阅读说明：本技术 用于酶促油脱胶的方法 (Process for enzymatic oil degumming ) 是由 查莹 阿里恩·塞恩 威廉·比杰勒威尔德 于 2019-05-06 设计创作，主要内容包括：本发明涉及一种用于减少三酰基甘油酯油中完整磷脂的量的方法,所述方法包括将所述油与具有磷脂酶A1活性的多肽一起孵育,其中所述多肽包含与SEQ ID NO:1的成熟氨基酸序列具有至少80％同一性的多肽。(The present invention relates to a method for reducing the amount of intact phospholipids in triacylglycerol oils, comprising incubating the oil with a polypeptide having phospholipase a1 activity, wherein the polypeptide comprises a polypeptide having at least 80% identity to the mature amino acid sequence of SEQ ID NO: 1.)

1. A method for reducing the amount of intact phospholipids in triacylglycerol oils, comprising incubating the oil with a polypeptide having phospholipase a1 activity, wherein the polypeptide comprises a polypeptide having at least 80% identity to the mature amino acid sequence of SEQ ID NO: 1.

2. The method of claim 1, wherein the mature amino acid sequence of SEQ ID NO 1 comprises amino acids 30 to 298 of SEQ ID NO 1.

3. The method of claim 1 or 2, wherein at least 85% of the amount of intact phospholipid is reduced.

4. The method of claim 1 or 3, wherein the polypeptide is capable of reducing the amount of the intact phospholipid originally present in the oil by at least 85% when the phospholipase A1 is incubated with the oil in an amount of 0.28mg active protein/kg oil for 4 hours at a temperature of 55 ℃, 60 ℃, 65 ℃ and/or 70 ℃.

5. The method of any one of claims 1 to 4, wherein the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol and/or phosphatidylcholine.

6. The method of any one of claims 1 to 5, further comprising the step of adding an acid to the oil.

7. The method of any one of claims 1 to 6, further comprising the step of adding water to the oil.

8. The method of any one of claims 1 to 7, further comprising the step of adding caustic to the oil.

9. The method according to any one of claims 6 to 8, wherein the step of adding acid, water and/or caustic is performed before incubating the oil with the phospholipase.

10. The method of any one of claims 1-9, further comprising incubating the oil with a polypeptide having phospholipase C activity, a polypeptide having phosphatidylinositol phospholipase C activity, and/or a polypeptide having phospholipase a2 activity.

11. The method of any one of claims 1 to 10, further comprising separating a phosphorous-containing component from the oil.

12. The method of any one of claims 1 to 11, wherein the oil comprises crude oil or water degummed oil.

13. The method of any one of claims 1 to 12, wherein the oil comprises a vegetable oil, an algal oil, an animal oil, or a fish oil.

14. A triacylglycerol oil comprising a polypeptide having phospholipase a1 activity, wherein the polypeptide comprises a polypeptide having at least 80% identity to the mature amino acid sequence of SEQ ID No. 1.

15. The oil of claim 14, further comprising a polypeptide having phospholipase C activity, a polypeptide having phosphatidylinositol phospholipase C activity, and/or a polypeptide having phospholipase a2 activity.

Technical Field

The present invention relates to a method for reducing the amount of phospholipids in triacylglycerol oils using an enzyme having phospholipase a1 activity.

Background

Crude vegetable oils obtained from pressing or solvent extraction processes are complex mixtures of triacylglycerols, phospholipids, sterols, tocopherols, free fatty acids, trace metals, and other trace compounds. In soybean oil processing, soybean kernels can be first flaked and then subjected to hexane extraction to obtain flaked oil (flake oil). In another generally known process, the kernel is first treated with a swelling agent and then extracted, thereby producing a swelling agent oil. The latter tends to result in higher oil yields but also in higher phospholipid contents. During the preparation of other oils, such as canola oil or rapeseed oil, the kernel is first pressed, resulting in a pressed oil fraction. The press cake can be further treated with a solvent to produce an extracted oil fraction, and the two fractions combined are referred to as a crude oil of canola (canola), rapeseed or sunflower.

In order to produce high quality edible oils, it is desirable to remove phospholipids, free fatty acids and trace metals. The most common methods used industrially are water or wet degumming, acid degumming, caustic refining and enzymatic degumming or refining. Typically, removal of phospholipids produces most of the losses associated with vegetable oil degumming. Since most phospholipid molecules have both a hydrophilic functional group and a lipophilic segment consisting of glycerol with two fatty acid chains, the phospholipid molecules tend to be excellent natural emulsifiers. It is therefore desirable to hydrolyse phospholipids to their lyso-or phospho (glycerophosphate) form in order to reduce the emulsifying properties. The main phospholipids in vegetable oils are Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI) and Phosphatidic Acid (PA).

Various methods for enzymatic degumming or enzymatic refining of vegetable oils using enzymes having phospholipase activity (e.g. phospholipase a1, phospholipase a2, phospholipase C) or phosphatidylinositol phospholipase C activity are known.

WO9705219 discloses a process for reducing the content of phosphorus containing components in vegetable oils using a phospholipase mixture from Aspergillus niger (Aspergillus niger) comprising a phospholipase a2 and/or a phospholipase a1 activity and a lysophospholipase activity.

EP0575133 teaches phospholipase a1(PLA1) enzymes from Aspergillus oryzae (Aspergillus oryzae) and Aspergillus niger strains and the use of these phospholipases for the preparation of lysophospholipids from, for example, phospholipid substrates of animal, plant and microbial origin. EP0575133 discloses that PLA1 from aspergillus niger has only about thirty percent (30%) of the residual activity after temperature treatment at 70 ℃ compared to the residual activity after temperature treatment at 50 ℃ and 60 ℃. PLA1 of aspergillus oryzae did not show any activity after treatment at a temperature of 70 ℃. EP0575133 does not teach a process for enzymatic degumming of edible oils.

US20160289658 discloses a phospholipase derived from Talaromyces leycettanus. The phospholipase showed relatively higher thermostability and showed higher activity at 70 ℃ compared to the commercial enzyme preparation Lecitase Ultra.

WO2011/051322 discloses a phospholipase from Aspergillus fumigatus (Aspergillus fumigatus) which hydrolyses phospholipids in soybean oil at temperatures of 55 ℃ and 60 ℃.

There is a need for an improved method for reducing the content of phospholipids in triacylglycerol oils using a phospholipase active over a wide temperature range.

Disclosure of Invention

The present invention relates to a method for reducing the amount of intact phospholipids in triacylglycerol oils, comprising incubating the oil with a polypeptide having phospholipase a1 activity, wherein the phospholipase a1 comprises a polypeptide having at least 80% identity to the mature amino acid sequence of SEQ ID No. 1. It has been found that phospholipase a1 having at least 80% identity with the mature amino acid of SEQ ID No. 1 is capable of reducing at least 85% of the phospholipids originally present in triacylglycerol oils in 4 hours over a wide temperature range of 55 ℃ to 70 ℃. The phospholipase a1 in the methods as disclosed herein is a phospholipase a1 that hydrolyzes at least 85%, 86%, 87%, 88%, 89% or at least 90% of the amount of intact phospholipid originally present in triacylglycerol oil when incubated with oil at a temperature between 55 ℃, 60 ℃, 65 ℃ and 70 ℃ for 4 hours in an amount of 0.28mg active protein/kg oil.

Definition of

A "mature polypeptide" is defined herein as a polypeptide that is in its final form and that is obtained after translation of mRNA into a polypeptide and post-translational modification of the polypeptide. Post-translational modifications include N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and removal of leader sequences (such as signal peptides, propeptides, and/or prepropeptides) by cleavage.

Phospholipids are also denoted as glycerophospholipids. As used herein, a phospholipid is an "intact" phospholipid and comprises a glycerol backbone containing two fatty acids and phosphoric acid. Phospholipids are also represented as diacylglycerides comprising phosphate groups.

Lysophospholipids are glycerol backbones comprising only one acyl (fatty acid) group and a phosphate group. Lysophospholipids may be formed after removal of the acyl group from the phospholipid by the action of phospholipase a1 and/or phospholipase a 2.

The terms triacylglycerol oil and triglyceride oil are used interchangeably herein. Triglycerides are esters derived from glycerol and three fatty acids. The triacylglycerol oil may be an edible oil and/or an oil used as biodiesel.

Sequence identity or sequence homology are used interchangeably herein. To determine the percent sequence homology or sequence identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes. To optimize the alignment between the two sequences, gaps can be introduced in either of the two sequences being compared. Such alignments can be performed over the full length of the sequences being compared. Alternatively, the alignment may be performed over a shorter length, for example over about 20, about 50, about 100 or more nucleotides/base or amino acids. Sequence identity is the percentage of identical matches between two sequences over the reported alignment region. Percent sequence identity between two amino acid sequences or between two nucleotide sequences can be determined using the Needleman and Wunsch algorithms for alignment of two sequences (Needleman, S.B. and Wunsch, C.D. (1970) J.mol.biol.48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by algorithms. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For The purposes of The present invention, The NEEDLE program from The EMBOSS package (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P.Longden, I.and Bleasby, A.trends in Genetics 16, (6), p.276. page 277, http:// EMBOSS. bioinformatics. nl /) was used. For protein sequences, EBLOSUM62 was used for the substitution matrix. Optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that when different algorithms are used, all of these different parameters will produce slightly different results, but the overall percent identity of the two sequences will not change significantly. The percentage of sequence identity between the query sequence and the sequence of the invention after alignment by the program NEEDLE as described above was calculated as follows: the number of corresponding positions in the alignment (which show the same amino acid or the same nucleotide in both sequences) is divided by the total length of the alignment minus the total number of gaps in the alignment. Identity, as defined herein, can be obtained from needled by using the NOBRIEF option and is labeled as "longest identity" in the output of the program.

The protein sequences disclosed herein can further be used as "query sequences" to perform searches on public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al, (1990) J.mol.biol.215: 403-10. A BLAST nucleotide search can be performed with the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gap alignments for comparison purposes, Gapped BLAST can be used, as described in Altschul et al, (1997) Nucleic Acids Res.25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See homepage http:// www.ncbi.nlm.nih.gov/, of the National Center for Biotechnology Information.

The term "variant" may refer to a polypeptide or a nucleic acid. Variants include substitutions, insertions, deletions, truncations, transversions and/or inversions at one or more positions relative to a reference sequence. Variants can be generated by, for example, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, as well as various other recombinant methods known to those skilled in the art. Variant genes of nucleic acids can be artificially synthesized by techniques known in the art.

Drawings

FIG. 1 is a schematic representation of the pGBTOPPLA-1 plasmid used to express the A.niger PLA1 enzyme.

Examples

Materials and methods

Molecular biology techniques

The techniques of Molecular biology known to the skilled worker are carried out in accordance with Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition, CSHL Press, Cold Spring Harbor, NY, 2001. Polymerase Chain Reaction (PCR) was performed on a thermal cycler using Phusion high fidelity DNA polymerase (Finnzymes OY, aspeo, Finland) according to the manufacturer's instructions.

Enzyme

PLC/PI-PLC is an enzyme cocktail comprising phospholipase C (SEQ ID NO 3) and phosphatidylinositol phospholipase C (SEQ ID NO:4)A compound (I) is provided.3G, an enzyme mixture comprising phospholipase C (SEQ ID NO:3), phosphatidylinositol phospholipase C (SEQ ID NO:4) and phospholipase A2 (porcine pancreas PLA2), commercially available from DSM.

From NovozymesUltra, a phospholipase a from Fusarium oxysporum (Fusarium oxysporum), from Sigma Aldrich.

PL XTRA, a phospholipase A from Aspergillus fumigatus (Aspergillus fumigatus), was obtained from AB Enzymes.

Strain construction

A strain of Aspergillus niger comprising a deletion of the gene encoding glucoamylase (glaA) and a deletion of the pepA gene (deposited at the CBS institute under accession number CBS 513.88) was constructed according to the method as described in EP 0635574B 1 and van den Hombergh et al, (1997) Eur J biochem.247(2):605-13), respectively. Subsequently, an oxalate deficient A.niger strain was constructed from an A.niger strain comprising Δ glaA, Δ pepA according to the method as described in WO2004/070022, thereby producing A.niger comprising a deletion of the glaA, pepA and oahA genes (Δ glaA, Δ pepA, Δ oahA).

Construction of a PLA 1-producing Aspergillus niger Strain

The A.niger PLA1 enzyme (with the coding sequence shown in SEQ ID NO:2, and the protein sequence shown in SEQ ID NO: 1) was selected for enzyme expression in A.niger (Δ glaA,. DELTA.pepA,. DELTA.oahA) strains.

The gene encoding PLA1 was prepared by gene synthesis and cloned into an aspergillus niger pGBTOP-12 expression vector under the control of a glucoamylase promoter using the techniques described in WO98/46772 and WO99/32617, thereby producing an aspergillus niger pGBTOPPLA-1 expression vector using the same techniques as described in WO98/46772 and WO99/32617 (fig. 1).

An enzyme-producing strain for the PLA1 enzyme was constructed by co-transforming the A.niger (. DELTA.glaA,. DELTA.pepA,. DELTA.oahA) strain with the vectors pGBAAS-1 and pGBTOPLA-1 containing the amdS selectable marker gene and then selecting transformants. The transformation and counter-selection procedures (as described in WO98/46772 and WO 99/32617) followed by strain selection resulted in (multi-copy) strains producing PLA1 protein. From all transformants of pGBTOPPLA-1, 1 high copy enzyme-producing strain within a background of (. DELTA.glaA,. DELTA.pepA,. DELTA.oahA) was selected, further replica-plated to obtain a single strain inoculum and designated strain PLA 1-1. In subsequent experiments, the PLA1-1 strain was used as the corresponding PLA 1-producing strain.

Aspergillus niger shake flask fermentation for PLA1 production

Fresh A.niger PLA1-1 spores were prepared. Four of the 500ml shake flasks with baffles had 20ml fermentation Medium 1 (10% w/v corn steep liquor solids, 1% w/v glucose. H₂O, 0.1% w/v NaH₂PO₄.H₂O, 0.05% w/v MgSO₄.7H₂O, 0.025% w/v Basildon, pH 5.8) 100ml shake flask 10⁷And (4) spores. These precultures were incubated at 34 ℃ and 170rpm for 16-24 hours. From the preculture, 10-15ml were used to inoculate a medium 2 (15% w/v maltose, 6% w/v soy peptone, 1.5% w/v (NH) with 100ml fermentation medium at 34 ℃ and 170rpm₄)₂SO₄0.1% w/v NaH₂PO₄.H₂O, 0.1% w/v MgSO₄.7H₂O, 0.1% w/v L-arginine, 8% w/v Tween-80, 2% w/v Basildon, 2% w/v MES, pH 5.1). After 7 days of culture, the cells were killed by addition of 3.5g/l sodium benzoate and holding at 30 ℃ for six hours. Subsequently, 10g/l CaCl2 and 45g/l perlite C25 were added to the broth. The filtration was carried out in one step using filter cloth and filters DE60/EKS P and K250 (Pall). The filter cake remaining on the filter was washed with 1.1l of sterile milliQ water. Followed by sterile filtration using a 0.22m GP Express PLUS membrane (Millipore). A filtrate comprising PLA1 was used in the examples.

Phospholipase A1(PLA1) Activity assay

The following solutions were prepared:

1) substrate solution: 1g of L- α -phosphatidylcholine (Sigma P3556, Zwijndrecht, the Netherlands) from egg yolk in a 2% triton X-100 solution.

2)0.2M acetate buffer pH 4.5

3) Stop solution: 1M HCl

A mixture of 500. mu.L of solution 1 and 300. mu.L of solution 2 was equilibrated at 37 ℃. The reaction was started by adding 100. mu.L of an enzyme solution with an activity between 0.05-1.0U/mL. After incubation at 37 ℃ for 10 min, the reaction was stopped by adding 100. mu.L of solution 3. Blank measurements were additionally performed by incubating the substrate without sample at 37 ℃ for 10 minutes. After addition of 100. mu.L of stop reagent, 100. mu.L of sample was added. The amount of free fatty acids formed in the samples and blanks was determined by following the instructions described in the package insert of the Wako HR series NEFA-HR (2) diagnostic kit (http:// www.wakodiagnostics.com/r _ NEFA. html). The activity was calculated as follows:

Δ FFA ═ FFA in sample-FFA in blank (μmol/mL)

Vt total volume after reaction stop (1mL)

Vs sample volume (0.1mL)

time t ═ incubation time (10 minutes)

df is the dilution factor of the sample

1U is defined as the amount of enzyme that releases one micromole of free fatty acid per minute under the conditions tested.

Phospholipase C (PLC) Activity assay

The substrate solution was composed of 10mM pNP-nitrophenylphosphorylcholine (product N83020 from Melford Laboratories Ltd, Ipswich, United Kingdom), 100mM MOPS buffer pH 7.3, 0.2% Triton X-100 and 1mM ZnSO₄And (4) forming. A mixture of 40. mu.L of sample (activity between 0.03-0.1U/mL) and 960. mu.L of substrate solution was incubated at 37 ℃For 30 minutes. The reaction was stopped by adding 1000. mu.L of a stop reagent containing 1M TRIS and 50mM EDTA adjusted to pH 10 with 0.5M NaOH. Blanks were made by adding a stop reagent before the enzyme sample. The Optical Density (OD) of the sample and blank was measured at 405 nm.

Calibration was performed by preparing pNP solutions in the above buffers at 0-0.5-1.0-2.0-2.9-4.0mM, respectively. 40 μ L of each standard solution was mixed with 960 μ L of substrate and 1000 μ L of stop reagent. The OD of each solution was measured at 405 nm. The slope of the calibration line is calculated by using linear regression.

The activity was calculated by using the following formula:

Δ Abs ═ a blank (a sample-a)

df is the dilution factor of the sample

Slope of p-nitrophenol calibration curve (mL/. mu. mol)

Measurement of time for incubation (30 min)

One unit is defined as the amount of enzyme that releases 1. mu. mol p-nitrophenol per minute under the test conditions (pH 7.3, 37 ℃).

Phosphatidylinositol phospholipase (PI-PLC) activity assay

The substrate solution consisted of 20mM 4-methylumbelliferyl inositol-1-phosphate, N-methyl-morpholine (BioSynth M-5717, Brussels, Belgium) dissolved in 200mM sodium phosphate buffer pH 7.5 also containing 0.1% Triton X-100. 140 μ L of substrate was equilibrated at 37 ℃. The reaction was started by adding 10. mu.L of a sample with an activity between 0.2U/mL and 1.0U/mL. The change in absorbance relative to the blank sample was measured at 380nm when incubated at 37 ℃. The slope of the linear part of the curve (Δ OD/time) was used as a measure of activity.

Calibration was performed by preparing 4-methylumbelliferone solutions in 200mM phosphate buffer, 0-1.0-2.0-3.0-4.0-5.0mM each. mu.L of each standard solution was mixed with 140. mu.L of substrate. The OD of each solution was measured at 380 nm. The slope of the calibration line is calculated by using linear regression.

The activity was calculated by using the following formula: U/mL ═ (. DELTA.abs/min)_{Sample (I)}-ΔAbs/min_{Blank space})×df/S

ΔAbs/min_{Sample (I)}Absorbance change per minute ═ of sample

Δ Abs/min blank ═ absorbance change per minute for buffer blank

df is the dilution factor of the sample

Slope of the calibration curve [ mL/micromole ] for S-4-methylumbelliferone

One unit is defined as the amount of enzyme that liberates 1. mu. mol of 4-methylumbelliferone from 18.7mM 4-methylumbelliferyl inositol-1-phosphate over a1 minute period at pH 7.5 and 37 ℃.

Use of³¹P NMR quantitative determination of phospholipid, lysophospholipid and glycerophosphate content

Accurately weigh 500-1000mg of oil into a suitable vial and add about 10g of cold acetone and mix well. The oil-acetone mixture was kept at 4 ℃ for at least 30 minutes and then centrifuged at 3000rpm for 10 minutes, after which the liquid phase was discarded. The pellet was resuspended in 500. mu.l buffer (containing 25g L-1 deoxycholic acid, 5.84g L-1EDTA, and 10.9g L-1TRIS, buffered to pH 9.0 using KOH) and 50. mu.L of internal standard solution (containing 10g L-1 triisopropyl phosphate in extraction buffer) was added.

Record 1D P on a Bruker Avance III HD spectrometer at a sample temperature of 300K³¹NMR spectroscopy, the Bruker Avance III HD spectrometer operating at a frequency of 31P of 161.97MHz was equipped with a nitrogen-cooled ultra-low temperature probe. Using the inverse gated pulse program with Waltz16 proton decoupling (ZGIG), 4 virtual scans and 128 scans of each spectrum were recorded using 90 degree pulses. An acquisition time of 3.37s, and a relaxation delay time of 11.5s were used.

The analyte concentration was calculated relative to triisopropyl phosphate.

Correction factors are applied to correct for incomplete relaxation of phosphorylcholine and phosphoethanolamine.

Quantitative determination of Diacylglycerols (DAG) in oil

The neutral lipid classes were separated using normal-phase High Performance Liquid Chromatography (HPLC) and the presence of diglycerides was determined using an Evaporative Light Scattering Detector (ELSD). The method has been improved from the official AOCS method Cd 11 d-96. The contents are expressed as percentages (wt%).

Example 1 degumming of crude Soybean oil and rapeseed oil at 55-70 ℃ Using PLA1

Two soybean crudes from american oilseed processors and two rapeseed crudes from european oilseed processors were used. 10g of oil was weighed into a vial and heated to 70 ℃, after which citric acid (as a 50% solution) was added to the oil until the final citric acid concentration in the oil was 500 ppm. The vial containing the citric acid-treated oil was incubated at 70 ℃ for at least 30 minutes.

After incubation, the temperature was adjusted and maintained at 55 ℃, 60 ℃, 65 ℃ and 70 ℃. When the temperature was stable, PLA1 produced as described above (0.28mg active protein/kg oil (1.25PLA units/g)) and water were mixed into the oil using an Ultra Turrax. The final water concentration in the oil was 3 wt%. The reaction was continued for 4 hours while the oil was kept mixed at 800ppm with a magnetic stirrer. Samples were taken after 4 hours of incubation and used as described above³¹P-NMR analyses for Phospholipid (PL) content, i.e., Phosphatidic Acid (PA), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), and Phosphatidylinositol (PI).

The results in tables 1 to 4 below show that PLA1 as disclosed herein reduced the content of all four phospholipids, PA, PC, PE and PI. More than 85% of the total amount of Phospholipids (PL) originally present in the oil was hydrolyzed after 4 hours reaction at temperatures of 55 deg.C, 60 deg.C, 65 deg.C and 70 deg.C.

Table 1 phospholipid content in soy crude oil before and after incubation with phospholipase a 1.

Table 2 phospholipid content in soy crude oil before and after incubation with phospholipase a 1.

Table 3 phospholipid content in rapeseed crude oil before and after incubation with phospholipase a 1.

Table 4 phospholipid content in rapeseed crude oil before and after incubation with phospholipase a 1.

Example 2 comparison of the Performance of PLA1 and commercial phospholipase A at 55 deg.C

Soybean crude oil and water degummed soybean oil from american oilseed processors were used. 10g of oil was weighed into a vial and heated to 70 ℃, after which citric acid (as a 50% solution) was added to the oil until the final citric acid concentration in the oil was 500 ppm. The vials containing the citric acid-treated oil were incubated at 70 ℃ for at least 30 minutes, after which the temperature was adjusted and maintained at 55 ℃.

When the temperature was stable, 25ppm of PLA1 produced as described above and water were mixed into the oil using an Ultra Turrax. The final water concentration in the oil was 3 wt%.

After treatment with citric acid and adjustment of the oil to 55 ℃ as described aboveUltra phospholipase A andbefore PL XTRA phospholipase a was incubated together, 2M NaOH was first added to the oil until a final concentration of 138ppm NaOH in the oil was reached. 25ppm of commercial enzyme and water were then mixed into the oil using an Ultra Turrax, the final in oilThe water concentration was 3 wt%.

After incubation at 55 ℃ for 4 hours while mixing at 800ppm using a magnetic stirrer, samples were taken and used as described above³¹P-NMR measures phospholipid content (i.e., Phosphatidic Acid (PA), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), and Phosphatidylinositol (PI)), lysophospholipid content (i.e., LysoPA (LPA), LysoPC (LPC), LysoPE (LPE), and LysoPI (LPI)), and glycerophosphate content (i.e., Glycerol PA (GPA), Glycerol PC (GPC), Glycerol PE (GPE), and Glycerol PI (GPI)).

The results in tables 5 and 6 show that the enzyme is compatible with two commercial enzymesUltra andPL XTRA reached lower levels of phospholipids PA, PC, PE and PI after 4 hours of reaction in soybean crude oil and water degummed soybean oil with a dose of 25ppm at 55 ℃ compared to PLA1 as disclosed herein under their optimal reaction conditions. Furthermore, the results in tables 5 and 6 indicate that PLA1 as disclosed herein shows lysophospholipase activity and is compatible with two commercial enzymesUltra andPL XTRA converted all 4 lysophospholipids to glycerophosphate esters at 55 ℃ in higher amounts in both the soybean crude oil and the water degummed soybean oil. Since glycerophosphate has a lower emulsifying power than lysophospholipid, conversion of lysophospholipid to glycerophosphate can improve the efficiency of oil-gel separation in the centrifugation step.

TABLE 5 content of phospholipids, lysophospholipids and glycerophosphates in soybean crude oil before and after 4 hours incubation with different phospholipases at 55 ℃

TABLE 6 content of phospholipids, lysophospholipids and glycerophosphates in water degummed Soybean oil before and after 4 hours incubation with different phospholipases at 55 ℃

Example 3 comparison of Performance between PLA1 and commercially available phospholipase A in crude Soybean oil at temperatures of 55 deg.C, 60 deg.C, and 65 deg.C

Soy crude oil from american oilseed processors was used. 10g of oil was weighed into a vial and heated to 70 ℃, after which citric acid (as a 50% solution) was added to the oil until the final citric acid concentration in the oil was 500 ppm. The vial containing the citric acid-treated oil was incubated at 70 ℃ for at least 30 minutes.

After incubation, the temperature was adjusted and maintained at 55 ℃, 60 ℃ and 65 ℃. When the temperature was stable, PLA1 produced as described above (0.25mg active protein/kg oil) and water were mixed into the oil using an Ultra Turrax. The final water concentration in the oil was 3 wt%.

To be andultra andPL XTRA was compared by first adding 2M NaOH to the citric acid treated oil until the final concentration of NaOH in the oil reached 138ppm to obtain optimal conditions for these enzymes. Subsequently, using an Ultra Turrax willUltra orPL XTRA (0.25mg active protein/kg oil) and water were mixed into the oil. The final water concentration in the oil was 3 wt%.

The reaction was continued for 4 hours while the oil was kept mixed at 800ppm with a magnetic stirrer. Samples were taken after 4 hours of incubation and used as described above³¹P-NMR analyses for phospholipid content, i.e., Phosphatidic Acid (PA), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), and Phosphatidylinositol (PI).

The results in tables 7 to 9 below show that the enzyme is compatible with commercial enzymesUltra andPL XTRA reached lower levels of phospholipids PA, PC, PE and PI after 4 hours of reaction in soybean crude oil at 55 ℃, 60 ℃, 65 ℃ as disclosed herein compared to aspergillus niger PLA1 under their optimal reaction conditions.

TABLE 7 phospholipid content in crude Soybean oil before and after 4 hours incubation with different phospholipases

<: not detected

TABLE 8 phospholipid content in crude Soybean oil before and after 4 hours incubation with different phospholipases

<: not detected

TABLE 9 phospholipid content in crude Soybean oil before and after 4 hours incubation with different phospholipases

Example 4 comparison of the Performance of PLA1 and commercially available phospholipase A in Water degummed Soybean oil at temperatures of 55 deg.C, 60 deg.C, and 65 deg.C

Water degummed soybean oil from american oilseed processors was used. 10g of oil was weighed into a vial and heated to 70 ℃, after which citric acid (as a 50% solution) was added to the oil until the final citric acid concentration in the oil was 500 ppm. The vial containing the citric acid-treated oil was incubated at 70 ℃ for at least 30 minutes.

After incubation, the temperature was adjusted and maintained at 55 ℃, 60 ℃ and 65 ℃. When the temperature was stable, PLA1 produced as described above (0.25mg active protein/kg oil) and water were mixed into the oil using an Ultra Turrax. The final water concentration in the oil was 3 wt%.

To be andultra andPL XTRA was compared by first adding 2M NaOH to the citric acid treated oil until the final concentration of NaOH in the oil reached 138ppm to obtain optimal conditions for these enzymes. Subsequently, using an Ultra Turrax willUltra orPL XTRA (0.25mg active protein/kg oil) and water were mixed into the oil. The final water concentration in the oil was 3 wt%.

The reaction was continued for 4 hours while the oil was kept mixed at 800ppm with a magnetic stirrer. Samples were taken after 4 hours of incubation and used as described above³¹P-NMR measures the phospholipid content, i.e., Phosphatidic Acid (PA), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), and Phosphatidylinositol (PI).

The results in tables 10 to 12 show that in PLA1,Ultra、Under the optimal reaction conditions of the three enzymes PL XTRA and the commercial enzymeUltra compared to PLA1 as disclosed herein, lower levels of phospholipids PA, PC, PE and PI were achieved in water degummed soybean oil after 4 hours of reaction at 55-65 ℃, whereas PLA1 reachedPL XTRA also resulted in low phospholipid levels at 60 ℃ and 65 ℃.

TABLE 10 phospholipid content in water degummed Soybean oil before and after 4 hours incubation with different phospholipases

<: not detected

TABLE 11 phospholipid content in water degummed Soybean oil before and after 4 hours incubation with different phospholipases

<: not detected

TABLE 12 phospholipid content in water degummed Soybean oil before and after 4 hours incubation with different phospholipases

<: not detected

Example 5: degumming using a PLC/PIPLC and PLA1 combination

The soy crude and rapeseed crude disclosed in example 1 were used. 10g of oil was weighed into a vial, which was heated to 70 ℃. For pretreatment, citric acid (50% solution) was added to the oil at a final citric acid concentration of 500ppm in the oil. The oil was incubated at 70 ℃ for at least 30 minutes, after which NaOH was added to a concentration of 138ppm NaOH. After pretreatment, the temperature was adjusted and maintained at 55 ℃.

The mixture of PLA1 and Purifine PLC/PI-PLC produced as disclosed above was added to the oil with water and mixed using an Ultra Turrax. The dose of PLA1 was 0.28mg active protein/kg oil and the dose of Purifine PLC/PI-PLC was 0.013U-PLC/g oil and 0.05U-PI-PLC/g oil, with a final water concentration of 3% by weight in the oil.

The reaction was carried out for 4 hours while the oil was kept mixed with a magnetic stirrer at 800 rpm. After incubation at 55 ℃ for 2 and 4 hours, samples were taken to determine the phospholipid content, i.e. Phosphatidic Acid (PA), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE) and Phosphatidylinositol (PI), and used³¹P-NMR analysis of the reaction product; diacylglycerol (DAG) was analyzed using HPLC.

The results in tables 13 and 14 show that PLA1 can be used at the same time as PLC/PI-PLC to further reduce the intact phospholipid content compared to PLC/PI-PLC alone. After 4 hours of PLA1+ PLC/PI-PLC reaction, > 90% of the phospholipids were hydrolyzed. When PLA1 was used simultaneously with PLC/PI-PLC, the formation of Diglyceride (DAG) was reduced, but without pretreatment, the reduction was limited to 5-15% of the total DAG formed.

TABLE 13 phospholipid content in Soy crude before and after incubation with phospholipase C, PI-PLC and phospholipase A1 at 55 deg.C

Is less than or equal to: not detected

TABLE 14 phospholipid content in rapeseed crude oil before and after incubation with phospholipase C and phospholipase A1 at 55 deg.C

Is less than or equal to: not detected

Example 6: use of Refining 3G and PLA1

Two soybean crudes from american oilseed processors were used. 10g of oil was weighed into a vial, which was heated to 60 ℃.

Will be provided with3G was added to the oil together with or in turn with water, PLA1 from Aspergillus niger produced as disclosed above. After addition of the enzyme, the oil mixture was mixed using an Ultra Turrax. When in useWhen 3G and PLA1 were added together, no pretreatment was performed. When in useWhen 3G and PLA1 are added sequentially, first, the mixture is mixed3G incubation for 2 hours, after which 500ppm citric acid was added, followed by PLA 1.

The dose of 3G was 0.013U-PLC/G oil and 0.05U-PI-PLC/G oil (150-. The final water concentration in the oil was 3 wt%.

The reaction was carried out for 4 hours while the oil was kept mixed with a magnetic stirrer at 800 rpm. After 2 and 4 hours of incubation at 60 ℃, samples were taken to determine the Phospholipid (PL) content, i.e. Phosphatidic Acid (PA), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE) and Phosphatidylinositol (PI), and used as disclosed above³¹The reaction product was analyzed by P-NMR. Diacylglycerol (DAG) was analyzed using HPLC as disclosed above.

The results in tables 15 and 16 show that the use of the individual compounds3G compared with the original standard when comparingWhen 3G was used in combination with PLA1, higher amounts of phospholipids were hydrolyzed. In some cases (e.g., soybean crude C), no pretreatment is required to bring the combination to low phosphorus levels, while in some other cases (e.g., soybean crude D), an acid needs to be added before PLA1 to achieve low phosphorus levels. When PLA1 is mixed withWhen 3G is used in combination, the formation of Diglycerides (DAG) is reduced, i.e. with only 3GThe total DAG formed after 3G incubation was reduced by 5-15% compared to DAG.

TABLE 15 reaction at 60 ℃ withPhospholipid content in soy crude C before and after 3G and PLA1 were incubated together. No pre-treatment was applied.

<: not detected

TABLE 16 reaction of at 60 deg.CPhospholipid content in soy crude D before and after incubation of 3G with PLA1(0.28mg active protein/kg oil in all experiments).

<: not detected

Instructions relating to the deposited microorganisms or other biological material

(PCT Rule 13bis)

Form PCT/RO/134(1998 month 7; 2004 month 1 reprint)

Sequence listing

<110> DSM IP Assets B.V.

<120> Process for enzymatic oil degumming

<130> 32910-WO-PCT

<150> EP18171015.3

<151> 2018-05-07

<160> 4

<170> BiSSAP 1.3.6

<210> 1

<211> 298

<212> PRT

<213> Aspergillus niger (Aspergillus niger)

<220>

<223> amino acid sequence of phospholipase A1

<400> 1

Met Phe Leu Arg Arg Glu Phe Gly Ala Val Ala Ala Leu Ser Val Leu

1 5 10 15

Ala His Ala Ala Pro Ala Pro Ala Pro Met Gln Arg Arg Asp Ile Ser

20 25 30

Ser Thr Val Leu Asp Asn Ile Asp Leu Phe Ala Gln Tyr Ser Ala Ala

35 40 45

Ala Tyr Cys Ser Ser Asn Ile Glu Ser Thr Gly Thr Thr Leu Thr Cys

50 55 60

Asp Val Gly Asn Cys Pro Leu Val Glu Ala Ala Gly Ala Thr Thr Ile

65 70 75 80

Asp Glu Phe Asp Asp Ser Ser Ser Tyr Gly Asp Pro Thr Gly Phe Ile

85 90 95

Ala Val Asp Pro Thr Asn Glu Leu Ile Val Leu Ser Phe Arg Gly Ser

100 105 110

Ser Asp Leu Ser Asn Trp Ile Ala Asp Leu Asp Phe Gly Leu Thr Ser

115 120 125

Val Ser Ser Ile Cys Asp Gly Cys Glu Met His Lys Gly Phe Tyr Glu

130 135 140

Ala Trp Glu Val Ile Ala Asp Thr Ile Thr Ser Lys Val Glu Ala Ala

145 150 155 160

Val Ser Ser Tyr Pro Asp Tyr Thr Leu Val Phe Thr Gly His Ser Tyr

165 170 175

Gly Ala Ala Leu Ala Ala Val Ala Ala Thr Val Leu Arg Asn Ala Gly

180 185 190

Tyr Thr Leu Asp Leu Tyr Asn Phe Gly Gln Pro Arg Ile Gly Asn Leu

195 200 205

Ala Leu Ala Asp Tyr Ile Thr Asp Gln Asn Met Gly Ser Asn Tyr Arg

210 215 220

Val Thr His Thr Asp Asp Ile Val Pro Lys Leu Pro Pro Glu Leu Leu

225 230 235 240

Gly Tyr His His Phe Ser Pro Glu Tyr Trp Ile Thr Ser Gly Asn Asp

245 250 255

Val Thr Val Thr Thr Ser Asp Val Thr Glu Val Val Gly Val Asp Ser

260 265 270

Thr Ala Gly Asn Asp Gly Thr Leu Leu Asp Ser Thr Thr Ala His Arg

275 280 285

Trp Tyr Thr Ile Tyr Ile Ser Glu Cys Ser

290 295

<210> 2

<211> 897

<212> DNA

<213> Aspergillus niger (Aspergillus niger)

<220>

<223> DNA sequence of phospholipase A1 (GenBank: XM _ 001393495)

<400> 2

atgtttctcc gcagggaatt tggggctgtt gcagccctat ctgtgctggc ccatgctgct 60

cccgcacctg ctccgatgca gcgtagagac atctcctcta ccgtcttgga caatatcgac 120

ctcttcgccc aatacagtgc agcagcttac tgctcctcca acatcgagtc caccggcacg 180

actctgacct gcgacgtagg caattgccct ctcgtcgagg cagccggtgc cacgaccatc 240

gatgagtttg acgacagcag cagctacggc gacccgacgg ggttcatcgc cgttgacccg 300

acgaacgagt taatcgttct gtctttccgg ggcagttccg acctctcgaa ctggattgcc 360

gacctagact tcggcctcac atccgtaagc agcatctgtg atggctgtga gatgcacaag 420

ggcttctacg aggcctggga agtcattgcc gacaccatca catccaaggt ggaggccgcc 480

gtctccagct atccggacta caccctcgtg ttcaccggac acagctacgg cgctgcattg 540

gcggctgtcg cggccaccgt gctccgcaac gccggataca ctcttgacct gtacaacttc 600

ggccagcccc gtattggcaa cctcgcctta gccgactaca tcaccgacca aaacatgggc 660

agcaactacc gcgtcacgca caccgatgac atcgtgccta agctgcctcc ggagctgctg 720

ggctaccacc acttcagtcc ggagtactgg atcaccagcg gcaatgatgt gacggtgaca 780

acgtcggacg tcaccgaggt cgtgggggtg gattcgacgg ctgggaatga cggcacgctg 840

cttgacagta cgactgccca tcggtggtac acgatctaca ttagtgaatg ctcgtag 897

<210> 3

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> phospholipase C from WO2005/086900

<400> 3

Trp Ser Ala Glu Asp Lys His Asn Glu Gly Ile Asn Ser His Leu Trp

1 5 10 15

Ile Val Asn Arg Ala Ile Asp Ile Met Ser Arg Asn Thr Thr Ile Val

20 25 30

Asn Pro Asn Glu Thr Ala Leu Leu Asn Glu Trp Arg Ala Asp Leu Glu

35 40 45

Asn Gly Ile Tyr Ser Ala Asp Tyr Glu Asn Pro Tyr Tyr Asp Asp Ser

50 55 60

Thr Tyr Ala Ser His Phe Tyr Asp Pro Asp Thr Gly Thr Thr Tyr Ile

65 70 75 80

Pro Phe Ala Lys His Ala Lys Glu Thr Gly Ala Lys Tyr Phe Asn Leu

85 90 95

Ala Gly Gln Ala Tyr Gln Asn Gln Asp Met Gln Gln Ala Phe Phe Tyr

100 105 110

Leu Gly Leu Ser Leu His Tyr Leu Gly Asp Val Asn Gln Pro Met His

115 120 125

Ala Ala Ser Phe Thr Asp Leu Ser Tyr Pro Met Gly Phe His Ser Lys

130 135 140

Tyr Glu Asn Phe Val Asp Thr Ile Lys Asn Asn Tyr Ile Val Ser Asp

145 150 155 160

Ser Asn Gly Tyr Trp Asn Trp Lys Gly Ala Asn Pro Glu Asp Trp Ile

165 170 175

Glu Gly Ala Ala Val Ala Ala Lys Gln Asp Tyr Pro Gly Val Val Asn

180 185 190

Asp Thr Thr Lys Asp Trp Phe Val Lys Ala Ala Val Ser Gln Glu Tyr

195 200 205

Ala Asp Lys Trp Arg Ala Glu Val Thr Pro Val Thr Gly Lys Arg Leu

210 215 220

Met Glu Ala Gln Arg Val Thr Ala Gly Tyr Ile His Leu Trp Phe Asp

225 230 235 240

Thr Tyr Val Asn Arg

245

<210> 4

<211> 298

<212> PRT

<213> Artificial sequence

<220>

<223> phosphatidylinositol phospholipase C from WO2011/046812

<400> 4

Met Ala Ser Ser Ile Asn Val Leu Glu Asn Trp Ser Arg Trp Met Lys

1 5 10 15

Pro Ile Asn Asp Asp Ile Pro Leu Ala Arg Ile Ser Ile Pro Gly Thr

20 25 30

His Asp Ser Gly Thr Phe Lys Leu Gln Asn Pro Ile Lys Gln Val Trp

35 40 45

Gly Met Thr Gln Glu Tyr Asp Phe Arg Tyr Gln Met Asp His Gly Ala

50 55 60

Arg Ile Phe Asp Ile Arg Gly Arg Leu Thr Asp Asp Asn Thr Ile Val

65 70 75 80

Leu His His Gly Pro Leu Tyr Leu Tyr Val Thr Leu His Glu Phe Ile

85 90 95

Asn Glu Ala Lys Gln Phe Leu Lys Asp Asn Pro Ser Glu Thr Ile Ile

100 105 110

Met Ser Leu Lys Lys Glu Tyr Glu Asp Met Lys Gly Ala Glu Ser Ser

115 120 125

Phe Ser Ser Thr Phe Glu Lys Asn Tyr Phe Arg Asp Pro Ile Phe Leu

130 135 140

Lys Thr Glu Gly Asn Ile Lys Leu Gly Asp Ala Arg Gly Lys Ile Val

145 150 155 160

Leu Leu Lys Arg Tyr Ser Gly Ser Asn Glu Ser Gly Gly Tyr Asn Phe

165 170 175

Phe Tyr Trp Pro Asp Asn Glu Thr Phe Thr Ser Thr Ile Asn Gly Asn

180 185 190

Val Asn Val Thr Val Gln Asp Lys Tyr Lys Val Ser Leu Asp Glu Lys

195 200 205

Ile Asn Ala Ile Lys Asp Thr Leu Asn Glu Thr Ile Asn Asn Ser Glu

210 215 220

Asp Val Asn His Leu Tyr Ile Asn Phe Thr Ser Leu Ser Ser Gly Gly

225 230 235 240

Thr Ala Trp Thr Ser Pro Tyr Tyr Tyr Ala Ser Arg Ile Asn Pro Glu

245 250 255

Ile Ala Asn Tyr Ile Lys Gln Lys Asn Pro Thr Arg Val Gly Trp Ile

260 265 270

Ile Gln Asp Phe Ile Asn Glu Lys Trp His Pro Leu Leu Tyr Gln Glu

275 280 285

Val Ile Asn Ala Asn Lys Ser Leu Val Lys

290 295

Detailed Description

The present invention relates to a method for reducing the amount of intact phospholipids in triacylglycerol oils, comprising incubating the oil with a polypeptide having phospholipase a1 activity, wherein the polypeptide comprises a polypeptide having at least 80% identity to the mature amino acid sequence of SEQ ID NO: 1. A polypeptide having a phospholipase activity with at least 80% identity to the mature amino acid sequence of SEQ ID No. 1 can be, for example, a polypeptide that is capable of reducing or reducing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or at least 90% of the phospholipids originally present in the oil when phospholipase a1 is incubated with the oil at a temperature of 55 ℃, 60 ℃, 65 ℃, and/or 70 ℃ for 4 hours in an amount of 0.28mg active protein/kg of oil in a method as disclosed herein.

The polypeptide having phospholipase a1 activity in the method for reducing the amount of intact phospholipids in triacylglycerol oil as disclosed herein can be a polypeptide that is capable of reducing or reducing at least 80%, 81%, 82%, 83%, 84%, or at least 85% of the phospholipids originally present in the oil when phospholipase a1 is incubated with the oil at a temperature of 55 ℃, 60 ℃, 65 ℃, and/or 70 ℃ for 4 hours in an amount of 0.1-0.4mg, e.g., 0.2-0.3mg or 0.28mg of active protein per kg of oil. The oil may also contain 500ppm citric acid and 3 wt% water.

Surprisingly, it was found that a polypeptide having phospholipase a1 activity in a method as disclosed herein is capable of reducing or reducing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or at least 90%, such as 85% to 99%, such as 86% to 98%, such as 87% to 97%, such as 88% to 96%, such as between 89% and 95%, such as between 90% and 94% of the phospholipids originally present in the oil over a wide temperature range between 55 ℃ and 70 ℃. The phospholipase a1 as disclosed herein can be incubated with the oil during a period of 4 hours at a temperature range of 55 ℃ to 70 ℃.

In one embodiment, the method for reducing the amount of intact phospholipids as disclosed herein is a method wherein the amount of intact phospholipids reduced is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or at least 90%, such as 85% to 99%, such as 86% to 98%, such as 87% to 97%, such as 88% to 96%, such as 89% to 95%, such as 90% to 94% of the amount of intact phospholipids originally present in the oil. Surprisingly, it was found that the amount of intact phospholipids decreased after incubating the oil with phospholipase a1 in an amount of 0.1mg active protein/kg oil to 0.4mg active protein/kg oil (e.g. 0.28mg active protein/kg oil) for 4 hours at a temperature of 55 ℃, 60 ℃, 65 ℃ and/or at 70 ℃.

Phosphorus components, such as phospholipids, lysophospholipids and phosphates, can be used, for example, as disclosed in the materials and methods section³¹P-NMR and/or HPLC.

The polypeptide having phospholipase a1 activity as used herein is phospholipase a1 according to enzyme classification e.c. 3.1.1.32. Phospholipase a1 is an enzyme that cleaves SN1 position of phospholipids to form lysophospholipids and fatty acids. Phospholipase a1 as used herein can also cleave lysophospholipids at position SN1, forming glycerophosphate and fatty acids. The phrases "phospholipase a 1" and "polypeptide having phospholipase a1 activity" are used interchangeably herein. The polypeptide having a phospholipase a1 activity as disclosed herein does not have a phospholipase a2 activity.

Incubating the oil with a polypeptide having phospholipase a1 activity in a method as disclosed herein comprises converting phospholipids in the oil to lysophospholipids and free fatty acids. Incubating the oil with a polypeptide having phospholipase a1 activity in a method as disclosed herein can further comprise converting phospholipids and/or lysophospholipids in the oil to lysophospholipids and/or glycerophosphates and free fatty acids.

Incubating the oil with the polypeptide having phospholipase a1 activity in the method as disclosed herein can be performed at a pH of 2 to 8, e.g., 3 to 7, e.g., 4 to 6.

Incubating the oil with the polypeptide having phospholipase a1 activity can be performed in the presence of an acid. Thus, a method as disclosed herein for reducing the amount of intact phospholipids in triacylglycerol oils comprises adding acid such that the amount of acid in the oil is from 100ppm to 1000ppm acid, for example from 200ppm to 900ppm acid, for example from 300ppm to 800ppm acid, for example from 400ppm to 600ppm acid. Suitable acids for use in the methods as disclosed herein may include citric acid, phosphoric acid, acetic acid, tartaric acid, and/or succinic acid, and any suitable mixtures thereof.

Water is typically present in the process as disclosed herein. The method as disclosed herein may further comprise adding water to the oil, for example adding water in an amount such that water is present in the oil in an amount of 0.5 to 5 wt.%, for example in an amount of 1 to 4 wt.%, for example 2 to 3 wt.%.

The incubation of the oil with the polypeptide having phospholipase a1 activity can be performed at a temperature of 40 ℃ to 75 ℃, e.g., a temperature of 45 ℃ to 70 ℃, e.g., a temperature of 50 ℃ to 65 ℃.

A suitable time period for incubating the edible oil with phospholipase a1 in the methods as disclosed herein is 0.5 to 10 hours, e.g., 1 to 8 hours, or 2 to 6 hours.

The oil was incubated with an appropriate amount of phospholipase a 1. A suitable amount of phospholipase is such that after 4 hours of incubation at a temperature of 55 ℃ to 70 ℃, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or at least 90%, 91%, 92%, 93%, 94% or at least 95% of the amount of intact phospholipid originally present in the triacylglycerol oil is reduced. For example, the amount is from 0.02mg active PLA1 protein/kg oil to 3mg active PLA1 protein/kg oil, e.g., between 0.05mg active PLA1 protein/kg oil to 1mg active PLA1 protein/kg oil, e.g., between 0.1mg active PLA1 protein/kg oil to 0.8mg active PLA1 protein/kg oil, e.g., 0.15mg active PLA1 protein/kg oil to 0.5mg active PLA1 protein/kg oil, e.g., 0.2mg active PLA1 protein/kg oil to 0.4mg active PLA1 protein/kg oil.

The phospholipase a1 may be derived from any suitable organism, for example from a fungus. Suitable fungi include filamentous fungi such as Aspergillus (Aspergillus), Talaromyces (Talaromyces), Trichoderma (Trichoderma), and yeasts such as Pichia (Pichia), Saccharomyces (Saccharomyces), e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans (Aspergillus nidulans), Talaromyces emersonii, Pichia pastoris (Pichia pastoris), Saccharomyces cerevisiae (Saccharomyces cerevisiae). The polypeptide having phospholipase A1 activity can be derived from Aspergillus niger.

The phospholipase a1 for use in the methods as disclosed herein can comprise a polypeptide having at least 80% identity with the mature amino acid sequence of SEQ ID No. 1, e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the mature amino acid sequence of SEQ ID No. 1. The polypeptide having phospholipase A1 activity can comprise or comprise the mature amino acid sequence of SEQ ID NO. 1. The mature amino acid sequence of SEQ ID NO. 1 comprises or contains amino acids 30 to 298 of SEQ ID NO. 1. The mature amino acid sequence of SEQ ID NO. 1 may further comprise or contain amino acids 29 to 297 of SEQ ID NO. 1, such as amino acids 28 to 296 of SEQ ID NO. 1, such as amino acids 31 to 298 of SEQ ID NO. 1, such as amino acids 32 to 297 of SEQ ID NO. 1. The mature amino acid sequence may further comprise one or more additional amino acids at the C-terminus of SEQ ID NO. 1.

The phospholipase a1 used in the methods as disclosed herein can be a naturally occurring polypeptide or a variant polypeptide.

The phospholipase a1 can be produced in any suitable host cell (e.g., prokaryotic or eukaryotic cell) that can be used to produce a polypeptide having phospholipase a1 activity as disclosed herein. The eukaryotic host cell may be a mammalian, insect, plant or fungal cell.

The fungal cell may for example be a yeast cell or a filamentous fungal cell, such as a saccharomyces, pichia, aspergillus, Trichoderma, e.g. saccharomyces cerevisiae, pichia pastoris, aspergillus niger, aspergillus oryzae, Trichoderma reesei (Trichoderma reesei) or Trichoderma viride (Trichoderma viride) cell. The techniques of Molecular biology known to the skilled worker are carried out in accordance with Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition, CSHL Press, Cold Spring Harbor, NY, 2001.

A host cell useful for producing phospholipase a1 as disclosed herein is cultured in a suitable fermentation medium that allows expression of phospholipase a 1. The person skilled in the art knows how to carry out a method for preparing a polypeptide having phospholipase a1 activity, depending on the host cell used. Suitable fermentation media typically comprise carbon and nitrogen sources. Typically, the pH of the fermentation medium is between 4 and 8. Suitable temperatures for culturing the host cells are typically between 25 ℃ and 60 ℃. The phospholipase a1 may be recovered from the fermentation medium by methods known in the art, e.g., by centrifugation, filtration and/or ultrafiltration.

The phospholipase a1 in the methods as disclosed herein may be a composition comprising the phospholipase a1 as disclosed herein, e.g., an aqueous composition or a solid composition comprising the phospholipase a1 as disclosed herein. The composition may be a fermentation broth, for example a fermentation broth from which cells and/or other components have been removed, for example by centrifugation, filtration or ultrafiltration.

The phospholipase a1 may also be pure or isolated phospholipase a1, i.e., a polypeptide having phospholipase a1 activity removed from at least one component with which it is naturally associated (e.g., other polypeptide materials).

Phospholipids in triacylglycerol oils include Phosphatidic Acid (PA), Phosphatidylethanolamine (PE), Phosphatidylinositol (PI) and Phosphatidylcholine (PC). It was surprisingly found that in the process as disclosed herein, the content or amount of these four phospholipids is reduced.

In one embodiment, the method as disclosed herein comprises the step of adding an acid to the oil. Suitable acids that may be added to the oil include citric acid, phosphoric acid, acetic acid, tartaric acid and/or succinic acid, and any suitable mixtures thereof.

In yet another embodiment, a method as disclosed herein comprises adding caustic to the oil. Suitable caustic bases may be, for example, potassium or sodium hydroxide, sodium silicate, sodium carbonate, calcium carbonate, sodium bicarbonate, ammonia, sodium citrate, or any suitable combination thereof.

The addition of acid, water and/or caustic may be performed during any suitable step in the process for reducing the amount of intact phospholipids in triacylglycerol oil as disclosed herein. The addition of acid, water and/or caustic may be performed before, during or after the incubation of the oil with the phospholipase. Preferably, the addition of acid, water and/or caustic is performed prior to incubating the oil with phospholipase a 1. The addition of caustic may be performed before or after the addition of acid, for example, the addition of caustic may be performed after the addition of acid.

The method as disclosed herein for reducing the amount of intact phospholipids in triacylglycerol oils may further comprise the step of pre-treating the oil in the presence of acid and/or caustic. The acid and/or caustic are as defined above. The pretreatment comprises incubating the oil in the presence of acid and/or caustic at a temperature of 50 ℃ to 75 ℃, for example 55 ℃ to 70 ℃. The pre-treatment may be performed during 1 minute to 2 hours, such as 5 minutes to 1 hour, such as 10 minutes to 40 minutes.

In one embodiment, the method as disclosed herein further comprises incubating the oil with an enzyme having phospholipase C activity, an enzyme having Phosphatidylinositol (PI) -phospholipase C activity, and/or an enzyme having phospholipase a2 activity.

Phospholipase c (plc) may be an enzyme from enzyme class number EC 3.1.4.3, which cleaves between the phosphate and glycerol groups of phospholipids, thereby producing diglyceride and phosphate compounds, such as phosphorylcholine or phosphoethanolamine. For example, a PLC is known from WO2005/086900, WO2012/062817 or WO 2016/162456. The PLC may be a polypeptide comprising the amino acid sequence of SEQ ID NO. 3, which is also disclosed on page 196 of WO 2005/086900. The phospholipase C can be a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence of SEQ ID No. 3.

The phospholipase C may also be phosphatidylinositol phospholipase C (PI-PLC). PI-PLC has a preference for cleaving phosphatidylinositol and can also act on other phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. The bacterial PI-PLC belongs to the enzyme classification EC 4.6.1.13. Suitable PI-PLC enzymes are disclosed, for example, in WO 2011/046812. Suitable PI-PLCs may comprise the amino acid sequence of SEQ ID No. 4, which corresponds to SEQ ID No. 8 disclosed in WO 2011/046812. Phosphatidylinositol phospholipase C can be a polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity to the amino acid sequence of SEQ ID No. 4.

Phospholipase a2(PLA2) releases fatty acids from the second carbon group of glycerol and belongs to the enzyme classification EC 3.1.1.4. PLA2 may be, for example, porcine pancreas PLA2, which may be expressed in a suitable host organism, such as an aspergillus species, e.g., aspergillus niger.

The method as disclosed herein may further comprise separating the phosphorus-containing component from the oil. The separation of the phosphorus-containing component may be performed by any suitable method known in the art, for example by centrifugation. The phosphorus-containing component includes phospholipids, lysophospholipids and glycerophosphates.

Any suitable triacylglycerol oil may be present or used in the methods for reducing phospholipids in edible oils as disclosed herein. The triacylglycerol oil may be a crude edible oil or a water degummed edible oil. Crude oil, also known as non-degummed oil, refers to pressed or extracted oil. The oil may be vegetable oil (vegetable oil/plant oil), animal oil, fish oil, or algae oil. The vegetable oil may be any suitable oil, such as soybean oil, rapeseed oil, canola oil, sunflower oil, palm kernel oil, coconut oil, sesame oil, olive oil, rice bran oil, cottonseed oil, corn oil, nut oils, such as almond oil, walnut oil, peanut oil, or mixtures thereof. The process for reducing the amount of intact phospholipids in triacylglycerol oils can also be expressed as an oil degumming or oil refining process.

The vegetable crude oil may comprise between 1ppm and 2000ppm, for example between 1ppm and 1000ppm, atomic phosphorus. The amount of atomic phosphorus indicates the amount of phospholipid.

Also disclosed herein is a triacylglycerol oil comprising a polypeptide having phospholipase a1 activity, wherein the polypeptide comprises a polypeptide having at least 80% identity to the mature amino acid sequence of SEQ ID No. 1. The triacylglycerol oil may be obtainable by a process as disclosed herein. All of the embodiments disclosed above for the process as disclosed herein apply to the triacylglycerol oil as disclosed herein.

The oil as disclosed herein can further comprise a polypeptide having phospholipase C activity, a polypeptide having phosphatidylinositol phospholipase C activity (PI-PLC), and/or a polypeptide having phospholipase a2 activity. The oil as disclosed herein may further comprise a polypeptide having phospholipase C activity, which polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence of SEQ ID No. 3; a polypeptide having phosphatidylinositol phospholipase C activity, which polypeptide has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99% identity to the amino acid sequence of SEQ ID NO. 4; and/or phospholipase a2 from porcine pancreas.