Immobilized cell for synthesizing lactulose and application

Abstract

The immobilized cell for synthesizing lactulose in the embodiment of the invention comprises a carrier and a host cell immobilized on the carrier, the carrier comprises diatomite, polyethyleneimine and trihydroxymethyl phosphine, and a cellobiose epimerase mutant is synthesized in the host cell; the mass ratio of the host cells to the diatomite is (0.05-3): 1, the ratio of the mass of the host cells to the volume of the polyethyleneimine is 1g: 2.5 mL-12.5 mL, and the ratio of the mass of the host cells to the volume of the trihydroxymethyl phosphine is 1g: 2.5 mL-12.5 mL; by controlling the proportion of the addition amount of the host cell, the diatomite, the polyethyleneimine and the trihydroxymethyl phosphorus in the immobilized cell, the immobilized cell has high stability and retains the catalytic activity of the cellobiose epimerase mutant at the same time, and the lactulose synthesis efficiency can be improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an immobilized cell for synthesizing lactulose and its application. Background Art

[0002] Lactulose is a non-digestible disaccharide. Its finished product is a clear yellow liquid with a sweetness comparable to lactose but less than sucrose, containing approximately 48% to 60% sucrose. It has a cool, mellow taste, low viscosity, low calories, high safety, and excellent stability. Furthermore, lactulose can increase the activity of bifidobacteria, acting as a potent bifidobacterium-promoting factor. Consequently, lactulose is widely used in the pharmaceutical, food, and animal feed industries.

[0003] Commercial lactulose is primarily produced chemically, but this method requires the addition of large amounts of catalysts and often requires intense reaction conditions, resulting in numerous byproducts. These mixtures significantly hinder subsequent product separation. Enzymatic lactulose production, by contrast, offers numerous advantages, including mild reaction conditions, safety, and hygiene, in line with low-carbon economic and environmental requirements.

[0004] Currently, the enzymes primarily used to catalyze the conversion of lactose to lactulose are glycoside hydrolases (EC3.2.1) and cellobiose epimerases (CEs, EC5.1.3.11). The β-galactosidase method requires the addition of lactose and fructose as substrates, produces a wide variety of byproducts, and has a low product yield (7.5-30%), making it unsuitable for industrialization. Cellobiose epimerases (CEs), on the other hand, can directly catalyze the conversion of lactose to lactulose, offering superior product yields compared to β-galactosidases, and have therefore attracted extensive research. Currently, enzymes such as Caldicellulosiruptorsaccharolyticus CE (CsCE), Caldicellulosiruptor obsidiansis CE (CoCE), Dictyoglomus turgidum CE (DtCE), and Dictyoglomus thermophilum CE (DitCE) have demonstrated 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 the by-product ipilimumab, while natural CE enzymes often have poor tolerance to long-term high temperature; however, the ipilimumab content in lactulose solutions in the pharmacopoeias of various countries is strictly restricted, so the ipilimumab content needs to be reduced in the reaction of synthesizing lactulose. Summary of the Invention

[0005] Based on this, the present application provides an immobilized cell for synthesizing lactulose and its application, so as to solve the technical problem in the prior art that is not conducive to improving the efficiency of lactulose synthesis.

[0006] In a first aspect, 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 carrier comprises diatomaceous earth, polyethyleneimine and trishydroxymethyl phosphine, and the host cell comprises a cellobiose epimerase mutant synthesized by the host cell, wherein the cellobiose epimerase mutant has an amino acid sequence as shown in SEQ ID NO: 1; the mass ratio of the host cell to the diatomaceous earth is 0.05 to 3:1, the ratio of the mass of the host cell to the volume of the polyethyleneimine is 1 g:2.5 mL to 12.5 mL, and the ratio of the mass of the host cell to the volume of the trishydroxymethyl phosphine is 1 g:2.5 mL to 12.5 mL.

[0007] Optionally, the mass ratio of the host cells to the diatomaceous earth is 1:2-4.

[0008] Optionally, the ratio of the mass of the host cells to the volume of the polyethyleneimine is 1 g:5.0 mL to 7.5 mL.

[0009] Optionally, the ratio of the mass of the host cells to the volume of the tris(hydroxymethyl)phosphine is 1 g:7.5 mL to 12.5 mL.

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned immobilized cells for synthesizing lactulose, comprising:

[0011] preparing a bacterial suspension by adding wet cells of host cells synthesizing a cellobiose epimerase mutant and a first buffer solution, wherein the pH value of the first buffer solution is 7.5 to 8.0;

[0012] adding diatomaceous earth and polyethyleneimine to the bacterial suspension in sequence to perform a first stirring reaction;

[0013] adding tris(hydroxymethyl)phosphine to the bacterial suspension to carry out a second stirring reaction;

[0014] The bacterial suspension is centrifuged and the precipitate is collected, and the precipitate is dried to obtain immobilized cell particles.

[0015] Optionally, the first stirring reaction time is 0.5 hours to 2.5 hours, and the second stirring reaction time is 0.5 hours to 2.5 hours.

[0016] Optionally, the first stirring reaction time is 0.5 hours to 1.5 hours, and the second stirring reaction time is 1.5 hours to 2.5 hours.

[0017] In a third aspect, the present invention provides a method for synthesizing lactulose, comprising:

[0018] The immobilized cells for synthesizing lactulose are added to a reaction solution containing lactose for catalysis, thereby converting lactose into lactulose.

[0019] Optionally, the concentration of the immobilized cells for synthesizing lactulose in the reaction solution is 1 mg / mL to 10 mg / mL, and the concentration of lactose in the reaction solution is 50 mg / mL to 500 mg / mL.

[0020] Optionally, the temperature of the catalytic reaction is 60° C. to 80° C., and the pH value of the catalytic reaction is 6 to 7.

[0021] The immobilized cells for synthesizing lactulose according to the embodiments of the present application include a carrier and host cells immobilized on the carrier, wherein the carrier includes diatomaceous earth, polyethyleneimine, and trihydroxymethyl phosphine, and the host cells synthesize a cellobiose epimerase mutant having an amino acid sequence as shown in SEQ ID NO: 1; the mass ratio of the host cells to the diatomaceous earth is 0.05 to 3:1, the ratio of the mass of the host cells to the volume of the polyethyleneimine is 1 g: 2.5 mL to 12.5 mL, and the ratio of the mass of the host cells to the volume of the trihydroxymethyl phosphine is 1 g: 2.5 mL to 12.5 mL; by controlling the ratio between the added amounts of the host cells, diatomaceous earth, polyethyleneimine, and trihydroxymethyl phosphine in the immobilized cells, the immobilized cells have high stability while retaining the catalytic activity of the cellobiose epimerase mutant, which is beneficial to improving the efficiency of lactulose synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The liquid chromatography standard curve of lactose in the examples of the present application is shown.

[0023] Figure 2 The liquid chromatography standard curve of lactulose in the examples of the present application is shown.

[0024] Figure 3 The liquid chromatography standard curve of lactose in the examples of the present application is shown.

[0025] Figure 4 The figure shows the liquid chromatography results of immobilized cell catalysis in the examples of the present application.

[0026] Figure 5 A comparison chart of the catalytic activities of Examples 1 to 5 of the present application is shown.

[0027] Figure 6 A comparison chart of the catalytic activities of Examples 6 to 10 of the present application is shown.

[0028] Figure 7 A comparison chart of the catalytic activities of Examples 11 to 15 of the present application is shown.

[0029] Figure 8 A comparison chart of the catalytic activities of Examples 16 to 20 of the present application is shown.

[0030] Figure 9 A comparison chart of the catalytic activities of Examples 21 to 25 of the present application is shown.

[0031] Figure 10 A comparison chart of the catalytic activities of Examples 26 to 30 of the present application is shown.

[0032] Figure 11 A comparison chart of the catalytic activities of Examples 31 to 35 of the present application is shown.

[0033] Figure 12 A comparison chart of the catalytic activities of Examples 36 to 47 of the present application is shown.

[0034] Figure 13 A comparison chart of catalytic activity at different reaction times of Example 41 of the present application is shown. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0036] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute a separate or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] 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.

[0040] The reaction principle formula for converting lactose into lactulose using the above-mentioned cellobiose epimerase or cellobiose epimerase mutant is as follows:

[0041]

[0042] It is described herein as "a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:". Obviously, a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:, wherein some of the sequences are deleted, modified, substituted, conservatively substituted or added, can also be used in this application, as long as it can exhibit the same or corresponding activity as the polypeptide, protein, mutant or enzyme having the amino acid sequence as shown in SEQ ID NO:. For example, it is not excluded that a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof is added before or after the "polypeptide, protein, mutant or enzyme having the amino acid sequence as shown in SEQ ID NO:". Moreover, a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:, when added with the above-mentioned sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof, also falls within the scope of this application, as long as it exhibits the same or corresponding activity as the amino acid sequence as shown in SEQ ID NO: after the addition of the above-mentioned sequence.

[0043] 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 carrier comprises diatomaceous earth, polyethyleneimine, and tris(hydroxymethyl)phosphine; the host cell comprises a cellobiose epimerase mutant synthesized by the host cell, and the cellobiose epimerase mutant has an amino acid sequence as shown in SEQ ID NO: 1.

[0044] The use of a carrier composed of diatomaceous earth, polyethyleneimine, and tris(hydroxymethyl)phosphine to immobilize host cells can enhance the stability of the cellobiose epimerase mutant synthesized by the host cells. Specifically, diatomaceous earth, primarily composed of silicon dioxide, has a large number of orderly arranged pores on its surface, which has a strong adsorption effect. The host cells are adsorbed on its surface, and the polyethyleneimine and tris(hydroxymethyl)phosphine can cross-link with the hydroxyl functional groups on the diatomaceous earth surface to form immobilized host cells.

[0045] The cellobiose epimerase mutant has two mutations in the amino acid sequence shown in SEQ ID NO: 2: a mutation of proline P at position 20 to serine S, and a mutation of asparagine N at position 184 to serine isoleucine I. The cellobiose epimerase mutant has greatly improved thermal stability, and using the cellobiose epimerase mutant to convert lactose into lactulose significantly reduces the content of the byproduct, epi-lactose.

[0046] Among them, during the process of immobilizing cells, the cross-linking of polyethyleneimine and trihydroxymethyl phosphine may lead to an increase in the thermal stability of the cellobiose epimerase mutant in the host cells, and may also lead to a decrease in the enzymatic activity of the cellobiose epimerase mutant in the host cells. It is necessary to control the ratio between the added amounts of host cells, diatomaceous earth, polyethyleneimine and trihydroxymethyl phosphine.

[0047] The mass ratio of the host cells to the diatomaceous earth is 0.05 to 3:1, the mass ratio of the host cells to the volume of polyethyleneimine is 1 g:2.5 mL to 12.5 mL, and the mass ratio of the host cells to the volume of tris(hydroxymethyl)phosphine is 1 g:2.5 mL to 12.5 mL. Through the interaction between the host cells, diatomaceous earth, polyethyleneimine, and tris(hydroxymethyl)phosphine, the adsorption of the host cells, the stability of the immobilized cells, and the activity of the enzymes in the host cells are synergistically enhanced, thereby improving the performance of the immobilized cells.

[0048] In this embodiment, by controlling the ratio of the added amounts of host cells, diatomaceous earth, polyethyleneimine, and tris(hydroxymethyl)phosphine in the immobilized cells, the immobilized cells can be made more stable while retaining the catalytic activity of the cellobiose epimerase mutant, thereby improving the efficiency of lactulose synthesis.

[0049] As an embodiment, the mass ratio of the host cells to the diatomaceous earth is 1:2 to 4. In this embodiment, controlling the ratio of the added amount of the host cells to the diatomaceous earth within the above range can further improve the catalytic activity of the immobilized cells.

[0050] As an embodiment, the ratio of the mass of the host cells to the volume of the polyethyleneimine is 1 g: 5.0 mL to 7.5 mL. In this embodiment, controlling the ratio of the added amount of host cells to polyethyleneimine within the above range can further improve the catalytic activity of the immobilized cells.

[0051] In one embodiment, the ratio of the mass of the host cells to the volume of the tris(hydroxymethyl)phosphine is 1 g:7.5 mL to 12.5 mL. In this embodiment, controlling the ratio of the host cells to the amount of tris(hydroxymethyl)phosphine within the above range can further improve the catalytic activity of the immobilized cells.

[0052] As an embodiment, a recombinant vector is provided, which includes the above-mentioned polynucleotide encoding the cellobiose epimerase mutant; the recombinant vector is then transformed into a host cell, so that the cellobiose epimerase mutant can be synthesized in the host cell of this embodiment.

[0053] The polynucleotide is a DNA chain or RNA chain formed by the polymerization of several nucleotides.

[0054] As long as the polynucleotide can encode the cellobiose epimerase mutant, any nucleotide in the polynucleotide may be chemically modified.

[0055] Wherein, recombinant vector refers to a DNA preparation, which contains the nucleic acid sequence of the polynucleotide encoding the cellobiose epimerase mutant, and it can also contain a control sequence, in which the nucleic acid sequence of the polynucleotide encoding the cellobiose epimerase mutant is operably linked to a suitable control sequence so that the cellobiose epimerase mutant can be expressed in a suitable host. Specifically, the control sequence can 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 being transformed into a suitable host cell, the recombinant vector can replicate or play a role independently of the host genome, or can be integrated into the genome itself to replicate or play a role.

[0056] Among them, there is no particular limitation on the type of recombinant vector, as long as it can replicate in the host cell, any vector known in the art can be used. For example, commonly used vectors in the art may include natural or recombinant plasmids, cosmids, viruses and phages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and the pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1 BAC vectors, etc. can be used.

[0057] Wherein, 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 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. 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 operably linked to the polynucleotide, a transcription termination element, a ribosomal domain, and a translation termination element.

[0058] The host cell may be of the genus Escherichia, Erwinia, Serratia, Providencia, Corynebacterium or Brevibacterium; illustratively, the host cell may be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or Aspergillus oryzae.

[0059] One embodiment of the present application provides a method for preparing the above-mentioned immobilized cells for synthesizing lactulose, comprising the following steps:

[0060] Step S11: preparing a bacterial suspension by mixing wet cells of host cells having synthesized a cellobiose epimerase mutant with a first buffer solution, wherein the pH value of the first buffer solution is 7.5-8.0;

[0061] The first buffer solution may be, for example, 50 mM Tris-HCl having a pH of 8.0. In the bacterial suspension formed during preparation, the concentration of host cells may be, for example, 0.5 to 6 g / L. Exemplarily, the concentration of host cells in the bacterial suspension may be, for example, 0.5 g / L, 1 g / L, 2 g / L, 4 g / L, or 6 g / L.

[0062] Step S12: adding diatomaceous earth and polyethyleneimine to the bacterial suspension in sequence to perform a first stirring reaction;

[0063] The concentration of diatomaceous earth in the bacterial suspension may be, for example, 2 to 10 g / L. For example, the concentration of diatomaceous earth in the bacterial suspension may be, for example, 2 g / L, 4 g / L, 6 g / L, 8 g / L or 10 g / L.

[0064] The volume fraction of polyethyleneimine in the bacterial suspension may be 0.5% to 2.5%. For example, the volume fraction of polyethyleneimine in the bacterial suspension may be 0.5%, 1%, 1.5%, 2%, or 2.5%.

[0065] During the first stirring reaction process, the host cells are adsorbed on the diatomaceous earth, and the polyethyleneimine undergoes a cross-linking reaction with the groups on the diatomaceous earth.

[0066] Step S13: adding trishydroxymethyl phosphine to the bacterial suspension to perform a second stirring reaction;

[0067] The volume fraction of tris(hydroxymethyl) phosphine in the bacterial suspension may be 0.5% to 2.5%. For example, the volume fraction of tris(hydroxymethyl) phosphine in the bacterial suspension may be 0.5%, 1%, 1.5%, 2%, or 2.5%.

[0068] During the second stirring reaction process, trimethylol phosphine undergoes a cross-linking reaction with the groups on the diatomaceous earth to obtain immobilized cells.

[0069] Step S14: centrifuging the bacterial suspension and collecting the precipitate, and drying the precipitate to obtain immobilized cell particles;

[0070] The bacterial suspension is centrifuged to obtain a precipitate and a supernatant, the supernatant is discarded, and the precipitate is collected. The precipitate is the immobilized cells, and the precipitate containing the immobilized cells is dried to obtain immobilized cell particles.

[0071] In the bacterial suspension and the resulting immobilized cell particles, the mass ratio of the host cells to the diatomaceous earth is 0.05 to 3:1, the mass ratio of the host cells to the volume of polyethyleneimine is 1 g:2.5 mL to 12.5 mL, and the mass ratio of the host cells to the volume of tris(hydroxymethyl)phosphine is 1 g:2.5 mL to 12.5 mL. By controlling the ratio of the added amounts of host cells, diatomaceous earth, polyethyleneimine, and tris(hydroxymethyl)phosphine in the immobilized cells, the immobilized cells can be made highly stable while retaining the catalytic activity of the cellobiose epimerase mutant, thereby improving the efficiency of lactulose synthesis.

[0072] As an embodiment, the first stirring reaction time is 0.5 hours to 2.5 hours, and the second stirring reaction time is 0.5 hours to 2.5 hours.

[0073] As an embodiment, the first stirring reaction time is 0.5 hours to 1.5 hours, and the second stirring reaction time is 1.5 hours to 2.5 hours.

[0074] One embodiment of the present application provides a method for synthesizing lactulose, comprising:

[0075] The immobilized cells for synthesizing lactulose are added to a reaction solution containing lactose for catalysis, thereby converting lactose into lactulose.

[0076] The above-mentioned immobilized cells for synthesizing lactulose are used to catalyze the conversion of lactose into lactulose, thereby improving the conversion efficiency of lactulose.

[0077] The catalytic system for the immobilized cells catalyzing lactose synthesis for lactulose synthesis may include a suitable excipient, which may include but is not limited to at least one of a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent. Exemplarily, the catalytic system for the immobilized cells catalyzing lactose synthesis for lactulose synthesis may include a buffer, which may include but is not limited to at least one of Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer.

[0078] As an embodiment, the concentration of the immobilized cells used to synthesize lactulose in the reaction solution is 1 mg / mL to 10 mg / mL. Exemplarily, the concentration of the immobilized cells can be 1 mg / mL, 2.5 mg / mL, 5 mg / mL, 7.5 mg / mL, 8 mg / mL, 9.5 mg / mL or 10 mg / mL.

[0079] As an embodiment, the concentration of lactose in the reaction solution is 50 mg / mL to 500 mg / mL. For example, the concentration of lactose can be 50 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL or 500 mg / mL.

[0080] As an embodiment, the temperature of the catalytic reaction is 60°C to 80°C. Exemplarily, the contact between the immobilized cells for synthesizing lactulose and lactose can be carried out at 60°C, 60°C, 65°C, 70°C, 75°C, 78°C, or 80°C.

[0081] As an embodiment, the pH value of the catalytic reaction is 6-7.

[0082] As an embodiment, the total reaction system can be 0.5 mL, 1 mL, 5 mL, 10 mL, 1 L, 3.5 L or 35 L.

[0083] Example

[0084] The genes encoding the cellobiose epimerase mutants in Examples 1 to 47 of the present application can be referred to as CsCE genes. The CsCE genes contain a nucleotide sequence corresponding to the amino acid sequence of the cellobiose epimerase mutants described above. In each example, the CsCE gene contains a nucleotide sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 1. The CsCE gene is constructed into the pET-28a plasmid to obtain the pET-28a-CsCE plasmid. The host cell used is Escherichia coli.

[0085] The steps of each embodiment in this application are as follows:

[0086] Protein expression

[0087] 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 of LB liquid medium (Kan+, 100 μg / mL) and culture it at 37°C, 220 rpm overnight to serve as the seed solution.

[0088] b. Transfer the seed solution into 50 mL LB liquid medium (Kan+, 100 μg / mL) and culture in a shaking incubator at 37°C and 220 rpm for reactivation.

[0089] c. Transfer the reactivated bacterial solution into 800 mL 2YT liquid medium (Kan + , 100 μg / mL) and cultured in a shaking incubator at 37°C and 220 rpm until the OD (Optical Density) 600 was approximately 0.6-0.8.

[0090] d. Lower the shaker temperature to 16°C–18°C. After the culture solution cools down, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14–16 h.

[0091] e. After expression is completed, the above culture solution is collected into a bottle and centrifuged in a precooled centrifuge at 4°C, 5500 rpm, for 10 minutes.

[0092] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the cells using a vortex shaker.

[0093] g. Centrifuge the resuspended cells again at 5500 rpm for 10 minutes. Discard the supernatant, add 30 mL of protein purification buffer, and resuspend the cells using a vortex shaker (no solid particles). Pour the suspension into a 50 mL centrifuge tube and store in a -80°C refrigerator.

[0094] The protein purification buffer was 50 mM sodium phosphate buffer, pH 7.

[0095] Immobilized cells

[0096] LB liquid medium (fermentation basal medium) composition: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, dissolved in deionized water and fixed to volume. LB solid medium was additionally added with agar powder 20 g / L, sterilized at 121 °C for 20 min, and set aside.

[0097] Basal culture medium: final concentration of peptone is 7 g / L, final concentration of yeast powder is 8 g / L, final concentration of glycerol is 3 g / L, final concentration of K2HPO4 is 2 g / L, final concentration of MgSO4·7H2O is 2 g / L, final concentration of NaCl is 1 g / L, and pH is 7.0.

[0098] Inoculate 500 μL of the recombinant plasmid-containing strain from the glycerol tube into 50 mL of LB medium (containing kanamycin at a final concentration of 50 μg / mL). Incubate at 37°C with constant shaking at 220 rpm for 5 hours to serve as the seed culture. Inoculate the culture at a 2% (volume fraction) inoculum into a 5 L fermentor containing 3 L of basal fermentation medium. When the OD600 reaches a certain value, cool the culture to 28°C and induce with IPTG or lactose for 14-16 hours.

[0099] The induced recombinant strain was centrifuged at 8000 rpm for 10 minutes to collect the cells, and the bacterial slurry was resuspended in buffer A (50 mM Tris-HCl pH 8.0). The cells were centrifuged again and the supernatant was removed to obtain the bacterial slurry.

[0100] Weigh the bacterial sludge and dissolve it in buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension. Add diatomaceous earth and then polyethyleneimine and react for 1 h. Then add tris(hydroxymethyl)phosphine and react for 2 h. Centrifuge at 3500 rpm for 5 min, remove the supernatant, and then wash with distilled water 2-3 times. Remove the solid particles and place them in a clean culture dish. Air-dry them in a fume hood for 12-14 h until completely dry. Weigh them, seal them, and store them at room temperature.

[0101] Enzyme-catalyzed reactions

[0102] In vitro enzyme-catalyzed reaction conditions:

[0103] Reaction buffer: Tris-HCl, sodium phosphate buffer, potassium phosphate buffer;

[0104] Reaction pH: 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5;

[0105] The final lactose concentrations were 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, and 500 g / L;

[0106] Amount of immobilized cells: 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL;

[0107] Reaction temperature: 50℃, 60℃, 70℃, 80℃, 90℃;

[0108] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h;

[0109] The total reaction system was 0.5 mL, 1 mL, 5 mL, and 10 mL;

[0110] In each example, immobilized cells and lactose were added to Tris-HCl at pH 7 to form a catalytic solution. The concentration of immobilized cells in the catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at 70°C for 2 hours, and the conversion activity of each example was measured.

[0111] Detection of lactose, lactulose and epilactose

[0112] Liquid phase detection conditions are as follows:

[0113] Mobile phase: 75% acetonitrile, 20% methanol, 5% water;

[0114] Liquid phase column: Shodex HILICpak VG-504E;

[0115] Temperature: 40℃;

[0116] Flow rate: 1 mL / min;

[0117] Injection volume: 10 μL.

[0118] The standard curve of lactose can be found in Figure 1 As shown, the standard curve of lactulose can be found in Figure 2 As shown, the standard curve of lactose can be found in Figure 3 As shown, the liquid phase diagram of the reaction of the embodiment is shown in Figure 4 As shown, the immobilized cells containing the cellobiose epimerase mutants in Examples 1 to 47 respectively converted lactose into lactulose, and all had lactose epimerization activity.

[0119] Example 1 to Example 5:

[0120] The bacterial sludge was weighed and dissolved in 10 mL of buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension. Diatomaceous earth (diatomaceous earth concentration in the bacterial suspension was 6 g / L) was added, and then 0.1 mL of polyethyleneimine was added and reacted for 1 h. Subsequently, 0.15 mL of tris(hydroxymethyl)phosphine was added and reacted for 2 h. The mixture was centrifuged at 3500 rpm for 5 min, the supernatant was removed, and the mixture was washed with distilled water 2-3 times. The solid particles were removed and placed in a clean culture dish. The mixture was air-dried in a fume hood for 12-14 h until completely dry. The mixture was weighed, sealed, and stored at room temperature.

[0121] Immobilized cells and lactose were added to Tris-HCl at pH 7 to form a catalytic solution. The concentration of immobilized cells in this catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at 70°C for 2 hours, and the conversion activity of each example was measured.

[0122] Table 1 Concentration of bacterial sludge in bacterial suspension

[0123] Bacterial sludge concentration in bacterial suspension Example 1 0.5g / L Example 2 1.0g / L Example 3 2.0g / L Example 4 4.0g / L Example 5 6.0g / L

[0124] The experimental results are as follows Figure 5 As shown in the data, when the bacterial sludge concentration was in the range of 0.5 g / L to 2 g / L during the preparation of immobilized cells, the catalytic activity of the immobilized cells increased with the increase of the bacterial sludge concentration; when the bacterial sludge concentration was in the range of 2 g / L to 6.0 g / L, the catalytic activity of the immobilized cells decreased with the increase of the bacterial sludge concentration.

[0125] Example 6 to Example 10:

[0126] Weigh the bacterial sludge and dissolve it in 10 mL of buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension with a bacterial sludge concentration of 2.0 g / L. Add diatomaceous earth and then add 0.1 mL of polyethyleneimine to react for 1 h; then add 0.15 mL of tris(hydroxymethyl)phosphine and react for 2 h; centrifuge at 3500 rpm for 5 min, remove the supernatant, and then wash with distilled water 2-3 times. Remove the solid particles and place them in a clean culture dish. Air-dry them in a fume hood for 12-14 h until completely dry, weigh them, seal them, and store them at room temperature.

[0127] Immobilized cells and lactose were added to Tris-HCl at pH 7 to form a catalytic solution. The concentration of immobilized cells in this catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at 70°C for 2 hours, and the conversion activity of each example was measured.

[0128] Table 2 Concentration of diatomaceous earth in bacterial suspension

[0129] Diatomaceous earth concentration in bacterial suspension Example 6 2.0g / L Example 7 4.0g / L Example 8 6.0g / L Example 9 8.0g / L Example 10 10.0g / L

[0130] The experimental results are as follows Figure 6 As shown, when the diatomaceous earth concentration was in the range of 2.0 g / L to 6.0 g / L during the preparation of immobilized cells, the catalytic activity of the immobilized cells increased with the increase of the diatomaceous earth concentration; when the diatomaceous earth concentration was in the range of 6.0 g / L to 10.0 g / L, the catalytic activity of the immobilized cells decreased with the increase of the diatomaceous earth concentration.

[0131] Example 11 to Example 15:

[0132] Weigh the bacterial sludge and dissolve it in 10 mL of buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension with a bacterial sludge concentration of 2.0 g / L; add diatomaceous earth (the concentration of diatomaceous earth in the bacterial suspension is 6 g / L), and then add polyethyleneimine to react for 1 h; then add 0.15 mL of trishydroxymethyl phosphine and react for 2 h; centrifuge at 3500 rpm for 5 min, remove the supernatant, and then wash with distilled water 2-3 times. Remove the solid particles and place them in a clean culture dish, air-dry them in a fume hood for 12-14 h until completely dry, weigh them, seal them, and store them at room temperature.

[0133] Immobilized cells and lactose were added to Tris-HCl at pH 7 to form a catalytic solution. The concentration of immobilized cells in this catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at 70°C for 2 hours, and the conversion activity of each example was measured.

[0134] Table 3 Concentration of polyethyleneimine in bacterial suspension

[0135] Polyethyleneimine volume fraction Example 11 0.5％ Example 12 1.0％ Example 13 1.5％ Example 14 2.0％ Example 15 2.5％

[0136] The experimental results are as follows Figure 7 As shown in the figure, when the volume fraction of polyethyleneimine was in the range of 0.5% to 1.0% during the preparation of immobilized cells, the catalytic activity of the immobilized cells increased with the increase of polyethyleneimine concentration; when the volume fraction of polyethyleneimine was in the range of 1.0% to 2.5%, the catalytic activity of the immobilized cells decreased with the increase of polyethyleneimine addition amount.

[0137] Example 16 to Example 20:

[0138] Weigh the bacterial sludge and dissolve it in 10 mL of buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension, wherein the concentration of the bacterial sludge in the bacterial suspension is 2.0 g / L; add diatomaceous earth (the concentration of diatomaceous earth in the bacterial suspension is 6 g / L), and then add 0.1 mL of polyethyleneimine; then add 0.15 mL of trishydroxymethyl phosphine, and react for 2 h; centrifuge at 3500 rpm for 5 min, remove the supernatant, and then wash with distilled water 2-3 times. Remove the solid particles and place them in a clean culture dish, air-dry them in a fume hood for 12-14 h until completely air-dried, weigh them, seal them, and store them at room temperature.

[0139] Immobilized cells and lactose were added to Tris-HCl at pH 7 to form a catalytic solution. The concentration of immobilized cells in this catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at 70°C for 2 hours, and the conversion activity of each example was measured.

[0140] Table 4 Crosslinking time of polyethyleneimine

[0141]

[0142]

[0143] The experimental results are as follows Figure 8 As shown, when the cross-linking time of polyethyleneimine in the preparation process of immobilized cells is in the range of 0.5 hours to 1.0 hours, the catalytic activity of the immobilized cells increases with the increase of the cross-linking time of polyethyleneimine; when the cross-linking time of polyethyleneimine is in the range of 1.0 hours to 2.5 hours, the catalytic activity of the immobilized cells decreases with the increase of the cross-linking time of polyethyleneimine.

[0144] Example 21 to Example 25:

[0145] Weigh the bacterial sludge and dissolve it in 10 mL of buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension with a bacterial sludge concentration of 2.0 g / L; add diatomaceous earth (the concentration of diatomaceous earth in the bacterial suspension is 6 g / L), and then add 0.1 mL of polyethyleneimine to react for 1 h; then add trimethylolphosphine and react for 2 h; centrifuge at 3500 rpm for 5 min, remove the supernatant, and then wash with distilled water 2-3 times. Remove the solid particles and place them in a clean culture dish, air-dry them in a fume hood for 12-14 h until completely dry, weigh them, seal them, and store them at room temperature.

[0146] Immobilized cells and lactose were added to Tris-HCl at pH 7 to form a catalytic solution. The concentration of immobilized cells in this catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at 70°C for 2 hours, and the conversion activity of each example was measured.

[0147] Table 5 Concentration of tris(hydroxymethyl)phosphine in bacterial suspension

[0148] Volume fraction of tris(hydroxymethyl)phosphine Example 21 0.5％ Example 22 1.0％ Example 23 1.5％ Example 24 2.0％ Example 25 2.5％

[0149] The experimental results are as follows Figure 9 As shown, when the volume fraction of trihydroxymethyl phosphine is in the range of 0.5% to 1.5% during the preparation of immobilized cells, the catalytic activity of the immobilized cells increases with the increase of trihydroxymethyl phosphine concentration; when the volume fraction of trihydroxymethyl phosphine is in the range of 1.5% to 2.5%, the catalytic activity of the immobilized cells decreases with the increase of the amount of trihydroxymethyl phosphine added.

[0150] Example 26 to Example 30:

[0151] Weigh the bacterial sludge and dissolve it in 10 mL of buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension, wherein the concentration of the bacterial sludge in the bacterial suspension is 2.0 g / L; add diatomaceous earth (the concentration of diatomaceous earth in the bacterial suspension is 6 g / L), then add 0.1 mL of polyethyleneimine, and react for 1 h; then add 0.15 mL of tris(hydroxymethyl)phosphine, and react for 2 h; centrifuge at 3500 rpm for 5 min, remove the supernatant, and then wash with distilled water 2-3 times, remove the solid particles, place them in a clean culture dish, air-dry them in a fume hood for 12-14 h, until completely air-dried, weigh them, seal them, and store them at room temperature.

[0152] Immobilized cells and lactose were added to Tris-HCl at pH 7 to form a catalytic solution. The concentration of immobilized cells in this catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at 70°C for 2 hours, and the conversion activity of each example was measured.

[0153] Table 6 Crosslinking time of tris(hydroxymethyl)phosphine

[0154] Tris(hydroxymethyl)phosphine cross-linking time Example 26 0.5 hours Example 27 1.0 hour Example 28 1.5 hours Example 29 2.0 hours Example 30 2.5 hours

[0155] The experimental results are as follows Figure 10 As shown, when the cross-linking time of tris(hydroxymethyl)phosphine during the preparation of immobilized cells is in the range of 0.5 hours to 2.0 hours, the catalytic activity of the immobilized cells increases with the increase of the cross-linking time of tris(hydroxymethyl)phosphine; when the cross-linking time of tris(hydroxymethyl)phosphine is in the range of 2.0 hours to 2.5 hours, the catalytic activity of the immobilized cells decreases with the increase of the cross-linking time of tris(hydroxymethyl)phosphine.

[0156] Example 31 to Example 35:

[0157] Weigh the bacterial sludge and dissolve it in 10 mL of buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension, wherein the concentration of the bacterial sludge in the bacterial suspension is 2.0 g / L; add diatomaceous earth (the concentration of diatomaceous earth in the bacterial suspension is 6 g / L), then add 0.1 mL of polyethyleneimine, and react for 1 h; then add 0.15 mL of tris(hydroxymethyl)phosphine, and react for 2 h; centrifuge at 3500 rpm for 5 min, remove the supernatant, and then wash with distilled water 2-3 times, remove the solid particles, place them in a clean culture dish, air-dry them in a fume hood for 12-14 h, until completely air-dried, weigh them, seal them, and store them at room temperature.

[0158] Immobilized cells and lactose were added to Tris-HCl at pH 7 to form a catalytic solution. The concentration of immobilized cells in this catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at the catalytic temperature for 2 hours, and the conversion activity of each example was measured.

[0159] Table 7 Catalytic Temperature

[0160] Catalytic temperature Example 31 50℃ Example 32 60℃ Example 33 70℃ Example 34 80℃ Example 35 90℃

[0161] The experimental results are as follows Figure 11 As shown, 70℃ is the optimal catalytic temperature.

[0162] Example 36 to Example 47:

[0163] Weigh the bacterial sludge and dissolve it in 10 mL of buffer A (50 mM Tris-HCl pH 8.0) to obtain a bacterial suspension, wherein the concentration of the bacterial sludge in the bacterial suspension is 2.0 g / L; add diatomaceous earth (the concentration of diatomaceous earth in the bacterial suspension is 6 g / L), then add 0.1 mL of polyethyleneimine, and react for 1 h; then add 0.15 mL of tris(hydroxymethyl)phosphine, and react for 2 h; centrifuge at 3500 rpm for 5 min, remove the supernatant, and then wash with distilled water 2-3 times, remove the solid particles, place them in a clean culture dish, air-dry them in a fume hood for 12-14 h, until completely air-dried, weigh them, seal them, and store them at room temperature.

[0164] Immobilized cells and lactose were added to Tris-HCl to form a catalytic solution. The concentration of immobilized cells in the catalytic solution was 3 mg / mL, and the concentration of lactose was 200 g / L. The catalytic solution was reacted at 70°C for 2 hours, and the conversion activity of each example was measured.

[0165] Table 8 pH value of catalytic solution

[0166]

[0167]

[0168] The experimental results are as follows Figure 12 As shown, 6.5 is the optimal catalytic pH value.

[0169] The immobilized cells in Example 41 were catalyzed ten times continuously, and the conversion activity of each catalysis was measured. The results are as follows: Figure 13 As shown, after ten uses, the immobilized cells still retained more than 50% of their activity.

[0170] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0171] The above embodiments merely represent preferred embodiments of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. An immobilized cell for synthesizing lactulose, characterized in that: The invention comprises a carrier and host cells immobilized on the carrier, wherein the carrier comprises diatomaceous earth, polyethyleneimine and tris(hydroxymethyl)phosphine, and the host cells synthesize a cellobiose epimerase mutant having an amino acid sequence as shown in SEQ ID NO: 1; the mass ratio of the host cells to the diatomaceous earth is 0.05 to 3:1, the ratio of the mass of the host cells to the volume of the polyethyleneimine is 1 g:2.5 mL to 12.5 mL, and the ratio of the mass of the host cells to the volume of the tris(hydroxymethyl)phosphine is 1 g:2.5 mL to 12.5 mL.

2. The immobilized cells for synthesizing lactulose according to claim 1, characterized in that The mass ratio of the host cells to the diatomaceous earth is 1:2-4.

3. The immobilized cells for synthesizing lactulose according to claim 1, characterized in that The ratio of the mass of the host cells to the volume of the polyethyleneimine is 1 g:5.0 mL to 7.5 mL.

4. The immobilized cells for synthesizing lactulose according to claim 1, characterized in that The ratio of the mass of the host cells to the volume of the tris(hydroxymethyl)phosphine is 1 g:7.5 mL to 12.5 mL.

5. A method for preparing immobilized cells for synthesizing lactulose according to any one of claims 1 to 4, characterized in that: include: preparing a bacterial suspension by adding wet cells of host cells synthesizing a cellobiose epimerase mutant and a first buffer solution, wherein the pH value of the first buffer solution is 7.5 to 8.0; adding diatomaceous earth and polyethyleneimine to the bacterial suspension in sequence to perform a first stirring reaction; adding tris(hydroxymethyl)phosphine to the bacterial suspension to carry out a second stirring reaction; The bacterial suspension is centrifuged and the precipitate is collected, and the precipitate is dried to obtain immobilized cell particles.

6. The method for preparing immobilized cells for synthesizing lactulose according to claim 5, characterized in that: The first stirring reaction time is 0.5 hours to 2.5 hours, and the second stirring reaction time is 0.5 hours to 2.5 hours.

7. The method for preparing immobilized cells for synthesizing lactulose according to claim 6, characterized in that: The first stirring reaction time is 0.5 hours to 1.5 hours, and the second stirring reaction time is 1.5 hours to 2.5 hours.

8. A method for synthesizing lactulose, characterized in that: include: The immobilized cells for synthesizing lactulose according to any one of claims 1 to 4 are added to a reaction solution containing lactose for catalysis to convert lactose into lactulose.

9. The method for synthesizing lactulose according to claim 8, characterized in that The concentration of the immobilized cells used for synthesizing lactulose in the reaction solution is 1 mg / mL to 10 mg / mL, and the concentration of lactose in the reaction solution is 50 mg / mL to 500 mg / mL.

10. The method for synthesizing lactulose according to claim 8, characterized in that: The temperature of the catalytic reaction is 60° C. to 80° C., and the pH value of the catalytic reaction is 6 to 7.

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