Acidic lipase mutant with improved thermal stability and activity and application thereof

Abstract

The invention discloses an acidic lipase mutant with improved thermal stability and activity and application thereof.A semi-rational design strategy is adopted, disulfide bond and amino acid site mutation are combined, the lipase mutant with improved thermal stability and activity is obtained, then a combinatorial optimization strategy is adopted, efficient expression of LIPR is achieved in pichia pastoris, and the lipase mutant with improved thermal stability and activity is obtained. The biodiesel preparation capacity of the LIPR is evaluated, and the biodiesel yield is obviously increased through a continuous flow system. According to the invention, the development of reusable bio-enzyme catalysts is facilitated, and the bio-enzyme catalysts are used for producing biodiesel and other chemicals.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and in particular relates to an acid lipase mutant with improved thermal stability and activity and application thereof. Background Art

[0002] Lipase (EC 3.1.1.3) is a proteinaceous acylglycerol hydrolase that is ubiquitous in nature. Under natural conditions, lipase can catalyze the production of free fatty acids, esters and glycerol of different chain lengths at the oil-water mixed interface, and participate in a variety of reactions such as transesterification, esterification, acidolysis and aminolysis in non-aqueous phase systems. At present, lipases have been widely used in industrial fields such as food, chemical, and pharmaceutical. However, the defects of lipases occasionally hinder their applicability in certain industrial applications, among which limited thermal stability is one of the recognized shortcomings. The high thermal stability of lipases is a key factor determining their efficacy in enzyme-mediated industrial processes, because the catalytic efficiency of lipases increases exponentially with increasing temperature until the protein denatures. Therefore, in addition to pursuing new enzymes with naturally high thermal stability, rational design aimed at improving the thermal stability of enzymes remains a major goal of researchers.

[0003] In recent years, researchers have isolated a fungal lipase, R.emersonii, from an Australian palm tree. The lipase has good tolerance to organic reagents, especially methanol, and can be used to produce biodiesel in the future. However, as a new type of acid lipase, no one has yet used its properties for industrial applications. This may be due to the low expression level of LIPR in the original strain and the high production cost. Therefore, it is particularly important to achieve efficient expression and secretion of LIPR.

[0004] Biodiesel (fatty acid methyl ester, FAME) is a renewable fuel with similar properties to conventional diesel, while being easily biodegradable, non-toxic, and environmentally friendly. In recent years, the production of biodiesel has attracted great interest among researchers, and a large number of studies have demonstrated the advantages and feasibility of biodiesel as an alternative to conventional energy fuels. Compared with the commonly used chemical acid-base catalysts, lipase-mediated transesterification of vegetable oils and microbial oils is a more environmentally friendly method for biodiesel production. In addition, current research on the production of biodiesel via transesterification is mainly carried out in batch reactors (i.e., flasks), which may limit the synthesis rate and productivity due to insufficient substrate mixing and low mass / heat transfer efficiency. In contrast, continuous flow systems can not only improve mass / heat transfer, reduce reaction volume, shorten reaction time, and increase spatial and temporal yields, but are also more conducive to continuous production and easier to scale up the process. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] One of the objects of the present invention is to provide an acid lipase mutant with improved thermostability and activity.

[0008] To solve the above technical problems, the present invention provides the following technical solution: an acid lipase mutant with improved thermal stability and activity, wherein the acid lipase mutant is a substitution selected from any one of the following (i) to (vi) in any one of the following mutant amino acid sequences (a1) to (a4):

[0009] (a1) A mutant in which both Ala at position 51 and Ser at position 110 of the amino acid sequence of lipase LIPR as shown in SEQ ID NO. 2 are mutated to Cys, and a disulfide bond is formed between the two Cys;

[0010] (a2) a mutant in which the Ala at position 126 and the Phe at position 238 of the amino acid sequence of the lipase LIPR as shown in SEQ ID NO. 2 are both mutated to Cys, and a disulfide bond is formed between the two Cys;

[0011] (a3) a mutant in which the Ala at position 131 and the Ala at position 140 of the amino acid sequence of the lipase LIPR as shown in SEQ ID NO. 2 are both mutated to Cys, and a disulfide bond is formed between the two Cys;

[0012] (a4) a mutant in which both Ser at position 255 and Val at position 261 of the amino acid sequence of the lipase LIPR as shown in SEQ ID NO. 2 are mutated to Cys, and a disulfide bond is formed between the two Cys;

[0013] (i) mutating the Glu at position 58 of the amino acid sequence of the mutant to Gly;

[0014] (ii) mutating Glu at position 139 of the amino acid sequence of the mutant to Leu;

[0015] (iii) mutating Asn at position 196 of the amino acid sequence of the mutant to Val;

[0016] (iv) mutating Asp at position 198 of the amino acid sequence of the mutant to Leu;

[0017] (v) mutating Asp at position 214 of the amino acid sequence of the mutant to Leu;

[0018] (vi) Glu at position 250 of the amino acid sequence of the mutant is mutated to Leu.

[0019] As a preferred embodiment of the acid lipase mutant with improved thermal stability and activity of the present invention, the acid lipase mutant is:

[0020] A mutant in which the Ala at position 126 and the Phe at position 238 of the amino acid sequence of the lipase LIPR as shown in SEQ ID NO.2 are both mutated to Cys, and a disulfide bond is formed between the two Cys; at the same time,

[0021] The Asp at position 214 of the amino acid sequence of the mutant was mutated to Leu.

[0022] As a preferred embodiment of the acid lipase mutant with improved thermal stability and activity of the present invention, the nucleotide sequence of the acid lipase mutant is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO.8.

[0023] Another object of the present invention is to provide a nucleic acid molecule encoding the acid lipase mutant as described above.

[0024] Another object of the present invention is to provide a biomaterial containing the nucleic acid molecule as described above, characterized in that the biomaterial comprises at least one of (b1) to (b11):

[0025] (b1) an expression cassette containing the nucleic acid molecule;

[0026] (b2) a recombinant vector containing the nucleic acid molecule;

[0027] (b3) a recombinant vector containing the expression cassette described in (b1);

[0028] (b4) a recombinant cell containing the nucleic acid molecule;

[0029] (b5) a recombinant cell containing the expression cassette described in (b1);

[0030] (b6) a recombinant cell containing the recombinant vector described in (b2);

[0031] (b7) a recombinant cell containing the recombinant vector described in (b3);

[0032] (b8) a recombinant microorganism containing the nucleic acid molecule;

[0033] (b9) a recombinant microorganism containing the expression cassette described in (b1);

[0034] (b10) a recombinant microorganism containing the recombinant vector described in (b2);

[0035] (b11) A recombinant microorganism containing the recombinant vector described in (b3).

[0036] As a preferred embodiment of the biomaterial of the present invention, the recombinant microorganism is Pichia pastoris, and the Pichia pastoris comprises at least one of (c1) to (c4):

[0037] (c1) replacing the α-factor signal peptide in the recombinant vector described in (b2) with the Mss signal peptide; the amino acid sequence of the Mss signal peptide is shown in SEQ ID NO.4;

[0038] (c2) the recombinant microorganism co-expresses a molecular chaperone PDI; the gene sequence of the molecular chaperone PDI is shown in SEQ ID NO.9;

[0039] (c3) the vacuolar sorting receptor Vps10-01 is knocked out in the recombinant microorganism; the gene sequence of the vacuolar sorting receptor Vps10-01 is shown in SEQ ID NO.10;

[0040] (c4) The dihydroxyacetone synthase gene DAS2 is overexpressed in the recombinant microorganism; the gene sequence of the dihydroxyacetone synthase gene DAS2 is shown in SEQ ID NO.11.

[0041] Another object of the present invention is to provide the use of the acid lipase mutant, the nucleic acid molecule or the biomaterial as described above in the preparation of biodiesel.

[0042] Another object of the present invention is to provide a method for preparing biodiesel, which uses the acid lipase mutant, the nucleic acid molecule or the biological material as described above to biocatalyze the preparation of biodiesel using olive oil and methanol as substrates.

[0043] As a preferred embodiment of the method for preparing biodiesel of the present invention, the crude enzyme of the acid lipase mutant is obtained by the acid lipase mutant as described in claim 1, the nucleic acid molecule as described in claim 2 or the biomaterial as described in claim 3, and the enzyme is immobilized by resin.

[0044] As a preferred embodiment of the method for preparing biodiesel of the present invention, the catalytic preparation of biodiesel is carried out in a continuous flow reactor, the solvent used is n-hexane, the amount of immobilized enzyme is 0.5-2.5 g, the reaction temperature is 35-55° C., the reaction time is 1-5 h, the oil-to-alcohol ratio is 1:3-7, and the flow rate of the continuous flow reactor is 5-25 mL / h.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The present invention uses a semi-rational design strategy combined with disulfide bond and amino acid site mutations to improve the thermal stability and activity of the lipase LIPR, and then adopts a combinatorial optimization strategy to achieve efficient expression of LIPR in Pichia pastoris. The present invention helps to develop reusable bio-enzyme catalysts for the production of biodiesel and other chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0048] Figure 1 The effects of combined optimized mutant strains on LIPR expression and activity; (A) shows the effects of different combined optimized mutant strains on LIPR activity, (B) shows the SDS-PAGE analysis of the supernatant of the recombinant strain GS54-LIPR at different induction times in a 5L fermenter, and (C) shows the LIPR activity, total supernatant protein and dry cell weight (DCW) during fermentation in a 5L fermenter.

[0049] Figure 2 The figure shows the effect of disulfide bond mutation on LIPR enzyme activity and thermal stability; wherein, (A) shows the effect of disulfide bond mutation on LIPR activity, and (B) shows the thermal stability of disulfide bond mutants with high enzyme activity.

[0050] Figure 3 is the half-life of different mutants at 55°C.

[0051] Figure 4 The effects of amino acid site mutations on LIPR enzyme activity and thermal stability; wherein, (A) is the effect of amino acid site mutations on LIPR activity, and (B) is the determination of thermal stability of amino acid site mutants.

[0052] Figure 5 Effect of reaction time on biodiesel production by LIPR.

[0053] Figure 6 Schematic diagram of lipase LIPR for biodiesel production in a continuous flow reactor.

[0054] Figure 7 Optimized reaction parameters for biodiesel in a continuous flow reactor; wherein (A) is reaction time, (B) is reaction temperature, (C) is enzyme dosage, (D) is the molar ratio of oil to methanol, and (E) is flow rate. DETAILED DESCRIPTION

[0055] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0056] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0057] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0058] For the convenience of describing the present invention, conventional and unconventional abbreviations of various amino acid residues are used. These abbreviations are familiar to those skilled in the art, but are listed below for clarity:

[0059] Asp = D = aspartic acid; Ala = A = alanine; Arg = R = arginine;

[0060] Asn = N = asparagine; Gly = G = glycine; Glu = E = glutamic acid;

[0061] Gln = Q = glutamine; His = H = histidine; Ile = I = isoleucine;

[0062] Leu = L = leucine; Lys = K = lysine; Met = M = methionine;

[0063] Phe = F = phenylalanine; Pro = P = proline; Ser = S = serine;

[0064] Thr=T=threonine; Trp=W=tryptophan; Tyr=Y=tyrosine;

[0065] Val = V = valine; Cys = C = cysteine.

[0066] The mutants described in the present invention are named according to the conventional naming method of those skilled in the art. For example, the mutant D214L indicates that the aspartic acid (D) at position 2214 of the amino acid sequence is site-directedly mutated to leucine (L).

[0067] The biological materials used in the present invention and their sources are shown in the table:

[0068] Escherichia coli Top 10, Pichia pastoris GS115, and plasmids pPIC9K and pPICZA were purchased from commercial sources. LA Taq DNA polymerase, restriction endonucleases, and In-Fusion PCR cloning kits were purchased from TaKaRa. All chemical reagents were purchased from Sangon Biotech (Shanghai) Co., Ltd., and all biochemical reagents were of analytical grade or higher quality.

[0069] Unless otherwise specified, other raw materials used in the examples were purchased commercially.

[0070] Example 1

[0071] (1) A novel acid lipase LIPR from R.emersoni (GenBank accession number: XP_013323577, nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2), based on the preference of Pichia codons, the base sequence encoding LIPR was optimized and artificially synthesized. It was inserted into the methanol-inducible secretory plasmid pPIC9K of Pichia pastoris, and the double enzyme digestion technology was used to achieve the insertion of LIPR into pPIC9K. The enzymes used for the double enzyme digestion were EcoRI and NotI, respectively. After the double enzyme digestion was completed, enzyme ligation was performed, and after purification, it was transformed into Escherichia coli DH5α competent cells, thereby successfully constructing the recombinant plasmid vector pPIC9K-LIPR.

[0072] (2) The expression vector pPIC9K-LIPR was linearized by SalI and electroporated into Pichia pastoris GS115, and plated on MD (20 g / L agar, 10 mL 1×YNB, 40 mL 50% glucose, 2 mL 500× biotin) plates. After 2-3 days, the transformants on the MD plates were gradually picked onto YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) plates containing different mass concentrations (0.5, 1.0, 2.0 and 3.0 mg / mL) of G418 geneticin to screen out the recombinant strain GS01-LIPR containing multiple copies of the gene.

[0073] (3) LIPR activity was detected by alkaline titration. In a 50 mL conical flask, 5 mL Tris-HCl (50 mM, pH 10.0) and 4 mL olive oil emulsion were added in sequence and preheated in a 40°C water bath for 5 min. 1 mL of preheated fermentation supernatant enzyme solution diluted appropriately was added to the experimental group, and 1 mL of preheated Tris-HCl was added to the blank group. After reacting in a 40°C water bath for 10 min, 15 mL of 95% ethanol solution was immediately added to terminate the reaction. Two drops of phenolphthalein indicator were added to the conical flask, and titrated with 0.05 M NaOH standard solution until the liquid turned slightly red and remained unchanged for 30 seconds, and the volume of NaOH standard solution consumed was recorded. The average value of three experiments was taken. The definition of the above-mentioned unit lipase LIPR enzyme activity is: the amount of enzyme required to produce 1 μmol of free fatty acids per minute under the above reaction conditions.

[0074] (4) The selected recombinant yeast strain GS01-LIPR was inoculated into 5 mL of YPD medium and cultured at 30°C for 12 h; the bacterial solution in YPD was transferred to 50 mL of BMGY medium (20 g / L peptone, 10 g / L yeast extract, 10 g / L glycerol, 3 g / L K2HPO4, 11.8 g / L KH2PO4, 10 mL 1×YNB, 2 mL 500× biotin) at a 4% inoculum volume and cultured in a shaking incubator at 30°C until OD 600 The cells in BMGY were collected by centrifugation, washed twice with 50 mL sterile water (precooled) and collected by centrifugation, resuspended in 50 mL BMMY (peptone 20 g / L, yeast extract 10 g / L, methanol 10 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, 10 mL 1×YNB, 2 mL 500×biotin) medium for fermentation, and 1% methanol was added every 24 h for induction. At the same time, the supernatant enzyme solution was taken and the protein expression of LIPR was detected by SDS-PAGE. The results are shown in the figure. Figure 1 As shown, the enzyme activity of GS01-LIPR reached 325U / mL.

[0075] Example 2

[0076] Effect of signal peptide on the expression level of lipase in Pichia pastoris

[0077] The secretory signal peptide MFH4 signal sequence (Mss) homologous to Pichia pastoris as shown in Table 1 was selected, and the signal peptide was amplified by designing the corresponding signal peptide primer as a template, and then connected to pPIC9K-LIPR after BamHI and EcoRI digestion to replace the α-factor signal peptide in the plasmid to construct the plasmid pPIC9K-Mss-LIP2.

[0078] Table 1

[0079]

[0080] Referring to the method of yeast transformation and screening of recombinant strains in Example 1, a recombinant yeast strain GS02-LIPR with optimized signal peptide was successfully constructed. Figure 1 It can be seen that the signal peptide Mss can increase the secretion activity of LIPR to 725U / mL, which is 2.2 times that of the starting strain.

[0081] Example 3

[0082] Effects of disulfide bonds on the thermal stability and activity of lipase

[0083] (1) 14 pairs of potential disulfide bond mutants were screened as shown in Table 2.

[0084] Table 2-

[0085]

[0086] (2) Using plasmid pPIC9K / Mss-LIPR as a template, primers were designed according to the disulfide mutation site to amplify the target gene. Subsequently, the plasmid pPIC9K was digested with BamHI and NotI, and then homologously recombined with the target gene using the In-Fusion PCR cloning kit to generate a disulfide bond mutation plasmid. The construction process of other disulfide bond mutation plasmids was the same. The mutant plasmid was transformed into Pichia pastoris GS115 by electroporation to obtain 14 recombinant strains carrying different disulfide bond mutations.

[0087] Referring to the yeast expression method in Example 1, the parent strain GS02-LIPR and 14 disulfide bond mutant strains were expressed. The results of enzyme activity determination are as follows Figure 2 As shown in A, four mutants (A51C-S110C, A126C-P238C, A131C-A140C, and S255C-V261C) showed higher enzyme activity compared to GS02-LIPR. In order to integrate beneficial mutations, we further selected these four mutants to determine the thermal stability of LIPR ( Figure 2 B). Among them, the mutant A126C-P238C has the highest thermal stability and can still maintain 45% of its original activity after incubation at 55°C for 3 hours, which is significantly better than GS02-LIPR (activity is only 28%). The half-life of GS02-LIPR enzyme at 55°C is only 101min, while the half-life of the mutant A126C-P238C enzyme reaches 166min ( Figure 3). In addition, the enzyme activity of mutant A126C-P238C reached 810U / mL, which was higher than that of GS02-LIPR. In view of the fact that GS08-LIPR (mutant A126C-P238C) showed the most favorable enzyme activity-thermal stability balance, amino acid site mutations were selected based on it. The nucleotide sequence of the disulfide bond mutant GS08-LIPR is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0088] Example 4

[0089] Effects of amino acid mutations on the thermal stability and activity of lipase

[0090] (1) 35 potential mutants shown in Table 3 were screened out.

[0091] Table 3

[0092]

[0093]

[0094] (2) Using the disulfide bond mutant plasmid pPIC9K / Mss-LIPR / A126C-P238C as a template, primers were designed according to the amino acid mutation site to amplify the target gene. The plasmid pPIC9K was digested with EcoRI and NotI, and then homologous recombination with the target gene was performed using the In-Fusion PCR cloning kit to generate an amino acid site mutant plasmid. The mutant plasmid was transformed into Pichia pastoris GS115 by electroporation to obtain a recombinant strain with amino acid site mutations. The construction process of other amino acid site mutant strains was the same.

[0095] (3) The enzyme activities of 35 strains with amino acid mutations were evaluated. Figure 4 A), the results showed that the enzyme activities of three mutants (E58G, N196V and D214L) were better than those of the parent strain GS08-LIPR. In addition, the thermal stability of 35 mutants was evaluated, of which four mutants (E139L, D198L, D214L and E250L) showed improved thermal stability relative to GS08-LIPR ( Figure 4 B). Although mutant E250L has the highest thermal stability, its enzyme activity is only 732U / mL, which is lower than GS08-LIPR. In contrast, mutant D214L has an enzyme activity of 885U / mL, and after incubation at 55℃ for 3h, its enzyme activity remains 58% of the original. In addition, the half-life of mutant D214L at 55℃ is 215min, which is much higher than GS08-LIPR ( Figure 3). It is noteworthy that the enzyme activity and thermal stability of mutant D214L were significantly improved compared with GS08-LIPR ( Figure 4 ). Considering that the enzyme activity and thermal stability must be balanced in the process of enzyme modification to be more conducive to industrial application, GS46-LIPR with the amino acid site mutation D214L was selected for subsequent research. The nucleotide sequence of the disulfide bond and amino acid site mutant GS46-LIPR is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8.

[0096] Example 5

[0097] Effects of Molecular Chaperones on the Expression Level of Lipase in Pichia pastoris

[0098] (1) Obtain the gene sequence of the molecular chaperone PDI (nucleotide sequence as shown in SEQ ID NO.9), design primers based on the gene sequence, use the extracted Pichia pastoris GS115 genome as a template, amplify the corresponding molecular chaperone fragment, and connect it to pPICZA after BamHI and EcoRI digestion to construct plasmid pPICZA-PDI. For the specific operation process, please refer to the literature Wang B, et al. Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion invitro. J Sci Food Agric. 2024 Jul; 104 (9): 5603-5613.

[0099] (2) Using the mutant GS46-LIPR as the starting strain, pPICZA-PDI was linearized with SacI, recovered and concentrated, and then electroporated into the recombinant strain GS46-LIPR to construct the recombinant yeast strain GS52-LIPR with optimized molecular chaperone co-expression.

[0100] (3) Referring to the yeast expression method in Example 1, the recombinant yeast strain GS52-LIPR was expressed. Figure 1 As shown, the molecular chaperone PDI can increase the secretory activity of LIPR to 1230 U / mL, which is 3.8 times that of the unoptimized LIPR. Through this example, a strain GS52-LIPR with the highest lipase activity was obtained, and it was selected for the next step of optimization research.

[0101] Example 6

[0102] Effect of Knockout of Vacuolar Sorting Receptor on the Expression Level of Lipase in Pichia pastoris

[0103] (1) Obtain the gene sequence of vacuolar sorting receptor Vps10-01 (nucleotide sequence as shown in SEQ ID NO.10), and design the corresponding sgRNA through the E-CRISPR website. Use restriction endonuclease BpiⅠ to cut the vector pCas9, obtain the gene fragment sgRNA by fusion PCR, and obtain the plasmid pCas9-sgRNAVps10-01 after double enzyme digestion. For the specific operation process, please refer to the literature Wang B, et al. Highly efficient expression of Rasamsonia emersoniilipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro. J Sci Food Agric. 2024 Jul; 104 (9): 5603-5613.

[0104] (2) Using the Pichia pastoris GS115 genome as a template, primers were used to amplify 1000 bp of the upstream and downstream gene fragments of the Vps10-01 gene. Next, fusion PCR was used to assemble a 2000 bp Vps10-01 donor DNA, and the PCR product was purified.

[0105] (3) 1 μg of circular pCas9-sgRNAVps10-01 plasmid and 5 μg of Donor DNA were mixed evenly with the recombinant yeast GS52-LIPR competent cells in an electroporation cup, and placed on ice for 15 min. The plasmid and Donor DNA were simultaneously transformed into yeast by electroporation (1500 V, 250 Ω). 1 mol / L D-sorbitol (precooled) was quickly added, and pre-cultured at 30°C for 2 h. The plasmid was spread on a YPD plate containing 200 μg / mL hygromycin and grown for 2-3 days. Primers (V 1300 -F / R) positive transformants were verified by Pichia colony PCR. The selected positive transformants were streaked twice on hygromycin plates, and then streaked on non-resistant YPD plates to remove the pCas9 plasmid in the cells until they could not grow on hygromycin plates. The genome of the selected positive transformants was extracted, and the positive transformants were verified again by PCR using the genome as a template to obtain the recombinant yeast strain GS53-LIPR with successful knockout of the Vps10-01 gene.

[0106] (4) Referring to the yeast expression method in Example 1, the knockout recombinant yeast strain GS53-LIPR was expressed. Figure 1 As shown, the recombinant strain GS53-LIPR with the Vps10-01 gene knocked out can increase the secretion activity of LIPR to 1470U / mL, which is 4.5 times the activity of the unoptimized LIPR. Through this example, a strain GS53-LIPR with the highest lipase activity was obtained, and it was selected for the next step of optimization research.

[0107] Example 7

[0108] Effect of overexpression of dihydroxyacetone synthase gene DAS2 on the expression level of lipase in Pichia pastoris

[0109] (1) Obtain the gene sequence of the dihydroxyacetone synthase gene DAS2 (nucleotide sequence as shown in SEQ ID NO.11), and on the basis of knocking out the Vps10-01 gene, fuse the expression cassette of the DAS2 gene with the Donor DNA fragment of Vps10-01 to obtain a Donor DNA element that overexpresses the DAS2 gene while knocking out the Vps10-01 gene. Referring to the gene knockout method in Example 6, a recombinant strain GS54-LIPR overexpressing the DAS2 gene was successfully constructed. For the specific operation process, please refer to the literature Wang B, et al. Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion invitro. J Sci Food Agric. 2024 Jul; 104 (9): 5603-5613.

[0110] (2) Referring to the yeast expression method in Example 1, the recombinant strain GS54-LIPR overexpressing the DAS2 gene was expressed. Figure 1 As shown, the recombinant strain GS54-LIPR overexpressing the DAS2 gene can increase the secretase activity of LIPR to 1635U / mL, which is 5 times that of the starting strain.

[0111] Comparative Example 1

[0112] Construction of combinatorially optimized recombinant strains without mutation-modified LIPR

[0113] (1) With reference to the combined optimization methods of LIPR in Examples 2, 5, 6 and 7, the strain GS01-LIPR without disulfide bond and amino acid site mutation was optimized. The recombinant strain GS55-LIPR was successfully constructed after optimization of signal peptide Mss, optimization of molecular chaperone PDI co-expression, knockout of vacuolar sorting receptor Vps10-01, and overexpression of dihydroxyacetone synthase gene DAS2.

[0114] (2) GS55-LIPR is a strain that has not undergone disulfide bond and amino acid site mutations, and its thermal stability is much lower than that of GS54-LIPR.

[0115] Example 8

[0116] Recombinant strain GS54-LIPR high-density fermentation

[0117] (1) The recombinant yeast strain GS54-LIPR was inoculated into 10 mL of YPD medium and cultured at 30°C for 12 h; the bacterial solution in YPD was transferred to 200 mL of BMGY medium at a 4% inoculum and cultured at 30°C for 24 h as the fermentation seed solution. The fermentation seed solution was inoculated into a 5 L fermentation tank containing 2 L of BSM (85% H3PO4 26.7 mL / L, CaSO4·2H2O 0.93 g / L, K2SO4 18.2 g / L, MgSO4·2H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 g / L, PTM 14 mL / L) inorganic salt medium, and the pH was adjusted by automatic feeding of 50% phosphoric acid and ammonia water. During the glycerol feeding culture stage, the pH was adjusted to 5.5 and the temperature was adjusted to 30°C. Glycerol was added by DO-star method. When the wet weight reached 260g / L or above, the addition of glycerol was stopped and starvation treatment was carried out for 30 minutes before entering the methanol addition induction stage. At this time, the temperature was adjusted to 27°C, methanol was slowly added to the culture medium, and the methanol content in the fermentation broth was detected by a methanol detector. Samples were taken every 24 hours to detect the cell dry weight (DCW), total protein content in the supernatant, and LIPR enzyme activity.

[0118] (2) The results of high-density fermentation of recombinant strains are as follows Figure 1 As shown in Figure C, with the increase of induction time, the yield of recombinant LIPR gradually increased. When the induction time reached 144h, the enzyme activity of LIPR was the highest, reaching 13620U / mL. Compared with shake flask fermentation, the enzyme activity increased by 8.3 times, achieving efficient expression and accumulation of LIPR in recombinant yeast. And with the increase of fermentation time, the total protein content and cell dry weight of the supernatant of the recombinant engineering strain also continued to increase. When the induction fermentation time was 168h, the cell dry weight reached a maximum of 170.3g / L. Since Pichia pastoris GS115 rarely secretes endogenous proteins to the extracellular space during the fermentation stage, the fermentation supernatant enzyme solution is basically recombinant LIP2 (such as Figure 1 B), which is beneficial to the separation and purification of acid lipase LIP2 for further industrial production.

[0119] Example 9

[0120] LIPR method for preparing biodiesel

[0121] (1) 100 mL of the fermentation supernatant in Example 8 was taken and concentrated using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. Subsequently, the crude enzyme sample was dialyzed using a dialysis bag with a molecular weight cutoff of 14 kDa, and finally freeze-dried using a freeze dryer and stored at -20°C.

[0122] (2) LIPR uses LXTE-1000 resin from Xi'an Lanxiao Technology Co., Ltd. to immobilize the enzyme. The immobilization method is as follows: First, the resin is washed with 0.2 M phosphate buffer, and then the enzyme and resin are mixed at a ratio of 40 mg enzyme to 1 g resin and incubated in a reactor at 20°C and 80 rpm for 18 h. After the reaction is completed, the immobilized enzyme is washed with 0.5 M NaCl buffer, and excess water is removed using a vacuum filtration device. The activity of the immobilized enzyme is evaluated using the alkaline titration point method.

[0123] (3) Biodiesel preparation conditions: 2 g of immobilized enzyme was placed in a 50 mL shaking flask. The reaction substrates were olive oil and methanol, of which 2 g of olive oil and a molar ratio of 6:1 were mixed with 7 mL of n-hexane. The entire reaction was carried out in a 50°C water bath shaker at a speed of 180 r / min. After the reaction, the sample was centrifuged at 9000 r / min for 5 min, and the upper liquid was taken for gas chromatography analysis. The yield of fatty acid methyl ester (FAME) was quantified using methyl ester of heptadecanoate as an internal standard. The yield of fatty acid methyl ester was calculated as the ratio of the total amount of methyl ester formed after the reaction to the total amount of fatty acids in the olive oil before the reaction.

[0124] (4) Figure 5 The biodiesel yields at different reaction times were different, with the highest yield at 24h. In addition, the biodiesel yield of the mutant GS54-LIPR at 24h was 52.2%, which was 4.3 times that of the unmutated GS55-LIPR, indicating that the disulfide bond and amino acid site mutations that improve the thermal stability of LIPR help enhance the yield of biodiesel.

[0125] Example 10

[0126] Biodiesel production in a continuous flow reactor

[0127] (1) The continuous flow reactor designed by the present invention is as follows Figure 6As shown, the immobilized enzyme and molecular sieve are filled in the inner layer of a jacketed chromatography column (10×200 mm, Beijing Henghui Instrument Co., Ltd.), and the outer layer is filled with constant temperature water circulating in a water bath to adjust the reaction temperature. There are two types of immobilized enzymes, namely, the combined optimized mutant GS54-LIPR and the unmutated combined optimized GS55-LIPR. The method for preparing biodiesel in a continuous flow reactor is as follows: First, fill the inner layer of the chromatography column with The immobilized lipase was prepared by mixing 100 μg of immobilized lipase with 400 μg of molecular sieve and 2 g of immobilized lipase. The substrates olive oil and methanol (dissolved in n-hexane) were pumped into the inner layer of the chromatography column at a specific flow rate. At the same time, constant temperature water was continuously pumped into the outer layer of the chromatography column. The entire reactor continued to react for 3 hours. After the reaction, the immobilized enzyme was washed with 0.5 M NaCl buffer and then dried by vacuum filtration. Finally, the activity of the immobilized enzyme was evaluated and then reintroduced into the continuous flow reactor for a new transesterification reaction.

[0128] (2) In order to determine the optimal reaction conditions of the continuous flow reactor, the enzyme dosage (0.5 g, 1.0 g, 1.5 g, 2.0 g and 2.5 g), reaction duration (1 h, 2 h, 3 h, 4 h and 5 h), reaction temperature (35 °C, 40 °C, 45 °C, 50 °C and 55 °C), oil to methanol molar ratio (1:3, 1:4, 1:5, 1:6 and 1:7) and flow rate (5 mL / h, 10 mL / h, 15 mL / h, 20 mL / h and 25 mL / h) were varied and optimized.

[0129] (3) First, Figure 7 A shows the effect of reaction temperature. The optimal reaction temperature of GS54-LIPR is 50°C, with a yield of 72.2%, while the optimal reaction temperature of GS55-LIPR is 45°C, with a yield of only 31.5%. It is worth noting that GS54-LIPR performed better than GS55-LIPR at all tested temperatures, which may be because the mutant improved the heat resistance of GS54-LIPR. At the same time, as the temperature continued to rise, the stability and durability of the enzyme also decreased, resulting in no increase in FAME production. In terms of reaction time, such as Figure 7 As shown in B, the maximum yield was achieved after 3 h of reaction, which greatly exceeded the yield after 24 h of reaction in the shake flask ( Figure 5 This result shows that the continuous flow reaction method has a higher time efficiency. When the amount of immobilized enzyme was increased from 0.5g to 2g, a significant upward trend in yield was observed ( Figure 7 C). In addition, the oil to methanol molar ratio is a key parameter in the biodiesel synthesis process. The results showed that the highest FAME yield was obtained at a molar ratio of 1:6 ( Figure 7D) Since the contact time between substrate and enzyme is controlled by flow rate, too fast flow rate may lead to incomplete substrate conversion, while too slow flow rate may promote reversible reaction and reduce yield. Therefore, flow rate is a key factor in continuous flow reaction system. Evaluation of FAME yield at different flow rates is as follows: Figure 7 As shown in Figure E, the optimal flow rate is 15 mL / h. Based on the above research, the optimal continuous flow reaction conditions are determined as follows: reaction time 3h, reaction temperature 50°C, immobilized enzyme 2g, oil to methanol molar ratio 1:6, flow rate 15 mL / h. Under the above conditions, the yield of biodiesel produced by GS54-LIPR reached 73.6%, which greatly exceeded the yield of 52.2% in the shake flask, thus confirming the ability of the continuous flow reaction system in enhancing enzyme-mediated reactions.

[0130] The present invention adopts a semi-rational design strategy, combined with disulfide bond and amino acid site mutations, to improve the thermal stability and activity of the lipase LIPR, and then adopts a combinatorial optimization strategy to achieve efficient expression of LIPR in Pichia pastoris, with an enzyme activity of 13620U / mL in a 5L high-density fermenter, which is the highest among the reported acid lipase enzyme activities. In addition, the biodiesel production capacity of LIPR was evaluated, and the biodiesel yield was significantly improved through a continuous flow system. The engineering described in the present invention provides new insights into enzyme optimization and helps to develop reusable bio-enzyme catalysts for the production of biodiesel and other chemicals.

[0131] SEQ ID NO:1

[0132] GAATTCATGTTCAAAAGTGCAGCCGTACGTGCAATAGCTTGCTTTGGGACTAACTGCTA

[0133] GTGTGTTAGCCGCTCCCGTTGAGTTAGGTAGAAGGGATGTCTCACAGGACCTTTTCGA

[0134] TCAGCTAAACTTGTTCGAGCAATATTCCGCAGCAGCATACTGTTCAGCTAATAACGAGG

[0135] CAAGTGCTGGAACTGCCATATCTTGTTCCGCCGGTAACTGTCCCTGGTACAACAAGC

[0136] AGGAGCCACCATTCTTTATTCATTCAACAATATCGGTTTCAGGCGACGTCACCGGATTTC

[0137] TGGCTTTAGACTCCACTAATCAGCTTATTGTACTGTCTTTTAGGGGTTCCGAAACGCTA

[0138] GAGAACTGGATTGCCGACTTAGAAGCCGACCTTGTTGATGCTTCAGCCATTTGCTCTG

[0139] GTTGCGAAGCTCATGATGGTTTTCTGAGTTCATGGAACTCCGTGGCCTCAACTTTAACC

[0140] TCCAAAATTTCTAGTGCAGTGAACGAGCACCCAAGTTATAAGCTAGTATTCACGGGCC

[0141] ACTCACTTGGAGCCGCATTAGCCACACTGGGCGCAGTCAGTTTAAGGGAATCAGGCTA

[0142] CAACATAGACTTATATAATTATGGCTGCCCTAGGGTAGGCAACACCGCACTAGCTGACT

[0143] TTATCACCACGCAGTCCGGCGGCACGAACTACCGTGTTACGCACTCCGATGACCCAGT

[0144] ACCCAAACTTCCTCCAAGAAGTTTTGGTTATTCACAGCCATCTCCTGAGTATTGGATTA

[0145] CTAGTGGCAATAATGTCACGGTCCAACCAAGTGATATAGAGGTTATCGAGGGTGTAGA

[0146] CTCAACCGCAGGAAACGACGGAACACCAGCCGGTTTAGATATAGATGCTCATAGGTGG

[0147] TACTTTGGACCCATTTCCGCTTGCTCC

[0148] SEQ ID NO:2

[0149] EFMFKSAAVRAIAALGLTASVLAAPVELGRRDVSQDLFDQLNLFEQYSAAAYCSANNEAS

[0150] AGTAISCSAGNCPLVQQAGATILYSFNNIGSGDVTGFLALDSTNQLIVLSFRGSETLENWIA

[0151] DLEADLVDASAICSGCEAHDGFLSSWNSVASTLTSKISSAVNEHPSYKLVFTGHSLGAALA

[0152] TLGAVSLRESGYNIDLYNYGCPRVGNTALADFITTQSGGTNYRVTHSDDPVPKLPPRSFGY

[0153] SQPSPEYWITSGNNVTVQPSDIEVIEGVDSTAGNDGTPAGLDIDAHRWYFGPISACS

[0154] SEQ ID NO:3

[0155] MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNG

[0156] LLFINTTIASIAAKEEGVSLEKREAEA

[0157] SEQ ID NO:4

[0158] MAIPRFPSIFIAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTN

[0159] NGLLEEAEAEAEPKFINTTIASIAAKEEGVSLEKREAEAYV

[0160] SEQ ID NO:5

[0161] GAATTCATGTTCAAAAGTGCAGCCGTACGTGCAATAGCTGCTTTGGGACTAACTGCTA

[0162] GTGTGTTAGCCGCTCCCGTTGAGTTAGGTAGAAGGGATGTCTCACAGGACCTTTTCGA

[0163] TCAGCTAAACTTGTTCGAGCAATATTCCGCAGCAGCATACTGTTCAGCTAATAACGAGG

[0164] CAAGTGCTGGAACTGCCATATCTTGTTCCGCCGGTAACTGTCCCCTGGTACAACAAGC

[0165] AGGAGCCACCATTCTTTATTCATTCAACAATATCGGTTCAGGCGACGTCACCGGATTTC

[0166] TGGCTTTAGACTCCACTAATCAGCTTATTGTACTGTCTTTTAGGGGTTCCGAAACGCTA

[0167] GAGAACTGGATTGCCGACTTAGAATGTGACCTTGTTGATGCTTCAGCCATTTGCTCTGG

[0168] TTGCGAAGCTCATGATGGTTTTCTGAGTTCATGGAACTCCGTGGCCTCAACTTTAACCT

[0169] CCAAAATTTCTAGTGCAGTGAACGAGCACCCAAGTTATAAGCTAGTATTCACGGGCCA

[0170] CTCACTTGGAGCCGCATTAGCCACACTGGGCGCAGTCAGTTTAAGGGAATCAGGCTAC

[0171] AACATAGACTTATATAATTATGGCTGCCCTAGGGTAGGCAACACCGCACTAGCTGACTT

[0172] TATCACCACGCAGTCCGGCGGCACGAACTACCGTGTTACGCACTCCGATGACCCAGTA

[0173] CCCAAACTTTGTCCAAGAAGTTTTGGTTATTCACAGCCATCTCCTGAGTATTGGATTAC

[0174] TAGTGGCAATAATGTCACGGTCCAACCAAGTGATATAGAGGTTATCGAGGGTGTAGAC

[0175] TCAACCGCAGGAAACGACGGAACACCAGCCGGTTTAGATATAGATGCTCATAGGTGGT

[0176] ACTTTGGACCCATTTCCGCTTGCTCC

[0177] SEQ ID NO:6

[0178] EFMFKSAAVRAIAALGLTASVLAAPVELGRRDVSQDLFDQLNLFEQYSAAAYCSANNEAS

[0179] AGTAISCSAGNCPLVQQAGATILYSFNNIGSGDVTGFLALDSTNQLIVLSFRGSETLENWIA

[0180] DLECDLVDASAICSGCEAHDGFLSSWNSVASTLTSKISSAVNEHPSYKLVFTGHSLGAALA

[0181] TLGAVSLRESGYNIDLYNYGCPRVGNTALADFITTQSGGTNYRVTHSDDPVPKLCPRSFGY

[0182] SQPSPEYWITSGNNVTVQPSDIEVIEGVDSTAGNDGTPAGLDIDAHRWYFGPISACS

[0183] SEQ ID NO:7

[0184] GAATTCATGTTCAAAAGTGCAGCCGTACGTGCAATAGCTGCTTTGGGACTAACTGCTA

[0185] GTGTGTTAGCCGCTCCCGTTGAGTTAGGTAGAAGGGATGTCTCACAGGACCTTTTCGA

[0186] TCAGCTAAACTTGTTCGAGCAATATTCCGCAGCAGCATACTGTTCAGCTAATAACGAGG

[0187] CAAGTGCTGGAACTGCCATATCTTGTTCCGCCGGTAACTGTCCCCTGGTACAACAAGC

[0188] AGGAGCCACCATTCTTTATTCATTCAACAATATCGGTTCAGGCGACGTCACCGGATTTC

[0189] TGGCTTTAGACTCCACTAATCAGCTTATTGTACTGTCTTTTAGGGGTTCCGAAACGCTA

[0190] GAGAACTGGATTGCCGACTTAGAATGTGACCTTGTTGATGCTTCAGCCATTTGCTCTGG

[0191] TTGCGAAGCTCATGATGGTTTTCTGAGTTCATGGAACTCCGTGGCCTCAACTTTAACCT

[0192] CCAAAATTTCTAGTGCAGTGAACGAGCACCCAAGTTATAAGCTAGTATTCACGGGCCA

[0193] CTCACTTGGAGCCGCATTAGCCACACTGGGCGCAGTCAGTTTAAGGGAATCAGGCTAC

[0194] AACATAGACTTATATAATTATGGCTGCCCTAGGGTAGGCAACACCGCACTAGCTCTTTTT

[0195] ATCACCACGCAGTCCGGCGGCACGAACTACCGTGTTACGCACTCCGATGACCCAGTAC

[0196] CCAAACTTTGTCCAAGAAGTTTTGGTTATTCACAGCCATCTCCTGAGTATTGGATTACT

[0197] AGTGGCAATAATGTCACGGTCCAACCAAGTGATATAGAGGTTATCGAGGGTGTAGACT

[0198] CAACCGCAGGAAACGACGGAACACCAGCCGGTTTAGATATAGATGCTCATAGGTGGTA

[0199] CTTTGGACCCATTTCCGCTTGCTCC

[0200] SEQ ID NO:8

[0201] EFMFKSAAVRAIAALGLTASVLAAPVELGRRDVSQDLFDQLNLFEQYSAAAYCSANNEAS

[0202] AGTAISCSAGNCPLVQQAGATILYSFNNIGSGDVTGFLALDSTNQLIVLSFRGSETLENWIA

[0203] DLECDLVDASAICSGCEAHDGFLSSWNSVASTLTSKISSAVNEHPSYKLVFTGHSLGAALA

[0204] TLGAVSLRESGYNIDLYNYGCPRVGNTALALFITTQSGGTNYRVTHSDDPVPKLCPRSFGY

[0205] SQPSPEYWITSGNNVTVQPSDIEVIEGVDSTAGNDGTPAGLDIDAHRWYFGPISACS

[0206] SEQ ID NO:9

[0207] CAATTCAACTGGAATATTAGAACTGTGGCAAGTATTTTGTCCGCTCTCACACTAGCACA

[0208] AGCAAGTGATCAGGAGGCTATTGCCCCAGAGGACTCTCATGTCGTCAAATTGACTGAA

[0209] GCCACTTTTGAGACTTTCATCACCAGTAATCCTCACGTTTTGGCAGAGTTTTTTGCCCC

[0210] TTGGTGTGGTCACTGTAAGAAGTTGGGCCCTGAACTTGTTTCTGCTGCCGAGATCTTA

[0211] AAGGACAATGAGCAGGTTAAGATTGCTCAAATTGATTGTACGGAGGAGAAGGAATTAT

[0212] GTCAAGGCTACGAAATTAAAGGGTATCCTACTTTGAAGGTGTTCCATGGTGAGGTTGA

[0213] GGTCCCAAGTGACTATCAAGGTCAAAGACAGAGCCAAAGCATTGTCAGCTATATGCTA

[0214] AAGCAGAGTTTACCCCCTGTCAGTGAAATCAATGCAACCAAAGATTTAGACGACACAA

[0215] TCGCCGAGGCAAAAGAGCCCGTGATTGTGCAAGTACTACCGGAAGATGCATCCAACTT

[0216] GGAATCTAACACCACATTTTACGGAGTTGCCGGTACTCTCAGAGAGAAATTCACTTTT

[0217] GTCTCCACTAAGTCTACTGATTATGCCAAAAAATACACTAGCGACTCGACTCCTGCCTA

[0218] TTCGCTTGTCAGACCTGGCGAGGAACCTAGTGTTTACTCTGGTGAGGAGTTAGATGAG

[0219] ACTCATTTGGTGCACTGGATTGATATTGAGTCCAAACCTCTATTTGGAGACATTGACGG

[0220] ATCCACCTTCAAATCATATGCTGAAGCTAACATCCCTTTAGCCTACTATTTCTATGAGAA

[0221] CGAAGAACAACGTGCTGCTGCTGCCGATATTATTAAACCTTTTGCTAAAGAGCAACGT

[0222] GGCAAAATTAACTTTGTTGGCTTAGATGCCGTTAAATTCGGTAAGCATGCCAAGAACTT

[0223] AAACATGGATGAAGAGAAACTCCCTCTATTTGTCATTCATGATTTGGTGAGCAACAAG

[0224] AAGTTTGGAGTTCCTCAAGACCAAGAATTGACGAACAAAGATGTGACCGAGCTGATT

[0225] GAGAATTCATCGCAGGAGAGGCAGAACCAATTGTGAAATCAGAGCCAATTCCAGAA

[0226] ATTCAAGAAGAGAAAGTCTTCAAGCTAGTCGGAAAGGCCCACGATGAAGTTGTCTTC

[0227] GATGAATCTAAAGATGTTCTAGTCAAGTACTACGCCCCTTGGTGTGGTCACTGTAAGAG

[0228] AATGGCTCCTGCTTATGAGGAATTGGCTACTCTTTACGCCAATGATGAGGATGCCTCTT

[0229] CAAAGGTTGTGATTGCAAAACTTGATCACACTTTGAACGATGTCGACAACGTTGATAT

[0230] TCAAGGTTATCCTACTTTGATCCTTTATCCAGCTGGTGATAAATCCAATCCTCAACTGTA

[0231] TGATGGATCTCGTGACCTAGAATCATTGGCTGAGTTTGTAAAGGAGAGAGGAACCCAC

[0232] AAAGTGGATGCCCTAGCACTCAGACCAGTCGAGGAAGAAAAGGAAGCTGAAGAAGA

[0233] AGCTGAAAGTGAGGCAGAGCTCACGACGAGCTT

[0234] SEQ ID NO:10

[0235] ATGTGGATTGAAAGAAACCTCATAGCGTCTATATTACTATTTTCAACTTCAGCTTATGCG

[0236] GCATTCAAACCCAGGATAGTTAAAGGAATTTGATGACCTTTTGAATCCAATATACTT

[0237] TAACGATTCATCGACAGTACTAGGTCTAGTAGATCAGACGCTGTTAATTTCCAACGATG

[0238] ATGGAAAATCATGGACTAACTTGCAGGAGGTTATTACACCTGGGGAAATTGATCCGCT

[0239] GACAATTGTAAACATTGAATTCAATCCATCCGCATCTAAGGCTTTTGTATTCACTGCTAG

[0240] TAAGCACTACCTTACTTTAGACAAAGGATCCACCTGGAAAGAATTTCAAATTCCTCTTG

[0241] AAAAATATGGTAACAGAATAGCCTACGACGTTGAGTTTAATTTTGTTAACGAAGAACAT

[0242] GCAATCATAAGAACAAGGTCTTGCAAACGTCGTTTTGATTGTAAGGATGAGTATTTTTA

[0243] TTCGTTAGATGACTTGCAAAGCGTTGACAAGATCACCATTTCTGACGAAATTGTCAATT

[0244] GCCAGTTTTCACAATCTTCCACTAGCTCAGATTCCCGCAAAAACGATGCCATCACTTGC

[0245] GTAACGCGTAAACTGGATTCCAACCGACACTTCTTGGAGTCGAACGTTCTGACAACCT

[0246] TGAACTTTTTCAAGGATGTTACTAGCTTGCCCGCCAGTGATCCATTAACTAAGATGCTT

[0247] ATCAAGGATATACGTGTTGTTCAAAATTACATTGTATTGTTTGTCAGTTCGGATAGATAC

[0248] AACAAATATTCACCCACTCTTCTTTTCATTTCCAAAGATGGAAATACGTTTAAGGAAGC

[0249] CAGTTTACCAGATTCTGAAGGTACATCACCGTCGGTGCACTTTTTGAAAAGTCCTAATC

[0250] CCAATTTGATAAGAGCAATTCGGCTAGGGAAAAAGAACTCACTAGATGGTGGTGGCTT

[0251] TTATTCAGAAGTTCTACAATCTGACTCTACAGGGTTACACTTTCACGTTCTTCTGGACC

[0252] ACTTAGAAGCAAATTTGCTTTCGTACTATCAAATAGAGAACTTAGCGAACCTTGAAGG

[0253] AATCTGGATTGCCAACCAAATCGACACTTCCAGCAAGTTTGGCTCAAAATCCGTTATA

[0254] ACATTTGATGCAGGTTTAACGTGGTCTCCTGTGACAGTAGATGAAGACGAAGATAAAA

[0255] GTTTGCACATCATTGCGTTTGCTGGTGAAAATAGCCTTTATGAGTCCAAGTTTCCGGTT

[0256] TCGACTCCAGGAATTGCCTTGAGGATAGGGCTTATTGGCGATAGTAGTGATGCACTTGA

[0257] TATTGGCAGCTATAGGACATTTTTAACCAGAGATGCAGGGCTAACATGGTCTCAAGTTT

[0258] TTGATAATGTCTCTGTTTGCGGCTTTGGAAACTATGGAAACATCATATTATGCTGTTCGT

[0259] ATGATCCACTACTTCGATCTGAGCCTTTGAAATTTCGTTATTCTTTGGATCAAGGTCTTA

[0260] ACTGGGAAAGTATTGATTTAGGCTTCAACGGAGTCGCTGTTGGCGTTTTGAACAATATA

[0261] GACAATAGCAGTCCTCAATTCCTTGTGATGACGATTGCCACGGATGGTAAGTCTTCAA

[0262] AGGCTCAGCATTTCTTGTATTCAGTTGATTTTTCTGATGCGTATGAGAAAAAATATGTG

[0263] ATGTTACAAAAGACGAATTATTTGAAGAATGGACGGGAAGAATAGATCCGGTGACGAA

[0264] GCTGCCTATTTGTGTTAACGGTCACAAGGAAAAATTCAGAAGACGGAAGGCTGACGC

[0265] TGAATGCTTCTCTGGTGAACTTTTTCAAGACCTAACTCCAATTGAAGAGCCATGTGATT

[0266] GTGATCCGGATATTGATTACGAATGTTCGCTTGGATTTGAGTTCGATGCAGAGTCTAAC

[0267] CGATGTGAGCCAAATTGTCAATCCTGTCCAGTCACTATTGTGTTGGGAAAAACTTAA

[0268] AGAGAAAAGTGAAAGTAGATAGAAAGTCGAAAGTTGCAGGCACAAAATGTAAAAAG

[0269] GATGTCAAACTTAAGGATAATTCTTTCACTTTAGACTGTTCCAAAACATCTGAACCAGA

[0270] TCTCAGCGAGCAAAGAATTGTTAGTACCACCATAAGCTTTGAAGGTCTCCAGTACAAT

[0271] ACATTTATTGAAACAGGGGACCAACACAACCCTTCTTGACGAAACAGTCATTTTTAAG

[0272] AACATCACTACGAACTGTGTACGTGTCTCATAACGGGGGAACAACTTTTGATAGAGTT

[0273] AGTATCGAAGATGATGTGTCATTTATTGACATCTATACAAACCATTACTTTCCAGATAAT

[0274] GTTTATTTGATCACTGATACAGATGAGCTGTACGTTTCGGATAATAGAGCTATCTCTTTC

[0275] CAGAAAGTTGACATGCCTTCAAGAGCTGGTTTGGAGCTTGGAGTTCGAGCTCTAACCT

[0276] TTCATAAGAGTGACCCTAACAAGTTTATTTGGTTCGGTGAGAAAGATTGTAACTCTATT

[0277] TTTGACAGAAGTTGTCAAACACAAGCTTATATTACGGAAGACAACGGCTTATCTTTCA

[0278] AGCCTCTTTTGGAAAATGTTAGATCATGTTACTTTGTTGGAACAACTTTTGATTCCAAG

[0279] CTGTATGATTTTGACCCGAACTTAATCTTTTGCGAGCAGAGAGTTCCAAATCAACGTTT

[0280] CTTGAAACTTGTAGCCAGTAAGGACTATTTCTATGATGACAAAGAAGAGCTGTATCCTA

[0281] AGATTATTGGAATTGCTACTACCATGAGCTTTGTTATCGTAGCGACTATCAACGAAGAC

[0282] AATAGATCATTGAAGGCGTTTATAACCGCGGATGGGTCTACTTTTGCGGAGCAATTGTT

[0283] TCCTGCAGATCTGGATTTTGGAAGAGAAGTAGCGTACACAGTTATTGACAATTGGGAA

[0284] TCAAAAACACCCAATTTCTTTTTCCATTTGACAACTTCTGAAGATAAAGATTTGGAATT

[0285] TGGAGCTTTACTGAAATCAAACTACAATGGAACAACCTATACGCTTGCTGCCAACAAT

[0286] GTCAATAGAAACGATAGAGGTTACGTTGACTATGAAATCGTTCTAAACTTAAACGGCAT

[0287] TGCTCTCATCAATACAGTTATTAACTCGAAGGAACTTGAATCCGAGCAGTCCCTTGAAA

[0288] CTGCTAAAAAACTGAAAACTCAAATAACGTACAACGACGGGTCTGAATGGGTGTATCT

[0289] GAAACCGCCAACCATTGATTCAGAAAAGAACAAGTTTTCGTGCGTCAAAGATAAGTT

[0290] GAGCTTGGAAAAATGCTCATTGAACCTCAAGGGTGCCACTGATCGGCCAGACAGCAG

[0291] AGACTCCATTTCTTCTGGTTCTGCTGTTGGTCTACTTTTTGGAGTAGGTAACGTTGGGG

[0292] AATACCTGAACCAAGATTCATCAGGTCTAGCATTGTATTTTTCGAAGGATGCGGGCATC

[0293] TCTTGGAAGGAGATTGCCAAAGGAGATTATATGTGGGAATTTGGAGATCAAGGAACAA

[0294] TCCTCGTAATTGTTGAGTTCAAGAAGAAGGTTGACACTTTGAAATACTCATTGGATGA

[0295] AGGAGAAACGTGGTTCGACTACAAGTTTGCAAATGAAAAAACATATGTTTTGGACCT

[0296] GCAACTGTGCCTTCAGATACTTCACGGAAGTTCATCATCCTCGCCAACAGAGGCGAGG

[0297] AGGGAGATCATGAAACTGTTGTTCACACAATAGACTTCAGTAAGGTTCACCAGCGTCA

[0298] ATGTTTATTGAATTTACAAGATAGTAACGCTGGTGATGATTTCGAATATTGGAGTCCGAA

[0299] GAACCCAAGCGCTGTTGACGGGTGTATGCTAGGGCATGAAGAGTCTTACCTAAAAAG

[0300] GATTGCATCCCACTCGGATTGTTTTATTGGGAACGCACCCCTATCAGAGAAATACAAAG

[0301] TGATTAAGAACTGCGCTTGCCACAAGGAGAGATTACGAATGTGATTACAATTTTGCTCTT

[0302] GCCAATGATGGAACTTGTAAATTGGTGGAAGGAGAGTCTCCTTTGGATTACTCTGAAG

[0303] TTTGTAGAAGGGATCCAACTTCCATTGAATATTTTTTGCCTACTGGGGTACAGAAAGGTG

[0304] GGATTGAGTACTTGTGAAGGCGGACTAGAACTGGATAATTGGAATCCCGTTCCATGTC

[0305] CAGGAAAAACCAGAGAATTCAATAGAAAATACGGCACCGGCGCCACCGATACAAGA

[0306] TTGTGGTCATAGTAGCAGTGCCTTTATTGGTTCTCTTGAGCGCCACTTGGTTCCTATATG

[0307] AGAAAGGAATAAAAAGGAATGGAGGTTTTGCCAGATTTGGAGTTATTCGATTAGGCGA

[0308] AGATGACGACGATGACTTGCAAATGATTGAGGAGAATAATACTGACAAAGTAGTCAAT

[0309] GTTGTAGTGAAAGGCCTCATTCATGCATTCAGAGCAGTTTTTGTGAGCTATTTATTTTTC

[0310] CGCAAACGTGCGGCCAAGATGTTTGGTGGATCGTCCTTTTCACACAGACACATATTGC

[0311] CTCAAGATGAGGATGCTCAAGCCTTTTTAGCCAGCGACTTGGAGTCAGAGAGTGGAG

[0312] AGCTTTTCCGATATGCAAGCGACGATGACGATGCCCGAGAGATTGACAGCGTGATCGA

[0313] GGGAGGAATTGATGTCGAAGACGACGACGAGGAGAATATCAATTTTGATTCCCGGTAGSEQ ID NO:11

[0314]

[0315] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An acid lipase mutant with improved thermal stability and activity, characterized in that: The acid lipase mutant is a substitution selected from any one of the following (i) to (vi) in any one of the following mutant amino acid sequences (a1) to (a4): (a1) A mutant in which both Ala at position 51 and Ser at position 110 of the amino acid sequence of lipase LIPR as shown in SEQ ID NO. 2 are mutated to Cys, and a disulfide bond is formed between the two Cys; (a2) a mutant in which the Ala at position 126 and the Phe at position 238 of the amino acid sequence of the lipase LIPR as shown in SEQ ID NO. 2 are both mutated to Cys, and a disulfide bond is formed between the two Cys; (a3) a mutant in which the Ala at position 131 and the Ala at position 140 of the amino acid sequence of the lipase LIPR as shown in SEQ ID NO. 2 are both mutated to Cys, and a disulfide bond is formed between the two Cys; (a4) a mutant in which both Ser at position 255 and Val at position 261 of the amino acid sequence of the lipase LIPR as shown in SEQ ID NO. 2 are mutated to Cys, and a disulfide bond is formed between the two Cys; (i) mutating the Glu at position 58 of the amino acid sequence of the mutant to Gly; (ii) mutating Glu at position 139 of the amino acid sequence of the mutant to Leu; (iii) mutating Asn at position 196 of the amino acid sequence of the mutant to Val; (iv) mutating Asp at position 198 of the amino acid sequence of the mutant to Leu; (v) mutating Asp at position 214 of the amino acid sequence of the mutant to Leu; (vi) Glu at position 250 of the amino acid sequence of the mutant is mutated to Leu.

2. The acid lipase mutant according to claim 1, characterized in that: The acid lipase mutant is: A mutant in which the Ala at position 126 and the Phe at position 238 of the amino acid sequence of the lipase LIPR as shown in SEQ ID NO.2 are both mutated to Cys, and a disulfide bond is formed between the two Cys; at the same time, The Asp at position 214 of the amino acid sequence of the mutant was mutated to Leu.

3. The acid lipase mutant according to claim 2, characterized in that: The nucleotide sequence of the acid lipase mutant is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.

8.

4. A nucleic acid molecule encoding the acid lipase mutant according to any one of claims 1 to 3.

5. A biological material containing the nucleic acid molecule according to claim 4, characterized in that: The biomaterial comprises at least one of (b1) to (b11): (b1) an expression cassette containing the nucleic acid molecule; (b2) a recombinant vector containing the nucleic acid molecule; (b3) a recombinant vector containing the expression cassette described in (b1); (b4) a recombinant cell containing the nucleic acid molecule; (b5) a recombinant cell containing the expression cassette described in (b1); (b6) a recombinant cell containing the recombinant vector described in (b2); (b7) a recombinant cell containing the recombinant vector described in (b3); (b8) a recombinant microorganism containing the nucleic acid molecule; (b9) a recombinant microorganism containing the expression cassette described in (b1); (b10) a recombinant microorganism containing the recombinant vector described in (b2); (b11) A recombinant microorganism containing the recombinant vector described in (b3).

6. The biomaterial according to claim 5, characterized in that: The recombinant microorganism is Pichia pastoris, and the Pichia pastoris comprises at least one of (c1) to (c4): (c1) replacing the α-factor signal peptide in the recombinant vector described in (b2) with the Mss signal peptide; the amino acid sequence of the Mss signal peptide is shown in SEQ ID NO.4; (c2) the recombinant microorganism co-expresses a molecular chaperone PDI; the gene sequence of the molecular chaperone PDI is shown in SEQ ID NO.9; (c3) the vacuolar sorting receptor Vps10-01 is knocked out in the recombinant microorganism; the gene sequence of the vacuolar sorting receptor Vps10-01 is shown in SEQ ID NO.10; (c4) The dihydroxyacetone synthase gene DAS2 is overexpressed in the recombinant microorganism; the gene sequence of the dihydroxyacetone synthase gene DAS2 is shown in SEQ ID NO.

11.

7. Use of the acid lipase mutant according to claim 1, the nucleic acid molecule according to claim 2 or the biomaterial according to claim 3 in the preparation of biodiesel.

8. A method for preparing biodiesel, characterized in that: Biodiesel is prepared by biocatalysis using olive oil and methanol as substrates through the acid lipase mutant according to claim 1, the nucleic acid molecule according to claim 2 or the biological material according to claim 3.

9. The preparation method according to claim 8, characterized in that: The crude enzyme of the acid lipase mutant is obtained by using the acid lipase mutant according to claim 1, the nucleic acid molecule according to claim 2 or the biological material according to claim 3, and the enzyme is immobilized by using a resin.

10. The preparation method according to claim 9, characterized in that: A continuous flow reactor is used, and the solvent for catalytic preparation of biodiesel is n-hexane.

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