Cellobiose epimerase mutant e250q, engineered bacteria and use thereof

Abstract

The application provides a cellobiose epimerase mutant E250Q, an engineering bacterium and application thereof, and belongs to the technical field of genetic engineering.The glutamic acid at the 250th position of the wild-type cellobiose epimerase amino acid sequence is mutated into glutamine to obtain the cellobiose epimerase mutant E250Q.The amino acid sequence of the cellobiose epimerase mutant of the application is shown in any one of SEQ ID NO.1-6.The conversion rate of lactose to lactulose reaches 67.1% by taking the recombinant bacterium or the cellobiose epimerase mutant E250Q as a catalyst and taking lactose as a substrate, and the yield of isomaltulose is only 4.0-5.7%, which is lower than the proportion of the by-product allowed by the Chinese Pharmacopoeia, and has a very high application value in the industrialized production of lactulose.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a cellobiose 2-epimerase mutant E250Q, an engineered bacterium and application thereof. BACKGROUND

[0002] Lactulose, molecular formula C 12 H 22 O 11 , molecular weight 342.3, CAS number 4618-18-2, alias 4-O-beta-D-galactopyranosyl-D-fructose, galactoside fructose, lactulose, etc., is a disaccharide formed by the combination of D-galactose and D-fructose with a beta-1, 4-glycosidic bond. Lactulose has the effects of treating constipation, preventing and treating hepatic encephalopathy and renal failure, and can also enhance the body's immunity and prevent the occurrence of cancer.

[0003] Commercial lactulose oral solution is a sugar concentrated solution in which lactulose accounts for the main proportion, and is an effective drug (over-the-counter drug OTC) for treating constipation and hepatic encephalopathy. At present, the industrial production of lactulose mainly relies on chemical catalysis and biological enzyme conversion. The chemical catalysis method usually uses extreme catalysts such as strong alkali, boric acid and sodium metaaluminate, and needs steps such as acid-base neutralization, ion exchange desalting and the like to obtain lactulose with qualified purity, the catalyst consumption is large, the cost is high, and it does not conform to the concept of green environmental protection. In the biological enzyme conversion method, cellobiose 2-epimerase (CE enzyme) is mainly used for catalysis, among which the most commonly used is cellobiose 2-epimerase (CSCE enzyme) derived from the thermophilic microorganism Caldicellulosiruptor saccharolyticus, which is the most reported and most widely used enzyme; in addition, cellobiose 2-epimerase derived from Dictyoglomus turgidum and Caldicellulosiruptor obsidiansis can also catalyze lactose to produce lactulose.

[0004] However, the lactulose conversion rate of most sources of cellobiose epimerase is not high, and even the main product is not lactulose, but the by-product epilactose. The structure of epilactose is similar to that of lactose and lactulose, and it is very difficult to separate. Therefore, the epilactose content of the lactulose product obtained by using cellobiose epimerase for catalysis exceeds the standard (≤7%), and cannot be sold as lactulose bulk drug or oral solution. In order to overcome this difficulty, Kim et al. proposed adding a chemical catalyst, boric acid, during biological catalysis, which can reduce the content of epilactose to 2%. However, boric acid is prohibited in the field of food and medicine, and it is very difficult and costly to completely remove it. Therefore, molecular biological modification of cellobiose epimerase is the only effective and fundamental means. Previous attempts have made certain molecular modifications to existing cellobiose epimerase in the hope of fundamentally improving the enzymatic properties. For example, the patent with the application number "202210645184.1" discloses a high-temperature-resistant cellobiose epimerase mutant. Although the enzyme mutant has the characteristics of high-temperature resistance, the yield of epilactose is 19.8%, which is lower than that of the unmodified enzyme, but still cannot meet the pharmacopoeia standard (≤7%). Therefore, it is of great significance to develop a cellobiose epimerase mutant with low epilactose yield for the lactulose industry. SUMMARY

[0005] The present application aims to provide a cellobiose epimerase mutant E250Q, an engineering bacterium and applications thereof, to solve the problem of high by-product yield during lactulose production.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a cellobiose epimerase mutant E250Q, in which the glutamic acid at position 250 of the wild-type cellobiose epimerase amino acid sequence is mutated to glutamine; the amino acid sequence of the cellobiose epimerase mutant E250Q is shown in any one of SEQ ID NO. 1-6.

[0008] The present application also provides a coding gene of the cellobiose epimerase mutant E250Q, and the nucleotide sequence of the coding gene is shown in any one of SEQ ID NO. 7-12.

[0009] The present application also provides an expression vector containing the coding gene.

[0010] The present application also provides an engineering bacterium containing the expression vector, and the engineering bacterium is Escherichia coli.

[0011] The application further provides a calcium alginate microsphere embedding the engineered bacteria, which is prepared by the following steps: mixing the wet cells of the engineered bacteria with a calcium alginate solution, dropping into a pre-cooled calcium chloride solution after stirring, and stirring for 8-12 hours to obtain the calcium alginate microsphere.

[0012] Preferably, the concentration of the wet cells of the engineered bacteria is 48-52 g / L, the concentration of the calcium alginate solution is 4% w / v, and the volume ratio of the wet cells of the engineered bacteria to the calcium alginate solution is 0.8-1.2:1.

[0013] Preferably, the concentration of the calcium chloride solution is 0.18-0.22 mol / L.

[0014] The application further provides an application of the cellobiose epimerase mutant E250Q, the engineered bacteria and / or the calcium alginate microsphere in catalyzing the preparation of lactulose from lactose.

[0015] Preferably, in the application, the catalytic reaction is carried out by taking the lactose as a substrate and taking the cellobiose epimerase mutant E250Q, the engineered bacteria or the calcium alginate microsphere as a catalyst to generate lactulose.

[0016] Preferably, the catalytic reaction condition is that the concentration of the lactose is 50-700 g / L, the addition amount of the calcium alginate microsphere is 5-200 g / L, the reaction temperature is 78-82 ℃, and the reaction time is 1-24 hours.

[0017] By adopting the above technical scheme, the application has the following beneficial effects: the application carries out site-specific mutation on the existing cellobiose epimerase, improves the catalytic performance, and the obtained mutant improves the lactulose conversion rate and reduces the eplactose yield. By taking the cellobiose epimerase mutant E250Q, the recombinant bacteria or the cellobiose epimerase mutant E250Q as a catalyst and taking the lactose as a substrate, the conversion rate of the conversion of the lactose to lactulose reaches 67.1%, and the eplactose yield is only 4.0-5.7%, which is lower than the proportion of the by-product allowed by the Chinese Pharmacopoeia, and the application has a very high application value in the industrialized production of lactulose. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 FIG. 2 is a protein SDS-PAGE map of the purified wild CSCE enzyme and enzyme mutant E250Q; lane M: standard protein molecular weight marker (unit kDa); lane 1: CSCE wild-type enzyme; lane 2: CSCE-E250Q mutant enzyme; lane 3: DTCE-E250Q; lane 4: DthermoCE-E250Q; lane 5: COCE-E250Q; lane 6: CBCE-E250Q; and lane 7: PPCE-E250Q.

[0019] Figure 2 HPLC profile of the product catalyzed by CSCE wild-type enzyme from lactose, wherein 1 represents lactose, 2 represents ipilactose, 3 represents lactulose, and the proportion of ipilactose in total sugar is 16.28%.

[0020] Figure 3 HPLC profile of the product catalyzed by CSCE mutant enzyme E250Q from lactose, wherein 1 represents lactose, 2 represents ipilactose, 3 represents lactulose, and the proportion of ipilactose in total sugar is 5.24% in the figure.

[0021] Figure 4 A schematic diagram of a reactor for catalytic reaction of immobilized recombinant E. coli cells.

[0022] Figure 5 Color comparison of the lactulose concentrated solution sample (A) prepared in Example 5 and the commercially available lactulose oral solution sample (B).

[0023] Figure 6 Effect of reaction batch on the relative catalytic activity of calcium alginate microspheres embedding recombinant E. coli cells. DETAILED DESCRIPTION

[0024] The present application provides a cellobiose epimerase mutant E250Q, wherein glutamic acid at position 250 of the wild-type cellobiose epimerase amino acid sequence is mutated to glutamine; the amino acid sequence of the cellobiose epimerase mutant is shown in any one of SEQ ID NO. 1-6.

[0025] The present application also provides a coding gene of the cellobiose epimerase mutant E250Q, and the nucleotide sequence of the coding gene is shown in any one of SEQ ID NO. 7-12.

[0026] The present application also provides an expression vector containing the coding gene.

[0027] The present application also provides an engineered bacterium containing the expression vector, and the engineered bacterium is E. coli.

[0028] The present application also provides a calcium alginate microsphere embedding the engineered bacterium, which is prepared by the following steps: mixing the engineered bacterium wet cells with a calcium alginate solution, stirring, and then dropping into a pre-cooled calcium chloride solution, stirring for 8-12 h to obtain the calcium alginate microsphere.

[0029] The application firstly mixes the wet cells of the engineering bacteria with a calcium alginate solution, the concentration of the wet cells of the engineering bacteria is 48-52 g / L, further preferably 49-51 g / L, more preferably 50 g / L; the concentration of the calcium alginate is 3.8-4.3% (w / v), further preferably 3.9-4.1% (w / v), more preferably 4% (w / v); the volume ratio of the wet cells of the engineering bacteria to the calcium alginate solution is 0.8-1.2:1, further preferably 0.9-1.1:1, more preferably 1:1. After mixing the wet cells of the engineering bacteria with the calcium alginate solution, the mixture is stirred uniformly, and then slowly dripped into a pre-cooled calcium chloride solution, the concentration of the calcium chloride solution is 0.18-0.22 mol / L, further preferably 0.19-0.21 mol / L, more preferably 0.2 mol / L. Then the mixture is hardened by magnetic stirring overnight at 4°C, washed with pre-cooled deionized water for three times, and the obtained calcium alginate immobilized microspheres are collected.

[0030] The application also provides the application of the cellobiose epimerase mutant E250Q, the engineering bacteria and / or the calcium alginate microspheres in catalyzing the preparation of lactulose from lactose. In the reaction system, lactose is used as the substrate, and the cellobiose epimerase mutant E250Q, the engineering bacteria or the calcium alginate microspheres are used as the catalyst to generate lactulose. The concentration of lactose in the reaction system is 50-700 g / L, further preferably 200-500 g / L, more preferably 300 g / L; the reaction temperature is 78-82°C, further preferably 79-81°C, more preferably 80°C; the reaction time is 1-24 h, further preferably 5-15 h, more preferably 10 h. If the cellobiose epimerase mutant E250Q is used as the catalyst in the reaction, the addition concentration of the cellobiose epimerase mutant E250Q is 1.8-2.2 g / L, further preferably 1.9-2.1 g / L, more preferably 2 g / L; if the engineering bacteria are used as the catalyst in the reaction, the addition concentration of the wet cells of the engineering bacteria is 48-52 g / L, further preferably 49-51 g / L, more preferably 50 g / L; if the calcium alginate microspheres are used as the catalyst in the reaction, the addition concentration of the calcium alginate microspheres is 5-200 g / L, further preferably 50-150 g / L, more preferably 100 g / L.

[0031] The technical solutions provided by the application are described in detail below in combination with the examples, but they should not be understood as limiting the protection scope of the application.

[0032] Example 1. Acquisition of cellobiose epimerase gene, construction of recombinant vector and site-directed mutation

[0033] 1. Acquisition of cellobiose epimerase gene

[0034] In order to obtain the encoding gene of cellobiose epimerase (CSCE) from Caldicellulosiruptor saccharolyticus, the DNA of the encoding gene was codon-optimized and fully synthesized by General Biosystems (Anhui) Co., Ltd., and the nucleotide sequence is shown as SEQ ID NO. 13. The primer was designed for PCR amplification with the DNA as a template, and the primer sequence contains the enzyme digestion sites NdeI and EcoRI at the upstream and downstream of the gene. The PCR amplification was performed by using the high-fidelity DNA polymerase of Takara, and the PCR reaction program was as follows: 98℃ for 10s, 55℃ for 15s, 72℃ for 1.5min; 30 cycles.

[0035] Upstream primer:

[0036] (underlined NdeI site)

[0037] Downstream primer: (underlined EcoRI site). The bold sequence is the matched part with the template.

[0038] The PCR amplification product was purified by using a DNA purification kit (MiniBEST DNA Fragment Purification Kit, TaKaRa) and then recovered and stored at -20℃.

[0039] The nucleotide sequence of the wild-type Caldicellulosiruptor saccharolyticus-derived cellobiose epimerase (CSCE) encoding gene (SEQ ID NO. 13):

[0040] ​

[0041] 2. Construction of recombinant vector of cellobiose epimerase mutant E250Q

[0042] The PCR amplification product and pET-28a plasmid were ligated, and the ligation method is as follows:

[0043] First, restriction enzymes NdeI and EcoRI (TaKaRa) were used for double digestion, and the double digestion system (50 μL) was as follows: 10 x H buffer 5 μL, PCR amplification product (or pET-28a plasmid) 25 μL, NdeI enzyme 1 μL, EcoRI enzyme 1 μL, double distilled water 18 μL; the enzyme digestion temperature was 37°C, and the enzyme digestion time was 5 h; the product was purified using a DNA purification kit (MiniBEST DNA Fragment Purification Kit, TaKaRa) and stored at -20°C. The enzyme digestion product was then ligated by T4 DNA ligase (purchased from the NEB website), and the ligation system (20 μL) was as follows: double-digested PCR amplification product 5 μL, double-digested pET-28a plasmid 5 μL, 10 x T4 DNA ligase buffer 2 μL, T4 DNA ligase 1 μL, double distilled water 7 μL. The ligation reaction temperature was 25°C, and the ligation reaction time was 12 h; after ligation, the reaction was transformed into E. coli Fast-T1 competent cells. The transformation method was as follows: 10 μL of the ligation product was added to 100 μL of E. coli Fast-T1 competent cells (Nanjing Novagen Biotech Co., Ltd.), incubated on ice for 20 min, heat shocked at 42°C for 90 s, then ice bathed for 5 min, 37°C cultured for 45 min, and 150 μL of bacterial liquid was spread on a 25 μg / mL kanamycin-resistant plate. After overnight culture, positive clones (referred to as E. coli Fast-T1-CSCE strain) were picked and sent to a sequencing company for sequencing, and the correct sequence clones were stored at -80°C.

[0044] 3. Site-directed mutagenesis of cellobiose epimerase gene

[0045] The E. coli Fast-T1-CSCE strain was cultured overnight in LB liquid medium containing 25 mg / L kanamycin, and the bacterial cells were centrifuged and extracted using a plasmid extraction kit (MiniBEST Plasmid Purification Kit, TaKaRa) to extract the plasmid pET-28a-CSCE as a template for subsequent site-directed mutagenesis experiments. The site-directed mutagenesis was performed by PCR, and the principle was to design a pair of sequence-complementary oligonucleotide primers near the coding gene site that needed to be mutated, and the specific steps were as follows:

[0046] The forward primer for E250Q site-directed mutagenesis of CSCE was:

[0047] TATGGTCACGATATT CAA GCGAGCTGGCTGCTG (SEQ ID NO. 16) (underlined is the mutation site),

[0048] Reverse primer for E250Q site-directed mutation of CSCE:

[0049] CAGCAGCCAGCTCGC TTG AATATCGTGACCATA (SEQ ID NO. 17) (underlined is the mutation site). PCR was performed using pET-28a-CSCE recombinant plasmid as template, using high-fidelity DNA polymerase (Takara) for reaction, and the PCR program was: 98℃ 10s, 55℃ 15s, 72℃ 7min; repeated for 16 cycles. After agarose gel electrophoresis analysis of the PCR product, the gel was cut and recovered for purification. The purified product was treated with restriction endonuclease DpnI at 37℃ for 12h to digest the template used for PCR; the treated product was transformed into E. coli BL21 (DE3), and single colonies that could grow on a 25mg / L kanamycin LB resistant plate were picked and named E. coli BL21 (DE3)-pET-28a / E250Q; sequencing was performed by a relevant gene technology company, and the sequence of the correct clone was stored at -80℃.

[0050] In this embodiment, the amino acid residue number is numbered with the first residue methionine of the wild-type CE enzyme as the first amino acid residue.

[0051] The amino acid sequence of the mutant E250Q of cellobiose epimerase (CSCE) from Caldicellulosiruptor saccharolyticus is shown in SEQ ID NO. 1:

[0052] MDITRFKEDLKAHLEEKIIPFWQSLKDDEFGGYYGYMDFNLNIDRKAQKGCILNSRILWFFSACYNVLKSEKCKEMAFHAFEFLKNKFWDKEYEGLFWSVSHKGVPVDVTKHVYVQAFGIYGLSEYYEASGDEEALHMAKRLFEILETKCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTNLYRLLKLDDVYEALEWIVRLFVDKIYKKGTGHFKVFCDDNWNELIKAVSYGHDIQASWLLDQAAKYLKDEKLKEEVEKLALEVAQITLKEAFDGQSLINEMIEDRIDRSKIWWVEAETVVGFFNAYQKTKEEKYLDAAIKTWEFIKEHLVDRRKNSEWLWKVNEDLEAVNMPIVEQWKCPYHNGRMCLEIIKRVD.

[0053] The nucleotide sequence of the gene encoding the E250Q mutant of the cellobiose epimerase (CSCE) from Caldicellulosiruptor saccharolyticus is shown in SEQ ID NO. 7:

[0054]

[0055] Example 2: Expression and purification of cellobiose epimerase mutant E250Q

[0056] The E. coli BL21(DE3)-pET-28a / E250Q strain was inoculated into LB liquid medium containing 25 mg / L kanamycin and cultured at 37 °C for 12 h, then transferred to fresh LB liquid medium containing 25 mg / L kanamycin and expanded at 37 °C for 4 h to OD 600 = 0.8; the temperature was lowered to 25 °C for continued culture, and IPTG was added at a final concentration of 0.5 mM for induction. After 24 h of expression, the bacterial cells were collected by centrifugation at 5000 rpm for 20 min. The expression of the target protein in the whole cells was detected by polyacrylamide gel electrophoresis (SDS-PAGE), and the specific steps were as follows:

[0057] The collected bacterial cells were broken by a high-pressure homogenizer, centrifuged at 12000 rpm at 4 °C for 20 min, and the precipitate was discarded. The broken supernatant was eluted through a nickel ion affinity column (model: Sepharose Fast Flow, GE Healthcare).

[0058] The affinity column was previously equilibrated with a buffer, then washed with a wash buffer, and then the cellobiose epimerase was eluted with an elution buffer. The eluted enzyme was desalted in a dialysis bag in 50 times the volume of dialysis solution at 4 °C. Finally, the enzyme solution was concentrated by absorbing water with polyethylene glycol powder with a molecular weight of 20000 Da, and the obtained enzyme solution can be used for subsequent biological catalysis.

[0059] The equilibration buffer comprises 10 mM HEPES buffer (pH 7.0) containing 10% w / v glycerol and 150 mM sodium chloride;

[0060] The wash buffer comprises 10 mM HEPES buffer (pH 7.0), 10% w / v glycerol, 50 mM imidazole, and 500 mM sodium chloride;

[0061] The elution buffer comprises 10 mM HEPES buffer (pH 7.0), 10% w / v glycerol, 200 mM imidazole, and 500 mM sodium chloride;

[0062] The dialysis solution is 10 mM HEPES buffer (pH 7.0) and 10% w / v glycerol.

[0063] The protein concentration of the enzyme solution was determined by the Bradford method using bovine serum albumin as a standard, and the result was 5.8 mg / mL. The purity of the enzyme was detected by SDS-PAGE electrophoresis, as shown in Figure 1 .

[0064] Example 3. Determination of enzyme activity of wild-type cellobiose epimerase and mutant E250Q enzyme and determination of the yield of epilactose

[0065] Example 3. Determination of enzyme activity of wild-type cellobiose epimerase and mutant E250Q enzyme and determination of the yield of epilactose

[0066] HPLC determination method: a 1200 liquid chromatograph (Agilent Technologies (China) Co., Ltd.) was used, a Ca 2+ type chromatographic column Rezex RCM-Monosaccharide (Phenomenex Co.) was used, the mobile phase was pure water, the column temperature was set to 80°C, the flow rate was 0.5 mL / min, and the HPLC detection results are shown in Figure 2 and Figure 3 The yield of lactose, epilactose, and lactulose was determined by using an external standard method according to the retention time and peak area of the peaks. The analytical pure standards of lactose, epilactose, and lactulose were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0067] Enzyme activity definition: the amount of enzyme that produces 1 μmol of lactulose per min is defined as 1 U, and the specific enzyme activity is the ratio of enzyme activity (U) to enzyme mass (mg). The yield of the byproduct epilactose and enzyme activity are shown in Table 1.

[0068] Table 1. Yield of epilactose and enzyme activity in the catalytic reaction of CSCE wild-type enzyme and mutant enzyme

[0069] Galactinol yield Stachydrine yield Specific enzyme activity (U / mg) CSCE wild-type enzyme 56.6％ 16.3％ 5.5 CSCE mutant enzyme E250Q 67.1％ 5.2％ 5.5

[0070] Example 4. Recombinant E. coli catalyzing lactose by cellobiose epimerase mutant E250Q

[0071] The E. coli BL21(DE3)-pET-28a / E250Q strain was fermented and induced for expression according to the method in Example 2, and the recombinant E. coli wet bacteria were obtained by centrifugation at 5000 rpm for 20 min. The recombinant E. coli was used to catalyze 700 g / L lactose at 80°C, and the addition amount of wet bacteria was 50 g wet bacteria / L, and the catalysis time was 3 h. After the reaction was completed, a reaction solution containing lactulose was prepared, and the reaction product was identified by HPLC, and the identification method was the same as in Example 3. HPLC detection showed that the proportion of lactulose in the reaction product reached 65%, the proportion of the byproduct epilactose was 5%, and the remaining lactose accounted for 30%.

[0072] The reaction solution is concentrated at 60°C under reduced pressure, and the reaction solution is concentrated to a brix of 75%. The reaction solution is cooled to room temperature overnight, and the next day, lactose crystals are removed by plate and frame filter. The obtained sugar solution is diluted with deionized water to a lactulose content of 667 g / L, which is a concentrated lactulose solution. The content of isomaltulose in the concentrated lactulose solution meets the standard of less than 7% in the Chinese Pharmacopoeia for lactulose oral solution.

[0073] The present application can also directly use the fermentation liquor for catalysis (without purification and without pH adjustment). The concentration of lactose in the catalytic system is 700 g / L, the addition amount of the fermentation liquor is 10% (v / v), the reaction temperature is 80°C, and the reaction time is 3 h. The catalytic effect is consistent with that of the recombinant E. coli wet cells.

[0074] The fermentation liquor is prepared as follows: the E. coli BL21 (DE3)-pET-28a / E250Q strain is inoculated into LB liquid medium containing 25 mg / L kanamycin, and cultured at 37°C for 12 h. Then, the culture is transferred into fresh LB liquid medium containing 25 mg / L kanamycin, and expandedly cultured at 37°C for 4 h to an OD 600 = 0.8. The temperature is lowered to 25°C for further culture, and 0.5 mM IPTG is added for induction. After 24 h of expression, the supernatant is collected by centrifugation at 5000 rpm for 20 min, which is the fermentation liquor.

[0075] Example 5. Preparation of calcium alginate immobilized microspheres embedded with recombinant E. coli and catalytic reaction thereof

[0076] The recombinant E. coli wet cells are obtained according to the method in Example 2. 50 g / L of the recombinant E. coli wet cells are added into an equal volume of 4% (w / v) calcium alginate solution, and stirred and mixed uniformly. Then, the mixture is slowly dripped into a 0.2 mol / L pre-cooled calcium chloride solution through a funnel, and magnetically stirred at 4°C overnight for hardening treatment. The calcium alginate immobilized microspheres are washed with pre-cooled deionized water for three times. The calcium alginate immobilized microspheres can be used for catalyzing lactulose from lactose, and are loaded into an immobilized enzyme reactor for batch catalytic reaction, as shown in Figure 4 .

[0077] The calcium alginate immobilized microspheres are used for catalyzing lactulose from lactose. The reaction system is as follows: 700 g / L lactose, and the addition amount of the calcium alginate immobilized microspheres is 100 g / L. The reaction temperature is 80°C, the pH is 7.0, and the catalytic time is 3.5 h. The concentrated lactulose solution is prepared according to the method in Example 4. The content of isomaltulose in the concentrated lactulose solution meets the standard of less than 7% in the Chinese Pharmacopoeia for lactulose oral solution. Moreover, the color of the concentrated lactulose solution is lighter than that of the commercially available lactulose oral solution of the same concentration, which indicates that the content of pigment impurities in the concentrated lactulose solution is lower. Figure 5 ). ​

[0078] The present application uses calcium alginate to embed recombinant E. coli cells to catalyze lactose in batches, to explore the relative catalytic ability of the immobilized cells. The reaction temperature is 80℃, the reaction time is 3.5h, and the initial activity of the first batch of catalysis is defined as 100%. The vertical coordinate is the relative catalytic activity of the immobilized cells, and the horizontal coordinate is the reaction batch of the immobilized cells. The relative catalytic ability of the immobilized cells is shown in Figure 6 Figure 6 It can be seen that after the immobilization treatment of the immobilized engineering bacteria cells, the immobilized cells can be used in multiple batches, and the enzyme activity decreases by less than 10% within 30 batches.

[0079] Example 6

[0080] In this embodiment, the glutamic acid at position 250 of the amino acid sequence of the cellobiose epimerase (DTCE) from the thermophilic bacteria Dictyoglomus turgidum is mutated to glutamine. The method in Example 3 is used to perform lactose catalytic reaction, and HPLC detection is performed after the catalysis is completed. The results are shown in Table 2.

[0081] Table 2 Effect of site-directed mutation in DTCE enzyme

[0082] Galactinol yield Stachydrine yield Specific enzyme activity DTCE wild-type enzyme 52.1％ 12.8％ 5.2 U / mg DTCEE250Q mutant enzyme 61.8％ 5.7％ 5.2 U / mg

[0083] The experimental results show that the DTCE E250Q mutant enzyme catalyzes lactose to obtain a mutation effect similar to that of the CSCE enzyme, and the proportion of the by-product allolactose is significantly reduced.

[0084] The amino acid sequence of the Dictyoglomus turgidum-derived cellobiose epimerase (DTCE) mutant E250Q is shown in SEQ ID NO. 2:

[0085] ​MDLKVLKSEIFEHLNNKIIPFWEELKDENNGGYISYVGFDLKPDPYAPKGLVLTSRILWFFSRLYNQLRKEEFINFADHSYKFLIKSFLDKENKGFYWMVDYKGEPIDKRKHLYGQAFVLYGLSEYYKATQKKESLDLALEIYKIIEEVCKNDVGYKEEFDEKWNPKENIIVSEYGIICERSMNTLLHILEAYTNLFTATYDQSIKKKIEDLIILFKEKIYDSKTNHLYVFFDKKMNPIIDAISYGHDIQATWLIDEALRYIDNNKLIKEMSEINLKIAEKVLEEAFESGSLLNERVRGIVDKNRIWWVQAEALVGFLNAYQKSKLDKFLKAVFELWEFIKDFLVDKRAQGEWFWKLDENYIPSPMPEVDLWKCPYHNGRMCLEVIKRI.

[0086] The nucleotide sequence of the gene encoding the mutant E250Q of the cellobiose epimerase (DTCE) from Dictyoglomus turgidum is shown in SEQ ID NO. 8:

[0087]

[0088] Example 7

[0089] In this example, the glutamic acid at position 250 of the amino acid sequence of the Dictyoglomus thermophilum-derived cellobiose epimerase (DthermoCE) was mutated to glutamine, and the catalytic reaction of lactose was carried out according to the method in Example 3. After the catalytic reaction was completed, HPLC detection was performed, and the isomaltulose yield and specific enzyme activity were calculated. The results are shown in Table 3.

[0090] Table 3 Effects obtained by site-directed mutagenesis in DthermoCE enzyme

[0091]

[0092] The experimental results show that the DthermoCE E250Q mutant enzyme catalyzes the reaction of lactose, and the proportion of the by-product isomaltulose is significantly reduced, similar to the mutation effect of the CSCE enzyme.

[0093] The amino acid sequence of the Dictyoglomus thermophilum-derived cellobiose epimerase (DthermoCE) mutant E250Q is shown in SEQ ID NO. 3:

[0094] MDLKHLKDEIFEQLNNKIIPFWENLKDENNGGYISYVGFDLKPDPYAPKGLVLTSRILWFFSRLYNQLRKEEFIEFADHAYEFLTDKLLDKENGGFFWIVDYKGDPLDKRKHLYGQAFALYGLSEYYKATKKKESLELSLELYKTIEERCKDNIGYKEEFDEKWTPKENIIVSEYGIICEKSMNTLLHLLEAYTNLFTATYDLKVRKELENLIILFKEKIYNPKTDHLYVFFDNKMKPIIDAISYGHDIQATWLIDEALRYIENNTLIKDMTEINLRIAERVLEEAFENNSLLNEKVRGEVNKDRVWWVQAEALLGFLNAYQKTKSDKFLKAVLSLWEFINNFLLDKRPNGEWFNKLDENCIPFPMPEVDLWKCPYHNGRMYLEVIRRI.

[0095] The nucleotide sequence of the gene encoding the Dictyoglomus thermophilum-derived cellobiose epimerase (DthermoCE) mutant E250Q is shown in SEQ ID NO. 9:

[0096]

[0097] Example 8

[0098] This example mutates the glutamic acid at position 250 of the amino acid sequence of the Caldicellulosiruptor obsidiansis-derived cellobiose epimerase (COCE) to glutamine, carries out a lactose catalytic reaction according to the method in Example 3, and after the catalysis is completed, carries out HPLC detection and calculates the allolactose yield and specific enzyme activity, and the results are shown in Table 4.

[0099] Table 4 Effects obtained by site-directed mutagenesis in COCE enzyme

[0100] Galactinol yield Stachydrine yield Specific enzyme activity COCE wild-type enzyme 53.8％ 11.0％ 5.3 U / mg COCEE250Q mutant enzyme 64.2％ 5.3％ 5.3 U / mg

[0101] The experimental results show that the COCE E250Q mutant enzyme carries out a lactose catalytic reaction, and the proportion of the by-product allolactose is significantly reduced, similar to the mutation effect of the CSCE enzyme.

[0102] The amino acid sequence of the Caldicellulosiruptor obsidiansis-derived cellobiose epimerase (COCE) mutant E250Q is shown in SEQ ID NO. 4:

[0103] MDITSFKKELKSHLEEKIIPFWQSLKDDEFGGYYGYMDFNLNINKKAQKGCILNSRILWFFSACYNVLKSEKCKELAFHAFEFLKNKFWDKDYEGLFWSVSLEGLPVDVTKHVYVQAFGIYGLSEYYEASGDKEALFLARKLFEILETRCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTNLYKVLKLDDVYEALEWLVRLFVEKIYKKGTGHFKVFCDDNWNELIKAVSYGHDIQASWLLDETAKYLRDEKLKEEVEKLTLEVAQVTLQEAFDGKSLINEKVEDRVDRSKIWWVEAETVVGFFNAYQKTKEEKYLDAAIKTWEFIKEYLVDKRKNSEWLWKVDENLNPVQMPIVEPWKCPYHNGRMCLEIIKRVG.

[0104] The nucleotide sequence of the gene encoding the Caldicellulosiruptor obsidiansis-derived cellobiose epimerase (COCE) E250Q mutant is shown in SEQ ID NO. 10:

[0105]

[0106] Example 9

[0107] This example mutates the glutamic acid at position 250 of the amino acid sequence of the Cellobiose Epimerase (CBCE) from Caldicellulosiruptor bescii to glutamine, carries out a lactose catalytic reaction according to the method in Example 3, and after the catalysis is completed, carries out HPLC detection and calculates the allolactose yield and specific enzyme activity, and the results are shown in Table 5.

[0108] Table 5 Effects obtained by site-directed mutagenesis in CBCE enzyme

[0109] Galactinol yield Stachydrine yield Specific enzyme activity CBCE wild-type enzyme 52.1％ 15.2％ 5.3 U / mg CBCEE250Q mutant enzyme 62.7％ 5.7％ 5.3 U / mg

[0110] The experimental results show that the CBCE E250Q mutant enzyme carries out a lactose catalytic reaction, and the proportion of the by-product allolactose is significantly reduced, similar to the mutation effect of the CSCE enzyme.

[0111] The amino acid sequence of the Cellobiose Epimerase (CBCE) E250Q mutant from Caldicellulosiruptor bescii is shown in SEQ ID NO. 5:

[0112] MDITKFKEDLKAHLEEKIVPFWQSLKDDEFGGYYGYMDFNLNIHRKAQKGCILNSRILWFFSACYNVLKNEKCKELAFHAFEFLKNKFWDKEYEGLFWNVSHKGVPVDMTKHVYVQAFGIYGLSEYYEASGDKEALQMAKKLFEILETKCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTNLYKVLRTKDVYEALEWIVRLFVDKIYKKGTGHFKVFCDDNWNELIKAVSYGHDIQASWLLDEAARYLKDEKLKEEVEKLTLEVAQVTLKEAFDGQSLINEMVEDRVDRSKIWWVEAETVVGFFNAYQKSKEEKFLDAAIKTWKFIEEHLVDKRKNSEWLWKVSEDLKALDMPIVEPWKCPYHNGRMCLEIIKRVG.

[0113] The nucleotide sequence of the gene encoding the Cellobiose Epimerase (CBCE) E250Q mutant from Caldicellulosiruptor bescii is shown in SEQ ID NO. 11:

[0114]

[0115] Example 10

[0116] In this example, the glutamic acid at position 250 of the Paenibacillus polymyxa-derived cellobiose epimerase (PPCE) amino acid sequence was mutated to glutamine, and the catalytic reaction of lactose was carried out according to the method in Example 3. After the catalytic reaction was completed, HPLC detection was performed, and the isoplatylactose yield and specific enzyme activity were calculated. The results are shown in Table 5.

[0117] Table 5 Effects obtained by site-directed mutagenesis in PPCE enzyme

[0118] Galactinol yield Stachydrine yield Specific enzyme activity PPCE wild-type enzyme 49.9％ 17.5％ 3.7 U / mg PPCEE250Q mutant enzyme 59.2％ 4.0％ 3.7 U / mg

[0119] The experimental results show that the PPCE E250Q mutant enzyme catalyzes the reaction of lactose, and the proportion of the by-product isoplatylactose is significantly reduced, similar to the mutation effect of the CSCE enzyme.

[0120] The amino acid sequence of the Paenibacillus polymyxa-derived cellobiose epimerase (PPCE) E250Q mutant is shown in SEQ ID NO. 6:

[0121] METLLDEIRQEWKEHILPFWLGLKDETHGGFYGEVDVGLHTHNQADKGGIATARLLWSFSAATRVTGENTYAEAARHAFTFLRDHLLDPLHGGMYWMVDYTGQPVDTCKHVYAQAFAIYALAEYACATDDPSALPLAMELFHLLEQKGYDPARQAYGEQYDRLWNTQPNELLSENGVTAHITMNTHIHVLEAYTQLLRVWPNEEVRDALTNVLDILYRRVYDASARRLGVFFDREWRSLLDLTSYGHDIQASWLIEDAMNVLGYYPSEYVDMVMDIANAVAERAVQPDGSLINEREGERVDTSRIWWVQAEGMVGFYNAFQRTNDERFLQIVRNLWAYTRQYIIDPRPGGEWFWSVQADGKPDAREIAGPWKCPYHNSRFCIEMLERMEDQ.

[0122] The nucleotide sequence of the gene encoding the Paenibacillus polymyxa-derived cellobiose epimerase (PPCE) E250Q mutant is shown in SEQ ID NO. 12:

[0123]

[0124] From the above examples, the present application provides cellobiose epimerase mutant E250Q, engineering bacteria and application thereof. The cellobiose epimerase mutant E250Q of the present application can reduce the yield of by-product epilactose when lactose is used to prepare lactulose.

[0125] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A cellobiose epimerase mutant E250Q, characterized in that, The glutamic acid at position 250 of the amino acid sequence of wild-type cellobiose epimerase is mutated into glutamine; the amino acid sequence of the cellobiose epimerase mutant is shown in SEQ ID NO.

1.

2. A gene encoding the cellobiose epimerase mutant E250Q according to claim 1, characterized in that, The nucleotide sequence of the coding gene is shown in SEQ ID NO.

7.

3. An expression vector, characterized by, The expression vector contains the coding gene of claim 2.

4. An engineered bacterium containing the expression vector of claim 3, wherein the engineered bacterium is Escherichia coli.

5. A calcium alginate microsphere embedding the engineered bacterium of claim 4, wherein, Preparation is carried out according to the following steps: The wet cells of the engineered bacterium are mixed with a calcium alginate solution, and after stirring, the mixture is dropped into a pre-cooled calcium chloride solution, and stirred for 8-12 hours to obtain the calcium alginate microspheres; The concentration of the wet cells of the engineered bacterium is 48-52 g / L, the concentration of the calcium alginate solution is 4% w / v, and the volume ratio of the wet cells of the engineered bacterium to the calcium alginate solution is 0.8-1.2:1; The concentration of the calcium chloride solution is 0.18-0.22 mol / L.

6. Use of the cellobiose epimerase mutant E250Q of claim 1, the engineered bacterium of claim 4, or the calcium alginate microspheres of claim 5 in catalyzing the preparation of lactulose from lactose.

7. Use according to claim 6, characterized in that, Lactulose is generated by catalytic reaction using lactose as the substrate and the cellobiose epimerase mutant E250Q of claim 1, the engineered bacterium of claim 4, or the calcium alginate microspheres of claim 5 as the catalyst.

8. Use according to claim 7, characterized in that, The conditions of the catalytic reaction are as follows: the concentration of lactose is 50-700 g / L, the reaction temperature is 78-82℃, and the reaction time is 1-24 hours.

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