2 kV, a capacitance of 25 μF, a resistance of 200 Ω, and a pulse time of 5.0 ms. Immediately after electroporation, 1 mL of ice-cold sorbitol solution was added, mixed well, and transferred to a 1.5 mL centrifuge tube. The mixture was cultured at 30 °C for 2 h. The transformed solution in the centrifuge tube was diluted to 100 μL and spread on YPD medium. After culturing overnight at 30 °C, single colonies were selected and verified using the 5-AOX and 3-AOX amplification techniques. S3. Construction of the pET51-Patatin recombinant vector: The target gene Patatin and the vector pET51b(+) were linearized using the restriction endonucleases XhoI and NotI, respectively. The target gene was collected and purified using a gel extraction kit. The target gene was ligated to the vector to obtain the pET51-Patatin recombinant vector. S4. Construction of the mutant recombinant plasmid: Using the pET51-Patatin expression vector as a template, PCR site-directed mutagenesis was performed. The mutants of Patatin were F108A, D286A, and M325A. The PCR reaction program was as follows: 30 cycles of amplification, denaturation at 95 °C for 10 s, annealing at 55 °C for 5 s, and extension at 72 °C for 15 s. After verification by 1% agarose gel electrophoresis, the PCR product was digested with Dpn I for 1 h, and the digested product was transferred into DH5α competent cells to obtain the Patatin mutant recombinant plasmid. S5. Recombinant expression of Patatin: The Patatin mutant recombinant plasmid was transferred into Pichia pastoris to obtain the expression strain. The expression strain was cultured in YPD liquid medium. When OD 600 = 2, the supernatant was discarded by centrifugation, and the cells were collected. S6. Purification: The cells were resuspended in BMGY medium and cultured for 24 h, then allowed to stand overnight. The upper layer of BMGY medium was discarded, and BMMY medium was added for induction for 72 h. Methanol was added every 24 h for induction. The cells were collected by centrifugation, resuspended in buffer, and lysed. The supernatant was collected by centrifugation and the Patatin mutant was purified through a nickel affinity column and further purified by gel filtration chromatography to obtain the purified Patatin mutant. S7. High-density fermentation of recombinant Pichia pastoris: Transfer the Patatin mutant recombinant plasmid into Pichia pastoris to obtain the expression strain; culture the expression strain in YPD liquid medium with an inoculation amount of 10.42%. When OD600 = 2, centrifuge to discard the supernatant and collect the cells; resuspend the cells with BMGY medium, culture and let stand overnight; discard the upper layer of the medium, add BMMY medium with a YNB content of 9.68% for induction for 72 h, add methanol for induction every 24 h to make the final concentration of methanol 0.44%, centrifuge to collect the cells, resuspend the cells in buffer to lyse the cells, centrifuge to collect the supernatant and purify the Patatin mutant through a nickel affinity column, and further purify by gel filtration chromatography to obtain the purified Patatin mutant. The high cell density fermentation process is divided into a basic culture stage (using glycerol as the carbon source, temperature 28 °C, rotation speed 500 r / min, pH 5.0), a fed-batch culture stage, and an induction stage (maintaining the dissolved oxygen at 20 - 30%, temperature 28 °C, pH 5.0 until the end of fermentation). The Patatin mutant prepared according to the above high cell density fermentation method can reach an expression level of 121 mg / L. Taking the methanol addition amount (0.0, 0.5, 1.0, 1.5, 2.0%), YNB content (5.0, 7.5, 10.0, 12.5, 15.0%), inoculation amount (1.0, 5.0, 10.0, 15.0, 20%) and culture time (48, 60, 72, 84, 96 h) as single factors to explore the effects of the above four factors on the production of Patatin by Pichia pastoris. Figure 1 Shows the effects of methanol addition amount, YNB addition amount, inoculation amount and culture time on Patatin expression. When the production of Patatin reaches its peak, the methanol addition amount is only 0.5% ( Figure 1 A), but when the effects of other factors are taken into account, the production of Patatin begins to decline, which is likely because the intake of methanol exceeds the standard, resulting in harmful effects. According to the research results, when the concentration of methanol exceeds 3.65%, the growth and reproduction of Pichia pastoris will be significantly hindered. Therefore, for the fermentation of Pichia pastoris, precise control must be carried out. When the YNB content reaches 10%, the enzyme activity reaches the highest, which is 131.37 U / mg, but as the YNB content increases, the enzyme activity will gradually decline ( Figure 1 B); Figure 1 In C, as the inoculation amount gradually increases, the enzyme production also gradually increases and reaches the maximum value when the inoculation amount is 10%; different culture times have little effect on the Patatin production, and the production can reach 110 U / mg ( Figure 1D). Based on single-factor experiments, the Box-Behnken model was adopted, with methanol concentration, YNB concentration, and inoculum size as influencing factors, which were divided into 3 levels, and response surface experiments were carried out (as Figure 2 ), and the optimal fermentation conditions were obtained as follows: methanol addition of 0.44%, YNB content of 9.68%, and inoculum size of 10.42%. The verification experiment found that when cultured under the optimal conditions optimized by response surface, the final Patatin enzyme activity was 130.21 U / mg. Effect of metal ions on Patatin enzyme activity Table 1 Effect of metal ions on Patatin enzyme activity Note: Different lowercase letters in each column represent significant differences (P < 0.05). As shown in Table 1, 1 mmol / L of Ca 2+ , Zn 2+ and Mg 2+ can significantly enhance the lipase activity of Patatin, while Fe 2+ , Fe 3+ and Cu 2+ at 1 mmol / L and above have an inhibitory effect on the lipase activity of Patatin. Application of potato Patatin mutants in hydrolyzing substrates with different chain lengths: I. Effects of pH, temperature, organic solvents, and surfactants on the lipase activity of Patatin Figure 3 In A, as the pH of the buffer solution increases, the lipase activity also increases significantly. When the pH value reaches 10.0, the lipase activity reaches 100%, indicating that the optimal reaction pH value of Patatin is 10.0; when the pH value is 4.0 - 6.0, the lipase activity decreases significantly, and its relative enzyme activity is about 30%. This may be due to the existence of an acidic environment, which causes some groups to dissociate, thereby hindering the binding of lipase to the substrate, and then reducing the reaction rate, resulting in a decrease in enzyme activity. Patatin has good alkali resistance and can be effectively applied in an alkaline environment to improve its function. Figure 3 In B, when the temperature ranges from 20 °C to 60 °C, the lipase activity first increases and then decreases. When the temperature reaches 35 °C, the catalytic activity of the lipase reaches the maximum of 100%, indicating that the optimal reaction temperature of Patatin is about 35 °C; under the conditions of 40 - 50 °C, the relative activities of the lipases are all higher than 40%, but when the temperature rises to 60 °C, their relative activities will decrease significantly, and the remaining relative activity is only about 20%; after treating the enzyme with 70 °C for 30 min, the relative enzyme activity is 0%, and Patatin is completely inactivated ( Figure 3C); Consisting of Figure 3 As can be seen from D, hydrophilic organic solvents such as methanol, ethanol, and isopropanol can significantly enhance the enzyme activity of lipase. Moreover, 30% methanol can increase the enzyme activity by more than 60%. Due to the high adsorption capacity on the surface of lipase, they are more easily adsorbed by organic solutes with high adsorption capacity, thus accelerating their binding with the substrate. Although both Triton X-100 and SDS can improve the enzyme activity to a certain extent, the promoting effect of SDS is more significant, and the relative enzyme activity reaches 124.52 ± 4.14%. Tween-80 and EDTA cannot significantly improve the enzyme activity, but Tween-20 can significantly reduce the enzyme activity. II. Substrate specificity of lipase Patatin and related properties of Patatin and its mutants Figure 4 In A, wild-type Patatin showed extremely strong hydrolysis ability when treating p-nitrophenyl butyrate (pNP-C4), while its activity was relatively low when treating p-nitrophenyl myristate (pNP-C14) and p-nitrophenyl palmitate (pNP-C16). Through research, it was found that lipase Patatin has a significantly better effect on treating short-chain p-nitrophenyl esters than other types of substrates, indicating that pNP-C4 is the ideal substrate for lipase Patatin, and it can more effectively treat p-nitrophenyl esters of the C4-C8 type. 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 using p-NPP (p-nitrophenyl phosphate) with different chain lengths as substrates ( Figure 4 In B, it was found by measurement that compared with other mutants, mutant D286A has stronger specificity for long-chain substrates. Figure 4 In C, the Tm values of Patatin and its mutants were measured by fluorescence quantitative PCR. The Tm value of Patatin was 66.18 °C, and the Tm values of the mutants all decreased to a certain extent compared with the wild type. The Tm value of F108A was 60.57 °C, the Tm value of D286A was 60.82 °C, and the Tm value of M325A was 62.34 °C; Figure 4 The circular dichroism spectrum in D shows that the structures of the three mutants after mutation are basically similar to that of the wild type. Therefore, it can be inferred that the mutation does not affect the protein folding. III. Flexibility changes of the dynamic loop 258-302 in the lipase Patatin D286A mutant Such as Figure 5As shown, compared with the wild-type lipase (WT), the D286A mutant led to a decrease in the ΔRMSF value of the dynamic loop 258 - 302, indicating a significant increase in the rigidity and a significant decrease in the flexibility of this domain; in addition, the RMSD of the D286A mutant with the pNP-C4 / pNP-C16 complex was significantly lower than that with the pNP-C4 / pNP-C16 complex (as Figure 6 A, B), indicating that the D286A mutant stabilized the binding of Patatin to the substrate. IV. Affinity of the Lipase Patatin D286A Mutant for the Long-chain Substrate pNP-C16 Figure 6 A shows the RMSD values of the lipase Patatin complex with p-nitrophenyl esters (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 with pNP-C16 increased significantly ( Figure 6 B), indicating an enhanced affinity between the D286A mutant Patatin and pNP-C16. The increase in the specific activity of the D286A mutant for pNP-C16 determined experimentally also supports this mechanism ( Figure 4 B). Figure 6 As can be seen in C, the D286A mutation disrupted the helical structure of domain 280 - 286, converting it into a flexible loop that can better regulate the long-chain substrate pNP-C16. This may be the key molecular mechanism for the high specificity of the D286A mutant for long-chain substrates. As described above, it is only a preferred embodiment of the present invention and does not impose any formal limitations on the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes, without departing from the spirit and technical solution of the present invention. Therefore, any simple modification, equivalent replacement, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A potato Patatin mutant, characterized in that: The Patatin mutant is Patatin F108A, Patatin D286A or Patatin M325A, and its amino acid sequence corresponds to SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3 respectively.

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 a host cell 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: The following steps are involved: S1. Construction of pET51-Patatin recombinant vector: linearize the target gene Patatin and the vector pET51b(+) using XhoI and NotI restriction endonucleases respectively, and then combine the target gene with the vector to obtain the pET51-Patatin recombinant vector; S2. Construction of mutant recombinant plasmid: Using the pET51-Patatin recombinant vector as a 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 transferred into Pichia pastoris to obtain an expression strain; the expression strain was cultured in YPD liquid medium, and when OD 600 =2, centrifuge and discard the supernatant to collect the bacteria; S4. Purification: Resuspend the bacteria in BMGY medium and let it stand overnight after cultivation; discard the upper medium, add BMMY medium for induction, add methanol for induction every 24 hours, collect the bacteria by centrifugation, resuspend the bacteria in buffer to break the bacteria, collect the supernatant by centrifugation and purify the Patatin mutant through a nickel affinity column, and further purify it 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 procedure in step S2 is: 30 cycles of amplification, deformation at 95°C for 10s, annealing at 55°C for 5s, and extension at 72°C for 15s.

6. The method for preparing a potato Patatin mutant according to claim 4, characterized in that: The high-density fermentation steps are as follows: transferring the Patatin mutant recombinant plasmid into Pichia pastoris to obtain an expression strain; culturing the expression strain in a YPD liquid culture medium with an inoculation amount of 10.42%, centrifuging and discarding the supernatant when OD600=2, and collecting the bacteria; resuspending the bacteria with a BMGY culture medium, and standing overnight after culturing; discarding the upper culture medium, adding a BMMY culture medium with a YNB content of 9.68% to induce for 72 hours, adding methanol for induction every 24 hours to make the final concentration of methanol 0.44%, centrifuging and collecting the bacteria, resuspending the bacteria in a buffer solution to break the bacteria, centrifuging and collecting the supernatant, and purifying the Patatin mutant through a nickel affinity column, and further purifying by gel filtration chromatography to obtain a purified Patatin mutant.

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

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