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Production method for l-lysine hydroxylase and hydroxy-l-lysine using same, and production method for hydroxy-l-pipecolic acid

一种赖氨酸羟化酶、赖氨酸羟化酶活性的技术，应用在生物化学设备和方法、氧化还原酶、酶等方向，能够解决尚未报道L-赖氨酸等问题

Active Publication Date: 2015-06-03
API CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no enzymes acting on L-lysine have been reported so far

Method used

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  • Production method for l-lysine hydroxylase and hydroxy-l-lysine using same, and production method for hydroxy-l-pipecolic acid
  • Production method for l-lysine hydroxylase and hydroxy-l-lysine using same, and production method for hydroxy-l-pipecolic acid
  • Production method for l-lysine hydroxylase and hydroxy-l-lysine using same, and production method for hydroxy-l-pipecolic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0184] Cloning of 2-oxoglutarate-dependent L-lysine hydroxylase gene

[0185] Based on the gene sequence (hyl-1, sequence No. 1), designed and synthesized the full-length primers hyl1_F (SEQ ID NO: 13) and hyl1_R (SEQ ID NO: 14) for amplifying the hyl-1 gene. Using the chromosomal DNA of Flavobacterium johnii as a template, PCR reaction was carried out according to conventional methods to obtain a DNA fragment of about 1.0 kbp.

[0186] Similarly, for Kineococcus radiotolerans NBRC101839 strain, Chitinophaga pinensis NBRC15968 strain, Chryseobacterium gleum NBRC15054 strain, and Niastella koreansis NBRC106392 strain The source of VioC homologues are Hyl-2 (GenBank Accession No. ABS05421, Sequence No. 4), Hyl-3 (GenBank Accession No. ACU60313, Sequence No. 6), Hyl-4 (GenBank Accession No. EFK34737, Sequence No. No. 8), Hyl-5 (GenBank Accession No. AEV99100, Sequence No. 10). Based on the gene sequences encoding each enzyme (hyl-2 (SEQ ID NO: 3), hyl-3 (SEQ ID NO: 5), h...

Embodiment 2

[0191] Activity Confirmation of 2-oxoglutarate-dependent L-lysine Hydroxylase Based on Resting Cell Response

[0192] Mix 5mM L-lysine, 10mM 2-oxoglutaric acid, 1mM L-ascorbic acid, 0.1mM ferrous sulfate, and the recombinant Escherichia coli obtained by the method according to Example 1 in a plastic tube, according to the turbidity OD 600 =10 way to mix the reaction solution. 0.5 ml of the prepared mixed solution was reacted at 30° C. and pH 7.0 for 3 hours. The reaction products were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA) and analyzed by HPLC. As a result, such as figure 1 , figure 2 As shown, it was confirmed that BL21(DE3) / pEHYL2 and BL21(DE3) / pJHYL6 produced consistent compounds when the retention time of the 3-hydroxylysine standard substance was 8.04 minutes. In addition, it was confirmed that BL21(DE3) / pEHYL1, BL21(DE3) / pEHYL3, BL21(DE3) / pEHYL4, and BL21(DE3) / pEHYL5 produced consistent 4-hydroxylysine standard substances at a rete...

Embodiment 3

[0196] Synthesis of (2S,3S)-3-hydroxylysine

[0197] Mix 35mL of 1M potassium phosphate buffer (pH7.0), 304mL of desalted water, 1.28g of L-lysine hydrochloride, 2.05g of 2-ketoglutarate, 0.14g of sodium L-ascorbate, and sulfuric acid in a 1L fermenter. 0.02g of ferrous iron, 0.35g of Adekanol LG109, and 8g of wet bacteria of recombinant Escherichia coli BL21(DE3) / pEHYL2 obtained according to the method of Example 1, at 30°C, pH7.0, agitation number 500rpm, air ventilation Under the condition of volume 2.0vvm, react for 17 hours. The end of the reaction was judged by confirming that the peak of L-lysine disappeared by HPLC analysis. By centrifugation and microfiltration, bacteria and bacteria residues were removed from the liquid after the reaction to obtain 390 g of filtrate.

[0198] In addition, the analysis conditions of L-lysine by HPLC are as follows.

[0199] Column: SUMICHIRAL OA-6100 (4.6mm×250mm) manufactured by Sumika Analytical Center, mobile phase: 1mM ...

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PUM

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Abstract

The present invention addresses the issue of providing a method whereby hydroxy-L-lysine can be efficiently produced. The present invention provides a production method for hydroxy-L-lysine, characterized by: causing a 2-oxoglutarate-dependent L-lysine hydroxylase, cells including same, a preparation of said cells, or a culture fluid obtained by cultivating said cells, to act on L-lysine; and generating the hydroxy-L-lysine indicated in general formula (I) (in the formula, R1, R2, and R3 each indicate a hydrogen atom or a hydroxyl group and at least one among R1, R2, and R3 indicates a hydroxyl group.)

Description

technical field [0001] The present invention relates to a method for producing hydroxy-L-lysine using a novel lysine hydroxylase and production of hydroxy-L-2-pipericolic acid using the obtained hydroxy-L-lysine Law. Background technique [0002] Hydroxy-L-lysine is a useful intermediate such as an intermediate of a drug. For example, it is known that (3R)-hydroxy-L-lysine can be used as a precursor of the protein kinase C inhibitor (-)-balanol (Non-Patent Document 1), and (5R)-hydroxy-L-lysine can be It is used as a precursor of Bengamide B which has antitumor activity (Non-Patent Document 2). In addition, it has also been reported that hydroxy-L-lysine can be used as a raw material of hydroxy-L-2-pipericolic acid (Non-Patent Documents 3 and 4). For example, (4R)-hydroxy-L-2-pipericolic acid can be used as a precursor of the HIV protease inhibitor palinavir (non-patent literature 5), (5S)-hydroxy-L-2-pipericolic acid and (5R) -Hydroxy-L-2-pipericolic acid can be used as...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12N9/02C12N15/09
CPCC12P13/08C12N9/0071C12P13/04C12P17/12C12Y114/11C12Y114/11004Y02P20/52
Inventor 木野邦器原良太郎三宅良磨川端润
Owner API CO LTD
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