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Method for separating and extracting 4-hydroxyisoleucine from fermentation liquor

A technology of hydroxyisoleucine and fermentation liquid, which is applied in the field of amino acid production by fermentation, can solve the problems of low conversion rate, environmental pollution, harsh reaction conditions, etc., and achieve good results

Inactive Publication Date: 2016-05-11
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, fenugreek seed extraction is mainly used to produce 4-HIL, however, the extraction efficiency of this method is low (0.091-0.6%) and the configuration of 4-HIL obtained by this method is more but only (2S,3R,4S)-4- HIL is biologically active
There are many reports on the synthesis of 4-HIL by chemical method, but it is mainly produced by multi-step chemical reaction with glucose, menthone or ethyl 2-methylacetoacetate as raw material. The method has harsh reaction conditions, many steps, difficult separation, The yield is low, and it is easy to cause environmental pollution
Enzymatic synthesis is based on hexanal, α-ketoglutarate and L-glutamic acid as substrates via 4-hydroxy-3-methyl-2-keto-pentanoic acid aldolase and branched chain amino acid aminotransferase. One-step catalytic synthesis, although this method has the advantages of short cycle, it is reported that it cannot meet the production requirements because of its low conversion rate and a large amount of by-products such as α-aminobutyric acid in the product

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] a. Take 25L of 4-hydroxyisoleucine fermentation broth (4-hydroxyisoleucine content 18.0g / L) and heat it at 80°C for more than 10 minutes, adjust the pH to 4.0 with hydrochloric acid, and enter the microfiltration membrane separation system for microfiltration , to obtain the 4-hydroxyisoleucine microfiltrate and bacterial protein from which the bacteria were removed; the microfiltration membrane separation system adopts a metal membrane separation system, the microfiltration membrane pore size is 50nm, and the operating temperature is 30-40°C. The difference between the pressure and the membrane exit pressure is 0.04Mpa. The filtration process is divided into several times with purified water for top washing, until the 4-hydroxyisoleucine content in the dope is lower than 2.0g / L, then stop the machine, and obtain the 4-hydroxyisoleucine that has been removed from the bacteria. Isoleucine microfiltrate. The recovery rate of 4-hydroxyisoleucine in the serum reaches 95.2%,...

Embodiment 2

[0028] Take 20L of 4-hydroxyisoleucine fermentation broth (4-hydroxyisoleucine content 20.3g / L) and heat it at 80°C for more than 10min, adjust the pH to 4.0 with hydrochloric acid, and carry out microfiltration sterilization according to the method of Example 1 Body and ultrafiltration to remove protein, adopt 3% ammonia water to elute the resin, collect the eluent with pH of 7.0-10.0, add 1.8% activated carbon decolorization of the eluent weight, temperature 55 ° C, stir for 25min, filter to obtain 4 -Hydroxyisoleucine decolorization solution, adjust the pH to 7.2 with hydrochloric acid, and then evaporate under reduced pressure to 12% of the original volume to obtain a suspension of 4-hydroxyisoleucine crystals, place it at 4°C for crystallization for 4 hours, and filter , to obtain 4-hydroxyisoleucine crystals, weighing 252.5g, the calculated yield was 62.2%, and the purity was 96.5%.

Embodiment 3

[0030]Take 28L of 4-hydroxyisoleucine fermentation broth (4-hydroxyisoleucine content 19.1g / L) and heat it at 80°C for more than 10min, adjust the pH to 4.0 with hydrochloric acid, and carry out microfiltration sterilization according to the method of Example 1 Body and ultrafiltration to remove protein, adopt 6% ammonia water to elute the resin, collect the eluent whose pH is 7.5-10.5, add 1.5% activated carbon decolorization of the eluent weight, temperature 50 ° C, stir and decolorize for 20min, filter to obtain 4 -Hydroxyisoleucine decolorization solution, adjust the pH to 7.2 with hydrochloric acid, and then evaporate under reduced pressure to 15% of the original volume to obtain a suspension of 4-hydroxyisoleucine crystals, place it at 4°C for crystallization for 4 hours, and filter , to obtain 4-hydroxyisoleucine crystals, weighing 322.5g, the calculated yield was 60.3%, and the purity was 96.8%.

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Abstract

The invention provides a method for separating and extracting 4-hydroxyisoleucine from fermentation liquor. The method comprises the steps that cross-flow filtration is conducted through a double-membrane (that is, a micro-filtration membrane and an ultra-filtration membrane) separation system to remove thalli, most protein and pigments in the fermentation liquor; the operation steps of heteroacid of ketoglutaric acid and the like removing conducted through a cation exchange column, powdered activated carbon decoloring, vacuum concentration, freezing crystallization, centrifugation to collect the product and finished product drying are conducted, the 4-hydroxyisoleucine crystal with the purity of 97.0% or above is obtained, and the yield reaches 65% or above. According to the method for separating and extracting 4-hydroxyisoleucine from the fermentation liquor, the micro-filtration membrane and the ultra-filtration membrane are adopted to filter thallus protein, the thallus protein is removed thoroughly, and filtered liquid is clear and colorless; the cation exchange technology is adopted, the impurities such as the ketoglutaric acid and the like which are difficult to separate are efficiently removed.

Description

technical field [0001] The invention relates to a method for separating and extracting 4-hydroxyisoleucine from fermentation liquid, and belongs to the technical field of amino acid production by fermentation. Background technique [0002] 4-Hydroxyisoleucine (4-Hydroxyisoleucine, 4-HIL) is a kind of L-isoleucine hydroxylate mainly present in the seeds of fenugreek plants. 4-HIL formula C 6 h 13 NO 3 , molecular weight 147.17, melting point greater than 200°C, boiling point 332°C, density 1.181g / cm 3 . Studies have shown that 4-hydroxyisoleucine has a glucose concentration-dependent activity of promoting insulin secretion and promoting the absorption of blood sugar by muscle cells, accelerating fat metabolism, lowering blood fat and protecting liver function. Therefore, as an ideal drug for effectively preventing and treating diabetes and obesity, 4-hydroxyisoleucine has broad application prospects and market demands. [0003] The production methods of 4-hydroxyisoleuc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C227/40C07C229/22
CPCC07C227/40
Inventor 陈宁麻杰徐庆阳范晓光谢希贤张成林李燕军
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY