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L-isoleucine hydroxylase gene as well as genetically engineered bacterium and application of L-isoleucine hydroxylase gene

A technology encoding isoleucine hydroxylase and isoleucine hydroxylase is applied in genetic engineering, application, oxidoreductase and other directions, can solve problems such as limited gene sequence, achieve low cost, reduce production cost, Simple and feasible effect

Active Publication Date: 2014-06-18
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently published gene sequence of L-isoleucine hydroxylase that can specifically catalyze the production of (2S, 3R, 4S)-4-hydroxyisoleucine from L-Ile is limited, and no microbial transformation has been used. Report on the Production of (2S, 3R, 4S)-4-Hydroxyisoleucine

Method used

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  • L-isoleucine hydroxylase gene as well as genetically engineered bacterium and application of L-isoleucine hydroxylase gene
  • L-isoleucine hydroxylase gene as well as genetically engineered bacterium and application of L-isoleucine hydroxylase gene
  • L-isoleucine hydroxylase gene as well as genetically engineered bacterium and application of L-isoleucine hydroxylase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Acquisition of the gene ido encoding L-isoleucine hydroxylase

[0051] The soil metagenome was extracted using the following methods:

[0052] Take 10g soil samples at 10cm below the topsoil in the garden of Tianjin University of Science and Technology and place them in a shaker flask, add 20mL DNA extraction solution (100mol / L Tris-HCl, 100mmol / L EDTA2Na; 100mmol / L Na3PO4, 1.5mol / L NaCl, 2% CTAB, pH8.0) and mix well. Add 500 μL of lysozyme (50 mg / mL, pH 8.0), mix well and then bathe in water at 37°C for 30 min. After being placed in an ultrasonic cleaner for 10 minutes, 1.5 g of sterile glass beads (diameter 1 mm) were added and shaken for 5 minutes. Add 40 μL of proteinase K (20 mg / mL), mix well, then add 2 mL of 20% SDS, bathe in water at 65 °C for 1 h, and shake gently every 15-20 min. Centrifuge at 10000 g for 10 min at room temperature, collect the supernatant and transfer to another centrifuge tube. Extract once each with equal volumes of phenol an...

Embodiment 2

[0059] Example 2: Construction of the recombinant plasmid pET-ido and functional analysis of the gene ido encoding L-isoleucine hydroxylase

[0060] The recombinant plasmid containing ido-3 was extracted and digested with restriction endonucleases NdeI and XhoI, followed by agarose electrophoresis, recovered after gel cutting, and connected to the expression vector pET-42a that had been cut with the same enzymes to obtain the recombinant plasmid pET-ido. It was transformed into E.coli BL21 (DE3) competent cells to obtain the recombinant strain BL-IDO.

[0061] The BL-IDO seed culture was inoculated into LB liquid medium, and 0.1mmol / L IPTG was used to induce expression for 4h. Take 1 mL of the culture and centrifuge at 10,000 g for 1 min at 4°C to collect the bacteria, and wash with 1 mL of buffer A (Na 2 HPO 4 12H 2 O2.9g / L, NaCl8.5g / L, NaH 2 PO 4 2H 2 (00.30g / L, pH7.4) Wash the bacterial pellet 3 times and resuspend with 1mL buffer A. Ultrasonic disrupt the above bact...

Embodiment 3

[0065] Embodiment 3: Construction of recombinant plasmid pXMJ-ido and recombinant strain CG-ido (see the construction process figure 1 )

[0066] Using the recombinant plasmid pET-ido as a template, use primers IDO-3 and IDO-4 to amplify its ido gene (see the electrophoretic pattern of the ido gene PCR product figure 2 );

[0067] IDO-3: 5'-CCG GTC GAC AAGGAAGCTAGATATGAAAATGAGTGGCTTTAGCATAG-3'(Sal Ⅰ)

[0068] IDO-4: 5'-CCG GAATTC TTATTTTGTCTCCTTATAAGAAAATGTTACTA-3'(EcoR Ⅰ)

[0069] The PCR conditions were: 94°C for 5 min for 1 cycle, 94°C for 30 s, 56°C for 30 s, 72°C for 1 min and 30 cycles, 72°C for 10 min and 1 cycle, and the reaction system was 100 μL. 10 μL of PCR products were detected by 1.5% agarose gel electrophoresis.

[0070]After recovery, the remaining 90 μL of the PCR amplification product was digested by SalⅠ and EcoRI respectively, and after being recovered by electrophoresis and gel cutting, it was connected to the expression vector pXMJ19 to obtain t...

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Abstract

The invention belongs to the technical field of biology and particularly relates to an L-isoleucine hydroxylase gene capable of specifically catalyzing L-isoleucine to generate (2S, 3R, 4S)-4-hydroxyisoleucine and a genetically engineered bacterium containing the gene. When the bacterium is used for producing (2S, 3R, 4S)-4-hydroxyisoleucine, defects such as low extraction ratio, difficulty in separation and purification, high raw material quality demand, high cost and the like existing in an extraction method can be overcome; the problems of harsh reaction conditions, more steps, difficulty in separation and low yield existing in a chemical synthesis method can be solved; the problems of low conversion rate and large-quantity alpha-aminobutyric acid and other byproduct production existing in an enzyme method can be solved, 4-hydroxyisoleucine with only one configuration, i.e., (2S, 3R, 4S)-4-hydroxyisoleucine, exists in a fermentation solution, and the conversion efficiency is further increased by using an L-isoleucine hydroxylase encoding gene over-expression method, so that the aims of simplifying processes and reducing the production cost are achieved.

Description

Technical field: [0001] The invention relates to an L-isoleucine hydroxylase gene capable of specifically catalyzing L-isoleucine (L-Ile) to generate (2S, 3R, 4S)-4-hydroxyisoleucine and containing The genetically engineered bacterium of the gene uses the strain to produce (2S, 3R, 4S)-4-hydroxyisoleucine with a single product and high conversion rate, and belongs to the field of biotechnology. Background technique: [0002] 4-Hydroxyisoleucine (4-Hydroxyisoleucine, 4-HIL) is a hydroxylated L-isoleucine mainly found in the seeds of fenugreek plants. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N1/21C12N15/77C12P13/04C12R1/15
Inventor 陈宁张成林谢希贤徐庆阳刘淑云刘远王鑫鑫
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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