Mutant lysophospholipase

Abstract

The present invention relates to a mutant lysophospholipase, and further relates to a gene encoding the enzyme, a vector comprising the gene, and a host cell. In addition, the present invention further relates to the use of the enzyme.

Description

Mutant lysophospholipase Technical Field

[0001] The present invention relates to a mutant lysophospholipase, a gene encoding the enzyme, a vector comprising the gene, and a host cell, and uses of the enzyme. Background Art

[0002] Two lipases have been reported in Aspergillus niger, designated lipaseA and lipaseB, or lipase1 and lipase2. LipaseB has unique properties. Zhu Shu-sen cloned and expressed lipaseB from Aspergillus niger A733 and found that its optimal temperature is 15°C and its optimal pH is 3.5-4.0, with no tolerance to temperatures exceeding 40°C. The enzyme is capable of hydrolyzing substrates with a chain length of pNPC4-pNPC18, with pNPC12 being the optimal substrate. However, the specific enzymatic activity of lipB is extremely low, reaching only 6.8 U / mg after purification.

[0003] Jiangke Yang et al. cloned and expressed lipase2 from Aspergillus niger CICC 4009. Although this lipase2 is highly homologous to the lipaseB cloned by Zhu Shu-sen, differing only by two amino acids, its properties differ to a certain extent. Its optimal substrates are pNPC8 and pNPC10, its optimal pH is less than 6.5, and its optimal temperature is 50°C. It cannot tolerate temperatures above 40°C.

[0004] In summary, the practical application of liapse2 or lipaseB derived from Aspergillus niger is limited. First, their specific activity is extremely low, their temperature tolerance is limited, and their optimal substrate is short-chain triglycerides. These enzymes are inferior to the more widely used lipases TL and RML, which can reach specific activities of 12,000 and 8,000 U / mg, respectively. TL can withstand temperatures of 60°C for 20 hours without inactivation, while RML can hydrolyze triglycerides containing a variety of long-chain fatty acids.

[0005] In the previous patent application CN202110707919.4, the inventors designed primers from the gene sequence of lipaseB of CBS513.88 from Aspergillus niger GIM 3.24 (AN02) and cloned the LPL (lysophospholipase) gene AN02-LPL of the above-mentioned Aspergillus niger strain. They found that AN02-LPL has very high phospholipase A1 activity and lysophospholipase activity, which can be used for oil degumming. Without the addition of alkali, the phosphorus content of crude oil can be reduced to 5ppm. It can be used for enzymatic degumming, and since no alkali is required, soap formation during degumming can be reduced. In addition, because AN02-LPL has extremely high lysophospholipase activity, it can be used in combination with phospholipase A2 to prepare glycerophosphatidylcholine (GPC) using soybean lecithin as raw material. It has the effect of strengthening the brain and preventing aging and is used in medicine and health products. However, when this gene was applied to pre-degummed soybean oil and rice, the degumming effect did not meet the requirements.

[0006] The art still needs to obtain lysophospholipases with higher enzyme activity and stability. Summary of the Invention

[0007] Based on previous work, the inventors developed a new mutant based on the mutant lysophospholipase developed in invention CN202110707919.4.

[0008] Specifically, the inventors obtained a mutant D94N of AN02-LPL by site-directed mutation of aspartic acid at position 94 of wild-type lysophospholipase. The enzyme activity loss of D94N after storage at 4°C for 2 weeks was reduced by 77% compared with AN02-LPL.

[0009] In addition, the inventors improved the specific enzyme activity towards phosphatidic acid by mutating the amino acids at positions 84 and 85 to arginine and threonine, respectively. The application effect of the enzyme in pre-degummed soybean oil degumming is better than that of the wild-type gene AN02-LPL, and the phosphorus content can be reduced to a lower level, meeting the requirements for physical refining.

[0010] The inventors also mutated lysine at position 111 to histidine, tyrosine at position 147 to leucine, serine at position 84 to arginine, and aspartic acid at position 85 to threonine based on AN02-LPL. The specific enzyme activity of the new mutant HLRT against phosphatidic acid was 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.

[0011] The inventors also mutated serine at position 84 to arginine and aspartic acid at position 85 to threonine based on the mutant polypeptide M1 of AN02-LPL (the mutation sites in the mature peptide are L86I, G187D, E209K, and A254D). The specific enzyme activity of the new mutant M1RT against phosphatidic acid was 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.

[0012] The inventors also mutated serine at position 84 to arginine and aspartic acid at position 85 to threonine based on the AN02-LPL gene. 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.

[0013] Based on the aforementioned WTRT, the inventors mutated aspartic acid at position 94 to tyrosine to obtain a new mutant, WTRT-Y. This new mutant still maintains high specific enzymatic activity against phosphatidic acid, increasing from 347 U / mg of AN02-LPL to 961 U / mg at pH 4.3; from 863 U / mg of AN02-LPL to 1803 U / mg at pH 4.8; and from 0 U / mg of AN02-LPL to 1212 U / mg at pH 5.5. This activity is essentially the same as that of the mutant WTRT (984 U / mg at pH 4.3, 2003 U / mg at pH 4.8, and 1342 U / mg at pH 5.5).

[0014] The enzyme activity of the mutant WTRT decreased from 29,921 U / ml to 20,076 U / ml after 2 weeks of storage at 4°C, while the protein stability of the mutant WTRT-Y was improved, with its enzyme activity only decreasing from 28,765 U / ml to 25,984 U / ml after 2 weeks of storage at 4°C, a 68% reduction compared to WTRT.

[0015] After the mutant WTRT expression strain was subcultured twice, the enzyme activity was only 37.5% of that of the unsubcultured strain, while the enzyme activity of the mutant WTRT-Y expression strain was consistent with that of the unsubcultured strain after being subcultured twice, with no instability.

[0016] Specifically, the present invention relates to the following aspects:

[0017] In one aspect, the present invention relates to a mutant lysophospholipase having one or more mutations in its amino acid sequence relative to a wild-type lysophospholipase and having increased specific activity or increased stability.

[0018] Those skilled in the art will appreciate that the wild-type 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 amino acid positions corresponding to positions 84 and 85 of SEQ ID NO: 1 relative to lysophospholipases known in the prior art.

[0019] For example, the sequence of the known lysophospholipase (manufactured by Novozymes) as the base sequence for the mutation may be:

[0020] Since SEQ ID NO: 8 has 27 more amino acids at the N-terminus than SEQ ID NO: 1, those skilled in the art will understand that the amino acid positions corresponding to positions 84 and 85 of SEQ ID NO: 1 refer to positions 111 and 112 of the sequence. Therefore, the present invention also relates to lysophospholipases having mutations at positions 111 and 112 of SEQ ID NO: 8.

[0021] As another example, the known lysophospholipase of the mutated base sequence is SEQ ID NO: 1 in CN2019800303753.

[0022] In one aspect, the present invention relates to a lysophospholipase having an amino acid sequence having a mutation at one or more positions selected from positions 84, 85 and 94 corresponding to SEQ ID NO: 1.

[0023] In one embodiment, the amino acid sequence has a mutation at position 94 relative to SEQ ID NO: 1. In one embodiment, the mutation is from aspartic acid to asparagine.

[0024] In one aspect, the present invention relates to a lysophospholipase having an amino acid sequence with mutations at positions 84 and 85 relative to SEQ ID NO: 1. In one embodiment, the mutations are a mutation of serine at position 84 to arginine and a mutation of aspartic acid at position 85 to threonine. In one embodiment, the lysophospholipase further comprises an additional mutation at one or more positions selected from the group consisting of 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 a mutation of lysine at position 111 to histidine and a mutation of tyrosine at position 147 to leucine. In one embodiment, the additional mutations are leucine to isoleucine at position 86, glycine to aspartic acid at position 187, glutamate to lysine at position 209, and alanine to aspartic acid at position 254.

[0025] In one embodiment, the amino acid sequence of the lysophospholipase of the present invention has mutations at positions corresponding to positions 84, 85, and 94 of SEQ ID NO: 1. In one embodiment, the mutations are serine at position 84 to arginine, aspartic acid at position 85 to threonine, and aspartic acid at position 94 to tyrosine.

[0026] In one embodiment, the amino acid sequence of the lysophospholipase is shown in SEQ ID NO: 2, 3, 5, 6 or 7.

[0027] On the other hand, the present invention relates to a nucleic acid molecule selected from: (a) a nucleotide sequence encoding the above-mentioned lysophospholipase; and (b) a nucleotide sequence complementary to the nucleotide sequence described in (a), and the complementarity can be partial or complete.

[0028] The skilled person will recognize that, due to the degeneracy of the genetic code, a variety of different nucleotide sequences may encode the same enzyme. Additionally, it will be appreciated that the skilled person is able to use conventional techniques to make nucleotide substitutions that do not affect the enzymatic activity encoded by the nucleotide sequence of the present invention, thereby reflecting the codon bias of any particular host organism used to express the enzyme of the present invention.

[0029] The present invention also provides a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the vector.

[0030] "Vector" refers to an extrachromosomal element that usually carries a gene that is not part of 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, genome-integrating sequences, phage or nucleotide sequences, linear or circular single-stranded or double-stranded DNA or RNA from any source, in which a number of nucleotide sequences have been joined or recombined into a specific construct that is capable of introducing a promoter fragment and DNA sequence for a selected gene product, along with appropriate 3' non-translated sequences, into a cell.

[0031] The gene and gene product of coding lysophospholipid hydrolase of the present invention can be expressed in heterologous host cells, for example bacterial cells, fungal cells, for example yeast cells, mammalian cells, insect cells and plant cells.The heterologous host cell for expressing nucleic acid molecules of the present invention can be a microbial host that is present in fungi or bacteria families and grows in wide temperature, pH value and solvent tolerance ranges.For example, it is expected that any bacterium, yeast and filamentous fungi can be the suitable host of expressing 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, and the like. cinetobacter), Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylococcus In one embodiment, the host cell is a fungal cell. In one embodiment, the host cell is a Pichia pastoris or Aspergillus niger cell.

[0032] Vectors that can be used to transform the above-mentioned host cells are well known in the art. Generally, the vector comprises sequences that direct the transcription and translation of the relevant genes, a selectable marker, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors comprise a 5' region of the gene containing a transcription initiation control and a 3' region of the DNA fragment that controls transcription termination.

[0033] In one aspect, the present invention also relates to a method for producing a lysophospholipase, comprising expressing a nucleic acid molecule encoding a lysophospholipase of the present invention in a host cell and recovering the resulting polypeptide.

[0034] A variety of culture methods can be applied to prepare the enzymes of the present invention. For example, large-scale production of specific gene products from recombinant microbial hosts can be performed by batch, fed-batch, and continuous culture methods.

[0035] Batch and fed-batch culture methods are commonly used and well known in the art, and examples can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, 2nd ed., Sinauer Associates, Inc., Sunderland, MA (1989)), and Deshpande, Mukund V., (Appl. Biochem. Biotechnol., 36:227-234 (1992).

[0036] The commercial production of enzyme of the present invention also can be carried out by continuous culture.Continuous culture is a kind of open system, wherein the substratum that sets is added in the bioreactor continuously, and shifts out equal amount of conditioned medium simultaneously and is used for processing.Continuous culture generally makes cell maintain the constant high liquid density that cell is mainly in the logarithmic growth phase.Perhaps, continuous culture can be carried out with immobilized cell, wherein adds carbon and nutrient continuously, and continuously takes out valuable product, by-product or waste from cell mass.Cell fixation can use a wide range of solid carriers to carry out, and described solid carrier is made up of natural material and / or synthetic material.

[0037] 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 at the desired pH, and then the cell slurry is suspended in an aqueous buffer at the desired pH and homogenized to produce a cell extract containing the desired enzyme.

[0038] The invention further relates to compositions comprising a fermentation broth, a fermentation supernatant and / or a fermentation concentrate comprising a lysophospholipase of the present invention or a host cell of the present invention. Enzyme compositions of the present invention may be in any form suitable 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 a host cell as the source of the enzyme. The enzyme composition may be a dry powder or granules, dust-free granules, a liquid, a stabilized liquid or a stabilized protected enzyme. The liquid enzyme composition may be stabilized, for example, by adding a stabilizer such as a sugar, a sugar alcohol or other polyol, and / or lactic acid or other organic acids, according to a desired process.

[0039] The present invention further relates to the fermentation broth, fermentation supernatant or fermentation concentrate of the host cell of the present invention.

[0040] 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 in oil degumming, the enzyme activity is significantly improved compared with the lysophospholipase of the prior art, thereby reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 shows the changes in enzyme activities of mutants WTRT and WTRT-Y after the pAOP-WTRT and pAOP-WTRT-Y strains were subcultured twice on PDA solid medium plates. DETAILED DESCRIPTION

[0042] Experimental Materials

[0043] 1. Experimental strains and plasmids

[0044] Strains: Pichia pastoris GS115 was purchased from Invitrogen, C18100; Aspergillus niger AN19 was purchased from China Industrial Microorganism Culture Collection Center, number: Aspergillus niger CICC2243.

[0045] SphI and HindIII restriction enzymes were purchased from NEB.

[0046] Lecithin (purity 98%) was purchased from Aladdin.

[0047] NEFA kit was purchased from Wako Pure Chemical Industries, Ltd., Japan.

[0048] Bradford kit was purchased from Shanghai Bioengineering Co., Ltd. Plasmid: pAOP-Eno plasmid was constructed in our laboratory, see below for details. pAO815 was purchased from Invitrogen, K1750-01.

[0049] The pAOP-Eno vector was constructed by the inventors with reference to the method of Molecular Cloning: A Laboratory Manual (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989). The specific process is as follows:

[0050] The RML gene (NCBI accession number: A34959) obtained by whole gene synthesis from Sangon Biotech (Shanghai) Co., Ltd., with the Aspergillus oryzae α-amylase signal peptide (NCBI sequence number: XM_001821384.2, 1-63bp sequence) was inserted into an expression cassette with the Aspergillus oryzae enolase promoter (NCBI sequence number: D63941.1, 215-734bp; containing 12 copies of enhancer sequence) and the Aspergillus niger saccharifying enzyme terminator (NCBI sequence number: AF214480.1, including the terminator sequence part) using SphI and HindIII restriction sites. The entire expression cassette was inserted into the multiple cloning site of the cloning vector pSP72 using BglII and XhoI. Finally, the PyrG expression gene from Aspergillus oryzae (NCBI sequence number: AB017705.1) was inserted into the vector using the XhoI restriction site, thereby constructing the RML gene expression vector pAOP-Eno.

[0051] 2. Culture media and solutions

[0052] Aspergillus niger screening plate: 1.34% yeast nitrogen base (YNB) containing ammonium sulfate but not amino acids, 2% maltose, 2% lecithin emulsion, 0.1 M citric acid-sodium citrate buffer pH 4.0, 2% agarose, 10 mM CaCl2.

[0053] PDA solid culture medium: purchased from BD, product number BD 213400.

[0054] YPD liquid medium: 1% yeast extract, 2% peptone, 2% glucose.

[0055] MGYS solid medium: 1.34% yeast nitrogen base (YNB) containing ammonium sulfate without amino acids, 1% glycerol, 1 M sorbitol, 4×10-5% D-biotin, and 2% agar.

[0056] BMM-soybean lecithin screening medium: 1.34% yeast nitrogen base (YNB) containing ammonium sulfate but no amino acids, 4×10-5% D-biotin, 0.5% methanol (added after sterilization), 2% soybean lecithin emulsion, 0.1M citric acid-sodium citrate buffer pH 6.6, 2% agar, and 10uM ZnSO4·7H2O.

[0057] --2% soybean lecithin emulsion: 2g soybean lecithin, 100ml H2O, homogenized in a high-speed homogenizer at 8000 rpm for 1 min.

[0058] BMGY liquid medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base (YNB) containing ammonium sulfate but not amino acids, 1% glycerol, 4×10-5% D-biotin, 0.1 M potassium dihydrogen phosphate-potassium hydrogen phosphate buffer, pH 6.0.

[0059] 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), 0.1 M citric acid-sodium citrate buffer, pH 6.6.

[0060] Improved Bradford protein concentration determination kit (purchased from Shanghai Shenggong Bioengineering Co., Ltd.)

[0061] PCR enzyme: HSDNA Polymerase (purchased from Takara Biotechnology (Dalian) Co., Ltd.)

[0062] NEFA kit (purchased from Wako Pure Chemical Industries, Ltd., Japan)

[0063] Phosphatidic acid was purchased from Aladdin Reagent.

[0064] Example 1: Construction of mutant D94N and determination of enzyme activity

[0065] 1. Construction of plasmid

[0066] 1.1 Construction of plasmid pAOP-Eno was as described above.

[0067] 1.2 Construction of pAOP-AN02-LPL and pAOP-D94N plasmids

[0068] The mature peptide of AN02-LPL was selected, as shown in SEQ ID NO: 1:

[0069] AN02-LPL (SEQ ID NO: 1)

[0070] Aspartic acid (D) at position 94 of AN02-LPL (SEQ ID NO: 1) was mutated to asparagine (N), and the mutated polypeptide was named D94N.

[0071] Amino acid sequence of the mature peptide of mutant D94N (SEQ ID NO: 2)

[0072] AN02-LPL and D94N were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for gene sequence synthesis and cloned into the pAOP-Eno plasmid to obtain the pAOP-AN02-LPL plasmid and the pAOP-D94N plasmid.

[0073] 2. Obtaining mutant strains of Aspergillus niger and determining enzyme activity

[0074] The pAOP-AN02-LPL plasmid and the pAOP-D94N plasmid were transformed into the Aspergillus niger AN19 strain, and the resulting strains were named 19-AN02-LPL and 19-D94N, respectively. The specific transformation methods are as follows:

[0075] (1) Spores of Aspergillus niger AN19 strain cultured on PDA solid medium (purchased from BD, catalog number BD 213400) were eluted with spore washing solution. The eluted spores were vortexed for 1 min and filtered through mircloth to prepare a uniform spore suspension.

[0076] (2) Inoculation 1×10 7 The 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 (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 at a ratio of 1 / 1000); FeSO4.7H2O 2.78 g / L, Na2.EDTA 3.73 g / L was added at a ratio of 1 / 100)) at 28°C, 200 rpm, and cultured for 42-48 h; the grown mycelia were collected by filtration using sterilized Mircloth (purchased from Milliproe); the collected mycelia were rinsed three times with a sterilized osmotic pressure stabilizer (0.6 mol / L MgSO4) and pressed dry.

[0077] (3) The mycelia were transferred to a 100 mL Erlenmeyer flask, and each 0.8 g of mycelia was resuspended in 10 mL of enzymatic solution (1% cellulase (purchased from Sigma, product number C1184-25KU), 1% lytic enzyme (purchased from Sigma, product number L1412-25G), 0.1% snailase (purchased from Biotech, product number A600870-0005)) and dispersed; 30°C, 60 rpm, 60-90 min (after 30 min, every 1 The mycelia hydrolyzed with the enzyme were filtered through Mircloth, rinsed with 0.6 mol / LMgSO4, and the filtrate was collected; the mixture was centrifuged at 1000 g for 10 min at 4°C, and the supernatant was discarded to obtain a protoplast pellet; the protoplast pellet was resuspended in 5 mL of pre-cooled 1.0 mol / L sorbitol solution, centrifuged at 800 g for 10 min at 4°C, and the supernatant was discarded; the protoplast pellet was resuspended in 1 mL of pre-cooled 1.0 mol / L sorbitol and placed in an ice bath for later use.

[0078] (4) The protoplasts were centrifuged and the volume was adjusted to 1×10 7 To 200 μL of protoplast suspension, add 5 μg DNA (pAOP-AN02-LPL plasmid or pAOP-D94N plasmid) and 50 μL PTC (40% w / v PEG4000, 50 mM CaCl2, 50 mM Tris-HCl, pH = 7.5) solution, gently tap to mix, and place on ice for 30 min; add 0.2 mL of PTC solution, mix, and then add 0.8 mL The PTC solution was mixed and incubated at room temperature for 30 min. The mixture was added 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) and mixed. The plate was spread on a regeneration plate containing regeneration medium (with the exception of 1.5% agar, other ingredients are the same as above) and cultured at 28°C for more than 3 days.

[0079] (5) The clones grown on the regeneration plate in step (4) above were picked onto screening plates. 100 transformants were screened for each strain, and 10 transformants that formed large white precipitation circles on the screening plates were selected for shake flask fermentation to compare the enzyme activity. The specific process is as follows:

[0080] (6) The 10 positive clones screened were spread on 3% (w / v) PDA solid medium (purchased from BD, catalog number BD 213400) and cultured at 28°C for about 3 days to form a large number of spores. The washed spores were vortexed for 1 min and filtered through mircloth to prepare a uniform spore suspension. 1×10 7 The spore suspension was added to a 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) Tween 80, trace elements) and cultured at 28°C, 200 rpm for 8 days. The enzyme activity was determined and the transformant with the highest enzyme activity was selected for enzyme activity comparison.

[0081] (7) Determination of enzyme activity of 19-AN02-LPL and 19-D94N strain fermentation broth before storage

[0082] Fermentation broths from strains 19-AN02-LPL and 19-D94N were used to determine enzyme activity using soybean lecithin as a substrate. The following procedure was used: 10 μl of fermentation broth was added to 90 μl of substrate solution. The mixture was incubated at 50°C for 10 minutes, inactivated at 95°C for 5 minutes, and centrifuged at 7000 rpm for 5 minutes. The supernatant (1 μl) was then added to 80 μl of Reagent A from the NEFA kit and incubated at 37°C for 10 minutes. The mixture was then incubated with 160 μl of Reagent B for another 10 minutes. The absorbance at 550 nm was measured to calculate the enzyme activity.

[0083] The substrate solution (9 mL) was composed of 5 mL of 4% w / v soybean 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.

[0084] The results showed that the enzyme activities of the fermentation broths of the 19-AN02-LPL strain and the 19-D94N strain were 47688 U / ml and 45457 U / ml, respectively.

[0085] (8) Determination of enzyme activity of 19-AN02-LPL and 19-D94N strain fermentation broth after storage

[0086] The fermentation broths of the 19-AN02-LPL and 19-D94N strains were stored at 4°C for 2 weeks, and the enzyme activities were determined using the same method as in step (7) above.

[0087] Results showed that after two weeks of storage, the enzyme activity in the fermentation broth of the 19-AN02-LPL strain decreased from 47,688 U / ml to 37,261 U / ml; whereas, the enzyme activity in the fermentation broth of the 19-D94N strain only decreased from 45,457 U / ml to 43,442 U / ml. Therefore, compared to AN02-LPL, D94N exhibited a 77% reduction in enzyme activity loss. This facilitates the stable storage of the enzyme and extends its useful life.

[0088] Example 2: Construction of mutant HLRT and specific enzyme activity against phosphatidic acid

[0089] The mature peptide of AN02-LPL was selected, as shown in SEQ ID NO: 1.

[0090] 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.

[0091] The amino acid sequence of the new mutant HLRT is as follows:

[0092] The amino acid sequences of AN02-LPL and HLRT were sent to Jinweizhi Bioengineering Co., Ltd. for gene sequence synthesis and cloned into the pAO815 vector to generate the P-AN02-LPL and P-HLRT plasmids. After linearization with BglII, the vectors were transformed into competent cells of the Pichia pastoris strain GS115 by electroporation. The transformants were plated on MGYS solid medium plates and cultured at 30°C for 3 days to obtain Pichia pastoris transformants of pic-AN02-LPL and pic-HLRT. Single colonies from the plates were picked and plated on BMM-soybean lecithin selection medium plates. Colonies with large white precipitates were selected and named pic-AN02-LPL and pic-HLRT.

[0093] pic-AN02-LPL and pic-HLRT were activated in YPD liquid medium, then inoculated into BMGY liquid medium and cultured overnight at 30°C with shaking at 220 rpm. The culture was transferred to BMMY liquid medium with an initial OD600 of 6.

[0094] First, the cells were induced with 2% methanol, and 1% methanol was added after 24 h and 32 h, and after 48 h and 56 h, and samples were taken after 72 h.

[0095] The resulting fermentation broth was desalted and concentrated 40-fold using ultrafiltration tubing with a 10 kDa molecular weight cutoff. The treated sample was added to a buffer solution (20 mM citric acid-sodium citrate buffer (pH 5.5)). The resulting enzyme solutions were designated ANO2-LPL and HLRT.

[0096] The enzyme activity was determined using phosphatidic acid as the substrate as follows:

[0097] 9 ml substrate: 5 ml 1% phosphatidic acid, 1 ml 20% Triton X-100, 2.5 ml 0.1 M citric acid-sodium citrate buffer (pH=4.3; 4.8; 5.5), 0.5 ml water.

[0098] Add 10 μl of diluted enzyme solution to 90 μl of substrate and incubate at 50°C for 10 minutes. Inactivate at 95°C for 5 minutes, centrifuge at 7000 rpm for 5 minutes, remove 1 μl of supernatant, add 80 μl of Reagent A from the NEFA kit, incubate at 37°C for 10 minutes, and then add 160 μl of Reagent B for 10 minutes. Measure absorbance at 550 nm to calculate enzyme activity.

[0099] The protein concentration of the fermentation broth was determined using a modified Bradford protein concentration assay kit, and the specific enzyme activities of AN02-LPL and HLRT against phosphatidic acid at different pH conditions were calculated.

[0100] 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.

[0101] Example 3: Degumming test of HLRT

[0102] Pre-degummed crude soybean oil was shaken, stirred, and heated to a constant temperature of 55°C. 50% citric acid was added to a final concentration of 650 ppm. The oil was sheared at 20,000 rpm for 1.5 minutes. Stirring was continued at 55°C for 1 hour. 30 ppm HLRT, 30 ppm AN02-LPL, and 60 ppm AN02-LPL were mixed with 1.8 ml of water and added simultaneously. The oil was sheared at 200,000 rpm for 1.5 minutes. After the enzyme-water mixture was added, the temperature was raised to 55°C and a timer was started. The reaction was continued for 4 hours. The enzyme was then inactivated by heating the oil to 85°C and maintaining the temperature for at least 8 minutes. The degummed oil was centrifuged and sampled for phosphorus content testing. A phosphorus content of less than 10 ppm met the standard.

[0103] Example 4: Construction of mutant M1RT and specific enzyme activity against phosphatidic acid

[0104] The mutant polypeptide M1 of AN02-LPL was prepared (the mutation sites are L86I, G187D, E209K, A254D based on the mature peptide). The sequence of M1 is shown below:

[0105] The serine at position 84 of M1 was mutated to arginine, and the aspartic acid at position 85 was mutated to threonine.

[0106] The amino acid sequence of the new mutant M1RT is as follows:

[0107] The M1RT amino acid sequence was sent to Jinweizhi Bioengineering Co., Ltd. for gene sequence synthesis and cloned into the pAO815 vector to generate the P-M1RT plasmid. After linearization with BglII, the vector was transformed into competent cells of the Pichia pastoris GS115 strain by electroporation. The transformant was plated on MGYS solid medium plates and cultured at 30°C for 3 days to obtain Pichia pastoris transformants of pic-M1RT. Single colonies from the plates were picked and plated on BMM-soybean lecithin selection medium plates. The colony with the largest white precipitate was selected and named pic-M1RT.

[0108] pic-M1RT was activated in YPD liquid medium, then inoculated into BMGY liquid medium and cultured overnight at 30°C with shaking at 220 rpm. The culture was transferred to BMMY liquid medium with an initial OD600 of 6.

[0109] First, the cells were induced with 2% methanol, and 1% methanol was added after 24 h and 32 h, and after 48 h and 56 h, and samples were taken after 72 h.

[0110] The resulting fermentation broth was ultrafiltered, desalted, and concentrated 40-fold using a 10 kDa molecular weight cutoff ultrafiltration tube. The treated sample was added to a buffer solution (20 mM citric acid-sodium citrate buffer (pH 5.5)). The resulting enzyme solution, designated M1RT, was assayed for enzyme activity using phosphatidic acid as a substrate using the following method:

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

[0112] Add 10 μl of diluted enzyme solution to 90 μl of substrate and incubate at 50°C for 10 minutes. Inactivate at 95°C for 5 minutes, centrifuge at 7000 rpm for 5 minutes, remove 1 μl of supernatant, add 80 μl of Reagent A from the NEFA kit, incubate at 37°C for 10 minutes, and then add 160 μl of Reagent B for 10 minutes. Measure absorbance at 550 nm to calculate enzyme activity.

[0113] The protein concentration of the fermentation broth was determined using a modified Bradford protein concentration assay kit, and the specific enzyme activity of M1RT against phosphatidic acid under different pH conditions was calculated.

[0114] 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.

[0115] Example 5: Construction of mutant WTRT and specific enzyme activity against phosphatidic acid

[0116] The mature peptide of AN02-LPL was selected, as shown in SEQ ID NO: 1.

[0117] The 84th serine was mutated to arginine, and the 85th aspartic acid was mutated to threonine to obtain the amino acid sequence of the new mutant WTRT as follows:

[0118] The WTRT amino acid sequence was sent to Jinweizhi Bioengineering Co., Ltd. for gene sequence synthesis and cloned into the pAOP-Eno vector to obtain the pAOP-WTRT plasmid, which was then transformed into the Aspergillus niger AN19 strain.

[0119] The conversion method is as follows:

[0120] The spores of Aspergillus niger cultured on PDA solid medium were eluted with spore washing solution, and the eluted spores were vortexed for 1 min and filtered through mircloth to prepare a uniform spore suspension; 1×10 7The spore suspension was added to a fermentation medium (2% glucose, 6% maltose, 7% sodium citrate, 1.5% ammonium sulfate, 4% Tryptic soybroth, 0.1% sodium dihydrogen phosphate, 0.1% magnesium sulfate, 0.07% Tween 80, trace elements) at 28°C, 200 rpm for 42-48 h; the grown mycelia were collected by filtration through sterilized Mircloth; the collected mycelia were rinsed three times with a sterilized osmotic pressure stabilizer (0.6 mol / L MgSO4) and pressed dry; the mycelia were transferred to a 100 mL conical flask, and each 0.8 g of mycelia was resuspended in 10 mL of enzymatic hydrolyzate and dispersed; the mixture was filtered through Mircloth at 30°C, 60 rpm for 60-90 min (observed every 10 min after 30 min); the protoplast mixture was filtered through Mircloth and then washed with 0.6 mol / L Rinse with MgSO4 and collect the filtrate; centrifuge at 1000g for 10 min at 4°C and discard the supernatant; resuspend the protoplast pellet in 5 mL of pre-cooled 1.0 mol / L sorbitol solution and centrifuge at 800g for 10 min at 4°C and discard the supernatant; resuspend the protoplast pellet in 1 mL of pre-cooled 1.0 mol / L sorbitol and place in an ice bath for later use. Centrifuge the protoplasts and adjust the concentration to 1×10 7 / mL; to 200 μL protoplast suspension, add 5 μg DNA and 50 μL PTC (40% PEG4000, 50mM CaCl2, 50mM Tris-HCl, pH=7.5) solution, gently tap to mix, and ice bath for 30 minutes; add 0.2mL PTC solution, mix, then add 0.8mL PTC solution, mix, and room temperature for 30 minutes; the above mixture is added to 5ml regeneration medium (0.2% KH2PO4, 0.1% KCl, 0.05% MgSO4·7H2O, 0.005% FeSO4·7H2O, 1M sucrose, 10mM acetamide, 20mM cesium chloride, 0.6% agar), mix; spread on regeneration medium (composition as above, 1.5% agar), 28°C, and culture for more than 3 days.

[0121] The positive clones grown on the regeneration medium were spread on PDA solid medium and cultured at 28°C for about 3 days until a large number of spores were formed. The washed spores were vortexed for 1 minute and filtered through mircloth to prepare a uniform spore suspension. 1×10 7The spore suspension was added to fermentation medium (2% glucose, 10% maltose, 7% sodium citrate, 1.5% ammonium sulfate, 4% Tryptic soybroth, 0.1% sodium dihydrogen phosphate, 0.1% magnesium sulfate, 0.07% Tween 80, trace elements) and cultured at 28°C, 200 rpm for 8 days. The enzyme activity was then determined.

[0122] Enzyme activity was determined using phosphatidic acid as a substrate using the same method as in Example 4. 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.

[0123] Example 6: Construction of mutant WTRT-Y

[0124] The mature peptide of AN02-LPL was selected, as shown in SEQ ID NO: 1.

[0125] The serine at position 84 was mutated to arginine, the aspartic acid at position 85 was mutated to threonine, and the aspartic acid at position 94 was mutated to tyrosine. The amino acid sequence of the new mutant WTRT-Y was as follows:

[0126] The WTRT-Y amino acid sequence was sent to Jinweizhi Bioengineering Co., Ltd. for gene sequence synthesis and cloned into the pAOP-Eno vector to obtain the pAOP-WTRT-Y plasmid.

[0127] Example 7: Comparison of enzyme activities of mutant WTRT-Y, AN02-LPL and mutant WTRT

[0128] The expression vectors pAOP-AN02-LPL (construction method see Example 1), pAOP-WTRT (construction method see Example 5), and pAOP-WTRT-Y were transformed into Aspergillus niger AN19E (see patent application CN202111598184.2). The transformation method is as follows:

[0129] Fresh Aspergillus niger AN19E spores were washed with spore wash solution, filtered through Miracloth to prepare a spore suspension, and adjusted to 1×10 7 Inoculate 1 mL of spore suspension into mycelial culture medium (2% tryptone, 1% yeast extract, 2% glucose, 0.3% uracil), culture at 28°C, 180 rpm, for 40 hours, and collect the grown mycelium by filtration through sterilized Miracloth.

[0130] The collected mycelia were rinsed three times with a sterilized osmotic pressure stabilizer (0.6M MgSO4, 10mM NaH2PO4, pH=5.8) and squeezed dry. The mycelia were transferred to a 100mL triangular flask and each 0.8g of mycelia was resuspended in 20mL of an enzymolysis solution (an enzymolysis solution containing 1% lyase, 1% cellulase, and 0.1% snailase prepared with an osmotic pressure stabilizer and sterilized by filtration through a 0.22μm microporous filter) and enzymolyzed for 60-90min at 30°C and 90rpm. The enzymolysis mixture of protoplasts was filtered through Miracloth, the filtrate was collected, and the mixture was centrifuged at 1000g for 10min at 4°C. The protoplast pellet was resuspended in 5mL of a pre-cooled 1.0mol / L sorbitol solution and centrifuged at 800g for 10min at 4°C. The supernatant was discarded. Then, the protoplasts were adjusted to 1×10 7 / mL, and keep on ice until use.

[0131] To 200 μL of protoplast suspension, add 10 μL of 1 μg / μL expression vector, then add 50 μL of PTC solution (40% PEG4000, 50 mM CaCl2, 50 mM Tris-HCl, pH = 7.5), mix thoroughly, and incubate on ice for 30 minutes. Then, add 0.2 mL of PTC solution, mix thoroughly, and then add 0.8 mL of PTC solution, mix thoroughly, and maintain at room temperature for 30 minutes.

[0132] The mixed solution was spread on regeneration medium (1% glucose, 0.6% NaNO3, 0.15% KH2PO4, 0.05% KCl, 0.05% MgSO4, 0.001% FeSO4, 1M sucrose, 2% agar powder) and cultured at 28°C for 7 days until colonies grew.

[0133] The colonies grown on the plates were transferred to Aspergillus niger screening plates and cultured at 28° C. for 3 days to obtain Aspergillus niger expression strains pAOP-AN02-LPL, pAOP-WTRT and pAOP-WTRT-Y of LPL.

[0134] The strains were fermented in shake flasks. The fermentation medium (2% glucose, 15% maltose, 7% sodium citrate, 1.5% ammonium sulfate, 4% TSB, 0.1% sodium dihydrogen phosphate, 0.1% magnesium sulfate, 0.07% Tween 80, trace elements) was sterilized by autoclaving at 115°C for 15 min. The fermentation conditions were 28°C, 220 rpm, 10 days, and an inoculum size of 1 × 10 7 spores / 50mL.

[0135] After shake flask fermentation, enzyme activity was determined. The fermentation broths of pAOP-AN02-LPL, pAOP-WTRT, and pAOP-WTRT-Y were diluted to an appropriate protein concentration, and the enzyme activity was determined using phosphatidic acid as a substrate, using the same method as in Example 4.

[0136] The results showed that the new mutant WTRT-Y still maintained high specific enzyme activity against phosphatidic acid, increasing from 347 U / mg of AN02-LPL to 961 U / mg at pH 4.3; from 863 U / mg of AN02-LPL to 1803 U / mg at pH 4.8; and from 0 U / mg of AN02-LPL to 1212 U / mg at pH 5.5. This activity was essentially the same as that of the mutant WTRT (984 U / mg at pH 4.3, 2003 U / mg at pH 4.8, and 1342 U / mg at pH 5.5).

[0137] Example 8: Evaluation of the anti-degradation stability of mutants WTRT and WTRT-Y

[0138] The supernatant of the fermentation enzyme solution of pAOP-WTRT and pAOP-WTRT-Y was microfiltered and stored at 4°C and -20°C for 2 weeks, respectively. The changes in residual lipase activity were then examined to evaluate the anti-degradation stability of the mutant gene.

[0139] The enzyme activity determination method is the same as in Example 4.

[0140] The results showed that the enzyme activity of the mutant WTRT decreased from 29,921 U / ml to 20,076 U / ml after two weeks of storage at 4°C, while the protein stability of the mutant WTRT-Y was improved, with its enzyme activity only decreasing from 28,765 U / ml to 25,984 U / ml after two weeks of storage at 4°C. This loss was reduced by 68% compared to WTRT.

[0141] Example 9: Strain stability assessment of mutants WTRT and WTRT-Y

[0142] The pAOP-WTRT and pAOP-WTRT-Y strains were subcultured twice on PDA solid culture plates, and shake flask fermentation was performed to investigate the changes in enzyme activity after subculture.

[0143] The results are shown in Figure 1. As shown in Figure 1, after the mutant WTRT was passaged twice, the enzyme activity was 37.5% of that of the unpassaged strain, while after the expression strain of the new mutant WTRT-Y was passaged twice, the enzyme activity was consistent with that of the unpassaged strain, showing no instability.

Claims

1. A mutant lysophospholipase having one or more mutations in its amino acid sequence relative to a wild-type lysophospholipase and having increased specific activity or increased stability.

2. The lysophospholipase of claim 1, which has a mutation in its amino acid sequence at one or more positions selected from positions 84, 85 and 94 corresponding to SEQ ID NO:

1.

3. The lysophospholipase of claim 2, wherein the amino acid sequence has a mutation at the position corresponding to position 94 of SEQ ID NO: 1, and preferably, the mutation is from aspartic acid to asparagine.

4. The lysophospholipase of claim 2, wherein the amino acid sequence has mutations at positions corresponding to positions 84 and 85 of SEQ ID NO: 1, preferably, the mutations are mutations of serine at position 84 to arginine, and mutations of aspartic acid at position 85 to threonine.

5. The lysophospholipase of claim 4, further comprising an additional mutation at one or more positions selected from 111, 147, 86, 187, 209 and 254 corresponding to SEQ ID NO: 1, preferably, the additional mutation is a mutation at positions 111 and 147, or a mutation at positions 86, 187, 209 and 254, more preferably, the additional mutation is a mutation of lysine at position 111 to histidine and tyrosine at position 147 to leucine; or a mutation of leucine at position 86 to isoleucine, glycine at position 187 to aspartic acid, glutamic acid at position 209 to lysine and alanine at position 254 to aspartic acid.

6. The lysophospholipase of claim 2, wherein the amino acid sequence has mutations at positions corresponding to positions 84, 85 and 94 of SEQ ID NO: 1; preferably, the mutations are serine at position 84 to arginine, aspartic acid at position 85 to threonine, and aspartic acid at position 94 to tyrosine.

7. The lysophospholipase of claim 1, comprising the amino acid sequence shown in SEQ ID NO: 2, 3, 5, 6 or 7.

8. A nucleic acid molecule selected from: (a) a nucleotide sequence encoding the lysophospholipase according to any one of claims 1 to 7; and (b) a nucleotide sequence complementary to the nucleotide sequence described in (a).

9. A vector comprising the nucleic acid molecule of claim 8.

10. A host cell comprising the nucleic acid molecule of claim 8, or the vector of claim 9; 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, more preferably a Pichia pastoris cell or an Aspergillus niger cell.

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

12. A composition comprising the lysophospholipase according to any one of claims 1 to 7 or the fermentation broth, fermentation supernatant and / or fermentation concentrate of the host cell according to claim 10.

13. The fermentation broth, fermentation supernatant or fermentation concentrate of the host cell according to claim 10.

14. Use of the lysophospholipase according to any one of claims 1 to 7, the composition according to claim 12, or the fermentation broth, fermentation supernatant and / or fermentation concentrate according to claim 13 in oil degumming.

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