Mutant CE-FA with improved structural isomerization catalytic activity

Abstract

The invention discloses a mutant CE-FA with improved structural isomerization catalytic activity, and belongs to the field of enzyme engineering. According to the invention, a mutant CsCE-Q371E of cellobiose epimerase (CsCE) derived from Caldicellosipurtor schcharlyticus is taken as a research object, and a mutant CsCE-FA of which the structural isomeric activity is improved by 3.37 times compared with that of the CsCE-Q371E is obtained; under a buffer system with the temperature of 80 DEG C, the pH value of 7.5 and 50 mM of HEPEs, the half-life period (t1 / 2) of CsCE-FA reaches 545 min and is increased by about 10 times and about 8.37 h compared with the half-life period (t1 / 2) (42.68 min) of CsCE-Q371E, a feasible scheme is provided for CE enzyme improvement, and the CsCE-FA has important significance in promoting enzymatic industrial synthesis of lactulose.

Description

TECHNICAL FIELD

[0001] The present application relates to a mutant CE-FA with improved structural isomerization catalytic activity, belonging to the field of enzyme engineering. BACKGROUND

[0002] Lactulose, as a functional oligosaccharide, is a disaccharide molecule composed of one molecule of galactose and one molecule of fructose connected by a beta-1, 4 glycosidic bond. This unique chemical structure endows lactulose with a series of remarkable physicochemical properties, including low heat value, no Maillard reaction, good acid and heat resistance, etc. It is precisely due to these properties that lactulose has been widely used in the food industry as a sweetener, emulsifier, stabilizer and thickening agent, etc., greatly improving the quality and taste of food. At present, the global market demand for lactulose is huge, and it is urgent to develop more efficient and environmentally friendly synthesis technology to meet the growing market demand.

[0003] In the preparation method of lactulose, chemical preparation usually requires relatively severe reaction conditions, specifically the use of high-concentration alkaline catalysts and operation at high temperature. Although this preparation method can obtain lactulose, the product obtained has a deep color and many by-products, affecting the purity and quality of the product. In contrast, the conditions for enzymatic catalysis of lactose to lactulose are much milder, with fewer by-products, and because of the mildness of the reaction conditions, there is no problem of heavy metal residues in the prepared lactulose product. In addition, the mild reaction conditions also avoid the destruction of sugar molecules and coking phenomenon, so that the final product is white in color and has no odor, and the sensory quality is significantly better than that of the product prepared by chemical method.

[0004] In the process of enzymatic catalysis, cellobiose epimerase (CE enzyme) can specifically catalyze the conversion of glucose residues in lactose molecules to fructose residues, thereby realizing the isomerization process of lactose to lactulose. However, the natural CE enzyme has certain limitations in structure, and its isomerization activity is usually low, which limits the conversion efficiency of lactulose. In order to overcome this problem, enzyme protein engineering uses directed evolution technology to modify CE enzyme, aiming to improve its catalytic activity, thereby promoting more efficient conversion from lactose to lactulose. The application of this technology not only improves the preparation efficiency of lactulose, but also provides a new technical path for the industrial production of lactulose. SUMMARY

[0005] [TECHNICAL PROBLEM] The present application constructs a mutant of CE through the directed evolution technology of random mutation, improves its structural isomerization activity, and the mutant constructed by the present application has good catalytic properties, which is of great significance for promoting the greenization of existing lactulose production process. Cs The mutant of CE constructed by the present application has good catalytic properties, which is of great significance for promoting the greenization of existing lactulose production process.

[0006] [Technical solutions] A first object of the present application is to provide a cellobiose epimerase mutant, which is a mutant of a cellobiose epimerase having an amino acid sequence represented by SEQ ID NO. 1 Cs Based on CE-Q371E, the following mutations are made: mutating the lysine at position 46 into tyrosine, mutating the glutamine at position 48 into asparagine, mutating the isoleucine at position 52 into valine, mutating the leucine at position 172 into valine, mutating the serine at position 173 into leucine, mutating the glutamic acid at position 174 into proline, mutating the phenylalanine at position 231 into alanine, mutating the lysine at position 356 into leucine, mutating the asparagine at position 358 into aspartic acid, mutating the aspartic acid at position 360 into serine, and mutating the glutamic acid at position 362 into asparagine; or, Based on CE-Q371E having an amino acid sequence represented by SEQ ID NO. 1 Cs mutating the glutamic acid at position 174 into proline, mutating the asparagine at position 175 into aspartic acid, and mutating the valine at position 177 into glutamine.

[0007] A second object of the present application is to provide a gene encoding the cellobiose epimerase mutant.

[0008] A third object of the present application is to provide an expression vector containing the gene.

[0009] A fourth object of the present application is to provide a genetically engineered bacterium containing the cellobiose epimerase mutant or the expression vector.

[0010] In an embodiment of the present application, the genetically engineered bacterium uses E. coli as a host cell.

[0011] In an embodiment of the present application, the E. coli includes BL21 (DE3).

[0012] In an embodiment of the present application, a pET series is used as an expression vector.

[0013] In an embodiment of the present application, the expression vector includes pET-28b.

[0014] A fifth object of the present application is to provide a method for improving the structural isomerization activity of cellobiose epimerase, which is a method for making the following mutations based on CE-Q371E having an amino acid sequence represented by SEQ ID NO. 1 Cs Based on CE-Q371E, the following mutations are made: the 52nd isoleucine is mutated into valine, the 172nd leucine is mutated into valine, the 173rd serine is mutated into leucine, the 174th glutamic acid is mutated into proline, the 231st phenylalanine is mutated into alanine, the 356th lysine is mutated into leucine, the 358th asparagine is mutated into aspartic acid, the 360th aspartic acid is mutated into serine, and the 362nd glutamic acid is mutated into asparagine; or, based on the amino acid sequence shown in SEQ ID NO. 1 Cs the 174th glutamic acid is mutated into proline, the 175th asparagine is mutated into aspartic acid, and the 177th valine is mutated into glutamic acid.

[0015] The application further provides a lactulose preparation method, which comprises adding the cellobiose epimerase mutant or the genetically engineered bacterium to lactose as a substrate for reaction.

[0016] In an embodiment of the application, the reaction condition is at pH 7-7.5 and 70-85℃.

[0017] The application further provides application of the cellobiose epimerase mutant, the genetically engineered bacterium or the method in preparation of lactulose or a lactulose-containing product.

[0018] The application further provides application of the cellobiose epimerase mutant, the genetically engineered bacterium or the method in preparation of food and medicine.

[0019] Beneficial effects: The application successfully obtains a mutant with improved catalytic performance by random mutation and modification of the cellobiose epimerase Cs CE-Q371E. Cs Compared with the cellobiose epimerase mutant Cs CE-Q371E, the structural isomerization activity of the cellobiose epimerase mutant Cs CE-Q371E is improved by 3.37 times, and the structural isomerization activity of the cellobiose epimerase mutant Cs CE--E174P-N175D-V177Q-Q371E is improved by 1.59 times compared with the cellobiose epimerase mutant Cs CE-Q371E. Cs The half-life (t 1 / 2The enzyme activity reached 545 min, and after treatment at 80℃ for 240 min, the relative enzyme activity was still maintained at over 70%, which is better than the half-life of CsCE-Q371E (t). 1 / 2 The reaction time was increased by approximately 10 times (42.68 min) to approximately 8.37 h, demonstrating both improved activity of the structural isomerase and good thermal stability. This invention provides a new, highly efficient catalyst for the industrial production of lactulose, not only solving the environmental pollution problems associated with traditional chemical methods for lactulose preparation but also overcoming the bottleneck of low catalytic efficiency of natural enzymes, thus opening up new avenues for the green manufacturing of lactulose. Attached Figure Description

[0020] Figure 1 The results of the initial screening of the mutant library of cellobiose epimerase of the present invention; Figure 2 These are the results of the secondary screening of cellobiose epimerase mutants of the present invention; Figure 3 Cs Half-life assessment of CE-Q371E and its mutants; (A) Cs CE-Q371E and its mutants Cs The decay constant of CE-FA at 80°C ( K d The vertical axis represents Ln of (Ut / Uo), where Ut is the residual activity of the enzyme after pre-culture, and Uo represents the initial activity of the enzyme; (B) Cs CE-Q371E and its mutants Cs Thermal stability of CE-FA. Detailed Implementation

[0021] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0022] Raw materials used in the examples: 1. Plasmid pET-28b- Cs CE-Q371E: The plasmid pET-28b- used in this embodiment Cs CE-Q371E discloses a construction method in patent publication number CN113564151A, which contains... Cs The gene fragment of CE-Q371E (amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2): The preparation of the mutant enzyme is disclosed in Example 2 of the specification (paragraph

[0073] ). Cs Primers were designed using pET-28b-CsCE as a template for CE-Q371E, and PCR was performed. CsMutated the CE-Q371 site to obtain a mutant plasmid.

[0023] 2. Primers: Cs CE-F: 5'-TGGTGCCGCGGCAGCCATATG-3'; Cs CE-R: 5'-GGTGGTGGTGGTGCTCGAGTTA-3'.

[0024] ZT-F: 5'-CATATGGCTGCCGCGGCACCA-3'; ZT-R: 5'-TAACTCGAGCACCACCACCACC-3'.

[0025] 3. Culture medium: LB liquid medium: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L; Kan + Add it when using it.

[0026] LB agar medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, 15 g / L agar; Kan + Add it when pouring the flat plate.

[0027] 4. Other reagents: Premix Taq (Baori Biotechnology Co., Ltd.), PrimerStar Max mix (Baori Biotechnology Co., Ltd.), Ezmax Ultra Universal CloneMix (Shanghai Tulugang Biotechnology Co., Ltd.); Lactose (Sinopharm Chemical Reagent Co., Ltd.), IPTG (Sangon Biotech Co., Ltd.), Lysozyme (≥20000 U / mg) purchased from Shanghai Titan Technology Co., Ltd.), Nickel ion affinity chromatography column (Sangon Biotech Co., Ltd.).

[0028] Example 1: Establishing a mutant library With plasmid pET-28b- Cs Using CE-Q371E as a template, primers were designed. Cs CE-F / R was obtained through PCR amplification. Cs Random mutant fragments of CE-Q371E. The random mutant PCR reaction system is shown in Table 1: Table 1 Random Mutation PCR Reaction System

[0029] With plasmid pET-28b- CsUsing CE-Q371E as a template, primers ZT-F / R were designed to amplify the plasmid vector fragment pET-28b by PCR. The PCR reaction system is shown in Table 2, and the PCR reaction procedure is shown in Table 3.

[0030] Table 2 Components of the vector PCR reaction system

[0031] Table 3 PCR reaction procedure

[0032] The plasmid vector fragment pET-28b and Cs The random mutant fragments of CE-Q371E were recovered by gel extraction and recombinant plasmids were constructed by homologous recombination (the homologous recombination reaction system is shown in Table 4, and the homologous recombination reaction procedure is shown in Table 5).

[0033] The constructed recombinant plasmid was transformed into [a new type of plasmid] using a thermal shock method. E. coli BL21(DE3), transformed cells were plated on LB (Kan + The plates were incubated upside down at 37°C for 16 h to construct a mutant library.

[0034] Table 4 Components of the Homologous Recombination Reaction System

[0035] Table 5 Homologous recombination reaction procedure

[0036] Example 2: Screening of mutant libraries (1) Initial screening: ① Mutant-induced expression LB (Kan) coated with the mutant library from Example 1 was used. + Single colonies from the plate were transferred one by one to 96-well plates, each well containing 500 μL (containing 50 μg / mL Kans). + LB liquid medium was incubated at 37°C and 250 rpm for 16 h to obtain seed culture; The seed solution in each well was diluted 100-fold and then transferred one by one to a 96-well deep-well plate. Each well contained 1 mL of the diluted seed solution and 500 μL of solution containing 50 μg / mL Kansin. + The culture was induced and cultured in 0.4 mM IPTG LB liquid medium at 25℃ and 250 rpm for 6 h. The bacterial culture from each well was collected. The remaining seed culture was stored at 4℃ for later use.

[0037] ② Bacterial cell lysis Centrifuge the bacterial culture from step ① at 4℃ and 4000 rpm for 15 min, and collect the bacterial cells. Resuspend the bacterial cells in 300 μL of HEPFS buffer (50 mM, pH 7.5) containing 5 mg / mL lysozyme, and incubate at 37℃ for 1 h. Centrifuge at 4000 rpm and 4℃ for 15 min, and collect the supernatant, which is the crude enzyme solution.

[0038] ③ Volume enzyme activity detection Add the crude enzyme solution extracted in step ② to a new 96-well deep plate containing 30 μL of HEPES buffer (50 mM, pH 7.5) with 40 g / L lactose dissolved in each well. Mix well using a microplate shaker and react in a 75°C water bath for 10 min, then stop the reaction by boiling for 5 min.

[0039] After cooling to room temperature, centrifuge at 4000 rpm and 4℃ for 15 min, collect the supernatant, dilute the supernatant twice with deionized water, take 50 μL of the diluted solution and add 150 μL of lactulose detection reagent, shake well, and incubate at 46℃ for 70 min.

[0040] Lactulose test reagent: 140 μL of 75% concentrated sulfuric acid and 10 μL of mixed colorimetric reagent (mixed colorimetric reagent: 2.5% (m / v) cysteine ​​hydrochloride and 0.08% tryptophan (m / v) dissolved in 10 mM hydrochloric acid).

[0041] The absorbance was measured at 518 nm using a microplate reader. The results are as follows: Figure 1 As shown.

[0042] Mutants with absorbance values ​​higher than wild-type enzymes were selected for rescreening.

[0043] (2) Secondary screening ① Overexpression The mutants initially screened in step (1) were sequenced, and the corresponding recombinant plasmids were then transferred into the expression host. E. coli Transformation solution was spread onto LB (Kan) competent cells. + Incubate on 5 mL / 50 mL LB agar plates at 37°C overnight; pick a single colony and incubate in 5 mL / 50 mL LB agar plates. + Seed culture was prepared by overnight incubation at 37°C and 200 rpm in liquid culture medium.

[0044] Take 1 mL of seed culture and inoculate it at a 1% (v / v) inoculation rate onto 40 mL / 100 mL of fresh LB (Kan) seed culture. + In liquid culture medium, incubate at 37°C with shaking at 200 rpm until the bacterial concentration reaches OD500. 600The concentration was 0.8. At this point, IPTG was added until the final concentration reached 0.2 mM. The culture was induced and cultured at 25℃ and 200 rpm for 18 h to obtain the bacterial culture.

[0045] ② Volume enzyme activity assay Take 5 mL of bacterial culture, centrifuge at 5500 rpm and 4℃ for 12 min, discard the supernatant, add 5 mL of HEPFS buffer (50 mM, pH 7.5) containing 5 mg / mL lysozyme to resuspend, and enzymatically digest at 37℃ for 1 h. Centrifuge at 4000 rpm and 4℃ for 15 min, and collect the supernatant, which is the crude enzyme solution.

[0046] Add 1 mL of crude enzyme solution to 1 mL of HEPES buffer (50 mM, pH 7.5) containing 400 mM lactose, react at 75 °C for 10 min, and then boil for 5 min to terminate the reaction.

[0047] Volumetric enzyme activity was determined using a colorimetric method. After cooling to room temperature, the enzyme was centrifuged at 4000 rpm and 4℃ for 15 min. The supernatant was collected and diluted 100-fold with deionized water. 50 μL of the diluted solution was added to 150 μL of lactulose assay reagent (2.8 mL of 75% concentrated sulfuric acid and 0.2 mL of mixed colorimetric reagent; mixed colorimetric reagent: 2.5% (m / v) cysteine ​​hydrochloride and 0.08% tryptophan (m / v) dissolved in 10 mM hydrochloric acid). The mixture was shaken well and incubated at 46℃ for 70 min. The absorbance was measured at 518 nm using a microplate reader.

[0048] Some of the screening results are as follows: Figure 2 As shown, the control strain ( Cs CE-Q371E) is strain number 1, and 17 (M1), 18 (M2), and 19 (M3) are effective strains with higher volumetric enzyme activity than the control.

[0049] (3) Enzyme activity assay The positive mutant with the highest volumetric enzyme activity was subjected to enzyme activity assay and gene sequencing, and the mutation site was determined by comparison with the original strain. The extracted plasmid was used as a template for the next round of mutation and screening. The specific steps are as follows: ①Induced expression The mutants screened in step (2) were sequenced, and the corresponding recombinant plasmids were then transferred into the expression host. E. coli Transformation of BL21 (DE3) competent cells was plated on LB (Kan+) plates and incubated overnight at 37°C; single colonies were picked and cultured in 50 mL LB (Kan+) plates. + Seed culture was prepared by overnight incubation at 37°C and 200 rpm in liquid culture medium.

[0050] Take 1 mL of seed culture and inoculate it at a 1% (v / v) inoculation rate into 100 mL of fresh LB (Kan) seed culture. + In liquid culture medium, the bacterial culture was carried out at 37℃ and 200 rpm with shaking until the bacterial concentration OD600 was 0.8. At this time, IPTG was added to the final concentration of 0.2 mM. The culture was then induced at 25℃ and 200 rpm for 18 h to obtain the bacterial solution.

[0051] Centrifuge the bacterial culture at 4℃ and 6000 rpm for 15 min, collect the bacterial cells, resuspend the bacterial cells in deionized water to remove residual culture medium, and centrifuge again at 4℃ and 6000 rpm for 10 min. The finally collected bacterial cells are stored in a -20℃ refrigerator for later use.

[0052] ② Enzyme purification The bacterial cells obtained in step ① were suspended and mixed in 20 mL of protein purification buffer (50 mM sodium phosphate, 200 mM sodium chloride, 10 mM imidazole, pH 7.5), and then sonicated in an ice bath to obtain crude enzyme solution. The crude enzyme solution was centrifuged at 4℃ and 12000 rpm for 15 min, the supernatant was collected, filtered through a 0.22 μm aqueous filter membrane, and the filtrate was collected.

[0053] Pack the column with 5 mL of nickel ion affinity chromatography packing material, wash with deionized water for 30 min (flow rate: 1 mL / min; 10 column volumes), and then equilibrate with protein purification buffer (50 mM sodium phosphate, 200 mM sodium chloride, 10 mM imidazole, pH 7.5) for half an hour. The filtrate was passed through a nickel-ion affinity chromatography column. First, impurities were removed using a washing buffer (50 mM sodium phosphate, 200 mM sodium chloride, 35 mM imidazole, pH 7.5). Then, elution was performed using an elution buffer (50 mM sodium phosphate, 200 mM sodium chloride, 25 mM imidazole, pH 7.5), and the eluent containing the target protein was collected. The eluent containing the target protein was dialyzed three times sequentially using 200, 100, and 50 mM HEPES (pH 7.5) at intervals of 2 h, 4 h, and 6 h. After dialysis, the purified enzyme solution in the dialysis bag was collected and stored at 4°C for later use.

[0054] ③ Enzyme activity assay Cs The catalytic activity of the structural isomers and epiomers of CE was evaluated by measuring their ability to catalyze the conversion of lactose to lactulose and to ipilactose, respectively. The reaction system consisted of 0.2 mg / mL purified enzyme solution, 0.2 M lactose, and 50 mM HEPES (pH 7.5) buffer. The reaction was incubated at 80 °C for 5 min, followed immediately by heating in a boiling water bath for 5 min to terminate the reaction.

[0055] Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of lactulose or ipilactose per minute under the test conditions.

[0056] ④ Half-life and thermal stability assessment Will Cs CE was incubated in HEPEs buffer (50 mM, pH 7.5) at 80°C for 0–240 min. Residual enzyme activity (structural isomeric activity) was measured every 30 min, with the initial activity before heat treatment considered as 100%. The half-life (t) was calculated using a first-order kinetic model. 1 / 2 ).

[0057] After multiple rounds of screening, effective mutants were obtained and their enzyme activity was measured. Table 8 Enzyme catalytic activity assay

[0058] in, M1 ( Cs The CE-E174D-Q371E mutant is... Cs Based on CE-Q371E, glutamic acid at position 174 is mutated to aspartic acid, and its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.4.

[0059] M2 ( Cs The CE-E174P-N175D-V177Q-Q371E mutant is... Cs Based on CE-Q371E, glutamic acid at position 174 was mutated to proline, asparagine at position 175 to aspartic acid, and valine at position 177 to glutamine. Its amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.6.

[0060] M3 ( Cs The mutant CE-K46Y-Q48N-I52V-L172V-S173L-E174P-F231A-K356L-N358D-D360S-E362N-Q371E is... CsBased on CE-Q371E, the following mutations were made: lysine at position 46 was replaced with tyrosine; glutamine at position 48 was replaced with asparagine; isoleucine at position 52 was replaced with valine; leucine at position 172 was replaced with valine; serine at position 173 was replaced with leucine; glutamic acid at position 174 was replaced with proline; phenylalanine at position 231 was replaced with alanine; lysine at position 356 was replaced with leucine; asparagine at position 358 was replaced with aspartic acid; aspartic acid at position 360 was replaced with serine; and glutamic acid at position 362 was replaced with asparagine. The M3 mutant was named... Cs CE-FA, whose amino acid sequence is shown in SEQ ID NO.7 and whose nucleotide sequence is shown in SEQ ID NO.8.

[0061] mutant Cs The CE-FA structure isomerization activity was increased by 3.37 times, and the enzyme activity catalyzing the structure isomerization of lactose was increased, making the mutant more suitable for lactulose production compared with CsCE-Q371E.

[0062] The specific results of the thermal stability analysis experiment show that ( Figure 3 Under conditions of 80℃ and pH 7.5 (50 mM HEPES buffer system), through continuous monitoring... Cs CE-Q371E and its mutants Cs The changes in residual enzyme activity of CE-FA can be clearly observed. Cs The half-life of CE-FA (t 1 / 2 It reached 545 minutes, compared to Cs The half-life (t) of CE-Q371E 1 / 2 The half-life of CsCE-FA was only 42.68 min, indicating that the half-life of CsCE-FA was increased by approximately 10-fold, reaching 8.37 hours. (Mutant) Cs CE-FA exhibits excellent thermal stability; even after treatment at 80°C for up to 240 min, its relative enzyme activity remains above 70%. This result fully demonstrates... Cs The extremely strong thermal stability of CE-FA under high temperature conditions (80℃) provides strong support for its potential in practical applications.

[0063] 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, characterized in that, The mutant is based on the CE enzyme with the amino acid sequence shown in SEQ ID NO.1, and the following mutations are performed: Mutate lysine at position 46 to tyrosine, glutamine at position 48 to asparagine, isoleucine at position 52 to valine, leucine at position 172 to valine, serine at position 173 to leucine, glutamic acid at position 174 to proline, phenylalanine at position 231 to alanine, lysine at position 356 to leucine, asparagine at position 358 to aspartic acid, aspartic acid at position 360 to serine, and glutamic acid at position 362 to asparagine; or, The amino acid sequence is as shown in SEQ ID NO.1 Cs Based on CE-Q371E, glutamic acid at position 174 is mutated to proline, asparagine at position 175 is mutated to aspartic acid, and valine at position 177 is mutated to glutamine.

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

3. A vector carrying the gene of claim 2.

4. Genetically engineered bacteria containing the cellobiose epimerase mutant of claim 1 or the vector of claim 3.

5. The genetically engineered bacteria as described in claim 4, characterized in that, Escherichia coli is the host.

6. A method for improving the structural isomerism activity of cellobiose epimerase, characterized in that, The method involves using an amino acid sequence as shown in SEQ ID NO.

1. Cs Based on CE-Q371E, the following mutations were performed: Mutate lysine at position 46 to tyrosine, glutamine at position 48 to asparagine, isoleucine at position 52 to valine, leucine at position 172 to valine, serine at position 173 to leucine, glutamic acid at position 174 to proline, phenylalanine at position 231 to alanine, lysine at position 356 to leucine, asparagine at position 358 to aspartic acid, aspartic acid at position 360 to serine, and glutamic acid at position 362 to asparagine; or, The amino acid sequence is as shown in SEQ ID NO.1 Cs Based on CE-Q371E, glutamic acid at position 174 is mutated to proline, asparagine at position 175 is mutated to aspartic acid, and valine at position 177 is mutated to glutamine.

7. A method for preparing lactulose, characterized in that, The method involves using lactose as a substrate, adding the cellobiose epimerase mutant of claim 1, or the genetically engineered bacteria of claim 4 or 5, to carry out the reaction.

8. The method according to claim 7, characterized in that, The reaction is carried out at pH 7-7.5 and 70-85°C.

9. The use of the cellobiose epimerase mutant of claim 1, or the genetically engineered bacteria of claim 4 or 5, or the method of claim 7 or 8 in the preparation of lactulose or products containing lactulose.

10. The use of the cellobiose epimerase mutant of claim 1, or the genetically engineered bacteria of claim 4 or 5, or the method of claim 7 or 8 in the preparation of food and pharmaceuticals.

Images