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Method for producing 4-hydroxy-L-isoleucine

A technology of hydroxyisoleucine and isoleucine, applied in oxidoreductase, fermentation and other directions, can solve the problem of no isoleucine hydroxylase reported

Inactive Publication Date: 2009-09-09
AJINOMOTO CO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on isoleucine hydroxylase derived from microorganisms

Method used

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  • Method for producing 4-hydroxy-L-isoleucine
  • Method for producing 4-hydroxy-L-isoleucine
  • Method for producing 4-hydroxy-L-isoleucine

Examples

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specific example

[0108] Specific examples include Bacillus thuringiensis (strain 2-e-2, strain AKU 238, strain NBRC3958, strain ATCC 35646, etc.), Bacillus licheniformis (strain AKU 223, strain IAM 11054, etc.), Bacillus sphaericus (strain AKU 227, strain NBRC 3526 etc.), Bacillus cereus strain ATCC 14579 and Bacillus weideri strain KBAB4. The strain 2-e-2 was named as the AJ110584 strain, and was deposited in the independent administrative agency, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary) (Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), and obtained accession number FERM BP-10688.

[0109] Among the above-mentioned microorganisms, those whose strain names start with AKU can be obtained from Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied LifeSciences, Graduate School of Agriculture, Kyoto University (Fermentation Physiology and Applied Microbiology Exp...

Embodiment 1

[0275] Example 1. Screening for strains with L-isoleucine hydroxylase

[0276] Screening of strains producing 4-hydroxyisoleucine and analysis of culture medium

[0277] The microorganisms having the ability to produce 4-hydroxyisoleucine were screened by using L-isoleucine as a substrate. In water, dissolve 0.4% (w / v) of soluble starch, 0.4% of yeast extract, 1% of malt extract (malt extract) and 0.2% of L-isoleucine, and then adjust the solution to pH7- 7.5 Obtain a medium and inoculate the medium with soil bacteria. After shaking culture at 28°C for 2 days, 4-hydroxyisoleucine was analyzed by amino acid analysis of the centrifuged supernatant.

[0278] Amino Acid Analysis Conditions

[0279] By using Waters AccQ-Tag TM method to detect 4-hydroxyisoleucine. The amino acids in 5 μl of the reaction mixture diluted to the appropriate concentration were derivatized in a conventional manner and the production of 4-hydroxyisoleucine was measured by HPLC analysis. ...

Embodiment 2

[0323] The separation and purification of embodiment 2.L-Ile hydroxylase

[0324] (1) Preparation of cell-free extract

[0325] Strain 2-e-2 was grown in a total volume of 2 L of AMKP production medium until OD 660 to 6.0, the cells were then washed with saline. The cells were suspended in a cell membrane treatment solution (10 mg / ml lysozyme (SIGMA), 5 mg / ml cellulase "ONOZUKA" R-10 (Yakult), Yatalase (Takara Bio), 1 mg / ml lysozyme (SIGMA), Soluble in 0.2M NaH 2 PO 4 and 0.6M KCl (pH5.5)), and then incubated at 30°C for 1 hour. The incubated cells were washed with physiological saline, then suspended in buffer A (50 mM HEPES (pH 7.0), 10% glycerol, 2 mM DTT, 1 mM EDTA, Complete (Roche)), and cooled with ice The cells were disrupted by using a sonicator (Branson). This treated suspension was centrifuged at 4°C and 18,500 xg for 60 minutes to obtain a supernatant. Subsequent isolation and purification steps were all carried out at 4°C or under ice-cooling condit...

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Abstract

The present invention provides a novel highly active L-isoleucine dioxygenase from Bacillus thuringiensis. A method for manufacturing (2S,3R,4S)-4-hydroxy-L-isoleucine or a salt thereof comprising reacting L-isoleucine in an aqueous solvent in the presence of L-isoleucine dioxygenase and isolating produced (2S,3R,4S)-4-hydroxy-L-isoleucine is also provided.

Description

technical field [0001] The present invention relates to the microbial industry, and in particular to novel dioxygenases and processes for the preparation of 4-hydroxy-L-isoleucine or salts thereof. Background technique [0002] 4-Hydroxy-L-isoleucine is an amino acid that can be extracted and purified from fenugreek seeds (Trigonella foenum-graecum L. leguminosae). 4-Hydroxy-L-isoleucine exhibits strongly interesting insulinotropic activity because its stimulatory effect is clearly dependent on the plasma glucose concentration in the medium, as in isolated perfused rat pancreas and human As demonstrated in pancreatic islets (Sauvaire, Y. et al., Diabetes, 47:206-210, (1998)). This glucose dependence was not confirmed for sulfonylureas (Drucker, D.J., Diabetes 47:159-169, (1998)), which are currently used in the treatment of type II diabetes [or non-insulin-dependent diabetes mellitus (NIDD or NIDDM)]. )(non-insulin-dependent diabetes(NIDD)mellitus(NIDDM))], hypoglycemia re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/06C12N9/02
Inventor 小寺智博瑟奇·V·斯米尔诺夫纳塔利亚·N·萨姆索诺瓦维罗尼卡·A·科特利亚罗瓦纳塔利亚·Y·拉什克维克尤里·I·科兹洛夫清水昌小川顺日比慎
Owner AJINOMOTO CO INC
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