Method for producing L-4-hydroxyisoleucine by microbial enzyme conversion method

A technology of hydroxyisoleucine and microbial enzymes, applied in the field of microbial enzyme conversion to produce L-4-hydroxyisoleucine, can solve the problem that the production of L-4-hydroxyisoleucine cannot meet domestic and foreign trade exports Demand, large demand for raw materials, difficulty in separation and purification, etc., to achieve the effect of reducing the specific growth rate of bacteria, facilitating protein expression, and simple extraction process

Active Publication Date: 2016-07-20
HENAN JULONG BIOLOGICAL ENG CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Limited by technology and cost, the output of L-4-hydroxyisoleucine in my country cannot meet the needs of domestic and foreign trade exports
Currently reported L-4-hydroxyisoleucine production methods include extraction, chemical synthesis, and enzymatic methods, but only the extraction method is used for industrial production and has disadvantages such as low extraction rate, difficult separation and purification, large demand for raw materials, and high cost. , the traditional chemical method, whether it is organic synthesis or chemical resolution, loses its competitiveness due to high production costs
In the biological method, the efficiency of the enzyme-catalyzed method is relatively high, but there are still problems of cumbersome operation, expensive raw materials, and high production costs, and there is currently a lack of relevant technology that can be used for industrialized large-scale production

Method used

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  • Method for producing L-4-hydroxyisoleucine by microbial enzyme conversion method

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Embodiment 1

[0039] A method for producing L-4-hydroxyisoleucine by a microbial enzyme conversion method, comprising the following steps:

[0040] (1) Introduce recombinant Escherichia coli bacteria capable of inducing the production of L-4-hydroxyisoleucine into a seed tank equipped with a seed tank culture medium, at a temperature of 36°C, pH 7.0, and dissolved oxygen at 25% Cultivate under the above conditions until the OD600 value of the bacterial solution is 14, that is, a mature Escherichia coli recombinant bacterium capable of inducing the production of L-4-hydroxyisoleucine is obtained;

[0041] In this embodiment, the Escherichia coli recombinant bacteria that can induce the production of L-4-hydroxyisoleucine are obtained through purchase; the construction method is to clone the gene whose gene sequence is shown in SEQNO.1; construct the gene into a plasmid pET28a, to obtain a recombinant plasmid, and then introduce the recombinant plasmid into Escherichia coli BL21 to obtain an ...

Embodiment 2

[0048] A method for producing L-4-hydroxyisoleucine by a microbial enzyme conversion method, comprising the following steps:

[0049] (1) Introduce recombinant Escherichia coli bacteria capable of inducing the production of L-4-hydroxyisoleucine into a seed tank with a seed tank culture medium, at a temperature of 37°C, pH 6.5, and control of dissolved oxygen at 25% Cultivate under the above conditions until the OD600 value of the bacterial solution is 12: obtain a mature Escherichia coli recombinant bacterium that can induce the production of L-4-hydroxyisoleucine;

[0050] Seed tank medium formula: glucose 20g / L, yeast powder 10g / L, peptone 5g / L, ammonium sulfate 2g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 1g / L, VB110mg / L, VH0.5mg / L L, ferrous sulfate 20mg / L, manganese sulfate 20mg / L, defoamer 0.4ml / L, and the rest is water.

[0051] (2) Put the mature strains into the fermenter, and cultivate them under the following conditions: ferment through liquid deep...

Embodiment 3

[0056] A method for producing L-4-hydroxyisoleucine by a microbial enzyme conversion method, comprising the following steps:

[0057] (1) Introduce recombinant Escherichia coli bacteria capable of inducing the production of L-4-hydroxyisoleucine into a seed tank equipped with a seed tank culture medium, at a temperature of 36°C, pH 6.8, and control of dissolved oxygen at 25% Cultivate under the above conditions until the OD600 value of the bacterial solution is 13: obtain a mature Escherichia coli recombinant bacterium that can induce the production of L-4-hydroxyisoleucine;

[0058] Seed tank medium formula: glucose 10g / L, yeast powder 8g / L, peptone 3g / L, ammonium sulfate 4g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 2g / L, VB115mg / L, VH5mg / L, Ferrous sulfate 30mg / L, manganese sulfate 30mg / L, defoamer 0.4ml / L, and the rest is water.

[0059] (2) Put the mature strains into the fermenter, and cultivate them under the following conditions: ferment through liquid de...

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Abstract

The invention relates to a method for producing L-4-hydroxyisoleucine by a microbial enzyme conversion method. The method comprises the following steps: 1, culturing a mature bacterial strain; 2, grafting mature escherichia coli recombinant bacteria which are capable of inducing production of L-4-hydroxyisoleucine into a fermentation tank, and carrying out fermentation culture, wherein the temperature of the fermentation process is controlled in two stages, i.e., 4 to 36 DEG C in the earlier stage and 30 to 32 DEG C in the later stage; 3, collecting thallus after fermentation, adding phosphate buffer and washing, then adding alpha-oxoglutarate, isoleucine, ferrous sulfate, magnesium sulfate and ascorbic acid, and carrying out forced ventilation for catalysis; 4, removing bacteria from the catalytic liquid with a ceramic membrane, filtering with an ultrafiltration membrane, decoloring with activated carbon, desalting with faintly acid cation exchange resin, adsorbing effluent with 732 cation exchange resin and eluting with ammonia water, carrying out evaporation concentration and recrystallization to obtain the L-4-hydroxyisoleucine. The preparation method disclosed by the invention is simple and efficient in reaction system and simple and feasible in extraction process. The conversion rate is up to over 95.4 percent, and the purity of the product can reach over 98 percent.

Description

technical field [0001] The invention relates to the technical field of microbial fermentation, in particular to a method for producing L-4-hydroxyisoleucine by a microbial enzyme conversion method. Background technique [0002] L-4-hydroxyisoleucine (4-hydroxyisoleucine) is a naturally occurring non-protein amino acid. Studies have found that L-4-hydroxyisoleucine has various effects such as promoting insulin secretion, improving insulin resistance and regulating blood lipids. L-4-hydroxyisoleucine can not only promote the uptake and utilization of glucose by peripheral tissues, but also reduce the glucose output of the liver to improve insulin resistance of peripheral tissues. Compared with drugs that have been used to treat T2DM, L-4-hydroxyisoleucine-regulated insulin synthesis is strictly dependent on glucose concentration. Therefore, L-4-hydroxyisoleucine does not cause side effects such as hypoglycemia. At the same time, it is also found that L-4-hydroxyisoleucine c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/06C12R1/19
CPCC12P13/06
Inventor 曹华杰刘帅李静岳贵龙张孟涛
Owner HENAN JULONG BIOLOGICAL ENG CO LTD
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