Method for using 4-hydroxyisoleucine to produce plasmids and strains and synthesizing method of 4-hydroxyisoleucine

Abstract

The invention relates to a synthesizing method of 4-hydroxyisoleucine. According to the method, isoleucine hydroxylase genes are screened out, recombinant expression plasmid is constructed, and fusion protein having an amino acid deaminase transmembrane domain and isoleucine hydroxylase is expressed, so that isoleucine hydroxylase is anchored to the surface of cells; then a complete cell catalysis technology is utilized to replace a cell thawing and breaking technology, a cell freezing technology is omitted, and freezing processing equipment is not needed; meanwhile, product separation difficulty caused when inclusion of the cells is spread to reaction liquid after the cells are broken is avoided, the production procedure is simplified, the energy consumption, the material consumption and the production cost are lowered by a large margin, the conversion ratio of 4-hydroxyisoleucine produced through the method reaches up to 99.0% or above, and the synthesizing method is more beneficial to industrialized production.

Description

technical field [0001] The invention relates to the field of compound biotechnology production, in particular to a 4-hydroxyisoleucine production plasmid and bacterial strain and a synthesis method of 4-hydroxyisoleucine. Background technique [0002] 4-hydroxyisoleucine (4-Hydroxyisoleucine, 4-HIL) is a non-protein amino acid mainly present in the seeds of fenugreek plants, accounting for about 80% of the total content of free amino acids in the seeds. The molecular formula of 4-HIL is C6H13NO3, the molecular weight is 147.17, the melting point is greater than 200°C, the boiling point is 332°C, and the density is 1.181g / cm3. Studies have shown that 4-hydroxyisoleucine has the functions of promoting insulin secretion, promoting muscle cell absorption of blood sugar, accelerating fat metabolism, reducing blood fat, anti-inflammation, anti-oxidation, anti-tumor, etc. It can effectively prevent and treat diabetes, and can be used as a therapeutic Ideal drug for obesity. There...

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Problems solved by technology

[0003] The production methods of 4-hydroxyisoleucine include extraction method, chemical synthesis method, enzymatic method and biological method. At present, the technology of industrial production of 4-hydroxyisoleucine is relatively mature, but it is mainly extracted from heavy fenugreek. Its product separation and purification is difficult, difficult to prepare, low product purity, serious environmental pollution, high cost of mass production, and more configurations of 4-hydroxyisoleucine obtained by this method, but only (2S, 3R, 4S)-4 -Hydroxyisoleucine has the above-mentioned biological activity, so that the method has an extremely low extraction rate (only 0.091-0.6%)

The total yield of this method is 39%, and the reaction conditions are harsh, there are many steps, and it is easy to cause environmental pollution, so it still stays in the laboratory stage

Enzymatic synthesis of 4-hydroxyisoleucine is a new method for the synthesis of 4-hydroxyisoleucine, which mainly utilizes isoleucine hydroxylase (IDO) with isoleucine and α-ketoglutarate and Fe 2+ reaction to produce 4-HIL, this method has high yield and fewer by-products, but this method needs to release intracellular isoleucine hydroxylase (IDO), so it needs to be processed by freeze-thawing and crushing, so this method The method requires a lot of energy consumption, and during the crushing process, the macromolecular substances in the cells will be released into the catalytic system with the cell rupture, which makes the separation and purification of downstream 4-HIL very difficult.

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