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CN-121991911-A - Nonspecific peroxygenase mutant and application thereof

CN121991911ACN 121991911 ACN121991911 ACN 121991911ACN-121991911-A

Abstract

The invention belongs to the technical field of enzyme engineering and biocatalysis, and particularly relates to a non-specific peroxygenase mutant and application thereof. Compared with the parent protein, the catalytic activity of the mutant protein is obviously improved, so that the production of 25-hydroxy vitamin D 3 or 25-hydroxy vitamin D 2 catalyzed by nonspecific peroxygenase is promoted, and a novel enzyme for production and a synthetic way are provided.

Inventors

  • MAO SHUHONG
  • WANG WENJING
  • CHEN XUN
  • QIN HUIMIN
  • LU FUPING

Assignees

  • 天津科技大学

Dates

Publication Date
20260508
Application Date
20251229

Claims (10)

  1. 1. A non-specific peroxygenase mutant, wherein the amino acid sequence of the mutant is one of: (1) The 113 rd amino acid of the amino acid sequence shown in SEQ ID NO. 1 is replaced by D; (2) The 113 th amino acid of the amino acid sequence shown in SEQ ID NO. 1 is replaced by D, and the 320 th amino acid is replaced by V by P; (3) The 113 rd amino acid of the amino acid sequence shown in SEQ ID NO. 1 is replaced by D, the 284 th amino acid is replaced by Y, and the 320 th amino acid is replaced by V; (4) The 70 th amino acid of the amino acid sequence shown in SEQ ID NO. 2 is replaced by D; (5) The 70 th amino acid of the amino acid sequence shown in SEQ ID NO.2 is replaced by D, and the 277 th amino acid is replaced by V by P; (6) The 70 th amino acid of the amino acid sequence shown in SEQ ID NO. 2 is replaced by D, the 241 st amino acid is replaced by Y, and the 277 th amino acid is replaced by V.
  2. 2. A gene encoding the nonspecific peroxygenase mutant of claim 1.
  3. 3. A recombinant vector comprising the coding gene of claim 2.
  4. 4. A host cell comprising the coding gene of claim 2 or the recombinant vector of claim 3.
  5. 5. Use of a non-specific peroxygenase mutant according to claim 1 wherein the mutant protein is used to catalyse the conversion of vitamin D 3 to 25-hydroxyvitamin D 3 and/or to catalyse the conversion of vitamin D 2 to 25-hydroxyvitamin D 2 .
  6. 6. A method for producing the non-specific peroxygenase mutant of claim 1, characterized in that it is subjected to gene recombination and expression by using a gene encoding the non-specific peroxygenase mutant or an expression vector comprising the encoding gene.
  7. 7. A method for preparing 25-hydroxy vitamin D, wherein the 25-hydroxy vitamin D comprises 25-hydroxy vitamin D 3 and/or 25-hydroxy vitamin D 2 , the method comprises the steps of adding hydrogen peroxide and the nonspecific peroxygenase mutant of claim 1 into an organic solvent-buffer solution reaction system, and preparing 25-hydroxy vitamin D 3 and/or 25-hydroxy vitamin D 2 by using vitamin D 3 and/or vitamin D 2 as substrates under the condition of suitable enzymatic reaction; The organic solvent is one or a mixture of more of acetone, methanol, ethanol and ethyl acetate, and the buffer solution is any one of phosphate buffer solution, citrate buffer solution and Tris-HCl buffer solution.
  8. 8. The method of claim 7, wherein the concentration of the substrate vitamin D 3 and/or vitamin D 2 in the reaction system is 0.1-50 mM, the concentration of the nonspecific peroxygenase mutant is 20-1760U/L, the concentration of the organic solvent is acetone, the volume ratio is 5-60%, and the hydrogen peroxide is continuously added in the reaction process at the adding rate of 0.1-2.5 mM h -1 ; The concentration of the buffer solution is 10-100 mM, and the pH is 4-8.5.
  9. 9. The method of claim 7, wherein the concentration of the substrate vitamin D 3 and/or vitamin D 2 in the reaction system is 0.4-20 mM, the concentration of the nonspecific peroxygenase mutant is 80-600U/L, the concentration of the organic solvent is acetone, the volume ratio is 20-50%, and the hydrogen peroxide is continuously added in the reaction process at the adding rate of 1.0-1.5 mM h -1 ; the concentration of the buffer solution is 50-100 mM, and the pH is 6-8.
  10. 10. The method according to claim 7 or 8 or 9, wherein the temperature of the reaction process of the method is 20-38 ℃ and the reaction time is 2-8 hours.

Description

Nonspecific peroxygenase mutant and application thereof Technical Field The invention belongs to the technical field of enzyme engineering and biocatalysis, and particularly relates to a non-specific peroxygenase mutant and application thereof. Background 25-Hydroxy vitamin D is a metabolite of vitamin D and is also the main active form of vitamin D in blood circulation, and the vitamin D directly enters blood without being metabolized by liver, and the biological potency is 3-5 times that of vitamin D and even higher. 25-hydroxy vitamin D plays an indispensable role in various physiological functions of the organism, and as a main active form of vitamin D, 25-hydroxy vitamin D is a main index for evaluating the nutritional status of human vitamin D, and the importance of the vitamin D is represented not only in the regulation of skeletal metabolism, but also in various fields such as the regulation of immune system, the exertion of antitumor effect, the protection of cardiovascular system and the like. In skeletal metabolism, 25-hydroxyvitamin D promotes calcium and phosphorus absorption and metabolism by further converting to an active form of 1, 25-dihydroxyvitamin D, maintaining skeletal health. In immunomodulation, it can enhance the anti-infective ability of the body by modulating the activity of immune cells, inhibiting excessive inflammatory reactions. In addition, research shows that the metabolite of 25-hydroxy vitamin D plays an important role in proliferation inhibition and apoptosis induction of tumor cells, and has potential anti-tumor value. Meanwhile, as a protecting factor for cardiovascular health, the 25-hydroxy vitamin D is helpful for maintaining vascular smooth muscle cell functions, regulating and controlling blood pressure and reducing the occurrence risk of cardiovascular diseases. Therefore, 25-hydroxyvitamin D is not only an important node of vitamin D metabolism, but also an important biological molecule for maintaining stable functions of multiple systems. At present, the main synthesis method of the 25-hydroxy vitamin D comprises chemical synthesis and biological synthesis, and the chemical method has various problems such as high organic solvent consumption, expensive partial reagents, environmental friendliness, more complicated steps and the like. The biological fermentation method is characterized in that a precursor vitamin D is added in the microbial fermentation process, and is converted into 25-hydroxy vitamin D by a specific enzyme in the microbial body, and the method has mild production conditions and is environment-friendly, but the problems of complex components of fermentation liquor, long reaction period and the like exist at present, and the method is generally limited by low activity, poor regioselectivity and insufficient stability of natural hydroxylase, so that the substrate conversion rate is low, the number of side hydroxylation products is large, the post-extraction is difficult, and the economic production requirements are difficult to meet. Therefore, the prior art still lacks a 25-hydroxy vitamin D synthesis path with simple route, mild condition, high efficiency and high selectivity, and needs to develop a new enzyme catalyst with excellent performance to break through the bottleneck. Nonspecific peroxygenases (Unspecific peroxygenase, UPOs) are a class of fungal source heme oxidases belonging to the peroxidase superfamily (EC 1.11.2.1). Compared with P450s, ‌ UPOs uses hydrogen peroxide as an electron acceptor and an oxygen donor, and can catalyze various oxidation reactions without cofactors, including hydroxylation of unactivated C-H bonds, olefin epoxidation, heteroatom oxidation, and the like. Disclosure of Invention In view of the shortcomings of the prior art, the invention aims to provide a non-specific peroxygenase (UPO) mutant and application thereof, wherein the catalytic activity of the mutant protein is obviously improved compared with that of a parent enzyme, so that the production of 25-hydroxy vitamin D (including D 3 or D 2) catalyzed by UPO is promoted. The technical scheme of the invention is as follows: The invention provides a mutant, the amino acid sequence of which is one of the following: (1) The 113 rd amino acid of the amino acid sequence shown in SEQ ID NO. 1 is replaced by D; (2) The 113 th amino acid of the amino acid sequence shown in SEQ ID NO. 1 is replaced by D, and the 320 th amino acid is replaced by V by P; (3) The 113 rd amino acid of the amino acid sequence shown in SEQ ID NO. 1 is replaced by D, the 284 th amino acid is replaced by Y, and the 320 th amino acid is replaced by V; (4) The 70 th amino acid of the amino acid sequence shown in SEQ ID NO. 2 is replaced by D; (5) The 70 th amino acid of the amino acid sequence shown in SEQ ID NO.2 is replaced by D, and the 277 th amino acid is replaced by V by P; (6) The 70 th amino acid of the amino acid sequence shown in SEQ ID NO. 2 is replaced by D, the 241 st amino