Polypeptide with phospholipase A activity and polynucleotide encoding the same

Abstract

The present invention relates to isolated polypeptides having phospholipase A activity and polynucleotides encoding these polypeptides. The present invention also relates to nucleic acid constructs, vectors and host cells comprising these polynucleotides as well as methods of producing and using these polypeptides.

Description

[0001] Reference to a sequence listing

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Background of the Invention Field of the Invention

[0004] The present invention relates to polypeptides having phospholipase A activity and polynucleotides encoding these polypeptides. The present invention also relates to nucleic acid constructs, vectors and host cells comprising these polynucleotides as well as methods for producing and using these polypeptides.

[0005] Related technical notes

[0006] Phospholipases have been isolated from a large number of organisms, such as animals, plants, bacteria, and fungi.

[0007] The lipase from Aspergillus niger (UNIPROT: B8YIE6) has an amino acid sequence that is 70% identical to the phospholipase of the present invention.

[0008] Phospholipases with A1 activity have been made available to the public by Novozymes A / S as LECITASE ULTRA ( Yang (Yang) et al., Food Technol. Biotechnol., 44(1): 101-104 (2006) ).

[0009] Several uses of phospholipases are known in the art, for example, the use of phospholipases in, for example, enzymatic degumming of vegetable oils (Yang et al., supra; US 5,264,367, Metallgesellschaft, ); treatment of starch hydrolysates (particularly from wheat starch) to improve filterability (EP 219,269, CPC International); as an additive to bread dough to improve the properties of dough and bread (US 4,567,046, Kyowa Hakko); and for the preparation of lysolecithin with special emulsifying properties.

[0010] Phospholipases can be applied to the degumming of vegetable oils to provide a refined, shelf-stable vegetable oil having a neutral taste and light color suitable for consumption. The degumming process involves the removal of phospholipid compounds, also known as phospholipids or "gums" from the triglyceride-rich oil fraction.

[0011] Traditionally, the degumming process is based on water extraction, treated with acid or corrosive treatment, followed by a separation process. Due to the emulsification of phospholipids, the degumming process results in the loss of oil, i.e., the loss of triglycerides. However, enzymatic degumming has become increasingly common. Enzymatic degumming is carried out on oil that has been degummed with water and on crude oil. In the water degumming process of edible oil, a part of phospholipids is left in the oil. That part is described with the general term "non-hydratable phospholipids" (NHP). In the process of producing oil, it is essential to remove the NHP content (US 5264367). In the enzymatic degumming process, NHP is converted into a water-soluble and water-extractable component by using phospholipase.

[0012] The most widely used commercial enzyme for industrial degumming of vegetable oils is the phospholipase LECITASE ULTRA from Novozymes A / S (Yang et al., supra). LECITASE ULTRA has a relatively high thermostability; however, it is best suited for degumming processes at temperatures up to 55°C.

[0013] Generally, by using acidic and corrosive chemicals to assist enzymatic degumming.In the initial degumming step, by metal and citrate chelating, the interpolation of citric acid or phosphoric acid has improved the hydration of the salt form of phospholipid.In the following enzymatic reaction, pH often needs to be partially neutralized (typically by adding sodium hydroxide) to adapt to the pH needs of the enzyme used.When using phospholipase LECITASE ULTRA, the optimum pH for this reaction is between 5.0 and 5.5 (referring to application form "Refining of vegetable oils-Improving degumming yield (Refining of vegetable oils-Improving degumming yield)"). However, within this pH range, during the enzymatic reaction, the calcium and / or magnesium released are combined with the buffer or chelated acid used to control reaction conditions, typically causing salt to form and causing the equipment pollution (US 7,713,727) because of the precipitation of the salt of calcium citrate and magnesium citrate.If enzymatic reaction can be carried out under lower pH, thus reducing the acid used for neutralization will be advantageous.

[0014] There is an ongoing need to provide novel phospholipases having improved properties, such as increased activity at high temperatures and / or low pH. The present invention relates to such novel polypeptides having phospholipase A activity and polynucleotides encoding these polypeptides. SUMMARY OF THE INVENTION

[0016] The inventors have identified a novel polypeptide having phospholipase A activity from Talaromyces leycettanus strain CBS398.68. The polypeptides of the invention show high thermostability and / or increased activity at high temperatures. This is an advantage because it allows industrial processes to be performed at higher temperatures. In enzymatic degumming, higher temperatures can increase the rate of enzymatic degumming of phospholipids present in the oil, facilitate separation of the oil phase and the aqueous phase after enzymatic degradation of the phospholipids, and / or reduce the viscosity of the oil - all of which result in shorter processing times and higher throughput of oil refining equipment.

[0017] Furthermore, the polypeptides of the present invention have unexpectedly high activity at low pH, which is an advantage, i.e., in enzymatic degumming of vegetable oils, where the need for pH adjustment is reduced, resulting in less deposition of precipitates on process equipment and thus saving reduced use of chemicals as well as reduced time for cleaning.

[0018] The peptides are active against most phospholipids, including phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC).

[0019] Therefore, the present invention provides novel polypeptides having phospholipase A activity and polynucleotides encoding these polypeptides.

[0020] In the first aspect, the present invention relates to an isolated polypeptide having phospholipase A activity, wherein the isolated polypeptide is selected from the group consisting of: (a) a polypeptide having at least 80% sequence identity with the mature polypeptide of SEQ ID NO: 2 or the polypeptide of SEQ ID NO: 3; (b) a polypeptide encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence, or (ii) the full-length complement of (i); (c) a polypeptide encoded by a polynucleotide that has at least 65% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence; (d) a variant of the mature polypeptide of SEQ ID NO: 2 or a variant of the polypeptide of SEQ ID NO: 3, the variant comprising substitutions, deletions, and / or insertions at one or more positions; and (e) a fragment of the polypeptide of (a), (b), (c), or (d), the fragment having phospholipase A activity.

[0021] In a second aspect, the present invention relates to an isolated nucleic acid, which is selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide having phospholipase A activity, wherein the polypeptide comprises the mature polypeptide of SEQ ID NO: 2 or the polypeptide of SEQ ID NO: 3; (b) a nucleotide sequence encoding a polypeptide having phospholipase A activity, wherein the polypeptide comprises an amino acid sequence having at least 80% identity to the mature polypeptide of SEQ ID NO: 2 or to the polypeptide of SEQ ID NO: 3; (c) a nucleotide sequence encoding a polypeptide having phospholipase A activity, wherein the nucleotide sequence comprises SEQ ID NO: 3. NO:1 mature polypeptide coding sequence or its cDNA sequence; (d) a nucleotide sequence having at least 80% sequence identity with the nucleotide sequence according to (a), (b), or (c); (e) a nucleotide sequence that hybridizes with the complementary strand of the nucleotide sequence according to (a), (b), (c), or (d) under high stringency conditions; (f) a subsequence of the nucleotide sequence according to (a), (b), (c), (d), or (e), having at least 100 nucleotides; (g) a sequence that, due to the degeneracy of the genetic code, is degenerate to a sequence as defined in any one of (a), (b), (c), (d), (e), or (f); and (h) a complementary strand of the nucleotide sequence according to (a), (b), (c), (d), (e), (f) or (g).

[0022] In a third aspect, the present invention relates to a nucleic acid construct or expression vector comprising the polynucleotide of the second aspect operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0023] In a fourth aspect, the invention relates to a recombinant host cell comprising the polynucleotide of the second aspect operably linked to one or more control sequences that direct the production of the polypeptide.

[0024] In a fourth aspect, the present invention relates to a method of producing a polypeptide of the first aspect, the method comprising: (a) cultivating a cell, which is in its wild-type form, producing the polypeptide under conditions conducive to the production of the polypeptide; and (b) recovering the polypeptide.

[0025] In a sixth aspect, the present invention relates to a method for producing a polypeptide having phospholipase A activity, the method comprising: (a) culturing the host cell of the fourth aspect under conditions conducive to production of the polypeptide; and (b) recovering the polypeptide.

[0026] In a seventh aspect, the present invention relates to a composition comprising (a) the polypeptide of the first aspect or a polypeptide obtainable by the method of any one of the fifth or sixth aspects; and (b) optionally an additional enzyme.

[0027] In an eighth aspect, the present invention relates to use of the polypeptide of the first aspect or any composition of the seventh aspect in hydrolyzing phospholipids.

[0028] In a ninth aspect, the present invention relates to a method for reducing the content of phosphorus-containing components in an edible oil, the method comprising contacting the oil with an aqueous solution of any polypeptide described in the first aspect, the aqueous solution being emulsified in the oil until the phosphorus content of the oil is reduced, and then separating the aqueous phase from the treated oil. Sequence Listing <110> Novozymes A / S <120> Polypeptide with phospholipase A activity and polynucleotide encoding the same <130> 12617-WO-PCT <150> 13160387.0 <151> 2013-03-21 <150> 13162328.2 <151> 2013-04-04 <160> 5 <170> PatentIn Version 3.5 <210> 1 <211> 1284 <212> DNA <213> Thermophilic thallus <220> <221> CDS <222> (101)..(182) <220> <221> CDS <222> (258)..(423) <220> <221> CDS <222> (485)..(821) <220> <221> CDS <222> (879)..(1181) <400> 1 atcgatggaa tgcagaggca agcaaatcc cagaccgtcc gcctgagctg gtttgcaaaa 60 gttttcatcg gccttttgct cgatctca gactgtcatc atg cac cgt cct ctc 115 Met His Arg Pro Leu 1 5 cag ttg tgg gct cct gca gcc ctg aca tcg ctg gtc acc gca gct 163 Gln Leu Trp Ala Leu Ala Leu Thr Ser Leu Val Thr Ala Ala Leu Pro 10 15 20 gct cca gtc ctg cgt cgt g gtaggctca tggaccttgt gccgaactat 212 Wing Pro Val Leu Arg Arg 25 aaaacaggag cgctgttcat atgcaggtct gactgttaca tcag at gtg tcc tcg 268 Asp Val Ser Ser 30 tct gtc ctg agt gag ctc gat ctc ttc gcg cag tac agt gcg gcc gca 316 Ser Val Leu Ser Glu Leu Asp Leu Phe Ala Gln Tyr Ser Ala Ala Ala Ala 35 40 45 tat tgc tct tcc aac att ggc tcc ccg gga acc aag ttg acg tgc agt Tyr Cys Ser Ser Asn Ile Gly Ser Pro Gly Thr Lys Leu Thr Cys Ser 50 55 60 gtg ggc aat tgc ccc cgg gta gag gct gcg gat acc gag aca tta att 412 Val Gly Asn Cys Pro Arg Val Glu Ala Ala Asp Thr Glu Thr Leu Ile 65 70 75 gag ttc aat ga gtaagtgata gacgattccg atcctcgttc cgctctgcac 463 Glu Phe Asn Glu 80 tctctgaaca ccacaatcta g g tct tca tct ttc ggc gac gtt act ggc tac 515 Ser Ser Ser Phe Gly Asp Val Thr Gly Tyr 85 90 att gcc gtg gac cga acc aac agc ctg ctc gtt ctg gcg ttc cga ggc 563 Ile Ala Val Asp Arg Thr Asn Ser Leu Leu Val Leu Ala Phe Arg Gly 95 100 105 agt agc act gtc tcc aac tgg gag gca gat ttg gac ttc ccg ttg act 611 Ser Ser Thr Val Ser Asn Trp Glu Ala Asp Leu Asp Phe Pro Leu Thr 110 115 120 125 gat gcc agc agt ctc tgt tcg ggc tgt gaa atc cac agt ggc ttc tgg 659 Asp Ala Ser Ser Leu Cys Ser Gly Cys Glu Ile His Ser Gly Phe Trp 130 135 140 gct gcc tgg cag acg gtt cag gcc agc atc acc tcg acg ctc gag tcg 707 Ala Ala Trp Gln Thr Val Gln Ala Ser Ile Thr Ser Thr Leu Glu Ser 145 150 155 gcc ata gcc agc tat ccc ggc tac acc ctg gtc ttc acc ggc cat agc 755 Ala Ile Ala Ser Tyr Pro Gly Tyr Thr Leu Val Phe Thr Gly His Ser 160 165 170 tat gga gct gcc ttg gct gca atc gcg gcc acg acg ttg cga aat gcc 803 Tyr Gly Ala Ala Leu Ala Ala Ile Ala Ala Thr Thr Leu Arg Asn Ala 175 180 185 gga tac acc atc cag ctg gtaagcgtcc cgccccaacc ataactcttc 851 Gly Tyr Thr Ile Gln Leu 190 195 ccgagctcac aacagtccgc gccaaag tat gac tac ggc cag cct cgc ctg ggc 905 Tyr Asp Tyr Gly Gln Pro Arg Leu Gly 200 aat ctg gca ttg gcc cag tac atc acc gcg cag acg caa ggc gcc aac 953 Asn Leu Ala Leu Ala Gln Tyr Ile Thr Ala Gln Thr Gln Gly Ala Asn 205 210 215 220 tac cgc gtc acg cac acc gac gac att gtc ccc aag ctt ccg cct gag 1001 Tyr Arg Val Thr His Thr Asp Asp Ile Val Pro Lys Leu Pro Pro Glu 225 230 235 cta ttt ggc tac cat cat ttc agt ccc gag tac tgg atc acc agt gga 1049 Leu Phe Gly Tyr His His Phe Ser Pro Glu Tyr Trp Ile Thr Ser Gly 240 245 250 gac aat gtg acg gtg acc acc tcc gat gtc caa gtt gtc act ggc atc 1097 Asp Asn Val Thr Val Thr Thr Ser Asp Val Gln Val Val Thr Gly Ile 255 260 265 gac tcg acc gct gga aat gat ggt acg ctt ctc gat agt aca tcg gcg 1145 Asp Ser Thr Ala Gly Asn Asp Gly Thr Leu Leu Asp Ser Thr Ser Ala 270 275 280 cat gac tgg tat att gtc tac atc gac ggg tgc gat taaagaagtc 1191 His Asp Trp Tyr Ile Val Tyr Ile Asp Gly Cys Asp 285 290 295 ggcctgcggt atgaatattt ggataaccta cgtatatata tggctccgac gctctaggta 1251 tctagcaaca atataaccct gaggggattt gaa 1284 <210>2 <211>296 <212>PRT <213>Thermomyces lanuginosus <400>2 Met His Arg Pro Leu Gln Leu Trp Ala Leu Ala Ala Leu Thr Ser Leu 1 5 10 15 Val Thr Ala Ala Pro Ala Pro Val Leu Arg Arg Asp Val Ser Ser Ser 20 25 30 Val Leu Ser Glu Leu Asp Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr 35 40 45 Cys Ser Ser Ash Ile Gly Ser Pro Gly Thr Lys Leu Thr Cys Ser Val 50 55 60 Gly Ash Cys Pro Arg Val Glu Ala Ala Asp Thr Glu Thr Leu Ile Glu 65 70 75 80 Phe Ash Glu Ser Ser Ser Phe Gly Asp Val Thr Gly Tyr Ile Ala Val 85 90 95 Asp Arg Thr Asn Ser Leu Leu Val Leu Ala Phe Arg Gly Ser Ser Thr 100 105 110 Val Ser Asn Trp Glu Ala Asp Leu Asp Phe Pro Leu Thr Asp Ala Ser 115 120 125 Ser Leu Cys Ser Gly Cys Glu Ile His Ser Gly Phe Trp Ala Ala Trp 130 135 140 Gln Thr Val Gln Ala Ser Ile Thr Ser Thr Leu Glu Ser Ala Ile Ala 145 150 155 160 Ser Tyr Pro Gly Tyr Thr Leu Val Phe Thr Gly His Ser Tyr Gly Ala 165 170 175 Ala Leu Ala Ala Ile Ala Ala Thr Thr Leu Arg Ash Ala Gly Tyr Thr 180 185 190 Ile Gln Leu Tyr Asp Tyr Gly Gln Pro Arg Leu Gly Ash Leu Ala Leu 195 200 205 Ala Gln Tyr Ile Thr Ala Gln Thr Gln Gly Ala Asn Tyr Arg Val Thr 210 215 220 His Thr Asp Asp Ile Val Pro Lys Leu Pro Pro Glu Leu Phe Gly Tyr 225 230 235 240 His His Phe Ser Pro Glu Tyr Trp Ile Thr Ser Gly Asp Asn Val Thr 245 250 255 Val Thr Thr Ser Asp Val Gln Val Val Thr Gly Ile Asp Ser Thr Ala 260 265 270 Gly Asn Asp Gly Thr Leu Leu Asp Ser Thr Ser Ala His Asp Trp Tyr 275 280 285 Ile Val Tyr Ile Asp Gly Cys Asp 290 295 <210>3 <211>277 <212>PRT <213>Thermomyces lanuginosus <400>3 Ala Pro Ala Pro Val Leu Arg Arg Asp Val Ser Ser Ser Val Leu Ser 1 5 10 15 Glu Leu Asp Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Ser Ser 20 25 30 Asn Ile Gly Ser Pro Gly Thr Lys Leu Thr Cys Ser Val Gly Asn Cys 35 40 45 Pro Arg Val Glu Ala Ala Asp Thr Glu Thr Leu Ile Glu Phe Ash Glu 50 55 60 Ser Ser Ser Phe Gly Asp Val Thr Gly Tyr Ile Ala Val Asp Arg Thr 65 70 75 80 Asn Ser Leu Leu Val Leu Ala Phe Arg Gly Ser Ser Thr Val Ser Asn 85 90 95 Trp Glu Ala Asp Leu Asp Phe Pro Leu Thr Asp Ala Ser Ser Leu Cys 100 105 110 Ser Gly Cys Glu Ile His Ser Gly Phe Trp Ala Ala Trp Gln Thr Val 115 120 125 Gln Ala Ser Ile Thr Ser Thr Leu Glu Ser Ala Ile Ala Ser Tyr Pro 130 135 140 Gly Tyr Thr Leu Val Phe Thr Gly His Ser Tyr Gly Ala Ala Leu Ala 145 150 155 160 Ala Ile Ala Ala Thr Thr Leu Arg Asn Ala Gly Tyr Thr Ile Gln Leu 165 170 175 Tyr Asp Tyr Gly Gln Pro Arg Leu Gly Asn Leu Ala Leu Ala Gln Tyr 180 185 190 Ile Thr Ala Gln Thr Gln Gly Ala Asn Tyr Arg Val Thr His Thr Asp 195 200 205 Asp Ile Val Pro Lys Leu Pro Pro Glu Leu Phe Gly Tyr His His Phe 210 215 220 Ser Pro Glu Tyr Trp Ile Thr Ser Gly Asp Asn Val Thr Val Thr Thr 225 230 235 240 Ser Asp Val Gln Val Val Thr Gly Ile Asp Ser Thr Ala Gly Asn Asp 245 250 255 Gly Thr Leu Leu Asp Ser Thr Ser Ala His Asp Trp Tyr Ile Val Tyr 260 265 270 Ile Asp Gly Cys Asp 275 <210>4 <211>891 <212>DNA <213>Thermomyces lanuginosus <400>4 atgcaccgtc ctctccagtt gtgggctctc gcagccctga catcgctggt caccgcagct 60 ccggctccag tcctgcgtcg tgatgtgtcc tcgtctgtcc tgagtgagct cgatctcttc 120 gcgcagtaca gtgcggccgc atattgctct tccaacattg gctccccggg aaccaagttg 180 acgtgcagtg tgggcaattg cccccgggta gaggctgcgg ataccgagac attaattgag 240 ttcaatgagt cttcatcttt cggcgacgtt actggctaca ttgccgtgga ccgaaccaac 300 agcctgctcg ttctggcgtt ccgaggcagt agcactgtct ccaactggga ggcagatttg 360 gacttcccgt tgactgatgc cagcagtctc tgttcgggct gtgaaatcca cagtggcttc 420 tgggctgcct ggcagacggt tcaggccagc atcacctcga cgctcgagtc ggccatagcc 480 agctatcccg gctacaccct ggtcttcacc ggccatagct atggagctgc cttggctgca 540 atcgcggcca cgacgttgcg aaatgccgga tacaccatcc agctgtatga ctacggccag 600 cctcgcctgg gcaatctggc attggcccag tacatcaccg cgcagacgca aggcgccaac 660 taccgcgtca cgcacaccga cgacattgtc cccaagcttc cgcctgagct atttggctac 720 catcatttca gtcccgagta ctggatcacc agtggagaca atgtgacggt gaccacctcc 780 gatgtccaag ttgtcactgg catcgactcg accgctggaa atgatggtac gcttctcgat 840 agtacatcgg cgcatgactg gtatattgtc tacatcgacg ggtgcgatta a 891 <210>5 <211>274 <212>PRT <213>Artificial Sequence <220> <223>Artificial Sequence <400>5 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Ala Asn Leu Asn Phe Trp 85 90 95 Leu Lys Lys Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gln Ala Thr Asp Ala Cys Asn Ala Gly Gly 260 265 270 Phe Ser

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Illustrated is what happens when different phospholipases cleave a phospholipid.

[0031] Figure 2 The reaction of a phospholipid with phospholipase A2 is described to form a lysophospholipid with a hydroxyl group at position C2 in the glycerol backbone and a free R2 fatty acid. A similar reaction with phospholipase A1 will cleave a fatty acid labeled with R1 to form a free R1 fatty acid and a lysophospholipid with a hydroxyl group at position C1 in the glycerol backbone.

[0032] Figure 3 The activity of Licitase Ultra and phospholipase of SEQ ID NO: 2 on PC plates from pH 3 to pH 7 is shown. The incubation time is 90 minutes, 150 minutes, 4 hours, 7 hours, 23 hours, 29 hours and 48 hours. The black circle represents the activity, the larger the circle, the higher the activity.

[0033] definition

[0034] Phospholipase A activity: In the context of the present invention, the term "phospholipase A activity" includes enzymes having phospholipase A1 and / or phospholipase A2 activity (A1 or A2, EC 3.1.1.32 or EC 3.1.1.4), i.e., hydrolysis activity for one or two carboxylic acid ester bonds in phospholipids such as lecithin. Phospholipases having both A1 and A2 activity are also referred to as phospholipase B.

[0035] For the purposes of the present invention, phospholipase A activity is determined according to the procedures described in the Materials and Methods section. In one aspect, the polypeptides of the invention have at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the phospholipase A activity of the mature polypeptide of SEQ ID NO: 2 and / or the polypeptide of SEQ ID NO: 3.

[0036] In addition to having phospholipase A activity, the polypeptides of the present invention may also have lipase activity.

[0037] Phospholipase C activity: Phospholipase C (EC 3.1.4.11) removes the phosphate moiety from phospholipids such as phosphatidylcholine to produce 1,2 diglycerides and phosphate esters.

[0038] Phospholipase D activity: Phospholipase D (EC 3.1.4.4) acts on phospholipids such as phosphatidylcholine and produces 1,2-glycerophosphodiester and a base group.

[0039] Lipase activity: The term "lipase activity" is defined herein as lipolytic activity that hydrolyzes carboxylate bonds in tributyrin, olein, pNP-butyrate, and pNP-palmitate (triglyceride lipase EC 3.1.1.3).

[0040] Thermal stability: In the context of the present invention, "thermal stability" is determined by differential scanning calorimetry (DSC) using the method described in Example 1. In one aspect, the polypeptide of the present invention has a denaturation temperature that is at least 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C or even 17°C higher than the denaturation temperature of LECITASE ULTRA, i.e., the polypeptide shown in SEQ ID NO:5.

[0041] Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation is naturally produced by mutation and can lead to polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode a polypeptide with an altered amino acid sequence. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0042] Catalytic domain: The term "catalytic domain" means the region of an enzyme that contains the catalytic machinery of the enzyme.

[0043] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription of a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The early, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature, spliced ​​mRNA.

[0044] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG or TTG) and ends with a stop codon (such as TAA, TAG or TGA). The coding sequence can be a genomic DNA, cDNA, synthetic DNA or a combination thereof.

[0045] Control sequences: The term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or exogenous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, and transcription and translation termination signals. For the purpose of introducing specific restriction sites that are conducive to connecting these control sequences to the coding region of a polynucleotide encoding a polypeptide, these control sequences may be provided with multiple linkers.

[0046] Crude oil: The term "crude oil" refers to (also referred to as non-degummed oil) an pressed or extracted oil or mixtures thereof from, for example, vegetable sources including but not limited to, acai oil, almond oil, palm kernel oil, blackcurrent seed oil, borage seed oil, canola oil, Oil, cashew oil, castor oil, coconut oil, coriander oil, corn oil, cottonseed oil, crambe oil, linseed oil, grapeseed oil, hazelnut oil, sesame oil, jatropha oil, jojoba oil, linseed oil, macadamia oil, mango kernel oil, meadowfoam seed oil, mustard oil, neatsfoot oil, olive oil, palm oil, palm kernel oil, palm olein, peanut oil, walnut oil, pine nut oil, pistachio oil, poppy seed oil, rapeseed oil, rice bran oil, safflower oil, camellia oil, sesame oil, avocado oil, soybean oil, sunflower oil, tall oil, camellia oil, walnut oil, various "natural" oils that have been genetically modified organisms (GMOs) or traditionally "breaded" to alter the fatty acid composition, such as high oleic, low oleic, or low saturated oils (high oleic rapeseed oil, low linolenic soybean oil, or high stearic sunflower oil).

[0047] Degummed oil: The term "degummed oil" refers to an oil obtained after removing non-hydratable phospholipids, hydratable phospholipids, and lecithin (collectively referred to as "gums") from the oil to produce a degummed oil or fat product that can be used in food production and / or non-food applications, such as biodiesel. In certain embodiments, the degummed oil has a phospholipid content of less than about 200 ppm phosphorus, less than about 150 ppm phosphorus, less than about 100 ppm phosphorus, less than about 50 ppm phosphorus, less than about 40 ppm phosphorus, less than about 30 ppm phosphorus, less than about 20 ppm phosphorus, less than about 15 ppm phosphorus, less than about 10 ppm phosphorus, less than about 7 ppm phosphorus, less than about 5 ppm phosphorus, less than about 3 ppm phosphorus, or less than about 1 ppm phosphorus.

[0048] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0049] Expression vector: The term "expression vector" means a linear or circular DNA molecule that includes a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0050] Fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids missing from the amino and / or carboxyl terminus of a mature polypeptide or domain, wherein the fragment has phospholipase A activity. Fragments according to the present invention have a size of greater than about 160 (e.g., amino acids 117-277 of SEQ ID NO: 3), preferably greater than 200 amino acid residues, preferably greater than 210 amino acid residues, more preferably greater than 220 amino acid residues, more preferably greater than 230 amino acid residues, more preferably greater than 240 amino acid residues, more preferably greater than 250 amino acid residues, more preferably greater than 260 amino acid residues (e.g., amino acids 18 to 277 or amino acids 1 to 259 of SEQ ID NO: 3), more preferably greater than 270 amino acid residues (e.g., amino acids 8 to 277 or amino acids 1 to 270 of SEQ ID NO: 3), and most preferably greater than 275 amino acid residues.

[0051] Host cell: The term "host cell" means any cell type susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0052] Isolated: The term "isolated" means a substance that is in a non-naturally occurring form or environment. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance; (2) any substance that is at least partially removed from one or more or all of the naturally occurring components with which it is associated in nature, including but not limited to any enzyme, variant, nucleic acid, protein, peptide or cofactor; (3) any substance that has been modified by the hand of man relative to that substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

[0053] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 20 to 296 of SEQ ID NO: 2 based on the program SignalP version 3 (Nielsen et al., 1997, Protein Engineering 10: 1-6). Amino acids 1 to 19 of SEQ ID NO: 2 are predicted to be signal peptides. The mature polypeptide is also shown as amino acids 1 to 277 in SEQ ID NO: 3. The N-terminal sequence of the polypeptide expressed in Aspergillus oryzae was analyzed using the Applied Biosystems Procise protein sequence system. It showed the following N-terminal sequence:

[0054] 35 kDa band with N-terminal DVSSSVL corresponding to residues 28-34 of SEQ ID NO: 2

[0055] 25 kDa band with N-terminal EADLDFP corresponding to residues 117-123 of SEQ ID NO: 2

[0056] 25 kDa band with an N-terminal LDFPLTD corresponding to residues 120-126 of SEQ ID NO: 2

[0057] It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus a host cell expressing a polynucleotide can produce a different mature polypeptide (e.g., having a different C-terminal and / or N-terminal amino acid) when compared to another host cell expressing the same polynucleotide. In one aspect, the mature polypeptide is amino acids 28 to 296 of SEQ ID NO: 2. In another aspect, the mature polypeptide is amino acids 117 to 296 of SEQ ID NO: 2 or amino acids 120 to 296 of SEQ ID NO: 2.

[0058] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having phospholipase A activity. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 1284 of SEQ ID NO: 1, or a cDNA sequence thereof. In one aspect, the cDNA sequence comprises or consists of nucleotides 101 to 182, nucleotides 258 to 423, nucleotides 485 to 821, and nucleotides 879 to 1184 of SEQ ID NO: 1. In one aspect, the cDNA sequence comprises or consists of nucleotides 1 to 891 of SEQ ID NO: 4. In one aspect, the cDNA is nucleotides 58 to 891 of SEQ ID NO: 4. Nucleotides 1 to 57 of SEQ ID NO: 4 encode a signal peptide.

[0059] Nucleic acid construct: The term "nucleic acid construct" means a single- or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or that is modified to contain a segment of nucleic acid in a manner that does not otherwise occur in nature, or that is synthetic, including one or more control sequences.

[0060] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0061] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle's notation "longest identity" (obtained using the -unsimplified option) was used as the percent identity and was calculated as follows:

[0062] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0063] For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needleman program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferred 5.0.0 version or updated version). The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output (using-non-simplified option to obtain) of the "longest consistency" of Needleman annotation is used as percentage consistency, and is calculated as follows:

[0064] (Number of identical DNA nucleotides x 100) / (length of alignment - total number of gaps in the alignment).

[0065] Strict conditions: Very low stringency conditions: The term "very low stringency conditions" means that for a probe of at least 100 nucleotides in length, standard Southern blotting procedures are followed, prehybridization and hybridization for 12 to 24 hours at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 25% formamide. The carrier material is finally washed three times at 45° C. for 15 minutes each time using 2X SSC, 0.2% SDS. Low stringency conditions : The term "low stringency conditions" means that for a probe of at least 100 nucleotides in length, standard Southern blotting procedures are followed, prehybridization and hybridization for 12 to 24 hours at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 25% formamide. The carrier material is finally washed three times at 50° C. for 15 minutes each time using 2X SSC, 0.2% SDS. Medium stringent conditions : The term "medium stringency conditions" means that for a probe of at least 100 nucleotides in length, standard Southern blotting procedures are followed, prehybridization and hybridization for 12 to 24 hours at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 35% formamide. The carrier material is finally washed three times at 55° C. for 15 minutes each time using 2X SSC, 0.2% SDS. middle- High stringency conditions : The term "medium-high stringency conditions" means that for a probe of at least 100 nucleotides in length, standard Southern blotting procedures are followed, prehybridization and hybridization for 12 to 24 hours at 42° C. in 5X SSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and or 35% formamide. The carrier material is finally washed three times at 60° C. with 2X SSC, 0.2% SDS, for 15 minutes each time. High stringency conditions : The term "high stringency conditions" means that for a probe of at least 100 nucleotides in length, standard Southern blotting procedures are followed, prehybridization and hybridization for 12 to 24 hours at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 50% formamide. The carrier material is finally washed three times at 65° C. with 2X SSC, 0.2% SDS, for 15 minutes each time. Very high stringency conditions : The term "very high stringency conditions" means that for a probe of at least 100 nucleotides in length, standard Southern blotting procedures are followed, prehybridization and hybridization for 12 to 24 hours at 42° C. in 5XSSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA and 50% formamide. The carrier material is finally washed three times at 70° C. for 15 minutes each time using 2X SSC, 0.2% SDS.

[0066] Subsequence: The term "subsequence" means a polynucleotide lacking one or more (eg, several) nucleotides from the 5' and / or 3' end of a mature polypeptide coding sequence or its cDNA sequence, wherein the subsequence encodes a fragment having phospholipase A activity.

[0067] Variant: The term "variant" means a polypeptide having phospholipase A activity comprising an alteration (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions. Substitution means that the amino acid occupying a position is replaced with a different amino acid; deletion means the amino acid occupying a position is removed; and insertion means the addition of an amino acid adjacent to and immediately following the amino acid occupying a position.

[0068] Detailed description of the invention

[0069] Polypeptide with phospholipase A activity

[0070] In one embodiment, the invention relates to isolated polypeptides having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO: 2 or with the polypeptide of SEQ ID NO: 3, and having phospholipase A activity. In one aspect, the polypeptides differ from the mature polypeptide of SEQ ID NO: 2 or with the polypeptide of SEQ ID NO: 3 by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a preferred embodiment, the phospholipase A activity is at least 70% of the phospholipase A activity of the polypeptide of SEQ ID NO: 3.

[0071] The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, or an allelic variant thereof, or is a fragment thereof having phospholipase A activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2 or the polypeptide of SEQ ID NO: 3.

[0072] In a preferred embodiment, the polypeptide of the present invention is more effective than Lecitase Ultra in reducing the phosphate content in crude oil when used at 70°C for 2 hours. Preferably, the polypeptide of the present invention is capable of reducing the phosphate content to less than 50 ppm phosphorus, preferably less than about 40 ppm phosphorus, more preferably less than about 30 ppm phosphorus, more preferably less than about 20 ppm phosphorus, more preferably less than about 15 ppm phosphorus, more preferably less than about 10 ppm phosphorus, more preferably less than about 7 ppm phosphorus, even more preferably less than about 5 ppm phosphorus, even more preferably less than about 3 ppm phosphorus or most preferably less than about 1 ppm phosphorus.

[0073] In another embodiment, the optimal pH range of the polypeptide of the present invention is between 1.5 and about 7.0, preferably 2.5 to 6, preferably 3.0 to 5.5, preferably from 3.5 to 5.0 and most preferably from 3.0 to 4.5.

[0074] In another embodiment, the polypeptide of the present invention is thermostable. Preferably, when determined by differential scanning calorimetry as described in Example 1, the thermal denaturation temperature is above 65°C, more preferably above 70°C, even more preferably above 75°C, and more preferably above 80°C. In another embodiment, the polypeptide of the present invention is equally thermostable at pH 4 and pH 5, for example, the DSC value changes by less than 5°C, more preferably less than 4°C, even more preferably less than 3°C and most preferably less than 2°C.

[0075] In another embodiment, the present invention relates to an isolated polypeptide having phospholipase A activity, which is encoded by a polynucleotide that hybridizes under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence, or (ii) the full-length complement of (i) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

[0076] The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, together with the polypeptide of SEQ ID NO: 2, or the polypeptide of SEQ ID NO: 3 or a fragment thereof, can be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having phospholipase A activity from strains of different genera or species according to methods well known in the art. In particular, such probes can be used to hybridize with genomic DNA or cDNA of cells of interest to identify and isolate the corresponding gene therein following standard Southern blotting procedures. Such probes can be significantly shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, or at least 800 nucleotides in length. Both DNA and RNA probes can be used. The probe is typically labeled (e.g., with a 32 P. 3 H. 35 S, biotin, or avidin) to detect the corresponding gene. The present invention encompasses such probes.

[0077] Genomic DNA or cDNA libraries prepared from such other strains can be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having phospholipase A activity. Genomic DNA or other DNA from such other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or separated DNA can be transferred to and immobilized on nitrocellulose or other suitable support material. To identify clones or DNA that hybridize to SEQ ID NO: 1 or a subsequence thereof, the support material is used in a Southern blot.

[0078] For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes under very low to very high stringency conditions to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence; (iii); (iv) its full-length complement; or (v) a subsequence thereof. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

[0079] In one aspect, the nucleic acid probe is nucleotides 1 to 100, nucleotides 1 to 200, nucleotides 1 to 250, or nucleotides 1 to 300 of SEQ ID NO: 1. In another aspect, the nucleic acid probe is a polynucleotide encoding the polypeptide of SEQ ID NO: 2; its mature polypeptide; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1.

[0080] In another embodiment, the present invention relates to an isolated polypeptide having phospholipase A activity, which is encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or to its cDNA sequence.

[0081] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 2 or variants of the polypeptide of SEQ ID NO: 3, which variants include substitutions, deletions and / or insertions at one or more (e.g., several) positions. In one embodiment, the number of amino acid substitutions, deletions and / or insertions introduced into the mature polypeptide of SEQ ID NO: 2 or the polypeptide of SEQ ID NO: 3 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. These amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions of typically 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by changing the net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0082] Examples of conservative substitutions are within the group consisting of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0083] Alternatively, the amino acid change has such a property that the physicochemical properties of the polypeptide are changed. For example, the amino acid change can increase the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0084] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for phospholipase A activity to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Mutations in putative contact site amino acids can also be combined with physical analysis of the structure, such as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, to determine the active site of the enzyme or other biological interactions. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Essential amino acids can also be identified by inference from alignments with related polypeptides.

[0085] The essential amino acids in the sequence of amino acids 1 to 296 of SEQ ID NO: 2 are located at positions H172, D228, and H285. In a preferred embodiment, these positions are retained in the polypeptide of the invention.

[0086] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science, 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA, 86:2152-2156; WO 95 / 17413 or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0087] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the importance of individual amino acid residues in a polypeptide to be rapidly determined.

[0088] Source of polypeptide with phospholipase A activity

[0089] The polypeptide having phospholipase A activity of the present invention can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from..." as used herein in conjunction with a given source should mean that the polypeptide encoded by the polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted outside the cell.

[0090] The polypeptide may be a fungal polypeptide. For example, the polypeptide may be a yeast polypeptide, such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide;or filamentous fungal polypeptides, such as Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes ermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporus the), Melanocarpus, Meripilus, Mucor, Myceliophthora, Nectria, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudoplectania Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella or Xylaria polypeptides. ;

[0091] In another aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.

[0092] In another aspect, the polypeptide is Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum), Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium grraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum), Fusarium torulosum, Fusariumtrichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimett, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptides.

[0093] In another aspect, the polypeptide is a polypeptide obtained from Nectria sp., e.g., Nectria cinnabarina, Nectria coccinea, Nectria ditissima, Nectria diversispora, Nectria eustromatica, Nectria foliicola, Nectria fragilis, Nectria fuckeliana, Nectria gallignena, Nectria haematococca, Nectria episphaeria, Nectria magnoliae, Nectria mammoidea var. rubi, Nectria mauritiicola, Nectria sphaeria, Nectria sphaeria var. peziza), Nectria pseudotrichia, Nectria punicea, Nectria radicicola and Nectria ramulariae.

[0094] It will be understood that for the above-mentioned species, the present invention encompasses both perfect and imperfect states, as well as other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. One of ordinary skill in the art will readily recognize the identity of appropriate equivalents.

[0095] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), the Netherlands Type Culture Collection (Centraalbureau Voor Schimmelcultures, CBS), and the Northern Regional Research Center (NRRL) of the Agricultural Research Service Type Collection (ARS).

[0096] The above-mentioned probe can be used to identify and obtain the polypeptide from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples directly obtained from natural materials (e.g., soil, compost, water, etc.). The technology for directly separating microorganisms and DNA from natural living environments is well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or a mixed DNA sample. Once the polynucleotide encoding the polypeptide is detected with one or more probes, the polynucleotide can be separated or cloned by using technology known to those of ordinary skill in the art (see, for example, Sambrook et al., 1989, the same).

[0097] Polynucleotide

[0098] The present invention also relates to isolated polynucleotides encoding the polypeptides of the present invention, as described herein.

[0099] Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. Cloning of polynucleotides from genomic DNA can be achieved, for example, by using well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with common structural features. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) can be used. These polynucleotides can be cloned by a strain of the genus Dactylum or a related organism, and therefore, for example, can be an allele or species variant of the polypeptide coding region of the polynucleotide.

[0100] Modification of the polynucleotide encoding the polypeptide of the present invention may be necessary to synthesize polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ from the polypeptide isolated from its natural source in some engineered manner, such as variants that differ in specific activity, thermostability, optimal pH, etc. These variants can be based on the polynucleotide presented in the form of the mature polypeptide coding sequence of SEQ ID NO: 1, or its cDNA sequence (e.g., a subsequence thereof), and / or by introducing nucleotide substitutions that do not change the amino acid sequence of the polypeptide, but correspond to the codon usage of the host organism intended for production of the enzyme, or by introducing nucleotide substitutions that can produce different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

[0101] Nucleic acid construct

[0102] The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0103] Polynucleotides can be manipulated in a variety of ways to provide expression of a polypeptide. Depending on the expression vector, manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0104] The control sequence can be a promoter, i.e., a polynucleotide that is recognized by the host cell to express a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0105] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900), Rhizomucor miehei (WO 00 / 56900), miehei) lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei β-xylosidase, and NA2-t pi promoter (a modified promoter from an Aspergillus neutral α-amylase gene in which the untranslated leader sequence is replaced by the untranslated leader sequence of an Aspergillus triose phosphate isomerase gene; non-limiting examples include a modified promoter from the gene for an Aspergillus niger neutral α-amylase in which the untranslated leader sequence is replaced by the untranslated leader sequence of an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant promoters, truncated promoters, and hybrid promoters thereof.

[0106] In yeast hosts, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0107] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3′-end of the polynucleotide encoding the polypeptide. Any terminator that functions in the host cell may be used in the present invention.

[0108] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0109] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanus et al., 1992, supra.

[0110] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0111] The control sequence can also be a leader sequence, a non-translated mRNA region that is important for host cell translation. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in a host cell can be used.

[0112] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0113] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0114] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that functions in the host cell may be used.

[0115] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0116] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0117] The control sequence can also be a signal peptide coding region that encodes a signal peptide that is connected to the N-terminus of the polypeptide and directs the polypeptide to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide can inherently include a signal peptide coding sequence that is naturally connected to the segment of the coding sequence of the encoded polypeptide in the translation reading frame. Alternatively, the 5'-end of the coding sequence can include a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally include a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide to enter the secretory pathway of the host cell can be used.

[0118] Effective signal peptide coding sequences for filamentous fungal host cells are those obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0119] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al. (1992), supra.

[0120] The control sequence can also be a propeptide coding sequence encoding a propeptide located at the N-terminal end of a polypeptide. The polypeptide generated is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptide is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), thermophilic myceliophthora laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease, and Saccharomyces cerevisiae α-factor.

[0121] Where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned immediately adjacent to the N-terminus of the polypeptide and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0122] It may also be desirable to add regulatory sequences that regulate the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to chemical or physical stimulation, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include lac, tac, and trp operon systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter, and Aspergillus oryzae glucoamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide will be operably connected to the regulatory sequence.

[0123] Expression vector

[0124] The present invention further relates to a recombinant expression vector comprising a polynucleotide of the present invention, a promoter, and a transcription and translation termination signal. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, and this recombinant expression vector can include one or more convenient restriction enzyme sites to allow the polynucleotides encoding the variant to be inserted or replaced at these sites. Alternatively, the polynucleotides can be expressed by inserting the polynucleotides or a nucleic acid construct comprising the polynucleotides into a suitable vector for expression. When producing the expression vector, the encoding sequence is located in the vector so that the encoding sequence is operably connected to the suitable control sequence for expression.

[0125] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause the expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0126] The carrier can be an autonomously replicating carrier, that is, a carrier existing as an extrachromosomal entity, and its replication is independent of chromosome replication, for example, a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The carrier can include any device for ensuring self-replication. Alternatively, the carrier can be such a carrier, when it is introduced into the host cell, is integrated into the genome and replicated with one or more chromosomes into which it has been integrated. In addition, a single carrier or plasmid or two or more carriers or plasmids (these carriers or plasmids contain the total DNA to be introduced into the genome of the host cell together) or a transposon can be used.

[0127] The vector preferably contains one or more selectable markers that allow easy selection of transformed cells, transfected cells, transduced cells or the like. A selectable marker is a gene whose product provides biocide resistance or virus resistance, heavy metal resistance, prototrophy of auxotrophic cells, etc.

[0128] Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as their equivalents. Preferred for use in Aspergillus cells are the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and the Streptomyces hygroscopicus bar gene. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0129] The vector preferably contains one or more elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0130] For integration into the host cell genome, the vector can rely on the sequence of the polynucleotide encoding polypeptide or any other element in the vector that is integrated into the genome by homologous or non-homologous recombination. Alternatively, the vector can include other polynucleotides for guiding one or more accurate positions in one or more chromosomes integrated into the host cell genome by homologous recombination. In order to increase the possibility of integration at the accurate position, the elements of these integrations should include a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to improve the possibility of homologous recombination. These integration elements can be any sequence homologous to the target sequence in the genome of the host cell. In addition, these integration elements can be non-coding polynucleotides or encoding polynucleotides. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0131] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0132] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00 / 24883). Isolation of the AMA1 gene and construction of a plasmid or vector comprising the gene can be accomplished according to the method disclosed in WO 00 / 24883.

[0133] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication; ARS1; ARS4; the combination of ARS1 and CEN3; and the combination of ARS4 and CEN6.

[0134] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase the production of a polypeptide. The increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by comprising an amplifiable selectable marker gene together with the polynucleotide, wherein cells comprising an amplified copy of the selectable marker gene and thus additional copies of the polynucleotide can be selected by culturing the cell in the presence of an appropriate selective agent.

[0135] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0136] Host cells

[0137] The present invention also relates to recombinant host cells, which include polynucleotides of the present invention, which are operably linked to one or more control sequences, which instruct the production of polypeptides of the present invention. Preferably, the polynucleotides are heterologous, meaning that they do not exist naturally in the host cell. The construct or vector comprising the polynucleotides is introduced into the host cell so that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The term "host cell" encompasses any progeny of a parent cell that is different from the parent cell due to mutations that occur during replication. The selection of host cells depends largely on the gene encoding the polypeptide and its source.

[0138] The host cell can be any cell useful for recombinant production of the polypeptides of the present invention, such as a prokaryotic cell or a eukaryotic cell.

[0139] The host cell may also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0140] The host cell may be a fungal cell. "Fungi" as used herein include Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all mitotic spore fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0141] The fungal host cell can be a yeast cell. "Yeast" as used herein includes yeasts that produce ascospores (Endosporales), basidiomyces and yeasts that belong to Fungi Deuteromyces (Blastomyces). Because the classification of yeast may change in the future, for purposes of the present invention, yeast should be defined as described in the biology and activities of yeast (Biology and Activities of Yeast) (Skinner, Passmore, and Davenport, ed., Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0142] The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0143] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subphylum Eumycota and Oomycota (as defined by Hawkesworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphae extension, and carbon catabolism is obligate aerobic. In contrast, the vegetative growth of yeast (such as Saccharomyces cerevisiae) is by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0144] The filamentous fungal host cell can be an Acremonium, Aspergillus, Bjerkandera, Ceroplastes, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neotrichia, Neurospora, Paecilomyces, Penicillium, Pseudocerviphora, Phlebia, Chytridium, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Toxicodendron, Trametes, or Trichoderma cell.

[0145] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis a eirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium feces, or Chrysosporium spp. merdarium), rent spores, Chrysosporium queenslandicum, tropical chrysosporium, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, rod-shaped fusarium, cereal fusarium, Kuwei fusarium, broadsword fusarium, graminearum fusarium, graminearum fusarium, heterospore fusarium, albizia fusarium, sharp fusarium, multi-branch fusarium, pink fusarium, elder fusarium, color fusarium, pseudo-branch spore fusarium, sulfur-colored fusarium, round fusarium, pseudo-spore fusarium, embellished fusarium, insolens humicola, soft hair humicola, rice black mold, thermophilic myceliophthora, rough chain spore, purple Penicillium, Phanerochaete chrysosporium, radiata fusarium, Pleurotus eryngii, eryngii, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0146] Fungal cells can be transformed in a manner known per se by methods involving protoplast formation, protoplast transformation, and cell wall regeneration. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474 and Christensen et al., 1988, Bio / Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96 / 00787. Yeast can be transformed using procedures described by, for example, Becker and Guarente, in Abelson, JN and Simon, MI, eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, PNAS 75:1920.

[0147] Generation method

[0148] The present invention also relates to a method for producing a polypeptide of the present invention, comprising (a) culturing a cell under conditions conducive to the production of the polypeptide, the cell producing the polypeptide in its wild-type form; and (b) recovering the polypeptide. In a preferred aspect, the cell is an Aspergillus cell. In a more preferred aspect, the cell is an Aspergillus oryzae or Aspergillus niger cell. The present invention also relates to a method for producing a polypeptide of the present invention, comprising (a) culturing a recombinant host cell of the present invention under conditions conducive to the production of the polypeptide; and (b) recovering the polypeptide.

[0149] These host cells are cultured in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, shake flask culture can be performed in a suitable medium and under conditions allowing expression and / or separation of the polypeptide, or small-scale or large-scale fermentation (including continuous, batch, batch feed, or solid-state fermentation) can be performed in a laboratory or industrial fermenter to culture cells. The culture is to use a program known in the art, and occurs in a suitable nutrient medium, which includes carbon and nitrogen sources and inorganic salts. Suitable medium can be obtained from commercial suppliers or can be prepared according to disclosed compositions (for example, in the catalog of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be directly recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

[0150] The polypeptide can be detected using methods known in the art that are specific for the polypeptide. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of the polypeptide.

[0151] The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

[0152] The polypeptide may be purified by a variety of procedures known in the art to obtain a substantially pure polypeptide, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, eds., VCH Publishers, New York, 1989).

[0153] In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the invention expressing the polypeptide is used as a source of the polypeptide.

[0154] Composition

[0155] The present invention also relates to compositions comprising the phospholipase A polypeptides of the present invention with additional components.

[0156] The composition may include a phospholipase A polypeptide of the invention as the major enzyme component, such as a monocomponent composition.

[0157] The present invention also relates to compositions comprising a mixture of a phospholipase A of the present invention and one or more additional phospholipase activities selected from the group consisting of PLA1, PLA2, PLC and PLD.

[0158] Alternatively, the composition may include additional enzymes such as an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, an α-glucosidase, a β-glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, a mannosidase, an oxidase, a pectinolytic enzyme, a peptidoglutaminase, a peroxidase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, or a xylanase. For example, the additional enzyme or enzymes can be produced by a microorganism belonging to the following genera: Aspergillus, such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae; Fusarium, such as Fusarium rod-sporangiformis, Fusarium cerealis, Fusarium kuwei, Fusarium machete, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium albizia, Fusarium oxysporum, Fusarium multibranched, Fusarium pink, Fusarium elderberry, Fusarium color, Fusarium sulfur color, Fusarium round, Fusarium pseudo-sporangiate, or Fusarium venenatum; Humicola, such as Humicola insolens or Humicola lanuginosus; or Trichoderma, such as Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0159] These compositions can be prepared according to methods known in the art and can be in the form of liquid or dry compositions. For example, the composition can be in the form of particles or microparticles. The polypeptide can be stabilized according to methods known in the art.

[0160] Phospholipid hydrolase of the present invention and the compositions comprising these can be prepared together with the component selected from the group consisting of the following: buffer, inorganic salt, solvent, inert solid and mixture thereof. Suitable buffer systems, for example, are made by the aqueous solution of salt or organic acid, amino acid, phosphate, amine or ammonia at a concentration between 0,01M and 1M at pH 2 to 10. Preferably, 0,1 to 0,2M citric acid, acetic acid, alkali metal salt of glycine and / or tris(hydroxymethyl) and ammonia hydrochloride at pH 4 to 8 are used. Preferably, the phospholipid hydrolase is dissolved in a buffered aqueous solution, for example glycine buffer, citric acid buffer, etc. The buffer comprising citrate is found to be very suitable, particularly sodium citrate buffer, preferably at neutral pH.

[0161] The composition of the present invention can include a phospholipase of the present invention fixed to a solid support. The solid support used in the present invention includes a gel. Some examples of gels include agarose, gelatin, glutaraldehyde, chitosan-treated glutaraldehyde, albumin glutaraldehyde, chitosan xanthan gum, toyopearl gel (polymer gel), alginate, alginate polylysine, carrageenan, agarose, glyoxyl agarose, magnetic agarose, dextran agarose, poly (carbamyl sulfonic acid) hydrogel, BSA-PEG hydrogel, phosphorylated polyvinyl alcohol (PVA), monoethylated-N-aminoethyl (MANA), amino, or any combination thereof. Another solid support used in the present invention is a resin or polymer. Some examples of resins or polymers include cellulose, acrylamide, nylon, rayon, polyester, anion exchange resin, AMBERLITE TM XAD-7, AMBERLITE TM XAD-8, AMBERLITE TM IRA-94、AMBERLITE TM IRC-50, polyvinyl, polyacrylic acid, polymethacrylate, or any combination thereof. Another type of solid support for use in the present invention is ceramic. Some examples include non-porous ceramic, porous ceramic, SiO2, AhO3. Another type of solid support for use in the present invention is glass. Some examples include non-porous glass, porous glass, aminopropyl glass, or any combination thereof. Another type of solid support that can be used is a microelectrode. One example is polyethyleneimine-coated magnetite. Graphite particles can be used as a solid support. Other exemplary solid supports used to practice the present invention include diatomaceous earth products and silicates. Some examples include Diatomaceous earth and SILASORB TM , and CELKA Synthetic calcium and magnesium silicates.

[0162] Some examples of methods for immobilizing enzymes include, for example, electrostatic droplet generation, electrochemical means, by adsorption, by covalent addition, by cross-linking, by chemical reaction or chemical process, by encapsulation, by entrapment, by calcium alginate or by poly-(2-hydroxymethyl methacrylate). Similar methods are described in Methods in Enzymology, Immobilized Enzymes and Cells, Part C, 1987, Academic Press, edited by SP Colowick and N.O. Kaplan, Vol. 136; and Immobilized Enzymes and Cells, 1997, Humana Press, edited by GF Bickerstaff, Series: Methods in Biotechnology, edited by JM Walker.

[0163] use

[0164] Phospholipid hydrolase of the present invention can be applied to the process of removing phospholipid from a kind of oil, for example, a kind of vegetable oil, animal oil or fat, tallow or animal fat.The application that can use phospholipid hydrolase of the present invention comprises i) the degumming of oil, for example, vegetable oil or edible vegetable oil, in a kind of process, this process is included in the hydrolysis phospholipid to release the triglyceride oil that is trapped in the gum fraction from water degumming, ii) in a kind of process, this process comprises the hydrolysis phospholipid to obtain the phospholipid emulsifier of improvement, wherein said phospholipid is lecithin specifically, iii) in a kind of process, this process is used to improve the filterability of the aqueous solution or slurry of carbohydrate source, this carbohydrate source comprises phospholipid, iv) in a kind of process, this process is used to produce animal feed product, v) in a kind of process, this process is used to produce biofuel, for example, biodiesel, vi) in a kind of process, this process is used to produce detergent product, and / or vii) in a kind of process, this process is used to make baked product, this process comprises adding phospholipid hydrolase in dough, and bakes dough to make baked product.

[0165] Degumming Phospholipid hydrolase of the present invention can be used for degumming oil, for example animal oil or fat, tallow, animal tallow or vegetable oil, that is, in the process of reducing the phospholipid content of edible oil.Referring to, for example, WO 2007 / 103005 and US 2008 / 0182322.Such process can be used for purifying any edible oil that comprises phospholipid, for example, vegetable oil such as soybean oil, rapeseed oil or sunflower oil or any other oil mentioned under the definition of crude oil.

[0166] One aspect of the present invention is a method, the method reduces the content of the phosphorus-containing component of edible oil by phospholipase treatment, the method is included in the condition that is enough to make enzyme and phospholipid react to form free fatty acid and lysophospholipid product, the oil is contacted with the aqueous solution of polypeptide of the present invention.Lysophospholipid is dissolved in water phase, causes the reduction of phosphorus content in oil.Free fatty acid will stay in oil.After phospholipase treatment, water phase is separated from processed oil.Phosphorus lipid is usually measured as " phosphorus content " in parts per million in oil.Table 1 has set forth the typical amount of the phospholipid present in main oil crops, and the distribution of the percentage of the phospholipid present in this oil accounted for by various functional groups.

[0167] Table 1: Typical levels and distribution of phospholipids for common oilseeds#tables0#

[0168] The phospholipase treatment can be carried out directly in the crude oil or after removing mucilage (mucilage) (e.g., by wet refining). In a preferred embodiment, the oil is selected from crude oil, water-degummed oil, acid-degummed oil and caustic refined oil.

[0169] After wet refining, at the beginning of the treatment with the phospholipase, the oil typically contains 50-250 ppm of phosphorus as phospholipids. In a preferred embodiment of the invention, the treatment reduces the phosphorus value, preferably to below 20 ppm, such as below 15 ppm, such as below 11 ppm, such as to below 10 ppm, below 9 ppm, below 8 ppm, below 7 ppm, below 6 ppm or even below 5 ppm.

[0170] The phospholipase treatment is carried out by dispersing an aqueous solution of the phospholipase, preferably in droplets with a mean diameter of less than 10 microns. The amount of water is preferably 0.5%-5% by weight relative to the oil. An emulsifier may optionally be added. Mechanical stirring may be used to maintain the emulsion. Stirring may be accomplished with a high shear mixer at a tip speed greater than 1400 cm / s.

[0171] In certain embodiments, a method for degumming oil that is suitable includes a) mixing an aqueous solution of acid with oil to obtain an acidic mixture with a pH of about 1 to 4, b) mixing alkali with an acidic mixture to obtain a reaction mixture with a pH of about 6-9, and c) degumming the reaction mixture with an enzyme of the present invention to obtain degummed oil. In certain embodiments, mixing in step a) and / or b) produces a kind of emulsion, which includes an average droplet size of about 15 microns to about 45 microns of water phase. In certain embodiments, mixing in step a) and / or b) produces a kind of emulsion, which includes at least 60% of water phase by volume, and the droplet size is between about 15 microns to about 45 microns, wherein the percentage of water phase is based on the total volume of water phase. Any acid considered suitable by those skilled in the art can be used in the method provided here. In certain embodiments, acid is selected from the group consisting of phosphoric acid, acetic acid, citric acid, tartaric acid, succinic acid, and mixtures thereof. Any acid considered suitable by those skilled in the art can be used in the method provided here. In certain embodiments, the base is selected from the group consisting of sodium hydroxide, potassium hydroxide, sodium silicate, sodium carbonate, calcium carbonate, and combinations thereof.

[0172] In a preferred embodiment, the phospholipase treatment can be carried out at a pH in the range of about 1.5 to about 7.0, preferably 2.5 to 6, preferably 3.0 to 5.5, preferably from 3.5 to 5.0, and most preferably from 3.0 to 4.5. The pH is measured in the emulsion or in the intermediate phase between the oil and the aqueous solution. Suitable temperatures are generally 30°C-80°C (particularly 30°C-70°C, 40°C-60°C, e.g., 50°C-55°C). In a preferred embodiment, the temperature of the oil is between 55°C and 80°C, more preferably between 60°C and 75°C and most preferably between 65°C and 70°C.

[0173] The reaction time is typically 1-12 hours (e.g., 1-6 hours or 1-3 hours, most preferably the reaction time is between 1.5 and 4 hours, even more preferably between 1.5 and 2 hours). The dosage of a suitable enzyme is typically 0.1-10 mg / liter (e.g., 0.5-5 / liter). The phospholipase treatment can be carried out in batches, such as in a stirred tank; or it can be continuous, such as a series of stirred tank reactors. The phospholipase treatment can be followed by separation of the aqueous phase and the oil phase. The separation can be carried out by conventional means, such as centrifugation. When using liquid fat, the aqueous phase will contain the phospholipase, and the enzyme can be reused to improve the economy of the process.

[0174] In addition to the phospholipases of the present invention, a further enzyme can be applied to the above-outlined degumming process. In a preferred embodiment, the further enzyme is a polypeptide having phospholipase A1, A2, B and / or C activity. A suitable polypeptide having phospholipase A1 activity can be LECITASE ULTRA available from Novozymes A / S. A suitable polypeptide having phospholipase C activity can be, for example, PURIFINE available from DSM or PLC's described in WO 2012 / 062817 and PCT / CN 2013 / 089106.

[0175] Phospholipid emulsifier Phospholipidase of the present invention can be used for partial hydrolysis of phospholipids, preferably lecithin, to obtain improved phospholipid emulsifiers. The application is further described in Ullmann's Encyclopedia of Industrial Chemistry (Publisher: VCH Weinheim (1996)), Japanese Patent 2794574, and JP-B 6-087751.

[0176] filter Phospholipases of the invention can be used to improve the filterability of aqueous solutions or slurries of carbohydrate sources by treating the carbohydrate source with the phospholipases. This is particularly useful for solutions containing slurries of starch hydrolysates, particularly wheat starch hydrolysates, which tend to be difficult to filter and tend to give turbid filtrates. The treatment can be accomplished similarly to EP 219,269 (CPC International).

[0177] Animal Feed Phospholipid hydrolase of the present invention can be used in a process for producing animal feed, and this process comprises mixing phospholipid hydrolase with feed material comprising at least one phospholipid.This process can be similar to EP 743 017 and finish.

[0178] Biodiesel When degumming is achieved in the same process, the phospholipidase of the present invention can be used in combination with one or more lipolytic enzymes to convert fats and oils into fatty acid alkyl esters. Such a process is described in US 8,012,724.

[0179] Detergent : The phospholipase of the present invention can be added to a detergent composition and thus the phospholipase is used as a component of a detergent composition.

[0180] The detergent composition may, for example, be formulated as a hand or machine laundry additive composition, including laundry additive compositions suitable for pretreatment of soiled fabrics and fabric softener compositions with added rinse aid, or as a detergent composition for use in general household hard surface cleaning operations, or as a detergent composition for hand or machine dishwashing operations.

[0181] Baking The phospholipases of the invention can be used to produce dough and baked products from dough, as well as to produce baking compositions and baking additives.

[0182] Typically the dough includes wheat meal or wheat flour and / or other types of meal, flour or starch, such as corn meal, corn starch, rye meal, rye flour, oat flour, oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potato flour or potato starch.

[0183] The dough can be fresh, frozen or par-baked.

[0184] The dough is typically a leavened dough or a dough that is to undergo leavening. The dough can be leavened in different ways, for example by adding a chemical leavening agent, such as sodium bicarbonate, or by adding an enzyme (leavening dough), but preferably the dough is leavened by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), such as a commercially available strain of S. cerevisiae.

[0185] The dough may also include other traditional dough ingredients, for example, proteins such as milk powder, gluten, and soy; eggs (whole eggs, egg yolks or egg whites); oxidants such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; amino acids such as L-cysteine; sugars; salts such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate.

[0186] The dough may include fat (triglycerides) such as granulated fat or shortening, but the invention is particularly applicable to a dough to which less than 1% by weight of fat is added, and particularly to a dough made without added fat.

[0187] The dough may further include an emulsifier such as mono- or diglycerides, diacetyl tartaric acid esters of mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetate esters of monoglycerides, polyoxyethylene stearates, or lysolecithin.

[0188] The dough may be used for any kind of baked product prepared from the dough, of a soft or crisp nature, of white, light or dark type. Examples are bread (especially white, wholemeal or rye bread), typically in the form of loaves or rolls, baguette type bread, pita bread, tortillas, crepes, pancakes, biscuits, crackers, cookies, pie crusts, crackers, steamed buns, pizza and the like.

[0189] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0190] Materials and Methods

[0191] Lipolytic activity (LU)

[0192] Lipolytic activity (lipase activity) can be determined using tributyrin as substrate. The method is based on the hydrolysis of tributyrin by the enzyme and the base consumption as a function of time to keep the pH constant during the hydrolysis.

[0193] One lipase unit (LU) is defined as the amount of enzyme that releases 1 micromole of titratable butyric acid per minute under standard conditions (i.e., at 30°C; pH 7.0; 0.1% w / v Gum Arabic as emulsifier and 0.16 M tributyrin as substrate). One KLU is 1000 LU.

[0194] Phospholipase A activity (LEU)

[0195] In the LEU assay, phospholipase A activity is determined from the ability to hydrolyze lecithin at pH 8.0, 40°C. The hydrolysis reaction can be followed by titration with NaOH for a reaction time of 2 minutes. The phospholipase from Fusarium oxysporum (LIPOPAN F) disclosed in WO 1998 / 26057 has an activity of 1540 LEU / mg enzyme protein and can be used as a standard.

[0196] Plate assay

[0197] A) The buffer was a mixture of 100 mM HEPES and 100 mM citrate with pH adjusted from pH 3.0 to pH 7.0.

[0198] B) 2% agarose (Litex HSA 1000) was prepared by mixing in buffer (A) and boiling for 5 minutes followed by cooling to approximately 60°C.

[0199] C) The substrate was L-alpha phosphatidylcholine, 95% from soy (Avanti 441601) dispersed in water (MilliQ) at 60°C for 1 min using an Ultra Turrax.

[0200] D) The purified enzyme solution of LECITASE ULTRA and the mature phospholipase of SEQ ID NO: 2 were diluted to 0.4 mg / ml.

[0201] Plates were cast by gently mixing a mixture of 5 ml substrate (C) and 5 ml agarose (B) into a culture dish with a diameter of 7 cm and cooled to room temperature before holes with a diameter of approximately 3 mm were punched by vacuum. Ten microliters of diluted enzyme (D) were added to each well before the plate was sealed with a sealing film and placed in an incubator at 55° C. for 48 hours. The plate was taken out for photography regularly.

[0202] N-terminal sequencing procedures

[0203] Using Applied Biosystems Protein sequencing system was used for N-terminal sequencing analysis. Purification was performed on precast 4%-20% SDS polyacrylamide gels (Life Technologies). The gels were run according to the manufacturer's instructions and blotted to PVDF membrane (Applied Biosystems). For N-terminal amino acid sequencing, the major protein band was excised and placed on The protein sequence determination system was placed in a blotting box. N-terminal sequencing was performed using a method run file for PVDF membrane samples (pulse liquid PVDF) according to the manufacturer's instructions. The N-terminal amino acid sequence can be inferred from the 7 chromatograms corresponding to amino acid residues 1 to 7 by comparing the retention time of the peaks in the chromatogram with the retention time of PTH amino acids in the standard chromatogram.

[0204] Enzymes

[0205] The enzyme preparation comprises a purified enzyme protein (TL PLA) of the phospholipase of the present invention, which is the mature polypeptide of SEQ ID NO: 2, such as amino acids 20 to 296 or 28 to 296 of SEQ ID NO: 2.

[0206] A commercial preparation available from Novozymes is LECITASE ULTRA. LECITASE ULTRA includes a protein engineered microbial polypeptide having both triglyceride lipase and phospholipase A activities. The polypeptide has the amino acid sequence shown in SEQ ID NO:5.

[0207] Example 1 Characterization of enzymes

[0208] The thermal stability of the phospholipases of the invention was determined by differential scanning calorimetry (DSC) using a VP-capillary differential scanning calorimeter (MicroCal Inc., Piscataway, NJ, USA). The thermal denaturation temperature Td (° C.) was obtained as the top of the denaturation peak (major endothermic peak) in the thermogram (Cp vs. T) obtained after heating an enzyme solution (approximately 0.5 mg / ml) in a buffer (50 mM sodium acetate at pH 4.0 or pH 5.0) at a constant programmed heating rate of 200 K / hr.

[0209] Sample solutions and reference solutions (approximately 0.2 ml) were loaded into the calorimeter from storage conditions at 10° C. (reference: buffer without enzyme) and thermally pre-equilibrated at 20° C. for 20 minutes before a DSC scan from 20° C. to 100° C. The denaturation temperatures were determined with an accuracy of approximately + / - 1° C. at 82° C. at pH 4.0 and at 81° C. at pH 5.0.

[0210] When expressed in Aspergillus oryzae, using the procedure described above, the polypeptide of SEQ ID NO: 2 was identified as having the following N-terminal sequence:

[0211] 35 kDa band with N-terminal DVSSSVL corresponding to residues 28-34 of SEQ ID NO: 2

[0212] 25 kDa band with N-terminal EADLDFP corresponding to residues 117-123 of SEQ ID NO: 2

[0213] 25 kDa band with an N-terminal LDFPLTD corresponding to residues 120-126 of SEQ ID NO: 2

[0214] Based on the plate assay described above, the phospholipase of the present invention exhibits activity in the following pH range between 3 and 5. For comparison, Lecitase Ultra exhibits weaker activity at pH 3 and 4 (see Figure 3 ).

[0215] Example 2: Degumming

[0216] Due to the thermostability of the phospholipases used in commercial enzymatic degumming, the process is usually carried out at a temperature not exceeding 55° C. However, the phospholipases of the invention are very thermostable, which allows the degumming process to be carried out at higher temperatures. In this example, soybean oil was degummed at a temperature of 70° C. using the phospholipases of the invention or the prior art phospholipases LECITASE ULTRA.

[0217] The oil was initially treated with orthophosphoric acid (85% solution) to convert the insoluble salts to a more hydratable form and obtain a pH suitable for the enzymes. The acid was used in an amount equal to 0.05% (100% pure orthophosphoric acid) based on the amount of oil.

[0218]

[0219] * The pH in the oil was determined by extraction of the water soluble acid from the oil followed by measurement of the pH of the aqueous phase.To improve the stability of the pH measurement the ionic strength of the aqueous phase was increased by adding 1% w / w KCl.

[0220] LECITASE ULTRA was dosed at the industry recommended amount of 30 mg formulated enzyme product / kg oil=30 ppm, and the phospholipases of the present invention were dosed at amounts equal to 34 mg enzyme product / kg oil=34 ppm, 67 mg enzyme product / kg oil=67 ppm, 224 mg enzyme product / kg oil=224 ppm and 537 mg enzyme product / kg oil=537 ppm.

[0221] The aliquot of 250g crude soybean oil in 500ml blue lid shake bottle is heated to 70 ℃ in the water bath with magnetic agitation at 200rpm. 85% phosphoric acid solution of 87 microlitres is applied to each shake bottle, and the mixture is high-shear mixed 10sec at 12.000rpm by Ultra-Turrax. After this, the mixture is hatched 15min at 70 ℃ and at 200rpm. Enzyme solution and MilliQ-water are applied to the total water addition (3% oil) of 7.5ml. By Ultra-Turrax, the mixture is high-shear mixed 10sec at 12.000rpm, and at 200rpm, hatched 2 hours at 70 ℃ with stirring. After the enzyme reaction, the product mixture is heated to more than 100 ℃ by microwave so that the enzyme is inactivated and promotes the separation of glue. By realizing the separation of oil and wet glue with 2000g centrifugal 5min.

[0222] The yield of free fatty acids (FFA) in the degummed oil was quantified by titration. The phosphorus content in the degummed oil was determined by ICP-OES. The results are shown in the table below.

[0223]

[0224] The greatest increase in FFA yield and the greatest reduction in phosphorus content were obtained with the phospholipase of the invention at pH ~4 at 70°C when compared to Lecitase Ultra.

[0225] Example 3 Phospholipase Hydrolysis Measured by Phospholipid Reduction

[0226] The phospholipases of the present invention and the phospholipases of LECITASE ULTRA were tested for their ability to hydrolyze the phospholipids phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC) in a low water environment at 50°C and at 70°C, and at pH 4.0 and 5.5. The test substrate was produced from soybean oil, which was spiked with phospholipids PA, PE, PI, and PC. The substrate included 50 mg phospholipids / 1 mL oil. In a thermoshaker, 250 microliters of the sample were incubated with the phospholipases of the present invention for 2 h at the desired temperature (50°C and 70°C). The phospholipases were applied as 10 microliters of an aqueous solution comprising an amount of purified enzyme protein equal to 100 mg / kg oil. In addition to the enzyme reactions, blank reactions were also incubated in which the enzyme solution was replaced by the addition of water.

[0227] pass 31 P NMR analysis samples. The principle of NMR analysis is to convert phospholipids into lysophospholipids. 31 The chemical shift of P was altered. The unconverted phospholipids were then quantified by integration. The data are presented as % of residual phospholipids relative to the blank reaction.

[0228]

[0229] Both enzymes have good activity on the phospholipids PA, PE, PI and PC. The maximum phosphorus reduction was achieved with the phospholipase of the invention, which reduced the phosphorus content to below the detection limit at 50°C and pH 5.5. At 70°C and pH 4.0, the phospholipase of the invention showed surprisingly high activity relative to LECITASE ULTRA.

Claims

1. An isolated polypeptide having phospholipase A activity, the isolated polypeptide consisting of SEQ ID NO: 2, or amino acids 28 to 296 of SEQ ID NO: 2, or a polypeptide of SEQ ID NO:

3.

2. An isolated polynucleotide encoding the isolated polypeptide having phospholipase A activity according to claim 1.

3. A nucleic acid construct or expression vector comprising the polynucleotide of claim 2 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

4. A recombinant host cell comprising the polynucleotide of claim 2 operably linked to one or more control sequences that direct the production of the polypeptide.

5. A method for producing the polypeptide according to claim 1, the method include: (a) cultivating a cell that produces the polypeptide in its wild-type form under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

6. A method for producing a polypeptide having phospholipase A activity, the method comprising: include: (a) culturing the host cell according to claim 4 under conditions conducive to production of the polypeptide; and (b) recovering the polypeptide.

7. A composition comprising: a) a polypeptide as claimed in claim 1 or a polypeptide obtainable by the method as claimed in any one of claims 5 or 6; and, b) optionally a further enzyme.

8. The composition of claim 7, wherein the additional enzyme is an enzyme selected from the group consisting of composition: Phospholipase A1, phospholipase A2, phospholipase B, phospholipase C, and phospholipase C.

9. Use of the polypeptide according to claim 1 or the composition according to any one of claims 7 or 8 in the process of hydrolyzing phospholipids.

10. Use of a polypeptide as claimed in claim 1 or a composition as claimed in any one of claims 7 or 8 in the production of a baking composition or baking additive or in the production of dough or baked products therefrom.

11. Use of the polypeptide according to claim 1 or the composition according to any one of claims 7 or 8 in reducing the phospholipid content of edible oil.

12. A method for reducing the content of phosphorus-containing components in an edible oil using a phospholipase treatment, the method comprising contacting the oil with an aqueous solution of a polypeptide as claimed in claim 1 until the phosphorus content of the oil is reduced, and then separating the aqueous phase from the treated oil.

13. The method according to claim 12, wherein the oil is selected from crude oil, water degummed oil and acid degummed oil.

14. A method according to claim 12 or 13, wherein the temperature of the oil is between 60°C and 75°C.

15. The method according to claim 12 or 13, wherein the phospholipase treatment is carried out at a pH between 3.0 and 5.5.

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