Degradation-resistant lysophospholipase mutants

Abstract

The invention belongs to the field of biological medicines, and relates to a degradation-resistant lysophospholipase mutant. The invention provides a lysophospholipase mutant. A mature polypeptide of the mutant is obtained by mutating 94-site aspartic acid into asparagine on the basis of a mature polypeptide of wild type lysophospholipase. The hemolytic esterase mutant obtained by a method for site-specific mutagenesis of 94-site aspartic acid has obviously improved storage stability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a lysophospholipase mutant resistant to degradation. Background Art

[0002] Two lipases, called lipase A and lipase B, or lipase 1 and lipase 2, have been reported in Aspergillus niger. Among them, the properties of lipase B are relatively special. Zhu Shu-sen cloned and expressed lipase B from Aspergillus niger A733 and found that the optimal temperature of lipase B is 15°C, the optimal pH is between 3.5 and 4.0, it cannot tolerate temperatures above 40°C, and it can hydrolyze substrates with pNPC4 - pNPC18 chain lengths, among which pNPC12 is the optimal substrate. However, the specific enzyme activity of lipB is extremely low, and the specific enzyme activity of purified lipB is only 6.8 U / mg.

[0003] Jiangke Yang et al. cloned and expressed lipas2 from Aspergillus niger CICC 4009. Although this lipase2 is highly homologous to lipase B cloned by Zhu Shu-sen, with only two amino acid differences, there are certain differences in properties. The optimal substrates are pNPC8 (p-nitrophenyl octanoate) and pNPC10 (p-nitrophenyl decanoate), the optimal pH is less than 6.5, the optimal temperature is 50°C, and it cannot tolerate temperatures above 40°C.

[0004] As described above, the practicality of lipase2 or lipase B derived from Aspergillus niger is not strong. Firstly, the specific enzyme activity is extremely low, the temperature tolerance is not high, and the applicable optimal substrates are triglycerides of short-chain fatty acids. It is not as good as the more widely used lipases TL and RML, whose specific enzyme activities can reach 12000 or 8000 U / mg. TL can tolerate a temperature of 60°C for 20 hours without inactivation, and RML can hydrolyze triglycerides of various long-chain fatty acids.

[0005] In Chinese Patent Application 202110707919.4, the inventors designed primers using the gene sequence of lipase B of CBS513.88 to clone the LPL gene AN02-LPL of the above-mentioned Aspergillus niger strain from Aspergillus niger GIM 3.24 (AN02). The Aspergillus niger strain AN02-LPL obtained by transforming the gene AN02-LPL has an enzyme with very high phospholipase A1 activity and lysophospholipase activity, which can be used for oil degumming. Without adding alkali, the phosphorus content of crude oil can be reduced to 5 ppm, and it can be used for enzymatic degumming. Moreover, since no alkali needs to be added, the formation of soap during degumming can be reduced. In addition, due to the extremely high lysophospholipase activity of AN02-LPL, it can be used in combination with phospholipase A2 to prepare glycerophosphocholine (GPC) using soybean phospholipid as a raw material, which has the effect of strengthening the brain and preventing aging and is used in medicine and health products. Summary of the Invention

[0006] In some embodiments, the present invention provides a lysophospholipase mutant, wherein the mature polypeptide of the mutant is an aspartic acid at position 94 of the mature polypeptide of the wild-type lysophospholipase mutated to asparagine. The mutation site can be named "D94N".

[0007] In some embodiments, the present invention obtained a mutant D94N of AN02LPL by site-directed mutagenesis of the aspartic acid at position 94 of the wild-type lysophospholipase, and its enzyme activity loss after storage at 4°C for 2 weeks was reduced by 77% compared to AN02LPL.

[0008] In some embodiments, compared with the wild-type lysophospholipase, the lysophospholipase mutant has improved anti-degradation ability.

[0009] In some embodiments, the mature polypeptide of the mutant is an aspartic acid at position 94 corresponding to the mature polypeptide of SEQ ID NO: 1 mutated to asparagine.

[0010] In some embodiments, the mature polypeptide of the mutant is based on SEQ ID NO: 1, with the aspartic acid at position 94 mutated to asparagine, and other site mutations that do not change the polypeptide properties are also covered within the scope of the present invention.

[0011] In some embodiments, the amino acid sequence of the mature polypeptide of the mutant is selected from SEQ ID NO: 2.

[0012] In some embodiments, the present invention provides a polypeptide comprising the sequence shown in SEQ ID NO: 2.

[0013] In some embodiments, the present invention provides a nucleic acid encoding the lysophospholipase mutant or the polypeptide.

[0014] In some embodiments, the present invention provides a vector, which comprises the nucleic acid described above or a nucleic acid complementary to its sequence.

[0015] In some embodiments, the vector is selected from plasmids, poxviruses, retroviruses, adenoviruses, herpesviruses, polioviruses, alphaviruses, baculoviruses, Sindbis viruses or bacterial vectors.

[0016] In some embodiments, the present invention provides a recombinant bacterium or host cell containing the vector described above.

[0017] In some embodiments, the cell is selected from bacterial cells, fungal cells, mammalian cells, insect cells or plant cells, but is not limited thereto.

[0018] In some embodiments, the fungal cells include yeast cells, mold cells, but are not limited thereto.

[0019] In some embodiments, the mold includes Aspergillus niger.

[0020] In some embodiments, the present invention provides a microorganism, which comprises any of the lysophospholipase mutants described above, or the polypeptide, or the vector.

[0021] In some embodiments, the microorganism is selected from Aspergillus niger.

[0022] In some embodiments, the present invention provides a kit comprising any of the lysophospholipase mutants described above or the polypeptide.

[0023] In some embodiments, the present invention provides a pharmaceutical composition comprising any of the lysophospholipase mutants described above or the polypeptide, and a pharmaceutically acceptable excipient.

[0024] In some embodiments, the present invention provides an expression system, which expresses the lysophospholipase mutant described above or the polypeptide, or the exogenous sequence integrated in its genome expresses the lysophospholipase mutant described above or the polypeptide, or the expression system contains the nucleic acid described above or the exogenous nucleic acid described above is integrated in its genome.

[0025] In some embodiments, the present invention provides the use of the lysophospholipase mutant described above, or the polypeptide, or the nucleic acid, or the vector, or the recombinant bacterium or host cell, or the microorganism, or the kit, or the pharmaceutical composition, or the expression system in the preparation of lysophospholipase, glycerophosphocholine, bread dough modification, starch hydrolysate treatment, or the preparation of drugs for anti-aging or treating brain diseases.

[0026] In some embodiments, the present invention provides a method for preparing glycerophosphocholine, comprising incubating the lysophospholipase mutant with a substrate for preparing glycerophosphocholine.

[0027] In some embodiments, the present invention provides a method for recombinantly producing the lysophospholipase mutant or the polypeptide, comprising the steps of: introducing the vector into a host cell to produce a transfected or infected host cell, culturing the transfected or infected host cell in vitro, recovering the cell culture and optionally purifying the produced mutant or polypeptide. Detailed implementation manners

[0028] The technical solutions of the present invention are further illustrated by the following specific examples. The specific examples do not represent a limitation to the protection scope of the present invention. Some non-essential modifications and adjustments made by others according to the concept of the present invention still fall within the protection scope of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0030] It should be understood that the present disclosure is not limited to the specific methods, protocols, reagents, etc. described herein, and can vary by itself. The terms used herein are for the purpose of describing specific embodiments or aspects only and are not intended to limit the scope of the present disclosure.

[0031] Unless otherwise specified, the mutations of the present invention include: natural mutations, forced mutations, or selective mutations, including but not limited to gene modifications, sequence insertions or deletions, or partial substitutions.

[0032] The "mutation" includes but is not limited to artificially mutating (mutagenesis or genetic engineering) a wild-type strain at a specific site. As is well known in the art, wild-type and mutant are also relative, and there are differences in nucleic acid or amino acid residue sequences in the wild-type strain itself.

[0033] As used herein, the term "and / or" means and encompasses any and all possible combinations of one or more of the associated listed items. When used in a list of two or more items, the term "and / or" means that any one of the listed items can be used alone or any combination of two or more of the listed items can be used. For example, if a composition, combination, construct, etc. is described as including (or comprising) components A, B, C, and / or D, the composition can include A alone; B alone; C alone; D alone; a combination of A and B; a combination of A and C; a combination of A and D; a combination of B and C; a combination of B and D; a combination of C and D; a combination of A, B, and C; a combination of A, B, and D; a combination of A, C, and D; a combination of B, C, and D; or A, B, C, and D used in combination.

[0034] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, and reference to "the fragment" includes reference to one or more fragments and equivalents thereof known to those of ordinary skill in the art, and so forth.

[0035] In addition, unless otherwise stated, the use of "or" means "and / or". Similarly, "comprising" and "including" are interchangeable and are not intended to be limiting.

[0036] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules with one or more free ends, nucleic acid molecules without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and various other polynucleotides known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the host cell after being introduced into the host cell and are thereby replicated along with the host genome. In addition, certain vectors are capable of directing the transcription or expression of the coding nucleotide sequences to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0037] Some vectors, which the present invention refers to as "recombinant expression vectors" (or simply "expression vectors"), are vectors, plasmids or vehicles designed such that the inserted nucleic acid sequence can be expressed after being transformed into a host. Generally speaking, the expression vectors used in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vectors. However, the present invention is intended to include other forms of such expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which serve the same function.

[0038] The term "plasmid" refers to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell and are often in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genomic integration sequences, phages, or nucleotide sequences from any source, linear, circular, or supercoiled, single-stranded or double-stranded DNA or RNA, and many of these nucleotide sequences have been ligated or recombined into unique structures that can introduce promoter fragments and DNA sequences for a selected gene product, as well as appropriate 3'-untranslated sequences, into cells.

[0039] As used herein, the term "transformation" refers to the process of introducing a gene into a host cell such that the gene can be expressed in the host cell. The transformed gene can include, without limitation, any gene, whether it is inserted into and located within the chromosome of the host cell or outside the chromosome, as long as it can be expressed in the host cell. The polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a polynucleotide construct containing all the necessary elements required for self-expression. The expression cassette can conventionally include a promoter operably linked to the gene, a transcription termination signal, a ribosome binding domain, and a translation termination signal. The expression cassette can be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide can be introduced into the host cell as it is or in the form of a polynucleotide construct and operably linked to the sequences necessary for its expression in the host cell, but is not limited thereto.

[0040] The microorganism can be any prokaryotic or eukaryotic microorganism as long as the microorganism includes the activity of the corresponding lysophospholipase variant or the microorganism is transformed to express the corresponding protein.

[0041] As used herein, the term "nucleic acid" or "polynucleotide" refers to a polymer of nucleotides that extends longitudinally by covalent bonds of nucleotide units, generally referring to a DNA or RNA strand of a certain length, and in the present disclosure, it refers to a nucleic acid encoding a lysophospholipase mutant. Due to codon degeneracy, the nucleic acid can have different nucleotide sequences encoding the same amino acid. Additionally, for optimized expression according to the host cell type, the nucleic acid can have a codon-optimized sequence.

[0042] In some embodiments herein, the host cell can be a unicellular microorganism or a non-unicellular microorganism. Unicellular microorganisms such as Gram-positive bacteria, including but not limited to Bacillus cells, such as, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus megaterium, Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus stearothermophilus, and Bacillus thuringiensis, etc.; or Streptomyces cells, such as Streptomyces violaceoruber; or Gram-negative bacteria, such as Escherichia coli and Pseudomonas.

[0043] As used herein, the term "transformant" refers to a host organism that has been transformed with heterologous or exogenous DNA. The recombinant bacterium of the present invention expresses an exogenous coding sequence or a gene encoding lysophospholipase. "Transformation" refers to the transfer of a DNA fragment into a host organism. The transferred DNA fragment can be integrated into the chromosome of the host organism or enter the host organism in a non-chromosomal manner (i.e., through a vector).

[0044] As used herein, the term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). As used herein, "LPL" refers to lysophospholipase.

[0045] In some embodiments herein, the host cell can also be a eukaryote, such as a mammalian, insect, plant, yeast, or fungal cell. In a preferred aspect, the host cell is a eukaryotic cell, and as used herein, "eukaryotic" includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, and Oomycota, etc.

[0046] As used herein, "specific activity of the enzyme" is the same as "specific enzyme activity", which refers to the number of enzyme activity units per milligram of protein.

[0047] Chinese Patent Application No. 202110707919.4 and Chinese Patent Application No. 202011434650.9 are incorporated herein by reference in their entirety.

[0048] Experimental materials:

[0049] SphI and HindIII restriction endonucleases were purchased from NEB.

[0050] Lecithin (purity 98%) was purchased from Aladdin.

[0051] The NEFA kit was purchased from Wako Pure Chemical Industries, Ltd.

[0052] The Bradford kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0053] Strain: Aspergillus niger AN19, purchased from China Center of Industrial Culture Collection, No.: Aspergillus niger (Aspergillus niger) CICC2243.

[0054] The fermentation medium formula for Aspergillus niger is: 2% (w / v) glucose, 10% (w / v) maltose, 7% (w / v) sodium citrate, 1.5% (w / v) ammonium sulfate, 4% (w / v) Tryptic soy broth, 0.1% (w / v) sodium dihydrogen phosphate, 0.1% (w / v) magnesium sulfate, 0.07% (w / v) Tween 80, trace elements (KI 0.83 g / L, H3BO3 6.2 g / L, MnSO4·4H2O 22.3 g / L, ZnSO4·7H2O 8.6 g / L, Na2MoO4·2H2O 0.25 g / L, CuSO4·5H2O 0.025 g / L, CoCl2·6H2O 0.025 g / L added at a ratio of 1 / 1000; FeSO4·7H2O 2.78 g / L, Na2·EDTA 3.73 g / L added at a ratio of 1 / 100).

[0055] Aspergillus niger screening plate: 1.34% yeast nitrogen base (YNB) with ammonium sulfate and without amino acids, 2% maltose, 2% lecithin emulsion, 0.1 M citric acid - sodium citrate buffer pH 4.0, 2% agarose, 10 mM CaCl2.

[0056] Example 1 Construction of mutants and determination of enzyme activity

[0057] 1. Construction of plasmids

[0058] 1.1 Construction of plasmid pAOP-Eno

[0059] The pAOP-Eno vector was constructed by the inventor referring to the method in "Molecular Cloning: A Laboratory Manual" (Third Edition, New York, Cold Spring Harbor Laboratory Press, New York: Cold Spring Harbor Laboratory Press, 1989). The specific process is as follows:

[0060] The RML gene (NCBI accession number: A34959) obtained by total gene synthesis from Sangon Biotech (Shanghai) Co., Ltd. was digested with SphI and HindIII restriction sites, and the Aspergillus oryzae α-amylase signal peptide (NCBI sequence number: XM_001821384.2, 1-63bp sequence) was inserted into the expression cassette with the Aspergillus oryzae enolase promoter (NCBI sequence number: D63941.1, 215-734bp; containing 12 copies of the enhancer sequence) and the Aspergillus niger glucoamylase terminator (NCBI sequence number: AF214480.1, the terminator sequence part). The entire expression cassette was inserted into the multiple cloning site of the cloning vector pSP72 with BglII and XhoI. Finally, the PyrG expression gene from Aspergillus oryzae (NCBI sequence number: AB017705.1) was inserted into the vector with the XhoI restriction site, thus constructing the RML gene expression vector pAOP-Eno. 1.2 Construction of pAOP-AN02LPL and pAOP-D94N plasmids

[0061] The mature peptide of AN02-LPL was selected as shown in SEQ ID NO: 1 below:

[0062] AN02-LPL (SEQ ID NO: 1)

[0063] DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDP

[0064] TGFIAVDPTNELIVLSFRGSSDLSNWIADL D FGLTSVSSICDGCEMHKGFYEAWEVIADTITS

[0065] KVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITG

[0066] QNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTAGN

[0067] DGTLLDSTTAHRWYTIYISECS

[0068] The aspartic acid (D) at position 94 of AN02L-P (SEQ ID NO: 1)L was mutated to asparagine (N), and the mutated polypeptide was named D94N.

[0069] Amino acid sequence of the mature peptide of mutant D94N (SEQ ID NO: 2)

[0070] DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDTSSYGDP

[0071] TGFIAVDPTNELIVLSFRGSSDLSNWIADL N FGLTSVSSICDGCEMHKGFYEAWEVIADTITS

[0072] KVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYNFGQPRIGNLALADYITG

[0073] QNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTAGN

[0074] DGTLLDSTTAHRWYTIYISECS

[0075] AN02-LPL and D94N were sent to GenScript Biotech Corporation in Suzhou for gene sequence synthesis and cloned into the pAOP-Eno plasmid to obtain the pAOP-AN02LPL plasmid and the pAOP-D94N plasmid.

[0076] 2. Obtaining of Aspergillus niger mutant strains and determination of enzyme activity

[0077] The pAOP-AN02LPL plasmid and the pAOP-D94N plasmid were respectively transformed into the Aspergillus niger AN19 strain, and the obtained strains were named 19-AN02LPL and 19-D94N respectively. The specific transformation method is as follows:

[0078] (1) The spores of the Aspergillus niger AN19 strain cultured on PDA solid medium (purchased from BD, catalog number BD 213400) were eluted with spore washing solution. After vortexing the eluted spores for 1 min, they were filtered through mircloth to prepare a uniform spore suspension.

[0079] (2) Inoculate 1×10 7A spore suspension was transferred to a fermentation medium (2% glucose, 6% maltose, 7% sodium citrate, 1.5% ammonium sulfate, 4% tryptic soy broth, 0.1% sodium dihydrogen phosphate, 0.1% magnesium sulfate, 0.07% Tween 80, trace elements (KI 0.83 g / L, H3BO3 6.2 g / L, MnSO4·4H2O 22.3 g / L, ZnSO4·7H2O 8.6 g / L, Na2MoO4·2H2O 0.25 g / L, CuSO4·5H2O 0.025 g / L, CoCl2·6H2O 0.025 g / L added at a ratio of 1 / 1000; FeSO4·7H2O 2.78 g / L, Na2·EDTA 3.73 g / L added at a ratio of 1 / 100)) and cultured at 28 °C and 200 rpm for 42 - 48 h; the grown mycelia were collected by filtration with sterilized Mircloth (purchased from Milliproe); the collected mycelia were rinsed three times with a sterilized osmotic stabilizer (0.6 mol / L MgSO4) and pressed dry.

[0080] (3) The mycelia were transferred to a 100 mL Erlenmeyer flask, and every 0.8 g of mycelial weight was resuspended in 10 mL of an enzymatic hydrolysis solution (1% cellulase (purchased from sigma, product number C1184 - 25KU), 1% lysing enzyme (purchased from sigema, product number L1412 - 25G), 0.1% snail enzyme (purchased from Sangon Biotech, product number A600870 - 0005)) and dispersed; at 30 °C and 60 rpm for 60 - 90 min (observed every 10 min after 30 min); the mycelia hydrolyzed by the enzyme were filtered with Mircloth, then rinsed with 0.6 mol / L MgSO4, and the filtrate was collected; centrifuged at 4 °C and 1000 g for 10 min, the supernatant was discarded to obtain a protoplast precipitate; the protoplast precipitate was resuspended in 5 mL of pre - cooled 1.0 mol / L sorbitol solution, centrifuged at 800 g and 4 °C for 10 min, and the supernatant was discarded; the protoplasts were resuspended in 1 mL of pre - cooled 1.0 mol / L sorbitol and kept on ice for use.

[0081] (4) The protoplasts were centrifuged and adjusted to 1×10 7cells / mL to obtain a protoplast suspension; to 200 μL of the protoplast suspension, add 5 μg of DNA (pAOP-AN02LPL plasmid or pAOP-D94N plasmid) and 50 μL of PTC (40% w / v PEG4000, 50 mM CaCl2, 50 mM Tris-HCl, pH = 7.5) solution, gently tap to mix, and incubate on ice for 30 min; add 0.2 mL of PTC solution, mix well, then add 0.8 mL of PTC solution, mix well, and incubate at room temperature for 30 min; add the above mixture to 5 ml of regeneration medium (0.2% (w / v) KH2PO4, 0.1% (w / v) KCl, 0.05% (w / v) MgSO4·7H2O, 0.005% (w / v) FeSO4·7H2O, 1 M sucrose, 10 mM acetamide, 20 mM cesium chloride, 0.6 w / v% agar), mix well; spread on a regeneration plate containing regeneration medium (same components as above except containing 1.5% agar), and culture at 28°C for more than 3 days.

[0082] (5) Pick the clones grown on the regeneration plate in the above step (4) to the screening plate, select 100 transformants for each strain, and pick 10 transformants that form a relatively large white precipitation circle on the screening plate for shake flask fermentation, and compare the enzyme activities. The specific process is as follows:

[0083] (6) Spread the 10 positive clones screened onto a 3% (w / v) PDA solid medium (purchased from BD, product number BD 213400), culture at 28°C for about 3 days until a large number of spores are formed, vortex the eluted spores for 1 min, and then filter through mircloth to prepare a uniform spore suspension; inoculate 1×10 7 spore suspensions into the fermentation medium (2% (w / v) glucose, 10% (w / v) maltose, 7% (w / v) sodium citrate, 1.5% (w / v) ammonium sulfate, 4% Tryptic soybroth, 0.1% (w / v) sodium dihydrogen phosphate, 0.1% (w / v) magnesium sulfate, 0.07% (w / v) Tween80, trace elements), culture at 28°C, 200 rpm for 8 days, perform enzyme activity determination, and select the transformant with the highest enzyme activity for enzyme activity comparison.

[0084] (7) Enzyme activity determination of the fermentation broth of 19-AN02LPL and 19-D94N strains before storage

[0085] Take the fermentation broths of strains 19-AN02LPL and 19-D94N, use soy lecithin as the substrate, and measure the enzyme activity. The method is as follows: Take 10 μl of the above fermentation broth and add it to 90 μl of the substrate solution, react at 50 °C for 10 min, inactivate at 95 °C for 5 min, centrifuge at 7000 rpm for 5 min, take 1 μl of the supernatant and add 80 μl of reagent A in the NEFA kit, react at 37 °C for 10 min, and then add 160 μl of reagent B and react for 10 min. Measure the absorbance at 550 nm, and thus calculate the enzyme activity.

[0086] Among them, the composition of the substrate solution (9 mL) is: composed of 5 mL of 4% w / v soy lecithin, 1 mL of 20% (v / v) Triton X-100, 2.5 mL of 0.1 M citric acid-sodium citrate buffer (pH = 4.3), and 0.5 mL of water.

[0087] The results show that the enzyme activities of the fermentation broths of strains 19-AN02LPL and 19-D94N are 47688 U / ml and 45457 U / ml respectively.

[0088] (8) Determination of the enzyme activity of the fermentation broths of strains 19-AN02LPL and 19-D94N after storage

[0089] Store the fermentation broths of strains 19-AN02LPL and 19-D94N at 4 °C for 2 weeks, and measure the enzyme activity by the same method as in step (7) above.

[0090] The results show that after 2 weeks of storage, the enzyme activity of the fermentation broth of strain 19-AN02LPL decreased from 47688 U / ml to 37261 U / ml; while the enzyme activity of the fermentation broth of strain 19-D94N only decreased from 45457 U / ml to 43442 U / ml. Therefore, compared with AN02LPL, the loss of enzyme activity of D94N decreased by 77%. This is beneficial to the stable storage of the enzyme and extends the usage period of the enzyme.

Claims

1. A lysophospholipase mutant, characterized in that, The mature polypeptide of the mutant has an aspartic acid at position 94 of the mature polypeptide of the wild-type lysophospholipase mutated to asparagine.

2. The lysophospholipase mutant according to claim 1, characterized in that, Compared with the wild-type lysolipase, the lysophospholipase mutant has improved anti-degradation ability; Preferably, the mature polypeptide of the mutant has an aspartic acid at position 94 corresponding to the mature polypeptide of SEQ ID NO: 1 mutated to asparagine; Preferably, the amino acid sequence of the mature polypeptide of the mutant is selected from SEQ ID NO:

2.

3. A polypeptide, characterized in that, It contains the sequence shown in SEQ ID NO:

2.

4. A nucleic acid, characterized in that, It encodes the lysophospholipase mutant according to any one of claims 1-2 or the polypeptide according to claim 3.

5. A carrier, characterized in that, The vector includes the nucleic acid according to claim 4 or a nucleic acid complementary to its sequence; Preferably, the vector is selected from plasmids, poxviruses, retroviruses, adenoviruses, herpesviruses, polioviruses, alphaviruses, baculoviruses, Sindbis viruses or bacterial vectors.

6. A recombinant bacterium or host cell containing the lysophospholipase mutant according to any one of claims 1-2, the polypeptide according to claim 3, the nucleic acid according to claim 4 or the vector according to claim 5; Preferably, the cell is selected from bacterial cells, fungal cells, mammalian cells, insect cells or plant cells; Preferably, the fungal cells include yeast cells or mold cells; Preferably, the mold includes Aspergillus niger.

7. A microorganism, characterized in that, The microorganism includes the lysophospholipase mutant according to any one of claims 1-2, the polypeptide according to claim 3, the nucleic acid according to claim 4 or the vector according to claim 5; Preferably, the microorganism is selected from Aspergillus niger.

8. A kit, characterized in that, It contains the lysophospholipase mutant according to any one of claims 1-2, or the polypeptide according to claim 3, the nucleic acid according to claim 4, the vector according to claim 5 or the recombinant bacterium or host cell according to claim 6.

9. A pharmaceutical composition, characterized in that, It contains the lysophospholipase mutant according to any one of claims 1-2, the polypeptide according to claim 3, the nucleic acid according to claim 4, the vector according to claim 5, the recombinant bacterium or host cell according to claim 6 or the microorganism according to claim 7 and a pharmaceutically acceptable excipient.

10. An expression system, characterized in that, The expression system expresses the lysophospholipase mutant according to any one of claims 1-2 or the polypeptide according to claim 3, or the exogenous sequence integrated in its genome expresses the lysophospholipase mutant according to any one of claims 1-2 or the polypeptide according to claim 3, or the expression system contains the nucleic acid according to claim 4 or the exogenous nucleic acid according to claim 4 integrated in its genome.

11. Use of the lysophospholipase mutant according to any one of claims 1-2, or the polypeptide according to claim 3, or the nucleic acid according to claim 4, or the vector according to claim 5, or the recombinant bacterium, or host cell according to claim 6, or the microorganism according to claim 7, or the kit according to claim 8, or the pharmaceutical composition according to claim 9, or the expression system according to claim 10 in the preparation of lysophospholipase, glycerophosphocholine, bread dough modification, starch hydrolysis product treatment, or preparation of a drug for anti-aging or treating brain diseases.

12. A method for preparing glycerophosphocholine, characterized in that, Including incubating the lysophospholipase mutant according to any one of claims 1-2 with a substrate for the preparation of glycerophosphocholine.

13. A method, characterized in that, For recombinantly producing the lysophospholipase mutant according to any one of claims 1-2 or the polypeptide according to claim 3, which is characterized by including the steps of: introducing the vector according to claim 5 into a host cell to produce a transfected or infected host cell, culturing the transfected or infected host cell in vitro, recovering the cell culture and optionally purifying the produced mutant or polypeptide.