Epimerase for synthesizing D-tagatose and its application

Abstract

The present application relates to the field of biotechnology, and in particular to an epimerase for synthesizing D-tagatose and its application, wherein the epimerase produces a mutation at position 100, or at position 125, or at position 127, or at position 131, or at position 268, or at position 270, or at position 271, or at position 308, or at position 339, or at position 340, or at position 342, or at position 366, or at position 402, or at position 437, or at position 440 in the amino acid sequence shown in SEQ ID NO. 142; through the above-mentioned method, the epimerase exhibits excellent epimerization activity at the 4th carbon position of D-fructose, can efficiently convert D-fructose into D-tagatose, and is beneficial to improving the synthesis efficiency of D-tagatose.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an isomerase 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 present 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 sucrose, and its glycemic index (GI) is 3, which is 5% of sucrose. Tagatose has a sweet taste similar to sucrose and has a variety of functions that are beneficial to health. In view of this, in a variety of products, tagatose can be used as an alternative sweetener that can meet health needs and taste.

[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 the whole cell to synthesize D-tagatose from scratch, the substrate loading is low, and the industrial production cost is high, which is not conducive to improving the synthesis efficiency of D-tagatose. Summary of the invention

[0004] In view of the above problems, the embodiments of the present application provide an epimerase for synthesizing D-tagatose and its application to solve the above technical problems that are not conducive to improving the synthesis efficiency of D-tagatose.

[0005] In a first aspect, the embodiments of the present application provide a diastereomerase for synthesizing D-tagatose, wherein the diastereomerase has an amino acid sequence as shown in any one of SEQ ID NO. 1 to SEQ ID NO. 141.

[0006] Optionally, the epimerase produces a mutation at position 100, or a mutation at position 125, or a mutation at position 127, or a mutation at position 131, or a mutation at position 268, or a mutation at position 270, or a mutation at position 271, or a mutation at position 308, or a mutation at position 339, or a mutation at position 340, or a mutation at position 342, or a mutation at position 366, or a mutation at position 402, or a mutation at position 437, or a mutation at position 440 in the amino acid sequence as shown in SEQ ID NO.142.

[0007] Optionally, the epimerase produces a mutation of serine S at position 125 shown in SEQ ID NO.142 to aspartic acid D, as well as a mutation at position 127, or a mutation at position 131, or a mutation at position 268, or a mutation at position 270, or a mutation at position 271, or a mutation at position 308, or a mutation at position 340, or a mutation at position 341, or a mutation at position 342, or a mutation at position 366, or a mutation at position 402, or a mutation at position 437, or a mutation at position 440.

[0008] Optionally, the epimerase produces a mutation of serine S at position 125 shown in SEQ ID NO.142 to aspartic acid D, a mutation of serine S at position 268 to alanine A, and 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 Mutation at position 177 or 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.

[0009] In a second aspect, an embodiment of the present application provides a polynucleotide encoding the above-mentioned epimerase.

[0010] In a third aspect, an embodiment of the present application provides a recombinant vector, which includes the above-mentioned polynucleotide.

[0011] In a fourth aspect, an embodiment of the present application provides a host cell, wherein the host cell comprises the above-mentioned recombinant vector.

[0012] In a fifth aspect, an embodiment of the present application provides a method for preparing a diastereomerase for synthesizing D-tagatose, comprising culturing a transformed host cell transformed with a recombinant vector, so that the diastereomerase accumulates in the bacteria of the transformed host cell, wherein the recombinant vector contains a nucleotide sequence encoding the above-mentioned diastereomerase.

[0013] Optionally, the recombinant vector comprises the nucleotide sequence shown in SEQ ID NO. 143, the nucleotide sequence encoding any one of the epimerases, and the nucleotide sequence shown in SEQ ID NO. 144 in sequence;

[0014] Alternatively, the recombinant vector comprises, in sequence, the nucleotide sequence shown in SEQ ID NO. 145, the nucleotide sequence encoding any one of the epimerases, and the nucleotide sequence shown in SEQ ID NO. 146.

[0015] In a sixth aspect, the present invention provides a method for synthesizing D-tagatose, comprising:

[0016] The above-mentioned epimerase, or a transformed host cell containing the above-mentioned epimerase, or a culture of the transformed host cell containing the above-mentioned epimerase is contacted with D-fructose to convert D-fructose into D-tagatose.

[0017] The epimerase and application for synthesizing D-tagatose provided in the embodiments of the present application, wherein the epimerase produces a mutation at at least one key site in the amino acid sequence shown in SEQ ID NO. 142 that is beneficial to 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 to improving the synthesis efficiency of D-tagatose.

[0018] These and other aspects of the present application will become more clearly understood in the description of the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The predicted protein three-dimensional structure of the wild-type epimerase amino acid sequence is shown.

[0020] Figure 2 A schematic diagram of the structure of the pET-28a-UxaE plasmid in the examples of the present application is shown.

[0021] Figure 3 A schematic diagram of the structure of the pET-22b-UxaE plasmid in the examples of the present application is shown.

[0022] Figure 4 The protein SDS-PAGE diagram of the epimerase in the examples of the present application is shown.

[0023] Figure 5 The liquid chromatography standard curve of D-tagatose in the examples of the present application is shown.

[0024] Figure 6 The liquid chromatography standard curve of D-fructose in the examples of the present application is shown.

[0025] Figure 7 The liquid chromatography result diagram of epimerase catalysis in the examples of the present application is shown. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0028] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] The terms "epimerase" and "epimerase for synthesizing D-tagatose" herein are both enzymes exhibiting D-fructose-4-epimerization activity, which can epimerize the 4th carbon position of D-fructose to convert D-fructose into D-tagatose.

[0031] The reaction principle formula for converting D-fructose into D-tagatose using the above-mentioned epimerase is as follows:

[0032] .

[0033] 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 the present application, as long as it can exhibit the same or corresponding activity as the polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO. For example, it is not excluded to add a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution before or after the "polypeptide, protein, mutant or enzyme having an 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, also falls within the scope of the present application, as long as it exhibits the same or corresponding activity as the amino acid sequence as shown in SEQ ID NO after the above-mentioned sequence is added.

[0034] The terms "tagatose" and "D-tagatose" are used interchangeably herein and both refer to a type of ketohexose in the monosaccharide.

[0035] The present application embodiment provides an epimerase for synthesizing D-tagatose, wherein the epimerase produces at least one mutation in the amino acid sequence shown in SEQ ID NO.142.

[0036] Among them, the amino acid sequence shown in SEQ ID NO. 142 can be obtained from GenBank of NCBI. Specifically, the amino acid sequence shown in SEQ ID NO. 142 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. 142 can be called a wild-type epimerase, and the enzyme formed by each mutated amino acid sequence can be called a mutant epimerase. The amino acid sequence shown in SEQ ID NO. 142 is input into the AlphaFold2 model to obtain the following: Figure 1 The predicted three-dimensional structure of the protein is shown.

[0037] right Figure 1The three-dimensional structure of the protein shown in SEQ ID NO. 142 was analyzed, and multiple key sites related to the binding of the substrate D-fructose and the activity of D-fructose-4-diamerization were selected as the research objects, including R100 (arginine R at position 100), Q123 (glutamine Q at position 123), Q124 (glutamine Q at position 124), S125, V126, R127, 340, H341, S342, G343, K346, H362, V363, K364, A366, G367, S402, Y403, H437, and Y440.

[0038] Saturation mutation screening was performed on the above-mentioned key sites to obtain an epimerase with improved D-fructose-4-diamerization activity.

[0039] In the epimerase for synthesizing D-tagatose of this embodiment, at least one of the following mutations occurs in the amino acid sequence shown in SEQ ID NO. 142: 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, mutation at position 185, mutation at position 186, mutation at position 187, mutation at position 188, mutation at position 189, mutation at position 190, mutation at position 191, mutation at position 192, mutation at position 193, mutation at position 194, mutation at position 195 83, 185, 266, 268, 270, 271, 299, 300, 302, 306, 308, 339, 340, 342, 343, 346, 362, 363, 364, 366, 367, 402, 403, 437, or 440. In the corresponding mutations, the amino acid residues at the corresponding positions in the amino acid sequence of SEQ ID NO. 142 are replaced by other amino acids.

[0040] 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.

[0041] In this embodiment, corresponding to the amino acid sequence shown in SEQ ID NO. 142, for example, position 100 (arginine R), position 125 (serine S), position 127 (arginine R), position 131 (arginine R), position 268 (serine S), position 270 (aspartic acid D), position 271 (glutamic acid E), position 308 (lysine K), position 339 (serine S), position 340 (leucine L), position 342 (serine S), position 366 (A), position 402 (serine S), position 437 (histidine H) and position 440 (tyrosine Y) are all key sites related to the catalytic activity of the diastereoisomerization of the 4th carbon position of D-fructose. By mutating the above-mentioned key sites, the resulting diastereomerase exhibits excellent diastereomerization activity at the 4th carbon position of D-fructose, and can efficiently convert D-fructose into D-tagatose, which is beneficial to improving the synthesis efficiency of D-tagatose.

[0042] As an embodiment, the epimerase for synthesizing D-tagatose of this embodiment has a mutation in the amino acid sequence shown in the corresponding SEQ ID NO. 142, 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.

[0043] In some embodiments, the epimerase has an amino acid sequence as shown in any one of SEQ ID NO. 1 to SEQ ID NO. 42.

[0044] In some embodiments, the epimerase corresponding to the amino acid sequence shown in SEQ ID NO.5, in which the serine S at position 125 is mutated to aspartic acid D, exhibits a greatly improved catalytic activity for the epimerization of the 4th carbon position of D-fructose.

[0045] As an embodiment, the epimerase for synthesizing D-tagatose in this example has two mutations in the amino acid sequence corresponding to SEQ ID NO. 142, one of which is a mutation of serine S at position 125 shown in SEQ ID NO. 142 to aspartic acid D, and the other mutation is one of the following: 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 340, a mutation at position 341, 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.

[0046] In some embodiments, the epimerase has an amino acid sequence as shown in any one of SEQ ID NO. 43 to SEQ ID NO. 80.

[0047] In some embodiments, the diastereomerase corresponding to the amino acid sequence shown in SEQ ID NO.52, which produces a mutation of serine S at position 125 to aspartic acid D and a mutation of serine S at position 268 to alanine A, exhibits a significantly improved catalytic activity for diastereomerization of the 4th carbon position of D-fructose.

[0048] As an embodiment, the epimerase for synthesizing D-tagatose in this example has three mutations in the amino acid sequence shown in SEQ ID NO. 142, the first mutation is that the serine S at position 125 shown in SEQ ID NO. 142 is mutated to aspartic acid D, the second mutation is that the serine S at position 268 shown in SEQ ID NO. 142 is mutated to alanine A, and the third mutation is one of the following: a mutation at position 123 or a mutation at position 124 or a mutation at position 126 or a mutation at position 129 or a mutation at position 130 or a mutation at position 132 or a mutation at position 133 or a mutation at position 134 or a mutation at position 136 or a mutation at position 159 or a mutation at position 163 or a mutation at position 164 or a mutation at position 177 The mutation at position 180 or the mutation at position 181 or the mutation at position 182 or the mutation at position 183 or the mutation at position 185 or the mutation at position 266 or the mutation at position 299 or the mutation at position 300 or the mutation at position 302 or the mutation at position 306 or the mutation at position 343 or the mutation at position 346 or the mutation at position 362 or the mutation at position 363 or the mutation at position 364 or the mutation at position 367.

[0049] In some embodiments, the epimerase has an amino acid sequence as shown in any one of SEQ ID NO. 81 to SEQ ID NO. 141.

[0050] In some embodiments, the diastereomerase corresponding to the amino acid sequence shown in SEQ ID NO.84, 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 glutamate E, exhibits a significantly improved catalytic activity for diastereomerization of the 4th carbon position of D-fructose.

[0051] In some embodiments, the diastereomerase corresponding to the amino acid sequence shown in SEQ ID NO.90, 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 asparagine N at position 129 to threonine T, exhibits a significantly improved catalytic activity for diastereomerization of the 4th carbon position of D-fructose.

[0052] In some embodiments, the diastereomerase corresponding to the amino acid sequence shown in SEQ ID NO.91, 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 asparagine N at position 129 to phenylalanine F, exhibits a significantly improved catalytic activity for diastereomerization of the 4th carbon position of D-fructose.

[0053] In some embodiments, the diastereomerase corresponding to the amino acid sequence shown in SEQ ID NO.94, 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 glutamate E at position 130 to glycine G, exhibits a significantly improved catalytic activity for diastereomerization of the 4th carbon position of D-fructose.

[0054] In some embodiments, the diastereomerase corresponding to the amino acid sequence shown in SEQ ID NO.116, 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 181 to methionine M, exhibits a significantly improved catalytic activity for diastereomerization of the 4th carbon position of D-fructose.

[0055] In some embodiments, the epimerase corresponding to the amino acid sequence shown in SEQ ID NO. 120, 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 aspartic acid D at position 183 to cysteine ​​C, exhibits a significant increase in the catalytic activity of epimerization of the 4th carbon position of D-fructose.

[0056] In some embodiments, the diastereomerase corresponding to the amino acid sequence shown in SEQ ID NO. 122, in which the serine S at position 125 is mutated to aspartic acid D, the serine S at position 268 is mutated to alanine A, and the serine S at position 185 is mutated to histidine H, exhibits a significant increase in the catalytic activity of diastereomerization of the 4th carbon position of D-fructose.

[0057] In some embodiments, the diastereomerase corresponding to the amino acid sequence shown in SEQ ID NO. 129, in which the serine S at position 125 is mutated to aspartic acid D, the serine S at position 268 is mutated to alanine A, and the leucine L at position 300 is mutated to threonine T, exhibits a significant increase in the catalytic activity of diastereomerization of the 4th carbon position of D-fructose.

[0058] 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.

[0059] The polynucleotide is a DNA chain or RNA chain formed by the polymerization of several nucleotides.

[0060] The polynucleotide only needs to encode the epimerase for synthesizing D-tagatose, and any nucleotide in the polynucleotide may be chemically modified.

[0061] The present application also provides a recombinant vector, comprising the above-mentioned polynucleotide encoding the epimerase for synthesizing D-tagatose.

[0062] Wherein, a recombinant vector refers to a DNA preparation containing a nucleic acid sequence of a polynucleotide encoding an epimerase for synthesizing D-tagatose, and it may also contain a control sequence, in which the nucleic acid sequence of a polynucleotide encoding an epimerase for synthesizing 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 may be replicated or function independently of the host genome, or may be integrated into the genome itself for replication or function.

[0063] Wherein, the type of recombinant vector is not particularly limited, as long as it can be replicated in the host cell, any vector known in the art can be used. Exemplarily, the commonly used vectors in the art may include plasmids, cosmids, viruses and phages in natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and 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.

[0064] As an embodiment, the recombinant vector comprises, in sequence, the nucleotide sequence shown in SEQ ID NO. 143, the nucleotide sequence encoding any epimerase, and the nucleotide sequence shown in SEQ ID NO. 144.

[0065] As an embodiment, the recombinant vector comprises, in sequence, the nucleotide sequence shown in SEQ ID NO. 145, the nucleotide sequence encoding any epimerase, and the nucleotide sequence shown in SEQ ID NO. 146.

[0066] The present application also provides a host cell, comprising the above-mentioned recombinant vector.

[0067] 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 introduced 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 located outside the chromosome of the host cell or inserted into the chromosome of the host cell and located outside the chromosome at the same time, and 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.

[0068] 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.

[0069] The present application also provides a method for preparing a diastereomerase for synthesizing D-tagatose, comprising the following steps: culturing a transformed host cell transformed with a recombinant vector to allow the diastereomerase to accumulate in the cells of the transformed host cell, wherein the recombinant vector contains a nucleotide sequence encoding the diastereomerase.

[0070] Among them, the transformed host cells transformed with the recombinant vector are grown under appropriate environmental conditions, and the host cell culture method may include but is not limited to batch culture, continuous culture or fed-batch culture. In order to achieve appropriate environmental conditions, the pH can be adjusted using alkaline compounds or acidic compounds 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 keep the culture environment in an aerobic state.

[0071] 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, carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils or fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as 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.

[0072] 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.

[0073] As an embodiment, the recombinant vector may include a nucleotide sequence corresponding to an amino acid sequence as shown in any one of SEQ ID NO. 1 to SEQ ID NO. 141. In some embodiments, the recombinant vector may include, in sequence, a nucleotide sequence as shown in SEQ ID NO. 143, a nucleotide sequence encoding any epimerase, and a nucleotide sequence as shown in SEQ ID NO. 144; or, the recombinant vector may include, in sequence, a nucleotide sequence as shown in SEQ ID NO. 145, a nucleotide sequence encoding any epimerase, and a nucleotide sequence as shown in SEQ ID NO. 146.

[0074] An embodiment of the present application provides a method for synthesizing D-tagatose, comprising:

[0075] 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.

[0076] Among them, using the above-mentioned epimerase to catalyze the conversion of D-fructose into D-tagatose can improve the conversion efficiency of D-tagatose.

[0077] Wherein, the catalytic system of epimerase catalyzing D-fructose may include a suitable excipient, and the excipient 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 of epimerase catalyzing D-fructose may include a buffer, and the buffer may include but is not limited to at least one of Tris-HCl, a sodium phosphate buffer and a potassium phosphate buffer.

[0078] Wherein, the catalytic system of epimerase catalyzing D-fructose may further include a metal salt, which 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. Further, the concentration of the metal salt may be 0.1 mM to 10 mM, for example, the concentration of the metal salt is 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM or 10 mM.

[0079] The contact between the epimerase and D-fructose may be carried out at pH 5.0 to 10.0. For example, the contact between the epimerase and D-fructose may be carried out at pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0 or pH 10.0.

[0080] The contact between the epimerase and D-fructose may be carried out at a temperature of 50°C to 90°C. For example, the contact between the epimerase and D-fructose may be carried out at 50°C, 60°C, 70°C, 80°C or 90°C.

[0081] 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.

[0082] As one embodiment, the transformed host cell containing the above-mentioned epimerase is contacted with D-fructose to produce D-tagatose, and the concentration of the host cell is 10 g / L to 200 g / L, for example, the concentration of the host cell 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.

[0083] Example

[0084] The coding gene of the epimerase of Examples 1 to 141 and Comparative Example 1 of the present application can be called UxaE gene, and the UxaE gene contains a nucleotide sequence corresponding to the amino acid sequence of the above-mentioned epimerase. For example, the UxaE gene in Examples 1 to 141 contains a nucleotide sequence corresponding to the amino acid sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 141, and the UxaE gene is constructed in the pET-28a plasmid to obtain Figure 2 The recombinant vector shown in FIG. 1 was used to construct the UxaE gene into the pET-22b plasmid to obtain Figure 3 The recombinant vector shown.

[0085] For example, Figure 2An example of the recombinant vector pET-28a-UxaE includes the nucleotide sequence shown in SEQ ID NO. 143, the nucleotide sequence encoding any epimerase, and the nucleotide sequence shown in SEQ ID NO. 144. Among them, 559 to 1374 as shown in SEQ ID NO. 143 are kanamycin resistance genes; 4983 to 5001 as shown in SEQ ID NO. 143 are T7 promoters; and 139 to 186 as shown in SEQ ID NO. 144 are T7 terminators.

[0086] For example, Figure 3 An example of the recombinant vector pET-22b-UxaE includes the nucleotide sequence shown in SEQ ID NO. 145, the nucleotide sequence encoding any epimerase, and the nucleotide sequence shown in SEQ ID NO. 146. Among them, 599 to 1459 as shown in SEQ ID NO. 145 are ampicillin resistance genes; 5118 to 5136 as shown in SEQ ID NO. 146 are T7 promoters; and 88 to 135 as shown in SEQ ID NO. 146 are T7 terminators.

[0087] The recombinant vector used in each embodiment 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 embodiment and comparative example 1 of the present application is Escherichia coli.

[0088] The steps of each embodiment and comparative example 1 in this application are as follows:

[0089] Protein expression

[0090] a. Transform pET-28a-UxaE into E. coli; pick 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 LB liquid medium (Kan+, 100 μg / mL), and culture it at 37°C and 220 rpm overnight as the seed solution.

[0091] b. Transfer the seed solution into 50 mL LB liquid medium (Kan+, 100 μg / mL) and incubate in a shaking incubator at 37°C and 220 rpm for reactivation.

[0092] c. Transfer the reactivated bacterial solution into 800 mL 2YT liquid medium (Kan+, 100 μg / mL) at a 1% inoculum volume and culture on a shaking incubator at 37°C and 220 rpm until the OD600 reaches approximately 0.6-0.8.

[0093] d. Lower the shaking temperature to 16 ℃-18 ℃. After the temperature of the cultured bacterial solution drops, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 hours.

[0094] e. After expression is completed, collect the above culture solution into a bottle, precool the centrifuge to 4℃, 5500 rpm, and centrifuge for 10 min.

[0095] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the cells using a vortex oscillator.

[0096] g. Centrifuge the resuspended cells again at 5500 rpm for 10 min. Pour off the supernatant, add 30 mL of protein purification buffer, resuspend the cells using a vortex oscillator (no solid particles), pour into a 50 mL centrifuge tube, and store in a -80 ℃ refrigerator.

[0097] Protein purification

[0098] a. Bacterial disruption: The collected bacterial solution is disrupted using a high-pressure low-temperature disruptor at a pressure of 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.

[0099] b. Centrifugation: Centrifuge the broken bacterial solution at 8000 rpm in a precooled 4°C centrifuge for 60 min. Take the precipitate and supernatant after centrifugation, prepare the sample, and collect the supernatant;

[0100] c. Purification: The supernatant is purified by nickel affinity chromatography. The specific steps are as follows:

[0101] (1) Column balancing: First wash with dd H2O for 2 column volumes, and then equilibrate the Ni affinity chromatography column with protein buffer for 1 column volume.

[0102] (2) Sampling: Take 50ul of the supernatant and slowly pass it through the Ni affinity chromatography column, flow through (can be repeated once) and take the first few drops of flow through sample.

[0103] (3) Elution of target protein: Use 30 mL of protein buffer containing 20 mM, 50 mM, 100 mM, 200 mM, or 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.

[0104] d. Concentration and replacement of liquid: The collected protein eluate containing the target protein was concentrated by centrifugation (4°C, 3400 r / min) using a 50 mL Amicon ultrafiltration tube (10 kDa, Millipore) to 1 mL. Then 10 mL of protein buffer was added and concentrated to 1 mL. This process was repeated once to ensure that the imidazole in the protein was removed and the purified protein Ceas was obtained.

[0105] Protein concentration determination

[0106] Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0107] First, use the protein's absorbance at 280 nm to make a preliminary determination of the protein concentration, and then dilute the protein concentration to 0.5-1 mg / mL based on the initial measured value. Prepare the reaction solution by mixing reagent A and reagent B in the BCA Protein Assay Kit in a ratio of 50:1. Take 200 uL of the reaction solution and place it in an ELISA plate. Add 25 uL of the diluted protein to the reaction solution, mix it with a gun and place it at 37°C for 30 min. Place the ELISA plate in an ELISA instrument to measure the absorbance at 562 nm, and process the data according to the protein standard curve to obtain the protein concentration. The protein SDS-PAGE gel image is shown below. Figure 4 shown.

[0108] Enzyme catalyzed reactions

[0109] In vitro enzyme catalytic reaction conditions:

[0110] Reaction buffer: at least one selected from Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer;

[0111] Reaction pH: pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, or pH 10;

[0112] The final fructose concentration was 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, or 500 g / L;

[0113] Metal salt type: ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, magnesium sulfate, or nickel sulfate;

[0114] The final concentration of the metal salt is 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM;

[0115] Protease amount: 1 mg / mL, 3 mg / mL, 5 mg / mL, or 10 mg / mL;

[0116] Reaction temperature: 50°C, 60°C, 70°C, 80°C, or 90°C;

[0117] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, or 24h;

[0118] The total reaction volume is 0.5 mL, 1 mL, 5 mL, or 10 mL.

[0119] Each embodiment and comparative example can adopt: magnesium sulfate, epimerase and D-fructose are added to Tris-HCl with a pH of 8 to form a catalytic solution; in the catalytic solution, the concentration of magnesium sulfate is 2mM, the concentration of epimerase is 3mg / mL, and the concentration of D-fructose is 200g / L. The catalytic solution is reacted at 60°C for 2 hours, and the conversion activity of each embodiment and comparative example is measured.

[0120] D-Tagatose detection

[0121] The liquid phase detection conditions are as follows:

[0122] Mobile phase: 80% acetonitrile, 20% water;

[0123] Liquid column Shimpack GIST, NH2;

[0124] Temperature: 40℃;

[0125] Flow rate: 0.5 mL / min;

[0126] Injection volume: 10uL.

[0127] The standard curve of D-tagatose can be found in Figure 5 As shown, the standard curve of D-fructose can be found in Figure 6 As shown, the liquid phase diagram of the reaction of the embodiment is shown in Figure 7 As 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.

[0128] The results of Examples 1 to 141 and Comparative Example 1 are specifically shown in Table 1, wherein the relative activity of the diasterase in Comparative Example 1 is 1, and the relative activities of the diasterase in Examples 1 to 141 are shown based on the relative activities of the diasterase in Comparative Example 1.

[0129] Table 1 Parameters of Examples 1 to 141 and Comparative Example 1

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136] The above is only an implementation method of the present application. It should be pointed out that a person skilled in the art can make improvements without departing from the inventive concept of the present application, but these improvements are within the scope of protection of the present application.

Claims

1. An epimerase for synthesizing D-tagatose, characterized in that The epimerase produces a mutation in which the serine S at position 125 shown in SEQ ID NO: 142 is mutated to aspartic acid D and a mutation in which the serine S at position 268 is mutated to alanine A, and the amino acid sequence of the epimerase is shown in SEQ ID NO: 84, SEQ ID NO: 90, SEQ ID NO: 120, SEQ ID NO: 129 or SEQ ID NO:

140.

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 is cultured to accumulate the epimerase in the cell of the transformed host cell, wherein the recombinant vector contains a nucleotide sequence encoding the epimerase according to claim 1.

6. The method for preparing epimerase for synthesizing D-tagatose according to claim 5, characterized in that: The recombinant vector comprises, in sequence, the nucleotide sequence shown in SEQ ID NO: 143, the nucleotide sequence encoding any one of the epimerases, and the nucleotide sequence shown in SEQ ID NO: 144; Alternatively, the recombinant vector comprises the nucleotide sequence shown in SEQ ID NO: 145, the nucleotide sequence encoding any one of the epimerases, and the nucleotide sequence shown in SEQ ID NO: 146 in sequence.

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 a transformed host cell containing the epimerase according to claim 1 is contacted with D-fructose to convert D-fructose into D-tagatose.

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