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CN-122012638-A - Oyster FMO1 protein and preparation method and application thereof

CN122012638ACN 122012638 ACN122012638 ACN 122012638ACN-122012638-A

Abstract

The invention relates to the technical field of bioengineering, in particular to a crassostrea gigas FMO1 protein and a preparation method and application thereof. The invention provides a method for efficiently preparing crassostrea gigas FMO1 protein, which realizes the identification of the sequence and the function of a key metabolic gene FMO1 generated by oxidation of hypotaurine, which is a key step of synthesis of crassostrea gigas taurine for the first time. Therefore, the invention can accurately identify the FMO1 candidate gene sequence of the crassostrea gigas and simultaneously realize the efficient and large-scale preparation of the FMO1 in-vitro recombinant protein of the crassostrea gigas. The invention also provides a method for detecting the activity of the crassostrea gigas FMO1 in-vitro recombinant protein in catalyzing the oxidation reaction of hypotaurine, and the method can evaluate the activity of FMO1 protein and the generation of taurine by monitoring the consumption of NADPH in a reaction system.

Inventors

  • QI HAIGANG
  • Pang Meiqian
  • WANG CHAOGANG
  • LI LI
  • ZHANG GUOFAN

Assignees

  • 中国科学院海洋研究所

Dates

Publication Date
20260512
Application Date
20260212

Claims (10)

  1. 1. The application of the crassostrea gigas FMO1 protein in catalyzing sulfinic acid oxidation reaction is characterized in that the crassostrea gigas FMO1 protein is encoded by a nucleotide sequence shown in SEQ ID NO. 1.
  2. 2. The application of the crassostrea gigas FMO1 protein in preparing products for catalyzing oxidation reaction of hypotaurine and/or producing taurine is characterized in that the crassostrea gigas FMO1 protein is encoded by a nucleotide sequence shown in SEQ ID NO. 1.
  3. 3. A product for catalyzing oxidation reaction of hypotaurine and/or producing taurine is characterized by comprising a crassostrea gigas FMO1 protein, wherein the crassostrea gigas FMO1 protein is encoded by a nucleotide sequence shown in SEQ ID NO. 1.
  4. 4. A method for preparing oyster FMO1 protein, which is characterized by comprising the following steps: (1) Downloading a multi-sequence comparison cryptohorse model of FMO gene family from a pfam database, and scanning a prediction coding gene protein sequence set of a crassostrea gigas genome by utilizing hmmer software to obtain a crassostrea gigas FMO candidate sequence; (2) Downloading a protein sequence coded by a human FMO gene from uniprot database, comparing the oyster FMO candidate sequence with the human FMO sequence in a multi-sequence manner, and constructing a gene evolutionary tree by adopting a maximum likelihood method to obtain a gene evolutionary tree file; (3) Selecting a crassostrea gigas FMO candidate sequence which is aggregated with a human FMO1 gene into one according to the gene evolution tree file and has the highest average expression level in the early development stage of crassostrea gigas FMO candidate sequence as a crassostrea gigas FMO1 gene, wherein the nucleotide sequence of the crassostrea gigas FMO1 gene is shown as SEQ ID NO. 1; (4) Connecting the oyster FMO1 gene to a pCMV-N-Myc vector through a seamless cloning method, and transferring the gene into a host cell to obtain recombinant bacteria; (5) Culturing the recombinant bacteria, and sequentially performing first centrifugation, re-suspension, second centrifugation, third centrifugation and fourth centrifugation on the culture obtained by the culture to obtain the crassostrea gigas FMO1 protein.
  5. 5. The method according to claim 4, wherein the genomic version number of crassostrea gigas is GCA_025765685.3.
  6. 6. The method according to claim 4, wherein the rotation speed of the first centrifuge is 1200rpm for 3min; the step of resuspension comprises the step of mixing the precipitate obtained by the first centrifugation with a buffer solution to obtain a resuspension; The second centrifugation comprises the steps of mixing the heavy suspension with sucrose and then carrying out the second centrifugation, wherein the rotation speed of the second centrifugation is 1200g and the time is 10 minutes; the third centrifugation comprises the step of carrying out third centrifugation on the supernatant obtained by the second centrifugation, wherein the rotation speed of the third centrifugation is 100000g, the temperature is 4 ℃ and the time is 60 minutes; the fourth centrifugation comprises the step of mixing the microsomal sediment obtained by the third centrifugation with the cell lysate and then carrying out the fourth centrifugation, wherein the rotation speed of the fourth centrifugation is 14000rpm, and the time is 5 minutes.
  7. 7. The method of claim 6, wherein the buffer comprises the following components: ; The cell lysate comprises Western and IP cell lysate.
  8. 8. A method for detecting the oxidation activity of the crassostrea gigas FMO1 protein catalyzed by hypotaurine, which is characterized by comprising the step of detecting absorbance after mixing and reacting the crassostrea gigas FMO1 protein, tris-HCl, EDTA, hypotaurine and NADPH obtained by the preparation method according to any one of claims 4-7.
  9. 9. The method of claim 8, wherein the temperature of the mixing reaction is 37 ℃ for 60 minutes.
  10. 10. The method of claim 8, wherein the wavelength of absorbance detection is 340nm.

Description

Oyster FMO1 protein and preparation method and application thereof Technical Field The invention relates to the technical field of bioengineering, in particular to a crassostrea gigas FMO1 protein and a preparation method and application thereof. Background The crassostrea gigas is rich in taurine, is a high-quality food material for supplementing natural taurine for human beings, adopts methods of bioinformatics, molecular biology and the like to screen key genes of oyster taurine metabolism and genetic control breeding elements, and is used for breeding new crassostrea gigas lines with high taurine content, thereby having great significance for oyster cultivation industry. According to the prior literature, the biosynthesis of oyster and other bivalve shellfish taurine mainly depends on a cysteine pathway, cysteine firstly generates cysteine under the action of cysteine dioxygenase, then the cysteine generates hypotaurine under the action of sulfinic acid alanine decarboxylase, and the hypotaurine is further oxidized by Flavin Monooxygenase (FMO) to generate taurine. FMO is a monooxygenase which takes flavin adenine dinucleotide as a prosthetic group and depends on reduced nicotinamide adenine dinucleotide phosphate, has the core function of catalyzing oxidation of substrates containing hetero atoms (N, S, P, se and the like), plays a key role in drug metabolism, poison detoxification and endogenous substance synthesis, and is a key enzyme for catalyzing oxidation of hypotaurine to generate taurine in mammals. The oxidation reaction of hypotaurine was proposed in the 60 s of the last century, but the enzyme that catalyzes this reaction was not formally identified until 2020. At present, protein preparation and enzyme activity function identification of oyster FMO1 genes are not reported. Disclosure of Invention The invention aims to provide a crassostrea gigas FMO1 protein, a preparation method and application thereof, so as to solve the problems in the prior art. In order to achieve the above object, the present invention provides the following solutions: The invention provides application of a crassostrea gigas FMO1 protein in catalyzing oxidation reaction of hypotaurine, wherein the crassostrea gigas FMO1 protein is obtained by encoding a nucleotide sequence shown in SEQ ID NO. 1. The invention provides application of a crassostrea gigas FMO1 protein in preparing a product for catalyzing oxidation reaction of hypotaurine and/or producing taurine, wherein the crassostrea gigas FMO1 protein is encoded by a nucleotide sequence shown as SEQ ID NO. 1. The invention provides a product for catalyzing oxidation reaction of hypotaurine and/or producing taurine, which comprises crassostrea gigas FMO1 protein, wherein the crassostrea gigas FMO1 protein is encoded by a nucleotide sequence shown as SEQ ID NO. 1. The invention provides a preparation method of a crassostrea gigas FMO1 protein, which comprises the following steps: (1) Downloading a multi-sequence comparison cryptohorse model of FMO gene family from a pfam database, and scanning a prediction coding gene protein sequence set of a crassostrea gigas genome by utilizing hmmer software to obtain a crassostrea gigas FMO candidate sequence; (2) Downloading a protein sequence coded by a human FMO gene from uniprot database, comparing the oyster FMO candidate sequence with the human FMO sequence in a multi-sequence manner, and constructing a gene evolutionary tree by adopting a maximum likelihood method to obtain a gene evolutionary tree file; (3) Selecting a crassostrea gigas FMO candidate sequence which is aggregated with a human FMO1 gene into one according to the gene evolution tree file and has the highest average expression level in the early development stage of crassostrea gigas FMO candidate sequence as a crassostrea gigas FMO1 gene, wherein the nucleotide sequence of the crassostrea gigas FMO1 gene is shown as SEQ ID NO. 1; (4) Connecting the oyster FMO1 gene to a pCMV-N-Myc vector through a seamless cloning method, and transferring the gene into a host cell to obtain recombinant bacteria; (5) Culturing the recombinant bacteria, and sequentially performing first centrifugation, re-suspension, second centrifugation, third centrifugation and fourth centrifugation on the culture obtained by the culture to obtain the crassostrea gigas FMO1 protein. Optionally, the gene version number of the crassostrea gigas genome is gca_025765685.3. Optionally, the rotation speed of the first centrifugation is 1200rpm for 3min; the step of resuspension comprises the step of mixing the precipitate obtained by the first centrifugation with a buffer solution to obtain a resuspension; The second centrifugation comprises the steps of mixing the heavy suspension with sucrose and then carrying out the second centrifugation, wherein the rotation speed of the second centrifugation is 1200g and the time is 10 minutes; the third centrifugation comprises the step of carrying out th