Cellobiose epimerase mutant E250Q, engineering bacteria and application thereof

Abstract

The invention provides a cellobiose epimerase mutant E250Q, an engineering bacterium and application of the cellobiose epimerase mutant E250Q, and belongs to the technical field of genetic engineering. According to the invention, glutamic acid at the 250th site of an amino acid sequence of wild cellobiose epimerase is mutated into glutamine, so that the cellobiose epimerase mutant E250Q is obtained. The amino acid sequence of the cellobiose epimerase mutant disclosed by the invention is shown as any one of SEQ ID NO.1-6. The invention further discloses a preparation method of the cellobiose epimerase mutant. According to the method, recombinant bacteria or cellobiose epimerase mutant E250Q is used as a catalyst, lactose is used as a substrate, the conversion rate of converting lactose to lactulose reaches 67.1%, the yield of brepinose is only 4.0-5.7% and is lower than the by-product proportion permitted by Chinese Pharmacopoeia, and the method has extremely high application value in industrial production of lactulose.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a cellobiose epimerase mutant E250Q, an engineering bacterium and applications thereof. Background Art

[0002] Lactulose, molecular formula C 12 H 22 O 11 , molecular weight 342.3, CAS number 4618-18-2, alias 4-O-β-D-galactopyranosyl-D-fructose, galactosyl fructose, lactulose, etc., is a disaccharide formed by the combination of D-galactose and D-fructose with β-1,4-glycosidic bonds. 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] Commercialized lactulose oral solution is a concentrated sugar solution with lactulose as the main proportion, and is an effective drug for treating constipation and hepatic encephalopathy (OTC drugs). At present, the industrial production of lactulose mainly relies on chemical catalysis and bioenzyme conversion. Chemical catalysis usually uses extreme catalysts such as strong alkali, boric acid, sodium aluminate, etc., and requires acid-base neutralization, ion exchange desalination and other steps to obtain lactulose of qualified purity. The catalyst usage is large, the cost is high, and it does not conform to the concept of green environmental protection. In the bio-enzymatic conversion method, cellobiose 2-epimerase (CE enzyme for short) is mainly used for catalysis. Among them, the most commonly used one is the cellobiose diastereomerase (CSCE enzyme for short) derived from the thermophilic microorganism Caldicellulosiruptorsaccharolyticus. This enzyme has the most reports and is the most widely used. In addition, cellobiose diastereomerase derived from Dictyoglomus turgidum and Caldicellulosiruptor obsidiansis can also catalyze lactose to produce lactulose.

[0004] However, the lactulose conversion rate of cellobiose epimerase from most sources is not high, and even the main product is not lactulose, but a byproduct such as epiliactose. The structure of epiliactose is similar to that of lactose and lactulose, and it is very difficult to separate. Therefore, the lactulose product obtained by catalysis using cellobiose epimerase has an excessive epiliactose content, which cannot meet the pharmacopoeia standard (≤7%) and cannot be sold as a lactulose bulk drug or oral liquid. In order to overcome this difficulty, Kim et al. proposed to add a chemical catalyst boric acid in the process of biocatalysis, which can reduce the content of epiliactose to 2%. However, boric acid is prohibited from being used in the field of food and medicine, and it is very difficult and costly to completely remove it. Therefore, molecular biological transformation of cellobiose epimerase is an effective fundamental means. Former researchers have conducted certain molecular transformation attempts on existing cellobiose epimerases, hoping to fundamentally improve the enzymatic properties. For example, the patent with 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 pilactose is 19.8%. Although the yield of pilactose is reduced compared with that before the transformation, it still cannot meet the pharmacopoeia standard (≤7%). Therefore, the development of a cellobiose epimerase mutant with a low yield of pilactose is of great significance to the lactulose industry. Summary of the invention

[0005] The purpose of the present invention is to provide a cellobiose epimerase mutant E250Q, an engineered bacterium and applications thereof, so as to solve the problem of high by-product yield when producing lactulose.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a cellobiose epimerase mutant E250Q, wherein the 250th glutamic acid in the amino acid sequence of wild cellobiose epimerase is mutated to glutamine; the amino acid sequence of the cellobiose epimerase mutant E250Q is shown in any one of SEQ ID NOs. 1 to 6.

[0008] The present invention also provides a gene encoding the cellobiose epimerase mutant E250Q, and the nucleotide sequence of the gene encoding is shown in any one of SEQ ID NOs. 7 to 12.

[0009] The present invention also provides an expression vector, which contains the encoding gene.

[0010] The present invention also provides an engineering bacterium comprising the expression vector, wherein the engineering bacterium is Escherichia coli.

[0011] The present invention also provides calcium alginate microspheres encapsulating the engineered bacteria, which are prepared according to the following steps: mixing the wet cells of the engineered bacteria with a calcium alginate solution, stirring and then dripping the mixture into a precooled calcium chloride solution, stirring for 8 to 12 hours, and obtaining the calcium alginate microspheres.

[0012] Preferably, the concentration of the engineered bacteria wet cells is 48-52 g / L, the concentration of the calcium alginate solution is 4% w / v, and the volume ratio of the engineered bacteria wet cells 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 present invention also provides the use of the cellobiose epimerase mutant E250Q, the engineering bacteria and / or the calcium alginate microspheres in catalyzing the preparation of lactulose from lactose.

[0015] Preferably, in the application, lactose is used as a substrate, and the cellobiose epimerase mutant E250Q or the engineered bacteria and the calcium alginate microspheres are used as catalysts to carry out a catalytic reaction to generate lactulose.

[0016] Preferably, the catalytic reaction conditions are: the concentration of lactose is 50-700 g / L, the addition amount of calcium alginate microspheres is 5-200 g / L, the reaction temperature is 78-82° C., and the reaction time is 1-24 h.

[0017] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention performs site-directed mutation on the existing cellobiose epimerase, improves the catalytic performance, and the obtained mutant improves the lactulose conversion rate while reducing the yield of pilactose. With cellobiose epimerase mutant E250Q, recombinant bacteria or cellobiose epimerase mutant E250Q as catalyst and lactose as substrate, the conversion rate of lactose to lactulose reaches 67.1%, while the yield of pilactose is only 4.0-5.7%, which is lower than the by-product ratio allowed by the Chinese Pharmacopoeia, and has extremely high application value in the industrial production of lactulose. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The SDS-PAGE profiles of proteins after purification of wild-type 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; Lane 7: PPCE-E250Q.

[0019] Figure 2 This is the HPLC spectrum of the products obtained by the catalysis of lactose by the wild-type CSCE enzyme, where 1 represents lactose, 2 represents epilactose, and 3 represents lactulose. The proportion of epilactose in the total sugars is 16.28%.

[0020] Figure 3 The HPLC graph of the product obtained by catalyzing lactose by the CSCE mutant enzyme E250Q is shown in Figure 1, where 1 represents lactose, 2 represents epilactose, and 3 represents lactulose. In the figure, epilactose accounts for 5.24% of the total sugars.

[0021] Figure 4 Schematic diagram of the reactor for catalytic reactions of immobilized recombinant Escherichia coli cells.

[0022] Figure 5 The 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 The effect of reaction batch on the relative catalytic activity of calcium alginate microspheres encapsulating recombinant Escherichia coli cells. DETAILED DESCRIPTION

[0024] The present invention provides a cellobiose epimerase mutant E250Q, wherein the 250th glutamic acid in the amino acid sequence of wild cellobiose epimerase is mutated to glutamine; the amino acid sequence of the cellobiose epimerase mutant is shown in any one of SEQ ID NOs. 1 to 6.

[0025] The present invention also provides a gene encoding the cellobiose epimerase mutant E250Q, and the nucleotide sequence of the gene encoding is shown in any one of SEQ ID NOs. 7 to 12.

[0026] The present invention also provides an expression vector, which contains the encoding gene.

[0027] The present invention also provides an engineering bacterium comprising the expression vector, wherein the engineering bacterium is Escherichia coli.

[0028] The present invention also provides calcium alginate microspheres for embedding the engineered bacteria, which are prepared according to the following steps: wet cells of the engineered bacteria are mixed with a calcium alginate solution, and the mixture is dripped into a precooled calcium chloride solution after stirring for 8 to 12 hours to obtain the calcium alginate microspheres.

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

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

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

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

[0033] 1. Acquisition of cellobiose epimerase gene

[0034] In order to obtain the gene encoding cellobiose epimerase (CSCE) from Caldicellulosiruptor saccharolyticus, the codon preference was optimized and the DNA encoding the gene was fully synthesized by General Biotechnology (Anhui) Co., Ltd. The nucleotide sequence is shown in SEQ ID NO.13. Using this DNA as a template, primers were designed for PCR amplification. The primer sequences included the restriction sites NdeI and EcoRI upstream and downstream of the gene. Takara's PCR amplification was performed using a high-fidelity DNA polymerase. The PCR reaction program was: 98°C for 10 s, 55°C for 15 s, and 72°C for 1.5 min; this was repeated for 30 cycles.

[0035] Upstream primer:

[0036]

[0037] (The NdeI site is underlined);

[0038] Downstream primer: (The underline is the EcoRI site.) The bold sequence is the part that matches the template.

[0039] The PCR amplification product was purified using a DNA purification kit (MiniBEST DNA Fragment Purification Kit, TaKaRa) and stored at -20°C.

[0040] The nucleotide sequence of the gene encoding the wild-type Caldicellulosiruptor saccharolyticus-derived cellobiose epimerase (CSCE) (SEQ ID NO.13):

[0041]

[0042] 2. Construction of the recombinant vector of cellobiose isomerase mutant E250Q

[0043] The PCR amplification product and the pET-28a plasmid were connected. The connection method is as follows:

[0044] First, restriction endonucleases NdeI and EcoRI (TaKaRa) were used for double digestion. The double digestion system (50 μL) was: 5 μL of 10×H buffer, 25 μL of PCR amplification product (or pET-28a plasmid), 1 μL of NdeI enzyme, 1 μL of EcoRI enzyme, and 18 μL of double distilled water; the digestion temperature was 37°C, and the digestion time was 5 h; the products were purified and recovered using a DNA purification kit (MiniBEST DNA Fragment Purification Kit, TaKaRa) and stored at -20°C. The digestion products were then connected using T4 DNA ligase (purchased from the NEB official website), and the connection system (20 μL) was: 5 μL of PCR amplification product after double digestion, 5 μL of pET-28a plasmid after double digestion, 2 μL of 10×T4 DNA ligase buffer, 1 μL of T4 DNA ligase, and 7 μL of double distilled water. The ligation reaction temperature was 25°C, and the ligation reaction time was 12h. After the ligation was completed, the reactants were 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 E. coli Fast-T1 competent cells (Nanjing Novozyme Biotechnology Co., Ltd.), incubated on ice for 20 min, transferred to 42°C heat shock for 90 s, followed by an ice bath for 5 min, cultured at 37°C for 45 min, and 150 μL of the bacterial solution was coated on a 25 μg / mL kanamycin resistance plate. After overnight, the positive clones (called E. coli Fast-T1-CSCE strains) were picked and sent to a sequencing company for sequencing. The clones with correct sequence alignment were stored at -80°C.

[0045] 3. Site-directed mutagenesis of the cellobiose isomerase gene

[0046] The E. coli Fast-T1-CSCE strain was cultured overnight in LB liquid medium containing 25 mg / L kanamycin resistance. After centrifugation to obtain the bacteria, the plasmid pET-28a-CSCE was extracted using a plasmid extraction kit (MiniBEST Plasmid Purification Kit, TaKaRa) as a template for subsequent site-directed mutagenesis experiments. Site-directed mutagenesis was performed using the PCR method. The principle is to design a pair of oligonucleotide primers with complementary sequences near the coding gene site to be mutated. The specific steps are as follows:

[0047] CSCE E250Q site-directed mutagenesis forward primer:

[0048] TATGGTCACGATATT CAA GCGAGCTGGCTGCTG (SEQ ID NO. 16) (the underline indicates the mutation site),

[0049] Reverse primer for E250Q site-directed mutagenesis of CSCE:

[0050] CAGCAGCCAGCTCGC TTG AATATCGTGACCATA (SEQ ID NO. 17) (underlined mutation sites). PCR used pET-28a-CSCE recombinant plasmid as template and used High-fidelity DNA polymerase (Takara) was used for the reaction. The PCR program was: 98°C for 10s, 55°C for 15s, and 72°C for 7min; 16 cycles were repeated. The PCR product was analyzed by agarose gel electrophoresis, and then the gel was cut and recovered for purification. The purified product was treated with restriction endonuclease DpnI at 37°C for 12h to digest the template used for PCR; the treated product was transformed into E. coli BL21 (DE3), and a single clone that could grow on a 25mg / L kanamycin LB resistance plate was selected, which was called E. coli BL21 (DE3)-pET-28a / E250Q; it was sent to a related gene technology company for sequencing, and the clone with the correct sequence alignment was stored at -80°C.

[0051] This embodiment stipulates that the amino acid residue numbering is based on the first residue methionine of the wild-type CE enzyme as the first amino acid residue.

[0052] The amino acid sequence of the cellobiose epimerase (CSCE) mutant E250Q from Caldicellulosiruptorsaccharolyticus is shown in SEQ ID NO.1:

[0053] MDITRFKEDLKAHLEEKIIPFWQSLKDDEFGGYYGYMDFNLNIDRKAQKGCILNSRILWFFSACYNVLKSEKCKEMAFHAFEFLKNKFWDKEYEGLFWSVSHKGVPVDVTKHVYVQAFGIYGLSEYYEASGDEEALHMAKRLFEILETKCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTN LYRLLKLDDVYEALEWIVRLFVDKIYKKGTGHFKVFCDDNWNELIKAVSYGHDIQASWLLDQAAKYLKDEKLKEEVEKLALEVAQITLKEAFDGQSLI NEMIEDRIDRSKIWWVEAETVVGFFNAYQKTKEEKYLDAAIKTWEFIKEHLVDRRKNSEWLWKVNEDLEAVNMPIVEQWKCPYHNGRMCLEIIKRVD.

[0054] The nucleotide sequence of the gene encoding the cellobiose epimerase (CSCE) E250Q mutant from Caldicellulosiruptorsaccharolyticus is shown in SEQ ID NO.7:

[0055]

[0056] Example 2: Expression and purification of cellobiose epimerase mutant E250Q

[0057] 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, it was transferred to fresh LB liquid medium containing 25 mg / L kanamycin and cultured at 37°C for 4 h until the OD 600 =0.8; cool to 25°C and continue culturing, add IPTG with a final concentration of 0.5 mM for induction, and after 24 hours of expression, collect the bacteria by centrifugation at 5000 rpm for 20 minutes. Use polyacrylamide gel electrophoresis (SDS-PAGE) to detect the expression of the target protein in the whole cell. The specific steps are as follows:

[0058] The collected bacteria were disrupted with a high-pressure homogenizer, centrifuged at 12,000 rpm and 4°C for 20 min, the precipitate was discarded, and the supernatant was eluted through a nickel ion affinity column (model: Sepharose Fast Flow, GE Healthcare).

[0059] The affinity column was pre-equilibrated with a buffer solution, then washed with a wash buffer solution, and the cellobiose epimerase was eluted with an eluent, and dialyzed in a 50-fold volume of a dialysate at 4°C using a dialysis bag to remove salt. Finally, a polyethylene glycol powder with a molecular weight of 20,000 Da was used to absorb water and concentrate the enzyme solution, and the obtained enzyme solution can be used for subsequent biocatalysis.

[0060] The equilibration buffer consisted of 10 mM HEPES buffer (pH 7.0) containing 10% w / v glycerol and 150 mM sodium chloride;

[0061] The wash buffer contained 10 mM HEPES buffer (pH 7.0), 10% w / v glycerol, 50 mM imidazole, and 500 mM sodium chloride;

[0062] The eluent contained 10 mM HEPES buffer (pH 7.0), 10% w / v glycerol, 200 mM imidazole, and 500 mM sodium chloride;

[0063] The dialysate was: 10 mM HEPES buffer (pH 7.0) and 10% w / v glycerol.

[0064] The protein concentration of the enzyme solution was determined by Bradford method using bovine serum albumin as the standard, and the result was 5.8 mg / mL. The purity of the enzyme was detected by SDS-PAGE electrophoresis. Figure 1 shown.

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

[0066] Lactose was prepared using lactose as substrate and the cellobiose epimerase mutant enzyme solution prepared in Example 4 and the CSCE wild-type enzyme as biocatalysts. The catalytic system was 0.5 mL: 50 g / L lactose, 10 mM HEPES buffer (pH 7.0), and 1 mg enzyme solution (prepared according to Example 2). The system was reacted at 80° C. and 400 rpm for 15 min, and the reaction product was identified by HPLC.

[0067] HPLC determination method: using 1200 liquid chromatograph (Agilent Technologies (China) Co., Ltd.), Ca 2+ The chromatographic column was Rezex RCM-Monosaccharide (Phenomenex), the mobile phase was pure water, the column temperature was set at 80°C, and the flow rate was 0.5 mL / min. The HPLC test results were as follows: Figure 2 and Figure 3 As shown. The external standard method was used to determine the yields of lactose, ipilactose, and lactulose based on the peak retention time and peak area. The analytically pure standards of lactose, ipilactose, and lactulose were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0068] Enzyme activity definition: The amount of enzyme that produces 1 μmol lactulose per minute is defined as 1U, and the specific enzyme activity is the ratio of enzyme activity (U) to enzyme mass (mg). The yield and enzyme activity of the byproduct epiratose are shown in Table 1.

[0069] Table 1 Ipilactose yield and enzyme activity in the catalytic reaction of CSCE wild-type enzyme and mutant enzyme

[0070] Lactulose yield Ipilactose 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

[0071] Example 4. Recombinant Escherichia coli of cellobiose epimerase mutant E250Q catalyzes lactose

[0072] According to the method in Example 2, the Escherichia coli BL21 (DE3) -pET-28a / E250Q strain was fermented and induced to express, and the recombinant Escherichia coli wet cells were obtained after centrifugation at 5000rpm for 20min. Recombinant Escherichia coli was used to catalyze 700g / L lactose at 80 ° C, the addition amount of wet cells was: 50g wet cells / L, the catalytic time was 3h, and 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 by-product epilactose accounted for 5%, and the remaining lactose accounted for 30%.

[0073] The reaction solution was concentrated by vacuum distillation at 60°C to a Brix of 75%, and then cooled to room temperature overnight for crystallization. The lactose crystals were removed by a plate and frame filter the next day, and the resulting sugar solution was diluted with deionized water to a lactulose content of 667 g / L, i.e., a concentrated lactulose solution, in which the content of epilactose met the standard of less than 7% in the lactulose oral solution of the Chinese Pharmacopoeia.

[0074] The present invention can also directly use fermentation broth for catalysis (no purification and no pH adjustment), the concentration of lactose in the catalytic system is 700 g / L, the amount of fermentation broth added 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 Escherichia coli wet cells.

[0075] The preparation method of the fermentation broth is as follows: the Escherichia coli BL21 (DE3)-pET-28a / E250Q strain is inoculated into LB liquid medium containing 25 mg / L kanamycin, cultured at 37°C for 12 h, then transferred to fresh LB liquid medium containing 25 mg / L kanamycin, and cultured at 37°C for 4 h until the OD 600 =0.8; cool to 25°C and continue culturing, add IPTG with a final concentration of 0.5 mM for induction, and after 24 hours of expression, centrifuge at 5000 rpm for 20 minutes, and collect the supernatant, which is the fermentation broth.

[0076] Example 5. Preparation of calcium alginate immobilized microspheres encapsulating recombinant Escherichia coli and its catalytic reaction

[0077] According to the method in Example 2, wet cells of recombinant Escherichia coli were obtained, and 50 g / L of wet cells of recombinant Escherichia coli were added to an equal volume of 4% (w / v) calcium alginate solution, stirred and mixed, and then slowly dripped into a 0.2 mol / L pre-cooled calcium chloride solution through a funnel, and hardened overnight under magnetic stirring at 4°C, and washed three times with pre-cooled deionized water to obtain calcium alginate immobilized microspheres. Calcium alginate immobilized microspheres can be used to catalyze lactose to prepare lactulose, and are loaded into an immobilized enzyme reactor for batch catalytic reaction, such as Figure 4 shown.

[0078] The above calcium alginate immobilized microspheres were used to catalyze lactose to prepare fructose. The reaction system was: 700 g / L lactose, 100 g / L calcium alginate immobilized microspheres, reaction temperature 80°C, pH 7.0, and catalytic time 3.5 h. A lactulose concentrated solution was prepared according to the method in Example 4, wherein the content of epilactose met the standard of less than 7% in the lactulose oral solution of the Chinese Pharmacopoeia, and the color of the lactulose concentrated solution was better than that of commercially available lactulose of the same concentration. Lactulose oral solution is lighter ( Figure 5 ), indicating that it contains lower levels of pigment impurities.

[0079] The present invention uses calcium alginate to embed recombinant Escherichia coli cells to catalyze lactose in multiple batches to explore the changes in the relative catalytic ability of immobilized cells. The reaction temperature of each batch is 80°C, the reaction time is 3.5h, and the initial activity of the first batch is defined as 100%. The ordinate is the relative catalytic activity of the immobilized cells, and the abscissa is the reaction batch of the immobilized cells. The changes in the relative catalytic ability of the immobilized cells are shown in Figure 2. Figure 6 As shown. Figure 6 It can be seen that the immobilized engineered bacterial cells can be used in multiple batches after being immobilized by calcium alginate embedding, and the enzyme activity decreases by less than 10% within 30 batches.

[0080] Example 6

[0081] In this example, the glutamic acid at position 250 of the amino acid sequence of cellobiose epimerase (DTCE) from the thermophilic bacterium Dictyoglomus turgidum was mutated to glutamyl, and the lactose catalysis reaction was carried out according to the method in Example 3. After the catalysis was completed, HPLC detection was performed, and the epililactose yield and specific enzyme activity were calculated. The results are shown in Table 2.

[0082] Table 2 Effects of site-directed mutagenesis in DTCE enzyme

[0083] Lactulose yield Ipilactose 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

[0084] The experimental results showed that the DTCE E250Q mutant enzyme obtained a mutation effect similar to that of the CSCE enzyme in the lactose catalysis reaction, and the proportion of the by-product epiratose was significantly reduced.

[0085] The amino acid sequence of the cellobiose epimerase (DTCE) mutant E250Q from Dictyoglomus turgidum is shown in SEQ ID NO.2:

[0086] MDLKVLKSEIFEHLNNKIIPFWEELKDENNGGYISYVGFDLKPDPYAPKGLVLTSRILWFFSRLYNQLRKEEFINFADHSYKFLIKSFLDKENKGFYWMVDYKGEPIDKRKHLYGQAFVLYGLSEYYKATQKKESLDLALEIYKIIEEVCKNDVGYKEEFDEKWNPKENIIVSEYGIICERSMNTLLHILEAYTN LFTATYDQSIKKKIEDLIILFKEKIYDSKTNHLYVFFDKKMNPIIDAISYGHDIQATWLIDEALRYIDNNKLIKEMSEINLKIAEKVLEEAFESGSLLNERVRGIVDKNRIWWVQAEALVGFLNAYQKSKLDKFLKAVFELWEFIKDFLVDKRAQGEWFWKLDENYIPSPMPEVDLWKCPYHNGRMCLEVIKRI.

[0087] The nucleotide sequence of the gene encoding the cellobiose epimerase (DTCE) mutant E250Q from Dictyoglomus turgidum is shown in SEQ ID NO.8:

[0088]

[0089] Example 7

[0090] In this example, the glutamic acid at position 250 of the amino acid sequence of cellobiose epimerase (DthermoCE) from Dictyoglomus thermophilum was mutated to glutamyl, and the lactose catalysis reaction was carried out according to the method in Example 3. After the catalysis was completed, HPLC detection was performed, and the pilolactose yield and specific enzyme activity were calculated. The results are shown in Table 3.

[0091] Table 3 Effects of site-directed mutagenesis in DthermoCE enzyme

[0092]

[0093] The experimental results showed that the DthermoCE E250Q mutant enzyme obtained a mutation effect similar to that of the CSCE enzyme in the lactose catalysis reaction, and the proportion of the by-product epiratose was significantly reduced.

[0094] The amino acid sequence of the cellobiose epimerase (DthermoCE) mutant E250Q from Dictyoglomus thermophilum is shown in SEQ ID NO.3:

[0095] MDLKHLKDEIFEQLNNKIIPFWENLKDENNGGYISYVGFDLKPDPYAPKGLVLTSRILWFFSRLYNQLRKEEFIEFADHAYEFLTDKLLDKENGGFFWIVDYKGDPLDKRKHLYGQAFALYGLSEYYKATKKKESLELSLELYKTIEERCKDNIGYKEEFDEKWTPKENIIVSEYGIICEKSMNTLLHLLEAYTN LFTATYDLKVRKELENLIILFKEKIYNPKTDHLYVFFDNKMKPIIDAISYGHDIQATWLIDEALRYIENNTLIKDMTEINLRIAERVLEEAFENNSLLNEKVRGEVNKDRVWWVQAEALLGFLNAYQKTKSDKFLKAVLSLWEFINNFLLDKRPNGEWFNKLDENCIPFPMPEVDLWKCPYHNGRMYLEVIRRI.

[0096] The nucleotide sequence of the gene encoding the cellobiose epimerase (DthermoCE) mutant E250Q from Dictyoglomus thermophilum is shown in SEQ ID NO.9:

[0097]

[0098] Example 8

[0099] In this example, the glutamic acid at position 250 of the amino acid sequence of cellobiose epimerase (COCE) from Caldicellulosiruptor obsidiansis was mutated to glutamyl, and the lactose catalysis reaction was carried out according to the method in Example 3. After the catalysis was completed, HPLC detection was performed, and the pilolactose yield and specific enzyme activity were calculated. The results are shown in Table 4.

[0100] Table 4 Effects of site-directed mutagenesis in COCE enzyme

[0101] Lactulose yield Ipilactose 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

[0102] The experimental results showed that the COCE E250Q mutant enzyme obtained a mutation effect similar to that of the CSCE enzyme in the lactose catalysis reaction, and the proportion of the by-product epiratose was significantly reduced.

[0103] The amino acid sequence of the cellobiose epimerase (COCE) mutant E250Q from Caldicellulosiruptor obsidiansis is shown in SEQ ID NO.4:

[0104] MDITSFKKELKSHLEEKIIPFWQSLKDDEFGGYYGYMDFNLNINKKAQKGCILNSRILWFFSACYNVLKSEKCKELAFHAFEFLKNKFWDKDYEGLFWSVSLEGLPVDVTKHVYVQAFGIYGLSEYYEASGDKEALFLARKLFEILETRCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTN LYKVLKLDDVYEALEWLVRLFVEKIYKKGTGHFKVFCDDNWNELIKAVSYGHDIQASWLLDETAKYLRDEKLKEEVEKLTLEVAQVTLQEAFDGKSLI NEKVEDRVDRSKIWWVEAETVVGFFNAYQKTKEEKYLDAAIKTWEFIKEYLVDKRKNSEWLWKVDENLNPVQMPIVEPWKCPYHNGRMCLEIIKRVG.

[0105] The nucleotide sequence of the gene encoding the cellobiose epimerase (COCE) E250Q mutant from Caldicellulosiruptor obsidiansis is shown in SEQ ID NO.10:

[0106]

[0107] Example 9

[0108] In this example, the glutamic acid at position 250 of the amino acid sequence of cellobiose epimerase (CBCE) from Caldicellulosiruptor bescii was mutated to glutamyl, and the lactose catalysis reaction was carried out according to the method in Example 3. After the catalysis was completed, HPLC detection was performed, and the pilolactose yield and specific enzyme activity were calculated. The results are shown in Table 5.

[0109] Table 5 Effects of site-directed mutagenesis in CBCE enzyme

[0110] Lactulose yield Yield of epiratose 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

[0111] The experimental results showed that the CBCE E250Q mutant enzyme obtained a mutation effect similar to that of the CSCE enzyme in the lactose catalysis reaction, and the proportion of the by-product epiratose was significantly reduced.

[0112] The amino acid sequence of the cellobiose epimerase (CBCE) E250Q mutant from Caldicellulosiruptor bescii is shown in SEQ ID NO.5:

[0113] MDITKFKEDLKAHLEEKIVPFWQSLKDDEFGGYYGYMDFNLNIHRKAQKGCILNSRILWFFSACYNVLKNEKCKELAFHAFEFLKNKFWDKEYEGLFWNVSHKGVPVDMTKHVYVQAFGIYGLSEYYEASGDKEALQMAKKLFEILETKCKRENGYTEQFERNWQEKENRFLSENGVIASKTMNTHLHVLESYTN LYKVLRTKDVYEALEWIVRLFVDKIYKKGTGHFKVFCDDNWNELIKAVSYGHDIQASWLLDEAARYLKDEKLKEEVEKLTLEVAQVTLKEAFDGQSLI NEMVEDRVDRSKIWWVEAETVVGFFNAYQKSKEEKFLDAAIKTWKFIEEHLVDKRKNSEWLWKVSEDLKALDMPIVEPWKCPYHNGRMCLEIIKRVG.

[0114] The nucleotide sequence of the gene encoding the cellobiose epimerase (CBCE) E250Q mutant from Caldicellulosiruptor bescii is shown in SEQ ID NO.11:

[0115]

[0116] Example 10

[0117] In this example, the glutamic acid at position 250 of the amino acid sequence of cellobiose epimerase (PPCE) from Paenibacillus polymyxa was mutated to glutamyl, and the lactose catalysis reaction was carried out according to the method in Example 3. After the catalysis was completed, HPLC detection was performed, and the pilolactose yield and specific enzyme activity were calculated. The results are shown in Table 5.

[0118] Table 5 Effects of site-directed mutagenesis in PPCE enzyme

[0119] Lactulose yield Yield of epiratose 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

[0120] The experimental results showed that the PPCE E250Q mutant enzyme obtained a mutation effect similar to that of the CSCE enzyme in the lactose catalysis reaction, and the proportion of the by-product epiratose was significantly reduced.

[0121] The amino acid sequence of the cellobiose epimerase (PPCE) E250Q mutant from Paenibacillus polymyxa is shown in SEQ ID NO.6:

[0122] METLLDEIRQEWKEHILPFWLGLKDETHGGFYGEVDVGLHTHNQADKGGIATARLLWSFSAATRVTGENTYAEAARHAFTFLRDHLLDPLHGGMYWMVDYTGQPVDTCKHVYAQAFAIYALAEYACATDDPSALPLAMELFHLLEQKGYDPARQAYGEQYDRLWNTQPNELLSENGVTAHITMNTHIHVLEAYTQL LRVWPNEEVRDALTNVLDILYRRVYDASARRLGVFFDREWRSLLDLTSYGHDIQASWLIEDAMNVLGYYPSEYVDMVMDIANAVAERAVQPDGSLINEREGERVDTSRIWWVQAEGMVGFYNAFQRTNDERFLQIVRNLWAYTRQYIIDPRPGGEWFWSVQADGKPDAREIAGPWKCPYHNSRFCIEMLEERMEDQ.

[0123] The nucleotide sequence of the gene encoding the cellobiose epimerase (PPCE) E250Q mutant from Paenibacillus polymyxa is shown in SEQ ID NO.12:

[0124]

[0125] As can be seen from the above examples, the present invention provides a cellobiose epimerase mutant E250Q, an engineered bacterium and its application. The cellobiose epimerase mutant E250Q of the present invention can reduce the yield of the byproduct epilactose when lactose is prepared from lactulose.

[0126] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cellobiose epimerase mutant E250Q, characterized in that The 250th glutamic acid in the amino acid sequence of wild cellobiose epimerase is mutated to glutamine; the amino acid sequence of the cellobiose epimerase mutant is shown in any one of SEQ ID NOs. 1 to 6.

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 any one of SEQ ID NOs. 7 to 12.

3. An expression vector, characterized in that: The expression vector contains the coding gene according to claim 2.

4. An engineered bacterium comprising the expression vector according to claim 3, wherein the engineered bacterium is Escherichia coli.

5. A calcium alginate microsphere encapsulating the engineered bacteria according to claim 4, characterized in that: The preparation is carried out according to the following steps: the wet cells of the engineered bacteria are mixed with the calcium alginate solution, and the mixture is dripped into the pre-cooled calcium chloride solution after stirring, and the mixture is stirred for 8 to 12 hours to obtain the calcium alginate microspheres.

6. The calcium alginate microspheres according to claim 5, characterized in that: The concentration of the engineered bacteria wet cells is 48-52 g / L, the concentration of the calcium alginate solution is 4% w / v, and the volume ratio of the engineered bacteria wet cells to the calcium alginate solution is 0.8-1.2:

1.

7. The calcium alginate microspheres according to claim 6, characterized in that: The concentration of the calcium chloride solution is 0.18-0.22 mol / L.

8. Use of the cellobiose epimerase mutant E250Q according to claim 1, the engineered bacteria according to claim 4 and / or the calcium alginate microspheres according to any one of claims 5 to 7 in catalyzing the preparation of lactulose from lactose.

9. The use according to claim 8, characterized in that: Lactose is used as a substrate, and the cellobiose epimerase mutant E250Q described in claim 1, the engineered bacteria described in claim 4, or the calcium alginate microspheres described in any one of claims 5 to 7 are used as catalysts to carry out a catalytic reaction to generate lactulose.

10. The use according to claim 9, characterized in that: The conditions of the catalytic reaction are: the concentration of lactose is 50-700 g / L, the reaction temperature is 78-82° C., and the reaction time is 1-24 h.

Images