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CN-121718507-B - Flavin monooxygenase mutant and application thereof in defluorination of 4-fluorophenol

CN121718507BCN 121718507 BCN121718507 BCN 121718507BCN-121718507-B

Abstract

The invention discloses a flavin monooxygenase mutant and application thereof in defluorination of 4-fluorophenol, wherein the flavin monooxygenase mutant is obtained by mutating serine at position 375 of an amino acid sequence shown in SEQ ID NO.2 into isoleucine (S375I) and mutating phenylalanine at position 441 into asparagine (F441N). The mutant has higher defluorination activity and regioselectivity, and is suitable for catalyzing the defluorination of 4-fluorophenol to generate hydroquinone. The invention also relates to a gene for encoding the mutant, a recombinant vector, recombinant genetically engineered bacteria and application thereof in degradation of environmental pollutants. Experiments show that the defluorination efficiency of the mutant to 4-fluorophenol is obviously improved, and the hydroquinone yield can reach 66% within 24 hours.

Inventors

  • REN XINKUN
  • ZHOU YINGYING
  • WANG ZHE
  • WANG JIAN
  • MENG SHUANGHE
  • WANG SHUAI

Assignees

  • 南京大学

Dates

Publication Date
20260512
Application Date
20260225

Claims (10)

  1. 1. A flavin monooxygenase mutant is characterized in that the mutant is obtained by carrying out combined mutation on 375 th and 441 th amino acid sequences shown in SEQ ID NO.2, wherein serine at 375 th is mutated into isoleucine S375I and phenylalanine at 441 st is mutated into asparagine F441N.
  2. 2. A gene encoding the flavin monooxygenase mutant of claim 1.
  3. 3. A recombinant vector comprising the gene of claim 2.
  4. 4. A recombinant vector according to claim 3, wherein the vector comprises a plasmid or phage.
  5. 5. A recombinant genetically engineered bacterium comprising the recombinant vector of claim 3 or 4.
  6. 6. Use of a flavin monooxygenase mutant according to claim 1, a gene according to claim 2, a recombinant vector according to claim 3 or 4 or a recombinant genetically engineered bacterium according to claim 5 for catalyzing defluorination of 4-fluorophenol to hydroquinone.
  7. 7. The method according to claim 6, wherein the method comprises the steps of fermenting and culturing the recombinant genetically engineered bacterium of the flavin monooxygenase mutant encoding gene to obtain crushed wet thalli as a catalyst, forming a reaction system by using a mixed solution of flavin reductase, FAD, glucose and glucose dehydrogenase, 4-fluorophenol, NAD + , catalase and kpi solution with pH of 7.0-8.0, reacting at 500-700 rpm and 18-37 ℃, obtaining a reaction solution containing hydroquinone after the reaction, and separating and purifying the reaction solution to obtain hydroquinone.
  8. 8. The use according to claim 7, wherein the kpi solution system is phosphate buffer, the pH being adjusted to 7.0-8.0 with phosphoric acid.
  9. 9. The use according to claim 7, wherein the catalyst is used in an amount of 10-40 g/L buffer based on the weight of wet cells, and the initial addition concentration of 4-fluorophenol is 0.5-5 mM.
  10. 10. The use according to claim 6, wherein the flavin monooxygenase mutant is catalysed by a crude enzyme solution after cell disruption or by an isolated and purified enzyme, or by an immobilized enzyme prepared by an immobilization technique or by an enzyme in immobilized cell form.

Description

Flavin monooxygenase mutant and application thereof in defluorination of 4-fluorophenol Technical Field The invention relates to an environment restoration microbial technology, in particular to a flavin monooxygenase mutant and application thereof in defluorination of 4-fluorophenol. Background 4-Fluorophenol (4-Fluorophenol) is an emerging persistent organic pollutant difficult to degrade and has pathogenic, oncogenic and mutagenic effects. Currently, with the rapid development of molecular biology and bioinformatics analysis technologies, functional bacteria identification and screening, functional gene mining and biological enzyme expression technologies have achieved relatively mature results in various pollutant biological treatment processes, and a conventional method thereof generally comprises screening, separating and purifying strains with degradation effects on specific pollutants from environmental samples with pollutants, mining genes with pollutant degradation functions through whole genome sequencing, and expressing required enzymes in specific vectors. However, the conventional method has low screening efficiency, the degradation effect of the original strain and the original enzyme on pollutants is weak, and meanwhile, the lack of the commonality of the cognition of the regional selectivity of the biological enzyme on the key action in the pollutant degradation process makes the further conversion of the pollutants in the metabolic pathway limited, so that the complete degradation is difficult to realize. The method for directly coupling ultraviolet light catalytic oxidation-biodegradation treatment of parachlorophenol in wastewater disclosed in Chinese patent CN 116495826A forms an ultraviolet light catalytic and biodegradation cooperative system by constructing a polyurethane sponge carrier for supporting lignin carbon-based composite catalyst and a biological membrane, wherein the degradation rate of the strain to 20 mg/kg of 2, 4-dichlorophenol in the soil is 53.90% within 10 days, the degradation rate of the strain in 10 days in a liquid culture medium is 59.65%, the preliminary degradation of pollutants is realized, the degradation period is long, the efficiency is limited, the adaptability to the pollutant concentration and the environmental condition is not strong, and the removal rate of total organic carbon of the system reaches 86.42% within 12 hours. Although the method obviously improves the degradation rate and the mineralization degree, the system construction and the operation cost are high, and the catalytic efficiency and the stability are limited by the performance of the photocatalytic material and the natural composition of a microbial community. Therefore, the development of the high-activity and high-stability halogenated aromatic hydrocarbon defluorination enzyme mutant obtained through directed evolution is significant for environmental bioremediation. Disclosure of Invention The invention aims to provide a flavin monooxygenase mutant with high defluorination efficiency and high regioselectivity, solve the problems of low degradation efficiency and lack of regioselectivity of 4-fluorophenol in the prior art, and realize efficient defluorination of 4-fluorophenol and further conversion in a metabolic pathway. The flavin monooxygenase mutant is obtained by carrying out joint mutation on 375 th and 441 th amino acid sequences shown in SEQ ID NO.2, wherein serine at 375 th is mutated into isoleucine (S375I) and phenylalanine at 441 st is mutated into asparagine (F441N). The invention discloses a gene for encoding the flavin monooxygenase mutant. The recombinant vector of the present invention comprises the gene. The recombinant vector of the present invention is not limited as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and/or eukaryotic cells, and the vector may be various vectors conventional in the art, such as various plasmids, phage or viral vectors, etc., preferably a pET22b (+) plasmid is used as an expression vector, and escherichia coli is used as an expression host (escherichia coli C43 cells or escherichia coli BL 21). The recombinant genetically engineered bacterium disclosed by the invention comprises the recombinant vector. The flavin monooxygenase mutant, gene, recombinant vector or recombinant genetic engineering bacteria are applied to catalyzing the defluorination of 4-fluorophenol to generate hydroquinone. Preferably, the application method comprises the steps of taking crushed wet thalli obtained by fermenting and culturing recombinant genetic engineering bacteria of flavin monooxygenase mutant encoding genes as a catalyst, forming a reaction system by using a mixed solution of flavin reductase, FAD, glucose and glucose dehydrogenase, 4-fluorophenol, NAD +, catalase and kpi solution with pH of 7.0-8.0, reacting at 500-700 rpm and 18-37 ℃, obtaining a reaction solution containing hydroquinone