CN113652409A - Novel glycyrrhetinic acid glucuronyl transferase mutant and application thereof - Google Patents
Novel glycyrrhetinic acid glucuronyl transferase mutant and application thereof Download PDFInfo
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- CN113652409A CN113652409A CN202110920494.5A CN202110920494A CN113652409A CN 113652409 A CN113652409 A CN 113652409A CN 202110920494 A CN202110920494 A CN 202110920494A CN 113652409 A CN113652409 A CN 113652409A
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Abstract
The invention discloses a novel glycyrrhetinic acid glucuronyl transferase mutant and application thereof, belonging to the technical field of biology. The amino acid sequence of the glycyrrhetinic acid glucuronyl transferase mutant consists of amino acid sequences shown as SEQ ID NO: 2, mutation formation of the sequence shown in the sequence table; the site of the sequence mutation is selected from one or more of the following amino acid residue sites: 395, 425, 430, 433, 437, 438, 441, 445 bits. The invention constructs the recombinant engineering bacteria of the glycyrrhetinic acid glucuronyl transferase mutant, the glycyrrhizic acid can be obtained by using the glycyrrhetinic acid as a substrate for fermentation and catalysis, and compared with the wild engineering bacteria, the glycyrrhetinic acid glucuronyl transferase mutant can catalyze the glycyrrhetinic acid to generate the glycyrrhizic acid with the output remarkably improved, the molar conversion rate of the glycyrrhetinic acid as the substrate is about 95 percent, and the glycyrrhizic acid output can be improved to more than 6-8 g/L.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a glycyrrhetinic acid glucuronyl transferase mutant, and construction and application of an engineering bacterium for synthesizing glycyrrhizic acid by using glycyrrhetinic acid as a substrate microorganism.
Background
The glycyrrhizic acid is an active component extracted from the liquorice and has the following biological activity, namely has a certain inhibition effect on the growth of cancer cells and can be used for treating liver cancer; has anti-hepatotoxicity effect, and can be used for treating toxic liver diseases; the content of glutamic-pyruvic transaminase can be effectively reduced, the function of cells is recovered, and the preparation has obvious effect on treating alcoholic liver or fatty liver; has jaundice treating effect, and can be used for treating icteric hepatitis. Therefore, how to rapidly synthesize glycyrrhizic acid becomes a problem of focusing on the modern society. At present, the main source of glycyrrhizic acid is original plants of liquorice, the planting period of the liquorice is long, the quality of the planted liquorice is uneven, the liquorice is seriously deficient due to excessive digging, the ecological environment is damaged, and the market demand cannot be met only by extracting the glycyrrhizic acid from the original plants of the liquorice, so that a new method for reasonably and efficiently obtaining the effective components of the liquorice is urgently needed.
Glycyrrhizic acid is prepared by converting glycyrrhetinic acid into glycyrrhizic acid through guanosine diphosphate dependent glucuronic acid transferase (UGAT), although glycyrrhizic acid can be prepared, the content of glycyrrhizic acid is extremely low, mainly because most of the enzymes are derived from eukaryotic cells, and eukaryotic cell source enzymes are generally insoluble when being expressed in prokaryotic cells, so that the yield of glycyrrhizic acid is influenced. The invention excavates UGAT which can be expressed in colon bacillus in a soluble way by a bioinformatics technology, obtains a glycyrrhetinic acid glucuronyl transferase mutant by mutating a specific site of wild glycyrrhetinic acid glucuronyl transferase, catalyzes glycyrrhetinic acid to produce glycyrrhizic acid by utilizing the mutant, and obviously improves the yield. Furthermore, the glycyrrhizic acid can be co-expressed with molecular chaperones to promote the correct folding of peptide chains, greatly increase the soluble expression of enzyme, further improve the yield of glycyrrhizic acid and provide support for large-scale industrial production of glycyrrhizic acid.
Disclosure of Invention
An object of the present invention is to provide a glycyrrhetinic acid glucuronyl transferase mutant.
The invention also aims to provide an engineering strain for producing glycyrrhizic acid by using glycyrrhetinic acid as a substrate and a construction method thereof.
The invention also aims to provide construction of the engineering bacteria for synthesizing glycyrrhizic acid by using glycyrrhetinic acid as a substrate and a method for producing glycyrrhizic acid by using glycyrrhetinic acid as a substrate.
The invention provides a glycyrrhetinic acid glucuronyl transferase mutant, which is an amino acid sequence of any one of the following:
a: the amino acid sequence of the glycyrrhetinic acid glucuronyl transferase mutant consists of amino acid sequences shown as SEQ ID NO: 2, mutation formation of the sequence shown in the figure; the site of the sequence mutation is selected from one or more of the following amino acid residue sites: 395, 425, 430, 433, 437, 438, 441, 445 bits.
b: the amino acid sequence of the glycyrrhetinic acid glucuronosyltransferase mutant has at least 95% of sequence identity with the amino acid sequence of a;
further, the amino acid sequence of the glycyrrhetinic acid glucuronyl transferase mutant has 98% or more sequence identity with the amino acid sequence of a; more preferably, the glycyrrhetinic acid glucuronosyltransferase mutant has an amino acid sequence having 99% or more sequence identity to the amino acid sequence of a;
c: the amino acid sequence of the glycyrrhetinic acid glucuronyl transferase mutant is formed by substituting, adding or deleting one or more amino acid residues at the C terminal and/or the N terminal of the amino acid sequence of a;
all the amino acid mutants have the function of catalyzing glycyrrhetinic acid to generate glycyrrhizic acid.
Preferably, the sequence mutation in a is selected from the following amino acid residue position mutation and the amino acid residue position mutation is shown as follows:
395 bits: asp, Ala;
425 bits: ser, Val, Leu, Ile;
430 bits: asp, Leu, Val, Met, Ala, Phe;
433 bits: ala, Asn;
437 bits: ser, Leu, Val, Met, Ala, Phe;
438 bit: asn;
441 bits: asp, Leu, Val, Met, Ala, Phe;
445 bits: ser and Asp.
Preferably, the sequence mutation in a is selected from the following amino acid residue position mutation and the amino acid residue position mutation is shown as follows:
395 bits: asp, and the amino acid sequence of the mutant is shown as SEQ ID No. 3;
425 bits: ser, the amino acid sequence of the mutant is shown in SEQ ID No. 5;
430 bits: asp, and the amino acid sequence of the mutant is shown as SEQ ID No. 7;
433 bits: ala, the amino acid sequence of the mutant is shown in SEQ ID No. 9;
437 bits: ser, the amino acid sequence of the mutant is shown in SEQ ID No. 11;
438 bit: asn, the amino acid sequence of the mutant is shown in SEQ ID No. 13;
441 bits: asp, and the amino acid sequence of the mutant is shown as SEQ ID No. 15;
445 bits: ser, and the amino acid sequence of the mutant is shown in SEQ ID No. 17.
The invention also provides a gene for coding the glycyrrhetinic acid glucuronyl transferase mutant.
According to the scheme, the gene sequence of the gene for coding the glycyrrhetinic acid glucuronosyltransferase mutant is shown as SEQ ID No.4 or SEQ ID No.6 or SEQ ID No.8 or SEQ ID No.10 or SEQ ID No.12 or SEQ ID No.14 or SEQ ID No.16 or SEQ ID No. 18.
The invention also provides a recombinant plasmid vector containing the glycyrrhetinic acid glucuronyl transferase mutant coding gene.
The invention provides a construction method of a recombinant plasmid vector, which takes constructed UGAT wild-type plasmid as a template, respectively adopts corresponding primers shown as the following to carry out PCR amplification, digests the obtained PCR product and removes the template to obtain the recombinant plasmid.
The invention also provides a recombinant gene engineering bacterium, which is obtained by transferring the recombinant plasmid vector into a host cell.
According to the scheme, in the recombinant genetic engineering bacteria, the host cell comprises the recombinant plasmid vector or a glycyrrhetinic acid glucuronyl transferase mutant coding gene integrated in the genome of the recombinant plasmid vector.
Preferably, the recombinant genetic engineering bacteria also contain molecular chaperones, wherein the molecular chaperones are one or more of pGro7, pG-KJE8, pKJE7, pGTf2 and pTf16, and are induced to express by L-arabinose.
The construction method comprises the following steps: the recombinant plasmid and different molecular chaperone plasmids are jointly transformed into a host cell, such as escherichia coli expression host bacteria BL21(DE3), and the recombinant genetic engineering bacteria containing the molecular chaperone are obtained.
The invention also provides the application of the glycyrrhetinic acid glucuronyl transferase mutant, the recombinant vector or the genetically engineered bacterium in the production of glycyrrhetinic acid glucuronyl transferase.
The invention also provides the application of the glycyrrhetinic acid glucuronyl transferase mutant, the recombinant vector or the genetically engineered bacterium in producing glycyrrhizic acid.
The invention also provides a method for producing glycyrrhizic acid, which comprises the following steps:
1) producing glycyrrhizic acid by using the glycyrrhetinic acid glucuronyl transferase mutant or the recombinant genetic engineering bacteria;
2) separating glycyrrhizic acid from the system of 1).
A biological conversion method of glycyrrhizic acid comprises the following steps:
(1) culturing any one of the engineering bacteria in a seed culture medium to obtain a seed solution;
(2) inoculating the seed liquid into a fermentation culture medium for fermentation culture;
(3) fermenting for a certain time, adding inducer for induction expression, inducing for a certain time, adding cell penetrating agent for treatment, adding substrate glycyrrhetinic acid and glucose for biotransformation, and synthesizing glycyrrhizic acid.
The seed culture solution contains antibiotics, and the antibiotics are correspondingly selected according to the selected plasmid resistance, and specifically can be one or more of chloramphenicol and kanamycin;
the seed liquid OD600＝5-10；
The seed culture medium comprises (by mass) 1% of tryptone, 1% of sodium chloride and 0.5% of yeast extract;
the culture conditions of the seed liquid are 28-40 ℃, 160-230rpm, preferably 37 ℃ and 225 rpm;
the culture time of the seed liquid is 10-15h, preferably 15 h;
the proportion of the seed liquid to the fermentation medium is 1-2%, and the optimal proportion is 2%;
the fermentation medium comprises (by weight) tryptone 1.2%, yeast extract 2.4%, glycerol 0.5%, and KH 0.231%2PO4And 1.64% K2HPO4·3H2O；
The culture conditions of the fermentation culture medium are 28-40 ℃, 160-230rpm, preferably 37 ℃ and 225 rpm;
the adding time of the inducer is 1-3h after fermentation culture, preferably 2 h;
the inducer is IPTG, and when recombinant genetic engineering bacteria containing molecular chaperones are used, the inducer also comprises L-arabinose;
the final concentration of IPTG is 0.1-10mM, preferably 1 mM;
the final concentration of the L-arabinose is 0.5-10mg/ml, preferably 4 mg/ml;
the condition for inducing expression is 16-37 ℃, preferably 28 ℃;
the cell penetrating agent is one or more of polymyxin B, lysozyme and CT, and preferably polymyxin B;
the adding time of the substrate is 6-15h after induction expression, preferably 10 h;
the glucose concentration of the reaction system is 1-100g/L, preferably 10 g/L;
the concentration of the substrate is 1-20 g/L; preferably 5 g/L;
the biotransformation time is 8-50h, preferably 28 h.
The invention discloses a novel glycyrrhetinic acid glucuronyl transferase mutant which is prepared by carrying out the following steps of: 2, carrying out specific site mutation on the glycyrrhetinic acid glucuronyl transferase amino acid sequence to obtain a plurality of mutants. Further discloses a recombinant vector containing the glycyrrhetinic acid glucuronyl transferase mutant coding gene and a recombinant gene engineering bacterium. Expressing the gene coding the glycyrrhetinic acid glucuronyl transferase mutant in a recombinant vector by a fermentation culture mode to obtain the glycyrrhetinic acid glucuronyl transferase. The process is easy to implement, the conditions are easy to control, and the method is suitable for popularization of industrial production.
UGAT is used for catalyzing glycyrrhetinic acid substrate to produce glycyrrhizic acid; meanwhile, the system and molecular chaperone plasmid are co-expressed, so that correct folding of a peptide chain is promoted, and soluble expression of enzyme in the system is enhanced.
Compared with the prior art, the invention has the following beneficial effects:
the glycyrrhetinic acid glucuronyl transferase mutant with improved activity can be obtained by mutating the specific site of the wild glycyrrhetinic acid glucuronyl transferase, and experiments prove that the yield of glycyrrhizic acid produced by the glycyrrhetinic acid glucuronyl transferase mutant is remarkably improved compared with that produced by the wild glycyrrhetinic acid glucuronyl transferase, and the yield of bioconversion glycyrrhizic acid reaches 6-8 g/L.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram showing construction of a wild-type glycyrrhetinic acid glucuronyl transferase recombinant plasmid PET28A-UGAT according to example 1.
FIG. 2 is the construction of engineering bacteria of recombinant plasmid pET28a-V433A and molecular chaperone pG-KJE8 in example 4.
FIG. 3 is the construction of the engineering bacteria of the recombinant plasmid pET28a-V433A and the molecular chaperone pGro7 in example 4.
FIG. 4 is the diagram of the construction of engineering bacteria of recombinant plasmid pET28a-V433A and molecular chaperone pG-Tf2 in example 4.
FIG. 5 is the construction of engineering bacteria of the recombinant plasmid pET28a-V433A and the molecular chaperone pkje7 in example 4.
FIG. 6 is the construction of engineering bacteria of recombinant plasmid pET28a-V433A and chaperone pTf16 in example 4.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
It is known to the person skilled in the art that if an enzyme is mutated in order to obtain a mutant with improved activity, it is crucial to find a site where the activity can be improved after the mutation. In the present invention, the amino acid sequence is as shown in SEQ ID NO: 2, the mutant of glycyrrhetinic acid glucuronyl transferase with improved activity is obtained by mutating the specific site of the human-derived wild type glycyrrhetinic acid glucuronyl transferase.
The invention is described in SEQ ID NO: 2, the glycyrrhetinic acid glucuronyl transferase mutant obtained by mutating one or more of the following sites of the amino acid sequence shown in the specification can improve the yield of glycyrrhizic acid: 395, 425, 430, 433, 437, 438, 441, 445 bits.
In the present invention, the terms "glycyrrhetinic acid glucuronyl transferase" or "glycyrrhetinic acid glucuronyl transferase mutant" or "glycyrrhetinic acid glucuronyl transferase of the present invention" are used with the same meaning, interchangeably herein, to mean a polypeptide obtained from an amino acid sequence such as SEQ ID NO: 2, a mutant of glycyrrhetinic acid glucuronyltransferase obtained by mutating at one or more of the above-mentioned sites is glycyrrhetinic acid glucuronyltransferase which has an activity of catalyzing glycyrrhetinic acid to produce glycyrrhizic acid and has an increased glycyrrhizic acid yield.
In view of the teaching of the present invention and the prior art, it will be further understood by those skilled in the art that the "glycyrrhetinic acid glucuronyl transferase mutant of the present invention" shall also include a variant thereof having the same or similar function as the "glycyrrhetinic acid glucuronyl transferase mutant of the present invention" but having an amino acid sequence slightly different from that of glycyrrhetinic acid glucuronyl transferase in the examples of the present invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 30, preferably 1 to 10, more preferably 1 to 6, still more preferably 1 to 3, most preferably 1) amino acids, and addition of one or more (usually up to 30, preferably up to 10, more preferably up to 6 or 3) amino acids at the C-terminus and/or N-terminus. For example, it is well known to those skilled in the art that substitutions with amino acids of similar or analogous properties, e.g., isoleucine and leucine, do not alter the function of the resulting protein. As another example, the addition of one or several amino acids at the C-terminus and/or N-terminus, such as a 6-His tag added for ease of isolation, will not generally alter the function of the resulting protein.
It will also be understood by those skilled in the art that the variant forms of the glycyrrhetinic acid glucuronyl transferase of the present invention described herein do not include reversion to the wild-type glycyrrhetinic acid glucuronyl transferase; in other words, the mutant glycyrrhetinic acid glucuronyl transferase according to the present invention is obtained by further mutating a mutant glycyrrhetinic acid glucuronyl transferase obtained in the examples of the present invention, but has a sequence corresponding to SEQ ID NO: 2 at the 395, 425, 430, 433, 437, 438, 441 and 445 positions is the same as that in the amino acid sequence of glycyrrhetinic acid glucuronyl transferase obtained in the examples of the present invention.
The term "corresponding to" as used herein has the meaning commonly understood by a person of ordinary skill in the art. Specifically, "corresponding to" means a position in two sequences that corresponds to a specified position in the other sequence after alignment by homology or sequence identity. Therefore, if a 6-His tag is added to one end of the amino acid sequence of glycyrrhetinic acid glucuronyl transferase obtained in the examples of the present invention, the mutant obtained has a sequence corresponding to SEQ ID NO: 2 may be position 401.
In particular embodiments, the homology or sequence identity may be 90% or more, preferably 95% to 98%, most preferably 99% or more.
Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: computer Molecular Biology (computerized Molecular Biology), Lesk, a.m. ed, oxford university press, new york, 1988; biological calculation: informatics and genomic Projects (Biocomputing: information and Genome Projects), Smith, d.w. eds, academic press, new york, 1993; computer Analysis of Sequence Data (Computer Analysis of Sequence Data), first part, Griffin, a.m. and Griffin, h.g. eds, Humana Press, new jersey, 1994; sequence Analysis in MolecuLar Biology (Sequence Analysis in MolecuLar Lar Biology), von Heinje, G., academic Press, 1987 and Sequence Analysis primers (Sequence Analysis Primer), Gribskov, M. and Devereux, J. eds M Stockton Press, New York, 1991 and Carllo, H. and Lipman, D., SIAM J. applied Math.48: 1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: GCG package (Devereux, J. et al, 1984), BLAST, Muscle, MAFFT and Clustal. The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altsch. mu.L, S. et al, NCBI NLM NIH Bethesda, Md.20894; Altsch. mu.L, S. et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.
Variants of the polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with DNA encoding the "Glycyrrhetinic acid glucuronosyltransferase mutant of the invention". The present invention also includes other polypeptides, such as fusion proteins comprising a "glycyrrhetinic acid glucuronyl transferase mutant of the present invention" or fragments thereof. In addition to the almost full-length polypeptide, the present invention shall also include the active fragment of the "glycyrrhetinic acid glucuronyl transferase mutant of the present invention". Typically, the fragment has at least about 20 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the amino acid sequence of the glycyrrhetinic acid glucuronyl transferase mutant of the present invention.
The present invention also provides analogs of glycyrrhetinic acid glucuronyl transferase. These analogs may differ from the natural "glycyrrhetinic acid glucuronyl transferase mutant of the present invention" in amino acid sequence, in modified form which does not affect the sequence, or in both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.
Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide such as acetylation, glycosylation or carboxylation, in vivo or in vitro. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.
In the present invention, the conservative variant polypeptide of "glycyrrhetinic acid glucuronyl transferase" refers to a polypeptide in which at most 20, preferably at most 10, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids having similar or similar properties, as compared with the amino acid sequence of the glycyrrhetinic acid glucuronyl transferase mutant in the present embodiment, but the conservative variant polypeptide still has the same or similar activity as the glycyrrhetinic acid glucuronyl transferase mutant in the present embodiment, i.e., the activity of catalyzing glycyrrhetinic acid to produce glycyrrhizic acid, and the glycyrrhizic acid production is significantly improved.
The protein of the present invention may be a recombinant protein, a natural protein, a synthetic protein, preferably a recombinant protein. The proteins of the invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells). Depending on the host used in the recombinant production protocol, the protein of the invention may be glycosylated or may be non-glycosylated. The proteins of the invention may or may not also include an initial methionine residue.
It will be understood by those skilled in the art that the "glycyrrhetinic acid glucuronyl transferase mutant" of the present invention also includes fragments, derivatives and analogs of the "glycyrrhetinic acid glucuronyl transferase mutant". A fragment, derivative or analogue of a polypeptide of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.
In view of the prior art in this field and the teaching of the present invention, those skilled in the art can easily obtain an active fragment of glycyrrhetinic acid glucuronyl transferase of the present invention. For example, a biologically active fragment of a "glycyrrhetinic acid glucuronyl transferase mutant" as used herein refers to a fragment of a "glycyrrhetinic acid glucuronyl transferase mutant", but which still retains all or part of the function of the full-length "glycyrrhetinic acid glucuronyl transferase mutant". Typically, the biologically active fragment retains at least 50% of the activity of the full-length "glycyrrhetinic acid glucuronyl transferase mutant". Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99% or 100% of the activity of the full-length "glycyrrhetinic acid glucuronyl transferase mutant".
Based on the teaching of the present invention and the prior art, it is also obvious to those skilled in the art that glycyrrhetinic acid glucuronyl transferase of the present invention can be prepared into other utilization forms such as immobilized enzymes.
On the basis of the glycyrrhetinic acid glucuronyl transferase of the invention, the invention also provides a polynucleotide sequence for encoding a glycyrrhetinic acid glucuronyl transferase mutant of the invention or a degenerate variant thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the nucleotide sequence encoding the glycyrrhetinic acid glucuronyl transferase mutant of the present embodiments or may be a degenerate variant. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence encoding a glycyrrhetinic acid glucuronyl transferase mutant according to the claims of the present invention, but differing from the nucleotide sequence encoding the glycyrrhetinic acid glucuronyl transferase mutant in the examples of the present invention.
In the present invention, the polynucleotide sequence encoding the "glycyrrhetinic acid glucuronosyltransferase mutant" may be inserted into a recombinant expression vector or genome. The term "expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.
The skilled person can use well-known methods to construct expression vectors containing the DNA sequence encoding the "Glycyrrhetinic acid glucuronosyltransferase mutant" and appropriate transcription/translation control signals, including in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or kanamycin or ampicillin resistance for E.coli.
Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
The host cell described herein includes a host cell comprising the above-described recombinant vector or having integrated on its genome the coding sequence of the "glycyrrhetinic acid glucuronyl transferase mutant" of the present invention. The host cell or the strain can efficiently express the novel glycyrrhetinic acid glucuronyl transferase with high catalytic performance, thereby improving the level of producing glycyrrhizic acid.
The host cell of the invention may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells. In particular embodiments, the strains include, but are not limited to: coli (e), corynebacterium glutamicum (corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis). In a preferred embodiment, the strain is escherichia coli (e.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. The recombinant polypeptide in the above method may be constitutively expressed or conditionally expressed, for example: after the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell disruption by osmosis, sonication, high-pressure homogenization, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
In view of the teachings of the present invention and the prior art, one of ordinary skill in the art will appreciate that the glycyrrhetinic acid glucuronyl transferase of the present invention, and its coding sequence, expression vector, host cell, can be used to catalyze the production of glycyrrhizic acid from glycyrrhetinic acid.
On the basis, the invention also provides a method for catalyzing glycyrrhetinic acid decarboxylation to generate glycyrrhizic acid by using the glycyrrhetinic acid glucuronyl transferase, the expression vector or the host cell. For example, in a specific embodiment, glycyrrhetinic acid can be catalyzed by culturing a host cell comprising an expression vector of the present invention or a coding sequence of a mutant glycyrrhetinic acid glucuronyl transferase of the present invention integrated on its genome to produce glycyrrhizic acid; and then obtaining the produced glycyrrhizic acid from the catalytic system.
The glycyrrhetinic acid used for catalyzing to produce glycyrrhizic acid can be glycyrrhetinic acid produced by host cells per se, and can also be exogenously added glycyrrhetinic acid.
The inventor has conducted extensive and intensive studies and unexpectedly found that a glycyrrhetinic acid glucuronyl transferase mutant with improved activity can be obtained by mutating a specific site of a wild type glycyrrhetinic acid glucuronyl transferase, thereby providing a basis for developing excellent glycyrrhetinic acid glucuronyl transferase to be further used for producing glycyrrhizic acid. The present invention has been completed based on this finding. The following will describe the acquisition and application of glycyrrhetinic acid glucuronyl transferase mutants in specific examples.
Example 1 construction of UGAT wild type Strain
UGAT comes from symbiotic algae Symbiodinium sp.KB8, and through codon optimization, artificially synthesized and optimized UGAT gene sequence is shown as SEQ ID No: 1, the UGAT gene was inserted between NdeI and XhoI sites of the pET28a (+) plasmid to obtain pET28a-UGAT expression vector (shown in fig. 1), which was transformed into e.coli DH5 α. Through sequencing verification, plasmids in the extracted positive clones are transformed into E.coli BL21(DE3), and E.coli BL21(DE3)/pET28a-UGAT wild-type engineering strains are obtained.
Example 2 obtaining of UGAT mutant strains
Through the rational analysis and design of UGAT genes, 8 single-point mutants are determined, the UGAT is subjected to site-directed mutagenesis by a homologous recombination method (see table 2), each mutant is provided with a pair of primers, the constructed pET28a-UGAT wild-type plasmid is used as a template, the corresponding primers are respectively adopted for PCR amplification (see table 3), and the amplification products are subjected to sequencing identification to respectively obtain 8 mutation sequences. After gene expression, UGAT 395 th site asparagine is mutated into aspartic acid, 425 th site histidine is mutated into serine, 430 th site asparagine is mutated into aspartic acid, 433 th site valine is mutated into alanine, 437 th site cysteine is mutated into serine, 438 th site glutamine is mutated into asparagine, 441 th site asparagine is mutated into aspartic acid, and 445 th site histidine is mutated into serine.
The amino acid sequence of the UGAT enzyme before mutation is shown as SEQ ID No: 2, respectively.
PCR amplification System (50. mu.L): template 1. mu.L, upstream and downstream primers 2.5. mu.L each, PrimeSTAR Max Premix (from takara) 25. mu.L, ddH2O 19μL。
TABLE 2 UGAT mutants
TABLE 3 primer sequences
The resulting PCR product was digested with Dpn I to remove the template. Coli DH 5. alpha. was transformed, the resulting transformants were sequenced from Beijing Onychosantha, and the plasmids with the correct sequencing of the 8 mutants were named pET28a-N395D, pET28a-H425S, pET28a-N430D, pET28a-V433A, pET28a-C437S, pET28a-Q438N, pET28a-N441D and pET28 a-H445S. Coli BL21(DE3), comprising the following steps: (1) take out the stored e.coli BL21(DE3) competent cells, lyse on ice; (2) add 3 μ L (about 1ng) plasmid solution into each tube, mix gently, stand on ice for 30 min; (3) placing the tube in a constant-temperature water bath at 42 ℃ for heat shock for 45 seconds; taking out the tube and placing on ice for 2 min; 0.5mL of LB medium incubated at 37 ℃ was added to each tube, shaking-cultured at 37 ℃ for 1 hour, and the culture was applied to LB plates with kanamycin resistance and cultured overnight in an inverted state at 37 ℃. Recombinant engineered strains E.coli BL21(DE3)/pET28a-N395D, E.coli BL21(DE3)/pET28a-H425S, E.coli BL21(DE3)/pET28a-N430D, E.coli BL21(DE3)/pET28a-V433A, E.coli BL21(DE3)/pET28a-C437S, E.coli BL21(DE3)/pET28a-Q438N, E.coli BL21(DE3)/pET28a-N441D and E.coli BL21(DE 387 3)/pET28a-H445S of the UGAT mutants are respectively obtained.
Example 3 glycyrrhizic acid production by UGAT wild type and mutant recombinant engineered strains
(1) Engineering bacteria E.coli BL21(DE3)/pET28a-N395D, E.coli BL D (DE D)/pET 28D-H425D, E.coli BL D (DE D)/pET 28D-N430D, E.coli BL D (DE D)/pET 28D-V433D, E.coli BL D (DE D)/pET 28D-C437D, E.coli BL D (DE D)/pET 28D-Q438D, E.coli BL D (DE D)/pET 28D-N441D and E.coli BL D (DE D)/pET 28D-H D are respectively cultured for 10H in a seed culture medium containing 50 mu g/mL of kanamycin to obtain a seed solution, and the OD of the seed solution600The culture medium (mass percent) of the seed is 1% of tryptone, 1% of sodium chloride and 0.5% of yeast extract, and the culture condition of the seed liquid is 37 ℃ and 225 rpm;
(2) inoculating 2% of the above seed solution to a solution containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, and 0.231% KH2PO4And 1.64% K2HPO4·3H2Performing fermentation culture in a fermentation culture medium (in percentage by mass) of O;
(3) after 2h of fermentation culture, adding 1mM IPTG for induction expression, wherein the temperature of the induction expression is 28 ℃, stopping fermentation after 10h of induction expression, adding a cell penetrating agent polymyxin B with the final concentration of 35 mug/mL for treatment for 30min, adding glycyrrhetinic acid with the final concentration of 5g/L and 10g/L glucose for solubilization by acetic acid, carrying out biotransformation for 28h, and detecting the yield of glycyrrhizic acid by using a gas phase mass spectrometer (Table 4).
TABLE 4 ratio of glycyrrhizic acid production by different recombinant engineering bacteria
Example 4 selection of chaperones
(1) The plasmid pET28a-V433A in the optimal mutant E.coli BL21(DE3)/pET28a-V433A is co-transformed with different molecular chaperone plasmids pG-KJE8, pGro7, pKJE7, pGTf2 and pTf16 (purchased from takara) into an Escherichia coli expression host bacterium BL21(DE3), and is coated on an LB solid culture medium containing 50 mu g/mL kanamycin antibiotic and 10 mu g/mL chloramphenicol antibiotic, an LB plate is cultured at 37 ℃ until a transformant grows out, a positive transformant is picked, and the expected engineering bacterium E.coli BL21(DE3)/pET28a-V433A and pG-KJE8 are obtained; coli BL21(DE3)/pET28a-V433A, pGro 7; coli BL21(DE3)/pET28a-V433A, pKJE 7; coli BL21(DE3)/pET28a-V433A, pGTf2, E coli BL21(DE3)/pET28a-V433A, pTf 16.
(2) Culturing the engineering bacteria in the step (1) in a seed culture medium containing 50 mu g/mL kanamycin and 20 mu g/mL antibiotics for 10 hours respectively to obtain a seed solution, wherein the OD600 of the seed solution is 5, the seed culture medium comprises 1% of tryptone, 1% of sodium chloride and 0.5% of yeast extract in percentage by mass, and the culture conditions of the seed solution are 37 ℃ and 225 rpm;
(3) inoculating 2% of the above seed solution to a solution containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, and 0.231% KH2PO4And 1.64% K2HPO4·3H2Performing fermentation culture in fermentation culture medium (mass percent) of O
(4) Fermenting and culturing for 2h, adding 1mM IPTG and 4mg/mL arabinose for induction expression at 28 deg.C for 10h, stopping fermentation, adding cell penetrating agent polymyxin B with final concentration of 35 μ g/mL for 30min, adding glycyrrhetinic acid with final concentration of 5g/L and glucose with final concentration of 10g/L for biotransformation for 28h, and detecting glycyrrhizic acid yield with gas phase mass spectrometer (Table 5)
TABLE 5 production ratios of glycyrrhizic acid added with different molecular chaperones
Therefore, the activity of the wild UGAT mutant is remarkably improved by mutating the specific site of the wild UGAT, so that the yield of glycyrrhizic acid produced by catalyzing glycyrrhetinic acid by the constructed recombinant engineering bacterium E.coli BL21(DE3)/pET28a-V433A is remarkably improved, as shown in Table 4; furthermore, the expression of UGAT is optimized by adding different molecular chaperone plasmids, so that the activity of UGAT is further improved, and the molar conversion rate can reach about 95%. The method provides practice for catalyzing glycyrrhetinic acid to produce glycyrrhizic acid by glycyrrhetinic acid glucuronyl transferase for industrialization, improving the production efficiency and reducing the production cost.
The vectors, genes and consumables described in the above examples are commercially available.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Nucleotide and amino acid sequence listing of the specification
< 110 > Hebei Weidakang Biotech Ltd
Less than 120, a new glycyrrhetinic acid glucuronyl transferase mutant and application thereof
＜160＞ 18
＜210＞ 1
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcaacgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggcacgttta cctgatgaat ctgatggttg ctcagctgtg ccagcgctat 1320
aacgaaattt accacgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
＜210＞ 2
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
MKLAVANYPR GEREVVLLTV QVGTESMSPL IWAVEKGALE SAREILNDLL TLRADRARYY 60
YGMEMLFTRH SDIISLLCTK APSLLPTVFD GLIWRSKNVK NGMRRANYYI ASLLRGEDGQ 120
LTDSLLDLIK QGDPEIICHP TVVFQADLLW IRLCCLPWAL TKLWFCATLV VYVMAEQQER 180
FTTIVCRAFL YIGSLGQLFA KHAYQTYRAV RQKQMTRLCC LPVPKYVLQT RQELTEVLLT 240
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFNVYGSD NYEQISVADE PMLKWIVMAF 420
AACWHVYLMN LMVAQLCQRY NEIYHDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 3
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
MKLAVANYPR GEREVVLLTV QVGTESMSPL IWAVEKGALE SAREILNDLL TLRADRARYY 60
YGMEMLFTRH SDIISLLCTK APSLLPTVFD GLIWRSKNVK NGMRRANYYI ASLLRGEDGQ 120
LTDSLLDLIK QGDPEIICHP TVVFQADLLW IRLCCLPWAL TKLWFCATLV VYVMAEQQER 180
FTTIVCRAFL YIGSLGQLFA KHAYQTYRAV RQKQMTRLCC LPVPKYVLQT RQELTEVLLT 240
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFDVYGSD NYEQISVADE PMLKWIVMAF 420
AACWHVYLMN LMVAQLCQRY NEIYHDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 4
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcgatgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggcacgttta cctgatgaat ctgatggttg ctcagctgtg ccagcgctat 1320
aacgaaattt accacgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
＜210＞ 5
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
MKLAVANYPR GEREVVLLTV QVGTESMSPL IWAVEKGALE SAREILNDLL TLRADRARYY 60
YGMEMLFTRH SDIISLLCTK APSLLPTVFD GLIWRSKNVK NGMRRANYYI ASLLRGEDGQ 120
LTDSLLDLIK QGDPEIICHP TVVFQADLLW IRLCCLPWAL TKLWFCATLV VYVMAEQQER 180
FTTIVCRAFL YIGSLGQLFA KHAYQTYRAV RQKQMTRLCC LPVPKYVLQT RQELTEVLLT 240
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFNVYGSD NYEQISVADE PMLKWIVMAF 420
AACWSVYLMN LMVAQLCQRY NEIYHDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 6
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcaacgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggagcgttta cctgatgaat ctgatggttg ctcagctgtg ccagcgctat 1320
aacgaaattt accacgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
＜210＞ 7
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFNVYGSD NYEQISVADE PMLKWIVMAF 420
AACWHVYLMD LMVAQLCQRY NEIYHDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 8
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcaacgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggcacgttta cctgatggat ctgatggttg ctcagctgtg ccagcgctat 1320
aacgaaattt accacgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
＜210＞ 9
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
YGMEMLFTRH SDIISLLCTK APSLLPTVFD GLIWRSKNVK NGMRRANYYI ASLLRGEDGQ 120
LTDSLLDLIK QGDPEIICHP TVVFQADLLW IRLCCLPWAL TKLWFCATLV VYVMAEQQER 180
FTTIVCRAFL YIGSLGQLFA KHAYQTYRAV RQKQMTRLCC LPVPKYVLQT RQELTEVLLT 240
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFNVYGSD NYEQISVADE PMLKWIVMAF 420
AACWHVYLMN LMAAQLCQRY NEIYHDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 10
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcaacgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggcacgttta cctgatgaat ctgatggcag ctcagctgtg ccagcgctat 1320
aacgaaattt accacgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
＜210＞ 11
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
MKLAVANYPR GEREVVLLTV QVGTESMSPL IWAVEKGALE SAREILNDLL TLRADRARYY 60
YGMEMLFTRH SDIISLLCTK APSLLPTVFD GLIWRSKNVK NGMRRANYYI ASLLRGEDGQ 120
LTDSLLDLIK QGDPEIICHP TVVFQADLLW IRLCCLPWAL TKLWFCATLV VYVMAEQQER 180
FTTIVCRAFL YIGSLGQLFA KHAYQTYRAV RQKQMTRLCC LPVPKYVLQT RQELTEVLLT 240
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFNVYGSD NYEQISVADE PMLKWIVMAF 420
AACWHVYLMN LMVAQLSQRY NEIYHDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 12
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcaacgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggcacgttta cctgatgaat ctgatggttg ctcagctgag ccagcgctat 1320
aacgaaattt accacgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
＜210＞ 13
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
MKLAVANYPR GEREVVLLTV QVGTESMSPL IWAVEKGALE SAREILNDLL TLRADRARYY 60
YGMEMLFTRH SDIISLLCTK APSLLPTVFD GLIWRSKNVK NGMRRANYYI ASLLRGEDGQ 120
LTDSLLDLIK QGDPEIICHP TVVFQADLLW IRLCCLPWAL TKLWFCATLV VYVMAEQQER 180
FTTIVCRAFL YIGSLGQLFA KHAYQTYRAV RQKQMTRLCC LPVPKYVLQT RQELTEVLLT 240
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFNVYGSD NYEQISVADE PMLKWIVMAF 420
AACWHVYLMN LMVAQLCNRY NEIYHDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 14
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcaacgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggcacgttta cctgatgaat ctgatggttg ctcagctgtg caatcgctat 1320
aacgaaattt accacgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
＜210＞ 15
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
MKLAVANYPR GEREVVLLTV QVGTESMSPL IWAVEKGALE SAREILNDLL TLRADRARYY 60
YGMEMLFTRH SDIISLLCTK APSLLPTVFD GLIWRSKNVK NGMRRANYYI ASLLRGEDGQ 120
LTDSLLDLIK QGDPEIICHP TVVFQADLLW IRLCCLPWAL TKLWFCATLV VYVMAEQQER 180
FTTIVCRAFL YIGSLGQLFA KHAYQTYRAV RQKQMTRLCC LPVPKYVLQT RQELTEVLLT 240
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFNVYGSD NYEQISVADE PMLKWIVMAF 420
AACWHVYLMN LMVAQLCQRY DEIYHDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 16
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcaacgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggcacgttta cctgatgaat ctgatggttg ctcagctgtg ccagcgctat 1320
gatgaaattt accacgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
＜210＞ 17
＜211＞ 782
＜212＞ PRT
< 213 > is artificially synthesized
MKLAVANYPR GEREVVLLTV QVGTESMSPL IWAVEKGALE SAREILNDLL TLRADRARYY 60
YGMEMLFTRH SDIISLLCTK APSLLPTVFD GLIWRSKNVK NGMRRANYYI ASLLRGEDGQ 120
LTDSLLDLIK QGDPEIICHP TVVFQADLLW IRLCCLPWAL TKLWFCATLV VYVMAEQQER 180
FTTIVCRAFL YIGSLGQLFA KHAYQTYRAV RQKQMTRLCC LPVPKYVLQT RQELTEVLLT 240
LLLMCLLLGC SRLNFLGIQG GISEWFVGSH RLCCEPVLHC LAVSSELLTN CCEYGEWQCH 300
LIRTYNRLAA FPMMLYFVLA SELVHLNVSL SVFSVICSCL MWEFILYVAV LAFFTAAFAS 360
AVACLPPSLG TDSIQMRDFF SWPLAFESLL SSAFNVYGSD NYEQISVADE PMLKWIVMAF 420
AACWHVYLMN LMVAQLCQRY NEIYSDARGN ARLTRGINIY ETSMPLISKK RWTAFVESLH 480
LEEACELDEG DNGPRGAVPT TEDPYDYLQY PKVELDRVQR YGGLANPALP WPSLEEAVDD 540
SAVGKLTRMT QAKFEEMDRL MVDMAIKLQV RPPGTSGGTK EYSAMHSEKR DESKMDGHEQ 600
DVGAAKETDI SKELAELSEE LPVNEATANE LVQERTEEGE AMSATPYDVN GTSVVSGSPS 660
VAEFEKKVAQ TGASIVGLDM GGADADSLEK KAREQKRILF LLLQELMTSP LEKELLLDRP 720
DVVMTDFATL AGCAVAQKLG IPLLVNLPGP ISLLRVFLGM VDTTTAVNFL GLHIARQRLS 780
PM 782
＜210＞ 18
＜211＞ 2349
＜212＞ DNA
< 213 > is artificially synthesized
atgaaactgg ctgtcgccaa ctatccacgt ggtgaacgtg aagttgtact gctgaccgtg 60
caggtaggca ccgagagcat gagcccgctg atttgggcgg tggagaaagg tgcgctggaa 120
tccgctcgtg aaatcctgaa cgatctgctg actctgcgtg cagatcgtgc tcgctattac 180
tatggcatgg agatgctgtt cacccgccac tctgacatca tttccctgct gtgcaccaaa 240
gcaccatctc tgctgccgac cgttttcgat ggcctgatct ggcgtagcaa gaacgtaaaa 300
aacggtatgc gccgtgctaa ctactatatc gcatctctgc tgcgtggcga agacggtcag 360
ctgaccgata gcctgctgga tctgatcaaa cagggtgacc cagagattat ctgtcatccg 420
acggttgttt tccaggcgga tctgctgtgg atccgtctgt gttgtctgcc gtgggcgctg 480
actaaactgt ggttttgtgc aaccctggtg gtttatgtga tggcggagca gcaggaacgt 540
ttcaccacga tcgtttgccg cgctttcctg tacatcggtt ccctgggcca gctgtttgct 600
aaacacgctt accagaccta ccgtgcagtc cgccaaaaac agatgactcg cctgtgctgc 660
ctgccggttc cgaagtacgt cctgcagacc cgtcaagaac tgaccgaggt tctgctgacc 720
ctgctgctga tgtgcctgct gctgggctgc tcccgtctga acttcctggg tattcagggc 780
ggcatctctg aatggttcgt tggtagccac cgtctgtgct gcgagccggt tctgcactgc 840
ctggctgtgt cttctgaact gctgactaac tgttgcgaat acggtgaatg gcagtgccat 900
ctgatccgta cttataaccg cctggcggca ttcccaatga tgctgtattt cgttctggcg 960
agcgaactgg ttcacctgaa cgttagcctg agcgttttca gcgtcatttg ctcctgcctg 1020
atgtgggaat ttatcctgta cgtagctgtc ctggcatttt ttaccgcagc gttcgcatct 1080
gctgttgcgt gtctgccgcc gtctctgggt actgattcca tccagatgcg cgactttttc 1140
tcttggccac tggcgttcga atctctgctg agctctgcat tcaacgtata tggctccgac 1200
aactacgaac aaatctctgt agcggatgaa ccgatgctga aatggatcgt tatggcgttc 1260
gcagcgtgtt ggcacgttta cctgatgaat ctgatggttg ctcagctgtg ccagcgctat 1320
aacgaaattt acagcgatgc acgtggtaac gcgcgtctga cccgtggtat caacatttac 1380
gaaacgtcta tgccgctgat cagcaaaaaa cgctggactg cgttcgttga atccctgcac 1440
ctggaagagg cgtgtgaact ggatgaaggt gataatggtc cgcgtggtgc tgttccgact 1500
accgaagatc catacgacta tctgcagtat cctaaagtgg aactggatcg tgtgcagcgc 1560
tatggtggtc tggctaatcc agcactgccg tggccttctc tggaagaagc ggtagatgat 1620
tctgctgtgg gcaaactgac tcgtatgact caggcaaaat tcgaagagat ggaccgtctg 1680
atggtcgaca tggccattaa gctgcaagtt cgtcctccgg gtacctctgg tggtaccaaa 1740
gaatattctg caatgcactc tgaaaaacgt gacgagtcta agatggacgg ccacgaacag 1800
gatgtcggcg ctgcaaaaga aaccgacatc tccaaggaac tggccgaact gtccgaagaa 1860
ctgccggtta acgaagccac cgcgaacgaa ctggttcagg agcgcaccga agaaggcgaa 1920
gctatgtctg cgaccccata cgacgtcaac ggtacctctg tcgtaagcgg ttccccgagc 1980
gttgcggaat tcgagaagaa ggttgctcag accggtgcgt ctatcgtggg tctggatatg 2040
ggtggcgcag atgcagattc cctggaaaaa aaagcgcgcg aacagaaacg catcctgttc 2100
ctgctgctgc aggaactgat gaccagcccg ctggaaaaag agctgctgct ggatcgcccg 2160
gacgtggtaa tgactgattt cgcaaccctg gcaggctgtg cggtagcaca gaaactgggt 2220
atcccactgc tggttaacct gccgggcccg atttctctgc tgcgcgtatt cctgggtatg 2280
gtcgacacca ctaccgccgt gaacttcctg ggtctgcaca tcgcacgtca gcgtctgtcc 2340
ccgatgtaa 2349
Claims (11)
1. A glycyrrhetinic acid glucuronyl transferase mutant, wherein the glycyrrhetinic acid glucuronyl transferase mutant comprises an amino acid sequence of any one of:
a: the amino acid sequence of the glycyrrhetinic acid glucuronyl transferase mutant consists of amino acid sequences shown as SEQ ID NO: 2, mutation formation of the sequence shown in the figure; the site of the sequence mutation is selected from one or more of the following amino acid residue sites: 395, 425, 430, 433, 437, 438, 441, 445;
b: the amino acid sequence of the glycyrrhetinic acid glucuronosyltransferase mutant has at least 95% of sequence identity with the amino acid sequence of a;
c: the amino acid sequence of the glycyrrhetinic acid glucuronosyltransferase mutant is formed by substituting, adding or deleting one or more amino acid residues at the C terminal and/or the N terminal of the amino acid sequence of a (mutation in the invention).
2. The glycyrrhetinic acid glucuronyl transferase mutant according to claim 1, wherein the sequence mutation in a is selected from one or more of the following amino acid residue positions mutated to the amino acid residue shown as follows:
395 bits: asp, Ala;
425 bits: ser, Val, Leu, Ile;
430 bits: asp, Leu, Val, Met, Ala, Phe;
433 bits: ala, Asn;
437 bits: ser, Leu, Val, Met, Ala, Phe;
438 bit: asn;
441 bits: asp, Leu, Val, Met, Ala, Phe;
445 bits: ser, Asp;
3. the glycyrrhetinic acid glucuronyl transferase mutant according to claim 2, wherein the sequence mutation in a is selected from one or more of the following amino acid residue positions mutated to the amino acid residue shown as follows:
395 bits: asp, and the amino acid sequence of the mutant is shown as SEQ ID No. 3;
425 bits: ser, the amino acid sequence of the mutant is shown in SEQ ID No. 5;
430 bits: asp, and the amino acid sequence of the mutant is shown as SEQ ID No. 7;
433 bits: ala, the amino acid sequence of the mutant is shown in SEQ ID No. 9;
437 bits: ser, the amino acid sequence of the mutant is shown in SEQ ID No. 11;
438 bit: asn, the amino acid sequence of the mutant is shown in SEQ ID No. 13;
441 bits: asp, and the amino acid sequence of the mutant is shown as SEQ ID No. 15;
445 bits: ser, and the amino acid sequence of the mutant is shown in SEQ ID No. 17.
4. A gene encoding the glycyrrhetinic acid glucuronyl transferase mutant of claim 1.
5. The gene according to claim 4, characterized in that: the gene sequence is shown in SEQ ID No.4 or SEQ ID No.6 or SEQ ID No.8 or SEQ ID No.10 or SEQ ID No.12 or SEQ ID No.14 or SEQ ID No.16 or SEQ ID No. 18.
6. A recombinant vector comprising the gene encoding glycyrrhetinic acid glucuronyl transferase mutant of claim 4.
7. A recombinant genetically engineered bacterium obtained by transferring the recombinant vector according to claim 5 into a host cell.
8. The recombinant genetically engineered bacterium of claim 7, further comprising a chaperone selected from one or more of pGro7, pG-KJE8, pKJE7, pGTf2 and pTf 16.
9. Use of the mutant glycyrrhetinic acid glucuronyl transferase according to any one of claims 1 to 3, the recombinant vector according to claim 5, or the genetically engineered bacterium according to claims 7 to 8 for the production of glycyrrhetinic acid glucuronyl transferase.
10. Use of the glycyrrhetinic acid glucuronyl transferase mutant according to any one of claims 1 to 3, the recombinant vector according to claim 5, or the genetically engineered bacterium according to claims 7 to 8 for the production of glycyrrhizic acid.
11. A method for producing glycyrrhizic acid is characterized by comprising the following steps:
1) producing glycyrrhizic acid by using the glycyrrhetinic acid glucuronyl transferase mutant according to any one of claims 1 to 3 or the recombinant genetically engineered bacterium according to any one of claims 6 to 7;
2) separating glycyrrhizic acid from the system of 1).
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