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CN-122012483-A - Tyrosine phenol lyase mutant and preparation method and application thereof

CN122012483ACN 122012483 ACN122012483 ACN 122012483ACN-122012483-A

Abstract

The invention discloses a tyrosine phenol lyase mutant and a preparation method and application thereof, and relates to the field of biology. The tyrosine phenol lyase mutant provided by the invention has the remarkable advantages of high catalytic activity and high phenol tolerance. In practical application, the high activity of the catalyst means that the enzyme dosage can be obviously reduced under the same catalytic condition, so that the production cost is effectively controlled, the production efficiency is improved, meanwhile, the excellent phenol tolerance can support the reaction system to adopt higher phenol concentration and faster feeding rate, the reaction time is shortened, the substrate conversion level and the final titer of the product can be further improved, and key support is provided for the large-scale and economic operation of related processes.

Inventors

  • CHENG LEIYU
  • PENG CHEN
  • HUANG GUODONG
  • WANG FUXIANG
  • Bai Junjian

Assignees

  • 黑龙江新和成生物科技有限公司
  • 浙江新和成股份有限公司

Dates

Publication Date
20260512
Application Date
20260213

Claims (10)

  1. 1. A mutant tyrosine phenol lyase having a mutation at any one or more of positions 49, 103, 186, 217, 385 of the amino acid sequence of a wild-type tyrosine phenol lyase; alternatively, the amino acid sequence of the wild-type tyrosine phenol lyase has at least 80% identity with the sequence shown in SEQ ID NO. 1.
  2. 2. The mutant according to claim 1, wherein the mutation at position 49 is T49S; And/or, the mutation at position 103 is E103K, E103Y or E103R; And/or, the mutation at 186 th position is L186C or L186T; and/or, the mutation at position 217 is R217D; And/or the 385 th mutation is S385T or S385A.
  3. 3. The mutant according to claim 2, wherein the mutant of the tyrosinase has any one of the following combinations of mutations T49S/E103Y/R217D, T S/E103R/S385T, T S/E103Y/R217D/S385A, E Y/L186C/S385T and T49S/E103R/L186C/R217D/S385T relative to the wild-type tyrosinase.
  4. 4. An isolated nucleic acid molecule encoding the tyrosine phenol lyase mutant of any one of claims 1-3.
  5. 5. A recombinant vector comprising the isolated nucleic acid molecule of claim 4.
  6. 6. A recombinant cell comprising the isolated nucleic acid molecule of claim 4 or the recombinant vector of claim 5; alternatively, the recombinant cell comprises a recombinant bacterium.
  7. 7. A catalyst comprising the tyrosine phenol lyase mutant of any one of claims 1 to 3; Optionally, the catalyst comprises recombinant cells or a culture thereof according to claim 6.
  8. 8. The method for preparing a mutant tyrosine phenol lyase according to any one of claims 1 to 3, comprising artificially synthesizing the mutant tyrosine phenol lyase or culturing the recombinant cell according to claim 6.
  9. 9. Use of a tyrosine phenol lyase mutant according to any of claims 1-3 or an isolated nucleic acid molecule according to claim 4 or a recombinant vector according to claim 5 or a recombinant cell according to claim 6 or a catalyst according to claim 7 for the preparation of L-tyrosine.
  10. 10. A method for preparing L-tyrosine, which is characterized by comprising the steps of adding the tyrosine phenol lyase mutant according to any one of claims 1-3 or the recombinant cell according to claim 6 or the catalyst according to claim 7 into a reaction system containing serine and phenol for catalytic reaction; optionally, the temperature of the catalytic reaction is 25-45 ℃ and the pH is 7-8; Optionally, in the reaction system, the concentration of serine is 60-80 g/L, the concentration of phenol is 6-10 g/L, and the concentration of the tyrosine phenol lyase mutant or the recombinant cell or the culture of the recombinant cell is 10-20 g/L; optionally, serine deaminase or a cell expressing serine deaminase, a surfactant and coenzyme PLP are also added to the reaction system; optionally, the surfactant is triton; Optionally, in the reaction system, the concentration of serine deaminase or bacteria expressing serine deaminase is 10-20 g/L, the concentration of triton is 0.1-2 g/L, and the concentration of coenzyme PLP is 0.01-1 g/L; Optionally, the preparation method further comprises the steps of after 30-90 min of the catalytic reaction, adding 40-60% (w/w) phenol solution in a flowing mode, and continuing the reaction for 9-15 h; optionally, the flow rate of the phenol solution is 12-16 g/h.

Description

Tyrosine phenol lyase mutant and preparation method and application thereof Technical Field The invention relates to the field of biology, in particular to a tyrosine phenol lyase mutant and a preparation method and application thereof. Background L-tyrosine is used as an essential amino acid for synthesizing proteins in animals, plays a key role in the growth and development of human beings and animals and in the metabolic process, and is widely applied to various industries such as food, feed, medicine, chemical industry, cosmetics and the like. Because of its efficacy in enhancing memory and mental acuity, it is often used as a dietary supplement and appetite suppressant, and at the same time, it is an important precursor of neurotransmitters such as dopamine, epinephrine, etc., which can participate in brain activity regulation. In addition, L-tyrosine has important applications in the pharmaceutical field as a synthetic precursor of high-value compounds, for example, p-hydroxy cinnamic acid in levodopa, nutraceuticals for treating parkinson's disease, and the like. At present, the production method of the L-tyrosine mainly comprises a hair hydrolysis method, a fermentation method and an enzyme catalysis method. The hair hydrolysis method extracts L-tyrosine by steps of hydrolyzing, decoloring, refining and the like on natural hair, but the method is gradually eliminated because the amino acid composition in the natural hair is complex, the separation and extraction difficulty of high-purity L-tyrosine is high, the yield is low, and the pollution to the environment is serious. The fermentation method utilizes microorganisms to synthesize L-tyrosine from the head by taking simple carbon sources such as glucose, glycerol and the like as substrates, but has the problems of long fermentation period, low acid production efficiency, high production cost, high extraction process pressure and the like, and is difficult to meet the industrial production requirement. The enzyme catalysis method is to utilize high-activity Tyrosine Phenol Lyase (TPL) to catalyze pyruvic acid, phenol and ammonium to synthesize L-tyrosine in the presence of coenzyme PLP. At present, the activity of TPL enzyme is difficult to meet the market demand, and low-activity TPL enzyme means that a large amount of thalli are needed to be put into the production process, and the high enzyme cost reduces the competitiveness of the product. In addition, TPL enzyme has poor phenol tolerance, for example, TPL enzyme derived from Rhodobacter capsulatus has an enzyme activity reduced by 90% or more at 7.5g/L (80 mM) of phenol, and TPL enzyme derived from Pantoea agglomerans has an enzyme activity of almost zero at 11.8g/L (125 mM) of initial concentration of phenol. Poor phenol tolerance makes TPL enzymes of many limitations in industrial applications. For example, the phenol concentration cannot be too high in the catalytic process, the phenol concentration needs to be detected in real time and the upper limit of the concentration needs to be controlled, so that the phenol with high concentration (local) has fatal influence on the enzyme, and the process difficulty is increased. In addition, the TPL enzyme is easy to be inactivated in the later period of the reaction, so that the dosage of the enzyme is often required to be increased in order to ensure the completion of the catalysis, or fresh enzyme liquid is added in the later period, thereby increasing the production cost. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a tyrosine phenol lyase mutant and a preparation method and application thereof. The invention is realized in the following way: In a first aspect, embodiments of the present invention provide a tyrosine phenol lyase mutant having a mutation at any one or more of positions 49, 103, 186, 217, 385 of the amino acid sequence of a wild-type tyrosine phenol lyase. In a second aspect, embodiments of the present invention provide an isolated nucleic acid molecule encoding a tyrosine phenol lyase mutant of the previous embodiments. In a third aspect, embodiments of the invention provide a recombinant vector comprising an isolated nucleic acid molecule as described in the previous embodiments. In a fourth aspect, embodiments of the invention provide a recombinant cell comprising an isolated nucleic acid molecule as described in the previous embodiments or a recombinant vector as described in the previous embodiments. In a fifth aspect, embodiments of the present invention provide a catalyst comprising the tyrosine phenol lyase mutant of the previous embodiments. In a sixth aspect, embodiments of the present invention provide a method for preparing a tyrosine phenol lyase mutant as described in the previous embodiments, comprising artificially synthesizing the tyrosine phenol lyase mutant or culturing the recombinant cell as described in the previous embodiments. In a seventh aspe