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DNA encoding novel enzyme having D-serine synthase activity, method of producing the enzyme and method of producing d-serine by using the same

A technology of serine and DNA sequence, applied in the field of preparation of D-serine, can solve the problems such as no

Active Publication Date: 2007-09-19
MITSUI CHEM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In addition, it has been reported that D-β-hydroxyamino acids can be prepared from glycine and aldehydes by using DTA from Xanthomonas, but there is no report on the production of D-β-hydroxyamino acids from formaldehyde using DTA from Xanthomonas. Report on synthesis of D-serine with glycine (see JP-A-5-168484)

Method used

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  • DNA encoding novel enzyme having D-serine synthase activity, method of producing the enzyme and method of producing d-serine by using the same
  • DNA encoding novel enzyme having D-serine synthase activity, method of producing the enzyme and method of producing d-serine by using the same

Examples

Experimental program
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Effect test

Embodiment 1

[0129] (Acquisition of the gene encoding DSA)

[0130] Achromobacter xyloseoxidans (ATCC9220), Achromobacter denitrificans (NBRC15125) and Achromobacter xyloseoxidans (NBRC13495) were inoculated in 50 ml of LB medium. After culturing overnight at 30°C, the bacteria were collected and lysed with a lysozyme solution containing 1 mg / ml. After treating the lysate with phenol, the DNA was precipitated by ethanol precipitation using the usual method. The resulting DNA pellet was recovered by winding it on a glass rod, washed, and used for PCR.

[0131] As primers for PCR, oligonucleotides (synthesized on request to Hokkaido System Science Co., Ltd.) having base sequences shown in SEQ ID NOs: 1 and 2 designed based on the known DTA gene were used. The above primers have restriction enzyme recognition sequences of KpnI and HindIII near the 5' end and near the 3' end, respectively.

[0132] Use 0.025ml of PCR reaction solution containing 6ng / μl of the chromosomal DNA of the above-me...

Embodiment 2

[0141] (Preparation method of DSA)

[0142] 0.5 mL of the transformant suspension prepared in Example 1 was disrupted in ice water for 5 minutes using a Bioruptor (manufactured by OLYMPUS). The broken solution of the transformant was centrifuged to prepare the broken solution. The disrupted solution was analyzed by SDS-polyacrylamide electrophoresis, and the results are shown in Figure 1.

[0143] 0.5 mL of 100 mM Tris-HCl buffer (pH 8.0) was added to the pellet, and the same analysis was performed on the cell residue.

[0144] Protein expressed at about 40 kDa was seen in the soluble fraction of all transformants and not in the insoluble fraction. The molecular weight almost coincides with the molecular weight of the amino acid sequence estimated from each gene.

Embodiment 3

[0146] (Purification of DSA and synthesis of D-serine by purification of DSA)

[0147] Centrifuge 10 mL of the disrupted MT-11015 solution prepared by the same method as in Example 2 at 10,000 rpm for 20 minutes to prepare an enzyme solution for removing cell residues. An anion exchange resin (HiTrap Q-XL, manufactured by Amersham) was adsorbed on the enzyme solution, and it was eluted with a linear gradient from 100 mM Tris-HCl buffer (pH 8.0) containing 10 mM magnesium chloride and 50 mM sodium chloride to 100 mM containing 10mM magnesium chloride and 500mM sodium chloride in Tris-HCl buffer (pH8.0). The hydrophobic chromatography resin (HiTrap Phenyl FF, manufactured by Amersham) was allowed to adsorb the active fraction, and it was eluted with a linear gradient from 100 mM Tris-HCl buffer (pH 8.0) containing 10 mM magnesium chloride saturated with ammonium sulfate to 100 mM Tris-HCl buffer (pH 8.0) containing 10 mM Magnesium chloride in Tris-HCl buffer (pH 8.0). It shoul...

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Abstract

A DNA encoding a novel enzyme which has an activity of synthesizing D-serine from glycine and formaldehyde; a recombinant DNA constructed by integrating the DNA into a vector; a transformant having been transformed by the recombinant DNA; and a method of producing D-serine from glycine and formaldehyde by using the above enzyme.

Description

technical field [0001] The present invention relates to a DNA encoding a novel enzyme capable of synthesizing D-serine from formaldehyde and glycine, a recombinant DNA formed by integrating the DNA into a vector, a transformant obtained by transforming the recombinant DNA, and having the ability to synthesize D-serine from formaldehyde and glycine. - Novel enzyme with activity of serine and method for preparing D-serine from formaldehyde and glycine using the enzyme. Background technique [0002] D-serine is known to be a very useful compound as a synthetic intermediate of pharmaceuticals such as D-cycloserine. [0003] At present, as an enzyme having the activity of synthesizing D-serine from formaldehyde and glycine, only D-threonine aldolase (hereinafter referred to as "DTA") from Arthrobacter sp.DK-19 has been disclosed (see Japanese Patent Laid-Open No. 58-116690). [0004] It has been reported that 50 mmol each of formaldehyde and glycine was used to react with DTA a...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N5/10C12N1/15C12N9/00C12N1/19C12P13/06C12N1/21B23P19/00B23P21/00B62D65/00
CPCC12N9/1014C12P13/06C12N5/10C12N9/00C12N15/09
Inventor 安乐城正秀崎友则渡边清一西田圭太长原清辉小糸光男
Owner MITSUI CHEM INC
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