CN120718979A - An in vitro biotransformation synthesis system for trehalose 6-phosphate and its application - Google Patents
An in vitro biotransformation synthesis system for trehalose 6-phosphate and its applicationInfo
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Abstract
The invention provides an in-vitro bioconversion synthesis system of trehalose 6-phosphate and application thereof, belonging to the technical field of biocatalysis. The method for synthesizing the trehalose-6-phosphate by in-vitro bioconversion provided by the invention takes the maltose and the polyphosphate which are cheap and easy to obtain as raw materials to synthesize the trehalose-6-phosphate, expensive cofactors such as ATP, UTP and the like are not required to be added in the reaction process, the production cost of the trehalose-6-phosphate is reduced, and the method has the advantages of low cost, low pollution and high yield.
Description
Technical Field
The invention belongs to the technical field of biocatalysis, and particularly relates to an in-vitro bioconversion synthesis system of trehalose 6-phosphate and application thereof.
Background
Trehalose 6-phosphate (Trehalose-phosphate, T6P) is an important physiological metabolite and has wide application value in the fields of biology, biochemistry, food industry and the like. Trehalose 6-phosphate is a key intermediate product of trehalose in plants and participates in regulating and controlling the growth and development process of the plants. Trehalose 6-phosphate has a variety of functions, and first, it is a signaling molecule involved in regulating plant nutrient metabolism and carbon metabolism. T6P can modulate metabolic pathways in plants, such as to promote photosynthesis, thereby affecting plant growth and development. Under the pressure condition, the trehalose 6-phosphate can promote the drought resistance, cold resistance, disease resistance and other stress resistance of plants, and maintain the normal growth of the plants. Second, trehalose 6-phosphate promotes sucrose metabolism in plants, and as the content of trehalose 6-phosphate increases, the plant's ability to utilize sucrose increases, and thus trehalose 6-phosphate can be considered an indicator of sucrose status in plants. In addition, the trehalose 6-phosphate also has the functions of regulating metabolism of plant starch, promoting flowering, embryogenesis, sprouting branching and the like, and can be called as a novel plant hormone for promoting yield increase of crops. Research shows that the trehalose 6-phosphate can be applied to crops in a spraying mode and the like, so that the yield and quality of the crops can be improved, and Jing Jigao is realized before the trehalose 6-phosphate is applied to agricultural production, but the trehalose 6-phosphate is limited by factors such as a production process and the like, so that the production cost of the trehalose 6-phosphate is high, and the industrial production is not realized yet. There is therefore a need to develop a low cost, efficient method for synthesizing trehalose 6-phosphate to meet the demand for trehalose 6-phosphate.
In 1998, junkoDoi et al, by means of yeast fermentation, synthesized trehalose6-phosphate (Bioscience, biotechnology, and Biochemistry,1998, 62 (4): 735-9.) with glucose as substrate, catalyzed by glucokinase (glucokinase, GK) to produce G6P, catalyzed by α -glucophosphoglycerate mutase (α -phosphoglucomutase, α -PGM) to produce α -glucose 1-phosphate (α -G1P), while another substrate UMP was reacted with ATP to produce urine UTP under the action of nucleoside monophosphate kinase (nucleosidemonophosphatekinase, NMK) and nucleoside diphosphate kinase (nucleosidediphosphatekinase, NDK), catalyzed by α -G1P to produce UDP-glucose (UDPG) under the action of UTP-monosaccharide 1-phosphouridine transferase (UTP-monosaccharide 1-phosphateuridylyltransferase, USP), catalyzed by α -gluco 6-phosphate synthase (trehalose 6-phosphatesynthase, TPS) to synthesize T6P with UDPG. The technology uses a large amount of ATP and UMP as substrates, the price is high, the yield of T6P is only 11%, and the yeast fermentation regulation is complex.
In contrast, the in vitro enzyme catalysis method is simpler and more efficient, and development of a new method which is not applicable to expensive raw materials such as ATP and UTP, only uses cheap and easily available raw materials, has low cost, low pollution and high yield and is suitable for large-scale production of trehalose 6-phosphate is needed.
Disclosure of Invention
Based on various problems existing in the current method for producing trehalose 6-phosphate, a novel method for producing trehalose 6-phosphate by in-vitro multienzyme catalysis is provided, which is efficient, environment-friendly and low in cost.
In order to solve the problems in the prior art, the invention aims to provide a method for preparing trehalose-6-phosphate by using maltose and polyphosphate as raw materials and utilizing in-vitro bioconversion. According to the invention, the trehalose 6-phosphate is efficiently synthesized by designing an in-vitro three-enzyme cascade catalytic reaction system, and expensive cofactors ATP and UTP are not needed for supplying energy and recycling, so that the high conversion rate utilization of raw materials and the high yield, low cost and suitability for large-scale production of products are realized.
The specific technical scheme is as follows:
[1] A method for synthesizing trehalose 6-phosphate, wherein maltose and polyphosphate are used as substrates to synthesize trehalose 6-phosphate;
the method comprises the following steps:
the first step of reaction, namely catalyzing maltose into glucose and beta-glucose 1-phosphoric acid;
the second step of reaction, converting glucose and polyphosphate into glucose 6-phosphoric acid;
thirdly, converting beta-glucose 1-phosphate and glucose 6-phosphate into trehalose 6-phosphate;
in the first step of reaction, glucose and beta-glucose 1-phosphate are produced by using the catalysis of maltose phosphorylase;
in the second step of reaction, catalyzing polyphosphate and glucose by polyphosphate glucokinase to generate glucose 6-phosphate;
in the third step of reaction, the trehalose 6-phosphate phosphorylase is utilized to catalyze and generate trehalose 6-phosphate;
The maltose phosphorylase comprises an amino acid sequence shown as SEQ ID NO.1 or an amino acid sequence with at least 97% or higher homology with the amino acid sequence shown as SEQ ID NO.1, the polyphosphate glucokinase comprises an amino acid sequence shown as SEQ ID NO.3 or an amino acid sequence with at least 97% or higher homology with the amino acid sequence shown as SEQ ID NO.3, and the trehalose 6-phosphate phosphorylase comprises an amino acid sequence shown as SEQ ID NO.5 or an amino acid sequence with at least 97% or higher homology with the amino acid sequence shown as SEQ ID NO. 5;
and/or the number of the groups of groups,
The maltose phosphorylase comprises an amino acid sequence shown as SEQ ID NO.7 or an amino acid sequence with at least 97% or higher homology with the amino acid sequence shown as SEQ ID NO.7, the polyphosphate glucokinase comprises an amino acid sequence shown as SEQ ID NO.9 or an amino acid sequence with at least 97% or higher homology with the amino acid sequence shown as SEQ ID NO.9, and the trehalose 6-phosphate phosphorylase comprises an amino acid sequence shown as SEQ ID NO.5 or an amino acid sequence with at least 97% or higher homology with the amino acid sequence shown as SEQ ID NO. 5.
[2] The method according to [1], wherein the reaction system further comprises a buffer solution, the buffer solution comprises a phosphate buffer solution, and the pH of the phosphate buffer solution is 6.5-8.0.
[3] The method according to [1] or [2], wherein the concentration of the substrate maltose is not less than 5 g/L, preferably 10 to 100 g/L.
[4] The method according to any one of [1] to [3], wherein the polyphosphate comprises a polyphosphate polymerized from 3, 6, 12, 24, 48 or 100 phosphoric acid molecules;
[5] The method according to any one of [1] to [4], wherein the polyphosphate comprises a sodium salt, a potassium salt and/or an ammonium salt.
[6] The method according to any one of [1] to [5], wherein the addition amounts of the maltose phosphorylase, polyphosphate glucokinase, trehalose 6-phosphate phosphorylase are 0.1 to 10U/mL, respectively.
[7] The method according to [6], wherein the amount of the pullulanase added is 1-5U/mL, the amount of the polyphosphate glucokinase added is 1-5U/mL, and the amount of the trehalose 6-phosphate phosphorylase added is 2-10U/mL.
[8] The method according to any one of [1] to [7], wherein the reaction system further comprises 1 to 50mM magnesium ion, and the reaction temperature is 25 to 40 ℃.
The invention provides a brand-new method for preparing trehalose-6-phosphate by using maltose and polyphosphate which are available at low cost through path design, the prepared trehalose-6-phosphate has high yield and high content without adding auxiliary factors such as ATP, UTP and the like in the reaction process, the high-purity trehalose-6-phosphate can be obtained by simply removing protein and salt ions, the purification process is simple, the production cost of the trehalose-6-phosphate can be reduced, the preparation process is environment-friendly, and the method has the advantages of low cost, low pollution and high yield.
Drawings
FIG. 1 shows the way in which a three enzyme system catalyzes the production of trehalose-6-phosphate from maltose and polyphosphate.
FIG. 2 is a SDS-PAGE diagram of the respective enzymes of the three enzyme system. Wherein M is a protein molecular weight standard, lane 1 is a maltose phosphorylase MP derived from enterococcus faecalis (Enterococcus faecalis), lane 2 is a maltose phosphorylase MP derived from Bacillus subtilis (Bacillus subtilis) 168, lane 3 is a polyglucose kinase PPGK derived from Mycobacterium tuberculosis (Mycobacterium tuberculosis), lane 4 is a polyglucose kinase PPGK derived from actinomyces thermophilus (Thermobifida fusca) YX, and lane 5 is a trehalose 6-phosphate phosphorylase TrePP derived from lactococcus lactis (Lactococcus lactis subsp.lactis) Il 1403.
FIG. 3A is a standard curve of trehalose 6-phosphate standard.
FIG. 3B is an ion chromatogram of trehalose 6-phosphate standard.
Detailed Description
Various exemplary embodiments, features and aspects of the invention are described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, well known methods, procedures, means, equipment and steps have not been described in detail so as not to obscure the present invention.
Unless otherwise indicated, all units used in this specification are units of international standard, and numerical values, ranges of values, etc. appearing in the present invention are understood to include systematic errors unavoidable in industrial production.
In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.
In this specification, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
In the present specification, the numerical range indicated by "numerical values A to B" means a range including the end point numerical values A, B.
As used herein, the term "and/or" encompasses all combinations of items connected by the term, and should be viewed as having been individually listed herein. For example, "a and/or B" encompasses "a", "a and B", and "B". For example, "A, B and/or C" encompasses "a", "B", "C", "a and B", "a and C", "B and C" and "a and B and C".
The term "comprising" is used herein to describe a sequence of a protein or nucleic acid, which may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein. Furthermore, it will be clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of a polypeptide may be retained in some practical situations (e.g., when expressed in a particular expression system) without substantially affecting the function of the polypeptide. Thus, in describing a particular polypeptide amino acid sequence in the present specification and claims, although it may not comprise a methionine encoded at the N-terminus by the initiation codon, a sequence comprising such methionine is also contemplated at this time, and correspondingly, the encoding nucleotide sequence may also comprise the initiation codon, and vice versa.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. For example, standard recombinant DNA and molecular cloning techniques for use in the present invention are well known to those skilled in the art and are more fully described in Sambrook, joseph Frank et al, "Molecular Cloning: A Laboratory Manual" (2001) (hereinafter "Sambrook"). Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
As used herein, the terms "polypeptide", "enzyme", "polypeptide or enzyme" or "polypeptide/enzyme" have the same meaning, which are interchangeable in this disclosure. The foregoing term refers to a polymer composed of and many amino acids through peptide bonds, which may or may not contain modifications such as phosphate and formyl.
In the present invention, sequence analysis software is typically used to measure sequence similarity of polypeptides. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, GCG software contains programs such as GAP and BESTFIT that can be used under default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as between homologous polypeptides from different organism species or between wild-type proteins and their mutant proteins. See, e.g., GCG version 6.1. The polypeptide sequences can also be compared using FASTA under default or recommended parameters, and the program in GCG version 6.1 FASTA (e.g., FASTA2 and FASTA 3) provides an alignment and percentage of sequence identity for the optimal overlap region between the query sequence and the search sequence. Another preferred algorithm when comparing sequences of the invention to databases containing a large number of sequences from different organisms is the computer program BLAST, in particular BLASTP or TBLASTN, using default parameters. See, for example, altschul et al (1990) journal of molecular biology 215:403-410 and (1997) Nucleic Acids research 25:3389-3402, each of which is incorporated herein by reference.
The term "transformation, transfection, transduction" in the present invention has the meaning commonly understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The transformation, transfection, transduction methods include any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate (CaPO 4) precipitation, calcium chloride (CaCl 2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.
The term "enzyme-catalyzed reaction" means a chemical reaction that proceeds under the action of a biocatalyst-enzyme.
As used herein, the term "suitable reaction conditions" refers to those conditions in an enzyme-catalyzed reaction system, such as the range of enzyme addition, substrate addition, temperature, pH, buffers, cofactors, etc., under which the enzyme-catalyzed reaction of the invention is capable of converting maltose into trehalose 6-phosphate. Some exemplary "suitable reaction conditions" are provided herein.
As used herein, the term "add-on" such as in "enzyme add-on" or "substrate add-on" refers to the concentration or amount of a component in the reaction mixture at the beginning of the reaction.
As used herein, the term "polyphosphate" refers to any salt that contains several P-O-P bonds created by corner sharing of the individual phosphate (PO 4) tetrahedra, thereby forming long chains. The term "PolyP n" is used synonymously, where n represents the average chain length of the number of phosphate residues, e.g., polyP 25 refers to polyphosphate having about 25 phosphate residues and PolyP 14 refers to polyphosphate having about 14 phosphate residues.
As used herein, "conversion" may also refer to the enzymatic conversion (or bioconversion) of a substrate(s) to the corresponding product(s). "percent conversion" refers to the percentage of substrate that is converted to product over a period of time under the indicated conditions. Thus, the "enzymatic activity" or "activity" of an amino sugar synthase polypeptide can be expressed as a "percent conversion" of a substrate to a product over a particular period of time.
As used herein, "culturing" refers to growing a population of microbial cells under any suitable conditions (e.g., using a liquid, gel, or solid medium), including but not limited to well plate culture, shake flask culture, batch culture, continuous culture, fed-batch culture, and the like, and various culture conditions such as temperature, time, and pH of the medium, and the like, may be appropriately adjusted according to the actual situation.
EC numbers or EC numbers are a set of numbered taxonomies made by the enzyme commission (Enzyme Commission) for enzymes, based on the chemical reactions catalyzed by each enzyme. This set of taxonomies also gives a suggested name for each enzyme, and is also known as enzyme commission nomenclature.
In the present invention, the use form of any one of the enzymes in the enzyme-catalyzed reaction system for preparing trehalose 6-phosphate may be varied, including but not limited to purified or partially purified enzymes, microorganisms expressing the enzymes, cultures of the microorganisms, or combinations thereof.
In the present invention, the enzymes of various functions in the enzyme-catalyzed reaction system for preparing trehalose 6-phosphate may be directly used as pure enzymes (i.e., free enzymes), or the pure enzymes may be immobilized (i.e., immobilized enzymes) to maintain stability and be recycled, or whole-cell microorganisms or cultures of whole-cell microorganisms (i.e., fermentation products) containing the enzymes of various functions may be directly used, or the whole cells may be immobilized (i.e., immobilized cells) to maintain stability and be recycled. In some embodiments of the invention, whole cells may be permeabilized, for example, by heat treatment to promote cell membrane permeability, in order to achieve a rapid response rate.
In the present invention, all enzymes useful in the present invention may be wild-type enzymes or enzymes that have been subjected to certain genetic modifications that enhance some properties of the enzyme, such as activity, e.g. substrate specificity, e.g. thermostability, etc. Enzymes having the desired properties derived from wild-type enzymes by genetic engineering techniques are referred to as "mutants", "mutant derivatives" or "mutants from the corresponding wild-type enzymes". In the present invention, these "mutants" having improved properties are included for the enzymes used in the practice of the present invention.
In the method for preparing trehalose 6-phosphate, an enzyme catalytic reaction system is established at a certain temperature to carry out reaction. Suitable temperatures depend on factors such as the nature and amount of the enzyme for various functions in the enzyme-catalyzed reaction system and the amount of substrate maltose.
Based on the existing problems, the invention provides a method for synthesizing trehalose 6-phosphate, which takes maltose and polyphosphate as substrates and converts the maltose and the polyphosphate into the trehalose 6-phosphate in a three-enzyme cascade catalytic reaction system.
In some embodiments, the three enzyme cascade catalytic reaction system consists of three enzymes, maltose phosphorylase (maltose phosphorylase, MP), polyphosphate glucokinase (polyphosphate glucokinase, PPGK), trehalose 6-phosphate phosphorylase (trehalose-phosphate phosphorylase, trePP), the reaction scheme is shown in fig. 1.
In some specific embodiments, MP catalyzes the hydrolysis of maltose to glucose and beta-glucose 1-phosphate (beta-G1P) using maltose as a substrate, the polyphosphoric acid-dependent PPGK catalyzes the production of glucose 6-phosphate (G6P) using glucose and polyphosphate as substrates, and the TrePP catalyzes the synthesis of trehalose 6-phosphate and inorganic phosphate, which are target products, using beta-G1P and G6P as substrates.
In some specific embodiments, the three enzyme cascade catalytic reaction system does not require ATP to provide energy, and the phosphate provided by the inorganic polyphosphate participates in the catalytic reaction.
In some embodiments, the MP is selected from the group consisting of a maltose phosphorylase having an EC number EC 2.4.1.8, optionally, the MP source includes, but is not limited to, E.coli (ESCHERICHIA COLI), bacillus sp, E.faecalis (Enterococcus faecalis), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus brevis (Levilactobacillus brevis), thermus thermophilus (Thermus thermophilus), pyrococcus sp (Pyrococcus horikoshii), pyrococcus furiosus (Pyrococcus furiosus), thermococcus baryococcus (Thermococcus barophilus), thermococcus extreme thermophilus (Thermococcus kodakarensis), thermotoga maritima (Thermotoga maritima), thermococcus maritimus (Thermococcus litoralis), thermoactinomyces thermophilus (Thermobifida fusca), and/or Methanopyrum first sulfide (Sulfolobus tokodaii).
In some preferred embodiments, the MP is derived from enterococcus faecalis (Enterococcus faecalis) or Bacillus subtilis168 (Bacillus subtilis) 168.
In some preferred embodiments, the MP comprises the amino acid sequence shown as SEQ ID NO.1 or the amino acid sequence shown as SEQ ID NO.7, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97% or at least 99% or more homology with the amino acid sequence shown as SEQ ID NO.1 or the amino acid sequence shown as SEQ ID NO. 7. Illustratively, the nucleotide sequences SEQ ID NO.2 and SEQ ID NO.8 encoding MP.
In some embodiments, the PPGK is selected from polyphosphate glucokinase having an EC number of EC 2.7.1.63, optionally the PPGK source includes, but is not limited to, mycobacterium tuberculosis (Mycobacterium tuberculosis), arthrobacter (Arthrobacter sp.), clostridium thermocellum (Hungateiclostridium thermocellum), thermus thermophilus (Thermus thermophilus), pyrococcus (Pyrococcus sp.), horikoshi's (Pyrococcus horikoshii), pyrococcus furiosus (Pyrococcus furiosus), thermococcus barophilum (Thermococcus barophilus), thermococcus extreme thermophilus (Thermococcus kodakarensis), thermotoga maritima (Thermotoga maritima), thermococcus maritimus (Thermococcus litoralis), thermoactinomyces thermophilus (Thermobifida fusca), thermomyces lanuginosus (Sulfolobus tokodaii), streptomyces murinus (Streptomyces murinus) and/or Bifidobacterium adolescentis (Bifidobacterium adolescentis)
In some preferred embodiments, the PPGK is derived from mycobacterium tuberculosis (Mycobacterium tuberculosis) or actinomycetes thermophilus (Thermobifida fusca) YX.
In some preferred embodiments, PPGK comprises the amino acid sequence shown as SEQ ID No.3 or the amino acid sequence shown as SEQ ID No.9, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97% or at least 99% or more homology to the amino acid sequence shown as SEQ ID No.3 or the amino acid sequence shown as SEQ ID No. 9. Illustratively, the nucleotide sequence encoding PPGK is set forth as SEQ ID NO.4 or as SEQ ID NO. 10.
In some embodiments, the TrePP is selected from trehalose 6-phosphate phosphorylase having EC number EC 2.4.1.216, optionally the TrePP source includes, but is not limited to, blue crab (CALLINECTES SAPIDUS), carnivorous genus (Carnobacterium sp.), lactobacillus pentosus (Lactiplantibacillus pentosus), lactococcus lactis (Lactococcus lactis), westernum (WEISSELLA CETI), pediococcus pentosaceus (Pediococcus pentosaceus), clostridium thermocellum (Hungateiclostridium thermocellum), thermus thermophilus (Thermus thermophilus), pyrococcus (Pyrococcus sp.), rhodococcus horii (Pyrococcus horikoshii), pyrococcus furiosus (Pyrococcus furiosus), pyrococcus barophilum (Thermococcus barophilus), pyrococcus maritimus (Thermococcus litoralis), and/or phyllobacterium roseum (Sulfolobus tokodaii).
In some preferred embodiments, the TrePP is derived from lactococcus Lactis subspecies Lactis (Lactococcus Lactis subsp. Lactis), more preferably the TrePP is derived from lactococcus Lactis subspecies Lactis (Lactococcus Lactis subsp. Lactis) Il1403.
In some preferred embodiments, the TrePP comprises the amino acid sequence as set forth in SEQ ID No.5, or comprises an amino acid sequence having at least 95%, at least 96%, at least 97% or at least 99% or more homology to the amino acid sequence set forth in SEQ ID No. 5. Illustratively, the nucleotide sequence encoding TrePP is shown in SEQ ID No. 6.
Further, the three enzyme cascade catalytic reaction is performed in a buffer solution.
In some preferred embodiments, the buffer solution comprises phosphate buffer, tris-HCl buffer, HEPES buffer, or the like. Preferably, the buffer is a phosphate buffer.
In some more preferred embodiments, the phosphate buffer is 5 to 300 mM sodium phosphate buffer, preferably 10 to 150 mM sodium phosphate buffer, more preferably the pH of the sodium phosphate buffer is 6.5 to 8.0.
In some embodiments of the invention, the methods of synthesizing trehalose 6-phosphate described herein can recycle inorganic phosphate, and/or at least one step of the methods of forming trehalose 6-phosphate comprises an energy-beneficial chemical reaction. In particular, inorganic phosphoric acid produced when TrePP catalyzes β -G1P and G6P to produce trehalose 6-phosphate can assist in the catalytic reaction of MP.
In some embodiments, the concentration of substrate maltose is not less than 5 g/L, preferably 10-100 g/L.
In some specific embodiments, the maltose phosphorylase, polyphosphate glucokinase, trehalose 6-phosphate phosphorylase are added in an amount of 0.1-10U/mL, preferably 1-5U/mL, polyphosphate glucokinase is added in an amount of 1-5U/mL, trehalose 6-phosphate phosphorylase is added in an amount of 2-10U/mL, respectively.
In some specific embodiments, the polyphosphate comprises a polyphosphate polymerized from 3, 6, 12, 24, 48, or 100 phosphoric acid molecules, the polyphosphate comprising a sodium salt, a potassium salt, and/or an ammonium salt. Illustratively, the polyphosphate is sodium hexametaphosphate at a concentration of 5 to 300 mM, preferably 15 to 150 mM.
Further, the reaction system also contains magnesium ions, which can be provided by magnesium chloride and/or magnesium sulfate, for example, one or a mixture of two of MgCl 2、MgSO4. In some exemplary embodiments, the magnesium ions are provided by MgSO 4 at a concentration of 1-50 mM, preferably 1-25 mM.
In some embodiments, the invention is conducted at a temperature ranging from about 20 ℃ to about 90 ℃, a pH ranging from about 5.0 to about 8.0, and/or from about 0.5 h to about 72 h. For example, the present invention is carried out under the condition that the angle C, pH is from 25 to 60 DEG to 6.5 to 8.0.
In some embodiments, the steps of the process for preparing trehalose 6-phosphate are performed in one bioreactor or the catalytic reactions of the intermediates and end products of the invention may also be performed separately in multiple bioreactors arranged in series.
Examples
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 construction of recombinant expression strains of related enzymes in a Tri-enzyme System
The maltose phosphorylase MP can be selected from enterococcus faecalis (Enterococcus faecalis) with the Uniprot accession number of Q836Y7, and the maltose phosphorylase MP can also be selected from bacillus subtilis (Bacillus subtilis) 168 with the Uniprot accession number of O06993;
Polyphosphate glucokinase PPGK may be selected from Mycobacterium tuberculosis (Mycobacterium tuberculosis) under Uniprot accession number P9WIN1, polyphosphate glucokinase PPGK may also be selected from Actinomyces thermophilus (Thermobifida fusca) under Uniprot accession number Q47NX5;
Trehalose 6-phosphate phosphorylase TrePP may be selected from the lactococcus lactis subspecies (Lactococcus lactis subsp. Lactis) Il1403 under Uniprot accession number Q9CID5.
The coding gene sequence of the enzyme is synthesized by a gene synthesis company and connected to a pET20b plasmid to construct a corresponding expression plasmid. The constructed plasmids are respectively transformed into competent cells of escherichia coli BL21 (DE 3), and strains carrying the recombinant plasmids are respectively obtained through plate screening of ampicillin-containing antibiotics. The specific process is that competent cells of the escherichia coli BL21 (DE 3) are taken out from a refrigerator at the temperature of-80 ℃ and placed on ice to be slowly melted, 5 mu L of recombinant plasmid is immediately added, the mixture is placed on ice for 30min after being gently mixed, then the mixture is placed on a water bath at the temperature of 42 ℃ for heat shock 90 s, and then the ice bath is 2 min. After adding 500. Mu.L of LB liquid medium under aseptic conditions and culturing 50 min in a shaking table of 220 rpm at 37℃to resuscitate the cells, the cells were uniformly spread on LB solid medium containing ampicillin (working concentration 100. Mu.g/mL), the plates were sealed with a sealing film and inverted and cultured overnight in a 37℃incubator.
Example 2 protein expression and purification of the respective enzymes in the Tri-enzyme System
From LB solid plates, the single clone strains of the transformants were picked up respectively and inoculated into 1L shake flasks containing 200 mL LB liquid medium (containing 100. Mu.g/mL ampicillin antibiotics) and cultured overnight, 37℃and 220 rpm were expanded to OD 600 of 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the culture was continued at 16℃for 16-20 h. The thalli are centrifugally collected at 5000 rpm,15 min,4 ℃ and the fermentation/shake flask fermentation supernatant is removed. The bacterial pellet was resuspended by adding an equal volume of 0.9% NaCl solution and centrifuged at 15min at 4℃and 5000 rpm to remove the supernatant. Adding lysis buffer (100 mM phosphate buffer (pH 7.0), 0.3M NaCl) to resuspend the bacterial suspension to reach the concentration of OD 600 about 50, crushing the bacterial suspension by using a high-pressure homogenizer or a ultrasonic crusher, centrifuging to collect supernatant after the lysis, filtering by using a 0.45 μm filter head, carrying out affinity adsorption on the expressed recombinant protein by using a nickel ion affinity chromatography column pre-balanced by the lysis buffer, washing 5 column volumes by using the lysis buffer containing 10mM imidazole after the loading is finished to remove the impurity proteins, and eluting the target protein by using the lysis buffer containing 100 mM imidazole. The eluent is replaced by 100 mM phosphate buffer (pH 7.0) in a short time to remove imidazole, and the high-concentration protein is obtained after ultrafiltration is completed and is stored. The concentration of the target protein was measured by the Bradford method (coomassie blue staining method), and the purity was measured by protein gel electrophoresis (fig. 2). Protein samples are typically stored by adding 5% glycerol to the sample, flash-freezing at-80 ℃, and sub-packaging.
Example 3 quantitative determination of trehalose 6-phosphate
When the target product is detected and quantified, the content of trehalose 6-phosphate is detected by adopting high-performance anion exchange chromatography and chromatographic columns Dionex Ion Pac TMAS-HC ANALYTICAL column (4 mm i.d. times. 250 mm;Thermo Fisher Scientific). The sample loading of the test sample was 10. Mu.L.
The retention time of the trehalose 6-phosphate standard (Shanghai Ala Biotechnology Co., ltd., cat# T339557) was 8.287 min as measured by high performance anion exchange chromatography, and a mother liquor solution of 0.1 g/L was prepared from the trehalose 6-phosphate standard, diluted to a standard solution of 0.001 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, and quantitative and molar conversion analysis of the product were performed by a standard curve (FIGS. 3A and 3B).
The calculation formula of the molar conversion rate of the trehalose 6-phosphate is the ratio of the molar concentration of the trehalose 6-phosphate in the final reaction system to the molar concentration of the maltose in the initial reaction system multiplied by 100%.
EXAMPLE 4 construction of a Trienzyme Cascade catalytic reaction System to catalyze maltose to synthesize trehalose 6-phosphate
In this example, the selected maltose phosphorylase MP was derived from Bacillus subtilis (Bacillus subtilis) 168 under Uniprot accession number O06993, the polyphosphate glucokinase PPGK was derived from actinomycetes thermophilus (Thermobifida fusca) YX under Uniprot accession number Q47NX5, and the trehalose 6-phosphate phosphorylase TrePP was derived from lactococcus lactis (Lactococcus lactis subsp. Lactis) Il1403 under Uniprot accession number Q9CID5. The corresponding enzymes were prepared by transformation and expression in the manner of examples 1 and 2 for the construction and reaction of a three-enzyme cascade catalytic reaction system.
A reaction system of 5 mL contained 50 mM mL of phosphate buffer (pH 7.5), 15: 15 mM Mg 2+, 15: 15 mM sodium hexametaphosphate, 10: 10 g/L maltose (molar concentration 29.2: 29.2 mM, maltose molecular weight 342: 342 g/mol), 2: 2U/mL maltose phosphorylase MP, 2: 2U/mL polyphosphate glucokinase PPGK, 2: 2U/mL trehalose 6-phosphate phosphorylase TrePP. The reaction was carried out in a constant temperature shaker at a reaction temperature of 37℃and a rotational speed of 200 rpm and sampled every 30: 30 min, with a reaction of about 2: 2h, and after the reaction was completed, the reaction was terminated by heating 10: 10min at 100℃and the protein was precipitated, and the supernatant was centrifuged at 12000: 12000 rpm and used for chromatography. The results showed that the yield of trehalose 6-phosphate was 12.07 g/L, the molar concentration was 28.6 mM (molecular weight 422 g/mol of trehalose 6-phosphate) and the molar conversion of trehalose 6-phosphate was 97.9%.
EXAMPLE 5 construction of a Trienzyme Cascade catalytic reaction System to catalyze maltose to synthesize trehalose 6-phosphate
In this example, the maltose phosphorylase MP was selected from enterococcus faecalis (Enterococcus faecalis) under Uniprot accession number Q836Y7, the polyphosphate glucokinase PPGK was selected from Mycobacterium tuberculosis (Mycobacterium tuberculosis) under Uniprot accession number P9WIN1, and the trehalose 6-phosphate phosphorylase TrePP was selected from lactococcus lactis (Lactococcus lactis subsp. Lactis) Il1403 under Uniprot accession number Q9CID5. The corresponding enzymes were prepared by transformation and expression in the manner of examples 1 and 2 for the construction and reaction of a three-enzyme cascade catalytic reaction system.
A reaction system of 5mL contained 50 mM mL of phosphate buffer (pH 7.5), 15: 15 mM Mg 2+, 15: 15 mM sodium hexametaphosphate, 10: 10 g/L maltose (molar concentration 29.2: 29.2 mM, maltose molecular weight 342: 342 g/mol), 2: 2U/mL maltose phosphorylase MP, 2: 2U/mL polyphosphate glucokinase PPGK, 2: 2U/mL trehalose 6-phosphate phosphorylase TrePP. The reaction was carried out in a constant temperature shaker at a reaction temperature of 37℃and a rotational speed of 200 rpm and sampled every 30: 30 min, with a reaction of about 2: 2h, and after the reaction was completed, the reaction was terminated by heating 10: 10 min at 100℃and the protein was precipitated, and the supernatant was centrifuged at 12000: 12000 rpm and used for chromatography. The supernatant after the reaction was examined, and the result showed that a chromatographic peak consistent with the standard was newly formed at a retention time 8.287 min, indicating that trehalose 6-phosphate was formed in the reaction solution. The results showed that the yield of trehalose 6-phosphate was 11.78 g/L, the molar concentration was 27.9 mM (molecular weight 422 g/mol of trehalose 6-phosphate) and the molar conversion of trehalose 6-phosphate was 95.5%.
SEQ ID NO.1 MP amino acid sequence, bacillus subtilis (Bacillus subtilis) 168,Uniprot ID:O06993
MINQRLFEIDEWKIKTNTFNKEHTRLLESLTSLANGYMGVRGNFEEGYSGDSHQGTYIAGVWFPDKTRVGWWKNGYPEYFGKVINAMNFMGIGLYVDGEKIDLHQNPIELFEVELNMKEGILRRSAVVRIQDKTVRIRSERFLSLAVKELCAIHYEAECLTGDAVITLVPYLDGNVANEDSNYQEQFWQEEAKGADSHSGHLAAKTIENPFGTPRFTVLAAMANETEGFVHESFKTTEMYVENRYSYQTKASLKKFVIVTTSRDFREEELLSKAKELLADVVENGYEDAKRRHTDRWKERWAKADIEIKGDEELQQGIRYNIFQLFSTYYGGDARLNIGPKGFTGEKYGGAAYWDTEAYAVPMYLATAEPEVTKNLLLYRYHQLEAAKRNAAKLGMKGALYPMVTFTGDECHNEWEITFEEIHRNGAICYAIYNYINYTGDRNYMEEYGIDVLVAVSRFWADRVHFSKRKNKYMIHGVTGPNEYENNVNNNWYTNVIAAWTLEYTLQSLESISAEKRRHLDVQEVELEVWREIIQHMYYPFSEELQIFVQHDTFLDKDLQTVDELDPAERPLYQNWSWDKILRSNFIKQADVLQGIYLFNDRFTMEEKRRNFEFYEPMTVHESSLSPSVHAILAAELKLEKKALELYKRTARLDLDNYNHDTEEGLHITSMTGSWLAIVHGFAGMRTANETLSFAPFLPKEWDEYSFNINYRNRLINVTVDEKRVIFELVKGEPLHMNVYEEPVVLQGRCERRTPNE
SEQ ID NO.2 MP nucleotide sequence, bacillus subtilis (Bacillus subtilis) 168
ATGATAAATCAGCGGTTATTTGAGATTGATGAATGGAAAATCAAAACAAATACATTTAATAAGGAGCATACACGGCTGCTGGAAAGCCTGACGTCTCTTGCCAATGGCTATATGGGGGTCAGAGGGAATTTTGAAGAAGGCTATTCAGGCGACAGTCACCAAGGCACATATATTGCAGGCGTGTGGTTCCCCGACAAAACGCGAGTAGGCTGGTGGAAAAACGGGTATCCAGAATATTTCGGAAAAGTGATCAATGCGATGAACTTTATGGGCATAGGCCTATATGTTGACGGTGAAAAAATCGATCTCCATCAAAACCCAATCGAATTATTTGAGGTAGAACTCAATATGAAAGAGGGGATTCTGCGGCGAAGCGCTGTTGTCCGCATTCAAGATAAAACCGTCAGAATCAGGTCAGAGCGGTTTCTTAGCCTTGCTGTAAAAGAACTCTGTGCGATTCATTATGAAGCGGAGTGCTTGACGGGAGATGCTGTCATTACGCTTGTTCCTTACCTGGATGGAAATGTGGCAAATGAAGATTCTAACTACCAAGAACAGTTTTGGCAGGAGGAAGCGAAAGGTGCTGATTCTCACAGCGGCCATTTAGCGGCAAAAACGATCGAAAATCCATTTGGAACACCGCGCTTCACGGTATTAGCCGCAATGGCAAACGAGACGGAGGGTTTTGTCCATGAAAGCTTTAAAACGACTGAAATGTATGTCGAGAATCGCTACAGTTATCAAACGAAGGCATCATTGAAAAAGTTTGTGATTGTCACGACTTCCCGTGATTTTCGGGAGGAAGAGCTTTTATCGAAAGCCAAGGAGCTTTTGGCGGATGTGGTAGAGAACGGCTATGAAGATGCAAAACGGAGGCACACTGATCGATGGAAGGAAAGATGGGCAAAAGCGGACATTGAGATTAAAGGAGATGAGGAGCTTCAACAAGGAATCCGCTACAATATCTTTCAGTTATTCTCGACATATTACGGGGGCGATGCCCGTTTGAATATCGGGCCGAAAGGATTTACTGGCGAGAAATATGGAGGTGCCGCATATTGGGATACTGAGGCGTACGCCGTTCCGATGTATTTGGCGACAGCCGAGCCGGAGGTGACGAAAAACCTGCTTTTGTATCGCTATCATCAGTTGGAGGCTGCCAAACGAAACGCTGCAAAATTGGGGATGAAGGGGGCACTTTATCCGATGGTGACGTTCACAGGTGATGAATGCCACAACGAATGGGAAATCACCTTTGAAGAAATTCACCGCAATGGCGCGATCTGTTATGCGATCTACAATTATATCAATTATACAGGCGACCGTAACTATATGGAAGAATACGGGATAGACGTACTTGTGGCAGTCAGCAGATTTTGGGCCGACCGTGTTCACTTCTCGAAACGAAAAAATAAGTATATGATCCATGGCGTCACAGGGCCGAATGAATACGAAAACAACGTTAACAACAATTGGTATACGAATGTCATTGCGGCTTGGACGTTGGAGTATACTCTACAAAGTCTCGAAAGTATCTCAGCGGAGAAACGCCGCCATCTGGATGTGCAGGAAGTAGAATTGGAAGTCTGGAGAGAAATCATCCAGCACATGTACTATCCATTTAGTGAAGAACTGCAAATTTTCGTTCAGCATGACACGTTCTTGGACAAAGACCTGCAAACAGTTGACGAATTAGATCCAGCGGAACGGCCTCTTTACCAGAATTGGTCATGGGACAAGATTCTCCGCTCCAATTTTATTAAGCAGGCAGATGTTCTCCAAGGCATTTATCTTTTTAATGACCGTTTTACAATGGAAGAAAAACGGCGAAATTTTGAATTTTATGAGCCGATGACTGTTCATGAATCAAGCCTATCGCCCTCTGTCCATGCGATTCTCGCAGCCGAACTCAAGCTGGAAAAGAAAGCGCTCGAATTATATAAGCGCACAGCAAGGCTTGATCTTGATAATTACAATCATGATACGGAAGAAGGCTTGCATATTACTTCAATGACGGGTAGCTGGCTGGCAATCGTTCATGGCTTTGCAGGCATGCGCACCGCGAATGAGACGCTGTCATTTGCTCCGTTTTTGCCGAAAGAATGGGACGAATATTCATTCAACATCAATTATCGAAATCGATTAATCAATGTGACGGTTGACGAAAAGCGCGTTATTTTTGAGCTTGTAAAAGGCGAGCCGCTGCACATGAACGTTTATGAGGAACCGGTTGTCCTACAGGGACGATGTGAAAGGAGAACGCCTAATGAGctcgagcaccaccaccaccaccactga
SEQ ID NO.3: PPGK amino acid sequence, actinomycetes thermophilus (Thermobifida fusca) YX, uniprot accession number Q47NX5
MASRGRVGLGIDIGGSGIKGAPVDLDRGTFVVDRVKIATPQPATPEAVAAVVAEIVTAFADDVPQDAPLGVTFPAVIQHGVARSAANVDRSWIGTNVEELLSAVTGRRVLVVNDADAAAMAEHRYGAASGVDGVVLLTTLGTGIGTAVLVDGVLLPNTEFGHLEIDGYDAETRASASAKERENLSYKEWAEERLQRYYSVIEDLLWPDLIVVGGGVSRKADKFLPHLRLRAPIVPAKLRNTAGIVGAAVLAAERLGGDRVSA
SEQ ID NO.4: PPGK nucleotide sequence, actinomycetes thermophilus (Thermobifida fusca) YX
Atggcatctcggggacgggtcgggctggggattgacatcgggggaagcgggatcaaaggcgcccctgtggacttggaccggggaacgttcgtggtggaccgggtcaagatcgctactccgcagcccgcaacccctgaggcggtggctgcggtggtggcggagatagtcaccgcgttcgccgacgatgtgccgcaggatgcaccgttgggggtgacgtttcccgcggtgatccagcacggggtggcgcgcagcgccgccaacgtggaccgctcgtggatcggcaccaacgtcgaggagctgctgtctgcggtgacggggcggcgggtgctggtggtcaacgacgctgacgccgcagcgatggcggagcaccgctacggcgctgcctcaggcgtcgacggggtggtgctgttgactactttgggtaccggtattggtacggcggtgctagtggacggggtgctgctccccaacacggagttcgggcacttggagatcgacggctacgacgctgagacccgggcctctgctagcgctaaggagcgcgagaacctctcctacaaggagtgggctgaggagcggctgcagcgctactactcggtgatcgaggatttgctgtggccggacttgatcgtggtgggcggcggggtcagccgcaaggcggacaagtttttgccgcatctccgcttgcgcgcgccgatcgtgccggcgaagttgcgcaataccgcggggatcgtgggtgcggccgtgctggccgcggagcggctggggggtgaccgggtc tctgcctga
SEQ ID No.5 TrePP amino acid sequence, lactococcus lactis subspecies (Lactococcus lactis subsp. Lacti) Il1403, uniprot ID: Q9CID5
MTEKDWIIQYDKKEVGKRSYGQESLMSLGNGYLGLRGAPLWSTCSDNHYPGLYVAGVFNRTSTEVAGHDVINEDMVNWPNPQLIKVYIDGELVDFEASVEKQATIDFKNALQIERYQVKLAKGNLTLVTTKFVDPINFHDFGFVGEIIADFSCKLRIETFTDGSVLNQNVERYRAFDSKEFEVTKISKGLLVAKTRTSEIELAIASKSFLNGLAFPKIDSENDEILAEAIEIDLQKNQEVQFDKTIVIASSYESKNPVEFVLTELSATSVSKIQENNTNYWEKVWSDADIVIESDHEDLQRMVRMNIFHIRQAAQHGANQFLDASVGSRGLTGEGYRGHIFWDEIFVLPYYAANEPETARDLLLYRINRLTAAQENAKVDGEKGAMFPWQSGLIGDEQSQFVHLNTVNNEWEPDNSRRQRHVSLAIVYNLWIYSQLTEDESILTDGGLDLIIETTKFWLNKAELGDDGRYHIDGVMGPDEYHEAYPGQEGGICDNAYTNLMLTWQLNWLTELSEKGFEIPKELLEKAQKVRKKLYLDIDENGVIAQYAKYFELKEVDFAAYEAKYGDIHRIDRLMKAEGISPDEYQVAKQADTLMLIYNLGQEHVTKLVKQLAYELPENWLKVNRDYYLARTVHGSTTSRPVFAGIDVKLGDFDEALDFLITAIGSDYYDIQGGTTAEGVHIGVMGETLEVIQNEFAGLSLREGQFAIAPYLPKSWTKLKFNQIFRGTKVEILIENGQLLLTASADLLTKVYDDEVQLKAGVQTKFDLK
SEQ ID NO.6 TrePP nucleotide sequence, lactococcus lactis subspecies (Lactococcus lactis subsp. Lactis) Il1403
ATGACCGAAAAAGATTGGATTATTCAGTATGATAAAAAAGAAGTGGGCAAACGCAGCTATGGCCAAGAAAGCCTGATGAGCCTGGGCAACGGCTATCTGGGCCTGCGCGGCGCGCCGCTGTGGAGCACCTGCAGCGATAACCATTATCCGGGCCTGTATGTGGCGGGCGTGTTTAACCGCACGAGCACCGAAGTGGCGGGCCATGATGTGATTAACGAAGATATGGTGAACTGGCCGAACCCGCAGCTGATTAAAGTGTATATTGATGGCGAACTGGTGGATTTTGAAGCGAGCGTGGAAAAACAAGCGACCATTGATTTTAAAAACGCGCTGCAGATTGAACGCTATCAAGTGAAACTGGCGAAAGGCAACCTGACCCTGGTGACCACCAAATTTGTGGACCCGATTAACTTTCATGATTTTGGCTTTGTGGGCGAAATTATTGCGGATTTTAGCTGCAAACTGCGCATTGAAACCTTTACCGATGGCAGCGTGCTGAATCAGAACGTGGAACGCTATCGCGCGTTTGATAGCAAAGAATTTGAAGTGACCAAAATTAGCAAAGGCCTGCTGGTGGCGAAAACCCGCACGAGCGAAATTGAACTGGCGATTGCGAGCAAAAGCTTTCTGAACGGCCTGGCGTTTCCGAAAATTGATAGCGAAAACGATGAAATTCTGGCGGAAGCGATTGAAATTGATCTGCAGAAAAACCAAGAAGTGCAGTTTGATAAAACCATTGTGATTGCGAGCAGCTATGAAAGCAAAAACCCGGTGGAATTTGTGTTAACCGAGCTGAGCGCGACGAGCGTGAGCAAAATTCAAGAAAACAACACCAACTATTGGGAAAAAGTGTGGAGCGATGCGGATATTGTGATTGAAAGCGATCATGAAGATCTGCAGCGCATGGTGCGCATGAACATTTTTCATATTCGCCAAGCGGCGCAGCATGGCGCGAATCAGTTTCTGGATGCGAGCGTGGGCAGCCGCGGCCTGACCGGCGAAGGCTATCGCGGCCATATTTTTTGGGATGAAATTTTTGTGCTGCCGTATTATGCGGCGAACGAACCGGAAACCGCGCGCGATCTGTTACTGTATCGCATTAACCGCCTGACCGCGGCGCAAGAAAACGCGAAAGTGGATGGCGAAAAAGGCGCGATGTTTCCGTGGCAGAGCGGCCTGATTGGCGATGAACAGAGTCAGTTTGTGCATCTGAACACCGTGAACAACGAATGGGAACCGGATAACAGCCGCCGTCAGCGCCATGTGAGCCTGGCGATTGTGTATAACCTGTGGATTTATAGTCAGCTGACCGAAGATGAAAGCATTCTGACCGATGGCGGCCTGGATCTGATTATTGAAACCACGAAATTTTGGCTGAACAAAGCGGAACTGGGCGATGATGGCCGCTATCATATCGATGGCGTGATGGGTCCGGATGAATACCACGAAGCGTATCCGGGCCAAGAAGGCGGCATTTGCGATAACGCGTATACCAACCTGATGCTGACCTGGCAGCTGAACTGGTTAACCGAACTGAGCGAAAAGGGCTTTGAAATTCCGAAAGAACTGCTGGAAAAAGCGCAGAAAGTGCGCAAAAAACTGTATCTGGATATTGATGAAAACGGCGTGATTGCGCAGTATGCGAAATATTTTGAACTGAAAGAAGTGGATTTTGCGGCGTATGAAGCGAAATATGGCGATATTCATCGCATTGATCGCCTGATGAAAGCGGAAGGCATTAGCCCGGATGAGTATCAAGTGGCGAAACAAGCGGATACCCTGATGCTGATTTATAACCTGGGCCAAGAACATGTGACCAAACTGGTGAAACAGCTGGCGTATGAACTGCCGGAAAACTGGCTGAAAGTGAACCGCGATTATTATCTGGCGCGCACCGTGCATGGCAGCACCACGAGCCGCCCGGTGTTTGCGGGCATTGATGTGAAACTGGGCGATTTTGATGAAGCGCTGGATTTTCTGATTACCGCGATTGGCAGCGATTATTATGATATTCAAGGCGGCACCACCGCGGAAGGCGTGCATATTGGCGTTATGGGCGAAACCCTGGAAGTGATTCAGAACGAATTTGCGGGCCTGAGCCTGCGCGAAGGTCAGTTTGCGATTGCGCCGTATCTGCCGAAAAGCTGGACCAAACTGAAATTTAATCAGATTTTTCGCGGCACCAAAGTGGAAATTCTGATTGAAAACGGTCAGCTGTTACTGACCGCGAGCGCGGATCTGCTGACCAAAGTGTATGATGATGAAGTGCAGCTGAAAGCGGGCGTGCAGACCAAATTTGATCTGAAACTCGAGCACCACCACCACCACCACTGA
SEQ ID NO.7 MP amino acid sequence, enterococcus faecalis (Enterococcus faecalis), uniprot ID Q836Y7
MKQIKRLFQIDPWKIRTTHLDKENLRLQESLTSIGNGYMGMRGNFEEHYSGDHHQGTYLAGVWYPDKTRVGWWKNGYPEYFGKVINAINFIAMDLQIDGQTIDLATTPYEDFSLELDMQNGVLSRQFTIQTPKNKVRFSFERFLSLEKKEAAYIHLTIEMLEGTGTITLHSKLDGDVQNEDSNYEEHFWEERAIETQETLGFVTTKTIPNNFEIERFTVTAGMRHFIDGASVVPTYTQQPLALTAELTVSLNEGETTAITKEVLVVTSRDVPETQQITRVNELFAEMTTLYPEAKAGQAAAWAKRWQLADVVIEGDDEAQQGIRFNLFQLFSTYYGEDDRLNIGPKGFTGEKYGGATYWDTEAYAVPLYLALAKPEVTKNLLKYRHNQLPQAIHNAQQQGLKGALYPMVTFTGVECHNEWEITFEEIHRNGAIAYAIYNYVNYTGDEDYLKDAGLEVLVAIARFWADRVHFSQRHKQYMIHGVTGPNEYENNINNNWYTNTIAAWVLRYTRESYLKFQEETTLKIADDELAKWADIVENMYFPVDNELGIFVQHDTFLDKDLMPVSDLPLSELPLNQHWSWDKILRSCFIKQADVLQGIYFFNDAFSLEEKRRNFNFYEPMTVHESSLSPSIHAVLAAELGMEEKAVEMYQRTARLDLDNYNNDTEDGLHITSMTGSWLAIVQGFAQMKTDHQQLKFAPFLPATWTAYSFHINYRNRLLFVEVAADQVAFTLLDGPAIPLTVYDQKYTLKDRLVLPIRKEEVHV
SEQ ID NO.8 MP nucleotide sequence, enterococcus faecalis (Enterococcus faecalis)
atgaaacaaatcaaacgtttattccaaattgatccttggaaaatccgtactacacacttagataaagaaaatttacgtttacaagaatcattaacaagcattggcaacggctatatggggatgcggggaaattttgaagaacactattctggcgaccaccatcaaggaacctatttagcaggtgtttggtatccagacaaaacacgtgttggttggtggaaaaatggttatcctgaatattttggtaaagtgattaacgcaattaatttcatcgcaatggacttacaaattgatggacaaacaattgatttagccactacaccttacgaagatttttctttagaattagacatgcaaaacggggttctttctcggcaattcaccatccaaacaccgaagaataaagtgcgcttctcttttgaacgttttttaagcttagaaaaaaaagaggcagcttacatccacttaacaattgaaatgctggaaggtacaggcacaatcactcttcactcaaaattggatggcgatgttcaaaatgaagacagcaattatgaagaacatttttgggaagaaagagctattgaaacacaagaaacacttggctttgttacgactaagaccattcctaacaactttgaaatagagcgttttacagtgactgcaggcatgcgccatttcatcgatggagcctctgttgtgcctacatatacacaacagcccttggctttaaccgcagaattgacagtgtctttaaacgaaggcgaaacgaccgccatcacaaaagaagtgcttgtcgttaccagtcgtgatgtgccagaaacgcaacaaatcacacgagtaaacgaattatttgcagaaatgactaccttgtaccctgaagcaaaagctgggcaagctgccgcttgggcaaaacgttggcaattagctgatgttgttattgaaggagatgacgaagcccaacaaggcattcgttttaatctgttccaacttttctcaacctactatggagaagacgatcgtttaaacatcggaccaaaaggctttacgggcgaaaaatatggaggtgccacatattgggatacagaagcatatgctgtcccactttacttggcgttggctaagcccgaagttactaaaaatctactgaaatatcgccacaatcaattgcctcaagccattcataatgcccaacaacaagggttaaagggtgcgctttacccaatggttacttttactggtgtcgaatgtcataatgagtgggaaatcacttttgaagagattcacagaaatggtgcgattgcttatgcgatttataattatgtcaactatacaggagacgaagattatctaaaagatgctggtctagaagtacttgtagcaatcgctcgtttttgggcagatcgggtccacttttcccaacgtcacaaacaatatatgattcatggcgtaacggggccaaatgaatatgaaaataatatcaataataactggtataccaataccatcgctgcgtgggttctacgttacacccgtgagtcttatttaaaattccaagaagaaacaacgctcaaaattgcagacgacgaattagctaaatgggcagacattgtagaaaatatgtacttcccagtggataacgaattaggcatttttgtacaacatgacactttcttagataaagatttaatgcctgtttctgatttaccacttagtgagctgcctttaaatcaacattggtcatgggataaaattttacggtcttgtttcattaaacaagccgatgttcttcaaggaatttatttcttcaatgatgccttttctttagaagaaaaacgccggaactttaacttttacgaaccaatgactgtccatgaatcttctctttcaccaagcattcatgctgtgttagctgctgaattaggcatggaagaaaaagcagtggaaatgtatcaacgaacagcgcgccttgacttagataattacaataatgatacagaagatggcttacacattacctcaatgactggtagttggctagcgattgttcaaggttttgcccaaatgaaaacggaccatcaacaactaaaatttgcaccctttttaccagcgacatggacagcctactcattccacattaattatcggaatcgtttactatttgttgaagttgcagcagaccaagtggccttcactttactcgacggtcctgcaattccattaactgtttacgatcaaaaatacacgttaaaagatcgattagttttaccgatcagaaaggaagaagttcatgtttaa
SEQ ID NO.9: PPGK amino acid sequence, mycobacterium tuberculosis (Mycobacterium tuberculosis), uniprot ID:P9WIN1
MTSTGPETSETPGATTQRHGFGIDVGGSGIKGGIVDLDTGQLIGDRIKLLTPQPATPLAVAKTIAEVVNGFGWRGPLGVTYPGVVTHGVVRTAANVDKSWIGTNARDTIGAELGGQQVTILNDADAAGLAETRYGAGKNNPGLVVLLTFGTGIGSAVIHNGTLIPNTEFGHLEVGGKEAEERAASSVKEKNDWTYPKWAKQVIRVLIAIENAIWPDLFIAGGGISRKADKWVPLLENRTPVVPAALQNTAGIVGAAMASVADTTH
SEQ ID NO.10: PPGK nucleotide sequence, mycobacterium tuberculosis (Mycobacterium tuberculosis)
atgaccagcaccggccccgagacgtccgaaacaccgggtgccacgacacagcgtcatggcttcggcatcgacgtcggcggcagcggcatcaagggcggaatcgtcgacttggacaccggccagctgatcggcgaccggatcaagctgctgaccccgcaaccggccactccgttggcggtcgccaaaaccatcgccgaggtcgtcaacggtttcggctggcggggtccgctgggggtgacctatcccggcgtcgtcactcacggcgtcgtccggaccgcggctaacgtggacaagtcctggatagggaccaacgcacgcgacactatcggcgccgagctgggcggtcagcaggtcaccatcctcaacgacgctgatgccgccgggctggccgagacacgctacggggccggcaagaacaaccctggcttagtggtactgctcacattcggaaccgggatcgggtccgcggtcatccacaacgggacgttgatacccaacaccgagttcggacatcttgaggtcggcggcaaggaagcggaggaaagggccgcctcctcggtaaaggaaaagaacgactggacctatccaaagtgggccaagcaggtgatacgcgtgctcatcgccatcgagaacgcgatctggcctgacctgttcatcgccggcggcggcatcagccgcaaggccgacaaatgggtgccgctactggaaaaccgcacaccagtagtgcccgcggccctgcagaacaccgccggaattgtcggtgcggccatggcctctgtcgcagatacgacgcactga
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.
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