Method for synthesizing L-tryptophan by immobilized enzyme

A technology of immobilized enzyme and immobilized enzyme carrier, which is applied in the direction of fermentation, can solve the problems of difficult separation and high cost, and achieve the effects of high catalytic efficiency, easy separation and low fermentation cost

Inactive Publication Date: 2011-08-03
NANKAI UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for synthesizing L-tryptophan with immobilized enzymes for t

Method used

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  • Method for synthesizing L-tryptophan by immobilized enzyme
  • Method for synthesizing L-tryptophan by immobilized enzyme
  • Method for synthesizing L-tryptophan by immobilized enzyme

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Construction of genetically engineered bacteria, induced expression of tryptophanase and preparation of crude enzyme solution

[0032] According to the tryptophanase gene sequence of Escherichia coli K12 strain, design upstream primer P1 of tryptophanase gene: 5'-CCG AAG CTT ATG GAA AAC TTT AAA CAT CTC C-3' and downstream primer P2: 5'-CCC GGA TCC TTA AAC TTC TTT CAG TTT TGC GG-3' Using the genomic DNA of Escherichia coli JM109 strain as a template, the tryptophanase gene was amplified by PCR, and the recombinant expression plasmid pET21a(+)-tnaA was constructed respectively.

[0033] The recombinant plasmid was transformed into Escherichia coli BL21(DE3), and the tryptophanase genetic engineering strain BL21(DE3)-pET21a(+)-tnaA was constructed.

[0034] Take a single colony of tryptophan engineered bacteria and inoculate it in LB medium, cultivate overnight at 37°C to obtain a saturated culture, inoculate the saturated culture at 1% in LB medium containing Amp (100 μ...

Embodiment 2

[0038] Determination of tryptophanase activity

[0039] In a 10 mL Erlenmeyer flask, add in order: 20 μL 0.2 mg / mL pyridoxal phosphate (PLP) solution, 10 μL 0.005 M reduced glutathione (GSH) solution, 270 μL obtained in Example 1 Tryptophan enzyme solution; 0.3 mL of the above solution was covered with 1 mL of toluene, incubated at 37 °C for 5 min, then added 100 μL of 5 mg / mL L-tryptophan solution, placed on a shaker at 37 °C for 10 min with gentle shaking , and then add 3 mL of indole chromogenic solution to stop the reaction, mix well, and measure the absorbance at 570 nm after standing for 30 min.

[0040] Definition of enzyme activity: under the above reaction conditions, the amount of enzyme required to catalyze the formation of 0.001 μmol indole at 37 °C for 10 minutes is an enzyme activity unit.

[0041] The expressed tryptophanase activity measured by the above method reached 3912.6 U / g, about 110 times of the tryptophanase activity of the original host bacteria itse...

Embodiment 3

[0043] Detection of L-Tryptophan in Reaction Solution by High Performance Liquid Chromatography

[0044] Immobilizing tryptophanase to obtain immobilized enzyme, using the immobilized enzyme as the enzyme source, using L-cysteine ​​and indole as the substrate, at 35 to 55 °C, the pH value is 6 to 11, After enzymatic conversion reaction for 1 to 5 hours, L-tryptophan is produced. Remove the immobilized enzyme from the catalyzed reaction liquid, measure the concentration of tryptophan generated by high performance liquid chromatography in the filtered supernatant, compare it with the tryptophan standard curve, and calculate the L-cysteine ​​in the substrate solution The molar conversion rate.

[0045] Chromatographic column: Nucleosil SA (250×4.6 mmol / L); injection volume: 20 mL.

[0046] Mobile phase: glacial acetic acid-triethylamine-water (0.1: 0.1: 99.8); flow rate: 1.0 mL / min; column temperature: 25 °C. UV detection wavelength: 280 nm.

[0047] ELSD detector parameter...

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Abstract

The invention discloses a method for synthesizing L-tryptophan by immobilized enzyme. In the method, immobilized tryptophanase is taken as an enzyme source, L-cysteine and indole are taken as substrates, the L-tryptophan is produced through enzymatic conversion, the immobilized enzyme is removed through filtration, and the L-cysteine is obtained through further separation. In the method, the tryptophanase is immobilized through GE resin, and the optimized catalysis reaction conditions are that: the pH is 8.5, the reaction time is 3 hours, the concentration of the substrates is 10g/L of L-cysteine and 10g/L of indole, the cell concentration of the enzyme source is 20mL/L, and the concentration of phosphopyridoxal coenzyme is 0.15g/L. Under the optimum reaction conditions, the conversion rate of the cysteine substrate is 81.5 percent. Because the immobilized enzyme is adopted for direct enzymatic synthesis, the method is easy and convenient to implement, high in catalysis efficiency and low in fermentation cost, and the products are easy to separate; therefore, the method has good industrial prospect in the field of industrial production of L-tryptophan.

Description

technical field [0001] The invention belongs to the technical field of amino acid production by biotechnology, and relates to the synthesis of L-tryptophan by an immobilized enzyme method. Background technique [0002] L-Tryptophan is one of the eight essential amino acids in human and animal life activities. It plays a very important role in the growth, development and metabolism of humans and animals. It is widely used in many aspects such as medicine, food and feed. The production of L-tryptophan mainly relied on chemical synthesis and protein hydrolysis at the beginning, but with the continuous deepening of research on the production of tryptophan by microbial methods, this method has become practical and is in a dominant position. Microbial methods can be roughly divided into direct fermentation method, microbial transformation method and enzymatic synthesis method. Wherein the enzymatic synthesis method utilizes the catalytic function of the tryptophan biosynthetic en...

Claims

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Application Information

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IPC IPC(8): C12P13/22
Inventor 张奇白钢侯洁白芳段静静
Owner NANKAI UNIV
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