Method for manufacturing cis-5-hydroxy-l-pipecolic acid

A manufacturing method, pipecolic acid hydroxylase technology, applied in the direction of biochemical equipment and methods, enzymes, oxidoreductases, etc., to achieve low-cost effects

Active Publication Date: 2017-08-29
API CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In Patent Document 3, it is reported that the gene of SmPH is changed in order to suppress the production of unnec

Method used

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  • Method for manufacturing cis-5-hydroxy-l-pipecolic acid
  • Method for manufacturing cis-5-hydroxy-l-pipecolic acid
  • Method for manufacturing cis-5-hydroxy-l-pipecolic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Cloning of α-ketoglutarate-dependent pipecolate hydroxylase gene

[0114] The putative L-proline cis-4-hydroxylase XdPH (GenBank accession number CDG16639, sequence number 4) encoding Xenorhabdus doucetiae FRM16 strain source was artificially synthesized by DNA2.0 company for expression in Escherichia coli The codon-optimized gene sequence (xdph_Ecodon, SEQ ID NO: 1) was inserted into pJExpress411 (DNA2.0) to create plasmid pJ411XdPH.

[0115] Similarly, cloning of a representative enzyme gene known to exhibit the 5-hydroxylation activity of pipecolic acid was also carried out. The L-picolic acid cis-5-hydroxylase SruPH (GenBank accession number EFV12517, sequence number 6) encoding Segniliparus rugosus NBRC101839 strain and the source of Sinorhizobium meliloti 1021 strain were artificially synthesized by DNA2.0 company. The codon-optimized gene sequences sruph_Ecodon (SEQ ID NO. 3) and smph_Ecodon of the L-proline cis-4-hydroxylase SmPH (GenBank accession number CAC...

Embodiment 2

[0117] Acquisition of α-ketoglutarate-dependent pipecolate hydroxylase gene expressing bacteria and confirmation of expression level

[0118] Next, Escherichia coli (Escherichia coli) BL21(DE3) (manufactured by Invitrogen) was transformed by a conventional method using each of the obtained plasmids to obtain recombinant Escherichia coli BL21(DE3) / pJ411XdPH, BL21(DE3) / pJ411SruPH, BL21(DE3 ) / pET24SmPH. In order to obtain bacterial cells expressing the introduced gene, each recombinant Escherichia coli was cultured at 28°C for 4 to 6 hours in liquid LB medium containing kanamycin and a lac promoter-inducing substance, and then incubated at 15°C. After further culturing for about 40 hours, bacterial cells were collected.

[0119] The obtained recombinant Escherichia coli was measured by turbidity OD 630 Suspended in 50mmol / L MES (2-morpholineethanesulfonic acid) buffer at pH 7 in a mode of about 10. 0.5 mL of this suspension was sonicated on ice, and then centrifuged at 12,00...

Embodiment 3

[0122] Activity confirmation of α-ketoglutarate-dependent pipecolate hydroxylase

[0123] 5mmol / L L-picolic acid, 10mmol / L α-ketoglutarate, 1mmol / L L-ascorbic acid, 0.5mmol / L iron sulfate, and the protein concentration of about 2mg / mL were added with Example 2 in a plastic tube. 0.2 mL of each crude enzyme solution obtained in was shaken at 30° C. for 1 hour.

[0124] The reaction product was derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA), and analyzed by UPLC-MS (manufactured by Waters). The result is as figure 2 As shown, it was confirmed that a compound corresponding to the retention time of the 5-hydroxypicolic acid standard of 5.3 minutes was produced from the solution reacted using each crude enzyme solution. The 5-hydroxypicolic acid production activity (U / g) exhibited by each enzyme liquid was calculated as 4.1 U / g, 8.9 U / g, and 3.1 U / g, respectively, in terms of the unit protein amount (g). The unit (U) here represents the ability to generate...

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Abstract

A method for manufacturing cis-5-hydroxy-L-pipecolic acid, characterized in that cis-5-hydroxy-L-pipecolic acid is produced by causing alpha-oxoglutarate-dependent L-pipecolic acid hydroxylase to act on L-pipecolic acid, the alpha -oxoglutarate-dependent L-pipecolic acid hydroxylase including a polypeptide indicated by (A), (B), or (C). (A) A polypeptide having an amino acid sequence represented by SEQ ID NO 4 or 11; (B) a polypeptide having an amino acid sequence in which one or several amino acids have been deleted, substituted, and/or added in an amino acid sequence represented by SEQ ID NO 4 or 11, and further having alpha-oxoglutarate-dependent L-pipecolic acid hydroxylase activity; or (C) a polypeptide having an amino acid sequence having 60% or greater identity with an amino acid sequence represented by SEQ ID NO 4 or 11, and further having alpha-oxoglutarate-dependent L-pipecolic acid hydroxylase activity.

Description

technical field [0001] The present invention relates to a method for producing cis-5-hydroxy-L-picolic acid using an enzyme capable of producing cis-5-hydroxy-L-picolic acid. Background technique [0002] cis-5-hydroxy-L-picolic acid (hereinafter sometimes referred to as "5OH-PA") is a compound useful as an intermediate of pharmaceuticals and the like. It is known that cis-5-hydroxy-L-picolic acid can be produced from L-picolic acid by a biological method. [0003] It has been reported that the BAB52605 protein derived from Mesorhizobium loti MAFF303099 in japonicus and the CAC47686 protein derived from Sinorhizobium meli loti 1021 (hereinafter sometimes referred to as "SmPH") have L-proline Ability to convert to cis-4-hydroxyproline (Patent Document 1). [0004] Although the BAB52605 protein has the ability to convert L-picolic acid into cis-5-hydroxy-L-picolic acid, its productivity is reported to be relatively low (Patent Document 2). [0005] Although the CAC47686 pro...

Claims

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Application Information

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IPC IPC(8): C12P17/12C12N9/02C12N15/09
CPCC12N15/09C12P17/12C12N9/0071C12Y114/11
Inventor 三宅良磨出来岛康方
Owner API CO LTD
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