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Strain for producing tyrosine ammonia lyase and application of strain

A technology of tyrosine and ammonia lyase, which is applied in the field of catalyzed preparation of p-hydroxycinnamic acid and its derivatives by tyrosine ammonia lyase, can solve the problem of poor enzyme stability of tyrosine ammonia lyase, poor tolerance of catalyzed products, and product Low concentration and other problems, to achieve the effect of facilitating extraction and separation operations, broadening the channel of strains, and simple medium components

Active Publication Date: 2020-07-17
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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  • Strain for producing tyrosine ammonia lyase and application of strain

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 at constant temperature Incubate 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 5g / 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 medium are so...

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 for producing tyrosine ammonia lyase and an application of the strain, and belongs to the field of biotechnology. The strain is classified and named as Acinetobacterjohnsonii, is preserved in the China General Microbiological Culture Collection Center on May 15, 2020, and has a preservation number of CGMCC No.19831. The strain provided by the invention has good hereditary stability and low culture cost, the generated tyrosine ammonia lyase has high enzyme activity, and the strain has a wide applicable substrate spectrum and can be directly used for direct conversion and synthesis of p-hydroxycinnamic acid and p-hydroxycinnamic acid derivatives. During catalytic synthesis, addition of a surfactant, an organic solvent and other membrane penetrating agents is not needed, introduction of exogenous impurities is avoided, extraction and separation operation is facilitated, and advantages are achieved for industrial application.

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 Applications(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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