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A tyrosine ammonia lyase-producing strain and its application

A tyrosine and ammonia lyase technology is applied in the field of tyrosine ammonia lyase catalyzed preparation of p-hydroxycinnamic acid and derivatives, and can solve the problems of poor stability of tyrosine ammonia lyase, poor tolerance of catalytic products, and poor product tolerance. problems such as low concentration, to achieve the effect of being conducive to extraction and separation operations, broadening the channels of bacteria, and simple medium components

Active Publication Date: 2020-08-21
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem that has always plagued people is the poor stability of tyrosine ammonia lyase, poor tolerance to catalytic products, and easy inactivation, resulting in low concentration of catalyzed products, and there is still a long way to go for industrial applications.

Method used

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  • A tyrosine ammonia lyase-producing strain and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Classification of Acinetobacter johnsonii strains

[0027] 1) Strain characteristics

[0028] The colony on the preservation medium was milky white, nearly round, with a smooth and moist surface, shiny, and neat edges; observed under a microscope, the bacteria were rod-shaped, unable to move, and Gram-negative; the physiological and biochemical indicators are shown in Table 1.

[0029] Table 1 Physiological and biochemical characteristics of strain Ytld-76

[0030] project Ytld-76 project Ytld-76 Oxidase - VP test - starch - Catalase test + Nitrate reduction - Citric acid utilization + Urease - gelatin hydrolysis -

[0031] 2) Strain identification

[0032] The length of the 16S rRNA gene sequence obtained by DNA extraction, PCR amplification and sequencing was 1446 bp, and the BLAST comparison was performed on GenBank and the phylogenetic tree was drawn ( figure 1 ), it was concluded that the base si...

Embodiment 2

[0040] Example 2 Bacterial strain cultivation and p-hydroxycinnamic acid synthesis

[0041] 1) Strain activation: pick strains to solid preservation medium: yeast extract powder 5 g / L, peptone 10 g / L, NaCl 5 g / L, agar 20 g / L, pH 7.0, streak inoculation in a constant temperature incubator Cultivate at 30°C for 48 hours;

[0042] 2) Preparation of liquid seeds: pick one ring of activated strains and inoculate them in a 500 mL Erlenmeyer flask containing 50 mL of liquid seed medium, seal the seal with eight layers of gauze, and culture at 30°C for 24 hours on a shaker at 200 r / min to obtain liquid seeds; Described liquid seed medium component and final concentration thereof: sodium acetate 5 g / L, (NH 4 ) 2 SO 4 1g / L, KH 2 PO 4 1g / L, MgSO 4 0.1g / L, peptone 5 g / L, yeast extract powder 1 g / L, pH 7.0;

[0043] 3) Liquid fermentation: add liquid seeds into the fermentation medium according to the inoculum size of 5%, the composition and final concentration of the fermentation...

Embodiment 3

[0045] Embodiment 3 catalyzes L-tyrosine to produce p-hydroxycinnamic acid

[0046] 1) Strain activation: pick the strains into the solid preservation medium, inoculate them in a constant temperature incubator at 30°C for 48 hours; the composition and final concentration of the solid preservation medium: yeast extract powder 5 g / L, peptone 10 g / L, NaCl 5 g / L, agar 20 g / L, pH 7.0;

[0047] 2) Preparation of liquid seeds: pick one ring of activated strains and inoculate them in a 500 mL Erlenmeyer flask containing 50 mL of liquid seed medium, seal the seal with eight layers of gauze, and culture at 30°C for 24 hours on a shaker at 200 r / min to obtain liquid seeds; Described liquid seed medium component: sodium acetate 8 g / L, (NH 4 ) 2 SO 4 3g / L, KH 2 PO 4 2g / L, MgSO 4 0.5g / L, peptone 5 g / L, yeast extract 2 g / L, pH 7.0,

[0048] 3) Liquid fermentation: according to the inoculum amount of 5%, the composition is sodium acetate 10 g / L, sodium citrate 3 g / L, (NH 4 ) 2 SO ...

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Abstract

The invention relates to a strain producing tyrosine ammonia-lyase and its application, which belongs to the field of biotechnology. The strain is classified and named as Acinetobacter johnsonii, and has been preserved in China's microbial strains on May 15, 2020 General Microbiology Center of the Preservation Management Committee, deposit number: CGMCC No.19831. The bacterial strain provided by the present invention has good genetic stability, low cultivation cost, high enzymatic activity of tyrosine ammonia-lyase, and wide applicable substrate spectrum. There is no need to add membrane-permeable agents such as surfactants and organic solvents during synthesis, avoiding the introduction of foreign impurities, which is conducive to extraction and separation operations, and has advantages in industrial applications.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a bacterial strain producing tyrosine ammonia lyase and a method for catalyzing the preparation of p-hydroxycinnamic acid and its derivatives by using the tyrosine ammonia lyase produced by the bacterial strain. Background technique [0002] 4-Hydroxycinnamic acid (p-coumaric acid), also known as p-coumaric acid, is widely found in plants in nature, and is one of the active ingredients of Hedyotis diffusa, Seaweed, and Eucommia leaves. It is used as a spice or acidifier in Widely used in the food industry, it can replace antibiotics in agriculture to treat viral or bacterial diseases in livestock and poultry; it is also an intermediate for the synthesis of important pharmaceutical active substances such as Kexinding, rhododendron, and esmolol, and has a very promising application prospect in the pharmaceutical field broad. In addition, the structural analogues of 4-hydrox...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P7/42C12R1/01
CPCC12N1/20C12P7/42C12N1/205C12R2001/01
Inventor 冯志彬张娟程仕伟葛宜和
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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