Epimerase for synthesizing D-tagatose and application
By mutation of key sites of epimerase, the efficiency of converting D-fructose into D-tagsose is improved, and the problems of unstable and high cost of production raw materials in the prior art are solved, thereby achieving efficient synthesis of D-tagsose.
Patent Information
- Application Number
- CN202510121994.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-03
- Publication Date
- 2025-08-29
AI Technical Summary
In the prior art, the biological preparation method of tagsose has problems such as unstable production raw materials, long reaction cycles and high industrial production costs, resulting in low synthesis efficiency.
The mutant epimerase was used to mutate the key sites of wild-type epimerase to improve the epimerization activity of D-fructose at the fourth carbon position to prepare and efficiently synthesize D-tagsose.
Through the application of mutant epimerase, the synthesis efficiency of D-tagsose is significantly improved, and efficient conversion of D-fructose into D-tagsose is achieved.
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Figure CN120555412A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of biotechnology, and in particular to an epimerase for synthesizing D-tagatose and its application. Background Art
[0002] Tagatose has a natural sweetness that is difficult to distinguish from sucrose, and also has physical properties similar to sucrose. Tagatose is a natural sweetener that is found in small amounts in foods such as milk, cheese, cocoa, and sweet fruits such as apples and oranges. The caloric value of tagatose is 1.5kcal / g, which is one-third of that of sucrose, and its glycemic index (GI) is 3, which is 5% of that of sucrose. Tagatose has a sweet taste similar to sucrose and has a variety of health-promoting functions. In view of this, tagatose can be used as an alternative sweetener that can meet health needs and taste in a variety of products.
[0003] At present, the biological preparation methods of tagatose mainly include the isomerase reaction method and the three-enzyme cascade method. The isomerase reaction method uses arabinose isomerase to catalyze D-galactose to produce D-tagatose. This method uses lactose as the production raw material. The supply of production raw materials is unstable and the reaction cycle is long, which is not conducive to improving the synthesis efficiency of D-tagatose. The three-enzyme cascade method requires whole cells to synthesize D-tagatose from scratch, with low substrate loading and high industrial production costs, which is not conducive to improving the synthesis efficiency of D-tagatose. Summary of the Invention
[0004] In view of the above problems, the present invention provides an epimerase for synthesizing D-tagatose and its application to solve the above technical problems that are not conducive to improving the efficiency of D-tagatose synthesis.
[0005] In a first aspect, the embodiments of the present application provide an epimerase for synthesizing D-tagatose, wherein the amino acid sequence of the epimerase is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
[0006] In a second aspect, an embodiment of the present application provides a polynucleotide encoding the above-mentioned epimerase.
[0007] In a third aspect, an embodiment of the present application provides a recombinant vector comprising the above-mentioned polynucleotide.
[0008] In a fourth aspect, an embodiment of the present application provides a host cell, wherein the host cell comprises the above-mentioned recombinant vector.
[0009] In a fifth aspect, an embodiment of the present application provides a method for preparing an epimerase for synthesizing D-tagatose, comprising: culturing a transformed host cell transformed with a recombinant vector, so that the epimerase accumulates in the cells of the transformed host cell, wherein the recombinant vector contains a nucleotide sequence encoding the above-mentioned epimerase.
[0010] Optionally, the recombinant vector comprises the nucleotide sequence shown in SEQ ID NO: 8, the nucleotide sequence encoding any one of the epimerases, and the nucleotide sequence shown in SEQ ID NO: 9 in sequence.
[0011] In a sixth aspect, the present invention provides a method for synthesizing D-tagatose, comprising:
[0012] The epimerase, the transformed host cell containing the epimerase, or the culture of the transformed host cell containing the epimerase is contacted with D-fructose to convert D-fructose into D-tagatose.
[0013] Optionally, host cells for synthesizing D-tagatose are added to a reaction solution containing D-fructose for fermentation, wherein the reaction solution comprises 0.1 mM to 10 mM of a metal salt and a first buffer, the metal salt being selected from at least one of ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, magnesium sulfate and nickel sulfate, and the first buffer being Tris-HCl buffer, sodium phosphate buffer or potassium phosphate buffer.
[0014] Optionally, the concentration of host cells in the reaction solution is 10 g / L to 200 g / L.
[0015] Optionally, the catalytic pH value is 5.0-10.0, and the catalytic temperature is 50°C-90°C.
[0016] The epimerase and its application for synthesizing D-tagatose provided in the examples of the present application are characterized by generating a mutation at at least one key site in the amino acid sequence shown in SEQ ID NO: 7 that is beneficial for improving the epimerization activity of the 4th carbon position of D-fructose. Through the above-mentioned method, the epimerase exhibits excellent epimerization activity of the 4th carbon position of D-fructose, can efficiently convert D-fructose into D-tagatose, and is beneficial for improving the efficiency of D-tagatose synthesis.
[0017] These and other aspects of the present application will become more readily apparent from the description of the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The predicted protein three-dimensional structure of the wild-type epimerase amino acid sequence is shown.
[0019] Figure 2 The schematic diagram of the structure of the pET-28a-UxaE plasmid in the examples of this application is shown.
[0020] Figure 3 A schematic diagram of the structure of the pET-22b-UxaE plasmid in the examples of this application is shown.
[0021] Figure 4 Shown is a protein SDS-PAGE diagram of the epimerase in the examples of this application.
[0022] Figure 5 The liquid chromatography standard curve of D-tagatose in the examples of the present application is shown.
[0023] Figure 6 The liquid chromatography standard curve of D-fructose in the examples of the present application is shown.
[0024] Figure 7 The figure shows the liquid chromatography results of epimerase catalysis in the examples of the present application.
[0025] Figure 8 The graph showing the change of the catalytic activity of the immobilized cells with the pH value in the examples of the present application is shown.
[0026] Figure 9 The graph shows the change of the catalytic activity of the immobilized cells with temperature in the examples of the present application.
[0027] Figure 10 The graph shows the change of the relative activity of immobilized cells with the number of catalysis times in the examples of the present application. DETAILED DESCRIPTION
[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.
[0029] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute a separate or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.
[0030] The experimental methods in the following examples are conventional methods unless otherwise specified.
[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0032] The terms "epimerase" and "epimerase for synthesizing D-tagatose" herein refer to enzymes that exhibit D-fructose-4-epimerization activity, which can epimerize the 4th carbon position of D-fructose to convert D-fructose into D-tagatose.
[0033] The reaction principle formula for converting D-fructose into D-tagatose using the above-mentioned epimerase is shown below:
[0034]
[0035] It is described herein as "a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO." Obviously, a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO., wherein some of the sequences are deleted, modified, substituted, conservatively substituted or added, can also be used in this application, as long as it can exhibit the same or corresponding activity as the polypeptide, protein, mutant or enzyme having the amino acid sequence as shown in SEQ ID NO. For example, it is not excluded that a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof is added before or after the "polypeptide, protein, mutant or enzyme having the amino acid sequence as shown in SEQ ID NO." Moreover, a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO., when added with the above-mentioned sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof, also falls within the scope of this application, as long as it exhibits the same or corresponding activity as the amino acid sequence as shown in SEQ ID NO. after the addition of the above-mentioned sequence.
[0036] The terms "tagatose" and "D-tagatose" are used interchangeably herein and both refer to a ketohexose monosaccharide.
[0037] The present embodiment provides an epimerase for synthesizing D-tagatose, wherein the epimerase has at least one mutation in the amino acid sequence shown in SEQ ID NO: 7.
[0038] Among them, the amino acid sequence shown in SEQ ID NO: 7 can be obtained from GenBank of NCBI. Specifically, the amino acid sequence shown in SEQ ID NO: 7 is derived from Thermotoga neapolitana, and the number in GenBank is WP_015918744.1. In this specification, the enzyme formed by the amino acid sequence shown in SEQ ID NO: 7 can be referred to as a wild-type epimerase, and the enzyme formed by each mutated amino acid sequence can be referred to as a mutant epimerase. The amino acid sequence shown in SEQ ID NO: 7 is input into the AlphaFold2 model, and the following is obtained: Figure 1 The predicted three-dimensional structure of the protein is shown.
[0039] right Figure 1 The three-dimensional structure of the protein was analyzed, and multiple key sites of the amino acid sequence shown in SEQ ID NO: 7 that are related to the binding of the substrate D-fructose and the activity of D-fructose-4-epimerization were selected as research objects, including R100 (arginine R at position 100), Q123 (glutamine Q at position 123), Q124 (glutamine Q at position 124), S125, V126, R127, E128, N129, E130, R131, T132, G133, R134, W136, and D159. , A160, D161, H162, V163, K164, F177, F180, T181, I182, D183, S185, E266, S268, D270, E271, A299, L300, F302, W306, K308, S339, L340, H341, S342, G343, K346, H362, V363, K364, A366, G367, S402, Y403, H437, and Y440.
[0040] Saturation mutation screening was performed on the above key sites to obtain an epimerase with improved D-fructose-4-epimerization activity.
[0041] In the epimerase for synthesizing D-tagatose of this embodiment, at least one of the following mutations occurs in the amino acid sequence of SEQ ID NO: 7: mutation at position 100, mutation at position 123, mutation at position 124, mutation at position 125, mutation at position 126, mutation at position 127, mutation at position 128, mutation at position 131, mutation at position 132, mutation at position 133, mutation at position 134, mutation at position 136, mutation at position 159, mutation at position 160, mutation at position 161, mutation at position 162, mutation at position 163, mutation at position 164, mutation at position 177, mutation at position 180, mutation at position 181, mutation at position 182, mutation at position 183, mutation at position 184, 83, mutation at position 185, mutation at position 266, mutation at position 268, mutation at position 270, mutation at position 271, mutation at position 299, mutation at position 300, mutation at position 302, mutation at position 306, mutation at position 308, mutation at position 339, mutation at position 340, mutation at position 342, mutation at position 343, mutation at position 346, mutation at position 362, mutation at position 363, mutation at position 364, mutation at position 366, mutation at position 367, mutation at position 402, mutation at position 403, mutation at position 437, or mutation at position 440. In the corresponding mutations, the amino acid residues at the corresponding positions in the amino acid sequence of SEQ ID NO: 7 are substituted by other amino acids.
[0042] In this embodiment, the above-mentioned "other amino acids" are not limited, as long as they are different from the amino acids corresponding to each position. Specifically, the other amino acids in the embodiments of the present application can be one or more amino acids selected from the following: non-polar amino acids glycine G, alanine A, valine V, leucine L, isoleucine I, methionine M, phenylalanine F, tryptophan W and proline P; polar amino acids serine S, threonine T, cysteine C, tyrosine Y, asparagine N and glutamine Q; acidic amino acids aspartic acid D and glutamic acid E; and basic amino acids lysine K, arginine R and histidine H, but are not limited thereto.
[0043] In this embodiment, positions 100 (arginine R), 125 (serine S), 127 (arginine R), 131 (arginine R), 268 (serine S), 270 (aspartic acid D), 271 (glutamic acid E), 308 (lysine K), 339 (serine S), 340 (leucine L), 342 (serine S), 366 (alanine A), 402 (serine S), 437 (histidine H), and 440 (tyrosine Y) in the amino acid sequence of SEQ ID NO: 7 are all key sites related to the catalytic activity of the epimerization of the fourth carbon position of D-fructose. By mutating the above-mentioned key sites, the resulting epimerase exhibits excellent epimerization activity at the 4th carbon position of D-fructose, and can efficiently convert D-fructose into D-tagatose, which is beneficial to improving the efficiency of D-tagatose synthesis.
[0044] As an embodiment, the epimerase for synthesizing D-tagatose of this example has a mutation in the amino acid sequence corresponding to SEQ ID NO: 7, and the mutation is a mutation at position 100, a mutation at position 125, a mutation at position 127, a mutation at position 131, a mutation at position 268, a mutation at position 270, a mutation at position 271, a mutation at position 308, a mutation at position 339, a mutation at position 340, a mutation at position 342, a mutation at position 366, a mutation at position 402, a mutation at position 437, or a mutation at position 440.
[0045] As an embodiment, the epimerase for synthesizing D-tagatose of this embodiment has three mutations in the amino acid sequence shown in SEQ ID NO: 7. The first mutation is a mutation of serine S at position 125 shown in SEQ ID NO: 7 to aspartic acid D, the second mutation is a mutation of serine S at position 268 shown in SEQ ID NO: 7 to alanine A, and the third mutation is one of the following: a mutation at position 123, a mutation at position 124, a mutation at position 126, a mutation at position 129, a mutation at position 130, a mutation at position 132, a mutation at position 133, a mutation at position 134, a mutation at position 136, a mutation at position 159, a mutation at position 163, a mutation at position 164, or a mutation at position 177. mutation at position 180 or mutation at position 181 or mutation at position 182 or mutation at position 183 or mutation at position 185 or mutation at position 266 or mutation at position 299 or mutation at position 300 or mutation at position 302 or mutation at position 306 or mutation at position 343 or mutation at position 346 or mutation at position 362 or mutation at position 363 or mutation at position 364 or mutation at position 367.
[0046] In some embodiments, the epimerase has an amino acid sequence as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 3, as shown in SEQ ID NO: 4, as shown in SEQ ID NO: 5, or as shown in SEQ ID NO: 6.
[0047] Among them, the epimerase corresponding to the amino acid sequence shown in SEQ ID NO: 1, which produces a mutation of serine S at position 125 to aspartic acid D, a mutation of serine S at position 268 to alanine A, and a mutation of glutamine Q at position 124 to lysine K, exhibits a significant improvement in the catalytic activity of epimerization of the 4th carbon position of D-fructose.
[0048] Among them, the epimerase corresponding to the amino acid sequence shown in SEQ ID NO: 2, which produces a mutation of serine S at position 125 to aspartic acid D, a mutation of serine S at position 268 to alanine A, and a mutation of threonine T at position 132 to alanine A, exhibits a significant improvement in the catalytic activity of epimerization of the 4th carbon position of D-fructose.
[0049] Among them, the epimerase corresponding to the amino acid sequence shown in SEQ ID NO: 3, which produces a mutation of serine S at position 125 to aspartic acid D, a mutation of serine S at position 268 to alanine A, and a mutation of glycine G at position 133 to valine V, exhibits a significant improvement in the catalytic activity of epimerization of the 4th carbon position of D-fructose.
[0050] Among them, the epimerase corresponding to the amino acid sequence shown in SEQ ID NO: 4, which produces a mutation of serine S at position 125 to aspartic acid D, a mutation of serine S at position 268 to alanine A, and a mutation of tryptophan W at position 136 to alanine A, exhibits a significant improvement in the catalytic activity of epimerization of the 4th carbon position of D-fructose.
[0051] Among them, the epimerase corresponding to the amino acid sequence shown in SEQ ID NO: 5, which produces a mutation of serine S at position 125 to aspartic acid D, a mutation of serine S at position 268 to alanine A, and a mutation of lysine K at position 164 to arginine R, exhibits a significant improvement in the catalytic activity of epimerization of the 4th carbon position of D-fructose.
[0052] Among them, the epimerase corresponding to the amino acid sequence shown in SEQ ID NO: 6, which produces a mutation of serine S at position 125 to aspartic acid D, a mutation of serine S at position 268 to alanine A, and a mutation of serine S at position 185 to histidine H, exhibits a significant improvement in the catalytic activity of epimerization of the 4th carbon position of D-fructose.
[0053] The present application also provides a polynucleotide encoding an epimerase for synthesizing D-tagatose, wherein the polynucleotide comprises a nucleotide sequence corresponding to the amino acid sequence of the epimerase.
[0054] The polynucleotide is a DNA chain or RNA chain formed by polymerization of several nucleotides.
[0055] The polynucleotide only needs to encode the epimerase for synthesizing D-tagatose, and any nucleotide in the polynucleotide may be chemically modified.
[0056] The present application also provides a recombinant vector comprising the aforementioned polynucleotide encoding the epimerase for synthesizing D-tagatose.
[0057] Among them, a recombinant vector refers to a DNA preparation containing a nucleic acid sequence encoding a polynucleotide for synthesizing an epimerase of D-tagatose, which may also contain a control sequence. In the recombinant vector, the nucleic acid sequence encoding a polynucleotide for synthesizing an epimerase of D-tagatose is operably linked to a suitable control sequence so that the epimerase for synthesizing D-tagatose can be expressed in a suitable host. Specifically, the control sequence may include but is not limited to a promoter capable of initiating transcription, any operator sequence for regulating transcription, a suitable mRNA ribosome binding site, and a sequence for controlling transcription and translation termination. After transformation into a suitable host cell, the recombinant vector can replicate or function independently of the host genome, or can be integrated into the genome itself to replicate or function.
[0058] Among them, there is no particular limitation on the type of recombinant vector, as long as it can replicate in the host cell, any vector known in the art can be used. For example, commonly used vectors in the art can include natural or recombinant plasmids, cosmids, viruses and phages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and the pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.
[0059] As an embodiment, the recombinant vector comprises, in sequence, the nucleotide sequence shown in SEQ ID NO: 8, a nucleotide sequence encoding any epimerase, and the nucleotide sequence shown in SEQ ID NO: 9.
[0060] The present application also provides a host cell comprising the above-mentioned recombinant vector.
[0061] Wherein, the above-mentioned recombinant vector is transformed into a host cell so that the epimerase for synthesizing D-tagatose can be synthesized in the host cell of the present embodiment. The recombinant vector is imported into the host cell, and the polynucleotide encoding the epimerase for synthesizing D-tagatose in the recombinant vector can be expressed in the host cell so that the host cell can synthesize the above-mentioned epimerase for synthesizing D-tagatose. The polynucleotide can be inserted into the chromosome of the host cell or be located outside the chromosome of the host cell or be inserted into the chromosome of the host cell and be located outside the chromosome at the same time. The polynucleotide can be DNA or RNA, as long as it can be expressed in the host cell. Exemplarily, the recombinant vector can be an expression cassette, including a promoter, a transcription termination element, a ribosomal domain, and a translation termination element operably connected to the polynucleotide.
[0062] As an embodiment, the host cell can be Escherichia, Erwinia, Serratia, Providencia, Corynebacterium or Brevibacterium; illustratively, the host cell can be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or Aspergillus oryzae.
[0063] The present application also provides a method for preparing an epimerase for synthesizing D-tagatose, comprising the following steps: culturing a transformed host cell transformed with a recombinant vector, wherein the recombinant vector contains a nucleotide sequence encoding the epimerase, so that the epimerase accumulates in the cells of the transformed host cell.
[0064] Among them, the transformed host cells transformed with the recombinant vector are grown under appropriate environmental conditions, and the methods of culturing the host cells may include but are not limited to batch culture, continuous culture or fed-batch culture. In order to achieve appropriate environmental conditions, the pH can be adjusted using an alkaline compound or an acidic compound to adjust the pH to an appropriate value, for example, the pH can be adjusted to 5 to 9, or the pH can be adjusted to 6 to 8, or the pH can be adjusted to 6.5 to 7.0. Exemplarily, the alkaline compound can be sodium hydroxide, potassium hydroxide or ammonia, and the acidic compound can be hydrochloric acid, phosphoric acid or sulfuric acid. In order to achieve appropriate environmental conditions, the temperature can be controlled at 25 to 60°C, or the temperature can be controlled at 30 to 45°C, or oxygen or an oxygen-containing gas mixture can be injected to maintain the culture environment in an aerobic state.
[0065] The carbon source contained in the host cell culture medium may include, but is not limited to, at least one of carbohydrates, oils or fats, fatty acids, alcohols, or organic acids. Examples of carbohydrates include glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose. Examples of oils or fats include soybean oil, sunflower oil, peanut oil, and coconut oil. Examples of fatty acids include palmitic acid, stearic acid, and linoleic acid. Examples of alcohols include glycerol and ethanol. Examples of organic acids include acetic acid. The phosphorus source contained in the host cell culture medium may include, but is not limited to, at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
[0066] The host cell culture medium may further include a metal salt, such as magnesium sulfate or iron sulfate, and / or the host cell culture medium may further include other substances required for growth, such as amino acids or vitamins.
[0067] In one embodiment, the recombinant vector may include a nucleotide sequence corresponding to the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6. In some embodiments, the recombinant vector may include, in sequence, the nucleotide sequence shown in SEQ ID NO: 8, a nucleotide sequence encoding any epimerase, and the nucleotide sequence shown in SEQ ID NO: 9.
[0068] One embodiment of the present application provides a method for synthesizing D-tagatose, comprising:
[0069] The epimerase, the transformed host cell containing the epimerase, or the culture of the transformed host cell containing the epimerase is contacted with D-fructose to convert D-fructose into D-tagatose.
[0070] The use of the above-mentioned epimerase to catalyze the conversion of D-fructose into D-tagatose can improve the conversion efficiency of D-tagatose.
[0071] The catalytic system for epimerase-catalyzed D-fructose conversion may include a suitable excipient, which may include but is not limited to at least one of a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent. Exemplarily, the catalytic system for epimerase-catalyzed D-fructose conversion may include a buffer, which may include but is not limited to at least one of Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer.
[0072] The catalytic system for epimerase-catalyzed D-fructose conversion may further include a metal salt, such as an ammonium salt, a nickel salt, an iron salt, a cobalt salt, a magnesium salt, or a manganese salt. For example, the metal salt may include at least one of ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, and nickel sulfate. Furthermore, the concentration of the metal salt may be 0.1 mM to 10 mM, such as 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM.
[0073] The contact between the epimerase and D-fructose can be performed at pH 5.0 to 10.0. For example, the contact between the epimerase and D-fructose can be performed at pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, or pH 10.0.
[0074] The contact between the epimerase and D-fructose can be carried out at a temperature of 50°C to 90°C. For example, the contact between the epimerase and D-fructose can be carried out at 50°C, 60°C, 70°C, 80°C or 90°C.
[0075] The contact time between the epimerase and D-fructose can be 2 hours to 24 hours. For example, the contact time between the epimerase and D-fructose can be 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.
[0076] In one embodiment, the transformed host cells containing the above-mentioned epimerase are contacted with D-fructose to produce D-tagatose, and the concentration of the host cells is 10 g / L to 200 g / L, for example, the concentration of the host cells can be 10 g / L, 25 g / L, 50 g / L, 75 g / L, 100 g / L, 150 g / L or 200 g / L; the concentration of D-fructose is 50 g / L to 500 g / L, for example, the concentration of D-fructose can be 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L or 500 g / L.
[0077] An embodiment of the present application also provides an immobilized cell, comprising a carrier and a host cell immobilized on the carrier, wherein the carrier comprises diatomaceous earth, polyethyleneimine, and tris(hydroxymethyl)phosphine; the host cell synthesizes an epimerase, and the epimerase has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 6.
[0078] Among them, the chemical composition of diatomaceous earth is mainly silicon dioxide, and its surface has a large number of orderly arranged pores, which has a strong adsorption effect, adsorbing the host cells on its surface. Polyethyleneimine and trihydroxymethyl phosphine can cross-link with the hydroxyl functional groups on the surface of diatomaceous earth to form immobilized host cells.
[0079] The mass ratio of diatomaceous earth to host cells is 0.03-0.15 g:0.5-6 g, the volume of polyethyleneimine is 0.075 mL-0.375 mL, and the volume of trishydroxymethyl phosphine is 0.075 mL-0.375 mL.
[0080] Illustratively, the host cells may be host cell sludge obtained by centrifuging wet cells of the host cells.
[0081] The present invention also provides a method for preparing immobilized cells, comprising the following steps:
[0082] Step S1: adding the host cells to a second buffer to obtain a reaction solution, and adding diatomaceous earth to the reaction solution;
[0083] The second buffer solution can be, for example, a Tris-HCl buffer solution at pH 8.0, the volume of the second buffer solution can be, for example, 15 mL. The host cells are the pellet collected by centrifugation of wet cells, and the mass of the host cells can be, for example, 0.5 g to 6 g. That is, the volume of the reaction solution is 15 mL, and the mass of the added diatomaceous earth can be, for example, 0.03 g to 0.15 g.
[0084] Step S2: adding polyethyleneimine to the reaction solution to perform a first reaction;
[0085] The volume ratio of polyethyleneimine to the reaction solution can be, for example, 0.5% to 2.5%, that is, the volume of polyethyleneimine is 0.075 mL to 0.375 mL.
[0086] The first reaction time may be 0.5 to 2.5 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours or 2.5 hours.
[0087] Step S3: adding tris(hydroxymethyl)phosphine to the reaction solution after the first reaction, performing a second reaction, and obtaining immobilized cells.
[0088] The volume ratio of trishydroxymethyl phosphine to the reaction solution can be, for example, 0.5% to 2.5%, that is, the volume of trishydroxymethyl phosphine is 0.075 mL to 0.375 mL.
[0089] The second reaction time may be 0.5 hours to 2.5 hours, for example, 0.5 hours, 1 hour, 1.5 hours, 2 hours or 2.5 hours.
[0090] After the second reaction, the reaction solution was centrifuged at 3500 rpm for 5 min, the supernatant was removed, and then washed with distilled water 2 to 3 times. The solid particles were taken out and placed in a clean culture dish. They were air-dried in a fume hood for 12 to 14 h until completely dry, weighed, sealed, and stored at room temperature.
[0091] One embodiment of the present application provides a method for synthesizing D-tagatose, comprising:
[0092] The immobilized cells are brought into contact with D-fructose to convert D-fructose into D-tagatose.
[0093] The catalytic system for the immobilized cells to catalyze the production of D-fructose may include a suitable excipient, which may include, but is not limited to, at least one of a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent. For example, the catalytic system for the immobilized cells to catalyze the production of D-fructose may include a buffer, which may include, but is not limited to, at least one of Tris-HCl, a sodium phosphate buffer, and a potassium phosphate buffer.
[0094] The catalytic system for the immobilized cells to catalyze the production of D-fructose may further include a metal salt. The metal salt may include, for example, an ammonium salt, a nickel salt, an iron salt, a cobalt salt, a magnesium salt, or a manganese salt. For example, the metal salt may include at least one of ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, and nickel sulfate. Furthermore, the concentration of the metal salt may be 0.1 mM to 10 mM, for example, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM.
[0095] The contact between the immobilized cells and D-fructose can be carried out at pH 4.0 to 10.0. For example, the contact between the immobilized enzyme and D-fructose can be carried out at pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0, or pH 10.0.
[0096] The contact between the immobilized cells and D-fructose can be carried out at a temperature of 50°C to 90°C. For example, the contact between the immobilized enzyme and D-fructose can be carried out at 50°C, 60°C, 70°C, 80°C or 90°C.
[0097] The contact time between the immobilized cells and D-fructose can be 2 hours to 24 hours. For example, the contact time between the immobilized enzyme and D-fructose can be 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.
[0098] The concentration of the immobilized cells may be 1 mg / mL to 10 mg / mL, for example, 1 mg / mL, 3 mg / mL, 5 mg / mL or 10 mg / mL.
[0099] Example
[0100] The encoding gene of the epimerase of Examples 1 to 6 and Comparative Example 1 of the present application can be referred to as the UxaE gene. The UxaE gene contains a nucleotide sequence corresponding to the amino acid sequence of the epimerase described above. For example, the UxaE gene in Examples 1 to 6 contains a nucleotide sequence corresponding to the amino acid sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 6. The UxaE gene is constructed into the pET-28a plasmid to obtain the following: Figure 2 The recombinant vector shown in FIG. 1 was constructed by inserting the UxaE gene into the pET-22b plasmid to obtain the following: Figure 3 The recombinant vector shown.
[0101] For example, Figure 2 An example of the recombinant vector, pET-28a-UxaE, includes, in sequence, the nucleotide sequence set forth in SEQ ID NO: 8, a nucleotide sequence encoding any epimerase, and the nucleotide sequence set forth in SEQ ID NO: 9. In SEQ ID NO: 8, positions 559 to 1374 represent a kanamycin resistance gene; positions 4983 to 5001 represent a T7 promoter; and positions 139 to 186 represent a T7 terminator.
[0102] The recombinant vector used in each example and comparative example 1 of the present application is a pET-28a plasmid containing the UxaE gene, hereinafter referred to as pET-28a-UxaE; the host cell used in each example and comparative example 1 of the present application is Escherichia coli.
[0103] The steps of each embodiment and comparative example 1 in this application are as follows:
[0104] Protein expression
[0105] a. Transform pET-28a-UxaE into E. coli; select a single clone of pET-28a-UxaE or a strain stored at -80°C and inoculate it into a small test tube containing 5 mL of LB liquid medium (Kan+, 100 μg / mL) and incubate overnight at 37°C, 220 rpm to serve as the seed solution.
[0106] b. Transfer the seed solution into 50 mL LB liquid medium (Kan+, 100 μg / mL) and culture in a shaking incubator at 37°C and 220 rpm for reactivation.
[0107] c. Transfer the reactivated bacterial solution into 800 mL of 2YT liquid medium (Kan+, 100 μg / mL) at a 1% inoculum volume and culture on a shaker at 37°C and 220 rpm until the OD600 reaches approximately 0.6-0.8.
[0108] d. Lower the shaker temperature to 16-18°C. After the culture solution cools down, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 hours.
[0109] e. After expression is completed, the above culture solution is collected into a bottle and centrifuged in a precooled centrifuge at 4°C, 5500 rpm, for 10 minutes.
[0110] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the cells using a vortex shaker.
[0111] g. Centrifuge the resuspended cells again at 5500 rpm for 10 minutes. Discard the supernatant, add 30 mL of protein purification buffer, and resuspend the cells using a vortex shaker (no solid particles). Pour the suspension into a 50 mL centrifuge tube and store in a -80°C refrigerator.
[0112] Protein purification
[0113] a. Cell disruption: The collected bacterial suspension was disrupted using a high-pressure, low-temperature disruptor at 800-1000 bar and 4°C for 3-5 minutes to fully lyse the cells, thereby releasing the expressed target protein and dissolving it in the protein buffer.
[0114] b. Centrifugation: Centrifuge the broken bacterial solution at 8000 rpm in a pre-cooled 4°C centrifuge for 60 minutes. Take the precipitate and supernatant after centrifugation, prepare the sample, and collect the supernatant;
[0115] c. Purification: The supernatant was purified by nickel affinity chromatography. The specific steps are as follows:
[0116] (1) Column equilibration: First wash with dd H2O for 2 column volumes, then equilibrate the Ni affinity chromatography column with protein buffer for 1 column volume.
[0117] (2) Sample loading: Take 50 μl of the supernatant and slowly pass it through the Ni affinity chromatography column. Allow it to flow through (can be repeated once) and take the first few drops of the flow-through sample.
[0118] (3) Elution of target protein: Use 30 mL of protein buffer containing 20 mM, 50 mM, 100 mM, 200 mM, and 300 mM imidazole to elute the bound impurities. Take the first few drops of flow-through sample, prepare the sample, and detect it by 12% SDS-PAGE.
[0119] d. Concentration and exchange of buffer: The collected protein eluate containing the target protein was centrifuged and concentrated (4°C, 3400 rpm) using a 50 mL Amicon ultrafiltration tube (10 kDa, Millipore) to 1 mL. 10 mL of protein buffer was then added and the eluate was concentrated to 1 mL. This process was repeated once to ensure the removal of imidazole from the protein, resulting in the purified protein Ceas.
[0120] Protein concentration determination
[0121] Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
[0122] First, use the protein absorbance at 280nm to perform a preliminary determination of the protein concentration, and then dilute the protein concentration to 0.5-1mg / mL based on the initial measured value. Prepare the reaction solution by mixing reagent A and reagent B in the BCA Protein Assay Kit at a ratio of 50:1. Take 200uL of reaction solution and place it in an ELISA plate. Add 25uL of diluted protein to the reaction solution, mix it with a gun, and place it at 37°C for 30 minutes. Place the ELISA plate in an ELISA instrument to measure the absorbance at 562nm, and process the data according to the protein standard curve to obtain the protein concentration. The protein SDS-PAGE gel is shown in the figure below. Figure 4 shown.
[0123] Enzyme-catalyzed reactions
[0124] In vitro enzyme-catalyzed reaction conditions:
[0125] Reaction buffer: at least one selected from Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer;
[0126] Reaction pH: pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, or pH 10;
[0127] The final fructose concentration was 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, or 500 g / L;
[0128] Metal salt type: ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, magnesium sulfate, or nickel sulfate;
[0129] The final concentration of the metal salt is 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM;
[0130] Protease amount: 1 mg / mL, 3 mg / mL, 5 mg / mL, or 10 mg / mL;
[0131] Reaction temperature: 50°C, 60°C, 70°C, 80°C, or 90°C;
[0132] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, or 24h;
[0133] The total reaction volume was 0.5 mL, 1 mL, 5 mL, or 10 mL.
[0134] Each Example and Comparative Example can be prepared by adding magnesium sulfate, epimerase, and D-fructose to Tris-HCl at pH 8 to form a catalytic solution; the concentration of magnesium sulfate in this catalytic solution is 2 mM, the concentration of epimerase is 3 mg / mL, and the concentration of D-fructose is 200 g / L. The catalytic solution is reacted at 60°C for 2 hours, and the conversion activity of each Example and Comparative Example is measured.
[0135] D-tagatose detection
[0136] Liquid phase detection conditions are as follows:
[0137] Mobile phase: 80% acetonitrile, 20% water;
[0138] Liquid phase column Shimpack GIST, NH2;
[0139] Temperature: 40℃;
[0140] Flow rate: 0.5 mL / min;
[0141] Injection volume: 10uL.
[0142] The standard curve of D-tagatose can be found in Figure 5 As shown, the standard curve of D-fructose is shown in Figure 6 As shown, the liquid phase diagram of the reaction of the embodiment is shown in Figure 7As shown, each epimerase in Examples 1 to 141 and Comparative Example 1 converts D-fructose into D-tagatose, and all have D-fructose-4-epimerization activity.
[0143] The results of Examples 1 to 6 and Comparative Example 1 are specifically shown in Table 1, wherein the relative activity of the epimerase in Comparative Example 1 is 1, and the relative activities of the epimerase in Examples 1 to 6 are shown based on the relative activity of the epimerase in Comparative Example 1.
[0144] Table 1 Parameters of Examples 1 to 6 and Comparative Example 1
[0145]
[0146] Immobilized cell preparation
[0147] The induced host cell wet bacteria were centrifuged at 8000 rpm for 10 minutes, the precipitate (bacteria) was collected, and the precipitate was resuspended in buffer A (50 mM Tris-HCl pH 8.0). After centrifugation again, the supernatant was removed to obtain a bacterial sludge precipitate. 2 g of bacterial sludge precipitate was weighed and dissolved in 15 mL of buffer A. 6 g / L of diatomaceous earth was added, and then a 1% volume fraction of polyethyleneimine (liquid) was added to react for 1 hour. Subsequently, a 1.5% volume fraction of THP aqueous solution was added and reacted for 2 hours. The mixture was centrifuged at 3500 rpm for 5 minutes, the supernatant was removed, and then washed with distilled water 2-3 times. The solid particles were taken out and placed in a clean culture dish. Air-dried in a fume hood for 12 to 14 hours until completely air-dried, weighed, sealed, and stored at room temperature.
[0148] Immobilized cell-catalyzed reactions
[0149] In vitro enzyme-catalyzed reaction conditions:
[0150] Reaction buffer: Tris-HCl, sodium phosphate buffer, potassium phosphate buffer;
[0151] Reaction pH: 4, 5, 6, 7, 8, 9, 10;
[0152] The final fructose concentrations were 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, and 500 g / L;
[0153] Metal salt types: ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, nickel sulfate;
[0154] The final concentrations of metal salts were 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM;
[0155] Amount of immobilized cells: 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL;
[0156] Reaction temperature: 50℃, 60℃, 70℃, 80℃, 90℃;
[0157] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h;
[0158] The total reaction system was 0.5 mL, 1 mL, 5 mL, and 10 mL.
[0159] Immobilized cell catalysis pH optimization experiment
[0160] The catalytic pH values in Examples 7 to 12 are different, but the other parameters are the same.
[0161] Table 2 Parameters of Examples 7 to 13
[0162]
[0163] The results are as follows Figure 8 As shown, the relative activities of the immobilized cells in Examples 7 to 13 are shown based on the relative activities of the epimerase in Example 11, which is 100%. The catalytic activity is good at a catalytic pH of 7.5 to 8.5.
[0164] Temperature optimization experiment for immobilized cell catalysis
[0165] The catalytic temperatures in Examples 14 to 18 are different, but the other parameters are the same.
[0166] Table 3 Parameters of Examples 13 to 18
[0167]
[0168] The results are as follows Figure 9 As shown, the relative activities of the immobilized cells in Examples 13 to 18 are shown based on the relative activities of the epimerase in Example 16, which is 100%. Good catalytic activity was observed at a catalytic temperature of 60°C to 80°C.
[0169] Immobilized cell cycle number experiment
[0170] Table 4 Parameters of Example 19
[0171]
[0172] The results are as follows Figure 10 As shown, after the immobilized cells were reused 7 times, the residual activity remained above 70%.
[0173] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0174] The above embodiments merely represent preferred embodiments of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.
Claims
1. An epimerase for synthesizing D-tagatose, characterized in that The amino acid sequence of the epimerase is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:
6.
2. A polynucleotide, characterized in that The polynucleotide encodes the epimerase according to claim 1.
3. A recombinant vector, characterized in that The recombinant vector comprises the polynucleotide according to claim 2.
4. A host cell, characterized in that The host cell comprises the recombinant vector according to claim 3.
5. A method for preparing an epimerase for synthesizing D-tagatose, characterized in that: include: The transformed host cell transformed with the recombinant vector contains a nucleotide sequence encoding the epimerase according to claim 1 and is cultured to accumulate the epimerase in the cell body of the transformed host cell.
6. The method for preparing epimerase for synthesizing D-tagatose according to claim 5, characterized in that: The recombinant vector sequentially comprises the nucleotide sequence shown in SEQ ID NO: 8, the nucleotide sequence encoding any one of the epimerases, and the nucleotide sequence shown in SEQ ID NO:
9.
7. A method for synthesizing D-tagatose, characterized in that: include: The epimerase according to claim 1, or a transformed host cell containing the epimerase according to claim 1, or a culture of the transformed host cell containing the epimerase according to claim 1 is contacted with D-fructose to convert D-fructose into D-tagatose.
8. The method for synthesizing D-tagatose according to claim 7, wherein Host cells for synthesizing D-tagatose are added to a reaction solution containing D-fructose for fermentation, wherein the reaction solution includes 0.1 mM to 10 mM of a metal salt and a first buffer, wherein the metal salt is selected from at least one of ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, magnesium sulfate, and nickel sulfate, and the first buffer is Tris-HCl buffer, sodium phosphate buffer, or potassium phosphate buffer.
9. The method for synthesizing D-tagatose according to claim 7, wherein The concentration of the host cells in the reaction solution is 10 g / L to 200 g / L.
10. The method for synthesizing D-tagatose according to claim 7, wherein The catalytic pH value is 5.0-10.0, and the catalytic temperature is 50°C-90°C.