Mutated lysophospholipase

Abstract

The invention relates to a mutated lysophospholipase. The invention also relates to a gene encoding the enzyme, and a vector and a host cell comprising the gene. In addition, the invention also relates to the application of the enzyme.

Description

Technical Field

[0001] The present invention relates to a mutated lysophospholipase. It also relates to a gene encoding the enzyme, a vector containing the gene, and a host cell. In addition, it also relates to the use of the enzyme. 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 optimum temperature of lipase B is 15 °C, the optimum pH is between 3.5 and 4.0, and it cannot tolerate temperatures exceeding 40 °C. This enzyme can hydrolyze substrates with chain lengths of pNPC4 - pNPC18, and pNPC12 is the optimum 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 optimum substrates are pNPC8 and pNPC10, the optimum pH is less than 6.5, the optimum temperature is 50 °C, and it cannot tolerate temperatures exceeding 40 °C.

[0004] In summary, the practicality of liapse2 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 optimum substrates are triglycerides of short-chain fatty acids. This enzyme 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 the previous patent application CN202110707919.4, the inventors designed primers using the gene sequence of lipase B of CBS513.88 to clone the LPL (lysophospholipase) gene AN02-LPL of the above-mentioned Aspergillus niger strain from Aspergillus niger GIM 3.24 (AN02), and found that AN02-LPL has very high phospholipase A1 activity and lysophospholipase activity, can be used for oil degumming. Without adding alkali, the phosphorus content of crude oil can be reduced to 5 ppm, can be used for enzymatic degumming, and 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 phospholipids as raw materials, which has the effect of strengthening the brain and preventing aging and is used in medicine and health products. However, when this gene is applied to pre-degummed soybean oil and rice, the degumming effect cannot meet the requirements.

[0006] There is still a need in the art to obtain lysophospholipases with higher specific enzyme activities. Summary of the Invention

[0007] Based on previous work, the inventors developed new mutants on the basis of the mutant lysophospholipase developed in Invention CN202110707919.4. Specifically, in the present invention, after mutating the 84th and 85th amino acids to arginine and threonine respectively, the specific enzyme activity towards phosphatidic acid was increased, and the application effect in pre-degummed soybean oil degumming was better than that of the wild-type gene AN02-LPL, and the phosphorus content could be reduced to a lower level, meeting the requirements for physical refining.

[0008] After the inventors mutated the 111th lysine to histidine, the 147th tyrosine to leucine, the 84th serine to arginine, and the 85th aspartic acid to threonine on the basis of AN02-LPL, the specific enzyme activity of the new mutant HLRT towards phosphatidic acid increased from 347 U / mg of AN02-LPL to 835 U / mg at pH 4.3; from 863 U / mg of AN02-LPL to 2903 U / mg at pH 4.8; and from 0 U / mg of AN02-LPL to 2527 U / mg at pH 5.5.

[0009] Based on the mutant polypeptide M1 of AN02-LPL (the mutant sites are L86I, G187D, E209K, A254D counted by mature peptide), after mutating serine at position 84 to arginine and aspartic acid at position 85 to threonine, the specific enzyme activity of the new mutant M1RT against phosphatidic acid increased from 347 U / mg of AN02-LPL to 1252 U / mg at pH 4.3; from 863 U / mg of AN02-LPL to 2956 U / mg at pH 4.8; and from 0 U / mg of AN02-LPL to 2972 U / mg at pH 5.5.

[0010] Based on the AN02-LPL gene, after mutating serine at position 84 to arginine and aspartic acid at position 85 to threonine, the specific enzyme activity of the new mutant WTRT against phosphatidic acid increased from 347 U / mg of AN02-LPL to 984 U / mg at pH 4.3; from 863 U / mg of AN02-LPL to 2003 U / mg at pH 4.8; and from 0 U / mg of AN02-LPL to 1342 U / mg at pH 5.5.

[0011] Specifically, the present invention relates to the following aspects:

[0012] On the one hand, the present invention relates to a lysophospholipase, the amino acid sequence of which has mutations at positions 84 and 85 relative to SEQ ID NO:1. In one embodiment, the mutations are serine at position 84 mutated to arginine and aspartic acid at position 85 mutated to threonine.

[0013] Those skilled in the art can understand that the lysophospholipase serving as the base sequence of the mutant lysophospholipase of the present invention is not limited to SEQ ID NO:1, and may also include other lysophospholipases known in the prior art. Specifically, the lysophospholipase of the present invention may have mutations at the amino acid positions corresponding to positions 84 and 85 of SEQ ID NO:1 relative to the lysophospholipases known in the prior art.

[0014] For example, the sequence of a known lysophospholipase (produced by Novozymes) that serves as the base sequence for mutation can be: MHRPLQLWALAALTSLVTAAPAPVLRRDVSSSVLSELDLFAQYSAAAYCSSNIGSPGTKLTCSVGNCPRVEAADTETLIEFNESSSFGDVTGYIAVDRTNSLLVLAFRGSSTVSNWEADLDFPLTDASSLCSGCEIHSGFWAAWQTVQASITSTLESAIASYPGYTLVFTGHSYGAALAAIAATTLRNAGYTIQLYDYGQPRLGNLALAQYITAQTQGANYRVTHTDDIVPKLPPELFGYHHFSPEYWITSGDNVTVTTSDVQVVTGIDSTAGNDGTLLDSTSAHDWYIVYIDGCD (SEQ ID NO:6).

[0015] Since SEQ ID NO:6 has 27 more amino acids at the N-terminus than SEQ ID NO:1, those skilled in the art can understand that the amino acid positions corresponding to positions 84 and 85 of SEQ ID NO:1 refer to positions 111 and 112 of this sequence. Accordingly, the present invention also relates to a lysophospholipase having mutations at positions 111 and 112 of SEQ ID NO:6.

[0016] As another example, the known lysophospholipase of the base sequence for mutation is SEQ ID NO:1 in CN2019800303753.

[0017] In one embodiment, the lysophospholipase further comprises additional mutations at one or more positions selected from 111, 147, 86, 187, 209, and 254. In one embodiment, the additional mutations are mutations at positions 111 and 147. In one embodiment, the additional mutations are mutations at positions 86, 187, 209, and 254. In one embodiment, the additional mutations are the mutation of lysine at position 111 to histidine and the mutation of tyrosine at position 147 to leucine. In one embodiment, the additional mutations are the mutation of leucine at position 86 to isoleucine, the mutation of glycine at position 187 to aspartic acid, the mutation of glutamic acid at position 209 to lysine, and the mutation of alanine at position 254 to aspartic acid.

[0018] In one embodiment, the amino acid sequence of the lysophospholipase is as shown in SEQ ID NO:2, 4, or 5.

[0019] On the other hand, the present invention relates to nucleic acid molecules selected from: (a) nucleotide sequences encoding the above-mentioned lysophospholipase; and (b) nucleotide sequences complementary to the nucleotide sequences described in (a), where the complementarity can be partial or complete.

[0020] Those skilled in the art will appreciate that due to the degeneracy of the genetic code, a variety of different nucleotide sequences can encode the same enzyme. Additionally, it can be recognized that those skilled in the art can perform nucleotide substitutions that do not affect the enzyme activity encoded by the nucleotide sequences of the present invention, which can reflect the codon bias of any particular host organism used to express the enzyme of the present invention.

[0021] The present invention also provides a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the vector.

[0022] "Vector" refers to an extrachromosomal element that usually carries genes not belonging to the central metabolism of the cell and is often in the form of a circular double-stranded DNA molecule. Such elements can be autonomously replicating sequences, genomic integration sequences, phages or nucleotide sequences from any source, linear or circular single-stranded or double-stranded DNA or RNA, many of which have been ligated or recombined into a specific construct that can introduce a promoter fragment and DNA sequence of the selected gene product together with a suitable 3' untranslated sequence into the cell.

[0023] The genes and gene products encoding the lysophospholipase of the present invention can be expressed in heterologous host cells, such as bacterial cells, fungal cells, such as yeast cells, mammalian cells, insect cells, and plant cells. The heterologous host cells for expressing the nucleic acid molecules of the present invention can be microbial hosts that are present in the fungal or bacterial families and grow within a wide range of temperature, pH, and solvent tolerance. For example, any bacteria, yeast, and filamentous fungi are expected to be suitable hosts for expressing the nucleic acid molecules of the present invention.Examples of host strains include, but are not limited to, bacterial, fungal or yeast species such as Pichia, Aspergillus, Trichoderma, Saccharomyces, Phaffia, Kluyveromyces, Yarrowia, Candida, Hansenula, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella and Myxococcus species. In one embodiment, the host cell is a fungal cell. In one embodiment, the host cell is a Pichia or Aspergillus niger cell.

[0024] Vectors that can be used to transform the above host cells are well known in the art. Generally, a vector contains sequences that direct transcription and translation of the relevant gene, optional markers, and sequences that permit autonomous replication or chromosomal integration. Suitable vectors contain a 5' region of the gene containing transcriptional initiation control and a 3' region of the DNA fragment that controls transcriptional termination.

[0025] On the one hand, the present invention also relates to a method for producing lysophospholipase, comprising expressing a nucleic acid molecule encoding the lysophospholipase of the present invention in a host cell and recovering the resulting polypeptide.

[0026] A variety of culture methods can be applied to prepare the enzyme of the present invention. For example, large-scale production of a specific gene product from a recombinant microbial host can be carried out by batch, fed-batch, and continuous culture methods.

[0027] Batch and fed-batch culture methods are commonly used and well known in the art, and examples can be found in the following literature: Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd edition, Sinauer Associates, Inc., Sunderland, MA (1989)), and Deshpande, Mukund V., (Appl. Biochem. Biotechnol., 36:227-234 (1992).

[0028] Commercial production of the enzyme of the present invention can also be carried out by continuous culture. Continuous culture is an open system in which a set medium is continuously added to a bioreactor and an equal amount of conditioned medium is removed for processing at the same time. Continuous culture generally maintains cells at a constant high liquid phase density in which the cells are mainly in the logarithmic growth phase. Alternatively, continuous culture can be carried out with immobilized cells, in which carbon and nutrients are continuously added and valuable products, by-products, or wastes are continuously removed from the cell mass. Cell immobilization can be carried out using a wide range of solid carriers, which are composed of natural materials and / or synthetic materials.

[0029] Recovery of the desired enzyme from batch fermentation, fed-batch fermentation, or continuous culture can be accomplished by any method known to those skilled in the art. For example, when the enzyme is produced intracellularly, the cell slurry is separated from the culture medium by centrifugation or membrane filtration, optionally washed with water or an aqueous buffer of the desired pH, and then the cell slurry in an aqueous buffer of the desired pH is suspended and homogenized to produce a cell extract containing the required enzyme.

[0030] The present invention also relates to a composition comprising the lysophospholipase of the present invention or the fermentation broth, fermentation supernatant and / or fermentation concentrate of the host cell of the present invention. The enzyme composition of the present invention can be in any suitable form for use, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cell debris, a semi-purified or purified enzyme composition, or the host cell as a source of the enzyme. The enzyme composition can be a dry powder or granule, a dust-free granule, a liquid, a stabilized liquid or a stabilized and protected enzyme. The liquid enzyme composition can be stabilized according to established processes, for example, by adding stabilizers such as sugars, sugar alcohols or other polyols, and / or lactic acid or other organic acids.

[0031] The present invention further relates to the fermentation broth, fermentation supernatant or fermentation concentrate of the host cell of the present invention.

[0032] The present invention also relates to the use of the lysophospholipase of the present invention in oil degumming. When the lysophospholipase of the present invention is used for oil degumming, compared with the lysophospholipase of the prior art, the specific enzyme activity is significantly improved, and the production cost is reduced. Detailed implementation mode

[0033] Experimental materials

[0034] 1. Experimental strains and plasmids

[0035] Strains: Pichia pastoris GS115 was purchased from Invitrogen, C18100; Aspergillus niger AN19 was purchased from the China Center for Industrial Culture Collection, No.: Aspergillus niger CICC2243.

[0036] Plasmids: The pAOP-Eno plasmid was constructed by our laboratory, and the details are as follows. pAO815 was purchased from Invitrogen, K1750-01.

[0037] The pAOP-Eno vector was constructed by the inventors 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:

[0038] The RML gene (NCBI accession number: A34959), which was obtained by total gene synthesis from Sangon Biotech (Shanghai) Co., Ltd., was digested with SphI and HindIII restriction sites and inserted into the expression cassette containing the Aspergillus oryzae enolase promoter (NCBI accession number: D63941.1, 215 - 734 bp; containing 12 copies of the enhancer sequence) and the Aspergillus niger glucoamylase terminator (NCBI accession number: AF214480.1, the terminator sequence part), with the signal peptide of Aspergillus oryzae α - amylase (NCBI sequence number: XM_001821384.2, 1 - 63 bp sequence). 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 accession number: AB017705.1) was inserted into the vector with the XhoI restriction site, thus constructing the RML gene expression vector pAOP - Eno.

[0039] 2.. Media and Solutions

[0040] PDA solid medium: PDA solid medium powder, 1.7% agar.

[0041] YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose.

[0042] MGYS solid medium: 1.34% yeast nitrogen base (YNB) with ammonium sulfate and without amino acids, 1% glycerol, 1 M sorbitol, 4×10 - 5% D - biotin, 2% agar.

[0043] BMM - soybean phospholipid screening medium: 1.34% yeast nitrogen base (YNB) with ammonium sulfate and without amino acids, 4×10 - 5% D - biotin, 0.5% methanol (added after sterilization), 2% soybean phospholipid emulsion, 0.1 M citric acid - sodium citrate buffer pH 6.6, 2% agar, adding 10 uM of ZnSO4·7H2O.

[0044] -- 2% soybean phospholipid emulsion: 2 g soybean phospholipid, 100 ml H2O, homogenized with a high - speed homogenizer at 8000 rpm for 1 min.

[0045] BMGY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB) with ammonium sulfate and without amino acids, 1% glycerol, 4×10 - 5% D - biotin, 0.1 M potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer pH 6.0.

[0046] BMMY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB) containing ammonium sulfate but not amino acids, 0.3% ZnSO4·7H2O, 0.5% methanol (added after sterilization), 4×10-5% D-biotin (added after sterilization), 0.1M citric acid-sodium citrate buffer pH 6.6. Aspergillus niger fermentation medium: 2% glucose, 10% 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,

[0047] Improved Bradford protein concentration determination kit (purchased from Shanghai Shenggong Biotechnology Co., Ltd.)

[0048] PCR enzyme: HSDNA Polymerase (purchased from Bao Biotechnology (Dalian) Co., Ltd.)

[0049] NEFA kit (purchased from Wako Pure Chemical Industries, Ltd., Japan)

[0050] Phosphatidic acid was purchased from Aladdin Reagents.

[0051] Example 1: Construction of mutant HLRT and specific enzyme activity against phosphatidic acid

[0052] The mature peptide of AN02-LPL was selected as follows

[0053] AN02-LPL

[0054] DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDT

[0055] SSYGDPTGFIAVDPTNELIVLSFRGSSDLSNWIADLDFGLTVSSICDGCEMHKGFY

[0056] EAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLY

[0057] NFGQPRIGNLALADYITGQNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSGNDVTVTTSDVTEVVGVDSTAGNDGTLLDSTTAHRWYTIYISECS(SEQ ID NO:1)

[0058] The lysine at position 111 was mutated to histidine, the tyrosine at position 147 was mutated to leucine, the serine at position 84 was mutated to arginine, and the aspartic acid at position 85 was mutated to threonine.

[0059] The amino acid sequence of the new mutant HLRT is as follows:

[0060] DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDT

[0061] SSYGDPTGFIAVDPTNELIVLSFRGSRTLSNWIADLDFGLTSVSSICDGCEMHHGFY

[0062] EAWEVIADTITSKVEAAVSSYPDYTLVFTGHSLGAALAAVAATVLRNAGYTLDLY

[0063] NFGQPRIGNLALADYITGQNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSG

[0064] NDVTVTTSDVTEVVGVDSTAGNDGTLLDSTTAHRWYTIYISECS(SEQ ID NO:2)

[0065] The amino acid sequences of AN02-LPL and HLRT were sent to Genewiz Bioengineering Co., Ltd. to synthesize the gene sequences, which were cloned into the pAO815 vector to obtain the plasmids P-AN02-LPL and P-HLRT. After linearization with BglII, the vectors were transformed into the competent cells of the Pichia pastoris GS115 strain by electroporation. The transformants were inoculated on the MGYS solid medium plate and cultured at 30 °C for 3 days to obtain the Pichia pastoris transformants of pic-AN02-LPL and pic-HLRT. Single colonies on the plate were picked and transferred to the BMM-soybean phospholipid screening medium plate, and the colonies with large white precipitation rings were selected and named pic-AN02-LPL and pic-HLRT.

[0066] Take pic-AN02-LPL and pic-HLRT, first activate them in YPD liquid medium, and then inoculate them into BMGY liquid medium and culture them overnight at 30 °C with shaking at 220 rpm. Transfer the culture to BMMY liquid medium with an initial OD600 of 6.

[0067] First, induce with 2% methanol, add 1% methanol at 24 h and 32 h respectively, add 1% methanol at 48 h and 56 h respectively, and take samples at 72 h.

[0068] The obtained fermentation broth was ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cut-off of 10 kDa and concentrated 40-fold. The treated sample was added to a buffer solution (20 mM citric acid-sodium citrate buffer (pH 5.5)). The resulting enzyme solution was designated as AN02-LPL and HLRT.

[0069] Using phosphatidic acid as the substrate, the enzyme activity was measured as follows:

[0070] 9 ml of substrate: 5 ml of 1% phosphatidic acid, 1 ml of 20% Triton X-100, 2.5 ml of 0.1 M citric acid-sodium citrate buffer (pH = 4.3; 4.8; 5.5), 0.5 ml of water.

[0071] 10 μl of the diluted enzyme solution + 90 μl of the substrate were reacted at 50 °C for 10 min, inactivated at 95 °C for 5 min, centrifuged at 7000 rpm for 5 min, 1 μl of the supernatant was taken and added to 80 μl of reagent A in the NEFA kit, reacted at 37 °C for 10 min, and then 160 μl of reagent B was added and reacted for 10 min. The absorbance at 550 nm was measured to calculate the enzyme activity.

[0072] The protein concentration of the fermentation broth was measured using a modified Bradford method protein concentration assay kit, and the specific enzyme activity of AN02-LPL and HLRT against phosphatidic acid under different pH conditions was calculated.

[0073] The specific enzyme activity of the new mutant HLRT against phosphatidic acid increased from 347 U / mg of AN02-LPL to 835 U / mg at pH 4.3; from 863 U / mg of AN02-LPL to 2903 U / mg at pH 4.8; and from 0 U / mg of AN02-LPL to 2527 U / mg at pH 5.5.

[0074] Example 2: Degumming test of HLRT

[0075] Take the predegummed soybean crude oil, shake, stir and heat it until the temperature is stable at 55 °C; add 50% citric acid to a final concentration of 650 ppm. Shear at 20000 rpm for 1.5 min; stir and react at 55 °C for 1 h; 30 ppm of HLRT, 30 ppm of AN02-LPL, and 60 ppm of AN02-LPL were each mixed with 1.8 ml of water and then added together, and sheared at 200000 rpm for 1.5 min; after adding the enzyme-water mixture, the temperature was raised to 55 °C and timing started. After reacting for 4 h, the temperature was raised to 85 °C to inactivate the enzyme, and maintained for at least > 8 min. After centrifugation to obtain the degummed oil, samples were taken for phosphorus content testing. A phosphorus content of less than 10 ppm meets the standard.

[0076]

[0077] Example 3: Construction of Mutant M1RT and Specific Enzyme Activity Against Phosphatidic Acid

[0078] Prepare the mutant polypeptide M1 of AN02-LPL (the mutation sites are L86I, G187D, E209K, A254D based on the mature peptide). The sequence of M1 is as follows:

[0079] DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDT

[0080] SSYGDPTGFIAVDPTNELIVLSFRGSSDISNWIADLDFGLTSVSSICDGCEMHKGFYE

[0081] AWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYN

[0082] Mutate serine at position 84 of M1 to arginine and aspartic acid at position 85 to threonine

[0083] The amino acid sequence of the new mutant M1RT is as follows:

[0084] DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDT

[0085] SSYGDPTGFIAVDPTNELIVLSFRGSRTISNWIADLDFGLTSVSSICDGCEMHKGFYE

[0086] AWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLYN

[0087] FGQPRIGNLALADYITDQNMGSNYRVTHTDDIVPKLPPKLLGYHHFSPEYWITSGN

[0088] DVTVTTSDVTEVVGVDSTDGNDGTLLDSTTAHRWYTIYISECS(SEQ ID NO:4)

[0089] The M1RT amino acid sequence was sent to GenScript Biotech Corporation for gene sequence synthesis and cloned into the pAO815

[0090] vector to obtain the P-M1RT plasmid. After linearization with BglII, the vector was transformed into competent cells of the Pichia pastoris GS115 strain by electroporation. The transformants were inoculated onto a solid MGYS medium plate and cultured at 30 °C for 3 days to obtain Pichia pastoris transformants of pic-M1RT. Single colonies on the plate were picked and transferred to a BMM-soybean phospholipid screening medium plate, and the colonies with large white precipitation rings were selected and named pic-M1RT.

[0091] Take pic-M1RT, first activate it in YPD liquid medium, and then inoculate it into BMGY liquid medium. Incubate it overnight at 30 °C with shaking at 220 rpm. Transfer the culture to BMMY liquid medium with an initial OD600 of 6.

[0092] First, induce with 2% methanol, add 1% methanol at 24 h and 32 h respectively, add 1% methanol at 48 h and 56 h respectively, and sample at 72 h.

[0093] The obtained fermentation broth was ultrafiltered, desalted and concentrated 40-fold using an ultrafiltration tube with a molecular weight cut-off of 10 kDa. The treated sample was added to a buffer (20 mM citric acid-sodium citrate buffer (pH 5.5)). The resulting enzyme solution was called M1RT. Using phosphatidic acid as the substrate, the enzyme activity was measured as follows:

[0094] 9 ml of substrate: 5 ml of 1% phosphatidic acid, 1 ml of 20% TritonX-100, 2.5 ml of 0.1 M citric acid-sodium citrate buffer (pH = 4.3; 4.8; 5.5).

[0095] 10 μl of diluted enzyme solution + 90 μl of substrate were reacted at 50 °C for 10 min, inactivated at 95 °C for 5 min, centrifuged at 7000 rpm for 5 min, 1 μl of the supernatant was taken and added to 80 μl of reagent A in the NEFA kit, reacted at 37 °C for 10 min, and then 160 μl of reagent B was added and reacted for 10 min. The absorbance at 550 nm was measured to calculate the enzyme activity.

[0096] The protein concentration of the fermentation broth was measured using a modified Bradford method protein concentration assay kit, and the specific enzyme activity of M1RT against phosphatidic acid under different pH conditions was calculated.

[0097] The specific enzyme activity of the new mutant M1RT against phosphatidic acid increased from 347 U / mg of AN02-LPL to 1252 U / mg at pH 4.3; from 863 U / mg of AN02-LPL to 2956 U / mg at pH 4.8; and from 0 U / mg of AN02-LPL to 2972 U / mg at pH 5.5.

[0098] Example 4: Construction of Mutant WTRT and Specific Enzyme Activity against Phosphatidic Acid

[0099] The mature peptide of AN02-LPL was selected, as shown in SEQ ID NO:1.

[0100] Serine at position 84 was mutated to arginine, and aspartic acid at position 85 was mutated to threonine, resulting in the following amino acid sequence of the new mutant WTRT:

[0101] DISSTVLDNIDLFAQYSAAAYCSSNIESTGTTLTCDVGNCPLVEAAGATTIDEFDDT

[0102] SSYGDPTGFIAVDPTNELIVLSFRGSRTLSNWIADLDFGLTSVSSICDGCEMHKGFY

[0103] EAWEVIADTITSKVEAAVSSYPDYTLVFTGHSYGAALAAVAATVLRNAGYTLDLY

[0104] NFGQPRIGNLALADYITGQNMGSNYRVTHTDDIVPKLPPELLGYHHFSPEYWITSG

[0105] NDVTVTTSDVTEVVGVDSTAGNDGTLLDSTTAHRWYTIYISECS(SEQ ID NO:5)

[0106] The amino acid sequence of WTRT was sent to Genewiz Bio-Engineering Co., Ltd. to synthesize the gene sequence, which was cloned into the pAOP-Eno vector to obtain the PAN-M1RT plasmid, and then transformed into the Aspergillus niger AN19 strain.

[0107] The transformation method is as follows:

[0108] The spores of the Aspergillus niger strain cultured on the PDA solid medium 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; inoculate 1×10 7A spore suspension was added to the 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), and cultured at 28°C and 200 rpm for 42 - 48 h; the grown mycelia were collected by filtration with sterilized Mircloth; the collected mycelia were rinsed three times with sterilized osmotic stabilizer (0.6 mol / L MgSO4) and pressed dry; the mycelia were transferred to a 100 mL Erlenmeyer flask, and every 0.8 g of mycelial weight was resuspended in 10 mL of enzyme solution and dispersed; at 30°C and 60 rpm for 60 - 90 min (observed every 10 min after 30 min); the protoplast mixture was 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, and the supernatant was discarded; the protoplast pellet 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. Take the protoplasts and centrifuge, and adjust to 1×10 7 cells / mL with pre-cooled STC (1.0 M Sorbitol, 50 mM CaCl2, 50 mM Tris-HCl, pH = 7.5) solution; to 200 μL of the protoplast suspension, add 5 μg of DNA and 50 μL of PTC (40% PEG4000, 50 mM CaCl2, 50 mM Tris-HCl, pH = 7.5) solution, gently tap and mix well, and keep 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% KH2PO4, 0.1% KCl, 0.05% MgSO4·7H2O, 0.005% FeSO4·7H2O, 1 M sucrose, 10 mM acetamide, 20 mM cesium chloride, 0.6% agar), and mix well; spread on the regeneration medium (same composition as above, 1.5% agar), and culture at 28°C for more than 3 days.

[0109] The positive clones grown on the regeneration medium were spread on the PDA solid medium and cultured at 28°C for about 3 days until a large number of spores were formed. After vortexing the eluted spores for 1 min, they were filtered through mircloth to prepare a uniform spore suspension; inoculate 1×10 7The spore suspension was added to the fermentation medium (2% glucose, 10% 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), and cultured at 28 °C and 200 rpm for 8 days, followed by enzyme activity assay.

[0110] Using phosphatidic acid as the substrate, the enzyme activity was measured as follows:

[0111] 9 ml of substrate: 5 ml of 1% phosphatidic acid, 1 ml of 20% Triton X-100, 2.5 ml of 0.1 M citrate-sodium citrate buffer (pH = 4.3; 4.8; 5.5).

[0112] 10 μl of the diluted enzyme solution + 90 μl of the substrate were reacted at 50 °C for 10 min, inactivated at 95 °C for 5 min, centrifuged at 7000 rpm for 5 min, 1 μl of the supernatant was taken and added with 80 μl of reagent A in the NEFA kit, reacted at 37 °C for 10 min, and then added with 160 μl of reagent B and reacted for 10 min. The absorbance at 550 nm was measured to calculate the enzyme activity.

[0113] The protein concentration of the fermentation broth was measured using a modified Bradford method protein concentration assay kit, and the specific enzyme activity of WTRT against phosphatidic acid under different pH conditions was calculated.

[0114] The specific enzyme activity of the new mutant WTRT against phosphatidic acid was increased from 347 U / mg of AN02-LPL to 984 U / mg at pH 4.3; from 863 U / mg of AN02-LPL to 2003 U / mg at pH 4.8; and from 0 U / mg of AN02-LPL to 1342 U / mg at pH 5.5.

Claims

1. A lysophospholipase, the amino acid sequence of which has mutations at positions 84 and 85 relative to SEQ ID NO:

1. Preferably, the mutations are the substitution of serine at position 84 with arginine and the substitution of aspartic acid at position 85 with threonine.

2. The lysophospholipase of claim 1, which further comprises additional mutations at one or more positions selected from positions 111, 147, 86, 187, 209, and 254. Preferably, the additional mutations are the mutations at positions 111 and 147, or the mutations at positions 86, 187, 209, and 254. More preferably, the additional mutations are the substitution of lysine at position 111 with histidine and the substitution of tyrosine at position 147 with leucine; or the substitution of leucine at position 86 with isoleucine, the substitution of glycine at position 187 with aspartic acid, the substitution of glutamic acid at position 209 with lysine, and the substitution of alanine at position 254 with aspartic acid.

3. The lysophospholipase of claim 1, the amino acid sequence of which is as shown in SEQ ID NO: 2, 4, or 5.

4. A nucleic acid molecule, selected from: (a) a nucleotide sequence encoding the lysophospholipase according to any one of claims 1 - 3; and (b) a nucleotide sequence complementary to the nucleotide sequence described in (a).

5. A vector, which comprises the nucleic acid molecule of claim 4.

6. A host cell, which comprises the nucleic acid molecule of claim 4, or the vector of claim 5; preferably, the host cell is selected from bacterial cells, fungal cells, mammalian cells, insect cells, and plant cells. More preferably, the host cell is a fungal cell, and even more preferably, it is a Pichia pastoris cell or an Aspergillus niger cell.

7. A method for producing lysophospholipase, comprising expressing a nucleic acid molecule encoding the lysophospholipase according to any one of claims 1 - 3 in a host cell and recovering the resulting polypeptide.

8. A composition, which comprises the lysophospholipase according to any one of claims 1 - 3 or the fermentation broth, fermentation supernatant, and / or fermentation concentrate of the host cell of claim 6.

9. The fermentation broth, fermentation supernatant, or fermentation concentrate of the host cell of claim 6.

10. Use of the lysophospholipase of claim 1, the composition of claim 8, or the fermentation broth, fermentation supernatant, and / or fermentation concentrate of claim 9 in oil degumming.

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