CN101379187A

CN101379187A - Novel phospholipase D

Info

Publication number: CN101379187A
Application number: CNA2007800043397A
Authority: CN
Inventors: 岩崎雄吾; 中野秀雄; 高桥哲也
Original assignee: Nagoya University NUC
Current assignee: Nagoya University NUC
Priority date: 2006-02-02
Filing date: 2007-02-01
Publication date: 2009-03-04
Also published as: WO2007089038A8; JPWO2007089038A1; JP5156951B2; JP2012187108A; WO2007089038A1; JP5420016B2

Abstract

A modified phospholipase D having an ability to synthesize phosphatidylinositol which contains: (A) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:2 by substitution of at least one of the amino acid residues at the 187-, 191- and 385-positions by other amino acid residue(s); or (B) an amino acid sequence of a polypeptide derived from the amino acid sequence as described in the above (A) by substitution, deletion, insertion and/or addition of at least one amino acid residue at a position different from the 187-, 191- and 385-positions and having an ability to synthesize phosphatidylinositol. By using this modified phospholipase D, phosphatidylinositol can be efficiently produced from a phospholipid. It is also intended to provide a method of screening a phospholipase D having an ability to synthesize an acylglycerophospholipid having glycol group.

Description

Novel phospholipase D

Technical field

The present invention relates to have the novel Phospholipase D of the ability of synthetic phospholipid acyl inositol.

Background technology

Phosphatide can be used for the preparation of composition, drug prescription and the liposome of emulsifying agent, makeup.Phosphatide from natural goods contains the mixtures such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositols (PI), phosphatidylserine (PS), phosphatidic acid (PA), sphingophospholipid conventionally.For separated specific phosphatide from natural phospholipid, can carry out the solvent of the extraction of methyl alcohol, ethanol, Virahol, hexane, chloroform equal solvent or recrystallization etc. and distinguish method, use the column chromatography for separation method of the sorbent materials such as silica gel, Lithium Oxide 98min, ion exchange resin, utilize CdCl ₂the partition method of the derivative of mixture or acetylate etc.

In natural goods (soybean lecithin etc.), carry out separation and the purifying of PI, this is for example as described in Japanese kokai publication hei 5-97873 communique and Japanese kokai publication hei 5-97872 communique.In Japanese kokai publication hei 5-97873 communique, recorded the lipid that contains PI has been dissolved in non-polar solvent, it is contacted with the aqueous polar solvent that contains that contains alkaline matter, then to non-polar solvent layer with do not contain the new of alkaline matter and carry out the distribution of liquid liquid containing aqueous polar solvent, PI is extracted into the method in moisture polar solvent, layer.In Japanese kokai publication hei 5-97872 communique, recorded the mixed phosphatide that contains PI has been dissolved in the mixed solvent of the lower alcohol that contains water or acetic acid solution and non-polar solvent, carried out the method for high performance liquid chromatography.Because aforesaid method needs a large amount of solvent etc., the high purityization of gained PI is more difficult, and high purity product are very expensive.

In addition, have the method for organic chemical synthesis PI, but route of synthesis contains the complicated route of synthesis in a lot of steps ground that comprises protection and deprotection, so efficiency is not high yet.

Also carried out preparing by enzyme process the trial of phosphatide in the past.Phospholipase D [EC 3.1.4.4] (below also referred to as " PLD ") is that catalysis generates the enzyme of PA and alcohol moiety by the phosphodiester bond hydrolysis of glyceryl phosphatide.Except this hydrolytic activity, PLD is the mutual conversion of the polar group of catalysis phosphatide also.This process quilt is called " phosphatidyl shift reaction ".Utilize phosphatidyl shift reaction, can be by the natural few phospholipid of phospholipid synthesis of natural amount being rich in.

Several natural phospholipids that resemble phosphatidyl glycerol (PG), PE or PS etc. are to adopt PLD catalysis phosphatidyl shift reaction, for example, by Yelkin TTS or PC synthetic (Japanese kokai publication hei 6-269287 communique, Japanese kokai publication hei 5-292981 communique, TOHKEMY 2002-51794 communique and TOHKEMY 2005-261362 communique).In Japanese kokai publication hei 6-269287 communique, recorded to use and take the microorganism of streptomyces and be the PLD of origin, under specific reaction conditions, carry out alkali permutoid reaction, obtain the method for target phosphatide, successfully generated PG, PE, PS and phosphatidyl propyl alcohol.In Japanese kokai publication hei 5-292981 communique, when the PLD by from microorganism carries out alkali transformationreation, acquisition target phosphatide, use sequestrant, successfully generated PG, PE and PS.Also generated PI, but alkali turnover ratio is low.In TOHKEMY 2002-51794 communique, record, not with an organic solvent, the phosphatide as raw material, the acceptor with hydroxyl, PLD and water are fully mixed, carry out homogenizing, at 15-65 ℃, carry out enzymatic reaction, successfully generated PS and PG.In TOHKEMY 2005-261362 communique, recorded the generation method of the phosphatidylserine that utilizes phosphatidyl shift reaction.

In Japanese kokai publication hei 3-87191 communique, recorded and used PLD to prepare the method for PI.In the method, using PLD with as the mixed phosphatide of raw material, do the used time, utilize PLD not with phosphatide in PI effect but be optionally hydrolyzed other phospholipid composition, obtain thus unreacted PI.The method must be after adding PLD, further to add alkali or acid phosphatase.

At present, character and the structure for PLD is studied (such as people such as R.Ulbrich-Hofmann, Biotechnology Letters,, 27 volumes, 535-544 page in 2005).In addition, in order to improve the validity of PLD, various changes (people such as TOHKEMY 2004-97011 communique and TOHKEMY 2005-80519 communique and Xi Chuan, Japanese chemurgy meeting 2004 annual general meetings lecture main ideas, 268 pages) have also been carried out.In TOHKEMY 2004-97011 communique and TOHKEMY 2005-80519 communique, recorded the stability-enhanced modification type PLD to organic solvent or heat.And the people such as Xi Chuan, Japanese chemurgy can 2004 annual general meetings lecture main ideas, record in 268 pages, have obtained having the phosphatide of naphthyl---and phosphatidyl-1-naphthols (P1NAP) has the variation PLD of degrading activity.

Summary of the invention

The object of the present invention is to provide and can be prepared efficiently by phosphatide the method for phosphatidylinositols (PI).The present invention also aims to provide the modification type PLD of the ability with synthetic PI.And, the object of the present invention is to provide the method for screening PLD, this PLD has the synthetic ability that has the acylglycerol phosphatide of glycol-based.

The invention provides the modification type Phospholipase D (below also referred to as " the modification type PLD of synthetic PI ") of the ability with synthetic phospholipid acyl inositol.The modification type PLD of this synthetic PI is the modification type Phospholipase D with the ability of synthetic phospholipid acyl inositol, and it contains

(A) at least one amino-acid residue of 187 of the aminoacid sequence shown in SEQ ID NO.2,191 and 385 s' amino-acid residue is replaced into the aminoacid sequence of other amino-acid residue gained; Or

(B) in the aminoacid sequence of this (A), at at least one amino-acid residue with being positioned at these 187,191 and 385 s' amino-acid residue different positions, there is displacement, disappearance, insert and/or additional, and there is the aminoacid sequence of polypeptide of the ability of synthetic phospholipid acyl inositol.

In one embodiment, 187 amino acids residues in aminoacid sequence shown in above-mentioned SEQ ID NO.2 are replaced into a kind of radical amino acid replacement that is selected from phenylalanine residue, serine residue, tyrosine residues, α-amino-isovaleric acid residue, glycine residue, arginine residues, histidine residues, asparagine residue and leucine residue, and/or

191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are replaced into a kind of amino-acid residue that is selected from arginine residues, threonine residues, Isoleucine residue, glycine residue, α-amino-isovaleric acid residue, phenylalanine residue, leucine residue, asparagicacid residue, methionine residues, tryptophan residue and alanine residue, and/or

385 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are replaced into a kind of amino-acid residue that is selected from cysteine residues, tryptophan residue, leucine residue, methionine residues, arginine residues, glutaminic acid residue, serine residue, phenylalanine residue, α-amino-isovaleric acid residue and proline residue.

In another embodiment, above-mentioned modification type Phospholipase D is selected from following:

(1) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is leucine residue;

(2) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are threonine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is cysteine residues;

(3) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are Isoleucine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tryptophan residue;

(4) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are glycine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(5) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is leucine residue;

(6) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(7) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tryptophan residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is methionine residues;

(8) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tryptophan residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is arginine residues;

(9) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are serine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is glutaminic acid residue;

(10) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are serine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is methionine residues;

(11) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are serine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is methionine residues;

(12) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(13) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is arginine residues;

(14) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(15) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(16) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is glutaminic acid residue;

(17) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are glycine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are asparagicacid residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is leucine residue;

(18) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tryptophan residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tryptophan residue;

(19) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is serine residue;

(20) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(21) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is phenylalanine residue;

(22) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are histidine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are methionine residues, and 385 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are the modification type Phospholipase D of α-amino-isovaleric acid residue;

(23) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(24) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are asparagine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are alanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(25) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are alanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(26) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are alanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is phenylalanine residue;

(27) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tyrosine residues;

(28) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tryptophan residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is proline residue;

(29) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type Phospholipase D that is tryptophan residue.

The present invention also provides the gene of the modification type PLD of the above-mentioned synthetic PI of coding.

The present invention provides again the method for preparing phosphatidylinositols or derivatives thereof.The step that the method comprises the modification type PLD reaction that makes raw material phosphatide, inositol or derivatives thereof, above-mentioned synthetic PI.

The present invention further provides the method for screening PLD, this PLD has the synthetic ability that has the acylglycerol phosphatide of glycol-based.The method includes the steps of:

Use raw material phosphatide, the compound with glycol-based, PLD, carry out phosphatidyl shift reaction, generate the step of the acylglycerol phosphatide with this glycol-based;

Make the acylglycerol phospholipid oxidation with this glycol-based generating, generate the step of aldehyde; And

Detect the step of this aldehyde generating.

Here, the generation of this aldehyde is the index that this PLD has the ability of synthetic this target acylglycerol phosphatide.

In one embodiment, the compound that has an above-mentioned glycol-based is inositol or derivatives thereof.

In another embodiment, in above-mentioned oxidation step, use the oxygenant that is selected from Periodic acid or its salt, lead tetraacetate and sodium bismuthate.

In yet another embodiment, the detection of above-mentioned aldehyde is that the hydrazone compound that reacts generation by detecting this aldehyde with hydrazine compound carries out.

According to the present invention, in the phosphatidyl shift reaction of acylglycerol phosphatide, can obtain thering is the PLD that the compound of large volume as inositol is imported to the ability in phosphatide.By using this PLD, can by phosphatide, prepare PI easy and efficiently.And according to the present invention, can promptly screen novel PLD, this novel PLD has the synthetic ability that has the acylglycerol phosphatide of glycol-based.

The best mode carrying out an invention

The present invention finds: the PLD with the ability of synthesizing the acylglycerol phosphatide with the large volume polar portion as inositol ring can be the PLD with following structure: the amino-acid residue of the specific position at the enzyme catalysis position of the PLD of unmodified type is replaced, its sterically hindered mitigation.

In this specification sheets, " unmodified type PLD " refers to the PLD of the synthesis capability of the acylglycerol phosphatide (for example phosphatidylinositols (PI)) without tool glycol-based.The natural type PLD that " unmodified type PLD " is included in natural generation in biology with the nucleic acid of this natural type of coding PLD is imported to the restructuring PLD that expresses in the biology different from origin originally and obtain.

In addition, the PLD (saltant type PLD) that has the variation the displacement of carrying out except obtaining the PLD of synthesis capability is also in the scope of this term.The mosaic type PLD of artificial preparation is also within the scope of this in addition.

In this specification sheets; the aminoacid sequence that the PLD for example, with acylglycerol phosphatide (PI) synthesis capability of tool glycol-based has in the PLD of above-mentioned unmodified type, the amino-acid residue of specific position changes; therefore, for convenience is also referred to as " modification type PLD "." modification type PLD " is for example the PLD with following aminoacid sequence: in the PLD of unmodified type, can obtain the aminoacid sequence of the replaced formation of amino acid of the PLD of the above-mentioned synthesis capability of tool.This amino-acid substitution can be the displacement of natural generation and the displacement of artificial preparation both.

(the modification type PLD with the ability of synthetic phospholipid acyl inositol)

The invention provides the modification type Phospholipase D (the modification type PLD of synthetic PI) of the ability with synthetic phospholipid acyl inositol.The modification type PLD of above-mentioned synthetic PI has following structure: the amino-acid residue of the specific position at the enzyme catalysis position of unmodified type PLD is replaced, its sterically hindered mitigation.In addition, up to now, not yet know the PLD with PI synthesis capability.

At least one amino-acid residue that the modification type PLD of synthetic PI of the present invention comprises 187,191 of aminoacid sequences shown in (A) SEQ ID NO.2 and 385 amino acids residues is replaced into the aminoacid sequence that other amino-acid residue obtains; Or (B) in the aminoacid sequence of this (A), at least one amino-acid residue of the position different from the amino-acid residue that is positioned at these 187,191 and 385 has displacement, disappearance, insert and/or additional, and has the aminoacid sequence of polypeptide of the ability of synthetic phospholipid acyl inositol.

In the present invention, as long as replaceable, disappearance, insertion and/or additional amino-acid residue number can form the enzyme with required enzymic activity, can be arbitrary number.For example, it can be the arbitrary number of the sequence with sequence identity of following explanation, be generally 250 or following, for example 150 or following, 100 or following or 50 or following, preferably 30 or below, more preferably 20 or following, further preferably 16 or following, further preferably 5 or following, further preferred 0-3 amino-acid residue again.Those skilled in the art can use site-directed mutagenesis method etc., import suitable displacement, disappearance, insertion and/or addition mutation, can change thus the structure of protein.In addition, amino acid whose sudden change occurs at occurring in nature, therefore, is not only the enzyme that amino acid artificial mutation is obtained, as long as have required enzymic activity, the enzyme that amino acid mutation has occurred at occurring in nature is also contained in above-mentioned PLD.The method and apparatus that determines whether to have required enzymic activity is that those skilled in the art are known, for example, can implement according to the method for following explanation.

In an embodiment, the modification type PLD of synthetic PI of the present invention can be:

187 amino acids residues in aminoacid sequence shown in SEQ ID NO.2 are replaced into a kind of radical amino acid replacement that is selected from phenylalanine residue, serine residue, tyrosine residues, α-amino-isovaleric acid residue, glycine residue, arginine residues, histidine residues, asparagine residue and leucine residue, and/or

More particularly, the modification type PLD of synthetic PI of the present invention has following modification type PLD:

(1) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is leucine residue;

(2) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are threonine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is cysteine residues;

(3) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are Isoleucine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tryptophan residue;

(4) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are glycine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(5) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is leucine residue;

(6) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(7) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tryptophan residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is methionine residues;

(8) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tryptophan residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is arginine residues;

(9) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are serine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is glutaminic acid residue;

(10) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are serine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is methionine residues;

(11) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are serine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is methionine residues;

(12) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are phenylalanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(13) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is arginine residues;

(14) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(15) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(16) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is glutaminic acid residue;

(17) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are glycine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are asparagicacid residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is leucine residue;

(18) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tryptophan residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tryptophan residue;

(19) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is serine residue;

(20) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(21) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is phenylalanine residue;

(22) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are histidine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are methionine residues, and 385 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are the modification type PLD of α-amino-isovaleric acid residue;

(23) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are arginine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(24) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are asparagine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are alanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(25) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are alanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(26) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are alanine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is phenylalanine residue;

(27) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are leucine residue, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tyrosine residues;

(28) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tryptophan residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is proline residue;

(29) 187 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are α-amino-isovaleric acid residue, 191 amino acids residues of the aminoacid sequence shown in above-mentioned SEQ ID NO.2 are tyrosine residues, and 385 amino acids residues of the aminoacid sequence shown in the above-mentioned SEQ ID NO.2 modification type PLD that is tryptophan residue.

The modification type PLD of synthetic PI of the present invention for example can be used the normally used site-directed mutagenesis method of those skilled in the art, and by unmodified type prepared by PLD.More particularly, can prepare in the following order: in the nucleic acid of the PLD of the aminoacid sequence that contains unmodified type PLD at coding, carry out occurring the mutagenesis of above-mentioned displacement, obtain the step of nucleic acid construct thing; Gained nucleic acid construct thing is imported in host, obtain the step of transformant; And cultivate gained transformant, with the form of the expression product of nucleic acid construct thing, obtain the step of the modification type PLD of above-mentioned synthetic PI.

Unmodified type PLD can be the PLD containing the aminoacid sequence shown in the SEQ ID NO.2 of ordered list.This aminoacid sequence is the aminoacid sequence from the PLD of antibiosis streptomycete (Streptomyces antibioticus) strain.In the present invention, so long as express PLD activity, can there is the sequence different from aminoacid sequence shown in SEQ ID NO.2.Above-mentioned different sequence can be 187 tryptophan residues in aminoacid sequence shown in SEQ ID NO.2,191 tyrosine residuess and the different sequence of amino-acid residue beyond 385 tyrosine residuess.For example, above-mentioned unmodified type PLD contains the polypeptide of aminoacid sequence that at least has 50% sequence identity with aminoacid sequence shown in above-mentioned SEQ ID NO.2 and show the polypeptide of PLD activity.Above-mentioned unmodified type PLD can be the polypeptide that contains following aminoacid sequence: in the aminoacid sequence shown in SEQ ID NO.2, at least there is displacement, disappearance, insertion and/or additional the displacement of PLD of tool synthesis capability (can obtain except) of an amino-acid residue, and show the aminoacid sequence of the polypeptide of PLD activity.

In this specification sheets, " the sequence identity " in aminoacid sequence is used the blastp program of BLAST, GENECYX supervisor to calculate.Condition now, such as the setting of expected value, field length (Wordsize), room (Gap) etc., can carry out according to the art usual method.In the present invention, above-mentioned sequence identity is at least 50%, and preferably at least 70%, more preferably at least 79%, further preferably at least 90%, further preferably at least 95%.

The activity of PLD can be evaluated by hydrolytic activity and/or phosphatidyl transfer activity.It is substrate that said hydrolyzed activity for example can be take 95% yolk phosphatidylcholine processed, and by the 0.2M acetate buffer of concentration of substrate 0.16%, (pH4.0 contains 10mM CaCl ₂, 1.3% Triton X-100) at 37 ℃ reaction, by calculating the now choline growing amount of per minute, evaluate.Here, the hydrolytic activity of the Yi Ge unit of PLD is the enzyme amount of the 1 μ mol choline that dissociates in one minute.Above-mentioned transfer activity for example can be usingd 95% yolk phosphatidylcholine processed and ethanol as substrate, and (pH4.0 contains 10mM CaCl to make the 0.2M acetate buffer of concentration of substrate 0.16% ₂, 1.3% TritonX-100) the choline growing amount that calculates per minute 37 ℃ of whens reaction evaluates.Here, the transfer activity of the Yi Ge unit of PLD is the required enzyme amount of 1 μ mol choline of dissociating in a minute.Above-mentioned transfer activity also can be used phosphatidyl p-NP and ethanol to carry out shift reaction, by measuring free p-NP, evaluate (such as people such as Hagishiuta, Anal.Biochem., 1999 years, 276 volumes (2) number, 161-165 page).

Above-mentioned unmodified type PLD is preferably used from the PLD that belongs to the microorganism of actinomycetes, particularly streptomyces (Streptomyces), streptoverticillium (Streptoverticillium), little monospore genus (Micromonospora), Nocardia (Nocardia), Nocardiopsis (Nocardiopsis), actinomadura (Actinomadura) etc.More particularly, there is antibiosis streptomycete, acidomycin streptomycete (Streptomycesacidomyceticus), streptomycete mutation AA586 (Streptomyces sp.AA586), streptomycete mutation PMF (Streptomyces sp.PMF), streptoverticillum (Streptoverticilliumcinnamoneum), (Streptomyces cinnamoneum IFO 12852), bronze little monospore (Micromonospora chalcea ATCC 12452), Nocardia intermedien (Nocardiamediterranei IFO 13142), reach loose Weir Nocardiopsis (Nocardiopsis dassonvilleiIFO 13908), the PLD of Actinomadura libanotica (Actinomadura libanotica IFO 14095) etc.

Mutagenesis for example can be undertaken by the normally used site-directed mutagenesis method in this area, and specifically, mutagenesis by gapped duplex method, Kunkel method, PCR etc. carries out.The nucleic acid of using in radical amino acid replacement can be in conjunction with designing for expressing the host's of the modification type PLD that synthesizes PI codon usage frequency.

The preparation of above-mentioned nucleic acid construct thing is to use conventional carrier according to used host.As carrier, for example, when host is intestinal bacteria, there are the plasmid vectors such as pUC18 and derivative thereof, pBR322 and derivative thereof, pBluescriptII and derivative thereof, pGEM and derivative thereof, the phage vectors such as λ ZAPII, λ gt11 etc., when host is yeast, there is pYAC derivative etc.Nucleic acid construct thing can contain the factors such as inducible promoter, selective marker gene, terminator.Express object polypeptide and can contain the sequence that can express with the form of His label polypeptide or GST fusion polypeptide, can make so the easy separation and purification of expression object polypeptide of nucleic acid construct thing.

The kind of amino acid whose displacement position and the amino-acid residue that may replace as mentioned above.

Nucleic acid construct thing can adopt the normally used method for transformation in this area, such as electroporation, calcium phosphate method, BEAE-dextran method etc. to host's importing, but is not limited to this.

Above-mentioned host is not particularly limited, such as having: intestinal bacteria, yeast, Bacillus subtilus, Pseudomonas aeruginosa, Salmonellas, L cell, COS cell, Chinese hamster ovary celI, sf9 cell etc.Intestinal bacteria specifically have HB101 strain, C600 strain, JM109 strain, BH5 α strain, BL21 (DE3) strain etc.Wherein from expressing the angle of controlling, consider preferably e. coli bl21 (DE3) strain.

The culture condition of transformant can suitably be set according to used host, carrier etc.Cultivate the substratum using and have Luria-Bertani (LB) substratum, SOC substratum, L substratum etc.Above-mentioned culture condition is specifically: use e. coli bl21 (DE3) strain as host, while using pETKmS1 as carrier, use the substratum of following embodiment 2, at 30 ℃, transformant is cultivated 8 hours, then sec.-propyl 1-sulfo--β-D-galactoside (IPTG) (1mM) is added in substratum, at 30 ℃, cultivate 16 hours.

The modification type PLD of the synthetic PI obtaining with the expression product form of nucleic acid construct thing can pass through conventional purification process, and from the culture of transformant, separation and purification obtains.Purification process has: saltout, ultra-high speed is centrifugal, ion-exchange chromatography, adsorpting column chromatography, hydrophobic chromatography, affinity column chromatography etc.

Above-mentioned purification process is appropriately combined, the modification type PLD of the synthetic PI of purifying, in each purification phase, by the evaluation method of said hydrolyzed activity and/or transfer activity, measure PLD active, and by conventional SDS-polyacrylamide gel electrophoresis and not denaturing polyacrylamide gel electrophoresis method evaluate the purification degrees of purified, can obtain expression product thus.In transfer activity evaluation assessment (routine phosphatidyl transfer activity evaluation assessment described above), use inositol as substrate, measure PLD active, can determine that thus gained expression product is the required PLD with PI synthesis capability.

Those skilled in the art can obtain the gene of the modification type PLD of the synthetic PI of coding according to the information of this specification sheets.The gene of the modification type PLD of the synthetic PI of coding is also contained in the present invention.Gene of the present invention, except the natural polynucleotide such as DNA, RNA, can be also the artificial molecule that contains artificial nucleotide derivative.Gene of the present invention can be the chimeric molecule of DNA-RNA.Use this gene, for example, adopt the recombinant technology of above-mentioned explanation, can prepare the modification type PLD of synthetic PI.

The modification type PLD of synthetic PI of the present invention in the phosphatidyl shift reaction of acylglycerol phosphatide, can by be difficult to import with unmodified type PLD large volume compound---inositol imports in phosphatide.Therefore, use the modification type PLD of synthetic PI of the present invention to prepare efficiently PI by raw material phosphatide.

(preparation method of phosphatidylinositols)

The present invention also provides the method for preparing phosphatidylinositols or derivatives thereof.The step that the method comprises the modification type PLD reaction that makes raw material phosphatide, inositol or derivatives thereof, synthetic PI.

The modification type PLD of the synthetic PI using in method of the present invention is the above-mentioned modification type PLD of synthetic PI arbitrarily.As mentioned above, this can be prepared by the microorganism of expressing PLD.

The usage quantity of the modification type PLD of the synthetic PI using in method of the present invention can be selected with respect to 1g raw material phosphatide in the scope of 10-200 unit.The Yi Ge unit of enzymic activity is the enzyme amount of soybean lecithin while dissociating 1 μ mol choline as substrate, when 0.01M Tris-malonic ester-NaOH damping fluid (pH9.5) of concentration of substrate 1% is reacted at 37 ℃, in one minute of take.

As long as the raw material phosphatide in method of the present invention can be used as the substrate of PLD, can be by natural extraction gained, can be also purifying gained or synthetic after extraction, all can use.Also can use commercially available or prepare according to known method.Such as having: soybean lecithin, skimmed soy beans Yelkin TTS, vegetable seed Yelkin TTS, sunflower lecithin etc. are from the Yelkin TTS of plant; Yolk lecithin, the Yelkin TTS from Yelkin TTS of sheep, ox etc. from animal; Yelkin TTS from fishery products such as squid, tunas; From Yelkin TTS of the microorganisms such as yeast etc.The moiety of these Yelkin TTS has: the single components such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidyl glycerol, lysolecithin, phosphatidic acid or their mixture etc.

In method of the present invention, inositol or derivatives thereof can be as the acceptor of phosphatidyl shift reaction.The inositol or derivatives thereof using as acceptor has: inositol, inositol monophosphate (such as a phosphoric acid, bisphosphate or Tripyrophosphoric acid), inositol polysaccharide (such as inositol mannoside) etc.

The mol ratio of raw material phosphatide and acceptor inositol or derivatives thereof can suitably be selected according to the kind of raw material phosphatide.Conventionally, with respect to 1mol PC, be applicable to using the acceptor of 5-50 times mole.

In method of the present invention, the reaction solvent using in phosphatidyl shift reaction can be used the mixed solvent of water solvent and organic solvent, also can use separately water solvent.Organic solvent has: the aliphatic hydrocrbons such as normal heptane, normal hexane, sherwood oil; The aliphatic hydrocrbon such as pentamethylene, hexanaphthene; The ethers such as diethyl ether, tetrahydrofuran (THF); The ester such as methyl acetate, ethyl acetate class; The halogenated hydrocarbon such as tetracol phenixin, chloroform.Water solvent is called water and aqueous buffer solution.Water is preferably used ion exchanged water, pure water or distilled water, also can use tap water.Aqueous buffer solution is such as having the acetate buffer of pH4-6, the phosphoric acid buffer of pH7-8 etc.

During mixed solvent that the reaction solvent using in method of the present invention is water solvent and organic solvent, the water solvent in reaction solvent and the ratio of mixture of organic solvent can suitably be selected according to the kind of used organic solvent.Conventionally, water solvent: organic solvent can mix use according to capacity ratio in the scope of 1:0.65-1:100.In order to suppress the raw material of side reaction---the phosphatidyl hydrolysis reaction of phosphatide, carry out target phosphatidyl shift reaction efficiently, preferably make water solvent content in reaction system 10 capacity % or following carrying out.The selection of organic solvent and ratio of mixture can be selected arbitrarily.

Organic solvent amount in the reaction solvent using in above-mentioned enzymatic reaction is preferably the 5-500 capacity times of the weight of the phosphatide using as raw material, and further preferred 10-100 capacity doubly.Lower than 5 capacity doubly, the viscosity increased of dissolving the solution of raw material substrate, causes reaction efficiency to reduce to organic molten amount.On the contrary, surpass 500 capacity doubly, phosphatidyl shift reaction deterioration of efficiency.

In method of the present invention, the preferred 10-60 ℃ of the temperature of phosphatidyl shift reaction, more preferably 25-45 ℃.Reaction required time changes according to enzyme amount or temperature of reaction, is approximately 0.5-48 hour.When the reaction solvent using in method of the present invention is the mixed solvent of water solvent and organic solvent, form two-phase system, therefore, for water layer and organic solvent layer are fully mixed, preferably in reaction, implement the processing such as suitable stirring, vibration.

After above-mentioned reaction, such as waiting to process by heating, make PLD inactivation, by standing processing, centrifuging etc., remove water layer, obtain organic solvent layer, under reduced pressure remove organic solvent, can obtain thus target acylglycerol phosphatide (for example phosphatidylinositols).And, gained acylglycerol phosphatide can be carried out to the processing such as solvent differentiation, silica gel classification, high performance liquid chromatography, can high purity ground purifying.

Or, after above-mentioned reaction, by standing processing or centrifuging, be divided into water layer and organic solvent layer, as required, in water layer, append PLD or various acceptor alcohol, mix with the new organic solvent that has dissolved raw material phosphatide, water layer is reacted for this repeatedly.Can, with above-mentioned same, from reacting, in the organic solvent layer of differentiation, obtain target acylglycerol phosphatide (for example phosphatidylinositols).

(screening method with the PLD of the ability of synthesizing the acylglycerol phosphatide that has glycol-based)

The present invention also provides the method for screening PLD, and this PLD has the synthetic ability that has the acylglycerol phosphatide of glycol-based.

As the object PLD screening in order to obtain the PLD of the ability with the synthetic acylglycerol phosphatide that has a glycol-based, being not particularly limited, for example, can be to have the polypeptide (PLD of variation) of the different aminoacid sequence of minority (for example 1-several (for example 2 or 3)) amino-acid residue from the aminoacid sequence of unmodified type PLD.Aforementioned polypeptides can be the polypeptide of artificial preparation.This can adopt the normally used site-directed mutagenesis method of those skilled in the art, specifically, adopts the preparations such as mutagenesis (such as overlapping PCR) of gapped duplex method, Kunkel method, PCR.Aforementioned polypeptides can be the polypeptide that the variation due to natural generation obtains.The PLD that supplies with screening can be the polypeptide of separating in can the biology of natural generation PLD, and wherein, whether described PLD has syntheticly has the ability of the acylglycerol phosphatide of glycol-based to be still not clear.

As the PLD that screens the variation of object, for example can obtain according to following order.Generate the DNA fragmentation of each peptide species of coding, wherein, described each peptide species have from the aminoacid sequence of unmodified type PLD (particularly natural type PLD) have minority (for example 1-several (for example 2 or 3)) at aminoacid sequences specific or that random site upper amino acid residue is different.These DNA fragmentations are connected with expression vector, can obtain nucleic acid construct thing.Gained nucleic acid construct thing is imported to host, obtain transformant.Cultivate gained transformant, as the expression product of nucleic acid construct thing, can obtain and generate the plasmid library that has the PLD of multiple variation than original PLD.The preparation of nucleic acid construct thing, to host's importing, the cultivation of transformant as mentioned above.Can screen the PLD of the variation of being expressed by plasmid library.

PLD is for example as follows, can supply with screening.The plasmid library of the gene that contains this PLD that encodes is imported in host, obtain transformant.Cultivate this transformant, for example, by gained colony transfer film (Nitrocellulose film).Here, this substratum should be preserved with the form of main flat board.Colony is grown on film, by the substratum that contains IPTG, make this PLD expression and secretion.Thus, the PLD of expression is adsorbed on this film.

In screening method of the present invention, the acceptor importing in acylglycerol phosphatide can be used the compound that contains glycol-based.Above-claimed cpd has carbohydrate (preferred oligosaccharides class (carbohydrate for example consisting of 1-10 sugar), inositol or derivatives thereof (for example inositol monophosphate (phosphoric acid, bisphosphate or Tripyrophosphoric acid)).Preferably use inositol or derivatives thereof.

The raw material phosphatide using in screening method of the present invention also as mentioned above, all can so long as can form the phosphatide of the substrate of PLD.

Use raw material phosphatide, acceptor and PLD to carry out phosphatidyl shift reaction.The preferred 10-60 ℃ of temperature of reaction, more preferably 25-40 ℃.Reaction required time changes according to temperature of reaction, is approximately 0.5-48 hour.While there is target P LD, can generate target acylglycerol phosphatide according to this reaction.

Above-mentioned phosphatidyl shift reaction for example can be by being immersed in the film of above-mentioned absorption PLD in the reacting solution that contains raw material phosphatide and acceptor and implementing.Preferably in this reacting solution, contain calcium salt.By above-mentioned phosphatidyl shift reaction, generate target acylglycerol phosphatide, form water-insoluble salt with the calcium ion coexisting in the aqueous solution, the position precipitation of enzymatic reaction has occurred on calcium film.This position refers to the position of the colony existence of expressing target P LD.

The glycol moiety of the acylglycerol phosphatide generating by phosphatidyl shift reaction, because oxidation is ftractureed, derives as aldehyde.Oxidation is to use oxygenant to carry out, as long as this oxygenant can make glycol moiety cracking, derive as aldehyde, can use any oxygenant.The preferred Periodic acid of above-mentioned oxygenant or its salt, lead tetraacetate and sodium bismuthate.Periodic acid or its salt for example have metaperiodic acid, potassium metaperiodate, sodium metaperiodate and ortho-periodic acid sodium.The preferred 10-60 ℃ of temperature of this oxidizing reaction, more preferably 20-40 ℃.Reaction required time changes according to temperature of reaction, is approximately 5-60 minute.

The method that above-mentioned aldehyde can adopt conventionally according to this area detects.The detection method of aldehyde for example can be utilized following reaction: make above-mentioned aldehyde and hydrazine compound (for example dinitrophenyl hydrazine, 7-nitro-2,1,3-Ben Bing oxadiazole (NBD)-hydrazine etc.) coupling, thus, this hydrazine forms hydrazone, but is not limited to this reaction.Also can utilize silver mirror reaction and expense Ling Shi reaction.For example, make NBD hydrazine and above-mentioned aldehyde reaction, NBD hydrazine forms NBD hydrazone, presents strong fluorescence.By measuring this fluorescence, can measure the existence of aldehyde.

By determining the existence of aldehyde, the existence of acylglycerol phosphatide aldehyde, that contain glycol moiety can be confirmed to have generated, then by phosphatidyl shift reaction, the existence of PLD can be confirmed, this PLD has the ability of the synthetic acylglycerol phosphatide containing glycol moiety.

While using the film of above-mentioned absorption PLD to carry out this series reaction, can on this film, detect the existence of aldehyde.Therefore, whether the existence detecting on film, can assert whether obtained the PLD with target synthesis capability.And, on the main flat board corresponding with this film, there is the transformant of expressing the PLD with this synthesis capability, therefore, can obtain the transformant of the gene of the PLD that possesses tool synthesis capability.And, by analyzing thering is the base sequence of gene of the PLD of synthesis capability, can identify the PLD with target synthesis capability.

Embodiment

Below provide embodiment, further illustrate the present invention, but the present invention being not limited by it.

In the present embodiment, the structure of recombinant vectors, the preparation of transformant, by transformant or clone, prepare the method that plasmid etc. can adopt conventionally according to fields such as molecular biology, physiotechnology, genetic engineerings and carry out.

(embodiment 1: the preparation in library)

By pPELB-PLD1 (according to people such as Y.Iwasaki, J.Ferment.Bioeng., nineteen ninety-five, 79 volumes, the record preparation of 417-421 page) with restriction enzyme XbaI and XhoI digestion, reclaim contain liposome binding site, from the pelB signal of pET22b (Novagen) with from the total length (1530bp of the PLD gene of antibiosis streptomycete; Base sequence is as shown in the SEQ IDNO.1 of sequence table) fragment, be inserted in advance the pBluescriptII KS with XbaI and XhoI digestion ⁺in (Japan's spinning is standby), preparation pPELB-PLD-KS.Used restriction enzyme ApaI digestion, together with the terminator codon of this PLD gene fragment around, removed this PLD gene downstream inverted repeats.On the other hand, by pSA1 (according to people such as Y.Iwasaki, Appl.Microbiol.Biotechnol., 1994,42 volumes, the record preparation of 290-299 page) with restriction enzyme BamHI and SalI digestion, cut the fragment that contains this PLD gene latter half, be inserted into the pBluescriptII KS digesting with BamHI and SalI in advance ⁺in (Japan's spinning is standby), obtain pSA1-BS.By restriction enzyme ApaI digestion for pSA1-BS, the terminator codon that cuts this PLD gene fragment around.The fragment that this is cut is connected with the pPELB-PLD-KS of ApaI digestion, then, with KpnI and XbaI, cuts off, and obtains the fragment that contains pelB signal and PLD full length gene (1530bp).By this fragment and the pBluescriptII KS cutting off with SalI and XbaI in advance ⁺plasmid (Japan's spinning is standby) connects, and obtains plasmid pBELB-PLD-KS II.

Use primer CAT-F1 (SEQ ID NO.3) and CAT-R1 (SEQ ID NO.4), the plasmid pHSG399 that contains cat gene (precious wine is made preparation) is formed to (each concentration is final concentration) by PCR[reaction solution: 10 * LA Taq is damping fluid (precious wine is made preparation, 1/10 capacity), 2.5mM MgCl for polysaccharase ₂, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, 10ng/ μ L plasmid PHSG399,0.5 μ M primer CAT-F1,0.5 μ M primer CAT-R1, (precious wine is made preparation to LA Taq archaeal dna polymerase, 0.025 unit/μ L)/temperature program(me): 94 ℃ after 1 minute, 94 ℃ 10 seconds-50 ℃ 10 seconds-72 ℃ 40 seconds, carry out 30 circulations, then be 72 ℃ of 7 minutes, 4 ℃ ∞] increase, with BglII digestion, obtain the fragment that contains cat.PETKmS1 (according to people such as Mishima N., Biotechnol.Prog.,, 13 volumes, the record preparation of 864-868 page in 1997) is cut off with Bgl II, by cat fragment Opposite direction connection, obtain plasmid pETKmS1-CAT.Then, this plasmid is cut off with SacI and XbaI, cut the fragment that contains cat reverse sequence and T7-lac promotor.By this fragment with the pPELB-PLD-KS II that SacI and XbaI cut off, be connected in advance, obtain plasmid pPELB-PLD-KSII-CAT (5581bp).This plasmid pPELB-PLD-KS II-CAT is used as the template of the overlapping PCR importing for the random amino acid displacement of fixing a point.

Take pPELB-PLD-KS II-CAT as template, use primer PL-F1 (SEQ ID NO.5) and OL-R1 (SEQ ID NO.6), by PCR[reaction solution, form (each concentration is final concentration): 10 * LA Taq is damping fluid (precious wine is made preparation, 1/10 capacity), 2.5mM MgCl for polysaccharase ₂, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, 10ng/ μ LpPELB-PLD-KS II-CAT, 0.5 μ M primer PL-F1, 0.5 μ M primer OL-R1, (precious wine is made preparation to LA TaqDNA polysaccharase, 0.025 unit/μ L)/temperature program(me): 94 ℃ after 2 minutes, 95 ℃ 10 seconds-60 ℃ 10 seconds-72 ℃ 1 minute 10 seconds, carry out 30 circulations, then be 72 ℃ 10 minutes, 4 ℃ of ∞ minute] increase, obtain the cat gene that contains this template plasmid, T7-lac promotor, liposome binding site, the amplified fragments of a part of pelB signal sequence and ripe PLD (fragment 1).

From the overall amino acid sequence of the mature protein of the PLD of antibiosis streptomycete as shown in the SEQ ID NO.2 of sequence table.Replaced in order to make from the 187th tryptophan residue of mature amino acid N-terminal and 191 tyrosine residuess of the PLD of antibiosis streptomycete, take pPELB-PLD-KSII-CAT as template, use primer OL-F1 (SEQ ID NO.7) and OL-R2 (SEQ ID NO.8), by PCR[reaction solution, form (each concentration is final concentration): 10 * LATaq damping fluid (precious wine is made preparation, 1/10 capacity), 2.5mM MgCl for polysaccharase ₂, 0.2mMdATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, 10ng/ μ LpPELB-PLD-KSII-CAT, 0.5 μ M primer OL-F1,0.5 μ M primer OL-R2, (precious wine is made preparation to LA TaqDNA polysaccharase, 0.025 unit/μ L)/temperature program(me): 94 ℃ after 2 minutes, 95 ℃ 10 seconds-67 ℃ 10 seconds-72 ℃ 30 seconds, carry out 25 circulations, then be 72 ℃ of 10 minutes, 4 ℃ ∞] increase, obtain having two amino acid variation positions the fragment (fragment 2) of various base sequences.

In order to make the 385th tyrosine residues of amino acid whose N-terminal of this maturation replaced, take pPELB-PLD-KSII-CAT as template, use primer pair OL-F2 (SEQ ID NO.9) and PL-R1 (SEQ ID NO.10), by PCR[reaction solution, form (each concentration is final concentration): 10 * LA Taq is damping fluid (precious wine is made preparation, 1/10 capacity), 2.5mM MgCl for polysaccharase ₂, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, 10ng/ μ LpPELB-PLD-KSII-CAT, 0.5 μ M primer OL-F2,0.5 μ M primer PL-R1, (precious wine is made preparation to LA TaqDNA polysaccharase, 0.025 unit/μ L)/temperature program(me): 94 ℃ after 2 minutes, 95 ℃ 10 seconds-62 ℃ 10 seconds-72 ℃ 30 seconds, carry out 25 circulations, then be 72 ℃ of 10 minutes, 4 ℃ ∞] increase, obtain having on an amino acid variation position, place the fragment (fragment 3) of various base sequences.

Then, utilize the lap of fragment 2 and fragment 3, make their hybridization, re-use primer OL-F1 (SEQ ID NO.7) and PL-R1 (SEQ ID NO.10), by PCR[reaction solution, form (each concentration is final concentration): 10 * LA Taq is damping fluid (precious wine is made preparation, 1/10 capacity), 2.5mM MgCl for polysaccharase ₂, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, 10ng/ μ L fragment 2,10ng/ μ L fragment 3,0.5 μ M primer OL-F1,0.5 μ M primer PL-R1, (precious wine is made preparation to LA Taq archaeal dna polymerase, 0.025 unit/μ L)/temperature program(me): 94 ℃ after 2 minutes, 95 ℃ 10 seconds-62 ℃ 10 seconds-72 ℃ 45 seconds, carry out 25 circulations, then be 72 ℃ of 10 minutes, 4 ℃ ∞] increase, obtain amplified fragments (fragment 4).

Then, utilize their lap, make fragment 1 and fragment 4 hybridization, do not add primer, [reaction solution forms (each concentration is final concentration): 10 * LA Taq is damping fluid (precious wine is made preparation, 1/10 capacity), 2.5mM MgCl for polysaccharase to carry out overlapping extension ₂, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, 0.2ng/ μ L fragment 1,0.2ng/ μ L fragment 4, (precious wine is made preparation to LA Taq archaeal dna polymerase, 0.05 unit/μ L)/temperature program(me): 94 ℃ after 5 minutes, 98 ℃ 10 seconds-59 ℃ 10 seconds-72 ℃ 2 minutes, carrying out 20 circulations, is then 4 ℃ of ∞].Then, the reaction solution of take after this overlapping extension is template, use primer PL-F1 (SEQ ID NO.5) and PL-R1 (SEQ ID NO.10) to carry out PCR[reaction solution composition (each concentration is final concentration): the reaction solution after overlapping extension (1/10 capacity), 10 * LATaq be damping fluid (precious wine is made preparation, 1/20 capacity), 2.5mM MgCl for polysaccharase ₂, 0.2mMdATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, 10ng/ μ L fragment 1,10ng/ μ L fragment 4,0.5 μ M primer PL-F1,0.5 μ M primer PL-R1, (precious wine is made preparation to LA Taq archaeal dna polymerase, 0.025 unit/μ L)/temperature program(me): 94 ℃ after 3 minutes, 95 ℃ 10 seconds-59 ℃ 10 seconds-72 ℃ 2 minutes, carry out 25 circulations, then be 72 ℃ of 10 minutes, 4 ℃ ∞], obtain amplified fragments.Like this, obtained having three amino acid variation positions the amplified fragments of various base sequences.

This amplified fragments is cut off with restriction enzyme SpeI and XhoI, and with restriction enzyme SpeI, be connected with the pEPKmS1-Term of SalI cut-out in advance, obtain expression vector.PEPKmS1-Term cuts off above-mentioned pEPKmS1-CAT with BamHI, by self, connect preparation.With this expression vector, transform intestinal bacteria (E.coli) DH5 α.The bacillus coli DH 5 alpha of this conversion is layered on to LB nutrient agar and (contains 50 μ g/mL kantlex and 30 μ g/mL paraxin; 10g/L Tryptones (デイ Off コ preparation), 5g/L yeast extract (デイ Off コ preparation), 10g/L NaCl, 15g/L agar (with the pure medicine preparation of light)) in, make its growth, form colony.On colony on nutrient agar, pour LB liquid nutrient medium (10g/L Tryptones (デイ Off コ preparation), 5g/L yeast extract (デイ Off コ preparation), 10g/L NaCl) into, scraping colony.By gained cell, prepare plasmid, with the form of plasmid library, obtain the mixture that contains variation PLD gene.

(embodiment 2: the expression of plasmid library)

Above-mentioned plasmid mixture is imported to as expressing with in host's e. coli bl21 (DE3), on the LB nutrient agar forming similarly to Example 1, grow at 37 ℃ 16 hours.On gained colony, place Nitrocellulose film (HybondC, the preparation of アマシヤ system), transfer printing colony.Then, film is transferred to synthetic medium ((quality that represents every 1L) composed as follows: 5g KH ₂pO ₄, 5g K ₂hPO ₄, 4.4g Na ₂hPO ₄, 3g (NH ₂) ₂sO ₄, 5g glucose, 3g MgSO ₄7H ₂o, 40mg FeSO ₄7H ₂o, 40mg CaCl ₂, 10mgMnSO ₄7H ₂o, 2.9mg CoCl ₂6H ₂o, 3mg CuSO ₄5H ₂o, 0.36mgNa ₂moO2H ₂o, 10mg H ₃bO ₃, 10mg ZnSO ₄7H ₂in O, add 1.5% agar gained), at 30 ℃, be incubated 8 hours, colony is grown on film.Colony on LB nutrient agar is kept in refrigerator as main flat board.Film is transferred in the above-mentioned synthetic medium that has added 1mM IPTG, at 30 ℃, is incubated 16 hours, on film, induce the expression of PLD.In this expression system, the PLD of expression is discharged into extracellular, is adsorbed on film.Use sonic washing machine, from this film, rinse the resistates of thalline.

(embodiment 3: the relevant clone's that the modification type PLD of synthetic PI expresses screening)

At 30 ℃, above-mentioned film is soaked 14 hours in substrate solution (10% soybean lecithin (preparation of SLP-PC70 Star Le-レシチ Application), 20% inositol, 2% calcium chloride and 50mM acetate buffer, pH5.6), on film, carry out enzymatic reaction.By this enzymatic reaction, generate the phosphatidylinositols (PI) as acidic phospholipid, they form water-insoluble salt with the calcium ion coexisting, the position precipitation reacting.After reaction, excessive substrate solution water is rinsed, by film in the 10% sodium periodate aqueous solution, at room temperature soak 10 minutes.By this sodium periodate, process, the glycol moiety oxidation cracking of the PI of precipitation, derives as aldehyde.Excessive sodium periodate solution water is rinsed, on film, be coated with the NBD-hydrazine aqueous solution (0.05% NBD-H, 10% DMSO).NBD-H and aldehyde reaction, form NBD-hydrazone, presents strong fluorescence.This fluorescence is observed under ultraviolet ray (365nm) is irradiated, identified bright spot.This spot is equivalent to the position that the PI on film exists.The colony consistent with this spot is separated from main flat board, obtain positive colony.Obtain thus 49 positive colonies (table 1).

(embodiment 4: the sequence of positive colony is determined)

Positive colony by embodiment 3 gained is prepared plasmid, determines the variation content of PLD gene by dideoxy method.Result is as shown in table 1.

Table 1

(embodiment 5: by the synthetic PI of modification type PLD)

By in table 1 liquid culture in the numbered synthetic medium (not containing agar) being cloned in embodiment 2 same compositions, at 30 ℃.After approximately 8 hours, add IPTG, final concentration is 1mM, the expression of induction PLD.Induce after 16 hours by the centrifugal thalline of removing, gained supernatant is carried out to the classification of sulfuric acid amine, the thick enzyme preparation of preparation PLD.

5mg dipalmitoyl phosphatidylcholine, 40mg myo-inositol, 4mg calcium chloride, 180 μ L50mM acetate buffers and 20 μ L (approximately 1 unit) the thick enzyme preparation of above-mentioned PLD is reacted while stirring 14 hours at 30 ℃.

The qualitative analysis of phosphatide is undertaken by the thin layer chromatography (TLC method) of following condition:

TLC plate ... silica gel 60 plates (preparation of メ Le Network)

Developing solvent ... chloroform: methyl alcohol: sherwood oil: acetic acid=4:2:3:1 (V/V/V/V)

Developing time ... approximately 20 minutes

Detect ... after expansion, the ethanolic soln of 0.1%2, the 7-dichlorofluorescein of spraying is then observed under 365nm uviolizing, or spraying Dittmer reagent, place, detect thus the spot of phosphatide.As a result, in the PLD being prepared by any clone, when using inositol as substrate, all detect the new spot that is equivalent to two palmityl PI.

Also carried out mass spectroscopy.Mass spectroscopy is to carry out stereometry by time of flight secondary ion massspectrometry instrument (TOF-SIMS, trade(brand)name PHITRIFT III, ア Le パ Star Network Off アイ preparation), by negative ion mode, is undertaken.As a result, in the PLD being prepared by any clone, while using inositol as substrate, all detect the ion that is equivalent to two palmityl PI.

产业实用性

本发明的新型PLD在酰基甘油磷脂的磷脂酰基转移法中可将如肌醇这样的大体积化合物导入到磷脂中，因此，使用该PLD，可简便且高效地从价格低、容易获得的磷脂(例如卵磷脂)制备PI。并且，本发明还提供可简便且迅速地筛选新型PLD的方法，该PLD具有合成有二醇基的酰基甘油磷脂的能力。

Sequence table

Claims

1. modification type Phospholipase D, this modification type Phospholipase D has the ability of synthetic phospholipid acyl inositol, and it contains

2. the modification type Phospholipase D of claim 1, wherein,

187 amino acids residues in aminoacid sequence shown in above-mentioned SEQ ID NO.2 are replaced into a kind of radical amino acid replacement that is selected from phenylalanine residue, serine residue, tyrosine residues, α-amino-isovaleric acid residue, glycine residue, arginine residues, histidine residues, asparagine residue and leucine residue, and/or

3. the modification type Phospholipase D of claim 1, this modification type Phospholipase D is selected from:

4. gene, the modification type Phospholipase D of any one in this genes encoding claim 1-3.

5. the method for preparing phosphatidylinositols or derivatives thereof, the step that the method comprises the modification type Phospholipase D reaction that makes any one in raw material phosphatide, inositol or derivatives thereof, claim 1-3.

6. screening has the method for the Phospholipase D of the ability of synthesizing the acylglycerol phosphatide that has glycol-based, and the method includes the steps of:

Use raw material phosphatide, the compound with glycol-based, Phospholipase D, carry out phosphatidyl shift reaction, generate the step of the acylglycerol phosphatide with this glycol-based;

Detect the step of this aldehyde generating;

Here, the generation of this aldehyde is the index that this Phospholipase D has the ability of synthetic this target acylglycerol phosphatide.

7. the method for claim 6, wherein, the compound with above-mentioned glycol-based is inositol or derivatives thereof.

8. claim 6 or 7 method, in above-mentioned oxidation step, used the oxygenant that is selected from Periodic acid or its salt, lead tetraacetate and sodium bismuthate.

9. the method for any one in claim 6-8, wherein, the detection of above-mentioned aldehyde is to react the hydrazone compound generating with hydrazine compound and carry out by detecting this aldehyde.