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CN-122012432-A - Phloretin enzyme activity related protein, and coding gene and application thereof

CN122012432ACN 122012432 ACN122012432 ACN 122012432ACN-122012432-A

Abstract

The invention discloses a phloretin enzyme activity related protein, and a coding gene and application thereof. The technical problem to be solved is how to provide a protein related to the activity of the phlorizin enzyme. Specifically disclosed is the use of any one of A1) the use of phloretin for catalyzing phlorizin to produce phlorizin and/or trilobatin and/or the use of A2) the use of phlorizin for catalyzing phlorizin to produce phlorizin and/or trilobatin products, A3) the use of phlorizin and/or the use of trilobatin products, B2) the fusion protein obtained by connecting the N terminal or/and the C terminal of B1) or B2) to a protein tag, wherein the amino acid sequence of the protein is the protein described by the sequence 2, and the fusion protein is obtained by substituting and/or deleting and/or adding the amino acid residues of the protein described by the B1) and has the same function as the protein described by the B1), wherein the protein has the identity of more than 80%. Can be used for industrial production.

Inventors

  • ZHOU JUNHUI
  • LIU JUAN
  • LI XIANG
  • LAN XIAOZHONG
  • JIANG CHAO
  • GAO JIAQI

Assignees

  • 中国中医科学院中药研究所

Dates

Publication Date
20260512
Application Date
20241111

Claims (10)

  1. 1. Use of a protein in any one of the following: A1 Catalyzing phloretin to generate phlorizin and/or trilobatin and/or preparing the phloretin to generate phlorizin and/or trilobatin products; a2 The use of the same for the production of phlorizin and/or for the preparation of a product for the production of phlorizin; A3 The use of the same for the production of trilobatin and/or for the preparation of a product for the production of trilobatin; The protein is any one of the following: b1 Amino acid sequence is a protein described in sequence 2; b2 A protein which has 80% or more identity and the same function as the protein represented by B1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein represented by B1); B3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag.
  2. 2. The use according to claim 1, wherein the protein is derived from mirabilis jalapa.
  3. 3. Use of a biological material related to the protein of claim 1 or 2 for any of the following: c1 Catalyzing phloretin to generate phlorizin and/or trilobatin and/or preparing the phloretin to generate phlorizin and/or trilobatin products; c2 The use of the same for the production of phlorizin and/or for the preparation of a product for the production of phlorizin; c3 The use of the same for the production of trilobatin and/or for the preparation of a product for the production of trilobatin; c4 Production of the protein of claim 1 or production of a product producing the protein of claim 1; The biomaterial is any one of the following D1) to D4): d1 A nucleic acid molecule encoding the protein of claim 1; d2 An expression cassette comprising D1) said nucleic acid molecule; d3 A recombinant vector comprising D1) said nucleic acid molecule, or a recombinant vector comprising D2) said expression cassette; d4 A recombinant microorganism comprising D1) said nucleic acid molecule, or a recombinant microorganism comprising D2) said expression cassette, or a recombinant microorganism comprising D3) said recombinant vector.
  4. 4. The use according to claim 3, wherein the nucleic acid molecule according to claim 3 is a DNA molecule according to sequence 1.
  5. 5. The preparation method is characterized by comprising a preparation method of phlorizin, a preparation method of trilobatin or a preparation method of phlorizin and trilobatin; The method for preparing phlorizin comprises the steps of reacting the protein or the phlorizin-producing product of claim 1 or 2 with phlorizin to obtain phlorizin; the method for preparing the trilobatin comprises the steps of reacting the protein or the product for producing the trilobatin in the claim 1 or 2 with phloretin to obtain the trilobatin; The method for preparing phlorizin and trilobatin comprises the step of reacting the protein or the product for producing phlorizin and trilobatin according to claim 1 or 2 with phloretin to obtain phlorizin and trilobatin.
  6. 6. The method of claim 5, wherein the reaction system of the reaction further comprises uridine diphosphate- β -O-D-glucose.
  7. 7. The method of claim 5, wherein the reaction system of the reaction is carried out at 30 ℃.
  8. 8. A reagent or kit comprising one or more of phloretin, the protein of claim 1 or 2, or the biological material of claim 3 or 4.
  9. 9. A product, characterized in that it is any one of the following: y1) a protein as described in claim 1 or 2; Y2) the biomaterial according to claim 3 or 4.
  10. 10. A product, characterized in that it is any one of the following: z1) a product containing the catalytic phloretin of claim 1, 2, 3 and/or 4 to produce phlorizin; Z2) a product containing the catalytic phloretin of claim 1, 2, 3 and/or 4 to form trilobatin; Z3) a product containing the catalytic phloretin of claim 1, 2, 3 and/or 4 for producing phlorizin and/or trilobatin.

Description

Phloretin enzyme activity related protein, and coding gene and application thereof Technical Field The invention specifically discloses a phloretin enzyme activity related protein, and a coding gene and application thereof. Background Mirabilis jalapa (Mirabilis himalaica), also known as Mirabilis jalapa (Oxybaphus himalaicus), is an annual herb of the genus Mirabilis of the family Mirabiliaceae. Is mainly distributed in high altitude areas such as Tibet, qinghai, gansu, sichuan and the like. Mirabilis jalapa is used as a root, is commonly used as Bazhu, zhiyaga, xia Reba, A Xia, and the like, is recorded in classical Tibetan medicine books such as YueWang medicine diagnosis, jingzhu Ben Cao and four medical classics, and is received in national Chinese herbal medicine Association and the drug Standard of the Ministry of health of the people's republic of China. Earlier researches show that the roots and leaves of Mirabilis himalaica contain dihydrochalcone compounds such as phloretin, phlorizin and trilobatin, and the compounds have strong antioxidant activity and can be used as natural sweetener. Disclosure of Invention The technical problem solved by the invention is how to provide a protein which has root Pi Sumei activity and can catalyze phloretin to generate phlorizin and/or trilobatin. In order to solve the above technical problems, the present invention provides the following applications. Use of a protein in any one of the following: A1 Catalyzing phloretin to generate phlorizin and/or trilobatin and/or preparing the phloretin to generate phlorizin and/or trilobatin products; a2 The use of the same for the production of phlorizin and/or for the preparation of a product for the production of phlorizin; A3 The use of the same for the production of trilobatin and/or for the preparation of a product for the production of trilobatin; The protein is any one of the following: b1 Amino acid sequence is a protein described in sequence 2; b2 A protein which has 80% or more identity and the same function as the protein represented by B1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein represented by B1); B3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of B1) or B2) with a protein tag. Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc. In the above proteins, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gapexistencecost, perresiduegapcost and Lambdaratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained. In the above protein, the 80% or more identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity. In the above protein, sequence 2 (SEQ ID No. 2) consists of 491 amino acid residues. This was designated as ohi.6618 protein. The coding gene is the Ohi.6618 gene. In order to facilitate purification and use of the protein of A1), a tag as shown in the following table may be attached to the amino-terminus or the carboxyl-terminus of a protein consisting of the amino acid sequences shown in positions 423 to 902 of sequence 2 in the sequence Listing. In the above application, the protein is derived from jasmine. In the above, the jasmine may be mirabilis jalapa. Any of the following applications of the biological material related to the above proteins: c1 Catalyzing phloretin to generate phlorizin and/or trilobatin and/or preparing the phloretin to generate phlorizin and/or trilobatin products; c2 The use of the same for the production of phlorizin and/or for the preparation of a product for the production of phlorizin; c3 The use of the same for the production of trilobatin and/or for the preparation of a product for the production of trilobatin; C4 Producing the above protein or preparing a product for producing the above protein; The biomaterial is any one of the following D1) to D4): D1 Nucleic acid molecules encoding the above proteins; d2 An expression cassette comprising D1) said nucleic acid molecule; d3 A recombinant vector comprising D1) said nucleic acid molecule, or a recombinant vector comprising D2) said expression cassette; d4 A recombinant microorganism comprising D1) said n