Cellobiose epimerase mutant and its application

Abstract

The present application relates to the field of biotechnology, and in particular to a cellobiose epimerase mutant and its application. The cellobiose epimerase mutant and its application provided in the embodiment of the present application is produced in at least one key site in the amino acid sequence shown in SEQ ID NO: 51, which produces a mutation that is beneficial to improving the catalytic activity of lactose epimerization to form lactulose; through the above-mentioned method, the cellobiose epimerase mutant exhibits excellent catalytic activity of lactose epimerization to form lactulose, can efficiently convert lactose into lactulose, and is beneficial to improving the efficiency of lactulose synthesis.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a cellobiose epimerase mutant and its application. Background Art

[0002] Lactulose is a disaccharide that is difficult to digest. Its finished product is a yellow clear liquid. Its sweetness is equivalent to lactose, but less than sucrose, about 48% to 60% sucrose. It has a cool taste, low viscosity, low calories, high safety and good stability. In addition, lactulose can increase the activity of bifidobacteria and is a good factor promoting bifidobacteria. Therefore, lactulose has been widely used in the pharmaceutical industry, food and animal feed fields.

[0003] Commercial lactulose is mainly prepared by chemical methods, but chemical methods require the addition of a large amount of catalysts, and the reaction conditions are generally quite intense, resulting in a large number of product byproducts. These mixtures bring great inconvenience to the subsequent product separation. In contrast, the preparation of lactulose by bioenzymatic method has many advantages, including mild reaction conditions, sanitation and safety, and meets the requirements of low-carbon economy and green environmental protection.

[0004] At present, the enzymes mainly used to catalyze lactose to produce lactulose are glycoside hydrolase (EC3.2.1) and cellobiose epimerase (CEs, EC5.1.3.11). The β-galactosidase method requires the addition of lactose and fructose as substrates, with a wide variety of byproducts and a low product yield (7.5-30%), which is not suitable for industrialization. Cellobiose epimerase (CEs) can directly catalyze the production of lactulose from single lactose, and the product yield is more advantageous than that of β-galactosidase, so it has been widely studied. At present, enzymes such as Caldicellulosiruptorsaccharolyticus CE (CsCE), Caldicellulosiruptor obsidiansis CE (CoCE), Dictyoglomus turgidum CE (DtCE) and Dictyoglomus thermophilum CE (DitCE) have the ability to synthesize lactulose. Among them, CsCE has been reported the most and is the most widely used. However, CE is still subject to the following limitations: high temperature is conducive to increasing the rate of isomerization reaction and reducing the production of byproduct ipililacose, while natural CE enzymes often have poor tolerance to long-term high temperature; but the ipililacose content in lactulose solutions in the pharmacopoeias of various countries is strictly restricted, so the ipililacose content needs to be reduced in the reaction of synthesizing lactulose. Summary of the invention

[0005] Based on this, the present application provides a cellobiose epimerase mutant and application to solve the above-mentioned technical problem that is not conducive to improving the efficiency of lactulose synthesis.

[0006] An embodiment of the present application provides a cellobiose epimerase mutant, wherein the cellobiose epimerase mutant produces a mutation at at least one key site in the amino acid sequence as shown in SEQ ID NO: 51, wherein the mutation at at least one key site is a mutation at position 20, or a mutation at position 64, or a mutation at position 167, or a mutation at position 168, or a mutation at position 169, or a mutation at position 170, or a mutation at position 175, or a mutation at position 179, or a mutation at position 184, or a mutation at position 192, or a mutation at position 212, or a mutation at position 257, or a mutation at position 297, or a mutation at position 298, or a mutation at position 302, or a mutation at position 310, or a mutation at position 313, or a mutation at position 367, or a mutation at position 373 in the amino acid sequence as shown in SEQ ID NO: 51, and the cellobiose epimerase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50.

[0007] One embodiment of the present application provides a polynucleotide encoding the above-mentioned cellobiose epimerase mutant.

[0008] An embodiment of the present application provides a recombinant vector, which includes the above-mentioned polynucleotide.

[0009] An embodiment of the present application provides a host cell, wherein the host cell comprises the above-mentioned recombinant vector.

[0010] An embodiment of the present application provides an immobilized cell for synthesizing lactulose, comprising a carrier and a host cell immobilized on the carrier, wherein the host cell synthesizes a cellobiose epimerase mutant, and the cellobiose epimerase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50.

[0011] An embodiment of the present application provides a method for preparing a cellobiose epimerase mutant, comprising: culturing a transformed host cell transformed with a recombinant vector to allow the cellobiose epimerase mutant to accumulate in the cells of the transformed host cell, wherein the recombinant vector contains a nucleotide sequence encoding the cellobiose epimerase mutant.

[0012] Optionally, the host cell is Escherichia coli or Bacillus subtilis.

[0013] An embodiment of the present application provides a method for synthesizing lactulose, characterized by comprising:

[0014] The cellobiose epimerase mutant or the transformed host cell containing the cellobiose epimerase mutant or the culture of the transformed host cell containing the cellobiose epimerase mutant is contacted with lactose to convert the lactose into lactulose.

[0015] Optionally, the cellobiose epimerase mutant or the transformed host cell containing the cellobiose epimerase mutant or the culture of the transformed host cell containing the cellobiose epimerase mutant is added to a reaction solution containing lactose for catalysis, and the reaction solution includes Tris-HCl buffer, sodium phosphate buffer or potassium phosphate buffer.

[0016] Optionally, the concentration of the cellobiose epimerase mutant in the reaction solution is 1 mg / mL to 10 mg / mL, and the catalytic temperature is 50°C to 85°C.

[0017] The present application provides a cellobiose epimerase mutant and its application. The cellobiose epimerase mutant produces a mutation in at least one key site in the amino acid sequence shown in SEQ ID NO: 51, which is beneficial to improving the catalytic activity of lactose epimerization to form lactulose. Through the above-mentioned method, the cellobiose epimerase mutant exhibits excellent catalytic activity of lactose epimerization to form lactulose, can efficiently convert lactose into lactulose, and is beneficial to improving the efficiency of lactulose synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The predicted protein three-dimensional structure of the wild-type cellobiose epimerase amino acid sequence is shown.

[0019] Figure 2 The protein SDS-PAGE diagram of the epimerase mutant in the examples of the present application is shown.

[0020] Figure 3 The liquid chromatography standard curve of lactose in the examples of the present application is shown.

[0021] Figure 4 The liquid chromatography standard curve of lactulose in the examples of the present application is shown.

[0022] Figure 5 The liquid chromatography standard curve of lactose in the examples of the present application is shown.

[0023] Figure 6 The liquid chromatography result diagram of the catalysis of the epimerase mutant in the examples of the present application is shown.

[0024] Figure 7 The OD graph of E. coli fermentation in the examples of the present application is shown.

[0025] Figure 8 The OD graph of Bacillus subtilis fermentation 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 "cellobiose epimerase" and "cellobiose epimerase mutant" herein are both enzymes that exhibit lactose epimerization activity, which is capable of converting lactose into lactulose.

[0031] The reaction principle formula for converting lactose into lactulose using the above-mentioned cellobiose epimerase or cellobiose epimerase mutant 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:", it is obvious that a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:, in which some sequences are deleted, modified, substituted, conservatively substituted or added, can also be used in the present application, as long as it can show 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 "a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:"; and a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:, when it is 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 shows the same or corresponding activity as the amino acid sequence as shown in SEQ ID NO: after the above-mentioned sequence is added.

[0034] An embodiment of the present application provides a cellobiose epimerase mutant, wherein the cellobiose epimerase mutant has at least one mutation in the amino acid sequence shown in SEQ ID NO:51.

[0035] The amino acid sequence shown in SEQ ID NO: 51 can be obtained from GenBank of NCBI. Specifically, the amino acid sequence shown in SEQ ID NO: 51 is derived from a thermophilic anaerobic cellulose degrading bacterium (Caldicellulosiruptor sp. DIB104C), and its number in GenBank is WP_271629184.1.

[0036] In this specification, the enzyme formed by the amino acid sequence shown in SEQ ID NO: 51 can be referred to as wild-type cellobiose epimerase, and the enzyme formed by each mutated amino acid sequence can be referred to as mutant cellobiose epimerase or cellobiose epimerase mutant. The amino acid sequence shown in SEQ ID NO: 51 is input into the AlphaFold2 model, and the following is obtained: Figure 1 Shown are the predicted three-dimensional structure of the protein and a schematic diagram of the docking of the protein three-dimensional structure with the substrate lactose.

[0037] right Figure 1The three-dimensional structure of the protein shown in SEQ ID NO: 51 was analyzed, and multiple key sites related to the binding of the substrate lactose and the diastereomerization activity of lactose in the amino acid sequence shown in SEQ ID NO: 51 were selected as the research objects, including P20 (proline P at position 20), C64 (cysteine ​​C at position 64), K167 (lysine K at position 167), E168 (glutamic acid E at position 168), N169 (asparagine N at position 169), R170 (arginine R at position 170), N175 (asparagine N at position 175), A179 (alanine A at position 179), N184 (asparagine N at position 184), S192 (serine S at position 192), I212 (isoleucine I at position 212), Q257 (glutamine Q at position 257), I297 (isoleucine I at position 297), E298 (glutamic acid E at position 298), D302 (aspartic acid D at position 302), E310 (glutamic acid E at position 310), T313 (threonine T at position 313), P367 (proline P at position 367), and K373 (lysine K at position 373).

[0038] Saturation mutation screening was performed on the above-mentioned key sites to obtain cellobiose epimerase mutants with improved lactose epimerization activity.

[0039] The cellobiose epimerase mutant of this embodiment produces a mutation at at least one key position in the amino acid sequence as shown in SEQ ID NO: 51, and the mutation at at least one key position is a mutation at position 20, or a mutation at position 64, or a mutation at position 167, or a mutation at position 168, or a mutation at position 169, or a mutation at position 170, or a mutation at position 175, or a mutation at position 179, or a mutation at position 184, or a mutation at position 192, or a mutation at position 212, or a mutation at position 257, or a mutation at position 297, or a mutation at position 298, or a mutation at position 302, or a mutation at position 310, or a mutation at position 313, or a mutation at position 367, or a mutation at position 373 in the amino acid sequence as shown in SEQ ID NO: 51. In the corresponding mutation, the amino acid residue at the corresponding position in the amino acid sequence as shown in SEQ ID NO: 51 is 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] The above 19 key sites are all key sites related to the catalytic activity of lactose epimerization. By mutating the above key sites, the cellobiose epimerase mutant produced exhibits excellent lactose epimerization activity, can efficiently convert lactose into lactulose, and is beneficial to improving the efficiency of lactulose synthesis.

[0042] In this example, the cellobiose epimerase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50.

[0043] Wherein, a mutation occurs in the amino acid sequence corresponding to SEQ ID NO: 51, and the mutation is a mutation at position 20, or a mutation at position 64, or a mutation at position 167, or a mutation at position 168, or a mutation at position 169, or a mutation at position 170, or a mutation at position 175, or a mutation at position 179, or a mutation at position 184, or a mutation at position 192, or a mutation at position 212, or a mutation at position 257, or a mutation at position 297, or a mutation at position 298, or a mutation at position 302, or a mutation at position 310, or a mutation at position 313, or a mutation at position 367, or a mutation at position 373. Furthermore, the catalytic activity of the cellobiose epimerase mutant in the epimerization of lactose to lactulose is greater than the lactose epimerization activity of the enzyme corresponding to the amino acid sequence as shown in SEQ ID NO: 51. The cellobiose epimerase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 32.

[0044] Among the cellobiose epimerase mutants produced with a mutation, the cellobiose epimerase mutant corresponding to the amino acid sequence shown in SEQ ID NO: 3 in which proline P at position 20 is mutated to serine S exhibits a greatly improved catalytic activity in the epimerization of lactose to lactulose.

[0045] Wherein, two mutations occur in the amino acid sequence corresponding to SEQ ID NO: 51, one of which is a mutation of proline P at position 20 of SEQ ID NO: 51 to serine S, and the other mutation is one of the following: a mutation at position 168, a mutation at position 169, a mutation at position 170, a mutation at position 175, a mutation at position 179, a mutation at position 184, a mutation at position 212, a mutation at position 257, a mutation at position 297, a mutation at position 298, a mutation at position 302, or a mutation at position 367 in the amino acid sequence of SEQ ID NO: 51. Moreover, the catalytic activity of the cellobiose epimerase mutant in the epimerization of lactose to lactulose is greater than the lactose epimerization activity of the enzyme corresponding to the amino acid sequence of SEQ ID NO: 51. The cellobiose epimerase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 33 to SEQ ID NO: 45.

[0046] Among the cellobiose epimerase mutants producing two mutations, the cellobiose epimerase mutant corresponding to the amino acid sequence shown in SEQ ID NO: 43 in which proline P at position 20 is mutated to serine S and proline P at position 367 is mutated to serine S exhibits a significant improvement in the catalytic activity of lactose epimerization to lactulose.

[0047] Wherein, three mutations occur in the amino acid sequence corresponding to SEQ ID NO: 51, wherein the first mutation is that proline P at position 20 of SEQ ID NO: 51 is mutated to serine S, the second mutation is that proline P at position 367 of SEQ ID NO: 51 is mutated to serine S, and the third mutation is one of the following: arginine R at position 170 of SEQ ID NO: 51 is mutated to phenylalanine F, arginine R at position 170 of SEQ ID NO: 51 is mutated to tyrosine Y, asparagine N at position 184 of SEQ ID NO: 51 is mutated to serine isoleucine I, isoleucine I at position 212 of SEQ ID NO: 51 is mutated to leucine L, or isoleucine I at position 297 of SEQ ID NO: 51 is mutated to cysteine ​​C. The cellobiose epimerase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 46 to SEQ ID NO: 50.

[0048] Among the cellobiose epimerase mutants with three mutations, the cellobiose epimerase mutant corresponding to the amino acid sequence shown in SEQ ID NO: 49, in which proline P at position 20 is mutated to serine S, proline P at position 367 is mutated to serine S, and asparagine N at position 184 is mutated to serine isoleucine I, shows a significant improvement in the catalytic activity of lactose epimerization to lactulose. In addition, the thermal stability of the cellobiose epimerase mutant corresponding to the amino acid sequence shown in SEQ ID NO: 49 is greatly improved, and the use of the cellobiose epimerase mutant corresponding to the amino acid sequence shown in SEQ ID NO: 49 to convert lactose into lactulose is conducive to greatly reducing the content of the byproduct epi-lactose.

[0049] The embodiments of the present application also provide a polynucleotide encoding a cellobiose epimerase mutant, wherein the polynucleotide comprises a nucleotide sequence corresponding to the amino acid sequence of the cellobiose epimerase mutant.

[0050] The polynucleotide has a nucleotide sequence corresponding to SEQ ID NO: 46 to SEQ ID NO: 50.

[0051] The polynucleotide is a DNA chain or RNA chain formed by the polymerization of several nucleotides.

[0052] As long as the polynucleotide can encode the cellobiose epimerase mutant, any nucleotide in the polynucleotide may be chemically modified.

[0053] The present application also provides a recombinant vector, comprising the above-mentioned polynucleotide encoding the cellobiose epimerase mutant.

[0054] Wherein, recombinant vector refers to a kind of DNA preparation, it contains the nucleic acid sequence of the polynucleotide of encoding cellobiose epimerase mutant, it can also contain control sequence, in the recombinant vector, the nucleic acid sequence of the polynucleotide of encoding cellobiose epimerase mutant is operably connected to suitable control sequence, so that cellobiose epimerase mutant can be expressed in suitable host. Specifically, control sequence can include but not limited to promoter that can start transcription, any operator sequence for regulating transcription, suitable mRNA ribosome binding site and sequence for controlling transcription and translation termination. After transformation into suitable host cell, recombinant vector can be replicated or play a role independently of host genome, or can be integrated into genome itself to be replicated or play a role.

[0055] 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.

[0056] The present application also provides a host cell, comprising the above-mentioned recombinant vector.

[0057] Wherein, the above-mentioned recombinant vector is transformed into a host cell so that the cellobiose epimerase mutant can be synthesized in the host cell of the present embodiment. The recombinant vector is introduced into a host cell, and the polynucleotide encoding the cellobiose epimerase mutant in the recombinant vector can be expressed in the host cell so that the host cell can synthesize the above-mentioned cellobiose epimerase mutant. The polynucleotide can be inserted into the chromosome of the host cell or be located outside the chromosome of the host cell or simultaneously inserted into the chromosome of the host cell and be located outside the chromosome, 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.

[0058] 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.

[0059] The embodiment of the present application provides an immobilized cell for synthesizing lactulose, comprising a carrier and a host cell immobilized on the carrier, wherein the host cell synthesizes a cellobiose epimerase mutant, and the cellobiose epimerase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50.

[0060] The immobilized cells used to synthesize lactulose are water-insoluble granular immobilized preparations, and the host cells synthesized with cellobiose diastereomerase mutants are fixed on a carrier. Compared with the diastereomerase in a free state, it can maintain higher activity and stability and can be reused.

[0061] In this embodiment, the cellobiose diastereomerase mutant that has been screened for mutations exhibits excellent lactose diastereomerization activity, and the host cell synthesized with the cellobiose diastereomerase mutant is fixed on a carrier, can maintain high activity and stability, and can be reused, and can efficiently convert lactose into lactulose, which is beneficial to improving the efficiency of lactulose synthesis.

[0062] As an embodiment, the carrier includes diatomaceous earth, glutaraldehyde and polyethyleneimine. 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, and the host cells are adsorbed on its surface. Glutaraldehyde and polyethyleneimine can cross-link with the hydroxyl functional groups on the surface of diatomaceous earth to form immobilized host cells.

[0063] In this embodiment, the host cells are fixed using a carrier formed by diatomaceous earth, glutaraldehyde and polyethyleneimine, which is beneficial to improving the activity recovery rate of the enzyme of the immobilized cells and improving the lactulose synthesis efficiency.

[0064] The host cell described in this embodiment is similar to the host cell embodiment described above. Please refer to the description of the host cell embodiment described above for details, and no further description is given here.

[0065] As an embodiment, in the immobilized cells for synthesizing lactulose, the mass ratio of the host cells to the carrier is 0.1 to 3:1.

[0066] An embodiment of the present application provides a method for preparing a cellobiose epimerase mutant, comprising the following steps: culturing a transformed host cell transformed with a recombinant vector to allow the cellobiose epimerase mutant to accumulate in the cells of the transformed host cell, wherein the recombinant vector contains a nucleotide sequence encoding the cellobiose epimerase mutant.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In one embodiment, the host cell is Escherichia coli.

[0071] In one embodiment, the host cell is Bacillus subtilis.

[0072] An embodiment of the present application provides a method for synthesizing lactulose, comprising:

[0073] The cellobiose epimerase mutant or the transformed host cell containing the cellobiose epimerase mutant or the culture of the transformed host cell containing the cellobiose epimerase mutant is contacted with lactose to convert the lactose into lactulose.

[0074] Among them, the use of the above-mentioned cellobiose epimerase mutant to catalyze the conversion of lactose into lactulose can improve the conversion efficiency of lactulose.

[0075] Wherein, the catalytic system of the epimerase mutant catalyzing lactose 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 the epimerase mutant catalyzing lactose 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.

[0076] Exemplarily, a cellobiose epimerase mutant or a transformed host cell containing the cellobiose epimerase mutant or a culture of a transformed host cell containing the cellobiose epimerase mutant is added to a reaction solution containing lactose for catalysis, and the reaction solution includes Tris-HCl buffer, sodium phosphate buffer or potassium phosphate buffer.

[0077] Illustratively, the concentration of the cellobiose epimerase mutant in the reaction solution is 1 mg / mL to 10 mg / mL, and the catalytic temperature is 50°C to 85°C.

[0078] The contact between the cellobiose epimerase mutant and lactose can be carried out at pH 4.0 to 10.0. For example, the contact between the cellobiose epimerase mutant and lactose 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.

[0079] Among them, the contact between the cellobiose epimerase mutant and lactose can be carried out at a temperature of 50°C to 85°C, for example, the contact between the cellobiose epimerase mutant and lactose can be carried out at 50°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C or 85°C.

[0080] The contact time between the cellobiose epimerase mutant and lactose can be 2 hours to 24 hours. For example, the contact time between the cellobiose epimerase mutant and lactose 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.

[0081] As an embodiment, the transformed host cells containing the above-mentioned epimerase mutant are contacted with lactose to produce lactulose, 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;

[0082] As an embodiment, the concentration of lactose is 50 g / L to 500 g / L, for example, the concentration of lactose can be 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L or 500 g / L.

[0083] As an embodiment, the concentration of the cellobiose epimerase mutant is 1 mg / mL to 10 mg / mL, for example, the concentration of the cellobiose epimerase mutant is 1 mg / mL, 3 mg / mL, 5 mg / mL or 10 mg / mL.

[0084] As an embodiment, the total reaction system can be 0.5 mL, 1 mL, 10 mL, 1 L, 3.5 L or 35 L.

[0085] Example

[0086] The encoding gene of the cellobiose epimerase mutant of Examples 1 to 50 of the present application and the encoding gene of the cellobiose epimerase of Comparative Example 1 can all be referred to as the CsCE gene, and the CsCE gene contains a nucleotide sequence corresponding to the amino acid sequence of the above-mentioned cellobiose epimerase mutant or cellobiose epimerase. For example, the CsCE gene in Examples 1 to 50 contains a nucleotide sequence corresponding to the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 50, and the CsCE gene is constructed in the pET-28a plasmid to obtain the pET-28a-CsCE plasmid, and the CsCE gene is constructed in the pET-22b plasmid to obtain the pET-22b-CsCE plasmid.

[0087] Exemplarily, pET-28a-CsCE includes a nucleotide sequence as shown in SEQ ID NO: 52, a nucleotide sequence encoding any cellobiose epimerase mutant, and a nucleotide sequence as shown in SEQ ID NO: 53. Among them, 559 to 1374 as shown in SEQ ID NO: 52 are kanamycin resistance genes; 4983 to 5001 as shown in SEQ ID NO: 52 are T7 promoters; and 91 to 138 as shown in SEQ ID NO: 53 are T7 terminators.

[0088] Exemplarily, pET-22b-CsCE includes a nucleotide sequence as shown in SEQ ID NO: 54, a nucleotide sequence encoding any cellobiose epimerase mutant, and a nucleotide sequence as shown in SEQ ID NO: 54. Wherein, 599 to 1459 as shown in SEQ ID NO: 54 are ampicillin resistance genes; 5117 to 5135 as shown in SEQ ID NO: 54 are T7 promoters; and 88 to 135 as shown in SEQ ID NO: 55 are T7 terminators.

[0089] The recombinant vector used in each example and comparative example 1 of the present application is a pET-28a plasmid containing a CsCE gene, hereinafter referred to as pET-28a-CsCE; the host cell used in each example and comparative example 1 of the present application is Escherichia coli.

[0090] The steps of each embodiment and comparative example 1 in this application are as follows:

[0091] Protein expression

[0092] a. Pick a single clone of pET-28a-CsCE 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 overnight at 37°C and 220 rpm as the seed solution.

[0093] 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.

[0094] c. Transfer the reactivated bacterial solution into 800 mL 2YT liquid culture medium (Kan+, 100 μg / mL) at a 1% inoculum volume and incubate in a shaking incubator at 37°C and 220 rpm until the OD (Optical Density) 600 reaches approximately 0.6-0.8.

[0095] 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 0.5mM and induce expression for 14~16h.

[0096] e. After expression is completed, collect the above culture solution into a bottle, precool the centrifuge to 4°C, 5500rpm, and centrifuge for 10 minutes.

[0097] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the cells using a vortex oscillator.

[0098] g. Centrifuge the resuspended cells again at 5500rpm for 10min. Pour off the supernatant, add 30mL of protein purification buffer, resuspend the cells with a vortex oscillator (no solid particles), pour into a 50mL centrifuge tube, and store in a -80℃ refrigerator.

[0099] The protein purification buffer was 50 mM sodium phosphate buffer, pH 7.

[0100] Protein purification

[0101] 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.

[0102] b. Centrifugation: Centrifuge the broken bacterial solution at 8000rpm in a precooled 4°C centrifuge for 60 minutes, take the precipitate and supernatant after centrifugation, prepare the sample, and collect the supernatant;

[0103] c. Purification: The supernatant is purified by nickel affinity chromatography. The specific steps are as follows:

[0104] (1) Column balancing: First wash with ddH2O for 2 column volumes, and then equilibrate the Ni affinity chromatography column with protein buffer for 1 column volume.

[0105] (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.

[0106] (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.

[0107] d. Concentration and replacement of liquid: The collected protein eluate containing the target protein is concentrated by centrifugation (4°C, 3400r / min) using a 50mL Amicon ultrafiltration tube (10kDa, Millipore) to 1mL. Then 10mL of protein buffer is added and concentrated to 1mL. This process is repeated once to ensure that the imidazole in the protein is removed and the purified protein Ceas is obtained.

[0108] Protein concentration determination

[0109] Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0110] First, use the absorbance of the protein at 280nm to make 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 preparing reagent A and reagent B in the BCA Protein Assay Kit at a ratio of 50:1. Take 200μL of the reaction solution and place it in an ELISA plate. Add 25μL of the 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 image is shown below. Figure 2 shown.

[0111] Enzyme catalyzed reactions

[0112] In vitro enzyme catalytic reaction conditions:

[0113] Reaction buffer: at least one selected from Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer;

[0114] Reaction pH: pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, or pH 10;

[0115] The final lactose concentration is 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, or 500 g / L;

[0116] Protease amount: 1 mg / mL, 3 mg / mL, 5 mg / mL, or 10 mg / mL;

[0117] Reaction temperature: 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C or 85°C;

[0118] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, or 24h;

[0119] The total reaction system is 0.5mL, 1mL, 10mL, 1L, 3.5L or 35L.

[0120] Each embodiment and comparative example can adopt: adding cellobiose epimerase mutant or cellobiose epimerase and lactose to Tris-HCl at pH 7 to form a catalytic solution; in the catalytic solution, the concentration of cellobiose epimerase mutant or cellobiose epimerase is 3 mg / mL, and the concentration of lactose is 200 g / L. The catalytic solution is reacted at 60° C. for 2 hours, and the conversion activity of each embodiment and comparative example is measured.

[0121] Detection of lactose, lactulose and epilactose

[0122] The liquid phase detection conditions are as follows:

[0123] Mobile phase: 75% acetonitrile, 20% methanol, 5% water;

[0124] Liquid column: Shodex HILICpak VG-504E;

[0125] Temperature: 40℃;

[0126] Flow rate: 1 mL / min;

[0127] Injection volume: 10 μL.

[0128] The standard curve of lactose can be found in Figure 3 The standard curve of lactulose is shown in Figure 4 As shown, the standard curve of lactose can be found in Figure 5 As shown, the liquid phase diagram of the reaction of the embodiment is shown in Figure 6 As shown, each epimerase (cellobiose epimerase mutant or cellobiose epimerase) in Examples 1 to 50 and Comparative Example 1 respectively converts lactose into lactulose, and all have lactose epimerization activity.

[0129] The relative activity changes of the cellobiose epimerase mutants were determined according to the liquid phase detection results of the examples and comparative examples, with the activity of the cellobiose epimerase of comparative example 1 being 100%.

[0130] Table 1 Parameters of Examples 1 to 50 and Comparative Example 1

[0131]

[0132]

[0133]

[0134] Experiment on Optimization of Fermentation Conditions of Escherichia coli

[0135] Transformation of E. coli:

[0136] 1) Take 100 μL of competent cells and place them on ice to slowly thaw, add 10 μL of ligation product, flick to mix, and place on ice for 30 min;

[0137] 2) Heat shock in a 42℃ water bath for 45 seconds, and immediately cool on ice for 2 minutes. Do not shake the centrifuge tube during this process.

[0138] 3) Add 900 μl LB liquid medium to each tube (without antibiotics) and culture at 37°C with shaking at 200 rpm for 1 h;

[0139] 4) Preparation of LB solid medium: When the sterilized LB solid medium cools to about 60°C, add Kan at a final concentration of 50 μg / mL, shake well, pour into a 90 mm culture dish, wait for it to completely solidify, and set aside.

[0140] 5) Centrifuge at 5000 rpm for 5 min, discard 900 μL of supernatant, and use the remaining 100 μL of culture medium to suspend the cells; the cell suspension is directly and evenly spread on the LB solid selection plate prepared above;

[0141] 6) Seal the plate with sealing film and invert it in a 37°C incubator for 12 to 16 hours until single colonies appear.

[0142] 7) The single clone was cultured into seed liquid, and the formula of the culture medium, the formula of the feed medium, the culture temperature and Ph, the induction concentration and the timing during the fermentation process were optimized to obtain a stable E. coli fermentation process. The OD graph of E. coli fermentation can be found in Figure 7 shown.

[0143] Experiment on Optimization of Fermentation Conditions of Bacillus subtilis

[0144] Transformation of Bacillus subtilis:

[0145] 1) Add 0.1-5 μg of plasmid DNA to 150 μL of melted competent cells and mix by inversion.

[0146] 2) Transfer to a pre-cooled 1 mm electroporation cup, place on ice for 5 minutes, place in an electroporation tank, and electroporate once at 2.0 KV.

[0147] 3) Transfer to a 1.5 ml EP sterile tube and add 1 ml of subtilis electric shock recovery solution and mix gently.

[0148] 4) Incubate at 37°C and 200 rpm for 3 h.

[0149] 5) Centrifuge at 4500 rpm for 5 min, discard the supernatant, keep 100 μL of liquid to resuspend the bacteria, spread all of it on a solid plate culture medium containing the corresponding resistance, and culture at 37°C for 16 to 48 h until positive colonies appear on the plate.

[0150] 6) Cultivate the single clone, optimize the culture medium formula, feed medium formula, culture temperature and pH conditions during the fermentation process, and obtain a stable Bacillus subtilis fermentation process. The OD graph of Bacillus subtilis fermentation can be found in Figure 8 shown.

[0151] Conditions for bacterial catalysis

[0152] The reaction buffer is selected from Tris-HCl, sodium phosphate buffer or potassium phosphate buffer;

[0153] Reaction pH: pH 4, pH 5, pH 6, pH 7, pH 8, pH 9 or pH 10;

[0154] The final lactose concentration was 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L or 500 g / L;

[0155] The amount of bacteria: 10g / L, 25g / L, 50g / L, 75g / L, 100g / L, 150g / L or 200g / L;

[0156] Reaction temperature: 50°C, 60°C, 70°C, 80°C or 90°C;

[0157] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h;

[0158] The total reaction system was 0.5mL, 1mL, 5mL, and 10mL.

[0159] Thermal stability test

[0160] Test conditions: Select one of the combination tests according to the bacterial catalytic conditions, such as 50mM sodium phosphate buffer, pH 7, lactose concentration 100g / L, bacterial dosage 50g / L, reaction temperature 70℃.

[0161] Thermal stability test: After the bacteria participating in the reaction were incubated at 80°C for 15 minutes, the (residual) enzyme activity was measured according to the test conditions. The initial enzyme activity of each enzyme was taken as 100%, and the enzyme activity after incubation was compared with it. The results are shown in Table 2.

[0162] Table 2 Determination of residual enzyme activity of Example 49 and Comparative Example 1

[0163]

[0164] Determination of the proportion of each component after reaction termination

[0165] When the reaction equilibrium is reached according to the test conditions, the proportion of each component in the reaction solution is shown in Table 3.

[0166] Table 3: Determination of the proportion of each component when the reaction of Example 49 and Comparative Example 1 reaches equilibrium

[0167]

[0168] The technical features of the above embodiments may 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.

[0169] The above embodiments only express the preferred implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A cellobiose epimerase mutant, characterized in that: The cellobiose epimerase mutant is produced by mutation of proline P at position 20 to serine S and mutation of proline P at position 367 to serine S in the amino acid sequence shown in SEQ ID NO: 51, and the amino acid sequence of the cellobiose epimerase mutant is any one of the amino acid sequences shown in SEQ ID NO: 46 to SEQ ID NO:

50.

2. A polynucleotide, characterized in that The polynucleotide encodes the cellobiose epimerase mutant 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. An immobilized cell for synthesizing lactulose, characterized in that: The invention comprises a carrier and a host cell immobilized on the carrier, wherein the host cell synthesizes a cellobiose epimerase mutant, and the amino acid sequence of the cellobiose epimerase mutant is any one of the amino acid sequences shown in SEQ ID NO: 46 to SEQ ID NO:

50.

6. A method for preparing a cellobiose epimerase mutant, characterized in that: include: The transformed host cell transformed with the recombinant vector is cultured to accumulate the cellobiose epimerase mutant in the cells of the transformed host cell, wherein the recombinant vector contains a nucleotide sequence encoding the cellobiose epimerase mutant according to claim 1.

7. The method for preparing a cellobiose epimerase mutant according to claim 6, characterized in that: The host cell is Escherichia coli or Bacillus subtilis.

8. A method for synthesizing lactulose, characterized in that: include: The cellobiose epimerase mutant according to claim 1, or a transformed host cell containing the cellobiose epimerase mutant according to claim 1, or a culture of a transformed host cell containing the cellobiose epimerase mutant according to claim 1 is contacted with lactose to convert lactose into lactulose.

9. The method for synthesizing lactulose according to claim 8, characterized in that: The cellobiose epimerase mutant or the transformed host cell containing the cellobiose epimerase mutant or the culture of the transformed host cell containing the cellobiose epimerase mutant is added to a reaction solution containing lactose for catalysis, and the reaction solution includes Tris-HCl buffer, sodium phosphate buffer or potassium phosphate buffer.

10. The method for synthesizing lactulose according to claim 9, characterized in that: The concentration of the cellobiose epimerase mutant in the reaction solution is 1 mg / mL to 10 mg / mL, and the catalytic temperature is 50° C. to 85° C.

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