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CN-121991912-A - Method for synthesizing high-value chemicals by enzyme-catalyzed aromatic hydrocarbon dearomatization

CN121991912ACN 121991912 ACN121991912 ACN 121991912ACN-121991912-A

Abstract

The invention discloses a method for synthesizing high-value chemicals by enzyme catalysis aromatic hydrocarbon dearomatization. In the method, a high-efficiency catalytic synthesis way is established by taking 2-methylnaphthalene as a substrate, hydrogen peroxide as an oxidant, ascorbic acid as a free radical scavenger and a non-specific peroxygenase mutant as a catalyst in a water phase system. Through rational design modification of the nonspecific peroxygenase, including point mutation of the 99 th or 119 th amino acid residue or truncation of the C terminal, the nonspecific peroxygenase mutant is obtained, and the tolerance and the catalytic efficiency of the enzyme to high-concentration aromatic hydrocarbon substrates are improved. The invention can realize the efficient synthesis of menaquinone by a green and mild biological enzyme method, and provides a key enzyme tool for the industrial biological manufacture of vitamin K3.

Inventors

  • ZHANG JIE
  • WANG YI
  • ZHANG WUYUAN

Assignees

  • 中国科学院天津工业生物技术研究所

Dates

Publication Date
20260508
Application Date
20251230

Claims (10)

  1. 1. A non-specific peroxygenase mutant characterized by any one of the following: m0, namely, the 60 th position of the amino acid sequence of the nonspecific peroxygenase MthUPO is mutated from leucine to phenylalanine, the 159 th position is mutated from serine to glycine and the 161 th position is mutated from alanine to phenylalanine; M1, mutating 99 th position of amino acid sequence of nonspecific peroxygenase MthUPO (M0) from phenylalanine to histidine; m2, mutating the 119 th amino acid sequence of nonspecific peroxygenase MthUPO (M0) from glycine to serine; m3, removing 24 amino acids at the C terminal of a nonspecific peroxisome MthUPO (M0) amino acid sequence; wherein the amino acid sequence of the nonspecific peroxygenase MthUPO is shown as SEQ ID NO. 1.
  2. 2. The non-specific peroxygenase mutant of claim 1 encoding gene.
  3. 3. The coding gene of claim 2, which is any one of the following: g0, namely mutating the 180 th position of a nucleotide sequence of the nonspecific peroxygenase MthUPO from a base A to a base T, mutating the 475 th position from the base T to a base G, mutating the 476 th position from the base C to the base G, mutating the 477 th position from the base C to the base T, mutating the 481 th position from the base G to the base T, mutating the 482 th position from the base C to the base T; G1, mutating 294 th from a base T to a base C in a nucleotide sequence of nonspecific peroxygenase MthUPO, mutating 295 th from a base T to a base A, mutating 296 th from a base T to a base C to obtain a DNA molecule; G2, namely a DNA molecule obtained by mutating 355 th position from a base G to a base T, 356 th position from a base G to a base C and 357 th position from a base A to a base T of a non-specific peroxygenase MthUPO nucleotide sequence; G3, deleting the nucleotide sequence from 661 to 735 of the nucleotide sequence of the nonspecific peroxygenase MthUPO to obtain a DNA molecule; Wherein the nucleotide sequence of the nonspecific peroxygenase MthUPO is shown as SEQ ID NO. 2.
  4. 4. A recombinant expression vector into which the coding gene according to claim 2 or 3 is inserted.
  5. 5. A recombinant engineering bacterium into which the recombinant expression vector according to claim 4 has been inserted the coding gene according to claim 2 or 3.
  6. 6. The recombinant engineering bacterium according to claim 5, wherein the engineering strain is a pichia pastoris recombinant engineering strain.
  7. 7. A method for preparing menaquinone by catalyzing dearomatization of 2-methylnaphthalene is characterized in that the 2-methylnaphthalene is taken as a raw material, and oxidation is carried out at a C-1 position under a reaction system formed by a cosolvent, an oxidant, a free radical scavenger, a phosphate buffer solution and the mutant according to claim 1 to obtain a dearomatization product menaquinone, namely vitamin K3.
  8. 8. The method of claim 7, wherein the co-solvent is acetone, acetonitrile, methanol or ethanol, the oxidizing agent is hydrogen peroxide, and the free radical scavenger is sorbic acid, ascorbic acid or NADH.
  9. 9. The method according to claim 7 or 8, wherein the concentration of 2-methylnaphthalene in the reaction system is 0.15 mM-300 mM, the volume fraction of cosolvent ethanol is 5-50%, the pH of phosphate buffer solution is 6-9, the hydrogen peroxide concentration is 4-150 mM, the temperature is 25-35 ℃, and the reaction time is 1-7 h.
  10. 10. Use of the mutant according to claim 1, the coding gene according to claim 2 or 3, the recombinant expression vector according to claim 4 or the recombinant engineering bacterium according to claim 5 or 6 for the preparation or synthesis of menaquinone.

Description

Method for synthesizing high-value chemicals by enzyme-catalyzed aromatic hydrocarbon dearomatization Technical Field The invention belongs to the technical field of biological enzyme catalysis, and particularly relates to a method for synthesizing high-value chemicals by catalyzing aromatic hydrocarbon dearomatization through enzyme. In particular to a method for synthesizing a dearomatization product menaquinone compound by catalyzing and synthesizing a non-specific peroxygenase MthUPO from Myceliophthora thermophila and further improving the tolerance of an organic solvent by using a rational design technology. Background Menadione (vitamin K3), english name Menadione, CAS number 58-27-5, molecular weight 172.2, yellow crystal, melting point 105-107 deg.C, is a key intermediate for synthesizing K vitamins, and has essential procoagulant physiological function as the cofactor for prothrombin synthesis in organism. In the feed industry, vitamin K3 is widely used as a core vitamin additive to prevent hemorrhagic diseases of livestock, poultry and aquatic animals caused by deficiency of vitamin K and ensure healthy growth of the animals. Currently, the commercial production of vitamin K3 relies on traditional chemical synthetic routes. The core step is that 2-methylnaphthalene is used as raw material, strong oxidant such as dichromate is adopted to carry out chemical oxidation under the condition of strong acid and heating. The process, although mature, has the inherent disadvantages of using heavy metal oxidants, producing a large amount of chromium-containing high-risk wastes, being harsh in reaction conditions (high temperature and strong acid), high in energy consumption, having excessive oxidation byproducts and the like. In addition, the method also has the defects of low catalytic efficiency and complex reaction conditions due to the adoption of a greener oxidant such as hydrogen peroxide or oxygen for catalysis and the like. These drawbacks are in serious opposition to the current green chemistry and sustainable development concepts, and the development of alternative synthetic routes that are environmentally friendly and mild in conditions has become an urgent industry need. The biological enzyme catalysis technology provides a new path with great potential for green synthesis of vitamin K3 with the outstanding advantages of high selectivity, mild condition, environmental friendliness and the like. Nonspecific peroxygenase as a highly efficient biocatalyst, exhibits unique advantages in oxidation reactions. Unlike P450 monooxygenases which rely on complex coenzymes and long electron transfer chains, the nonspecific peroxygenases employed in the present invention are capable of catalyzing oxidation reactions directly using hydrogen peroxide as a clean oxygen source and electron acceptor. The electron transfer path is shortened, molecular oxygen (O 2) is not needed to participate, and thus, side reactions and complicated mass transfer limitation related to oxygen activation are avoided. The enzyme catalysis process is usually carried out in a water phase isothermal and solvent system with normal temperature and near neutral pH, the reaction condition is extremely green and mild, and the oxidative conversion of atomic economy can be realized theoretically. Based on the method, a brand new vitamin K3 biocatalysis synthesis process is developed by utilizing non-specific peroxygenase, so that the method is expected to radically overcome a plurality of defects of the existing chemical method, and has important theoretical value and application prospect. Disclosure of Invention The invention aims to solve the defects of the traditional method for chemically synthesizing menaquinone. The invention discloses a nonspecific peroxygenase capable of catalyzing 2-methylnaphthalene to generate menadione under green temperature and environment-friendly conditions, and improves the catalytic performance of the menadione catalytic synthesis by optimizing a reaction system and reasonably modifying the reaction system, so as to establish a biosynthesis method for efficiently and catalytically synthesizing menadione. The invention adopts the following technical scheme: the invention provides a nonspecific peroxygenase mutant, which is any one of the following: m0, namely, the 60 th position of the amino acid sequence of the nonspecific peroxygenase MthUPO is mutated from leucine to phenylalanine, the 159 th position is mutated from serine to glycine and the 161 th position is mutated from alanine to phenylalanine; M1, mutating 99 th position of amino acid sequence of nonspecific peroxygenase MthUPO (M0) from phenylalanine to histidine; m2, mutating the 119 th amino acid sequence of nonspecific peroxygenase MthUPO (M0) from glycine to serine; m3, removing 24 amino acids at the C terminal of a nonspecific peroxisome MthUPO (M0) amino acid sequence; wherein the amino acid sequence of the nonspecific peroxygenase MthUPO is s