WO2007089038A1

WO2007089038A1 - Novel phospholipase d

Info

Publication number: WO2007089038A1
Application number: PCT/JP2007/052172
Authority: WO
Inventors: Yugo Iwasaki; Hideo Nakano; Tetsuya Takahashi
Original assignee: National University Corporation Nagoya University
Priority date: 2006-02-02
Filing date: 2007-02-01
Publication date: 2007-08-09
Also published as: JP5156951B2; WO2007089038A8; JPWO2007089038A1; JP5420016B2; CN101379187A; JP2012187108A

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 thismodified 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

Description New phospholipase D Technical field

The present invention relates to a novel phospholipase D having the ability to synthesize phosphatidylinositol. The background technology

Phospholipids can be used as emulsifiers, cosmetic ingredients, pharmaceutical formulations, and for the preparation of liposomes. Naturally derived phospholipids are usually phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), sphingomyelin, etc. Consists of a mixture. To fractionate specific phospholipids from natural phospholipids, solvent fractionation methods such as extraction and recrystallization with solvents such as methanol, ethanol, isopropyl alcohol, hexane and chloroform, silica gel, alumina, column chromatography fractionation using an adsorbent such as ion exchange resins, fractionation is carried out by use of derivatives, such as C d C 1 ₂ complex Ya Asechiru product.

Separation and purification of PI from natural products (soybean lecithin and the like) is described in, for example, Japanese Patent Application Laid-Open Nos. 5-98773 and 5-97872. In JP-A-5-9 7 8 7 3, a lipid containing PI is dissolved in a nonpolar solvent, and after contacting this with a hydrous polar solvent containing a basic substance, a nonpolar solvent layer is formed. A method is described in which a liquid containing a basic substance, liquid-liquid partition with a new hydrous polar solvent, and PI is extracted into the hydrous polar solvent layer. JP-A-5-9 7 8

7 No. 2 contains mixed phospholipids containing PI, water or aqueous acetic acid. In other words, a method is described in which it is dissolved in a mixed solvent of a lower alcohol and a nonpolar solvent and used for high-speed liquid chromatography. Such a method requires a large amount of solvent, so that it is difficult to obtain high purity PI, and high-purity products are extremely expensive.

There is also a method of organically synthesizing PI, but it is not very efficient because it is a complex synthetic route consisting of many steps including protection and deprotection.

Attempts to enzymatically produce phospholipids have also been made. Phospholipase D [EC 3. 1. 4. 4] (hereinafter also referred to as “PLD”) is an enzyme that catalyzes the production of PA and alcohol moieties by hydrolyzing the phosphodiester bonds of glycerophospholipids. In addition to this hydrolytic activity, PLD also catalyzes the interconversion of phospholipid polar groups. This process is called “phosphatidyl group transfer reaction”. Using the phosphatidyl group transfer reaction, it is possible to synthesize naturally-occurring phospholipids from naturally abundant phospholipids.

Some natural phospholipids such as phosphatidylglycerol (PG), PE, or PS are synthesized from lecithin or PC using PLD-catalyzed phosphatidyl group transfer reaction (see, for example, JP-A-6-269287, JP-A-5-292981, JP-A-2002-51794, and JP-A-2005-261362). JP-A-6-269287 describes a method for obtaining a target phospholipid by performing a base exchange reaction under specific reaction conditions using PLD originating from a microorganism belonging to the genus Streptomyces. , PS, and phosphatidylpropanol have been successfully produced. Japanese Patent Laid-Open No. 5-292981 describes a method using a chelating agent for obtaining a target phospholipid by a base conversion reaction with a microorganism-derived PLD, and succeeded in producing PG, PE, and PS. is doing. PI is also produced, but the base conversion rate seems to be low. In JP-A-2002-51794, raw materials and A method of performing an enzymatic reaction at 15 to 65 ° C by thoroughly mixing and homogenizing a phospholipid, a hydroxyl group-accepting receptor, PLD, and water without using an organic solvent is described. PG generation is successful. Japanese Unexamined Patent Application Publication No. 2005-2 61362 describes a method for producing phosphatidylserine using a phosphatidyl group transfer reaction.

Japanese Patent Application Laid-Open No. 3-87191 describes a method for producing PI using PLD. In this method, when PLD is allowed to act on the mixed phospholipid of the raw material, PLD does not act on PI in the phospholipid, but utilizes the selective hydrolysis of other phospholipid components, Collect unreacted PI. 'This method requires the addition of alkaline or acid phosphatase after the addition of PLD. .

PLD has been studied for its properties and structure (for example, R. Ulbrich-Hof 匪 n et al., Biotechnology Letters, 2005, 27 卷, pp.53 5-544). In addition, PLD has been modified in various ways to enhance its usefulness (for example, JP 2004-97011 A and JP 2005 8 0519 A1 and Nishikawa et al., Japanese Society of Agricultural Chemistry 2004). (Summary of the conference, page 268). JP-A-2004-97011 and JP-A-2005-80519 describe modified PLDs with improved stability to organic solvents and heat. In addition, Nishikawa et al., Abstract of the 2004 Annual Meeting of the Japanese Society for Agricultural Chemistry, page 268, describes the acquisition of mutant PLDs that have the ability to degrade phosphatidyl-1-naphthol (P 1NAP), a phospholipid having a naphthyl group. Are listed. Disclosure of the invention

An object of the present invention is to provide a method capable of efficiently producing phosphatidylinositol (PI) from phospholipids. The present invention also synthesizes PI. Another object of the present invention is to provide a method for screening a PLD having the ability to synthesize a acylglycose phospholipid having a darlicol group.

The present invention provides a modified phospholipase D (hereinafter also referred to as “PI synthesis modified PLD”) having the ability to synthesize phosphatidylinositol. The PI synthesis modified PLD is a modified phospholipase D having the ability to synthesize phosphatidylinositol,

(A) An amino acid in which at least one amino acid residue of the amino acid residues at positions 1 87, 19 1 and 3 85 of the amino acid sequence shown in SEQ ID NO: 2 is substituted with another amino acid residue An array; or

(B) In the amino acid sequence of (A), at least one amino acid residue at a position different from the amino acid residues located at positions 1 87, 19 1 and 3 85 The amino acid sequence of a polypeptide having the following substitutions, deletions, insertions, and / or additions and having the ability to synthesize phosphatidylinositol. ,

In one embodiment, the amino acid residue at position 187 in the amino acid sequence shown in column No. 2 above is a phenylalanine residue, a serine residue, a tyrosine residue, a parin residue, or a glycine residue. Substituted with one amino acid residue selected from the group consisting of a group, an arginine residue, a histidine residue, an asparagine residue, and a leucine residue, and / or

In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residue at position 1 91 is an argyen residue, a threonine residue, an isoleucine residue, a glycine residue, a parin residue, a phenylalanine residue, a leucine residue, Substituted with one amino acid residue selected from the group consisting of aspartic acid residue, methionine residue, tribtophan residue, and alanine residue, and / or

Amino acid residue at positions 3 85 in the amino acid sequence shown in SEQ ID NO: 2 Is selected from the group consisting of cysteine residues, tributophane residues, leucine residues, methionine residues, arginine residues, glutamic acid residues, serine residues, phenylalanine residues, valine residues, and proline residues. One amino acid residue is substituted.

In a further embodiment, the modified phospholipase D is selected from the group consisting of:

(1) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase, D, wherein the group is an arginine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosin residue;

(2) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase D which is a threonine residue and wherein the amino acid residue at position 3.85 in the amino acid sequence shown in SEQ ID NO: 2 is a cysteine residue;

(3) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the group is an isoleucine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue;

(4) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D wherein is a glycine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue; (5) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D wherein is a valine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a leucine residue;

(6) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase D wherein the group is a valine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(7) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the amino acid residue at position 1 91 in the amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase D, which is a tyrosine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a methionine residue;

(8) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the amino acid residue at position 1 91 in the amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase D, which is a tyrosine residue, and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue;

(9) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue, and the amino acid residue at position 1 1 9 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D in which is a tyrosine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a dartamic acid residue;

(1 0) Amino acid at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 The amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid sequence shown in SEQ ID NO: 2 3 8 Modified phospholipase D in which the amino acid residue at position 5 is a methiyun residue;

(1 1) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the group is a phenylalanine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a methionine residue;

(1 2) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and the amino acid residue at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase D, wherein the group is a phenylalanine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosin residue;

(1.3) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and the amino acid sequence shown in SEQ ID NO: 2 at position 1 1 in 1 1 A modified phospholipase D in which the amino acid residue is a mouth residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue;

(14) 'The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and the amino acid at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the residue is a leucine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(15) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a palin residue, and the amino acid sequence shown in SEQ ID NO: 2 1 9 wherein the amino acid residue at position 1 is a tyrosine residue, and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue; modified phospholipase D;

(16) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a valine residue, and the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 2 Modified phospholipase D, wherein the residue is a tyrosine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a glutamic acid residue;

(1 7) The amino acid residue at position 18.7 in the amino acid sequence shown in SEQ ID NO: 2 is a glycine residue, and the amino acid sequence shown in SEQ ID NO: 2 in position 1 A modified phospholipase D in which the amino acid residue is an aspartic acid residue, and the amino acid residue at position 385 in the amino acid sequence represented by SEQ ID NO: 2 is a leucine residue;

(1 8) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a palin residue, and the amino acid at position 1 1 in amino acid sequence shown in SEQ ID NO: 2 Modified phospholipase D, wherein the residue is a tryptophan residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue;

(19) The amino acid residue at position 18.7 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid residue at position 1-19 in the amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase D which is a tyrosine residue and wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue;

(20) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 is The group is a tyrosine residue, and the above SEQ ID NO: A modified phospholipase D in which the amino acid residue at position 3 85 in the amino acid sequence shown in 2 is a tyrosine residue;

(2 1) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid at position 1 91 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the residue is a tyrosine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue;

(2 2) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a histidine residue, and the amino acid residue at position 1 in amino acid sequence shown in SEQ ID NO: 2 Modified phospholipase D, wherein is a methionine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a palin residue;

(2 3) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the residue of amino acid at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the group is an Argyyun residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(2 4) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a asparagine residue, and the amino acid residue at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein is alanine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(25) The amino acid residue at position 18.7 in the amino acid sequence shown in SEQ ID NO: 2 is a mouth isine residue, and the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 2 The residue is an alanine residue, and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue. A modified phospholipase D;

(2 6) The amino acid residue at position 1807 in the amino acid sequence shown in SEQ ID NO: 2 is a leucine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein is alanine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue;

(2 7) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a mouth cisine residue, and 1 9 1 position in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D in which the amino acid residue is a mouth cisin residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(28) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a tributophane residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 Is a tyrosine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a proline ¾ group;

(29) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a valine residue, and amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 2 Modified phospholipase 0, wherein the acid residue is a tyrosine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tributophane residue.

The present invention also provides a gene encoding the P I synthesis modified PLD. The present invention also provides a method for producing phosphatidylinositol or a derivative thereof. The method includes a step of reacting a raw material phospholipid, inositol or a derivative thereof, and the PI synthesis modified PLD.

Furthermore, the present invention synthesizes a glycyl mouth phospholipid having a glycol group. A method of screening for PLDs having the ability to The method comprises a step of producing a phosphatidyl group transfer reaction using a raw material phospholipid, a compound having a glycol group, and PLD to produce a acylglyceport phospholipid having the glycol group;

Oxidizing the generated acylglycose phospholipid having the glycol group to produce an aldehyde; and

Detecting the generated aldehyde. Here, the production of the aldehyde is an indicator that the PLD has the ability to synthesize the target acylglycose phospholipid. '

In one embodiment, the compound having a glycol group is inositol or a derivative thereof.

In another embodiment, in the oxidation step, an oxidizing agent selected from the group consisting of periodic acid or a salt thereof, lead tetraacetate, and sodium bismuthate is used.

In yet another embodiment, the aldehyde is detected by detecting a hydrazone compound produced by reacting the aldehyde with a hydrazine compound.

According to this effort, PLD having the ability to introduce a bulky compound such as inositol into the phospholipid in the phosphatidyl group transfer reaction of the acyl glyce 'mouth phospholipid can be obtained. By using this PLD, PI can be easily and efficiently produced from phospholipids. Furthermore, according to the present invention, it is possible to rapidly screen for a novel PLD having the ability to synthesize a acyldaricerophospholipid having a glycol group. BEST MODE FOR CARRYING OUT THE INVENTION

Acylglycerophospholipids with bulky polar moieties such as inositol rings PLD with the ability to synthesize PLD has a structure in which the steric hindrance is thought to be mitigated by substituting an amino acid residue at a specific position in the enzyme catalytic site of unmodified PLD. It was found that this could be

As used herein, “unmodified PLD” refers to a PLD that does not have the ability to synthesize a glycosyl glyceport phospholipid having a glycol group (eg, phosphatidylinositol (P I)). “Unmodified PLD” refers to a naturally occurring PLD naturally occurring in an organism and a nucleic acid obtained by introducing and expressing a nucleic acid encoding the natural PLD in an organism different from the original origin. PLD is included. Also included within the scope of this term are PLDs with mutations other than substitutions aimed at obtaining PLDs that acquire synthetic ability (mutant PLDs). Also included are artificially produced chimeric P and LD.

In the present specification, a PLD having the ability to synthesize a glycylmouth phospholipid having a glycol group (for example, PI) has an amino acid sequence in which an amino acid residue at a specific position is changed in the unmodified PLD. Therefore, for convenience, it is also called “modified PLD”. The “modified PLD” is, for example, a PLD having an amino acid sequence in which amino acid substitution has been performed to obtain a PLD that acquires the above-mentioned synthesis ability from an unmodified PLD. This amino acid substitution can be both a naturally occurring substitution and an artificially created substitution. (Modified PLD having the ability to synthesize phosphatidylinositol) The present invention provides a modified phospholipase D (? 1 synthesis modified? 0) having the ability to synthesize phosphatidylinositol. The PI synthetic modified PLD has a structure in which the steric hindrance is considered to be relaxed by replacing an amino acid residue at a specific position of the enzyme catalytic site of the unmodified PLD. To date, no PLD with PI synthesis ability is known.

The PI synthesis modified PL D of the present invention comprises: (A) the amino acid represented by SEQ ID NO: 2 An amino acid sequence wherein at least one amino acid residue of the amino acid residues at positions 1 87, 19 1 and 3 85 of the sequence is substituted with another amino acid residue; or (B) said ( In the amino acid sequence of A), substitution, deletion, insertion of at least one amino acid residue at a position different from the amino acid residues located at positions 1 87, 19 1 and 3 85 And an amino acid sequence of a polypeptide having Z or an addition and having the ability to synthesize phosphatidylinositol.

In the present invention, the number of amino acid residues that can be substituted, deleted, inserted and / or added is an arbitrary number as long as the enzyme has a desired enzyme activity. For example, it can be any number that results in the sequences described below. Usually 2500 or less, for example 15.0 or less, 1100 or less, or 50 or less, preferably 30 or less, more preferably 20 or less, more preferably 16 or less, even more preferably 5 or less Even more preferred are 0 to 3 amino acid residues. A person skilled in the art can modify the protein structure by introducing appropriate substitution, deletion, insertion, and / or addition mutations using, for example, site-specific mutagenesis. . In addition, since amino acid mutations may occur in nature, not only enzymes that have artificially mutated amino acids, but also enzymes in which amino acids have been mutated in nature have the desired PLD as long as they have the desired enzyme activity. included. Methods and means for determining the presence or absence of a desired enzyme activity are well known to those skilled in the art and can be performed, for example, based on the methods described below.

In one embodiment, the PI synthesis modified PLD of the present invention comprises

The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, a serine residue, a tyrosine residue, a parin residue, a glycine residue, an arginine residue, a histidine residue, Substituted with one amino acid residue selected from the group consisting of asparagine residues and oral isine residues, and / or Or

The amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, a threonine residue, an isoleucine residue, a glycine residue, a Paris residue, a phenylalanine residue, or a leucine residue. Substituted with one amino acid residue selected from the group consisting of an aspartic acid residue, a methionine residue, a tryptophan residue, and a alanine residue, and Z or

The amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a cysteine residue, tryptophan residue, leucine residue, methionine residue, arginine residue, glutamic acid residue, serine residue, Substituted with one amino acid residue selected from the group consisting of a luranin residue, a norin residue, and a proline residue. .

More specifically, the PI synthesis modified PLD of the present invention includes the following modified PLD:

(1) The amino acid residue at position 18 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is A modified PLD, which is an arginine residue and the amino acid residue at positions 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(2) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified PLD, wherein is a threonine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a cysteine residue;

(3) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is An isoleucine residue and in SEQ ID NO: 2 IS A modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown is a tryptophan residue;

(4) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a glycine residue. A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(5) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a valine residue A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a mouth isine residue;

(6) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a valine residue And a modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(7) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, And a modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a methionine residue;

(8) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, And the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, Modified P LD;

(9) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue, the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and SEQ ID NO: A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in 2 is a glutamic acid residue;

(10) the amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue, the amino acid residue at position 19 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and A modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a methionine residue;

(11) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is a ferranin residue, And a modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a methionine residue;

(12) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(13) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a leucine residue, and the sequence A modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown in No. 2 is an arginine residue; (14) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is a leucine residue, and the sequence A modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown in No. 2 is a tyrosine residue;

(15) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a palin residue, the amino acid residue at position 19 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and the sequence A modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown in No. 2 is a tyrosine residue;

(16) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a palin residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue. A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a glutamic acid residue;

(17) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a glycine residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 is an aspartic acid residue. A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a leucine residue;

(18) The amino acid residue at position 187 in the amino acid sequence shown in SEQ ID NO: 2 is a valine residue, the amino acid residue at position 19 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the sequence A modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown in No. 2 is a tryptophan residue;

(19) Amino acid at position 187 in the amino acid sequence shown in SEQ ID NO: 2 The residue is an arginine residue, the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 A modified PLD wherein the group is a serine residue;

(20) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(2 1) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue;

(2 2) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a histidine residue, and the amino acid residue at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 is a methionine residue A modified PLD wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a palin residue;

(2 3) 'The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is an algin residue, and the amino acid residue at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 is A modified PLD which is an arginine residue and wherein the amino acid residue at positions 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(2 4) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a wasparagine residue, and in the amino acid sequence shown in SEQ ID NO: 2, 1 9 A modified PLD in which the amino acid residue at position 1 is an alanine residue and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(2 5) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a leucine residue, and 1 in the amino acid sequence shown in SEQ ID NO: 2

9 Modified PLD, wherein the amino acid residue at position 1 is an alanine residue, and the amino acid residue at positions 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(26) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a mouth isine residue, and 1 in the amino acid sequence shown in SEQ ID NO: 2

9 Modified PLD, wherein the amino acid residue at position 1 is an alanine residue, and the amino acid residue at positions 3 and 8 in the amino acid sequence shown in SEQ ID NO: 2 is a phenol residue;

(2 7) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a mouth residue, and the amino acid at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified PLD in which the residue is a mouth residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(28) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 is A modified PLD that is a tyrosine residue and in which the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a proline residue; and

(29) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a valine residue, and the amino acid at position 1 91 in the amino acid sequence shown in SEQ ID NO: 2 The residue is a tyrosine residue and in SEQ ID NO: 2 above A modified PLD in which the amino acid residue at position 385 in the amino acid sequence shown is a tryptophan residue.

The PI synthesis modified PLD of the present invention can be produced from unmodified PLD using, for example, a site-directed mutagenesis method commonly used by those skilled in the art. More specifically, a step of obtaining a nucleic acid construct by introducing a mutation to cause the above-described substitution in a nuclear acid encoding a PLD containing the amino acid sequence of unmodified PLD; And obtaining a transformant; and culturing the obtained transformant to obtain the PI synthesis modified PLD as an expression product of a nucleic acid construct.

Examples of the unmodified PLD include a PLD containing the amino acid sequence shown in SEQ ID NO: 2 in the Sequence Listing. This amino acid sequence is the amino acid sequence of PLD derived from Streptomyces antibioticus strain. Further, in the present invention, as long as it exhibits PLD activity, it may have a sequence different from the amino acid sequence shown in SEQ ID NO: 2. The above different sequences are sequences in which amino acid residues other than the 187th tributofan residue, the 191st tyrosine residue, and the 385th tyrosine residue in the amino acid sequence shown in SEQ ID NO: 2 are different. obtain. For example, the unmodified PLD is a polypeptide having an amino acid sequence of a polypeptide having at least 50% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 and exhibiting PLD activity. Can be chido. The unmodified PLD has at least one amino acid residue substitution, deletion, insertion, and / or addition in the amino acid sequence shown in SEQ ID NO: 2 (however, the ability to synthesize is acquired). And a polypeptide containing the amino acid sequence of a polypeptide exhibiting PLD activity.

In this specification, the “sequence identity” in the amino acid sequence is calculated using a program such as 61 ^ 83-3 lastp program, GENETYX, etc. It is. Conditions such as the expected value, word size, gap, etc. can be set by a general method in this technical field. In the present invention, the sequence identity is at least 50%, preferably at least 70%, more preferably at least 79%, more preferably at least 90%, and even more preferably at least 95%.

The activity of PLD can be assessed by hydrolysis activity and phosphatidyl group transfer activity. The hydrolysis activity is, for example, 95% egg yolk phosphatidylcholine as a substrate, and a substrate concentration of 0.16% of 0.2 M acetate buffer (pH 4.0, 101 & 1 ^. & 01 ₂ , 1.3 % Of Triton X-100) can be evaluated by calculating the amount of choline produced per minute when reacted at 37 ° C. Here, one unit of PLD hydrolysis activity is the amount of enzyme that releases 1 μπιο1 of choline per minute. For example, 95% egg yolk phosphatidylcholine and ethanol is used as the substrate, and the substrate concentration is 0.16. / ₀ 0.21 ^ acetate buffer (1) 114. 0, C a C 1 2 of .1 Omm, 1. The included) 3% Tr ITON X- 100 when reacted at 37 ° C It can be evaluated by calculating the amount of choline produced per minute. Here, one unit of the transfer activity of PLD is the amount of enzyme that liberates 1 wmo 1 choline per minute. The above transfer activity can also be evaluated by performing a transfer reaction using phosphatidylparanitrophenol and ethanol and measuring the released para-trophenol (for example, Hagishiuta et al., Anal. Biochem. 1999, 276 (2), pp.161-165).

Examples of the unmodified PLD include actinomycetes, in particular, the genus Streptomyces, the genus Streptoverticillium, the genus Micromonospora, the genus Nocardia, PLD derived from microorganisms belonging to the genus Nocardiopsis, Actinomadura, etc. is preferably used. More Physically, Streptomyces antib ioticus, Streptomyces acidomyceticus, Streptomyces acidomyceticus AA5 8 6 species (Streptomyces sp. A A586 ), Streptomyces spm PMF (Streptomyces sp. PMF), Streptovertici Ilium cinnamoneum (Streptomyces cinnamoneum IF; 012852), Micro CC

12452), Nocardia mediterranei IFO 13142, Nocardiopsis dassonvillei IFO 13908, and Actinomadura libanotic a IFO 14095.

Mutation can be performed by, for example, a site-specific mutagenesis method commonly used by those skilled in the art, specifically, a gapped duplex method, a Kunkel method, a PCR mutagenesis method, and the like. . The nucleic acid used for substitution of amino acid residues can be designed according to the codon usage of the host for expressing PI synthesis modified PLD.

For the preparation of the nucleic acid construct, a conventional vector can be used depending on the host to be used. Examples of vectors include plasmid vectors such as pUC 18 and its derivatives, p BR 3 22 and its derivatives, p B 1 uescript II and its derivatives, p GEM and its derivatives, when the host is Escherichia coli, λ Examples include phage vectors such as ZAP II and λ gt 11, and pYAC derivatives when the host is yeast. The nucleic acid construct may contain factors such as an inducible promoter, a selection marker gene, and a terminator. The nucleic acid construct may contain a sequence that allows the polypeptide to be expressed to be expressed as a His tag polypeptide or a GST fusion polypeptide so that the polypeptide to be expressed can be easily isolated and purified. . The position of amino acid substitution and the kind of amino acid residue to be substituted are as described above.

For introduction of the nucleic acid construct into the host, a transformation method commonly used by those skilled in the art, for example, but not limited to, an electroporation method, a calcium phosphate method, a D EAE-dextran method, and the like can be used.

Although it does not specifically limit as said host, For example, colon_bacillus | E._coli, yeast, Bacillus subtilis, green bacterium, Salmonella, L cell, COS cell, CHO cell, sf9 cell etc. are mentioned. Specific examples of E. coli include HB 101 strain, C600 strain, JM109 strain, DH5a strain, BL21 (DE3) strain, and the like. Among these, from the viewpoint of expression control, the 6-enteric strain BL 21 (DE 3) is preferable. The culture conditions for the transformant can be appropriately set according to the host, vector, etc. to be used. Examples of the medium used for the culture include Luria-Bertani (LB) medium, SOC medium, and L medium. As the culture conditions, specifically, when E. coli BL 21 (DE3) strain is used as the host and pETKmS 1 is used as the vector, the transformant is prepared using the medium described in Example 2 below. Incubate at 30 ° C for 8 hours, and then add isopropyl 1-thio-j3-D-galactoside (IPTG) (1 mM) to the medium and incubate at 30 for 16 hours.

The PI synthetic modified PL D obtained as the expression product of the nucleic acid construct can be isolated and purified from the transformant culture by a general purification method. Examples of the purification method include salting out, ultracentrifugation, ion exchange column chromatography, adsorption power ram chromatography, hydrophobic column chromatography, and affinity column chromatography.

By appropriately combining the above purification methods, PI synthesis-modified PL D is purified, and at each purification stage, PLD activity is measured by the above-described method for evaluating hydrolysis activity and Z or transfer activity, and conventional SDS— Polyacrylamide gel electric The expression product can be obtained by evaluating the purity of the purified product by electrophoresis method or native polyacrylamide gel electrophoresis. The obtained expression product has a desired PI synthesis ability by measuring PLD activity 1 "using inositol as a substrate in the transfer activity evaluation method (for example, the above-described phosphatidyl transfer activity evaluation method). It can be confirmed that it is PL D.

The person skilled in the art, based on the information described in this application, 1 synthetic modified type? And a gene encoding 0 can be obtained. A gene encoding PI synthetic modified PL D is also included in the present invention. The gene of the present invention can be an artificial molecule including an artificial nucleotide derivative in addition to a natural polynucleotide such as DNA or RNA. The gene of the present invention may be a DNA-RNA chimeric molecule. By using this gene, for example, a PI synthesis modified PLD can be produced using a rough change technique as described above.

The PI synthetic modified PLD of the present invention can introduce inositol, which is a bulky compound, which was difficult to introduce with an unmodified PLD, into the phospholipid in the phosphatidyl group transfer reaction of the acylglycose phospholipid. Therefore, PI can be efficiently produced from raw phospholipids using the PI synthesis modified PLD of the present invention.

(Method for producing phosphatidylinositol)

The present invention also provides a method for producing phosphatidylinositol or a derivative thereof. This method includes a step of reacting a raw material phospholipid, inositol or a derivative thereof, and a PI synthesis modified PLD.

The PI synthesis modified PLD used in the method of the present invention is any of the above PI synthesis modified PLDs. This can be prepared from a microorganism that expresses PLD, as described above.

The amount of PI synthesis modified PLD used in the method of the present invention can be selected in the range of 10 to 200 units relative to the raw material phospholipid lg. In addition, fermentation One unit of elementary activity is 1% when reacted with 0.31 M Tris- maleate-NaOH buffer solution (pH 5.5) at a substrate concentration of 1% soy lecithin at 37 ° C. The amount of enzyme that releases 1 / mo 1 of choline in a minute.

As a raw material phospholipid in the method of the present invention, any one extracted from nature, purified after extraction, or synthesized can be used as long as it can serve as a substrate for PLD. A commercially available product or a product prepared by a known method may be used. For example, lecithins derived from plants such as soybean lecithin, defatted soybean lecithin, rapeseed lecithin, sunflower lecithin; lecithins derived from animals such as egg yolk lecithin, sheep, lecithin; There may be lecithin derived from microorganisms such as yeast. Examples of these lecithin components include phosphatidylcholine, phosphatidylethanolo-reamine, phosphatidylserine, phosphatidylglyceoseol, lysolecithin, phosphatidic acid, and the like, or a mixture thereof.

In the method of the present invention, inositol or a derivative thereof is used as a receptor for phosphatidyl group transfer reaction. Examples of inositol or a derivative thereof used as a receptor include inositol, inositol phosphate (eg, monophosphate, bisphosphate, or polyphosphate), inositol dalican (eg, inositol mannoside), and the like.

The molar ratio between the raw phospholipid and the receptor inositol or its derivative must be appropriately selected depending on the type of raw phospholipid. In general, it is appropriate to use 5 to 50 moles of receptor per mole of PC.

In the method of the present invention, the reaction solvent used for the phosphatidyl group transfer reaction may be a mixed solvent of an aqueous solvent and an organic solvent, or an aqueous solvent alone. Examples of the organic solvent include aliphatic hydrocarbons such as n-heptane, n-hexane, and petroleum ether; cyclic aliphatic hydrocarbons such as cyclopentane and cyclohexane; Examples include ethers such as jetyl ether and tetrahydrofuran; esters such as methyl acetate and ethyl acetate; and halogenated hydrocarbons such as carbon tetrachloride and chloroform. The aqueous solvent refers to water and an aqueous buffer solution. As water, ion-exchanged water, purified water, or distilled water is preferably used, and tap water can also be used. As the aqueous buffer, for example, an acetic acid buffer having a pH of 4 to 6, a phosphate buffer having a pH of 7 to 8, and the like are preferably used.

When the reaction solvent used in the method of the present invention is a mixed solvent of an aqueous solvent and an organic solvent, the mixing ratio of the aqueous solvent and the organic solvent in the reaction solvent is appropriately selected according to the type of the organic solvent to be used. can do. In general, an aqueous solvent: organic solvent can be used by mixing in a volume ratio of 1: 0.65 to 1: 100. In order to suppress the hydrolysis reaction of the phosphatidyl group of the raw material phospholipid, which is a side reaction, and to efficiently perform the target phosphatidyl group transfer reaction, the content of the aqueous solvent in the reaction system should be 10% by volume or less. Is preferred. The selection of organic solvents and the mixing ratio can be arbitrarily selected. .

The amount of the organic solvent in the reaction solvent used for the enzyme reaction is preferably 5 to 500 volume times, more preferably 10 to 100 volume times the weight of the phospholipid used as a raw material. If the amount of the organic solvent is less than 5 times, the viscosity of the solution in which the raw material substrate is dissolved becomes high and the reaction efficiency is lowered. On the other hand, if it exceeds 500 volume times, the phosphatidyl group transfer reaction efficiency is deteriorated.

In the method of the present invention, the temperature of the phosphatidyl group transfer reaction is preferably 10 to 60 ° C, more preferably 25 to 45 ° C. The time required for the reaction varies depending on the amount of enzyme and the reaction temperature, but is generally 0.5 to 48 hours. When the reaction solvent used in the method of the present invention is a mixed solvent of an aqueous solvent and an organic solvent, a two-phase system is formed. Therefore, in order to sufficiently mix the aqueous layer and the organic solvent layer, the reaction is performed during the reaction. It is preferable to perform treatment such as stirring and shaking as appropriate.

After the above reaction, for example, the PLD is deactivated by a treatment such as heating, and is allowed to stand. The water layer is removed by a heart separation method or the like to obtain an organic solvent layer, and the organic solvent is removed under reduced pressure to obtain the desired acylglycose phospholipid (for example, phosphatidylinositol). Furthermore, the obtained acylglyceport phospholipid can be purified to high purity by subjecting it to solvent fractionation, silica gel fractionation, high performance liquid chromatography, and the like.

Alternatively, after the above reaction, the aqueous layer and the organic solvent layer are separated by stationary treatment, centrifugation, etc., and if necessary, PLD and various receptor alcohols are added to the aqueous layer to create new raw materials. By mixing the phospholipid with the dissolved organic solvent, the aqueous layer can be used repeatedly for this reaction. From the organic solvent layer separated after the reaction, the desired acylglycose phospholipid (for example, phosphatidylinositol) can be obtained in the same manner as described above.

(Screening method for PLD having the ability to synthesize a glycyl-mouth phospholipid having a darlicol group)

The present invention also provides a method for screening for PLD having the ability to synthesize a acylglycose phospholipid having a glycol group.

The PLD to be screened to obtain a PLD having the ability to synthesize a glycyl-mouth phospholipid having a glycol group is not particularly limited. For example, the amino acid sequence of an unmodified PLD is a small number (for example, 1 to Several (eg, up to 2 or 3) amino acid residues may be polypeptides (modified PLDs) having different amino acid sequences. Such a polypeptide can be an artificially produced polypeptide. This includes site-specific mutagenesis methods commonly used by those skilled in the art, specifically, the gapped duplex method (gappeddup 1 ex) method, the Kunkel method (K unke 1 method), and the PGR mutagenesis method (for example, overlapping PCR). Or the like. Such polypeptides are derived from naturally occurring mutations. But it can be. The PLD subjected to the screening can also be a polypeptide isolated from an organism that can naturally produce PLD that is unclear whether it has the ability to synthesize a glycosylphospholipid having a glycol group.

The modified PLD to be screened can be obtained, for example, by the following procedure. Amino acid sequences that differ in amino acid residues at a specific or random position in a few (eg, 1 to a few (eg, 2 or 3)) from the amino acid sequence of an unmodified PLD (particularly, native PL D) DNA fragments encoding various polypeptides having These DNA fragments are ligated to an expression vector to obtain a nucleic acid construct. The obtained nucleic acid construct is introduced into a host to obtain a transformant. The obtained transformant is cultured to obtain a plasmid library in which a variety of modified I ³ LDs are produced as the expression product of the nucleic acid construct. Production of the nucleic acid construct, introduction into the host, and culture of the transformant are as described above. Screening can be performed for modified PLD expressed by the plasmid library. .

PLD can be subjected to screening, for example, as follows. A plasmid library containing the gene encoding the PLD is introduced into a host to obtain a transformant. This transformant is cultured, and the obtained colonies are transferred to a membrane (for example, a nitrocellulose membrane). Here, the medium should be stored as a master plate. Colonies are grown on the membrane, and the PLD is expressed and secreted in a medium containing I PTG. As a result, the expressed PL D is adsorbed on the membrane.

In the screening method of the present invention, a compound containing a glycol group can be used as a receptor introduced into the acylglycose phospholipid. Examples of such compounds include saccharides (preferably oligosaccharides (for example, saccharides composed of 1 to 10 saccharides), inositol or derivatives thereof (for example, inositol phosphates (for example, monophosphate, bisphosphate, or polyphosphorus)). Acid))) It is done. Preferably, inositol or a derivative thereof is used.

As described above, the starting phospholipid used in the screening method of the present invention may be any phospholipid that can serve as a PLD substrate. A phosphatidyl group transfer reaction is performed using raw phospholipids, receptors, and PLD. The reaction temperature is preferably 10 to 60 ° C, more preferably 25 to 40 ° C. The time required for the reaction varies depending on the reaction temperature, but is generally 0.5 to 48 hours. In the presence of the target P L D, this reaction yields the target acyl glyceport phospholipid.

The phosphatidyl group transfer reaction can be carried out, for example, by immersing the PLD adsorption membrane in a reaction solution containing a raw material phospholipid and a receptor. This reaction aqueous solution preferably contains a calcium salt. When the target acylglycose phospholipid is produced by the above phosphatidyl group transfer reaction, calcium ions coexisting in the aqueous solution and water-insoluble salts are formed, and deposited on the membrane where the enzyme reaction has occurred. This place means the place where the colony expressing the target PLD was present.

The glycol moiety of the acylglycerophospholipid produced by the phosphatidyl transfer reaction can be cleaved by oxidation and derivatized to aldehyde. The oxidant is formed using an oxidant, and any oxidant can be used as long as the oxidant can cleave the glycol moiety to induce aldehyde. As such an oxidizing agent, preferably, periodic acid or a salt thereof, lead tetraacetate, and sodium bismuth can be used. Examples of periodic acid or a salt thereof include metaperiodic acid, potassium metaperiodate, sodium metaperiodate, and sodium orthoperiodate. The temperature of this oxidation reaction is preferably 10 to 60 ° C, more preferably 20 to 40 ° C. The time required for the reaction varies depending on the reaction temperature, but is generally from 5 to 60 minutes.

The above aldehyde can be detected by techniques commonly used by those skilled in the art. Arde As a method for detecting hydride, for example, by coupling the above aldehyde with a hydrazine compound (eg, dinitrophenylhydrazine, 7_nitro-1,2,1,3-benzoxadiazole (NBD) monohydrazine, etc.). A reaction in which the hydrazine becomes a hydrazone can be used, but the reaction is not limited thereto. The silver mirror reaction can also be used. For example, when NBD hydrazine is reacted with the aldehyde, NBD hydrazine becomes NBD hydrazone and exhibits strong fluorescence. The presence of aldehyde can be detected by measuring this fluorescence.

Determination of the presence of aldehydes leads to the presence of acylglycose phospholipids containing glycol moieties that have generated aldehydes, and, in addition, the ability to synthesize glycosyl phospholipids containing glycol moieties via phosphatidyl group transfer reactions. The existence of PLD can be recognized.

When these series of reactions are performed using the above PLD adsorption membrane, the presence of aldehyde can be detected on the membrane. Therefore, whether or not PLD having the desired synthesis ability has been obtained can be identified by the presence or absence of detection in the membrane. Furthermore, since a transformant expressing PLD having this synthesis ability exists in the master plate corresponding to this membrane, a transformant having a PLD gene having synthesis ability can be obtained. Furthermore, by analyzing the nucleotide sequence of a gene having a synthetic ability, a target PLD having a synthetic ability can be identified. Example

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

In this example, the construction of recombinant vectors, the production of transformants, the preparation of plasmids from transformed cells or clones, etc. In accordance with methods commonly used by those skilled in the field of engineering and genetic engineering,

(Example 1: Library creation)

PELB-P LD 1 (prepared as described in Y. Iwasaki et al., J. Ferment. Bioeng., 1995, 79 卷, pp. 417 421) was prepared using restriction enzymes Xb a I and X ho

Digested with I, ribosome binding site, pelB signal derived from ET22b (Novagen) and full length of PLD gene derived from Streptomyces antibioticus (1530 bp; nucleotide sequence is as shown in SEQ ID NO: 1 in the sequence listing) ) Was collected and inserted into pBluescriptll KS ⁺ (Toyobo) previously digested with XbaI and XhoI to prepare pPELB- PLD-KS. This was digested with the restriction enzyme Apa I, and the reverse repeat sequence downstream of the PLD gene was removed together with a fragment around the stop codon of the PLD gene. On the other hand, p SAl (prepared as described in Y. Iwasaki et al., Appl. Microbiol. Biotechnol., 1994, 42 ，, pp. 290-299) was digested with restriction enzymes BamHI and Sa1I. Then, a fragment containing the latter half of the PLD gene was excised and inserted into pBluescript II SK ⁺ (manufactured by Toyobo) previously digested with BamHI and Sa1I to obtain pSAl-BS. pSA 1-BS was digested with the restriction enzyme Ap a I, and the fragment around the stop codon of the PLD gene was excised. This excised fragment is ligated with pPELB-PLD-KS digested with A pa I, then cut with Kpn I and XbaI, and a fragment containing the pelB signal and the full length of the PLD gene (1530 bp) is obtained. Obtained. This fragment was ligated with pBluescript II KS ⁺ plasmid (manufactured by Toyobo Co., Ltd.) previously cut with Sa1I and XbaI to obtain plasmid pPELB-PLD-KSII.

PCR using the plasmid _P HSG399 (Takara Shuzo) containing the cat gene with primers CAT-F1 (SEQ ID NO: 3) and CAT-R1 (SEQ ID NO: 4): 10XLA Taq polymerase Buffer (Takara Shuzo, 1/10 volume), MgCl ₂ (2.5 mM), dATP (0.2 mM), dGTP (0.2 mM), dCTP (0.2 mM), d TTP (0.2mM), Plasmid pHSG399 (lOng / ^ L), Primer CAT- Fl (0.5 μΜ), Primer CAT-Rl (0.5μΜ), LA TaqDNA Polymerase (Takara Shuzo, 0.0.25υηΐΐ ₈ /// ί ) / Temperature program: 94 ° C for 1 minute, then 94 10 seconds – 50 ° ○ 10 seconds, 7 2 ° C for 40 seconds for 30 cycles, followed by 7 2 ° C for 7 minutes, 4 ° C∞}, Digestion with B g 1 II gave a fragment containing cat. pETKmSl (prepared based on the description of Mishima N. et al., Biotechnol. Prog., 1997, 13 pp. 864-868) was digested with B g 1 II, and the cat gene fragment was ligated in the reverse direction. Plasmid pETKmS 卜 CAT was obtained. The plasmid was then cut with SacI and XbaI to excise a fragment containing the cat reverse sequence and the T7-lac promoter. This fragment was ligated to pPELB- PLD-KS II previously cut with Sac I and Xba I to obtain plasmid pPELB- PLD-KS Π-CAT (5 5 8 1 ρ). This plasmid pPELB- PLD-KSII-CAT was used as a type of overlapping PCR for introducing site-specific random amino acid substitutions.

pPELB-PLD-KS II-CAT in a saddle shape and using primers PL-F1 (SEQ ID NO: 5) and 0L-R1 (SEQ ID NO: 6) PCR (reaction solution composition (each concentration is the final concentration)): 10 0XLA Taq polymerase buffer (Takara Shuzo, 1/10 volume), MgCl ₂ (2.5 mM), d ATP (0.2 mM), dGTP (0.2 mM), dCTP (0.2 mM), dTTP (0.2 mM), pPELB — PL D-KSII-CAT (lOng / ^ L), Primer PL- Fl (0.5μΜ), Primer 0L-Rl (0.5Μ), LA TaqDNA Polymerase (Takara Shuzo, 0.025Units / μL) / Temperature Program: 94 After 5 minutes at ° C, 9 5 ° C1 0 seconds — 60. C1 0 sec — 7 2 ° C 1 min 10 sec amplified by 30 cycles, 72 ° C10 0 min, 4 ∞ min}, and the cat gene, T7-lac promoter, ribosome binding site of this saddle type plasmid, An amplified fragment (fragment 1) containing the pelB signal sequence and part of the mature PLD was obtained.

The complete amino acid sequence of the mature protein of Streptomyces anti-Pioticus-derived PLD is as shown in SEQ ID NO: 2 in the sequence listing. This strept From the N-terminus of the mature amino acid of Myces antibioticus PLD, pPELB- PLD- KSII- CAT is truncated so that the 7th tryptophan residue and the 191st tyrosine residue are replaced. PCR using primers 0L-F1 (SEQ ID NO: 7) and 0L-R2 (SEQ ID NO: 8) (reaction solution composition (each concentration is the final concentration): 10XLA Taq polymerase buffer (Takara Shuzo, 1/10 volume) ), MgCl ₂ (2.5mM), dATP (0.2mM), dGTP (0.2mM), dCTP (0.2mM), dTTP (0.2niM), pPELB- PLD-KSII-CAT (lOng // zL), primer OL- Fl (0.5 μM), Primer 0L-R2 (0.5 μM), LA TaqDNA Polymerase (Takara Shuzo, 0.025Units / μL) / Temperature program: 94 ° C for 2 minutes, 95 ° C for 10 seconds 67 ° C 10 s — 72 ° C 30 s, 25 cycles, followed by amplification at 72 ° C 7 min, 4 ° C∞}, fragments with various nucleotide sequences at the two amino acid mutation sites (fragment 2 ) It was. In addition, pPELB- PLD-KSII-CAT is in a saddle shape so that the 385th tyrosine residue from the N-terminus of this mature amino acid is substituted, and primer pair OL-F2 (SEQ ID NO: 9) and PL-R1 ( PC.R {reaction solution composition (each concentration is final concentration): 10XLA Taq polymerase buffer (Takara Shuzo, 1/10 volume), Mg Cl ₂ (2.5 mM), dATP (0.2 mM), dGTP (0.2mM), dCTP (0.2mM), dTTP (0.2mM), pPELB- PLD-KSII-CAT (lOng // L), primer OL-F2 (0.5M), primer PL-Rl ( 0.5μΜ), LA TaqDNA polymerase (manufactured by Takara Shuzo Co., Ltd., 0.025Units / μ °) 2Temperature program: 94 ° C for 2 minutes, 95 ° C for 10 seconds and 1 second at 62 ° C for 10 seconds—72 ° C for 30 seconds, followed by 72 ° C10 Min, 4 ° C∞},

Fragments (fragment 3) having various nucleotide sequences at one amino acid mutation site were obtained.

Next, fragment 2 and fragment 3 are hybridized using their overlapping parts, and PCR (reaction solution composition) is performed using primers OL-F1 (SEQ ID NO: 7) and PL-R1 (SEQ ID NO: 10). (each concentration final concentration): 10XLA Taq polymerase buffer (manufactured by Takara Shuzo, 1/10 volume), M _g Cl ₂ (2.5 mM), dATP (0.2mM), dGTP (0.2mM), dCTP (0.2mM), dTTP (0.2mM), Fragment 2 (10ng / L), Fragment 3 (lOng / ^ L), Primer OL-F1 (0.5μΜ), Primer PL-Rl (θ.5 μΜ), LA TaqDNA polymerase (Takara Shuzo, 0.025Units / ^ L) Temperature program: 9 4 ° C 2 minutes, 9 5 ° C 1 0 seconds 1 6 2 ° C 10 seconds-7 2 ° C4 5 seconds 25 cycles followed by 7 2 ° C 10 minutes, 4 ° C ∞} to obtain an amplified fragment (fragment 4).

Subsequently, Fragment 1 and Fragment 4 are hybridized using the overlap part, and overlap extension reaction without adding a primer (reaction solution composition (each concentration is final concentration)): 10XLA Taq Polymer Buffer solution (Takara Shuzo, 1/10 volume), MgCl ₂ (2.5 mM), dATP (0.2 mM), dGT P (0.2 mM), dCTP (0.2 mM), dTTP (0.2 mM), Fragment 1 (0.2 ng / L), Fragment 4 (0.2ng / μL), LA TaqDNA Polymerase (Takara Shuzo Co., Ltd., 0.05Units / μL) Non-temperature program: 9 4 ° C 5 minutes, then 9 8 ° C 10 seconds 5 9 ° C 1 0 seconds 1 2 2 ° C 2 minutes 20 cycles followed by 4 ° C∞}. Subsequently, the reaction solution after the overlap extension reaction is made into a saddle type, and the primer composition P L-F1 (SEQ ID NO: 5) and PL-R1 (SEQ ID NO. Concentration): Reaction volume after overlap extension reaction 1/2 volume, 10 XLA Taq polymerase buffer (Takara Shuzo, 1/20 volume), MgCl ₂ ( _2.5 mM), dATP (0.2 mM), dGTP (0.2 mM), dCTP (0.2mM), dTTP (0.2mM), Fragment 1 (10ng /// L), Fragment 4 (lOng / z L), Primer P L-Fl (0.5 μM), Primer PL-Rl ( 0.5 / zM), LA TaqDNA Polymerase (Takara Shuzo, 0.025Units / L) / Temperature program: 94 ° C for 3 minutes, then 9 8 ° C for 10 seconds 1 5 9 ° C for 10 seconds _ 7 2 ° C for 2 minutes 30 cycles, followed by 7 2 ° C 10 minutes, 4 ° C ∞}, gave amplified fragments. Thus, amplified fragments having various base sequences at three amino acid mutation sites were obtained.

This amplified fragment is cleaved with restriction enzymes Spe I and Xho I, 31) An expression vector was obtained by ligation to pETKmS 卜 Term which had been cut by 6 1 and 3 & 11. pETKmSl-Term was created by cutting the above pETKmSmCAT with BamHI and self-ligating. This expression vector was used to transform E. coli (i) DH5α. This transformed Escherichia coli DH5α is added to LB agar medium (containing 50 / ig / ml kanamycin and SOjUg / ml chloramphenicol;

10 g / L tryptone (Difco), 5 _g / L yeast extract (Difco), 10 g / L NaCl, 15 g / L agar (Wako Pure Chemical)) were grown and colonies were formed. Colonies on the agar medium were spread with LB liquid medium (10 g / L tryptone (Difco), 5 g / L yeast extract (Difco), 10 g / L NaCl), and the colonies were scraped. A plasmid was prepared from the obtained cells, and a mixture containing the mutated PLD gene was obtained as one plasmid library.

(Example 2: Expression of plasmid library)

The above plasmid mixture was introduced into E. coli BL 21 (DE3), which is an expression host, and grown on an LB agar medium having the same composition as described in Example 1 at 37 ° C. for 16 hours. A nitrocellulose membrane (Hy bond C; manufactured by Amersham) was placed on the obtained colonies to transfer the colonies. Next, synthetic medium (composition is as follows (mass per liter is shown): KH ₂ P0 ₄ 5 g, K ₂ HPO 4 5 g, Na ₂ HP0 ₄ 4.4 g, (NH ₂ ) ₂ S0 ₄ 3 g, Gnore course 5 g, Mg S0 ₄ '7H ₂ 0 3 g, F e S0 ₄ ' 7H ₂ 0 40 mg, C a

_{C 12 40mg, Mn SO 4 '} 7H 2 O 10mg, Co C l 2' 6H 2 O 2. 9mg, Cu S0 4 · 5H 2 0 3 mg, N a 2 M o O · 2 H 2 O 0. 36mg, _{_{H 3 B0 3 l Omg, Z}} n S0 4 - 7H 2 0 l Omg 1. those supplemented with 5% agar) were transferred film on the colonies 8 hours while keeping to film at 30 ° C Grow. Colonies on the LB agar medium were stored in a refrigerator as a master plate. Membrane on the above synthetic medium supplemented with 1 mM IPTG And incubated at 30 ° C for 16 hours to induce the expression of PLD on the membrane. In this expression system, the expressed PLD is released out of the cell and adsorbed onto the membrane. Bacterial residues were washed away from this membrane using an ultrasonic cleaner.

(Example 3: Screening of clones for expression of PI synthetic modified PLD)

14 hours at 30 ° C in the substrate solution (10% soy lecithin (SLP-PC70, manufactured by Tsuru lecithin industry), 20% inositol, 2% calcium chloride, and 50 mM acetate buffer, H5.6) Immersion was performed and an enzyme reaction was performed on the membrane. When this enzyme reaction produces phosphatidylinositol (P I), which is an acidic phospholipid, these form a water-insoluble salt with the coexisting calcium ions and deposit at the place where the reaction takes place. After the reaction, excess substrate solution was washed away with water, and the membrane was immersed in a 10% aqueous sodium periodate solution at room temperature for 10 minutes. By this sodium periodate treatment, the deposited glycol part of PI is oxidatively cleaved and induced to aldehyde. Excess sodium periodate solution was washed away with water, and an aqueous NBD-hydrazine solution (0.05% NBD-H, 10% DMSO) was applied to the membrane. NBD-H reacts with aldehyde to form NBD-hydrazone and exhibits strong fluorescence. This fluorescence was observed under ultraviolet (365 ηm) irradiation, and bright spots were identified. This spot corresponds to the location where PI on the membrane was present. A colony matching this spot was isolated from the master plate to obtain a positive clone. This yielded 49 positive clones (Table 1).

(Example 4: Sequencing of positive clones)

A plasmid was prepared from the positive clone obtained in Example 3, and the mutation content of the PLD gene was determined by the dideoxy method. The results are shown in Table 1. Table 1 Amino acids

Clone 187i Trp 19 "Tyr 385 Tyr positive so.

Gene code coded gene code coded fe dark coded number of clones amino acid amino acid amino acid

1 TTC Phe AGG Arg TAC Tyr 19

2 TTC Phe ACG Thr Dc GC Cys 1

3 TTC Phe ATC lie TGG Trp 1

4 TTC Phe GGC Gly TAC Tyr ί

5 TTC Phe GTG Val CTC Leu 1

6 TTC Phe GTG Val TAC Tyr 1

7 TGG Trp TAC Tyr ATG Met 1

8 TGG Trp TAG Tyr CGG Arg 1

9 TCG Ser TAG Tyr GAG Glu 1

10 TCC Ser AGG Arg ATG Met 1

11 TCC Ser TTC Phe ATG Met 1

12 TAC Tyr TTC Phe TAC Tyr 1

13 TAC Tyr TTG Leu AGG Arg 1

14 TAG Tyr TTG Leu TAC Tyr 1

15 GTG Val TAC Tyr TAG Tyr 1

16 GTC Val TAC Tyr GAC Glu 1

17 GGC Gly GAC Asp CTG Leu 1

18 GCG Val TGG Trp TGG Trp 1

19 CGG Arg TAC Tyr AGC Ser 1

20 CGG Arg TAC Tyr TAC Tyr

21 CGG Arg TAC Tyr TTC Phe 1

22 CAC His ATG Met GTC Val 1

23 AGG Arg AGG Arg TAG Tyr 1

24 AAC Asn GCG Ala TAC Tyr 1

25 TTG Leu GCG Ala TAC Tyr 1

26 TTG Leu GCG Ala TTC Phe 1

27 TTG. Leu TTG Leu TAC Tyr 1

28 TGG Trp TAC Tyr CCG Pro 1

29 GTG Val TAC Tyr TGG Trp 1

49 (Example 5: PI synthesis by modified PLD)

All numbered clones in Table 1 were liquid cultured at 30 ° C. in a synthetic medium (excluding agar) having the same composition as that described in Example 2. After about 8 hours, IPTG was added to a final concentration of ImM to induce PLD expression. The supernatant obtained by sterilization by centrifugation 16 hours after induction was fractionated with ammonium sulfate to prepare a PLD crude enzyme product.

Dipanolemitoyl phosphatidylcholine 5mg, myo_inositonore 40mg, calcium chloride 4mg, 50mM acetate buffer solution 180L and above ΡLD crude enzyme product 20L (approx. 1 unit) with stirring at 30 ° C for 14 hours I let you.

Phospholipid qualitative analysis was performed by thin layer chromatography (TLC method) under the following conditions:

TLC plate ··· Silica gel 60 plate (Merck)

Developing solvent · · 'Black mouth form: Methanol: Petroleum Itel: Acetic acid-4: 2: 3: 1 (V / V / V / V)

Deployment time · · · About 20 minutes

Detection ··········· 0.1% 2, 7-dichlorofluorescein ethanol solution after mist fogging, observation power under 365 nm UV irradiation Phospholipid spots were detected. As a result, in the PLD prepared from any clone, a new spot corresponding to dipalmitoyl PI was detected when inositol was used as a substrate. It was also analyzed by mass spectrometry. Mass spectrometry was performed using a time-of-flight secondary ion mass spectrometer (TOF—S IMS, trade name PHITRIFT III, ULVAC-FINE) and negative ion mode. As a result, in the PLD prepared from any of the clones, ions corresponding to dipalmitoyl PI were detected when inositol was used as a substrate. Industrial applicability

Since the novel PLD of the present invention can introduce a bulky compound such as inositol into the phospholipid in the phosphatidyl group transfer reaction of the silk, lyserophospholipid, it can be obtained at low cost using this PLD. For example, PI can be easily and efficiently produced from lecithin). Furthermore, according to the present invention, there is also provided a method for easily and rapidly screening for a novel PLD having the ability to synthesize a acylglyceport phospholipid having a glycol group.

Claims

The scope of the claims

1. A modified phospholipase D having the ability to synthesize phosphatidylinositol, comprising:

(B) in the amino acid sequence of (A), substitution of at least one amino acid residue at a position different from the amino acid residues located at positions 1 87, 19 1 and 3 85; A modified phospholipase 0 having a deletion, insertion, and an amino acid sequence of a polypeptide having a Z or addition and having the ability to synthesize phosphatidylinositol.

2. The amino acid residue at position 18 in the amino acid sequence shown in SEQ ID NO: 2 is phenylalanine residue, serine residue, tyrosine residue, valine residue, glycine residue, arginine residue, histidine Substituted with one amino acid residue selected from the group consisting of residues, asparagine residues, and leucine residues, and / or

1 9 1st amino acid residue in the amino acid sequence shown in SEQ ID NO: 2 Force Arginine residue, Threonine residue, Isoleucine residue, Glycine residue, Palin residue, Phenylalanine residue, Leucine residue, Aspartic acid Substituted with one amino acid residue selected from the group consisting of a residue, a methionine residue, a tryptophan residue, and an alanine residue, and or

Amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 1 Cysteine residue, tryptophan residue, leucine residue, methionine residue, arginine residue, glutamic acid residue, serine residue, phenol A luranin residue, Substituted with one amino acid residue selected from the group consisting of a valine residue and a proline residue,

The modified phospholipase 13 according to claim 1. 3. The modified phospholipase D of claim 1, selected from the group consisting of:

(1) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D wherein is an arginine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosin residue;

(2) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein is a threonine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a cysteine residue;

(3) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase D which is an isoleucine residue and wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue;

(4) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a vinylalanine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 Is a glycine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, modified phospholipase D; (5) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a furanine residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 The group is a palin residue, and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: + 2 is a leucine residue.

5 groups, modified phospholipase D;

(6) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 Is a valine residue, and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue.

Modified phospholipase D, which is an L0 group;

(7) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the amino acid residue at position 1 91 in the amino acid sequence shown in SEQ ID NO: 2 Is a tyrosine residue, and the amino acid residue at positions 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is methionine.

Modified phospholipase D, which is an L5 residue;

(8) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is tyrosine And the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is arginine.

Modified phospholipase D, which is 20 residues;

(9) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue, and the amino acid residue at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 is Is a tyrosine residue, and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a glutamic acid residue.

15 modified phospholipase D;

(1 0) amino acid at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 The nonacid residue is a serine residue, the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and 3 in the amino acid sequence shown in SEQ ID NO: 2 8 Modified phospholipase D wherein the amino acid residue at position 5 is a methionine residue;

(1 1) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue, and the amino acid at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the residue is a ferroalanine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a methionine residue;

(1 2) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and 1 9 1 position in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D wherein the amino acid residue is a phenylalanine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosin residue;

(1.3) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and the amino acid residue at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 is A modified phospholipase D, wherein the group is a mouth isine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue;

(14) 'The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue, and the amino acid at position 1 1 9 in the amino acid sequence shown in SEQ ID NO: 2 Modified phospholipase D, wherein the residue is a leucine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(16) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a palin residue, and amino acid at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D wherein the acid residue is a tyrosine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a glutamic acid residue;

(1 7) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a glycine residue, and the amino acid residue at position 1 1 9 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the group is an aspartic acid residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a leucine residue;

(1 8) In the amino acid sequence shown in SEQ ID NO: 2, the amino acid residue at position 1 87 is a valine residue, and in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D wherein the amino acid residue at the position is a tryptophan residue and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue;

(19) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 is tyrosine A modified phospholipase D, wherein the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a serine residue;

(20) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 The group is a tyrosine residue, and said SEQ ID NO: A modified phospholipase D in which the amino acid residue at position 3 85 in the amino acid sequence shown in 2 is a tyrosine residue;

(2 1) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid residue at position 1 91 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein t is a tyrosine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue;

(2 2) The amino acid residue at position 1807 in the amino acid sequence shown in SEQ ID NO: 2 is a histidine residue, and the amino acid at position 1191 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the group is a methionine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a palin residue;

(23) The amino acid residue at position 1807 in the amino acid sequence shown in SEQ ID NO: 2 is an arginine residue, and the amino acid residue at position 191 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D wherein is an arginine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(2 4) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a asparagine residue, and the amino acid residue at position 1 91 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D wherein the group is an alanine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(25) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a mouth isine residue, and the amino acid at position 1 in the amino acid sequence shown in SEQ ID NO: 2 The residue is an alanine residue, and the amino acid residue at position 3 85 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue. A modified phospholipase D;

(2 6) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a mouth residue, and the amino acid at position 1 1 in the amino acid sequence shown in SEQ ID NO: 2 A modified phospholipase D, wherein the residue is an alanine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a phenylalanine residue;

(2 7) The amino acid residue at position 1 87 in the amino acid sequence shown in SEQ ID NO: 2 is a mouth isine residue, and the amino acid residue at position 1 in the amino acid sequence shown in SEQ ID NO: 2 Modified phospholipase D, wherein the amino acid residue is a leucine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tyrosine residue;

(28) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a tryptophan residue, and the amino acid residue at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 is A modified phosphorylase D, which is a tyrosine residue, and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a proline residue; and

(29) The amino acid residue at position 18 7 in the amino acid sequence shown in SEQ ID NO: 2 is a valine residue, and amino acid at position 19 1 in the amino acid sequence shown in SEQ ID NO: 2 Modified phospholipase 0, wherein the acid residue is a tyrosine residue and the amino acid residue at position 385 in the amino acid sequence shown in SEQ ID NO: 2 is a tributophane residue.

4. A gene encoding the modified phospholipase D according to any one of claims 1 to 3.

5. A process for producing phosphatidylinositol or a derivative thereof, A method comprising a step of reacting a raw material phospholipid, inositol or a derivative thereof, and the modified phospholipase D according to any one of claims 1 to 3.

6. A method for screening phospholipase D, which has the ability to synthesize a glycylmouth phospholipid having a dallicol group, comprising:

A step of causing a phosphatidyl group transfer reaction using a raw material phospholipid, a compound having a glycol group, and phospholipase D to produce an acylglyceport phospholipid having the glycol group;

'Detecting the produced aldehyde

Wherein the production of the aldehyde is an indication that the phospholipase D has the ability to synthesize the desired acylglycose phospholipid.

7. The method according to claim 6, wherein the compound having a glycol group is inositol or a derivative thereof.

8. The method according to claim 6 or 7, wherein an oxidizing agent selected from the group consisting of periodic acid or a salt thereof, lead tetraacetate, and sodium bismuthate is used in the oxidation step.

9. The method according to any one of claims 6 to 8, wherein the aldehyde is detected by detecting a hydrazone compound produced by reacting the aldehyde with a hydrazine compound.