Cellobiose epimerase mutant with high lactulose production and application thereof

Abstract

The application discloses a cellobiose epimerase mutant with high lactulose yield and application thereof, and belongs to the technical field of enzyme engineering. Dictyoglomus The cellobiose epimerase (referred to as Disp-WT enzyme) derived from microorganism Bifidobacterium longum sp. is used as a parent, a gene mutation technology is used, a histidine His at the 245th position is replaced by glutamic acid Glu, and a mutant H245E is obtained.The optimal catalytic condition of the mutant enzyme H245E does not change, but the isomerase activity of the enzyme at 85 DEG C is increased to 254 % of the original, and the generation amount of epi-lactose is greatly reduced.This finding has important research value for researching more cellobiose epimerases with low isomerase activity.

Description

Technical Field

[0001] This invention relates to a cellobiose epimerase mutant that produces high levels of lactulose and its applications, belonging to the field of enzyme engineering technology. Background Technology

[0002] Lactose (4-O-β-D-galactopyranosyl-D-glucose; C 12 H 22 O 11 Lactose is a disaccharide composed of galactose and glucose, a carbohydrate commonly found in the milk of mammals. Both of its constituent monosaccharides are six-membered cyclic aldoses. Lactose is hydrolyzed in water, and its glucose moiety hydrolyzes into an open-ring form, exhibiting reducing properties. Only a very small portion (<0.1%) of lactose in solution is in the open-ring state, but this portion is extremely important as it is chemically reactive and prone to chemical changes such as the Maillard reaction. Compared to other disaccharides, lactose has very low solubility. At 25°C, its solubility is only one-tenth that of sucrose. However, despite its low solubility, lactose does not easily crystallize; crystallization only occurs when the supersaturation exceeds 2.1. Crystalline lactose exists in a closed-ring form, with two terminal isomers: α-lactose and β-lactose. Crystallized α-lactose exists as a monohydrate and is the main form of lactose found in dried foods. β-lactose crystallizes in anhydrous form.

[0003] Lactulose (4-O-β-D-galactopyranosyl-D-fructose; C 12 H 22 O 11 Lactulose (Lactulose) is composed of galactose and fructose linked by β-1,4 glycosidic bonds. It is a white, powdery solid at room temperature, exhibiting good stability and safety. It is readily soluble in water and has a lower sweetness than lactose. Lactulose is not easily digested in the small intestine, but it increases intestinal peristalsis and absorbs water, promoting the passage of food through the intestines. It has been used as a laxative for over half a century. In the human colon, lactulose provides energy for lactobacilli and bifidobacteria. During metabolism, it produces organic acids such as lactic acid and formic acid, which can regulate the balance of intestinal flora and promote mineral absorption. Furthermore, lactulose has various beneficial effects, including treating hepatic encephalopathy, lowering blood ammonia levels, promoting the production of B vitamins, and resisting endotoxins. Due to these properties, lactulose can also be used as a functional food additive. For example, formula milk powder containing 0.5% lactulose can stimulate the growth of bifidobacteria in the infant's intestines, while formula milk powder containing 1% lactulose has a laxative effect. Moreover, the addition of lactulose does not affect the storage quality of the formula milk powder. Yogurt containing lactulose can also relieve constipation in young children, and lactulose is more effective than soy fiber and lacto-oligosaccharides. Because it promotes the growth of Bifidobacteria, lactulose can shorten the fermentation time of yogurt.

[0004] Currently, most industrial methods for preparing lactulose are based on the Lobry de Bruyn-Van Ekenstein reaction. Commonly used catalysts include hydroxides, tertiary amine compounds, boric acid, and potassium carbonate. This reaction uses lactose as a raw material and produces products including lactose, lactulose, and ipilactose, with subsequent separation processes being complex.

[0005] In the food industry, enzymatic methods for preparing functional sugars offer significant advantages over traditional chemical methods. Enzymatic preparation not only boasts high conversion rates, strong specificity, and simple operation procedures, but also demonstrates superior safety and environmental friendliness compared to chemical methods, making it the current trend in functional sugar production. Cellobiose epimerase is currently the only effective biocatalyst for preparing ipilactose and is also the most specific biocatalyst for lactulose production. Compared to other lactulose-producing enzymes, cellobiose epimerase does not require the addition of fructose as a co-substrate to lactose, exhibits higher conversion efficiency, and produces fewer byproducts.

[0006] Although enzymes that can produce lactulose already exist, cellobiose epimerase has low specific enzyme activity, and the high yield of the byproduct ipilactose increases the cost of subsequent separation and purification. Therefore, molecular modification is needed to improve enzyme activity and reduce the yield of the byproduct ipilactose. Summary of the Invention

[0007] The present invention provides a cellobiose epimerase mutant, which is obtained by mutating the 245th amino acid of the cellobiose epimerase as shown in SEQ ID NO. 2.

[0008] In one embodiment of the present invention, the nucleotide sequence encoding the cellobiose epimerase is shown in SEQ ID NO. 1.

[0009] In one embodiment of the present invention, the cellobiose epimerase is derived from microorganisms. Dictyoglomus sp. (GeneBank accession number is PMQ01814.1).

[0010] In one embodiment of the present invention, the cellobiose epimerase mutant is obtained by mutating histidine at position 245 of the cellobiose epimerase shown in SEQ ID NO. 2 to glutamic acid; and is named: H245E.

[0011] In one embodiment of the present invention, the amino acid sequence of the mutant H245E is shown in SEQ ID NO.4.

[0012] SEQ ID NO.4:

[0013] MEELKREVIEHLNNKIIPFWESLIDKKYGGFIGFVDFDLNRYPYAPKSCVLQTRILWFFSSAYNFKKDESLLEYAEHAYEFVINHIWDKEKEGLFWMVNHDGSSLDTRKHVYAQAFGIYALSEYYSAVKDSKALDLAIKLFEILENKCRDDYAYWEEFERDWTKKENIILGEYNVKPIRSMNSLLHILEAYT NLYKVWKDPFLKERLINIINLFKDKIYNFESNHFEVFLDSNWTPQIPAISYGEDIEGTWLLDLALETIGEMRSDIDEMNIKIAETVLKEGFEKSSLINEKAGDKIDKSRVWWVQAEALVGFLNAYNKTKDQKFLKAVTNLWDFIKNNLVDKRENSEWFSRLDEHLKPVKLPIVEPWKCPYHNGRMCIEVGRRI

[0014] In one embodiment of the present invention, the nucleotide sequence of the gene encoding the mutant H245E is shown in SEQ ID NO.3.

[0015] SEQ ID NO.3:

[0016]

[0017] In one embodiment of the present invention, the cellobiose epimerase is derived from microorganisms. Dictyoglomus sp. (GeneBank accession number is PMQ01814.1).

[0018] The present invention also provides a gene encoding the above-mentioned cellobiose epimerase mutant.

[0019] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0020] In one embodiment of the present invention, the recombinant vector uses pET-22b(+) as the expression vector.

[0021] The present invention also provides a recombinant cell expressing the above-mentioned cellobiose epimerase mutant, or carrying the above-mentioned gene, or carrying the above-mentioned recombinant vector.

[0022] In one embodiment of the present invention, the recombinant cells use bacteria or fungi as expression hosts.

[0023] This invention provides a recombinant enzyme catalyst containing the above-mentioned cellobiose epimerase mutant, which is any one of the following forms:

[0024] (1) Cultivate recombinant expression transformants containing the cellobiose epimerase mutant, and isolate transformant cells containing the enzyme of the recombinant cellobiose epimerase mutant;

[0025] (2) Cultivate recombinant expression transformants containing the cellobiose epimerase mutant, isolate transformant cells containing the recombinant cellobiose epimerase mutant enzyme, and break the transformant cells containing the recombinant cellobiose epimerase mutant enzyme to obtain cell lysate;

[0026] (3) Cultivate recombinant expression transformants containing the cellobiose epimerase mutant, isolate transformant cells containing the recombinant cellobiose epimerase mutant enzyme, break the transformant cells containing the recombinant cellobiose epimerase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant cellobiose epimerase mutant enzyme to obtain lyophilized enzyme powder.

[0027] This invention provides a method for improving the activity of cellobiose epimerase, wherein the method involves mutating histidine at position 245 of the cellobiose epimerase, as shown in SEQ ID NO. 2, to glutamic acid.

[0028] This invention provides a method for increasing the lactulose yield of cellobiose epimerase and reducing the byproduct ipilactose, wherein the method involves mutating histidine at position 245 of the cellobiose epimerase, as shown in SEQ ID NO. 2, to glutamic acid.

[0029] The present invention also provides a recombinant Escherichia coli expressing the above-mentioned cellobiose epimerase mutant.

[0030] In one embodiment of the present invention, the recombinant Escherichia coli is used as E. coli BL21(DE3) was used as the expression host, and pET-22b(+) was used as the expression vector.

[0031] This invention also provides a method for preparing the above-mentioned cellobiose epimerase mutant H245E, the specific steps of which are as follows:

[0032] (1) Utilizing sources Dictyoglomus Protein sequence homology modeling of cellobiose epimerase sp. to determine mutation sites;

[0033] (2) Design site-directed mutagenesis primers for mutants, and use the vector pET-22b(+)-Disp-WT carrying the cellobiose epimerase gene as a template to construct the mutant plasmid pET-22b(+)-H245E by site-directed mutagenesis;

[0034] (3) Transform Escherichia coli with the mutant plasmid pET-22b(+)-H245E. E. coli BL21(DE3), selected positive single clones after verification for fermentation culture;

[0035] (4) Centrifuge the bacterial cells, resuspend them, sonicate to break them up, and purify them by nickel ion affinity chromatography to obtain the mutant enzyme H245E.

[0036] The present invention also provides a method for increasing lactulose production and reducing the byproduct ipilactose, wherein the method comprises adding the above-mentioned mutant, or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinase catalyst, or the above-mentioned recombinant Escherichia coli to a reaction system containing lactose for reaction preparation.

[0037] In one embodiment of the present invention, the amount of the cellobiose epimerase mutant added is 0.05~2 mg / mL;

[0038] Preferably, the reaction conditions are: pH 7.0-8.0, temperature 75-85℃, and reaction time 15-120 min;

[0039] Preferably, the concentration of the substrate lactose is 100-800 mM.

[0040] In one embodiment of the present invention, the concentration of lactose in the above reaction system is 200 mM.

[0041] In one embodiment of the present invention, the amount of the cellobiose epimerase mutant added to the above reaction system is 0.1 mg / mL.

[0042] In one embodiment of the present invention, the reaction conditions of the reaction system are: pH 7.5, temperature 85°C, and reaction time 30 min.

[0043] The present invention also provides a method for preparing lactulose, wherein the above-mentioned mutant, or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinase catalyst, or the above-mentioned recombinant Escherichia coli is added to a reaction system containing lactose to prepare the lactulose.

[0044] In one embodiment of the present invention, the concentration of lactose in the above reaction system is 200 mM.

[0045] In one embodiment of the present invention, the amount of the cellobiose epimerase mutant added to the above reaction system is 0.1 mg / mL.

[0046] In one embodiment of the present invention, the reaction conditions of the reaction system are: pH 7.5, temperature 85°C, and reaction time 30 min.

[0047] The present invention also provides the use of the above-mentioned cellobiose epimerase mutant, or the above-mentioned gene, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned recombinant Escherichia coli in the preparation of products containing lactulose.

[0048] Beneficial effects

[0049] This invention provides a cellobiose epimerase mutant H245E. Compared with the wild-type enzyme Disp-WT, the optimal catalytic conditions of the cellobiose epimerase mutant H245E have not changed, but the isomerase activity of the mutant at 85°C is increased to 254% of the original, and the production of ipilactose is significantly reduced.

[0050] When this mutant was applied to the industrial production of lactulose, the yield per unit time was significantly improved, which further provides a favorable foundation for the industrial application of cellobiose epimerase. Attached Figure Description

[0051] Figure 1 The percentage of substrate and product after the original enzyme Disp-WT and the mutant enzyme reacted at 85 ℃ for 60 min.

[0052] Figure 2 Agarose gel electrophoresis analysis of mutant and original enzyme pure enzyme solution, where the leftmost band is the marker. Detailed Implementation

[0053] The culture media involved in the following examples are as follows:

[0054] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride.

[0055] LB solid medium: Add 15 g / L agar to LB liquid medium.

[0056] The detection methods involved in the following embodiments are as follows:

[0057] Lactulose content detection: The reaction products were detected using high performance liquid chromatography.

[0058] Column: Shodex® VG-50 4E; Mobile phase: a mixture of water, methanol, and acetonitrile (5:20:75, v / v); Column temperature: 40℃; Flow rate: 1 mL / min.

[0059] Example 1: Method for preparing Disp-WT enzyme mutant

[0060] The specific steps are as follows:

[0061] (1) Construction of recombinant plasmid pET-22b(+)-Disp-WT:

[0062] according to Dictyoglomus sp. (GeneBank accession number: PMQ01814.1), the gene fragment Disp-WT (nucleotide sequence shown in SEQ ID NO. 1) for synthesizing cellobiose epimerase, and ligated to the pET-22b(+) restriction site. Nde I and Xho Between I and II, the recombinant plasmid pET-22b(+)-Disp-WT was obtained.

[0063] The gene sequence (SEQ ID NO.1) encoding the wild-type cellobiose epimerase (Disp-WT) is shown below:

[0064]

[0065] The amino acid sequence (SEQ ID NO.2) of the wild-type cellobiose epimerase (Disp-WT) is shown below:

[0066] MEELKREVIEHLNNKIIPFWESLIDKKYGGFIGFVDFDLNRYPYAPKSCVLQTRILWFFSSAYNFKKDESLLEYAEHAYEFVINHIWDKEKEGLFWMVNHDGSSLDTRKHVYAQAFGIYALSEYYSAVKDSKALDLAIKLFEILENKCRDDYAYWEEFERDWTKKENIILGEYNVKPIRSMNSLLHILEAYT NLYKVWKDPFLKERLINIINLFKDKIYNFESNHFEVFLDSNWTPQIPAISYGHDIEGTWLLDLALETIGEMRSDIDEMNIKIAETVLKEGFEKSSLINEKAGDKIDKSRVWWVQAEALVGFLNAYNKTKDQKFLKAVTNLWDFIKNNLVDKRENSEWFSRLDEHLKPVKLPIVEPWKCPYHNGRMCIEVGRRI

[0067] (2) Construction of pET-22b(+)-H245E mutant plasmid:

[0068] Using pET-22b(+)-Disp-WT plasmid as a template, H245E, H245A, and A293L site-directed mutations were introduced via PCR. Sequencing verification results showed that no random mutations occurred except at the required mutation sites. Therefore, the mutant plasmids pET-22b(+)-H245E, pET-22b(+)-H245A, and pET-22b(+)-A293L were successfully constructed.

[0069] The H245E mutant primers are shown below: (Underlined text indicates mutants)

[0070] H245E forward mutation primers:

[0071] 5'- TCCTATGGAGAAGATATA GAA GGTACATGGCTTTTGGATTTAG -3 (SEQ ID NO.5);

[0072] H245E reverse mutation primer:

[0073] 5'- TATATC TTCTCCATAGGAGATTGCAGGTATCTGAGGTG -3' (SEQ ID NO. 6);

[0074] H245A forward mutation primers:

[0075] 5'- TCCTATGGAGAAGATATA GCG GGTACATGGCTTTTGGATTTAG -3 (SEQ ID NO.7);

[0076] H245A reverse mutation primer:

[0077] 5'- TATATC CGC TCCATAGGAGATTGCAGGTATCTGAGGTG -3' (SEQ ID NO.8);

[0078] A293L forward mutation primer:

[0079] TTAATGAAAA A CTG GGAGAC AAGATTGATA AATC (SEQ ID NO.9)

[0080] A293L reverse mutation primer:

[0081] TTGTCTCC CAG TTTTTCATT AATAAGACTG GATT (SEQ ID NO.10)

[0082] The composition of the PCR reaction system is shown in Table 1, using the cloning vector pET-22b(+)-Disp-WT carrying the cellobiose epimerase target gene as a template.

[0083] Table 1: Composition of the PCR reaction system

[0084]

[0085] The PCR amplification conditions were as follows: 95 °C pre-denaturation for 3 min; followed by 95 °C denaturation for 0.5 min, 56 °C annealing for 0.5 min, and 72 °C extension for 3.5 min, for 26 cycles; and finally, incubation at 72 °C for 5 min.

[0086] The PCR amplification products were detected by agarose gel electrophoresis and purified by gel extraction.

[0087] The PCR amplification products, purified by agarose gel electrophoresis, were then subjected to restriction endonuclease... Nde I and XhoAfter digestion with enzyme I, the cells were ligated into the vector pET-22b(+) and transformed into E. coli DH5α competent cells. After overnight culture in LB solid medium containing 50 µg / mL ampicillin, single clones were picked and cultured in LB liquid medium containing 50 µg / mL ampicillin. The mutant plasmids pET-22b(+)-H245E, pET-22b(+)-H245A, and pET-22b(+)-A293L were extracted and transformed into host E. coli BL21(DE3) competent cells. The mutant plasmids were identified as correct mutations by sequencing, and the plasmids pET-22b(+)-H245E, pET-22b(+)-H245A, and pET-22b(+)-A293L containing the mutants were prepared.

[0088] Example 2: Expression and purification method of cellobiose epimerase mutants H245E, H245A, and A293L

[0089] The specific steps are as follows:

[0090] (1) The pET-22b(+)-Disp-WT prepared in Example 1 and the plasmids pET-22b(+)-H245E, pET-22b(+)-H245A and pET-22b(+)-A293L containing mutants were transformed into Escherichia coli BL21(DE3) cells. Positive transformants were picked and cultured overnight in LB liquid medium at 37 ℃ and 200 rpm in shake flasks. Then they were inoculated into LB liquid medium and cultured at 37 ℃ until the OD600 value was 0.6~0.8. The temperature was then lowered to 28 ℃, and IPTG was added to a final concentration of 1 mM to induce fermentation for 6 h to obtain the fermentation broth.

[0091] (2) The prepared fermentation broth was centrifuged at 4 ℃ and 10000 rpm for 20 min, and the precipitate (cells) was collected. 20 mL of buffer (50 mM PIPES, 200 mM NaCl, pH adjusted to 7 with HCl) was added to the cells to fully resuspend them. Then, the centrifuge tube was placed in an ice bath and then placed in an ultrasonic cell disruptor. The ultrasonic disruption conditions were: working time 1 ls, stop time 2 s, for a total of 20 min. The obtained disrupted solution was centrifuged at low temperature and high speed at 4 ℃ and 10000 rpm for 30 min to obtain crude enzyme solution. It was filtered through a 0.45 μm microporous membrane for later use.

[0092] Crude enzyme solutions containing wild-type Disp-WT, mutant H245E, mutant H245A, and mutant A293L were prepared respectively.

[0093] (3) Obtaining pure enzyme solution containing mutant

[0094] To prepare a nickel ion affinity chromatography column, first, at room temperature, use a constant flow pump to pump deionized water into the column to rinse it (approximately 6-12 column volumes). Then, equilibrate the column environment with buffer A (500 mmol / L NaCl, 50 mM PIPES, pH 7.5). When the pH of the effluent at the bottom of the column matches that of buffer A pumped into the column (approximately 5 column volumes of buffer are required), add the crude enzyme solution obtained in step (2) into the column. First, wash away any contaminating proteins with buffer B (500 mmol / L NaCl, 50 mmol / L imidazole, 50 mM PIPES, pH 7.5) until baseline equilibration. Then, elute with elution buffer containing a high concentration of imidazole (500 mmol / L NaCl, 500 mmol / L imidazole, 50 mM PIPES, pH 7.5). Collect the elution buffer containing the absorption peak to obtain the target protein.

[0095] Pure enzyme solutions containing Disp-WT, H245E, H245A mutant, and A293L mutant were prepared respectively. The prepared H245E and wild-type WT pure enzyme solutions were analyzed by agarose gel electrophoresis (e.g., Figure 2 As shown in the figure, the results show that the relative molecular mass of the purified enzyme is around 40 kDa and is a single band, which is consistent with the calculated molecular mass.

[0096] The prepared pure enzyme solutions were diluted to a final concentration of 1 mg / mL.

[0097] Example 3: Enzyme activity determination of cellobiose epimerase

[0098] The method for detecting the enzyme activity of the mutant of the present invention is as follows:

[0099] (1) Preparation of α-lactose PIPES solution: Weigh 50 mM piperazine-1,4-diethanesulfonic acid (PIPES) into water, add NaOH to adjust the pH to dissolve the PIPES, then continue to add NaOH to make the pH 7.5, and then make up to 1 L. Weigh 250 mM α-lactose, dissolve it completely in the prepared PIPES and make up to 1 L.

[0100] (2) The reaction system is 1 mL. 200 µL of the pure enzyme solution prepared in Example 2 was added to 800 µL of α-lactose PIPES solution (250 mM) to obtain the reaction system. The reaction system was placed at 85 °C for 30 min. 100 µL of hydrochloric acid (1.5 mol / L) was added to terminate the reaction. The reaction products were detected and the enzyme activity was calculated. The results are shown in Table 2. The enzyme activity here means the amount of enzyme required to produce 1 μmol of lactulose per minute.

[0101] 1 U of total enzyme activity is defined as the amount of enzyme required to produce 1 μmol of lactulose per minute under pH 7.5 conditions. The amount of lactulose synthesized is determined using HPLC, and the enzyme activity is calculated.

[0102] Table 2: Enzyme activity of wild-type enzymes and different mutants at 85℃

[0103]

[0104] Compared with the wild-type enzyme WT, the optimal catalytic conditions of the mutant enzyme H245E remained unchanged, but the isomeric enzyme activity at 85°C increased to 254%.

[0105] Example 4: Application of cellobiose epimerase mutant

[0106] Most industrial methods for preparing lactulose are based on the Lobry de Bruyn-Van Ekenstein reaction. Commonly used catalysts include hydroxides, tertiary amine compounds, boric acid, and potassium carbonate. This reaction uses lactose as a raw material and produces various monosaccharides, including lactose, lactulose, and ipilactose, with complex subsequent separation processes. The co-catalytic production of lactulose using β-galactosidase and glucose isomerase requires fructose as a co-substrate and has low conversion efficiency, necessitating the separation of numerous byproducts during lactulose extraction. Cellobiose epimerase, on the other hand, only requires lactose as a substrate, does not require fructose as a co-substrate, and has high conversion efficiency. The specific steps are as follows:

[0107] Reaction system (1 mL):

[0108] The pure enzyme solutions prepared in Example 2, consisting of 0.2 mL (0.94 U) containing wild-type Disp-WT, H245E, mutant H245A, or mutant A293L, were added to a system containing 0.8 mL of lactose for reaction. The final lactose concentration was 200 mM. The reaction system was then placed at 85°C for 30 min. The results are shown in Table 3. Figure 1 As shown.

[0109] Table 3: Products after reaction with different cellobiose epimerases

[0110]

[0111] The results showed that, compared with the wild-type enzyme, H245E produced more lactulose per unit time, while the mutant produced significantly less ipilactose per unit time.

[0112] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A cellobiose epimerase mutant for producing lactulose, characterized in that, The mutant is obtained by mutating histidine at position 245 of cellobiose epimerase, as shown in SEQ ID NO. 2, to glutamic acid.

2. The gene encoding the cellobiose epimerase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. A recombinant cell, characterized in that, The recombinant cells express the cellobiose epimerase mutant of claim 1, or the recombinant cells carry the gene of claim 2, or the recombinant cells carry the recombinant vector of claim 3, wherein the recombinant cells are bacteria or fungi as expression hosts.

5. A recombinant enzyme catalyst containing the cellobiose epimerase mutant of claim 1, characterized in that, It is any of the following forms: (1) Culture recombinant expression transformants containing the cellobiose epimerase mutant and isolate transformant cells containing the recombinant cellobiose epimerase mutant enzyme; (2) Cultivate recombinant expression transformants containing the cellobiose epimerase mutant, isolate transformant cells containing the recombinant cellobiose epimerase mutant enzyme, and break the transformant cells containing the recombinant cellobiose epimerase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the cellobiose epimerase mutant, isolate transformant cells containing the recombinant cellobiose epimerase mutant enzyme, break the transformant cells containing the recombinant cellobiose epimerase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant cellobiose epimerase mutant enzyme to obtain lyophilized enzyme powder.

6. A method for increasing the activity of cellobiose epimerase or increasing the yield of lactulose prepared using cellobiose epimerase and reducing the byproduct ipilactose, characterized in that, The method involves mutating histidine at position 245 of the cellobiose epimerase, as shown in SEQ ID NO.2, to glutamic acid.

7. A method for increasing lactulose yield and reducing the byproduct ipilactose, characterized in that, The method involves adding the cellobiose epimerase mutant of claim 1, the recombinant cell of claim 4, or the recombinase catalyst of claim 5 to a reaction system containing lactose for reaction preparation.

8. A method for preparing lactulose, characterized in that, The method involves adding the cellobiose epimerase mutant of claim 1, the recombinant cell of claim 4, or the recombinase catalyst of claim 5 to a reaction system containing lactose to prepare the product.

9. The method according to claim 8, characterized in that, The amount of cellobiose epimerase mutant added is 0.05~2 mg / mL; the reaction conditions are pH 7.0-8.0, temperature 75-85℃, and reaction time 15-120 min; the concentration of lactose is 100-800 mM.

10. The use of the mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 4, or the recombinase catalyst of claim 5 in the preparation of lactulose or lactulose-containing products.

Images