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Novel phospholipase D

A technology of phospholipase and phosphatidylinositol, applied in the field of phospholipase D, can solve the problems of low alkali conversion rate and the like

Inactive Publication Date: 2009-03-04
NAGOYA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PI is also produced, but with low base conversion

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0144] (Example 1: Preparation of library)

[0145] pPELB-PLD1 (prepared according to Y.Iwasaki et al., J.Ferment.Bioeng., 1995, Vol. 79, pages 417-421) was digested with restriction enzymes XbaI and XhoI, and the liposome-binding site, The pelB signal from pET22b (Novagen) and the fragment of the full-length (1530bp; base sequence as shown in SEQ ID NO.1 of the sequence listing) of the PLD gene from the resistant Streptomyces were inserted into the pBluescriptII KS digested with XbaI and XhoI in advance +(manufactured by Toyobo), pPELB-PLD-KS was prepared. This was digested with the restriction enzyme ApaI, together with the fragment around the stop codon of the PLD gene, the inverted repeat sequence downstream of the PLD gene was removed. On the other hand, pSA1 (prepared according to Y.Iwasaki et al., Appl.Microbiol.Biotechnol., 1994, volume 42, pages 290-299) was digested with restriction enzymes BamHI and SalI, and the second half of the PLD gene containing it was excise...

Embodiment 2

[0153] (Example 2: Expression of plasmid library)

[0154] The above-mentioned plasmid mixture was introduced into Escherichia coli BL21(DE3) as an expression host, and grown on LB agar medium having the same composition as in Example 1 at 37°C for 16 hours. A nitrocellulose membrane (HybondC, manufactured by Amasyam) was placed on the obtained colonies, and the colonies were transferred. Next, the membrane is transferred to a synthetic medium (the composition is as follows (indicates the quality per 1 L): 5g KH 2 PO 4 , 5g K 2 HPO 4 , 4.4g Na 2 HPO 4 , 3g (NH 2 ) 2 SO 4 , 5g glucose, 3g MgSO 4 ·7H 2 O, 40mg FeSO 4 ·7H 2 O, 40mg CaCl 2 , 10mgMnSO 4 ·7H 2 O, 2.9mg CoCl 2 ·6H 2 O, 3mg CuSO 4 ·5H 2 O, 0.36mgNa 2 MoO·2H 2 O, 10 mg H3 BO 3 , 10mg ZnSO 4 ·7H 2 1.5% agar was added to O) and incubated at 30°C for 8 hours to allow colonies to grow on the membrane. Colonies on LB agar medium were kept in the refrigerator as master plates. The membrane was trans...

Embodiment 3

[0155] (Example 3: Screening of clones expressing modified PLDs that synthesize PI)

[0156] The above membrane was immersed in a substrate solution (10% soybean lecithin (manufactured by SLP-PC70ツルレシチン), 20% inositol, 2% calcium chloride and 50 mM acetate buffer, pH 5.6) at 30°C For 14 hours, the enzymatic reaction was carried out on the membrane. Phosphatidylinositol (PI), which is an acidic phospholipid, is produced by this enzymatic reaction, and these form water-insoluble salts with coexisting calcium ions, and precipitate at the site where the reaction occurs. After the reaction, the excess substrate solution was rinsed with water, and the membrane was immersed in a 10% aqueous sodium periodate solution at room temperature for 10 minutes. By this sodium periodate treatment, the diol moiety of the precipitated PI is oxidatively cleaved to derivatize to an aldehyde. The excess sodium periodate solution was rinsed with water, and the membrane was coated with an aqueous NB...

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PUM

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Abstract

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

Description

technical field [0001] The present invention relates to novel phospholipase D having the ability to synthesize phosphatidylinositol. Background technique [0002] Phospholipids are used in emulsifiers, cosmetic ingredients, pharmaceutical formulations, and in the preparation of liposomes. Phospholipids derived from natural products usually contain mixtures of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), sphingomyelin and the like. In order to separate specific phospholipids from natural phospholipids, solvent separation methods such as extraction with solvents such as methanol, ethanol, isopropanol, hexane, and chloroform or recrystallization can be performed, and adsorption agents such as silica gel, lithium oxide, and ion exchange resin can be used. Column chromatography using CdCl 2 Separation of derivatives such as complexes or acetylates. [0003] Isolation and purification of PI ...

Claims

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Application Information

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IPC IPC(8): C12N15/00C12N9/16C12N15/09C12P9/00C12Q1/34C12P7/6481
CPCC12P7/6481C12P9/00C12N9/16C12Q1/44
Inventor 岩崎雄吾中野秀雄高桥哲也
Owner NAGOYA UNIVERSITY
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