CN106701712B

CN106701712B - Novel phospholipase

Info

Publication number: CN106701712B
Application number: CN201510778847.7A
Authority: CN
Inventors: 戴小军; 苏斐; 牛其文
Original assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Current assignee: Wilmar Shanghai Biotechnology Research and Development Center Co Ltd
Priority date: 2015-11-13
Filing date: 2015-11-13
Publication date: 2021-05-28
Anticipated expiration: 2035-11-13
Also published as: CN106701712A

Abstract

The invention relates to a novel phospholipase and a coding gene thereof. Also relates to a vector containing the gene and a host cell. The phospholipase provided by the invention comprises or consists of a sequence selected from the group consisting of: (a) 2 and (b) a sequence obtained by substituting, deleting or adding at least one amino acid from the sequence in (a), wherein the sequence still maintains the function of the SEQ ID NO. 2. The phospholipase provided by the invention has the capability of hydrolyzing acyl ester of phosphatidylethanolamine but not hydrolyzing phosphatidylcholine.

Description

Novel phospholipase

Technical Field

The invention relates to a novel phospholipase and a coding gene thereof. Also relates to a vector containing the gene and a host cell.

Background

Phospholipases are enzymes that hydrolyze phospholipids and are classified into several major groups according to their hydrolysis position. Phospholipase A1(PLA1) hydrolyzes to free fatty acid at sn-1 position; phospholipase A2(PLA2) hydrolyzes to release fatty acid at sn-2 position; phospholipase B (PLB) hydrolyzes to release fatty acid at sn-1 and sn-2 positions; phospholipase C (PLC) hydrolyses the glycerol-phosphate ester bond, releasing diacylglycerol and phosphate ester; phospholipase d (pld) hydrolyses phospho-base ester bonds, releasing phosphatidic acid and base (choline, ethanolamine or inositol). Phospholipases are widely used in phospholipid removal, phospholipid modification, and the like.

Lecithin (phosphatidylcholine, PC) is the most commonly used phospholipid. It is a natural surfactant, has a variety of important physiological functions, and is widely used in emulsifying machines, wetting agents, dispersants, stabilizers, drug carriers, and for directly treating cardiovascular diseases. Lecithin is mainly derived from soybean and egg yolk. The industrial-grade lecithin is mostly from oil residue generated after degumming of crude oil, the yield of soybean is rich, and the content of unsaturated fatty acid in the soybean lecithin is higher than that of egg yolk lecithin. In addition to lecithin, however, cephalin (phosphatidylethanolamine, PE), Phosphatidylserine (PS), phosphatidic acid, fatty acid salts, neutral oil, protein, and other impurities are present in the oil residue. Lecithin with high purity is required for treatment, fine chemical engineering, drug carriers and even high-grade food, but the lecithin generally exists with other phospholipids, has similar dissolution property and is difficult to separate. Lecithin is prepared from soy, typically by first preparing a crude phospholipid, which is then purified as required. The method comprises the steps of purifying lecithin; a metal salt precipitation method; performing a supercritical extraction method; fourthly, thin-layer chromatography; fifthly, carrying out column chromatography; sixthly, a membrane separation method. [ extraction, purification and analysis of Phosphatidylcholine, Luozhen, Master thesis ]. The most common industrial method is ethanol extraction [ separation and purification method of high-purity lecithin, Zhangxiuqing Yuan, grain and oil processing and food machinery, 2006,4 th, 45-48], PC is dissolved in ethanol, PE is dissolved in hot ethanol [ extraction and purification and physiological activity analysis of phospholipid, thesis ], but ethanol enrichment can only concentrate PC content to 45.3% [ extraction and purification research of soybean phosphatidylcholine, Geyuqin, thesis ] can reach 95% purity through column chromatography. If an enzyme which can hydrolyze the cephalin but not the lecithin exists, the purification of the lecithin is obviously simpler, and the method has low consumption and low pollution.

Glycerol Phosphoethanolamine (GPE) is a water-soluble phospholipid metabolite naturally existing in vivo, mainly exists in brain and liver, and is an important precursor for biosynthesis of phospholipid components such as PC, PE, PS and the like in vivo. GPE has shown significant memory and learning-mimicking abilities in laboratory animal tests, and in human clinical tests, it has influenced mental intelligence test results, commonly used neural scale measurements, and neurophysiological parameters. It can be used for treating various diseases with nervous and mental function deficiency, such as mental activity retardation, memory decline with age, emotional lability, and mental weakness. Therefore, GPE is often used as a main active ingredient of a medicament, and is mutually assisted by a proper carrier, so as to be used for treating chronic brain nervous tissue syndrome caused by cerebral degeneration or cerebrovascular insufficiency. At present, PC and PE in phospholipid are mixed and are difficult to distinguish, so that pure GPE is difficult to prepare, the alcoholysis of a mixture of PC and PE is generally catalyzed by sodium methoxide, and then GPE is separated by ion exchange resin, so that the efficiency is low. If there is an enzyme that transiently hydrolyzes cephalin, it can be used to hydrolyze mixed phospholipids, releasing GPE. The preparation work is simpler and more efficient.

Disclosure of Invention

The object of the present invention is to provide a novel phospholipase having a hydrolysis activity of acyl ester of phosphatidylethanolamine but not a hydrolysis activity of phosphatidylcholine. Which comprises or consists of a sequence selected from: (a) an amino acid sequence as shown in SEQ ID NO 2, and

(b) the sequence obtained by the sequence of (a) after at least one amino acid substitution, deletion or addition, wherein the substitution is preferably amino acid conservative substitution, and the sequence still maintains the function of SEQ ID NO. 2.

In one embodiment, the phospholipase of the invention has a sequence as shown in SEQ ID NO 2. In addition, SEQ ID NO 2 of the invention may also have one or several deletions, insertions or substitutions of amino acid residues, which modifications result in a silent change or in a functionally equivalent enzyme. In the case of maintaining enzymatic activity, the amino acid substitutions designed may be based on similarity in polarity, charge, solubility, hydrophilicity, hydrophobicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar heads that have similar hydrophilicity include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tryptophan.

Conservative substitutions may be made according to the following table. Amino acids belonging to the same partition in the second column may be substituted for each other, and in a preferred case amino acids in the same row in the third column may be substituted for each other:

the invention includes sequences obtained by deletion, insertion or conservative substitution of SEQ ID NO. 2, which still retain their activity. It should be considered as protected by the equivalent scheme of SEQ ID NO. 2.

The invention also relates to a nucleic acid molecule selected from the group consisting of: (1) a nucleotide sequence encoding the phospholipase of the invention or a nucleotide sequence complementary to the nucleotide sequence of (1).

In one embodiment, the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO 1.

The skilled person will appreciate that due to the degeneracy of the genetic code, a plurality of different nucleotide sequences may encode the same enzyme. In addition, it will be appreciated that the skilled person is able to use conventional techniques to perform nucleotide substitutions that do not affect the activity of the enzyme encoded by the nucleotide sequence of the invention, and as such may reflect the codon bias of any particular host organism used to express the enzyme of the invention. Thus, the invention also includes sequences that are at least 90% identical, more preferably at least 95% identical, and even more preferably at least 98% identical to SEQ ID NOs 1 and 2 set forth herein, and which still retain the desired activity. It should be considered protected as equivalent to SEQ ID NO 1 and 2. Comparison of amino acid and nucleic acid sequence identity may be performed using computer programs routine in the art.

The invention also provides a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the vector. The host cell can be Escherichia coli, Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, Aspergillus niger, Aspergillus oryzae, Thraustochytrium, Schizochytrium, etc. In another embodiment, the bacterial cell is a pichia cell.

The invention also relates to the use of the aforementioned polypeptide, polynucleotide, expression vector or host cell for the preparation of phospholipase.

The present invention also relates to a method for enhancing the activity of the phospholipase of the invention comprising adding magnesium, calcium, copper, manganese or zinc ions to the enzyme preparation, preferably magnesium sulfate, calcium chloride, copper sulfate, manganese chloride or zinc sulfate.

Drawings

FIG. 1 is a photograph showing the detection of the recovered PCR product of phospholipase gene by electrophoresis.

FIG. 2 is a photograph of electrophoresis of pPIC9k vector containing phospholipase gene for enzyme digestion test.

FIG. 3 is a schematic diagram of the structure of pPIC9k vector containing the phospholipase gene.

FIG. 4 is a photograph of PCR test electrophoresis of recombinant Pichia pastoris colonies, UD is the amplification product of the phospholipase gene, and AOXAlf is the amplification product of the entire expression cassette.

FIG. 5 is a photograph of a thin layer chromatography of an n-hexane extract after hydrolysis of mixed phospholipids, lecithin and cephalin by a crude enzyme solution. "calcium", "magnesium" and "zinc" mean crude enzyme solutions obtained by fermentation using g1-9k strain and to which calcium, magnesium, or zinc ions are added in the reaction system, respectively. "calcium-" "magnesium-" zinc- "means that the crude enzyme solution obtained by fermentation using the 9k-1# strain was used and calcium, magnesium, or zinc ions were added in the reaction system, respectively. PLA1, PLA2 refer to the hydrolysis of cephalins using the commercial enzymes PLA1 or PLA2, respectively; DAG is a1, 3-diglyceride standard. FFA is an oleic acid standard.

FIG. 6 shows the results of thin-layer neutral developed chromatography of soybean mixed phospholipids, lecithin and cephalin by the fermentation of crude enzyme solutions of g1-9k-1# and g1-9k-2# bacteria. The negative is the hydrolysis result of the crude enzyme liquid obtained by the fermentation of the 9k-1# bacteria.

FIG. 7 is a thin-layer polarity development chromatogram of crude enzyme solution hydrolyzed lecithin and cephalin products obtained by fermenting the g1-9k-1# strain. The negative is the crude enzyme liquid obtained by fermenting the 9k-1# strain. A1, A2 refer to the products of the hydrolysis of cephalins using the commercial enzymes PLA1 or PLA2, respectively. PE is cephalin.

FIG. 8 is a photograph of a thin layer chromatography neutral developed chromatogram of a crude enzyme solution obtained by fermentation of 1-9k-1# strain after hydrolysis of a mixture of cephalin, lecithin and soybean phospholipid.

Detailed Description

The invention amplifies the phosphatidase gene of schizochytrium by a PCR method, connects the phosphatidase gene with an expression vector, then uses methanol to induce expression in pichia pastoris, collects and concentrates fermentation supernatant fluid. Soybean mixed phospholipid, lecithin and cephalin are respectively used as substrates, and the activity of the expression product phospholipase is detected.

The phospholipase is well expressed in pichia pastoris, and the activity of the phospholipase is detected.

The phospholipase of the invention comprises or consists of a sequence selected from the group consisting of: (a) an amino acid sequence as shown in SEQ ID NO 2, and

2 can be substituted, deleted or added by 1-20 amino acids. Preferably, 1-15, 1-10, 1-5 or 1-3 amino acid substitutions, deletions or additions are made.

The invention also relates to a nucleic acid molecule selected from the group consisting of: (1) a nucleotide sequence encoding the phospholipase of the invention or a nucleotide sequence complementary to the nucleotide sequence of (1). In one embodiment, the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID NO 1.

The invention also includes sequences that have a certain percentage identity to SEQ ID NOs 1 and 2 set forth herein, and still retain the desired activity.

Percent identity is the relationship between two or more polypeptide sequences or between two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match of such linear sequences. "identity" can be readily calculated by known methods, including but not limited to those described below:Computational Molecular Biology(Lesk，A.M.，Ed.)Oxford University Press，NY(1988)；Biocomputing:Informatics and Genome Projects(Smith，D.W.，Ed.)Academic Press，NY(1993)；Computer Analysis of equence Data，Part I(Griffin, a.m., and Griffin, h.g., Eds.) human Press, NJ (1994);Sequence Analysis in Molecular Biology(von Heinje，G.，ed.)Academic Press (1987); andSequence Analysis Primer(Gribskov, M.and Devereux, J., eds.) Stockton Press, NY (1991). Methods of determining identity are codified in publicly available computer programs. Sequence alignment and percent identity calculation the Megalign program of the LASERGENE bioinformatics calculation Software package (DNASTAR Inc., Madison, Wis.), the AlignX program of Vector NTI v.7.0 (Informatx, Inc., Bethesda, MD), or EMBOSS Open Software Suite (EMBL-EBI; Rice et al, Trends in Genetics 16, (6): 276-. Sequence multiple alignments can be performed using the Clustal alignment method (i.e., CLUSTALW; e.g., version 1.83) (Higgins and Sharp, CABIOS, 5: 151-.

Suitable nucleotide or amino acid sequences have at least about 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 98% identity to the sequences reported herein.

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

"vector" refers to an extrachromosomal element that typically 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 may be autonomously replicating sequences, genome integrating sequences, bacteriophage or nucleotide sequences, linear or circular single-or double-stranded DNA or RNA, derived from any source, many of which have been joined or recombined into a construct capable of introducing a promoter fragment of a selected gene product and the DNA sequence into a cell along with appropriate 3' untranslated sequence.

The genes and gene products of the sequences of the invention may be produced in heterologous host cells, particularly microbial host cells. Preferred heterologous host cells for expression of the genes and nucleic acid molecules of the invention are microbial hosts which are present in the fungal or bacterial family and which grow over a wide range of temperatures, pH values and solvent tolerance. For example, it is contemplated that any bacteria, yeast, and filamentous fungi may be suitable hosts for expression of the nucleic acid molecules of the present invention. Examples of host strains include, but are not limited to, bacteria, fungi or yeast species such as Aspergillus (Aspergillus), Trichoderma (Trichoderma), Saccharomyces (Saccharomyces), Pichia (Pichia), Phaffia (Phaffia), Kluyveromyces (Kluyveromyces), Yarrowia (Yarrowia), Candida (Candida), Hansenula (Hansenula), Salmonella (Salmonella), Bacillus (Bacillus), Acinetobacter (Acinetobacter), Zymomonas (Zymomonas), Agrobacterium (Agrobacterium), Erythromobacter (Erythromobacter), Chloromyces (Chlorobium), Chromobacterium (Chromobacterium), Flavobacterium (Flavobacterium), Cytophaga (Cytophaga), Rhodobacter (Rhodobacter), Rhodococcus (Streptococcus), Streptomyces (Corynebacterium), Corynebacterium (Corynebacterium), Escherichia (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium) and Escherichia) are used in the strains of the strain (Corynebacterium), Bacillus) and the strain (Corynebacterium) may be, Pantoea (Pantoea), Pseudomonas (Pseudomonas), Sphingomonas (Sphingomonas), Methylomonas (Methylomonas), Methylobacterium (Methylobacter), Methylococcus (Methylococcus), Methylosinus (Methylosine), Methylomicrobium (Methylomicium), Methylocystis (Methylocystis), Alcaligenes (Alcaligenes), Synechocystis (Synechocystis), Synechococcus (Synechococcus), Anabaena (Anabaena), Thiobacillus (Thiobacillus), Methanobacterium (Methanobacterium), Klebsiella (Klebsiella), and Myxococcus (Myxococcus) species. The host cell may be Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Aspergillus niger, Aspergillus oryzae, Thraustochytrium, Schizochytrium, etc. In one embodiment, the host cell is a pichia cell.

Vectors useful for transforming the above host cells are well known in the art. Typically, the vector comprises sequences directing transcription and translation of the gene of interest, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a 5 'region of a gene containing transcriptional initiation controls and a 3' region of a DNA fragment that controls transcriptional termination.

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

Batch and fed-batch culture methods are common and well known in the art, and examples can be found in the following documents: thomas D.Brock inBiotechnology:A Textbook of Industrial MicrobiologySecond edition, Sinauer Associates, Inc., Sunderland, MA (1989)), and Desmopande, Mukund V. (appl. biochem. Biotechnol., 36: 227-.

Commercial production of the enzyme of the invention can also be carried out by continuous culture. Continuous culture is an open system in which a set amount of culture medium is continuously added to a bioreactor and an equal amount of conditioned medium is simultaneously removed for processing. Continuous culture generally maintains cells at a constant high liquid phase density where the cells are predominantly in logarithmic growth phase. Alternatively, continuous culture can be performed with immobilized cells, wherein carbon and nutrients are continuously added and valuable products, by-products or waste are continuously removed from the cell pellet. Cell immobilization can be carried out using a wide range of solid supports, consisting of natural and/or synthetic materials.

Recovery of the desired enzyme from a 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 desired pH, and then the cell slurry in the aqueous buffer of desired pH is suspended and homogenized to produce a cell extract containing the desired enzyme.

The enzyme of the invention may be stored in glycerol. Experiments show that the glycerol and low temperature do not inhibit the enzyme activity.

The enzyme of the present invention may also be stored in the form of a solid powder. For example, the cell extract may optionally be filtered through a suitable filter aid such as diatomaceous earth or silica to remove cell debris prior to the heat treatment step used to precipitate the undesired proteins from the enzyme solution. The solution containing the desired enzyme can then be separated from the precipitated cell debris and proteins by membrane filtration or centrifugation, and the resulting partially purified enzyme solution enriched by additional membrane filtration, then optionally mixed with a suitable excipient (e.g., maltodextrin, trehalose, sucrose, lactose, sorbitol, mannitol phosphate buffer, citrate buffer, or mixtures thereof) and spray dried to produce a solid powder containing the desired enzyme.

The inventors have also found that the activity of the enzymes of the invention can be mentioned by the addition of certain ions. For example, in order to enhance the activity thereof, magnesium ion, calcium ion, copper ion, manganese ion or zinc ion may be added to the enzyme preparation, and preferably, magnesium sulfate, calcium chloride, copper sulfate, manganese chloride or zinc sulfate is added to the enzyme preparation.

The enzyme of the invention is separated from the schizochytrium limacinum genome, so that the enzyme can be well expressed in the schizochytrium limacinum.

When an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. When a range is defined, it is not intended that the range be limited to the specific values recited.

The following examples are provided to illustrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the methods disclosed herein, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the methods disclosed herein.

Reagents used in the examples:

the pPIC9k vector and the expression host bacterium Pichia pastoris SMD1168 were purchased from Invitrogen corporation.

PLA1, Lecite Ultra A1 was purchased from Novovin, PLA2 was purchased from Sigma under the Cat number P8685.

Pure lecithin and cephalin were purchased from alatin.

The first embodiment is as follows:

schizochytrium sp.ATCC 20888 was inoculated into 50ml YPD liquid medium (1% yeast powder, 2% peptone, 2% glucose) for 48h, centrifuged at 4000rpm at 4 ℃ for 5min to collect the cells and washed with deionized water 2 times, the cells were ground in liquid nitrogen, and Genomic DNA was extracted using Takara MiniBEST Universal Genomic DNA extraction kit.

Primers gU (SEQ ID NO:3) and gD (SEQ ID NO:4) were synthesized, and the phospholipase gene was amplified from the schizochytrium genome using the pair of primers, and had NdeI at the 5 'end and Not I restriction sites at the 3' end. The PCR system is 50ul and comprises the following components: 13.5ul of water, 25ul of 2 XGC I buffer, 6ul of dNTPs, 2ul of each primer (20. mu.M stock solution), 0.5ul of LA-Tag (Takara Co., Ltd.), and 1ul of template. The PCR amplification procedure was: denaturation at 98 ℃ for 5 min; 12 cycles of thermal asymmetric PCR: 95 ℃ 30s, 63 ℃ 30s, 72 ℃ 1min, 95 ℃ 30s, 44 ℃ 30s, 72 ℃ 1 min; 10min at 72 ℃.

The PCR product was purified using the Omega Cycle Pure kit, and the purified product was checked by 1% agarose gel electrophoresis, and the results are shown in FIG. 1.

The PCR product was digested in 37 ℃ water bath with EcoRI and NotI from NEB for 2h, the digestion system was: 20. mu.l of water, 10. mu.l of 4# buffer, 60. mu.l of PCR product, 5. mu.l of EcoRI, 5. mu.l of NotI.

The pPIC9k vector was digested simultaneously with EcoRI and NotI from NEB for 2h in a water bath at 37 ℃ in a volume of 60. mu.l, 10. mu.l for 4# buffer, 20. mu.l for plasmid, 5. mu.l for EcoRI and 5. mu.l for NotI.

The PCR product after double digestion and the pPIC9k vector after double digestion were purified using the Omega Cycle Pure kit, and the purified product was eluted with deionized water to 30. mu.l.

The PCR product after double digestion and the pPIC9k vector after double digestion are connected by using T4DNA ligase of Fermentas company, and the connecting system is as follows: 15.5. mu.l of water, 2. mu.l of 10 XT 4DNA ligase buffer, 2. mu.l of gene fragment, 0.5. mu.l of vector fragment. The ligation conditions were 16 ℃ for 2 h.

10ul of the ligation product was added to 200ul of E.coli DH5 alpha competent cells (Takara D9057), gently mixed, heat-shocked in a water bath at 42 ℃ for 90 seconds after ice-bath for 30 minutes, immediately ice-bathed for 2 minutes and added with 800ul of fresh LB medium (10 g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.0), shake-cultured at 200rpm in a shaker at 37 ℃ for 40 minutes, spread on an LB plate (1.8% agar powder in LB medium, 50ug/ml ampicillin-containing medium), and cultured overnight in a 37 ℃ incubator. The next day, a single colony was picked and inoculated into 5ml of LB medium (containing 50ug/ml ampicillin), and cultured overnight with shaking at 37 ℃ on a shaker at 200 rpm.

The extracted plasmid was obtained using Axygen plasmid extraction kit, and double restriction enzyme digestion was performed using NdeI and EcoRI restriction enzymes from NEB, 20. mu.l of the digestion system, 5. mu.l of plasmid, 1. mu.l of each enzyme, 2. mu.l of 4# buffer, and 11. mu.l of water. The enzyme was digested in a 37 ℃ water bath for 1h, and the cleavage products were checked by agarose gel electrophoresis, the results of which are shown in FIG. 2.

From the results of FIG. 2, a fragment corresponding to the size of the gene appeared at position 1.5k, indicating that the gene had been inserted into the pPIC9k vector. The plasmid is subjected to Shanghai's biological sequencing test, and the result shows that the insertion sequence is shown as SEQ ID NO. 1, the insertion sequence corresponds to the amino acid sequence shown as SEQ ID NO. 2, the obtained gene is named as pld8, the pPIC9k vector containing the gene is named as g1-pPIC9k, and the structure of the vector is shown as figure 3.

Blastp analysis was performed on GeneBanK and the results of the alignment showed that the closest result to this gene was Ricinus communis-derived phosphoipase d beta, putative with a maximum similarity of only 41%.

The g1-pPIC9k and pPIC9k vectors were linearized in water (55. mu.l, plasmid 60. mu.l, buffer 4# 15. mu.l, SalI-HF) with the NEB SalI-HF restriction enzyme (2 h) in a water bath at 37 ℃. Purified using Omega Cycle Pure kit and eluted to 20. mu.l with deionized water.

Inoculating a strain liquid of a pichia pastoris SMD1168 strain into a glycerol storage tube, streaking the strain liquid with an inoculating loop, inoculating the strain liquid onto a YPD (2% glucose, 2% peptone, 1% yeast powder, 2% agar powder, pH6.8) flat plate, culturing the strain liquid in an incubator at 28 ℃ for 48 hours until obvious bacterial colonies appear, then inoculating a single strain of the pichia pastoris SMD1168 into the YPD liquid, and culturing the strain liquid overnight. The cells were collected by centrifugation at 4000rpm for 5min at 4 ℃ and the g1-pPIC9k and pPIC9k empty vectors linearized with SacI restriction enzymes from NEB were transferred to Pichia pastoris by the method described in the literature [ Shixuan Wu & Geoffrey J.Letchworth. high efficiency transformation by side electrophoresis of Pichia pastoris with dithioless acid and dithio reaction, 2004,36(1):152 ], the linearized vector was 35. mu.l water, 5. mu.l SacI enzyme, 10. mu.l plasmid 1# buffer, 50. mu.l plasmid, the enzyme was digested overnight at 37 ℃ and then recovered using a omega PCR kit and eluted with 30. mu.l deionized water. The transformed yeast was cultured in an MD plate (1.34% YNB, 4X 10-5% biotin, 2% glucose) in an incubator at 28 ℃ for 2 days.

Several colonies were picked from g1-pPIC9k screening plates and from pPIC9k empty vector transformation screening plates, inoculated into YPD medium and cultured overnight at 28 ℃ with shaking at 200 rpm. And (5) freezing and storing the thalli for later use.

The recombinant strain thallus is quick frozen in liquid nitrogen and then transferred to grinding, liquid nitrogen is added for grinding, and then genome DNA is extracted by using a MiniBEST Universal Genomic DNA extraction kit of Takara company. This genome was used as a template for PCR amplification verification. The PCR system was 25. mu.l, and consisted of 17. mu.l of water, 2.5. mu.l of 10 XBuffer, 2. mu.l of dNTPs, 1. mu.l each of primers (20. mu.M stock solution), 0.3. mu.l of rTag, and 1. mu.l of template. The PCR amplification procedure was: denaturation at 98 ℃ for 5 min; 30 cycles of PCR: at 95 ℃ for 40s, 50 ℃ for 30s and 72 ℃ for 2 min; 10m at 72 ℃. Wherein,

gU and gD are used as primer pairs to amplify exogenous gene phospholipase pld8 (UD);

the full expression cassette (AOXAlf) is amplified and identified by using alfac (shown as SEQ ID NO: 5) and 3' AOX (shown as SEQ ID NO: 6) as primer pairs.

The amplification product is checked by 1% agarose gel electrophoresis, and the result shows that 5 recombinant bacteria in total amplify target bands UD and AOXAlf, which indicates that the g1-pPIC9k vector has been inserted into the genome of the corresponding strain and is named as g1-9k-1#, g1-9k-2#, g1-9k-3#, g1-9k-5# and g1-9k-5#, wherein the electrophoresis result of g1-9k-1# is shown in FIG. 4. The empty vector transformed Pichia pastoris was named 9 k.

Example 2: activity assay

Inoculating positive recombinant bacterium g1-9k-1# and control 9k to 50ml YPD culture solution, and shake culturing at 28 ℃ and 200rpm overnight. After centrifugation at 400rpm for 2min to remove the supernatant, the cells were resuspended in 50ml of BMMY culture medium (1% yeast extract, 2% peptone, 100mM potassium phosphate pH6.0, 1.34% YNB, 4X 10-5% biotin, 0.5% methanol), cultured at 28 ℃ with 200rpm in a shaker, and induced by adding 250. mu.l of anhydrous methanol every 24 hours. After 96 hours of methanol-induced expression, the fermentation supernatant was collected by centrifugation at 12000rpm at 4 ℃ for 10min, filtered through a 0.22 μm filter, and then subjected to ultrafiltration using a Millipore 10K ultrafiltration membrane to displace the buffer, followed by concentration of 50ml of the fermentation broth to 5 ml.

1. Experiment of hydrolysis of phospholipids:

5% of soybean mixed phospholipid, lecithin and cephalin are used as substrates, and the reaction is carried out in a 1.5ml Eppendorf tube by the following reaction system: 200. mu.l of 5% phospholipid + 40. mu.l of 250mM salt solution + 100. mu.l of enzyme solution + 200. mu.l of 200mM pH8.5Tris-HCl buffer + 460. mu.l of water. The reaction condition is that shaking table oscillation is carried out at the temperature of 37 ℃ and the speed of 200rpm overnight, wherein, the positive system is enzyme liquid obtained by fermentation of g1-9k-1# bacteria, and the negative system is enzyme liquid obtained by fermentation of 9k bacteria. After the reaction was completed, 400. mu.l of n-hexane was added to the reaction tube, and the tube was placed on an inverted homogenizer to mix for 1 hour and centrifuged at 12000rpm at room temperature for 1 min. And taking a top n-hexane layer for thin layer chromatography analysis. Spot size 5 μ l, development conditions: chloroform: diethyl ether: glacial acetic acid 82:18: 2. spreading the high-efficiency thin layer plate in a chromatography tank for 30min, taking out, naturally drying, and fumigating in iodine tank to obtain the result shown in FIG. 5.

According to the result of fig. 5, the recombinant bacterial enzyme solution containing the target gene can release Free Fatty Acid (FFA) from mixed phospholipid and cephalin, calcium ions and zinc ions are beneficial to enzyme activity, and zinc ions are better.

2. pH test

The enzyme solutions of g1-9k-1# and g1-9k-2# were used for the experiments. The enzymatic hydrolysis was carried out in a 2ml Eppendorf tube, the reaction system was as follows: 200. mu.l 5% phospholipid + 40. mu.l 250mM saline solution + 100. mu.l enzyme solution + 200. mu.l buffer + 460. mu.l water; reacting enzyme liquid obtained by fermenting the two strains under the condition of pH5.5 (acetate); only 1# reacted at pH8.5 (Tris). Wherein the phospholipids used are soybean mixed phospholipid, lecithin and cephalin. The reaction conditions were shaking overnight at 28 ℃ on a shaker. After the reaction, 500. mu.l of n-hexane was added to the reaction tube, the mixture was inverted and mixed for 1 hour, and then centrifuged at 12000rpm for 2 minutes, and the upper n-hexane phase was removed to conduct thin layer chromatography. Sampling 5 mul of each sample, wherein the high-efficiency silica gel plate developing agent is as follows: chloroform: diethyl ether: acetic acid 82:18: 2.

After the sample is developed, the thin layer plate is taken out, dried at room temperature, placed in an iodine steam dye vat for fumigating and dyeing for 10min, and taken out for photographing. Fig. 6 shows the result of the thin layer development. Wherein the 51-calcium is hydrolysis system containing calcium under pH5.5 system, mixed phospholipid is used as substrate, and fermentation supernatant of 1# bacteria is used as enzyme solution. 52-calcium is a hydrolysis system containing calcium under a pH5.5 system, mixed phospholipid is used as a substrate, and fermentation supernatant of 2# bacteria is used as an enzyme solution. 81-calcium is a hydrolysis system containing calcium under a pH8.0 system, mixed phospholipid is used as a substrate, and fermentation supernatant of 1# bacterium is used as an enzyme solution. 51-calcium is a hydrolysis system containing calcium under a pH5.5 system, pure lecithin is used as a substrate, and fermentation supernatant of the 1# bacterium is used as an enzyme solution. 52-calcium is a hydrolysis system containing calcium under a pH5.5 system, pure lecithin is used as a substrate, and fermentation supernatant of 2# bacteria is used as an enzyme solution. 81-calcium is a hydrolysis system containing calcium under a pH8 system, pure lecithin is used as a substrate, and fermentation supernatant of the 1# bacterium is used as enzyme liquid. 51-calcium is a hydrolysis system containing magnesium under a system pH5.5, pure lecithin is used as a substrate, and fermentation supernatant of the 1# bacterium is used as an enzyme solution.

DAG is pure 1, 3-diglyceride.

51-magnesium is a hydrolysis system containing magnesium under a system pH5.5, mixed phospholipid is used as a substrate, and fermentation supernatant of the 1# bacterium is used as an enzyme solution. 52-magnesium is a hydrolysis system containing magnesium under a system pH5.5, mixed phospholipid is used as a substrate, and fermentation supernatant of the 2# bacterium is used as enzyme liquid. 81-magnesium is a hydrolysis system containing magnesium under a system pH of 8.0, mixed phospholipid is used as a substrate, and fermentation supernatant of the 1# bacterium is used as an enzyme solution. 51-brain zinc is a zinc-containing hydrolysis system under a system with pH5.5, pure cephalin is used as a substrate, and fermentation supernatant of the 1# bacterium is used as enzyme liquid. 52-brain zinc is a hydrolysis system containing calcium under a pH5.5 system, pure cephalin is used as a substrate, and fermentation supernatant of 2# bacteria is used as enzyme solution. 81-brain zinc is a hydrolysis system containing calcium under a pH8 system, pure cephalin is used as a substrate, and fermentation supernatant of the 1# bacterium is used as enzyme liquid. 51-brain calcium is a hydrolysis system containing calcium under a pH5.5 system, pure cephalin is used as a substrate, and fermentation supernatant of the strain No. 1 is used as enzyme solution. 52-brain calcium is a hydrolysis system containing calcium under a pH5.5 system, pure cephalin is used as a substrate, and fermentation supernatant of the 2# bacterium is used as enzyme liquid. 81-brain calcium is a hydrolysis system containing calcium under a pH8 system, pure cephalin is used as a substrate, and fermentation supernatant of the 1# bacterium is used as enzyme liquid. 5 minus is a zinc-containing hydrolysis system under a pH5.5 system, soybean mixed phospholipid is used as a substrate, and 9k bacteria are used for fermenting to obtain an enzyme solution. 8 negative is a zinc-containing hydrolysis system under a pH8 system, soybean mixed phospholipid is used as a substrate, and 9k bacteria are used for fermenting the obtained enzyme liquid. The foremost band of the lamellae is free fatty acid.

According to the results in FIG. 6, the hydrolysis of mixed phospholipids, the 81-calcium system, released free fatty acids in amounts significantly exceeding 51-calcium, indicating that the conditions of pH8 are more suitable: the hydrolysis effect of 81-calcium is better than that of 51-calcium, 52-calcium, 51-magnesium and 52-magnesium. The effect of 81-brain zinc is better than that of 51-brain zinc, 52-brain zinc, 51-brain calcium, 52-brain calcium and 81-brain calcium. The enzyme solutions obtained from neither the 1# nor the 2# bacteria were able to degrade lecithin. The results of treating the mixed phospholipid with the enzyme solution obtained by 9k bacteria fermentation are shown in the 5 th minus and the 8 th minus.

The reaction was carried out in a 1.5ml tube at pH8.5, and four enzymes of recombinant enzyme solution, control enzyme solution, PLA1 and PLA2 were used for comparison. And (3) developing a thin layer of the reacted extract under a polar condition, wherein the developing system is chloroform: methanol: ammonia water 65: 35: the results are shown in FIG. 7.

According to the polarity development results shown in FIG. 7, the enzyme solution obtained in the fermentation of # g1-9k-1 could not treat lecithin, but hydrolyzed cephalin. As a result of the hydrolysis, lysophosphatidylethanolamine remained in PLA2, but the enzyme and PLA1 did not exist.

The above results show that the recombinant yeast of the present invention, which can produce an enzyme capable of acting on phospholipids after methanol induction, requires calcium ion or zinc ion for its activity and can react under both acidic and alkaline conditions. The enzyme is PLA1 or PLB.

3. Titration

The enzyme activity was detected by titration. The reaction is enzyme solution obtained by expressing g1-9k bacteria, and the negative control is enzyme solution obtained by 9k recombinant bacteria. After overnight reaction, the pH was adjusted to 7.5 with 0.05M NaOH. The reaction system was 4ml of 5% mixed phospholipid + 800. mu.l of 250mM calcium or zinc salt + 5ml of enzyme solution + 9.8ml of water + 400. mu.l of 1M Tris-HCl buffer pH 7.5. The reaction conditions were temperature 40 ℃ shaking table 100rpm for 16 h.

The speed of the enzyme liquid releasing free fatty acid is as follows: 1.5 mu m/h.ml; 1.1 μm/h.ml of zinc system

4. Effects of other ions

Hydrolyzing a substrate by using a crude enzyme solution obtained by fermenting the strain g1-9k-1# under the ion conditions shown in the figure 8, and detecting the enzyme activity by TLC, wherein the hydrolysis system is as follows: 610. mu.l of water, 10mM of metal, 100. mu.l of 5% PC or 200. mu.l of 2.5% PE, 200. mu.l of enzyme solution, 50. mu.l of 1M Tris pH 7.5. A neutral developing system: chloroform: diethyl ether: the volume ratio of acetic acid was 82:18: 2. The ions are selected from sodium chloride, potassium chloride, ammonium sulfate, calcium chloride, zinc sulfate, copper sulfate, manganese chloride, nickel sulfate, cobalt chloride, and ferric chloride.

According to the results of FIG. 8, the enzyme solution did not have the activity of hydrolyzing lecithin in the presence of any salt ion. The enzyme produced by the recombinant bacterium is active in a calcium, magnesium, zinc, copper and manganese system by taking cephalin as a substrate. The mixed phospholipid is used as a substrate and is active in a calcium and zinc system.

Claims

1. The amino acid sequence of the phospholipase is shown in SEQ ID NO. 2.

2. A nucleic acid molecule selected from: (1) a nucleotide sequence encoding the phospholipase of claim 1 or a nucleotide sequence complementary to the nucleotide sequence of (1).

3. The nucleic acid molecule of claim 2, having the nucleotide sequence shown in SEQ ID NO 1.

4. A vector comprising the nucleic acid molecule of claim 2 or 3.

5. The vector of claim 4, further comprising a regulatory sequence that regulates expression of the nucleic acid molecule, wherein the nucleic acid molecule is operably linked to the regulatory sequence.

6. A host cell which is a microbial host cell comprising the nucleic acid molecule of claim 2 or 3, or the vector of claim 4 or 5.

7. The host cell of claim 6, which is a Pichia cell.

8. Use of the amino acid sequence of claim 1, the nucleic acid molecule of any one of claims 2 or 3, the vector of claim 4 or 5, or the host cell of claim 6 or 7 for the preparation of a phospholipase.

9. The method for enhancing the activity of the phospholipase of claim 1 comprising adding magnesium, calcium, copper, manganese or zinc ions to the enzyme preparation.

10. The method of claim 9, comprising adding magnesium sulfate, calcium chloride, copper sulfate, manganese chloride, or zinc sulfate to the enzyme preparation.