CN101223274A - Novel phospholipid processing agent - Google Patents

Abstract

To provide a novel phospholipase B (PLB) having a poor hydrolytic activity on phosphatidylinositol (PI) among diacyl-phospholipids, and to provide a process for producing PI and glycerylphosphorylcholine (GPC) from a phospholipid mixture by exploiting the substrate specificity of the enzyme. [MEANS FOR SOLVING PROBLEMS] A phospholipid processing agent comprising an enzyme having such a PLB activity that is highly effective on a phospholipid other than PI (e.g., phosphatidylcholine (lecithin) and poorly effective on PI.

Description

Novel phospholipid processing agent

Technical field

The present invention relates to a kind of novel phospholipid processing agent, described phospholipid processing agent has high phospholipase B (PLB) activity, and phosphatide is had substrate specificity.

Background technology

Phospholipid hydrolase is the general name of enzyme that can hydrolytic phosphatide.Phosphatide (glyceryl phosphatide) has by ester bond and is connected to lipid acid on the hydroxyl of the α position of glycerine and β position, also has simultaneously by phosphate group to be connected to choline, thanomin or inositol etc. on the hydroxyl of another α position of glycerine.

The enzyme of the lipid acid ester bond on the α position of the glycerine group of hydrolysis glyceryl phosphatide is commonly referred to as phospholipase A1.The enzyme of the fatty acid ester group on the β position of hydrolysis glycerine group is commonly referred to as Phospholipase A2.In addition, have phospholipase A1 activity and the active enzyme of Phospholipase A2 simultaneously and be commonly referred to as phospholipase B (PLB).

In addition, the phosphatide that only removes a fatty acid acyl α position and the β position fatty acid acyl from phosphatide is commonly referred to as lysophospholipid.The endonuclease capable of the lipid acid ester bond that hydrolysis stays after with the lysophospholipid effect produces the degradation production identical with above-mentioned PLB.Therefore, described enzyme also can be included in the PLB family.

On the other hand, the enzyme with glycerine group in the phosphatide and the ester linkage hydrolyzing between the phosphate group is commonly referred to as Phospholipase C (PLC).In addition, the hydrolysis enzyme of connecting key that has a phosphate group of choline or thanomin etc. is commonly referred to as Phospholipase D (PLD).

As mentioned above, PLB also has phospholipase activity.Known PLB is present in mould, intestinal bacteria and the yeast etc. of animal, plant, Penicillium notatum.A little less than the effect very of PLB to the phosphatide of diacyl form, but very strong to the effect of lysolecithin.Therefore, PLB never is used for the common phosphatide of effective hydrolysis.In addition, for those known PLB, report is only arranged, and they can act on various phosphatide, for example phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositols (non-patent literature 1 and 2).

Existing various reports have been reported that phosphatidylinositols is included in the soybean lecithin and are closely related with the signal transduction of cell.In recent years, it is reported that the absorption phosphatidylinositols can reduce triacylglycerol (TG) level and the level (non-patent literature 3) that improves HDL-C (high density lipoprotein cholesterol) in the blood.In the art, the physiological derivative of the physiological function of phosphatidylinositols and phosphatidylinositols such as hemolytic phosphatidyl inositol and glycerine phosphinylidyne inositol just receive publicity.

In addition, according to another report, the hemolytic phosphatidyl inositol has anti-mildew effect (patent documentation 1).

Attempted the phosphatide effect being obtained phosphatidylinositols in the enzyme spcificity mode by PLC or PLD.Yet the purity of gained phosphatidylinositols is very low.Therefore, this area need have more optionally that method prepares phosphatidylinositols (patent documentation 2).Non-patent literature 2 been has also has been recorded and narrated if allow PLC act on phosphatide except that the phosphatidyl inositol, will be when about 30% percent hydrolysis termination reaction.

Patent documentation 1: Japanese kokai publication hei 06-256366 communique

Patent documentation 2: Japanese kokai publication sho 62-48390 communique

Non-patent literature 1:Biochimica Biophysica Acta (1974) 369,245～253

Non-patent literature 2:Biochimica Biophysica Acta (1975) 403,412～424

Non-patent literature 3:Biochem.J (2004) 382, and 441～449

Non-patent literature 4:Jim W.Burgess etc., Journal of Lipid Research (46), 350～355

Summary of the invention

Problem to be solved by this invention

An object of the present invention is to provide a kind of phospholipid processing agent, described phospholipid processing agent has high PLB activity, and phosphatide is had substrate specificity; The present invention also provides a kind of method that can prepare high-purity phospholipid acyl inositol and glycerophosphoryl choline effectively.

Solve the means of described problem

The inventor addresses the above problem to have carried out deep research; found that; enzyme from mycocandida (Candida) has high PLB activity to the phosphatide of diacyl form surprisingly; and described enzyme unexpectedly has such specificity to phosphatide: the phosphatide in the described enzyme selectivity ground hydrolyzed soy phosphatide (it is a mixture of phospholipids) except that the phosphatidyl inositol; utilize described specificity, the inventor has found the method that can make high-purity phospholipid acyl inositol and glycerophosphoryl choline effectively.At last, the inventor has finished the present invention based on described discovery.Be usually used in foodstuffs industry and medicinal product with the lipase in the raw material production from the described enzyme of mycocandida.Yet, only reported that described enzyme has the what is called " lipase activity " that acts on neutral lipid.Do not report that described enzyme has the PLB activity.

That is, the present invention relates to following every:

＜1 〉, a kind of phospholipid processing agent, described phospholipid processing agent comprises: from mycocandida (Candida) and have the active enzyme of phospholipase B (PLB).

＜2 〉, a kind of phospholipid processing agent, described phospholipid processing agent comprises: have the active enzyme of phospholipase B (PLB), described enzyme only is regardless of the phosphatidylinositols of separating in the mixture of phospholipids basically.

＜3 〉, as item＜1 or＜2〉described phospholipid processing agent, wherein, have the active enzyme of phospholipase B (PLB) and also have lipase activity.

＜4 〉, as item＜1～＜3〉each described phospholipid processing agent, wherein, have the active described enzyme of PLB and have following physicochemical property:

(1) effect: with phosphatide be hydrolyzed into 2 mol ratios free fatty acids and etc. the effect of glycerophosphoryl choline of mol ratio;

(2) molecular weight: 53,000 ± 3,000 (according to the SDS electrophoresis technique determining);

(3) iso-electric point: pH 4.21 ± 0.2;

(4) best pH: about pH 5.5～6.5;

(5) pH stability: about pH 5～9 (handling 90 minutes for 37 ℃);

(6) stability: 55 ℃ (handling 10 minutes) at pH 5; With

(7) substrate specificity: to the specific activity of phosphatidylinositols is below 10% of specific activity to phosphatidylcholine.

＜5 〉, a kind of phospholipid processing agent, described phospholipid processing agent comprises the enzyme that the nutrient solution by column candiyeast (Candida cylindracea) obtains.

＜6 〉, a kind of phospholipid processing agent, described phospholipid processing agent obtains by following steps:

(1) cultivates the column candiyeast;

(2) nutrient solution of concentrated column candiyeast;

(3) make the enzyme precipitation with organic solvent;

(4) with hydrophobic chromatography the rough enzyme solution that step (3) obtains is purified; With

(5) enzyme that step (4) is obtained with ion exchange chromatography separates and purifies.

＜7 〉, a kind of phospholipid processing agent, described phospholipid processing agent comprises at least a enzyme that is selected from the following enzyme: the enzyme with aminoacid sequence of SEQ ID No.1; Be more than 75% and have the active enzyme of PLB with the homology of the aminoacid sequence of SEQ ID No.1; Have disappearance in the aminoacid sequence of SEQ ID No.1, replace or increased one or more amino acid whose aminoacid sequences and had the active enzyme of PLB.

＜8 〉, a kind of phospholipid processing agent, described phospholipid processing agent comprises at least a enzyme that is selected from the following enzyme: the enzyme with aminoacid sequence of SEQ ID No.2; Be more than 75% and have the active enzyme of PLB with the homology of the aminoacid sequence of SEQ ID No.2; Have disappearance in the aminoacid sequence of SEQ ID No.2, replace or increased one or more amino acid whose aminoacid sequences and had the active enzyme of PLB.

＜9 〉, a kind of method of making the pure and mild glycerophosphoryl choline of phosphatidyl-4, described method comprises to be made＜1～＜8 in each described phospholipid processing agent act on mixture of phospholipids.

＜10 〉, a kind of method of making phosphatidylinositols, described method comprises to be made＜1～＜8 in each described phospholipid processing agent act on mixture of phospholipids.

＜11 〉, a kind of method of making phosphatidylinositols, said method comprising the steps of:

(1) make claim＜1 〉～＜8 in each described phospholipid processing agent act on mixture of phospholipids;

(2) with an organic solvent extract phosphatidylinositols; With

(3) by water-soluble or water miscibility alkyl-carbonyl alkyl solvent treatment, precipitation also reclaims phosphatidylinositols.

＜12 〉, as item＜9～＜11 in the method for each described manufacturing phosphatidylinositols, wherein, described mixture of phospholipids is from soybean.

＜13 〉, a kind of phosphatidylinositols, described phosphatidylinositols is by item＜10 〉～＜12 in each described manufacture method obtain, and the purity that has in total phospholipids is 50 moles more than the %.

＜14 〉, a kind of method of making glycerophosphoryl choline, described method comprises to be made＜1～＜8 in each described phospholipid processing agent act on mixture of phospholipids.

＜15 〉, a kind of method of making glycerophosphoryl choline, said method comprising the steps of:

(1) make claim＜1 〉～＜8 in each described phospholipid processing agent act on mixture of phospholipids;

(2) usefulness contains the solvent extraction of organic solvent and removes lipid composition, and glycerophosphoryl choline is collected in the water layer; With

(3) the described phospholipid processing agent that plays a role in the step (1) is adsorbed on the gac, and removes described phospholipid processing agent.

＜16 〉, as item＜9,＜14 or＜15〉described manufacturing glycerophosphoryl choline method, wherein, described mixture of phospholipids is from soybean.

＜17 〉, a kind of glycerophosphoryl choline, described glycerophosphoryl choline is by item＜14 〉～＜16 in each described manufacture method obtain, and have the above glycerophosphoryl choline purity of 55 weight %.

＜18 〉, the application of item＜13〉described phosphatidylinositols in preparation high purity Phosphoric acid glycerol esters inositol.

＜19 〉, the application of item＜17〉described glycerophosphoryl choline in preparation high purity cholinphospholipide.

＜20 〉, contain by item＜10 〉～＜12 in food, medicine or the makeup of the described phosphatidylinositols made of each described manufacture method.

＜21 〉, contain＜14～＜16 in food, medicine or the makeup of the described glycerophosphoryl choline made of each described manufacture method.

＜22 〉, a kind of hemolytic phosphatidyl inositol, described hemolytic phosphatidyl inositol is to have phospholipase A1 or the active enzyme of Phospholipase A2 acts on by item＜10 by making 〉～＜12 in the described phosphatidylinositols of each described manufacture method manufacturing obtain.

＜23 〉, a kind of method of making the hemolytic phosphatidyl inositol, described method comprises making to have phospholipase A1 or the active enzyme of Phospholipase A2 acts on by item＜10 〉～＜12 in the described phosphatidylinositols made of each described manufacture method.

＜24 〉, contain by item＜23 food, medicine or the makeup of the hemolytic phosphatidyl inositol made of described manufacture method.

＜25 〉, a kind of glycerine phosphinylidyne inositol, described glycerine phosphinylidyne inositol is by the active enzyme of phospholipase B that has that phosphatidylinositols is had an abundant effect is acted on by item＜10 〉～＜12 in the described phosphatidylinositols of each described manufacture method manufacturing obtain.

＜26 〉, a kind of method of making glycerine phosphinylidyne inositol, described method comprises acts on by item＜10 the active enzyme of phospholipase B that has that phosphatidylinositols is had an abundant effect 〉～＜12 in the described phosphatidylinositols made of each described manufacture method.

＜27 〉, contain by item＜26 food, medicine or the makeup of the described glycerine phosphinylidyne inositol made of described manufacture method.

＜28 〉, food, medicine or makeup, described food, medicine or makeup contain the two or more materials that are selected from the group that following material forms: by item＜10 〉～＜12 in the phosphatidylinositols made of each described manufacture method; By item＜14 〉～＜16 in the glycerophosphoryl choline made of each described manufacture method; By item＜23〉the hemolytic phosphatidyl inositol made of described manufacture method; With by＜26〉the described manufacture method glycerine phosphinylidyne inositols of making.

Effect of the present invention

Use phospholipid processing agent of the present invention to make and can prepare highly purified phosphatidylinositols, hemolytic phosphatidyl inositol, glycerophosphoryl choline and glycerine phosphinylidyne inositol etc., these materials can be used as functional phosphatide or functional phosphatide raw material.

Description of drawings

Fig. 1 has shown the color atlas that is obtained by the gel filtration chromatography as the PLB of the phospholipid processing agent of the embodiment of the invention 4.

Fig. 2 has shown the best pH as the PLB of the phospholipid processing agent of the embodiment of the invention 5.

Fig. 3 has shown the pH stability result as the PLB of the phospholipid processing agent of the embodiment of the invention 5.

Fig. 4 has shown the thermostability result as the PLB of the phospholipid processing agent of the embodiment of the invention 5.

Fig. 5 has shown the electric electrophoresis result such as grade as the PLB of the phospholipid processing agent of the embodiment of the invention 5.

Embodiment

Formation of the present invention and preferred embodiment hereinafter will be described in further detail.

1, as the phospholipase B (PLB) of phospholipid processing agent

In this manual, term " PLB " is meant the enzyme of the synthetic and fatty acid ester exchange of hydrolysis, the fatty acid ester of the α position that is used to carry out phosphatide and the fatty acid ester on the β position.PLB of the present invention comprises any enzyme of the lipid acid ester bond that acts on lysophospholipid and hydrolysed residue.In addition, term " phosphatide processing " is meant such reaction, the synthetic or fatty acid ester exchange of for example hydrolysis of phosphatide, fatty acid ester etc.Described term refers to that preferably described hydrolysis or fatty acid ester synthesize, and more preferably refer to described hydrolysis.In another preferred mode, described term refers to that described fatty acid ester is synthetic.In addition, machining agent of the present invention not only comprises PLB itself, but also comprises the PLB that is added with such as enzyme stabilizers such as sugar and pH buffer reagent.In addition, described phospholipid processing agent can provide with forms such as dried powder or liquid by the mode that is similar to conventional enzyme.

The invention provides and have the active phospholipid processing agent of PLB, described phospholipid processing agent only is regardless of the phosphatidylinositols of separating in the mixture of phospholipids basically, but other components of decomposing described mixture.

With regard to phospholipid processing agent, phrase " only is regardless of basically and separates the phosphatidyl inositol " and is meant and is being limited to below 10% on respect to the specific activity (relative reactivity) of phosphatidylcholine as the phosphatidylinositols in the mixture of phospholipids of substrate.Mean its specific activity and be preferably below 7%, more preferably below 5%, especially be preferably below 3%, most preferably be below 1%.This phrase refers to that also the following of its specific activity is limited to more than 0.01%, more preferably more than 0.1%.In addition, this phrase refers to that also phosphatide except that phosphatidyl inositol and phosphatidylcholine (for example phosphatidylethanolamine, phosphatidylserine or phosphatidic acid (PA) etc.) is with respect to following being limited to more than 15% of the specific activity (relative reactivity) of phosphatidylcholine.Mean its specific activity and be preferably more than 20%, more preferably more than 25%, especially be preferably more than 30%, most preferably be more than 40%.Represent its specific activity on be limited to below 200%, be preferably below 150%, more preferably below 100%.

In addition, the PLB as phospholipid processing agent of the present invention preferably has following character:

1) effect: with phosphatide (for example Yelkin TTS) be hydrolyzed into 2 mol ratios free fatty acids and etc. the effect of glycerophosphoryl choline of mol ratio; With acting on tri-glyceride it is hydrolyzed to 3 mol ratios free fatty acids and etc. the effect of glycerine of mol ratio;

2) molecular weight: 53,000 ± 3,000 (according to the SDS electrophoresis technique determining);

3) according to etc. the iso-electric point that records of electric electrophoretic method: pH 4.21 ± 0.2;

4) optimum response pH: about pH 5.5～6.5;

5) pH stability: about pH 5～9 (handling 90 minutes for 37 ℃);

6) thermostability: 55 ℃ (handling 10 minutes) at pH 5; With

7) substrate specificity: to the specific activity of phosphatidylinositols is below 10% of specific activity to phosphatidylcholine.

Source (root) as the PLB of phospholipid processing agent of the present invention is not particularly limited, as long as described PLB has above-mentioned characteristic.When described PLB was natural origin, described PLB was preferably microbe-derived, more preferably from mycocandida, more specifically said so from the column candiyeast.In addition, can be based on the enzyme of genetic modification of the enzyme of above-mentioned natural origin as the PLB of phospholipid processing agent of the present invention.The PLB that makes by gene recombination technology is included among the PLB as phospholipid processing agent of the present invention, and preferably it has above-mentioned characteristic.

In addition, described PLB comprises enzyme itself with the aminoacid sequence shown in SEQ ID No.1 or 2 or the aminoacid sequence that has 75% above homology with the aminoacid sequence shown in SEQ ID No.1 or 2.Have the active any enzyme of PLB and also all be included among the PLB as phospholipid processing agent of the present invention, and such enzyme is not particularly limited, active as long as it has a PLB.Yet, be preferably more than 80% with respect to the homology of the aminoacid sequence shown in the SEQ ID No.1, more preferably more than 85%, more preferably more than 90%, especially be preferably more than 95%, most preferably be more than 98%.In addition, preferred described phospholipid processing agent only is regardless of basically and separates the phosphatidyl inositol.Term " only is regardless of basically and separates the phosphatidyl inositol " and aforesaid term synonym.

The term " homology " that the present invention uses is meant the homology of aminoacid sequence or dna nucleotide sequence, and it can adopt computer to pass through currently known methods (as sequence relatively) and measure.Used analysis software is GENETEX WIN 5.2 (by Software Co., Ltd makes).The illustrative computer program of using when measuring homology can be the GCG routine package (Devereux that comprises GAP, J. etc., 387 (1984)) or blast program bag (NCBI Nucleic Acids Research 12 (12):, or Altschul, S.F. etc., J.Mol.Biol., 215:403～410 (1990)) and the Smith-Waterman algorithm, but specifically be not defined in these.

In addition, comprise having same function and have disappearance in the aminoacid sequence shown in SEQ ID No.1 or 2, replace or increased by one to a plurality of amino acid whose aminoacid sequences and have the active modified enzyme arbitrarily of PLB as the PLB of phospholipid processing agent of the present invention.The lower limit of disappearance, displacement or the amino acid quantity that increases is preferably (but specifically not being limited to) more than one, and more preferably more than two, and the upper limit is preferably below 25, more preferably below 20, more preferably below 15, especially be preferably below 10, most preferably be below 5.In addition, preferred described phospholipid processing agent only is regardless of basically and separates the phosphatidyl inositol.Term " only is regardless of basically and separates the phosphatidyl inositol " and aforesaid term synonym.

The aminoacid sequence of well known 5 kinds of different lipase from the column candiyeast.Any natural lipase can be used as phospholipid processing agent of the present invention.Even it is disappearance, replace or increased by one and also can be used for following purposes to a plurality of amino acid whose enzymes, active as long as described enzyme has a PLB.

Simultaneously, phospholipid processing agent of the present invention preferably also has lipase activity.Described phospholipid processing agent can act on tri-glyceride and with described tri-glyceride be hydrolyzed into 3 mol ratios free fatty acids and etc. the glycerine of mol ratio.If described phospholipid processing agent is applied to the mixture of phosphatide and tri-glyceride, they can be subjected to hydrolysis simultaneously.

2, make the method for PLB

Be used for the PLB of phospholipid processing agent of the present invention can be easily with commercially available from the form of the enzyme product of mycocandida and use.As selection, described enzyme can be prepared by the product that obtains by the Institute of Micro-biology of cultivating mycocandida.The preferred embodiment of the microorganism of mycocandida includes but not limited to the strain of column candiyeast.The bacterial strain of described generation PLB can be the mutant strain of handling with ultraviolet rays and chemical mutagen.In addition, can use by importing the genetic recombinants that host cell makes with the aftermentioned PLB gene of form that can high level expression.

The nutrient solution component and the culture condition of microorganism culturing are not particularly limited, as long as bacterial strain can generate PLB.The culture condition that is fit to described enzyme generation is generally under aerobic conditions in moderate heated culture temperature culture condition in nutrient medium.When with described strain culturing in any described condition following time, can observe the generation of good bacterial growth and enzyme.Specifically, for example, the carbon source that is used for nutrient solution comprises glucose, sucrose, fructose, lactose, maltose, W-Gum and yam starch.The example of nitrogenous source comprises wheat bran, peptone, yeast extract, soyflour, defatted soyflour, casein, cottonseed meal, degreasing cottonseed meal, gelatin, each seed amino acid (for example L-glutamic acid), ammonium sulfate and urea etc.Described carbon source and nitrogenous source can suitably be used in combination.Wherein, preferred examples comprises the combination of soyflour and glucose or the combination of defatted soyflour and glucose.The example of other components that can use in described nutrient solution has such as inorganic salt such as salt, sodium phosphate, sal epsom and calcium chloride, or the like.Wherein can preferably use salt, phosphoric acid salt or magnesium salts.With the yeast incubation of mycocandida in the aseptic culture fluid that contains carbon source, nitrogenous source and inorganic salt, incubation under aerobic conditions then.The heated culture temperature that adopts is 22 ℃ to 33 ℃, is preferably 26 ℃ to 30 ℃, especially is preferably 27 ℃ to 28 ℃.The incubation time is 30 hours to 60 hours, and this is according to ventilating, shake or heated culture temperature being decided.Can monitor the PLB activity of supernatant liquor, when reaching stationary phase, stop incubation then.

Used microorganism can secrete under above-mentioned culture condition and generates the enzyme PLB that is used for phospholipid processing agent of the present invention among the present invention.Supernatant liquor as the nutrient solution of the material that is used to prepare described enzyme can obtain by separating with thalline.Can be by adopting salt precipitation that ammonium sulfate carries out and the precipitation that adopts organic solvent such as acetone or alcohol to carry out to purify to be included in the PLB in the culture supernatant that obtains by centrifugation and filter, but preferably purify by acetone precipitation.Can before described precipitation program, concentrate enzyme by ultrafiltration.In addition, if desired, can adopt the program of knowing such as ion exchange chromatography, gel filtration method, hydrophobic chromatography and affinity chromatography etc., obtain enzyme through purifying by described enzyme being purified and/or being concentrated into required level.The purity that is included in the enzyme in the phospholipid processing agent used in the present invention is high more, just can get over and decompose phosphatide effectively.Yet, according to described purpose, even the enzyme of rough state also can use.Even if described enzyme is the enzyme fraction A that is fractionated to by ion exchange chromatography and the mixture of B, such enzyme also is operable.

When being when having the modified enzyme of same function as the PLB of phospholipid processing agent of the present invention, those skilled in the art can according to sequence table in coding have the relevant information of the base sequence of SEQ ID No.3 gene of enzyme of the aminoacid sequence shown in the SEQ ID No.1 and obtain such enzyme.It should be noted that gene recombination technology can be according to currently known methods (for example, gene recombination technology handbook, as Sambrook, " the Molecular Cloning-A Laboratory Manual " of J. etc., Cold Spring Harbor Laboratory, NY, 1989) carry out.

For example, can obtain modified enzyme by the following method: according to sequence table in the relevant information of SEQ IDNo.3 gene order, design suitable primer or suitable probe.Adopt described primer or probe and carry out polymerase chain reaction (PCR) method or hybrid method, obtain target gene thus from the sample of target live organism.Then, by commonly used in the genetic modification such as inducing locus specificity sudden change method methods such as (Mark, D.F., Proc.Natl.Acad.Sci.USA, 81,5662～5666,1984) to carry out genetic modification.Then, in such as the expression system that adopts yeast belong (Saccharomyces) or Pichia (Pichia) to be fit to, express modified gene, to obtain modified enzyme as host's expression system etc.Can adopt the method for describing among the embodiment 2 to confirm whether modified enzyme has the PLB activity.The phospholipid processing agent that preferably contains modified enzyme only is regardless of the phosphatidylinositols of separating in the mixture of phospholipids basically.In addition, preferred described phospholipid processing agent has lipase activity.

3, make the method for phosphatidylinositols (PI) and/or glycerophosphoryl choline (GPC)

The invention provides the method for making highly purified PI and GPC simultaneously effectively.In addition, the present invention also provides the method for making highly purified PI or GPC separately and effectively.Phospholipid processing agent of the present invention has the phosphatide specificity that is used for the phosphatide of selective hydrolysis mixture of phospholipids except PI.Therefore, can optionally make highly purified PI.In addition, adopt phospholipid processing agent of the present invention to decompose and comprise the soybean phospholipid of PC, can when making PI, produce highly purified GPC as main ingredient.Acquisition is useful especially as the high purity PI and the GPC of functional phosphatide or functional phosphatide raw material.

PI can also obtain (seeing aforesaid patent documentation 2) by making PLC act on phosphatide, but this method can not cause the generation of GPC.The GPC that generates as reaction product by the present invention estimates can be as the material that prevents dementia etc., and therefore, the present invention who has utilized the effect of PLB equally also is favourable in this.

Hereinafter will describe the method for making each component in detail.

(a) method of manufacturing phosphatidylinositols (PI)

PLB as phospholipid processing agent of the present invention is particularly useful in the application of the naturally occurring phosphatide of processing.The invention provides a kind of method of making high purity PI effectively.Phosphatide is the structural constituent of cytolemma, and responsible important physical function.Specifically, PI participates in the born of the same parents in the cytolemma/the extracellular signal transduction closely.Phosphatide source of supply in medicine/food applications mainly is soybean, egg yolk or fish-egg etc.These phosphatide obtain as the mixture such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA) and phosphatidylinositols multiple molecular substances such as (PI) usually.In order from these mixture of phospholipids, to isolate PI, adopt the chromatography of organic solvent fractionating process, use such as carriers such as silica gel or aluminum oxide etc. usually.These methods can obtain more competent PC of content and PE effectively, but are enough to far from obtain such as phospholipid fractions such as low levels PI.Also need to use a large amount of organic solvents simultaneously such as halogens such as chloroforms.Therefore, the purposes of products therefrom is limited.In order to prepare PI by the phosphatide that contains PC, PE, PS, PA, PI effectively, optionally hydrolysis except PI such as phosphatide such as PC, PE, PA and PS, so that PI stays.

By making phospholipid processing agent of the present invention act on mixture of phospholipids, optionally from mixture of phospholipids, stay PI separately, can obtain PI effectively.Can use homogenizer etc. to make mixture of phospholipids be dispersed in the water in advance or force to stir the aqueous solution that described mixture of phospholipids prepares homogeneous.The concentration of phosphatide can be any concentration, as long as PLB can act on the mixture of phospholipids that is dispersed in the water, and described concentration is in 1%～20%, is preferably 5%～10%, especially is preferably 6%～8% scope.The pH of reaction can be pH arbitrarily, as long as PLB can act on mixture of phospholipids, and described pH preferably is in 3～10, the about pH 5.5～6.5 when perhaps being in the PLB activity and reaching the highest.Described pH especially is preferably about pH 6.

The preferred buffer reagent that uses makes reaction remain on constant pH.The type of buffer reagent is not particularly limited, as long as it has the surge capability that is in pH 5.5～6.5 scopes.From the angle of food uses, preferably use acetate buffer.In addition, can be by in reaction, suitably adding NaOH solution, with the pH regulator of reaction soln in preferred range.

The concentration that is used as the PLB of phospholipid processing agent of the present invention can be 1,000 units/kg phosphatide～100,000, and 000 units/kg phosphatide is preferably 2,000 units/kg phosphatide～5,000,000 units/kg phosphatide.

Used PLB can add with the form of the aqueous solution, perhaps can be for being fixed on the form on insoluble carrier such as diatomite (Celite) or the ion exchange resin.

Enzyme reaction can be carried out at arbitrary temp, as long as the PLB non-inactivation.Upper temperature limit is preferably below 60 ℃, and more preferably below 45 ℃, and the lower limit of temperature is preferably more than 10 ℃, more preferably more than 30 ℃.

Reaction times is different because of the enzyme reaction condition, is generally 1 hour～150 hours.Can come termination reaction according to qualitative assurance to the substrate residual quantity.

After reaction finishes, can heat-treat or pH handles to wait and makes the PLB inactivation, this is without any problem.

Silica gel thin-layer chromatography (TLC) method is simple, the easiest method that confirms the hydrolysis reaction state.When using TLC to use the iodine development process quantitatively to confirm the substrate residual quantity then, can be when almost not seeing the point that any phosphatide of indication except PI is arranged on the TLC termination reaction.

In deriving from the product of described reaction, free fatty acids, glycerophosphoryl choline (GPC), glycerophosphorylethanolamine (GPE), glyceryl phosphatide acid (GP) and residual unreacted PI exist with mixture.Organic solvents such as employing such as chloroform, ethanol, methyl alcohol and hexane can reclaim described PI from described mixture.From the angle of food uses, especially preferably use ethanol, hexane or its mixed solvent.In order to improve the liquid separation in the extraction step, can before extracting, change the pH of reaction.

Free fatty acids and PI dissolve in the organic solvent.GPC and GPE etc. are insoluble to such as in the organic solvents such as hexane, but in the water soluble.By such as the vacuum concentration supervisor, from the PI that organic solvent layer, reclaims and free fatty acids, isolate described organic solvent.The blended alkyl-carbonyl solvent of water insoluble dissolubility of PI or water.Free fatty acids dissolves in any described solvent.Therefore, PI can be used as the precipitation recovery.Water-soluble or water miscibility alkyl-carbonyl solvent can be acetone or methyl ethyl ketone etc.Preferred examples is an acetone.Can reclaim PI by the solid-liquid separable programming of centrifugation or filter method.

Mixture of phospholipids is not particularly limited.Can use phosphatide from egg yolk, from such as the phosphatide of fish-eggs such as salmon roe or from the phosphatide of soybean.From the angle of the stability of phosphatide source of supply, preferably from the phosphatide of egg yolk with from the phosphatide of soybean, especially preferably from the phosphatide of soybean.Phospholipid processing agent of the present invention can also have strong lipase activity except having the PLB activity.Yet for prepared PI and glycerophosphoryl choline by soybean phospholipid for, having lipase activity is not problem.Soybean phospholipid also can be used as the mixture supply with tri-glyceride.When using such mixture as raw material, can while hydrolysis tri-glyceride and phosphatide.In this, it is very important having lipase activity and PLB activity simultaneously.When the active height of PLB and lipase activity is low or when not possessing lipase activity, can add extra lipase to decompose tri-glyceride and phosphatide simultaneously.

According to aforesaid method of the present invention, can provide highly purified PI.The purity of PI is not particularly limited, as long as PI has high purity.Yet the lower limit of the purity of PI in total phospholipids is preferably 50 moles more than the %, more preferably 60 moles more than the %, and more preferably 70 moles more than the %, especially be preferably 80 moles more than the %, most preferably be 85 moles more than the %.Highly purified PI can be used as functional phosphatide.Because PI comprises the inositol that is rich in hydroxyl in the molecule, therefore with such as other phosphatide such as PC and PE, compare, PI demonstrates good dispersiveness or solvability in water, for example PI has the solvability that extremely is different from other lipid compositions, therefore, the advantage of PI is that it can be used as phosphatide and is widely used in the various uses.

(b) manufacturing of glycerophosphoryl choline (GPC)

PLB as phospholipid processing agent of the present invention is exceedingly useful in the application of the naturally occurring phosphatide of processing.Therefore, the invention provides a kind of method of making high purity GPC effectively.

Make method that phospholipid processing agent of the present invention acts on mixture of phospholipids identical with method in the method for making above-mentioned PI.Reaction times can be 5 hours～20 hours usually.Reaction can be stopped when the concentration of GPC in reactant reaches maximum, but this depends on used enzyme concn.

Can be to adding aforesaid identical organic solvent, for example chloroform, ethanol, methyl alcohol or hexane by phospholipid processing agent of the present invention is acted in the reaction soln of the PLB that mixture of phospholipids obtains.The remaining water layer in extracting liq component (comprising free fatty acids) back contains GPC, GPE, GP or free enzyme.Directly the aqueous solution that contains GPC, GPE or GP is carried out vacuum concentration, obtain syrup-like solution thus.From removing the angle of tinting material component and phospholipid processing agent of the present invention, preferably before concentrating, contain the aqueous solution of GPC, GPE or GP with activated carbon treatment.Can use Zeo-karb etc. to obtain highly purified GPC.

The phospholipid composite that uses in the method for manufacturing GPC of the present invention is not particularly limited, as long as it has high PC content.Can use phosphatide from egg yolk, from such as the phosphatide of fish-eggs such as salmon roe or from the phosphatide of soybean.From the angle of the stability of phosphatide source of supply, preferably from the phosphatide of egg yolk with from the phosphatide of soybean.Especially preferred is phosphatide from soybean.Aforesaid method of the present invention provides highly purified GPC.The purity of GPC is not particularly limited, so long as high purity gets final product.The lower limit of GPC purity is preferably more than the 45 weight %, more preferably more than the 50 weight %.

4, make the method for hemolytic phosphatidyl inositol

The method of making the hemolytic phosphatidyl inositol below will be described.

The method of the highly purified hemolytic phosphatidyl inositol of manufacturing of the present invention comprises the steps:

Step (1): use to have phospholipase A1 activity or the active enzyme of Phospholipase A2 to the be hydrolyzed step of reaction of high purity PI; With

Step (2): the mixed solvent (wherein free fatty acids dissolving and hemolytic phosphatidyl inositol can not dissolve) that contains one or more kinds of solvents by use washs and removes free fatty acids, and the hemolytic phosphatidyl inositol is separated to reclaim the step of hemolytic phosphatidyl inositol with free fatty acids as reaction product.

Hereinafter will be described in detail.

In the manufacturing of hemolytic phosphatidyl inositol of the present invention, use have the phospholipase A1 activity or the active enzyme of Phospholipase A2 is not particularly limited, thereby as long as described enzyme be to act on the enzyme that PI is converted into PI the hemolytic phosphatidyl inositol.The example of described enzyme comprises: be included in Phospholipase A2 in the enzyme mixture (from the pancreatin of pig pancreas) (by Novozym Co., the Lectitase that Ltd. makes or by Genencor Kyowa Co., the Lipomod 699L that Ltd. makes); Phospholipase A2 (by Genencor Kyowa Co., the Lizomax PF that Ltd. makes) from Streptomyces violaceoruber (streptomyces violaceoruber); From the phospholipase A1 of aspergillus oryzae (Aspergillus oryzae) (by SankyoLifeTech Co., the Phospholipase A1 that Ltd. makes) etc.Wherein, preferred examples is included in Phospholipase A2 in the enzyme mixture (from the pancreatin of pig pancreas) (by Novozym Co., Ltd. the Lectitase of Zhi Zaoing) with from the phospholipase A1 (by Sankyo LifeTech Co., the Phospholipase A1 that Ltd. makes) of aspergillus oryzae.

Enzyme reaction in the hemolytic phosphatidyl inositol of the present invention manufacturing can adopt the mode that enzyme and substrate is in contact with one another at aqueous medium or under wet condition to carry out.The pH of reaction is not particularly limited, as long as described pH is in the scope that can carry out enzyme reaction.For example, preferred pH scope is pH 6.5～9.0.

Temperature of reaction is not particularly limited, as long as described temperature is in the scope that can carry out enzyme reaction.For example, described temperature preferably is in 10 ℃～70 ℃ the scope, more preferably is in 30 ℃～60 ℃ the scope.

Can be when reaction reaches required state termination reaction, in required state, use for example efficient liquid phase chromatographic analysis, thin-layer chromatographic analysis to follow the tracks of to be fit to the reaction of the compound of following the tracks of reaction, for example increase of the minimizing of PI or hemolytic phosphatidyl inositol.Required state can be for example reaction terminating state, steady state or make the state of the hemolytic phosphatidyl inositol of capacity, and this can determine according to industrial different angles.

The example in reaction times can be 1 hour to 10 days.

Used enzyme amount can be the amount that abundant reaction is carried out of being enough in the reaction, but from industrial angle, uses to surpass the amount that needs and do not have advantage.For example, when use 1, during the enzyme of 000U/g, with respect to substrate, preferred embodiment can be 0.05 weight %～50 weight %.In addition, can add calcium ion (calcium chloride) etc. as enzyme stabilizers.In addition, after stopping enzyme reaction, can adopt ordinary method to make enzyme deactivation.

The inactivation means can be thermal treatment (handling 10 minutes to 2 hours at 50 ℃～90 ℃) or pH processing (handling 10 minutes to 2 hours at pH 1～4 or pH 8～12) etc.In addition, after the termination reaction or after making enzyme deactivation, can use can dissolved fat acid but can not dissolve the solvent of hemolytic phosphatidyl inositol such as acetone washs and removes the free fatty acids that is generated.

5, make the method for glycerine phosphinylidyne inositol

The method of making glycerine phosphinylidyne inositol will be described below.

The method of manufacturing high purity glycerine phosphinylidyne inositol of the present invention may further comprise the steps:

Step (1): use to have the step that the active enzyme of phospholipase B is hydrolyzed and reacts high purity PI; With

Step (2): glycerine phosphinylidyne inositol is separated with free fatty acids (reaction product) to reclaim the step of glycerine phosphinylidyne inositol.

Hereinafter will be described in detail described method.

The used active enzyme of phospholipase B that has is not particularly limited in glycerine phosphinylidyne inositol preparation of the present invention, as long as it can act on PI fully and PI is changed into glycerine phosphinylidyne inositol.The example of described enzyme comprises the enzyme from Penicillium notatum (Penicillium) genus and yeast etc.

Enzyme reaction of the present invention can adopt the mode that enzyme and substrate is in contact with one another at aqueous medium or under wet condition to carry out.The pH of reaction is not particularly limited, as long as described pH is in the scope that can carry out enzyme reaction.For example, preferred pH scope is pH 3.5～8.0.When using phospholipase B from a mould (Penicillium notatum) as enzyme, especially preferably about pH4.0～5.0.

Temperature of reaction is not particularly limited, as long as described temperature is in the scope that can carry out enzyme reaction.For example, described temperature preferably is in 10 ℃～70 ℃ the scope, more preferably is in 30 ℃～60 ℃ the scope.

Can be when reaction reaches required state termination reaction in addition, in required state, use that for example efficient liquid phase chromatographic analysis, thin-layer chromatographic analysis are followed the tracks of the compound reaction that is fit to follow the tracks of reaction, for example increase of the minimizing of PI or glycerine phosphinylidyne inositol.Required state can be for example reaction terminating state, steady state or make the state of the glycerine phosphinylidyne inositol of capacity, and this can determine according to industrial different angles.The example in reaction times can be 1 hour to 10 days.

Used enzyme amount can be the amount that reacts fully and carry out of being enough in the reaction, but from industrial angle, uses to surpass the amount that needs and do not have advantage.For example, when use 3, during the enzyme of 800U/g, with respect to substrate, preferred embodiment can be 0.05 weight %～5 weight %.In addition, can add calcium ion (calcium chloride etc.) as enzyme stabilizers.In addition, after stopping enzyme reaction, can adopt ordinary method to make enzyme deactivation.The inactivation means can be thermal treatment (handling 10 minutes to 2 hours at 50 ℃～90 ℃) or pH processing (handling 10 minutes to 2 hours at pH 1～4 or pH 8～12) etc.

In addition, after the termination reaction or after making enzyme deactivation, can use to extract such as the hexane equal solvent and remove the free fatty acids that is generated, the vacuum concentration water layer is preserved or is preserved as lyophilized powder with the aqueous solution form that contains high density glycerine phosphinylidyne inositol more then.As selection, can directly the glycerine phosphinylidyne inositol in the water layer be adsorbed in ion exchange resin etc. and wash then.After this, use the free fatty acids of further purifying of the high salt concentration solution with various pH.In addition, if desired, any colouring component can be adsorbed onto on the gac to decolour.

6, the application of high purity PI and high purity GPC

Used fully the PLB of hydrolysis PI by the PI that makes the method acquisition of PI of the present invention from a mould or Dictyostelium discoidoum, then can be as the raw material in the preparation of high purity glycerine phosphinylidyne inositol.In addition, the GPC that method by manufacturing of the present invention GPC obtains can be as the raw material that synthesizes at the chemosynthesis of the GPC by adopting lipid acid or fatty acid ester or enzyme process ester when making the highly purified cholinphospholipide (that is functional phosphatide) that contains lipid acid.

7, the food, medicine or the makeup that contain phosphatidylinositols (PI), glycerophosphoryl choline (GPC), hemolytic phosphatidyl inositol or glycerine phosphinylidyne inositol

The content of the PI in food and the medicine, GPC, hemolytic phosphatidyl inositol and glycerine phosphinylidyne inositol is not particularly limited.The content of each in these materials can be preferably corresponding to 1mg/ days～10,000mg/ days, be preferably 10mg/ days～5,000mg/ days, more preferably 30mg/ days～1, the intake of 000mg/ days PI, GPC, hemolytic phosphatidyl inositol or glycerine phosphinylidyne inositol.

The food that contains PI of the present invention, GPC, hemolytic phosphatidyl inositol or glycerine phosphinylidyne inositol is unrestricted, as long as they contain described arbitrarily component.The example of described food comprises that enriching substance (for example, be dispersed in, granule, soft capsule, hard capsule, tablet, chewable tablet, fast disintegrant, syrup, solution etc.), beverage (for example, tea, soda pop, lactic drink and sports beverages etc.), sweet goods (for example, rubber sugar (gummy), jelly, chewing gum, chocolate, cooky and preserved fruit etc.), oil, oil/fatty foods (for example, mayonnaise, seasonings, butter, cream and oleomargarine etc.), tomato-sauce, sauce, liquid food, dairy products (for example, milk, sour milk and cheese etc.), bread, noodles (for example, Japanese noodle, Fagopyrum esculentum Moench, hand-pulled noodles, spaghetti, fried flour, flat face (kisimen), vegetarian noodles (soumen), cool braised noodle (hiyamugi) and ground rice etc.), soup (for example, miso soup, corn soup and consomme soup etc.) and be used to be sprinkling upon dry seasoning matter on the rice etc.

If desired; contain PI of the present invention; GPC; can add various arbitrarily nutrition in the food of hemolytic phosphatidyl inositol or glycerine phosphinylidyne inositol; various vitaminss (for example; vitamin A; VITMAIN B1; Wei ShengsuB2; vitamin B6; vitamin B12; vitamins C; vitamins D; vitamin-E and vitamin K); various mineral substance (for example; magnesium; zinc; iron; sodium; potassium; selenium; titanium oxide); food fiber; various carbohydrates (for example; Mierocrystalline cellulose; dextrin and chitin); various polyunsaturated fatty acids (for example; arachidonic acid; docosahexenoic acid; timnodonic acid and clupanodonic acid); various conjugated fatty acidss (for example; conjugated linolic acid; conjugate linolenic acid; the conjugation arachidonic acid; conjugation DHA; conjugation EPA and conjugation DPA); various phosphatide (for example; Yelkin TTS; PA; PS; PE; phosphatidyl glycerol; PC and phosphatidyl DHA); various glycolipids (for example; cerebroside); various types of carotene (for example; β-Hu Luobusu; Lyeopene; astaxanthin; β-cryptoxanthin; capsanthin; lutein and zeaxanthin); various types of flavones (for example; Mongolian oak skin ketone; digicitrine element and isoflavones); each seed amino acid (for example; glycine; Serine; L-Ala; glutamine; Xie Ansuan; leucine; Isoleucine; Methionin; arginine; aspartic acid; L-glutamic acid; tryptophane; phenylalanine; Histidine; proline(Pro); methionine(Met) and halfcystine); other various nutrition (for example; the Coenzyme Q10 99.0 of oxidised form; the Coenzyme Q10 99.0 of reduction form; carnitine; sesamin; alpha-lipoic acid; inositol; the D-chiro-inositol; pine camphor; taurine; glycosamine; chondroitin sulfate; S-adenosylmethionine; curcumine; γ-orizanol; gsh; γ-An Jidingsuan; synephrine; Pyrroloquinoline quinone; catechin and capsaicine); various dispersion agents; stablizer (as various emulsifying agents); various sweeting agents (for example; Sorbitol Powder and sucrose); various flavoring ingredients (for example, citric acid and oxysuccinic acid); condiment; royal jelly; honey; beeswax; propolis; Larch agaric; genseng and Fructus piperis nigrum extract (bioperine) etc.In addition, can add such as any herbal medicine such as peppermint, Citrus bergamia, camomile and lavandula angustifolia.In addition, can mix such as arbitrary substances such as the very small glucoside of sheep (teanin), trans-dehydroandrosterone and melatonins.

The medicine that contains PI of the present invention, GPC, hemolytic phosphatidyl inositol or glycerine phosphinylidyne inositol is unrestricted, as long as it contains these components arbitrarily.The preparation of medicine comprise be dispersed in, granule, pill, soft capsule, hard capsule, tablet, chewable tablet, fast disintegrant, syrup, solution, suspension agent, suppository, ointment, emulsifiable paste, gel, mucilage, inhalation and injection etc.These preparations can prepare according to conventional methods.Yet,, therefore it can be dissolved in such as in vegetables oil and the non-arbitrarily hydrophilic solvent of animal wet goods because PI is water insoluble.As selection, can use homogenizer (high pressure homogenisers) that PI and emulsifying agent (a kind of dispersion agent) or tensio-active agent are disperseed together or be emulsified in the aqueous solution.In addition, in order to improve the adsorptivity of PI, can under the situation that has or do not exist vehicle (for example Sudan Gum-arabic, dextrin or casein etc.), use PI PI being crushed to after averageparticle is about 1 micron.

The additive that can use when preparation for example comprises: soybean oil, Thistle oil, sweet oil, germ oil, sunflower oil, tallow, animal oil such as sardine oil; Polyvalent alcohol such as polyoxyethylene glycol, propylene glycol, glycerine and Sorbitol Powder; The fatty acid ester of tensio-active agent such as sorbitan aliphatic ester, sucrose fatty ester, glycerine and polyglycerol fatty acid glyceryl ester; Such as vehicle such as pure water, lactose, starch, Microcrystalline Cellulose, D-seminose, maltose, Yelkin TTS, Sudan Gum-arabic, dextrin, sorbitol solution and saccharide solutions; Sweeting agent; Tinting material; The pH regulator agent; And seasonings.In addition, liquid preparation can adopt when administration and be dissolved or suspended in the water or the form in other suitable media arbitrarily.In addition, can adopt method coated tablets or the granule of knowing.

When using with the form of injection, preferably with intravenously, intraperitoneal, intramuscular, subcutaneous, in skin, intraarticular, synovial membrane, in the alveolar, in the periosteum, in hypogloeeis or the oral cavity, use.Wherein, especially preferred is that intravenously is used or intraperitoneal is used.It can be that any one in (porous administration) used in instillation and infiltration that intravenously is used.

The makeup that contain PI of the present invention, GPC, hemolytic phosphatidyl inositol or glycerine phosphinylidyne inositol comprise emulsifiable paste, emulsus lotion, washing lotion, micro emulsion essence, apply paste and prickly-heat powder etc.Can also sneak into perfume compound etc.

Describe the present invention below with reference to embodiment, but the present invention is not limited to following embodiment.

Embodiment

Embodiment 1 makes the method for PLB

The nutrient solution of the column candiyeast strain ATCC14830 of 100ml preincubation in same medium on the aseptic inoculation in the fermentor tank of 30 liters of volumes that 20 liters of aseptic culture mediums are housed (28 ℃ incubation 4 days), described aseptic culture medium contains 3% degreasing cottonseed meal, 0.3% salt, 0.2% dipotassium hydrogen phosphate, 0.2% potassiumphosphate, 0.1% sal epsom and 0.3% defoamer.When stirring, carry out incubation and with sterile air ventilate (20 liters/minute) at 28 ℃ with 300rpm.Confirm that after 50 hours PLB has reached maximum activity (0.2U/ml).

With 5,000rpm obtains 16 liters supernatant liquor to gained medium centrifugal 10 minutes.By the ultrafiltration and concentration supernatant liquor.With 5,000rpm is to by adding centrifugal 10 minutes of sediment that 9 liters of freezing acetone form with collecting precipitation in 3 liters gained enriched material, then with described resolution of precipitate 2 liters of 10mM Tris hydrochloride buffers (hereinafter referred is Tris-HCl) that contain 2M sodium-chlor (pH7.5) in.By the centrifugal insoluble substance of removing, make it the pillar (column volume of filling with octyl sepharose then by in advance; 300ml) adsorb PLB.Then, with 10mMTris-HCl (pH 7.5) that contains 2M sodium-chlor and 10mM Tris-HCl (pH 7.5) washing pillar.Be adsorbed on PLB (purifying) on the pillar with the 10mM Tris-HCl that contains 2%Adecatol SO 120 (pH 7.5) wash-out with hydrophobic chromatography.Then, make elutriant (1 liter) by using 10mM Tris-HCl (pH7.5) equilibrated DEAE-agarose pillar (column volume in advance; 200ml) with absorption PLB.Using above-mentioned damping fluid washing pillar, is the sodium-chlor wash-out enzyme of 0～0.3M then with concentration gradient.Obtain PLB enzyme fraction A at about 0.1M sodium-chlor place, obtain PLB enzyme fraction B (adopting ion-exchange chromatography to separate purifies) at about 0.25M sodium-chlor place.

Adopt Protein Sequencer to measure the N-terminal aminoacid sequence of enzyme fraction A and enzyme fraction B.As a result of, the aminoacid sequence of enzyme fraction A is a SEQ ID No.1 sequence, and the aminoacid sequence of enzyme fraction B is a SEQ ID No.2 sequence.In addition, use GENETEX WIN 5.2 (by Software Co., Ltd. makes) to carry out homology analysis, with the homology of enzyme analysis fraction B with respect to enzyme fraction A, the result is 88.5%.

Embodiment 2 measures the active method of PLB

The enzymic activity of PLB can detect by the enzyme process of enforcement mensuration by the GPC of phosphatidylcholine generation.Promptly, 37 ℃ with the reaction soln preheating of 0.5ml 2 minutes to 3 minutes, described reaction soln is made up of the following material in the 1M MES-NaOH damping fluid that contains 3%Triton X-100 (hereinafter referred is MES-NaOH) (pH 6) that is dissolved in 0.5ml: the 10mM egg phospholipids phatidylcholine of 0.05ml, 0.025ml 1M calcium chloride, 0.05ml 0.2%TODB, 0.05ml the amino antipyrine of 0.2%4-, 0.1ml 50U/ml monoglyceride lipase, 0.1ml 300U/m Phosphoric acid glycerol esters oxydase, 0.025ml 6U/ml GPC phosphodiesterase and the 100U/ml peroxidase of 0.05ml.Subsequently, use 10mM Tris-HCl (pH7.5) the diluting reaction solution that contains 0.05%BSA, in reaction soln, add the PLB solution (0.03U/ml～0.15U/ml), it is diluted with initiation reaction of 25 μ l then with identical dilution buffer liquid.After 10 minutes, add the 0.5%SDS termination reaction of 1ml, measure its absorbancy then at the 550nm place.

It should be noted that the activity that per minute is discharged the GPC of 1 mmole is defined as a unit.

Embodiment 3 measures the method for lipase activity

By using monoglyceride lipase will use diglyceride to be converted into グ Le セ リ Application, measure by utilizing enzymatic assays to carry out lipase activity then as the monoglyceride that substrate generates.Promptly, the enzyme solution that the 10mMTris-HCl to contain 0.05%BSA of adding 25 μ l dilutes in the reaction soln of 0.5ml (0.03U/ml～0.15U/ml), then 37 ℃ of reactions of carrying out 10 minutes, described reaction soln is made up of the following material among the 1MMES-NaOH that contains 3%Triton X-100 that is dissolved in 0.1ml (pH 6.0): 10mM 1,2 diglyceride of 0.05ml, 0.025ml 0.5M calcium chloride, 0.025ml the 0.05M magnesium chloride, 0.05ml 0.05MATP, 0.05ml 10U/ml monoglyceride lipase, 0.05ml the 5U/ml glycerol kinase, 0.25ml 400U/ml Phosphoric acid glycerol esters oxydase, 0.025ml the peroxidase of 100U/ml, 0.025ml 0.3%TOOS and the amino antipyrine of the 0.3%4-of 0.025ml.Afterwards, use the 0.5%SDS termination reaction, measure its absorbancy then at the 550nm place.It should be noted that the activity that per minute is discharged the glycerine of 1 mmole is defined as the lipase activity of a unit.

The purification that embodiment 4 is undertaken by the gel filtration chromatography of PLB fraction A

Concentrate the PLB organized enzyme fraction A that obtains by embodiment 1 by the centrifugal ultrafiltration device.Afterwards, concentrated solution is carried out gel filtration chromatography (SuperDex 75) (in advance with 10mM Tris-HCl (pH 7.5) balance that contains 0.5M sodium-chlor), with 0.5ml/ minute flow velocity separation, obtain active fraction thus then.

In each fraction, obtain PLB activity and lipase activity by method as described in embodiment 2 and 3.Use the absorbancy at 280nm place to measure protein concn.The result of PLB activity, lipase activity and the protein concn of each fraction as shown in Figure 1.

As a result, the active elution mode of wherein measuring at the 280nm place of protein concn, lipase activity and PLB is coincide mutually.Discovery has lipase activity and the active albumen of PLB is the same enzyme albumen.

Embodiment 5PLB character

5-1 (the best pH of reaction)

In the composition of the reaction soln shown in the embodiment 2, use acetate buffer (hereinafter referred is Acetate), PIPES-NaOH damping fluid (hereinafter referred is PIPES-NaOH) or MES-NaOH to measure the enzymic activity of enzyme fraction A under various pH.Obtained the active relative reactivity that enzyme fraction A obtains with respect to use MES-NaOH pH 6.0 in each damping fluid.The result as shown in Figure 2.

As shown in Figure 2, MES-NaOH shows maximum enzymic activity at about pH 6.In addition, estimate that also PIPES-NaOH and Acetate also show maximum enzymic activity at about pH 6.

5-2 (pH stability)

Enzyme fraction A dissolving made be 5U/ml in each damping fluid in its Acetate, MES-NaOH, Tris-HCl or the Bicine-NaOH damping fluid (hereinafter referred is Bicine-NaOH), left standstill 90 minutes at 37 ℃ then at 10mM.Afterwards, measure the PLB activity, then measure under each pH with respect to the active relative residual activity in Acetate (pH 5) according to the method for embodiment 2.The result as shown in Figure 3.

As shown in Figure 3, find in the wide range of pH 5～pH 8, to have pH stability.

5-3 (thermostability)

Enzyme fraction A dissolving made be 5U/ml among its MES-NaOH (pH 6.0), adopt the method shown in the embodiment 2 then, after 10 minutes, carry out the PLB determination of activity at 0～70 ℃ temperature range internal heating at 10mM.The result as shown in Figure 4.When the PLB activity of the enzyme fraction A that cold storage is preserved is defined as 100%, be more than 90% with 55 ℃ the relative residual activity of Temperature Treatment at the most.Find that thus enzyme fraction A is the thermostability enzyme.

5-4 (substrate specificity)

By as the active method of embodiment 2 described mensuration PLB, measure two kinds of different phospholipid processing agents that obtain as ion exchange chromatography by embodiment 1 each PLB (enzyme fraction A and enzyme fraction B), about the activity of corresponding phosphatide with respect to PC.In other words, different with the response composite of the Yelkin TTS shown in the embodiment 2 is, uses PE, PI or PA to measure relative reactivity with respect to PC in determination of activity.The result is as shown in table 1.As a result of, enzyme fraction A demonstrates the less relative reactivity with respect to PC (comparing with other phosphatide) simultaneously with enzyme fraction B.Find that phospholipid processing agent of the present invention has substrate specificity, make activity to PI less than activity to any other phosphatide.The result of table 1 shows that each fraction has the PLB activity, and therefore described phospholipid processing agent can be the combination of two kinds of fractions.

Table 1

5-5 (molecular weight)

The enzyme fraction A through purifying that obtains by embodiment 1 described method is carried out the SDS-polyacrylamide gel electrophoresis, obtain single band.With reference to the known albumen that serves as a mark of molecular weight, the molecular weight of described product is 53 kilodaltons.

5-6 (iso-electric point)

Adopt and use both sexes dielectric carrier (Carrier Ampholyte), the enzyme fraction A through purifying that obtains by embodiment 1 described method is carried out the isoelectric point determination of enzyme fraction A by forming the electric electrophoretic method such as grade that the pH gradient is carried out.In addition, adopt the absorbancy at 280nm place that the enzyme fraction A through purifying is carried out determination of protein concentration.The iso-electric point of enzyme fraction A and the relation between the enzyme concn are as shown in Figure 5.As a result of, the described proteinic pH of the absorbance measurement at employing 280nm place and lipase activity and PLB activity fit like a glove, and described pH value (iso-electric point) is 4.21.Can find out obviously that from Fig. 5 described protein demonstrates the peak identical with PLB and lipase, find out that thus PLB and lipase are identical zymoprotein.

The quantitative test of embodiment 6PI

The PI solution that adds 0.02ml to the staining fluid of 0.5ml, thus 37 ℃ of reactions of carrying out 10 minutes, described staining fluid contains the 1M Tris-HCl (pH 8) of 0.1ml, 0.05ml 10mMNAD, 0.05ml the 10mM magnesium chloride, 0.05ml 2%Triton X-100,0.05ml 50U/ml PI specificity lecithinase C, 0.05ml the 50U/ml alkaline phosphatase, 0.05ml the 50U/ml myo-Inositol dehydrogenase, 0.05ml 5% nitroblue tetrazolium(NBT) and the pure water of 0.15ml, the specimen (1mg/ml～3mg/ml) be dissolved in 2%TritonX-100 make of described PI solution by containing PI.Use the 0.5%SDS termination reaction of 0.5ml, measure the absorbancy at 550nm place then.With the inositol aqueous solution calibration of concentration known, the amount of quantitative analysis PI then.

The quantitative analysis of embodiment 7GPC

Adding 25 μ l in the reaction soln of 0.5ml contains the sample of GPC (0.3mg/ml～0.9mg/ml), described reaction soln contains the amino antipyrine of 0.2%4-, the 6U/ml glycerophosphoryl choline phosphodiesterase of 0.025ml, the 100U/ml peroxidase of 0.05ml, the 200U/ml E.C. 1.1.99.1 of 0.05ml and the pure water of 0.15ml of 0.2%TODB, 0.05ml of 1M calcium chloride, the 0.05ml of 1M Tris-HCl (pH 7.5), the 0.025ml of 0.1ml., after 10 minutes the 0.5%SDS of 0.5ml is joined in the reaction soln 37 ℃ of reactions, measure the absorbancy at 550nm place then.The aqueous choline base solution of concentration known is used for calibration, quantitative analysis GPC then.

Embodiment 8 makes the method for PI

When stirring, 40g is suspended among the 10mMAcetate (pH 5.8) of 400ml from the phosphatide of soybean, add then the conduct phospholipid processing agent of the present invention of 1,400 unit PLB (the enzyme fraction A that obtains among the embodiment 1) thus 45 ℃ of initiation reactions.After 20 hours, termination reaction adds the ethanol of 200ml and the hexane of 400ml then, then stirs 1 hour.By centrifugation, hexane layer and water layer is separated from one another, reclaim organic solvent layer thus.Under reduced pressure concentrate the organic solvent that is reclaimed, in concentrated solution, add the acetone of 150ml then.Collect and dry gained precipitation, obtain to contain the powder (8.2g) of high purity PI thus.According to Standard Oiland Fat Analytical Test Method (establishing 1996 by JAPAN Oil Chemists ' Society), measure the purity of gained phosphatide.As a result, the purity with respect to total phospholipids is 86.8 moles of %.

Embodiment 9 makes the method for GPC

GPC, GPE and GP are included in by in the water layer that the PLB reactant in the embodiment 8 described methods is carried out the organic solvent extraction acquisition.Make the pillar (column volume: 50ml), thus obtain colourless pass through liquid of water layer of 380ml by being filled with gac.Under reduced pressure concentrate described liquid, obtain the GPC solution of 40ml thus.According to the gpc analysis described in the embodiment 2, the purity of GPC is 55 weight %.

Embodiment 10 makes the method for hemolytic phosphatidyl inositol

In the PI that 10g obtains, add the water of 20ml in embodiment 8, fully stir then, then add 100mg by Sankyo LifeTech Co., and the phospholipase A1 that Ltd. makes (11,900U/g).When stirring, carry out enzyme reaction then in 50 ℃.After reaction beginning 24 hours, 80 ℃ of processing reaction solution 30 minutes, termination reaction thus.Dry reaction solution adds 100ml acetone and fully stirring, obtains solid subsequently after filtration.Dry then described solid obtains 6g hemolytic phosphatidyl inositol thus.According to the result that TLC analyzes, confirm unreacted PI and be removed as the free fatty acids of byproduct of reaction.

TLC carrier: Silica Gel60, layer thickness is 2mm (by Merck Co., Ltd. makes), developping agent: chloroform: methyl alcohol: water=65: 35: 4, dyeing process: iodine staining, Rf value: hemolytic phosphatidyl inositol (0.12), PI (0.28), free fatty acids (0.81).

Embodiment 11 is made the method for phospholipase B by a mould

Based on currently known methods, be prepared as follows phospholipase B by a mould.The point mould (IFO-4640) of 100ml preincubation in taper culturing bottle (volume is 500ml) transferred to 20 liters of nutrient solution (3.5% corn steep liquor, 5.5% lactose, 0.7% potassiumphosphate, 0.3% sal epsom, 0.5% lime carbonate and 0.25% soybean oil through the autoclave sterilization, pH 5.4) in, then under aerobic conditions in 26 ℃ of incubations 4 days.After stopping incubation, obtain thalline by filtering.In described thalline, add 10 liters of pure water, used the homogenizer homogenizing then 10 minutes,, filter subsequently to obtain the enzyme of dissolvingization.The result obtains 8 liters of rough enzyme solution.The pillar that makes the cellulosic fibre of described filtrate by being filled with the 2kg palmitoylation washs pillar with pure water then with adsorptive enzyme, then with containing the 1mM phosphoric acid buffer (pH 7.0) of 0.5%AdecatolSO120 and 0.2mM EDTA with the enzyme wash-out.Elutriant (10 liters) is loaded in the Q-Sepharose ion exchange column, thus adsorptive enzyme.With the 1mM phosphoric acid buffer that contains 0.2mMEDTA (pH 7.0) washing pillar, then with containing the 10mM phosphate buffered saline buffer (pH 7.0) of 0.2M NaCl and 2mM EDTA with the enzyme wash-out.Afterwards, enzyme is dialysed, carry out desalination and freeze-drying then, obtain enzyme powder (3800U/g) thus with the 10mM phosphate buffered saline buffer (pH 7.0) that contains 2mM-EDTA.

Embodiment 12 makes the method for glycerine phosphinylidyne inositol

The 2mM Acetate (pH4.0) that adds 100ml in embodiment 8 in the PI that 10g obtains fully stirs then, then adds the phospholipase B of 200mg as being obtained by a mould as described in the embodiment 11, thereby carry out enzyme reaction in 40 ℃ when stirring.After 24 hours, handle 30 minutes with termination reaction from the reaction beginning at 80 ℃.In reaction soln, add the hexane of 50ml, stir then, then leave standstill to remove hexane layer.Then, reclaim water layer.Make tank by being pre-charged with the pillar of gac, collect the liquid that passes through then, then carry out freeze-drying.The result obtains 8g glycerine phosphinylidyne inositol.

Food products preparation example 1 contains the oleomargarine of phosphatidylinositols (PI)

The PI that obtains among the embodiment 8 is added in the vegetables oil, makes it to become the oleomargarine of 5 weight %, stir with emulsor etc. then.Prepare oleomargarine by ordinary method.

Food products preparation example 2 contains the bread of phosphatidylinositols (PI)

PI, 15g sugar, 2g salt and the fatty milk powder of 5g that 1g is obtained in embodiment 8 are dissolved in the 70g hot water, add two eggs and thorough mixing then.Described mixture is added in the mixture of 130g whole meal flour and 2g dry yeast, then knead with hand.About 30g butter is joined in the described mixture, and further knead described mixture, make 30 spherical doughs that are used for swiss roll then.Afterwards, after fermentation, apply the surface of dough with the egg of smashing.Dough 180 ℃ oven for baking 15 minutes, is obtained swiss roll thus.

Food products preparation example 3 contains the Japanese noodle (Japanese wheat noodles) of phosphatidylinositols (PI)

With respect to the 400g whole meal flour, the PI that 2g is obtained in embodiment 8 joins in 200g water and the 20g salt, then kneads and allows described mixture leave standstill time enough.Then, stretch hand-pulled noodles group and be cut into the wide small pieces of about 6mm, obtain Japanese noodle thus.

Food products preparation example 4 contains the beverage of phosphatidylinositols (PI)

The PI that 30g is obtained in embodiment 8 is suspended in the sweet oil of 5 times of volumes, is heated to 50 ℃ then, obtains oil phase thus.10g is joined in the 90g glycerine as the glycerol fatty acid ester of emulsifying agent, be heated to 70 ℃ and dissolving then.When stirring, add above-mentioned oil phase gradually to described solution.Use the described mixture of emulsifier unit emulsification under high pressure and obtain through the emulsive composition.Then, 180ml water is joined 20g in the emulsive composition, also stir, obtain containing the beverage of PI thus.

Formulation example 1 contains the tablet of phosphatidylinositols (PI)

The PI 120g that in embodiment 8, obtains

Microcrystalline Cellulose 330g

Calcium carboxymethylcellulose 15g

Hydroxypropylcellulose 10g

Pure water 60ml

Adopt ordinary method to mix above-mentioned composition, dry then, then add the 10g Magnesium Stearate.Described mixture is made tablet, obtain the 100mg tablet thus, every tablet contains the PI of 20mg amount.

Formulation example 2 contains the soft capsule of phosphatidylinositols (PI)

The PI that will obtain in embodiment 8 is suspended in the sweet oil of 5 times of volumes, and thorough mixing is even then.Then, utilize the capsule tucker, obtain the capsule that content is 300mg with described mixture filled capsules.

The makeup preparation example contains the medicinal cream (makeup) of phosphatidylinositols (PI)

The PI that will obtain in embodiment 8 joins and makes in the white vaseline that its amount is 10 weight %, stirs together with aromatics etc. then.Make medicinal cream by ordinary method.

Food products preparation example 5 contains the oleomargarine of hemolytic phosphatidyl inositol

The hemolytic phosphatidyl inositol that will obtain in embodiment 10 joins in the vegetables oil, makes it to become the oleomargarine of 5 weight %, stirs with emulsor etc. then.Make oleomargarine by ordinary method.

Food products preparation example 6 contains the bread of hemolytic phosphatidyl inositol

The milk powder that hemolytic phosphatidyl inositol, 15g sugar, 2g salt and the 5g that 1g is obtained in embodiment 10 contains fat is dissolved in the 70g hot water, adds two eggs and thorough mixing then.Described mixture is added in the mixture of 130g whole meal flour and 2g dry yeast, then knead with hand.About 30g butter is joined in the described mixture, and further knead described mixture, make 30 spherical doughs that are used for swiss roll then.Afterwards, after fermentation, apply the surface of dough with the egg of smashing.Dough 180 ℃ oven for baking 15 minutes, is obtained swiss roll thus.

Food products preparation example 7 contains the Japanese noodle (Japanese wheat noodles) of hemolytic phosphatidyl inositol

With respect to the 400g whole meal flour, the hemolytic phosphatidyl inositol that 2g is obtained in embodiment 10 joins in 200g water and the 20g salt, then kneads, and allows described mixture leave standstill time enough then.Then, stretch hand-pulled noodles group and be cut into the wide small pieces of about 6mm, obtain Japanese noodle thus.

Food products preparation example 8 contains the beverage of hemolytic phosphatidyl inositol

The hemolytic phosphatidyl inositol that 30g is obtained in embodiment 10 is suspended in the sweet oil of 5 times of volumes, is heated to 50 ℃ then, obtains oil phase thus.10g is joined in the 90g glycerine as the glycerol fatty acid ester of emulsifying agent, be heated to 70 ℃ and dissolving then.When stirring, add above-mentioned oil phase gradually to described solution.Use the described mixture of emulsifier unit emulsification under high pressure and obtain through the emulsive composition.Then, 180ml water is joined 20g in the emulsive composition, also stir, obtain containing the beverage of hemolytic phosphatidyl inositol thus.

Formulation example 3 contains the tablet of hemolytic phosphatidyl inositol

The hemolytic phosphatidyl inositol 120g that in embodiment 10, obtains

Microcrystalline Cellulose 330g

Calcium carboxymethylcellulose 15g

Hydroxypropylcellulose 10g

Pure water 60ml

Adopt ordinary method to mix above-mentioned composition, dry then, then add the 10g Magnesium Stearate.Described mixture is made tablet, obtain the 100mg tablet thus, every tablet contains the hemolytic phosphatidyl inositol of 20mg amount.

Formulation example 4 contains the soft capsule of hemolytic phosphatidyl inositol

The hemolytic phosphatidyl inositol that will obtain in embodiment 10 is suspended in the sweet oil of 5 times of volumes, and thorough mixing is even then.Then, utilize the capsule tucker, obtain the capsule that content is 300mg with described mixture filled capsules.

The makeup preparation example contains the emulsifiable paste (makeup) of hemolytic phosphatidyl inositol

The hemolytic phosphatidyl inositol that will obtain in embodiment 10 joins and makes in the white vaseline that its amount is 10 weight %, stirs together with aromatics etc. then.Make emulsifiable paste by ordinary method.

Food products preparation example 9 contains the oleomargarine of glycerine phosphinylidyne inositol

The glycerine phosphinylidyne inositol that obtains among the embodiment 12 is added in the vegetables oil, makes it to become the oleomargarine of 5 weight %, stir with emulsor etc. then.Prepare oleomargarine by ordinary method.

Food products preparation example 10 contains the bread of glycerine phosphinylidyne inositol

Glycerine phosphinylidyne inositol, 15g sugar, 2g salt and the fatty milk powder of 5g that 1g is obtained in embodiment 12 are dissolved in the 70g hot water, add two eggs and thorough mixing then.Described mixture is added in the mixture of 130g whole meal flour and 2g dry yeast, then knead with hand.About 30g butter is joined in the described mixture, and further knead described mixture, make 30 spherical doughs that are used for swiss roll then.Afterwards, after fermentation, apply the surface of dough with the egg of smashing.Dough 180 ℃ oven for baking 15 minutes, is obtained swiss roll thus.

Food products preparation example 11 contains the Japanese noodle (Japanese wheat noodles) of glycerine phosphinylidyne inositol

With respect to the 400g whole meal flour, the glycerine phosphinylidyne inositol that 2g is obtained in embodiment 12 joins in 200g water and the 20g salt, then kneads, and allows described mixture leave standstill time enough then.Then, stretch hand-pulled noodles group and be cut into the wide small pieces of about 6mm, obtain Japanese noodle thus.

Food products preparation example 12 contains the beverage of glycerine phosphinylidyne inositol

The glycerine phosphinylidyne inositol that 30g is obtained in embodiment 12 is suspended in the sweet oil of 5 times of volumes, is heated to 50 ℃ then, obtains oil phase thus.10g is joined in the 90g glycerine as the glycerol fatty acid ester of emulsifying agent, be heated to 70 ℃ and dissolving then.When stirring, add above-mentioned oil phase gradually to described solution.Use the described mixture of emulsifier unit emulsification under high pressure and obtain through the emulsive composition.Then, 180ml water is joined 20g in the emulsive composition, also stir, obtain containing the beverage of glycerine phosphinylidyne inositol thus.

Formulation example 5 contains the tablet of glycerine phosphinylidyne inositol

The glycerine phosphinylidyne inositol 120g that in embodiment 12, obtains

Microcrystalline Cellulose 330g

Calcium carboxymethylcellulose 15g

Hydroxypropylcellulose 10g

Pure water 60ml

Adopt ordinary method to mix above-mentioned composition, dry then, then add the 10g Magnesium Stearate.Described mixture is made tablet, obtain the 100mg tablet thus, every tablet contains the glycerine phosphinylidyne inositol of 20mg amount.

Formulation example 6 contains the soft capsule of glycerine phosphinylidyne inositol

The glycerine phosphinylidyne inositol that will obtain in embodiment 12 is suspended in the sweet oil of 5 times of volumes, and thorough mixing is even then.Then, utilize the capsule tucker, obtain the capsule that content is 300mg with described mixture filled capsules.

The makeup preparation example contains the emulsifiable paste (makeup) of glycerine phosphinylidyne inositol

The glycerine phosphinylidyne inositol that will obtain in embodiment 12 joins and makes in the white vaseline that its amount is 10 weight %, stirs together with aromatics etc. then.Make emulsifiable paste by ordinary method.

Industrial applicibility

Phospholipid processing agent of the present invention is suitable for using from the phosphatide of the soybean function as raw material The manufacturing of property phosphatide.

Sequence table

＜110〉Asahi Kasei Pharma Corp

＜120〉contain the novel agent of phospholipase B

<130>F106074

<140>

<141>

<150>JP2005-208913

<151>2005-7-19

<150>JP2006-057141

<151>2006-3-3

<160>3

<170>PatentIn?Ver.2.1

<210>1

<211>549

<212>PRT

＜213〉candiyeast

<220>

＜223〉contriver: the luxuriant row in modern village

<400>1

Met?Glu?Leu?Ala?Leu?Ala?Leu?Leu?Leu?Ile?Ala?Ser?Val?Ala?Ala?Ala

1 5 10 15

Pro?Thr?Ala?Thr?Leu?Ala?Asn?Gly?Asp?Thr?Ile?Thr?Gly?Leu?Asn?Ala

20 25 30

Ile?Ile?Asn?Glu?Ala?Phe?Leu?Gly?Ile?Pro?Phe?Ala?Glu?Pro?Pro?Val

35 40 45

Gly?Asn?Leu?Arg?Phe?Lys?Asp?Pro?Val?Pro?Tyr?Ser?Gly?Ser?Leu?Asp

50 55 60

Gly?Gln?Lys?Phe?Thr?Leu?Tyr?Gly?Pro?Leu?Cys?Met?Gln?Gln?Asn?Pro

65 70 75 80

Glu?Gly?Thr?Tyr?Glu?Glu?Asn?Leu?Pro?Lys?Ala?Ala?Leu?Asp?Leu?Val

85 90 95

Met?Gln?Ser?Lys?Val?Phe?Glu?Ala?Val?Leu?Pro?Leu?Ser?Glu?Asp?Cys

100 105 110

Leu?Thr?Ile?Asn?Val?Val?Arg?Pro?Pro?Gly?Thr?Lys?Ala?Gly?Ala?Asn

115 120 125

Leu?Pro?Val?Met?Leu?Trp?Ile?Phe?Gly?Gly?Gly?Phe?Glu?Val?Gly?Gly

130 135 140

Thr?Ser?Thr?Phe?Pro?Pro?Ala?Gln?Met?Ile?Thr?Lys?Ser?Ile?Ala?Met

145 150 155 160

Gly?Lys?Pro?Ile?Ile?His?Val?Ser?Val?Asn?Tyr?Arg?Val?Ser?Ser?Trp

165 170 175

Gly?Phe?Leu?Ala?Gly?Asp?Glu?Ile?Lys?Ala?Glu?Gly?Ser?Ala?Asn?Ala

180 185 190

Gly?Leu?Lys?Asp?Gln?Arg?Leu?Gly?Met?Gln?Trp?Val?Ala?Asp?Asn?Ile

195 200 205

Ala?Ala?Phe?Gly?Gly?Asp?Pro?Thr?Lys?Val?Thr?Ile?Phe?Gly?Glu?Leu

210 215 220

Ala?Gly?Ser?Met?Ser?Val?Met?Cys?His?Ile?Leu?Trp?Asn?Asp?Gly?Asp

225 230 235 240

Asn?Thr?Tyr?Lys?Gly?Lys?Pro?Leu?Phe?Arg?Ala?Gly?Ile?Met?Gln?Leu

245 250 255

Gly?Ala?Met?Val?Pro?Leu?Asp?Ala?Val?Asp?Gly?Ile?Tyr?Gly?Asn?Glu

260 265 270

Ile?Phe?Asp?Leu?Leu?Ala?Ser?Asn?Ala?Gly?Cys?Gly?Ser?Ala?Ser?Asp

275 280 285

Lys?Leu?Ala?Cys?Leu?Arg?Gly?Val?Leu?Ser?Asp?Thr?Leu?Glu?Asp?Ala

290 295 300

Thr?Asn?Asn?Thr?Pro?Gly?Phe?Leu?Ala?Tyr?Ser?Ser?Leu?Arg?Leu?Leu

305 310 315 320

Tyr?Leu?Pro?Arg?Pro?Asp?Gly?Val?Asn?Ile?Thr?Asp?Asp?Met?Tyr?Ala

325 330 335

Leu?Val?Arg?Glu?Gly?Lys?Tyr?Ala?Asn?Ile?Pro?Val?Ile?Ile?Gly?Asp

340 345 350

Gln?Asn?Asp?Glu?Gly?Thr?Phe?Phe?Gly?Thr?Leu?Leu?Leu?Asn?Val?Thr

355 360 365

Thr?Asp?Ala?Gln?Ala?Arg?Glu?Tyr?Phe?Lys?Gln?Leu?Phe?Val?His?Ala

370 375 380

Ser?Asp?Ala?Glu?Ile?Asp?Thr?Leu?Met?Thr?Ala?Tyr?Pro?Gly?Asp?Ile

385 390 395 400

Thr?Gln?Gly?Leu?Pro?Phe?Asp?Thr?Gly?Ile?Leu?Asn?Ala?Leu?Thr?Pro

405 410 415

Gln?Phe?Lys?Arg?Ile?Leu?Ala?Val?Leu?Gly?Asp?Leu?Gly?Phe?Thr?Leu

420 425 430

Ala?Arg?Arg?Tyr?Phe?Leu?Asn?His?Tyr?Thr?Gly?Gly?Thr?Lys?Tyr?Ser

435 440 445

Phe?Leu?Leu?Lys?Gln?Leu?Leu?Gly?Leu?Pro?Val?Leu?Gly?Thr?Phe?His

450 455 460

Ser?Asn?Asp?Ile?Val?Phe?Gln?Asp?Tyr?Leu?Leu?Gly?Ser?Gly?Ser?Leu

465 470 475 480

Ile?Tyr?Asn?Asn?Ala?Phe?Ile?Ala?Phe?Ala?Thr?Asp?Leu?Asp?Pro?Asn

485 490 495

Thr?Ala?Gly?Leu?Leu?Val?Lys?Trp?Pro?Glu?Tyr?Thr?Ser?Ser?Leu?Gln

500 505 510

Leu?Gly?Asn?Asn?Leu?Met?Met?Ile?Asn?Ala?Leu?Gly?Leu?Tyr?Thr?Gly

515 520 525

Lys?Asp?Asn?Phe?Arg?Thr?Ala?Gly?Tyr?Asp?Ala?Leu?Phe?Ser?Asn?Pro

530 535 540

Pro?Leu?Phe?Phe?Val

545

<210>2

<211>549

<212>PRT

＜213〉candiyeast

<220>

＜223〉contriver: the luxuriant row in modern village

<400>2

Met?Lys?Leu?Ala?Leu?Ala?Leu?Leu?Leu?Ile?Ala?Ser?Val?Ala?Ala?Ala

1 5 10 15

Pro?Thr?Ala?Lys?Leu?Ala?Asn?Gly?Asp?Thr?Ile?Thr?Gly?Leu?Asn?Ala

20 25 30

Ile?Ile?Asn?Glu?Ala?Phe?Leu?Gly?Ile?Pro?Phe?Ala?Glu?Pro?Pro?Val

35 40 45

Gly?Asn?Leu?Arg?Phe?Lys?Asp?Pro?Val?Pro?Tyr?Ser?Gly?Ser?Leu?Asn

50 55 60

Gly?Gln?Lys?Phe?Thr?Leu?Tyr?Gly?Pro?Leu?Cys?Met?Gln?Gln?Asn?Pro

65 70 75 80

Glu?Gly?Thr?Phe?Glu?Glu?Asn?Leu?Gly?Lys?Thr?Ala?Leu?Asp?Leu?Val

85 90 95

Met?Gln?Ser?Lys?Val?Phe?Gln?Ala?Val?Leu?Pro?Gln?Ser?Glu?Asp?Cys

100 105 110

Leu?Thr?Ile?Asn?Val?Val?Arg?Pro?Pro?Gly?Thr?Lys?Ala?Gly?Ala?Asn

115 120 125

Leu?Pro?Val?Met?Leu?Trp?Ile?Phe?Gly?Gly?Gly?Phe?Glu?Ile?Gly?Ser

130 135 140

Pro?Thr?Ile?Phe?Pro?Pro?Ala?Gln?Met?Val?Thr?Lys?Ser?Val?Leu?Met

145 150 155 160

Gly?Lys?Pro?Ile?Ile?His?Val?Ala?Val?Asn?Tyr?Arg?Val?Ala?Ser?Trp

165 170 175

Gly?Phe?Leu?Ala?Gly?Asp?Asp?Ile?Lys?Ala?Glu?Gly?Ser?Gly?Asn?Ala

180 185 190

Gly?Leu?Lys?Asp?Gln?Arg?Leu?Gly?Met?Gln?Trp?Val?Ala?Asp?Asn?Ile

195 200 205

Ala?Gly?Phe?Gly?Gly?Asp?Pro?Ser?Lys?Val?Thr?Ile?Phe?Gly?Glu?Leu

210 215 220

Ala?Gly?Ser?Met?Ser?Val?Leu?Cys?His?Leu?Ile?Trp?Asn?Asp?Gly?Asp

225 230 235v240

Asn?Thr?Tyr?Lys?Gly?Lys?Pro?Leu?Phe?Arg?Ala?Gly?Ile?Met?Gln?Leu

245 250 255

Gly?Ala?Met?Val?Pro?Leu?Asp?Pro?Val?Asp?Gly?Thr?Tyr?Gly?Asn?Glu

260 265 270

Ile?Tyr?Asp?Leu?Phe?Val?Ser?Ser?Ala?Gly?Cys?Gly?Ser?Ala?Ser?Asp

275 280 285

Lys?Leu?Ala?Cys?Leu?Arg?Ser?Ala?Leu?Ser?Asp?Thr?Leu?Leu?Asp?Ala

290 295 300

Thr?Asn?Asn?Thr?Pro?Gly?Phe?Leu?Ala?Tyr?Ser?Ser?Leu?Arg?Leu?Leu

305 310 315 320

Tyr?Leu?Pro?Arg?Pro?Asp?Gly?Lys?Asn?Ile?Thr?Asp?Asp?Met?Tyr?Lys

325 330 335

Leu?Val?Arg?Asp?Gly?Lys?Tyr?Ala?Ser?Val?Pro?Val?Ile?Ile?Gly?Asp

340 345 350

Gln?Asn?Asp?Glu?Gly?Thr?Ile?Phe?Gly?Leu?Leu?Leu?Leu?Asn?Val?Thr

355 360 365

Thr?Asn?Ala?Gln?Ala?Arg?Ala?Tyr?Phe?Lys?Gln?Leu?Phe?Ile?His?Ala

370 375 380

Ser?Asp?Ala?Glu?Ile?Asp?Thr?Leu?Met?Ala?Ala?Tyr?Pro?Gln?Asp?Ile

385 390 395 400

Thr?Gln?Gly?Leu?Pro?Phe?Asp?Thr?Gly?Ile?Phe?Asn?Ala?Ile?Thr?Pro

405 410 415

Gln?Phe?Lys?Arg?Ile?Leu?Ala?Val?Leu?Gly?Asp?Leu?Ala?Phe?Ile?His

420 425 430

Ala?Arg?Arg?Tyr?Phe?Leu?Asn?His?Phe?Gln?Gly?Gly?Thr?Lys?Tyr?Ser

435 440 445

Phe?Leu?Leu?Lys?Gln?Leu?Leu?Gly?Leu?Pro?Ile?Met?Gly?Thr?Phe?His

450 455 460

Ala?Asn?Asp?Ile?Val?Trp?Gln?Asp?Tyr?Leu?Leu?Gly?Ser?Gly?Ser?Val

465 470 475 480

Ile?Tyr?Asn?Asn?Ala?Phe?Ile?Ala?Phe?Ala?Thr?Asp?Leu?Asp?Pro?Asn

485 490 495

Thr?Ala?Gly?Leu?Leu?Val?Asn?Trp?Pro?Lys?Tyr?Thr?Ser?Ser?Leu?Gln

500 505 510

Leu?Gly?Asn?Asn?Leu?Met?Met?Ile?Asn?Ala?Leu?Gly?Leu?Tyr?Thr?Gly

515 520 525

Lys?Asp?Asn?Phe?Arg?Thr?Ala?Gly?Tyr?Asp?Ala?Leu?Met?Thr?Asn?Pro

530 535 540

Leu?Leu?Phe?Phe?Val

545

<210>3

<211>1650

<212>DNA

＜213〉candiyeast

<220>

＜223〉contriver: the luxuriant row in modern village

<400>3

atggagctcg?ctcttgcgct?cctgctcatt?gcctcggtgg?ctgctgcccc?caccgccacg?60

ctcgccaacg?gcgacaccat?caccggtctc?aacgccatca?tcaacgaggc?gttcctcggc?120

attccctttg?ccgagccgcc?ggtgggcaac?ctccgcttca?aggaccccgt?gccgtactcc?180

ggctcgctcg?atggccagaa?gttcacgctg?tacggcccgc?tgtgcatgca?gcagaacccc?240

gagggcacct?acgaggagaa?cctccccaag?gcagcgctcg?acttggtgat?gcagtccaag?300

gtgtttgagg?cggtgctgcc?gctgagcgag?gactgtctca?ccatcaacgt?ggtgcggccg?360

ccgggcacca?aggcgggtgc?caacctcccg?gtgatgctct?ggatctttgg?cggcgggttt?420

gaggtgggtg?gcaccagcac?cttccctccc?gcccagatga?tcaccaagag?cattgccatg?480

ggcaagccca?tcatccacgt?gagcgtcaac?taccgcgtgt?cgtcgtgggg?gttcttggct?540

ggcgacgaga?tcaaggccga?gggcagtgcc?aacgccggtt?tgaaggacca?gcgcttgggc?600

atgcagtggg?tggcggacaa?cattgcggcg?tttggcggcg?acccgaccaa?ggtgaccatc?660

tttggcgagc?tggcgggcag?catgtcggtc?atgtgccaca?ttctctggaa?cgacggcgac?720

aacacgtaca?agggcaagcc?gctcttccgc?gcgggcatca?tgcagctggg?ggccatggtg?780

ccgctggacg?ccgtggacgg?catctacggc?aacgagatct?ttgacctctt?ggcgtcgaac?840

gcgggctgcg?gcagcgccag?cgacaagctt?gcgtgcttgc?gcggtgtgct?gagcgacacg?900

ttggaggacg?ccaccaacaa?cacccctggg?ttcttggcgt?actcctcgtt?gcggttgctg?960

tacctccccc?ggcccgacgg?cgtgaacatc?accgacgaca?tgtacgcctt?ggtgcgcgag?1020

ggcaagtatg?ccaacatccc?tgtgatcatc?ggcgaccaga?acgacgaggg?caccttcttt?1080

ggcaccctgc?tgttgaacgt?gaccacggat?gcccaggccc?gcgagtactt?caagcagctg?1140

tttgtccacg?ccagcgacgc?ggagatcgac?acgttgatga?cggcgtaccc?cggcgacatc?1200

acccagggcc?tgccgttcga?cacgggtatt?ctcaacgccc?tcaccccgca?gttcaagaga?1260

atcctggcgg?tgctcggcga?ccttggcttt?acgcttgctc?gtcgctactt?cctcaaccac?1320

tacaccggcg?gcaccaagta?ctcattcctc?ctgaagcagc?tcctgggctt?gccggtgctc?1380

ggaacgttcc?actccaacga?cattgtcttc?caggactact?tgttgggcag?cggctcgctc?1440

atctacaaca?acgcgttcat?tgcgtttgcc?acggacttgg?accccaacac?cgcggggttg?1500

ttggtgaagt?ggcccgagta?caccagcagc?ctgcagctgg?gcaacaactt?gatgatgatc?1560

aacgccttgg?gcttgtacac?cggcaaggac?aacttccgca?ccgccggcta?cgacgcgttg?1620

ttctccaacc?cgccgctgtt?ctttgtgtaa 1650

Claims (28)

1. phospholipid processing agent, described phospholipid processing agent contains from mycocandida (Candida) and has the active enzyme of phospholipase B (PLB).

2. phospholipid processing agent, described phospholipid processing agent contain (PLB) the active enzyme that has phospholipase B, and described enzyme only is regardless of the phosphatidylinositols of separating in the mixture of phospholipids basically.

3. phospholipid processing agent as claimed in claim 1 or 2 wherein, has the active enzyme of phospholipase B (PLB) and also has lipase activity.

4. as each described phospholipid processing agent of claim 1～3, wherein, have the active enzyme of phospholipase B and have following physicochemical property:

(1) effect: with phosphatide be hydrolyzed into 2 mol ratios free fatty acids and etc. the effect of glycerophosphoryl choline of mol ratio;

(2) molecular weight: 53,000 ± 3,000 (according to the SDS electrophoresis technique determining);

(3) iso-electric point: pH4.21 ± 0.2;

(4) best pH: about pH 5.5～6.5;

(5) pH stability: about pH 5～9 (handling 90 minutes for 37 ℃);

(6) stability: 55 ℃ (handling 10 minutes) at pH 5; With

(7) substrate specificity: to the specific activity of phosphatidylinositols is below 10% of specific activity to phosphatidylcholine.

5. phospholipid processing agent, described phospholipid processing agent contain the enzyme that the nutrient solution by column candiyeast (Candidacylindracea) obtains.

6. phospholipid processing agent, described phospholipid processing agent obtains by following steps:

(1) cultivates the column candiyeast;

(2) nutrient solution of concentrated described column candiyeast;

(3) make the enzyme precipitation with organic solvent;

(4) with hydrophobic chromatography the rough enzyme solution that step (3) obtains is purified; With

(5) enzyme that step (4) is obtained with ion exchange chromatography separates and purifies.

7. phospholipid processing agent, described phospholipid processing agent contains at least a enzyme that is selected from the following enzyme: the enzyme with aminoacid sequence of SEQ ID No.1; Be more than 75% and have the active enzyme of phospholipase B with the homology of the aminoacid sequence of SEQ ID No.1; Have disappearance in the aminoacid sequence of SEQ ID No.1, replace or increased one or more amino acid whose aminoacid sequences and had the active enzyme of phospholipase B.

8. phospholipid processing agent, described phospholipid processing agent contains at least a enzyme that is selected from the following enzyme: the enzyme with aminoacid sequence of SEQ ID No.2; Be more than 75% and have the active enzyme of phospholipase B with the homology of the aminoacid sequence of SEQ ID No.2; Have disappearance in the aminoacid sequence of SEQ ID No.2, replace or increased one or more amino acid whose aminoacid sequences and had the active enzyme of phospholipase B.

9. method of making the pure and mild glycerophosphoryl choline of phosphatidyl-4, described method comprises makes each described phospholipid processing agent of claim 1～8 act on mixture of phospholipids.

10. method of making phosphatidylinositols, described method comprises makes each described phospholipid processing agent of claim 1～8 act on mixture of phospholipids.

11. a method of making phosphatidylinositols said method comprising the steps of:

(1) make each described phospholipid processing agent of claim 1～8 act on mixture of phospholipids;

(2) with an organic solvent extract phosphatidylinositols; With

(3) by water-soluble or water miscibility alkyl-carbonyl alkyl solvent treatment, precipitation also reclaims phosphatidylinositols.

12. as the method for each described manufacturing phosphatidylinositols of claim 9～11, wherein, described mixture of phospholipids is from soybean.

13. a phosphatidylinositols, described phosphatidylinositols obtains by each described manufacture method of claim 10～12, and wherein, the purity of described phosphatidylinositols in total phospholipids is 50 moles more than the %.

14. comprising, a method of making glycerophosphoryl choline, described method make each described phospholipid processing agent of claim 1～8 act on mixture of phospholipids.

15. a method of making glycerophosphoryl choline said method comprising the steps of:

(1) make each described phospholipid processing agent of claim 1～8 act on mixture of phospholipids;

(2) usefulness contains the solvent extraction of organic solvent and removes lipid composition, and glycerophosphoryl choline is collected in the water layer; With

(3) phospholipid processing agent that plays a role in the step (1) is adsorbed on the gac, and removes described phospholipid processing agent.

16. as the method for claim 9,14 or 15 described manufacturing glycerophosphoryl cholines, wherein, described mixture of phospholipids is from soybean.

17. a glycerophosphoryl choline, described glycerophosphoryl choline obtains by each described manufacture method of claim 14～16, and has the above glycerophosphoryl choline purity of 55 weight %.

18. the application of the described phosphatidylinositols of claim 13 in preparation high purity Phosphoric acid glycerol esters inositol.

19. the application of the described glycerophosphoryl choline of claim 17 in preparation high purity cholinphospholipide.

20. contain food, medicine or the makeup of the phosphatidylinositols of making by each described manufacture method of claim 10～12.

21. contain food, medicine or the makeup of the glycerophosphoryl choline of making by each described manufacture method of claim 14～16.

22. a hemolytic phosphatidyl inositol, described hemolytic phosphatidyl inositol are to have the phosphatidylinositols that phospholipase A1 or the active enzyme of Phospholipase A2 act on by each described manufacture method manufacturing of claim 10～12 and obtain by making.

Have phospholipase A1 or the active enzyme of Phospholipase A2 and act on the phosphatidylinositols of making by each described manufacture method of claim 10～12 23. a method of making the hemolytic phosphatidyl inositol, described method comprise making.

24. contain food, medicine or the makeup of the hemolytic phosphatidyl inositol of making by the described manufacture method of claim 23.

25. a glycerine phosphinylidyne inositol, described glycerine phosphinylidyne inositol are by the phosphatidylinositols that the active enzyme of phospholipase B acts on by each described manufacture method manufacturing of claim 10～12 that has that phosphatidylinositols is had an abundant effect is obtained.

26. comprising, a method of making glycerine phosphinylidyne inositol, described method make the active enzyme of phospholipase B that has that phosphatidylinositols is had an abundant effect act on the phosphatidylinositols of making by each described manufacture method of claim 10～12.

27. contain food, medicine or the makeup of the glycerine phosphinylidyne inositol of making by the described manufacture method of claim 26.

28. a food, medicine or makeup, described food, medicine or makeup contain the two or more materials in the group that is selected from following material composition: by the phosphatidylinositols of each described manufacture method manufacturing of claim 10～12; Glycerophosphoryl choline by each described manufacture method manufacturing of claim 14～16; Hemolytic phosphatidyl inositol by the described manufacture method manufacturing of claim 23; With the glycerine phosphinylidyne inositol of making by the described manufacture method of claim 26.

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