A cellobiose epimerase mutant, engineered bacteria and its application

Abstract

This invention discloses a cellobiose epimerase mutant, engineered bacteria, and its applications. The mutant is obtained by mutating leucine at position 227 of the cellobiose epimerase to valine. The nucleotide sequence of the cellobiose epimerase is shown in SEQ ID NO.1. This invention involves site-directed mutagenesis of existing cellobiose epimerases, maximizing the catalytic performance of the natural enzyme. The resulting mutant exhibits a high lactulose conversion rate. Under optimized conditions, using crude enzyme solution from recombinant bacteria as a catalyst and lactose as a substrate, the conversion rate from lactose to lactulose reaches 70%–73.3%, while the yield of ipilactose is only 7–10%, lower than the byproduct ratio allowed by the Chinese Pharmacopoeia. This demonstrates the potential for large-scale application and has extremely high application value in the industrial production of lactulose.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering, specifically to a cellobiose epimerase mutant, engineered bacteria, and its applications. Background Technology

[0002] Lactulose is a functional disaccharide formed by the linkage of D-galactose and D-fructose through a β-1,4 glycosidic bond, with a sweetness approximately 48%-62% that of sucrose. Studies have shown that the β-glycosidic bond of lactulose cannot be hydrolyzed by digestive enzymes in mammals, and therefore cannot be absorbed by the human body. However, it can promote the growth of beneficial bacteria such as Bifidobacteria in the gut and inhibit the growth of harmful microorganisms, effectively maintaining the balance of the gastrointestinal ecosystem. It can effectively relieve constipation and improve immunity. Lactulose can also maintain blood glucose and insulin levels and can be used as an adjunct to the treatment of diabetes. Furthermore, it can reduce blood ammonia concentration in patients with hepatic encephalopathy and alleviate their condition.

[0003] The main methods for producing lactulose include chemical isomerization and enzymatic conversion. Currently, commercially available lactulose is primarily prepared under alkaline conditions via chemical isomerization. Chemical lactulose production requires strongly alkaline conditions, resulting in numerous byproducts and complex desalting processes, making it relatively difficult to obtain high-purity lactulose and contradicting the trend towards green production.

[0004] Compared to chemical methods, the enzymatic conversion method for producing lactulose has milder reaction conditions, requires no large amounts of chemical reagents, eliminates the need to consider the separation of metal ions, and causes no environmental pollution, making the product more readily accepted by the market. Currently, the main enzymatic methods include β-galactosidase and cellobiose epimerase (CEase). β-galactosidase uses lactose and fructose as substrates, hydrolyzing lactose into galactose and glucose. Then, through transglycosidic function, the galactose groups condense with fructose to form lactulose. The final product generally includes lactulose, glucose, galactose, fructose, and lactose. However, the yield of lactulose is low, it contains many impurities, and its separation and purification are difficult and costly, limiting its industrialization potential. Cellobiose epimerase, using lactose as the sole substrate, catalyzes the isomerization of β-1,4-glycosidic linked glucose residues to fructose residues, thereby producing lactulose and ipilactose. It is currently the most efficient enzyme for lactulose production. Cellobiose epimerase produces a certain proportion of ipilactose during the catalytic conversion reaction. The ratio of ipilactose to lactulose is generally 0.20 to 0.26. The Chinese Pharmacopoeia stipulates that the ipilactose content should be less than 10%. Therefore, reducing the ipilactose content is an urgent problem to be solved when applying the cellobiose epimerase method.

[0005] Existing technologies for reducing ipilulose content mainly include increasing reaction temperature, adding boric acid to the reaction system, and refining the fermentation broth. While increasing reaction temperature can improve lactulose yield, relying solely on temperature increases still doesn't meet the pharmacopoeia-specified standard (less than 10%) for ipilulose content produced by enzymatic methods. Therefore, other methods are needed to further reduce ipilulose content. Boron is a prohibited ingredient in food, and refining methods increase production procedures and costs. For these reasons, large-scale industrial production of lactulose using enzymatic methods has not yet been achieved. Summary of the Invention

[0006] The first objective of this invention is to provide a cellobiose epimerase mutant, which is obtained by mutating leucine at position 227 of the cellobiose epimerase to valine;

[0007] The nucleotide sequence of the cellobiose epimerase is shown in SEQ ID NO.1.

[0008] A second objective of the present invention is to provide an expression vector containing the above-mentioned nucleotide sequence.

[0009] In a preferred embodiment, the expression vector is pET28a(+).

[0010] A third objective of this invention is to provide recombinant engineered bacteria containing the aforementioned expression vector. Specifically, the recombinant expression plasmid obtained by introducing the mutant coding gene into the expression vector is transferred into a host cell to obtain the recombinant engineered bacteria.

[0011] In a preferred embodiment, the recombinant engineered bacteria are constructed using Escherichia coli as the host bacterium.

[0012] A fourth objective of this invention is to provide the application of the above-mentioned cellobiose epimerase mutant in the fermentation of lactulose.

[0013] In a preferred embodiment, the cellobiose epimerase mutant is used as a catalyst and lactose is used as a substrate to carry out the conversion reaction at 75-80°C.

[0014] In a preferred embodiment, the cellobiose epimerase mutant is used as a catalyst in the form of a pure enzyme or a crude enzyme solution;

[0015] The crude enzyme solution is prepared as follows: a recombinant engineered bacterium is constructed using the cellobiose epimerase mutant as the target gene. After induction culture, the recombinant engineered bacterium is collected as wet cells. The wet cells are then resuspended, sonicated, and the supernatant is collected, which is the crude enzyme solution. It is preferable to use the crude enzyme solution, as the enzyme purification process is cumbersome, has a low yield, and exhibits significant enzyme activity reduction during purification. For industrial applications, only the crude enzyme solution is required.

[0016] In a preferred embodiment, the concentration of the substrate lactose is 100–400 g / L.

[0017] In a preferred embodiment, the enzyme dosage is 16–25 U / g lactose.

[0018] In a preferred embodiment, the conversion reaction is carried out at pH 6.0–7.0 (preferably pH 6.5) and 100–200 rpm for 5–10 h (preferably 6 h) to obtain lactulose.

[0019] The cellobiose epimer mutant described in this invention is obtained by mutating a single amino acid in the cellobiose epimerase of *Rhodothermus marinus*, thereby enhancing its ability to catalyze the isomerization of lactose to lactulose. First, the cellobiose epimerase encoding gene (SEQ ID NO.1) is ligated into the expression vector pET28a(+) to construct a recombinant expression plasmid. Then, the recombinant expression plasmid is transformed into *E. coli* BL21(DE3). Using the recombinant expression plasmid containing the cellobiose epimerase gene as a template, gene modification is performed promptly through site-directed mutagenesis, and then the recombinant expression plasmid is transformed into *E. coli* BL21(DE3). This yields *E. coli* BL21(DE3) genetically engineered bacteria containing the cellobiose epimerase mutant gene. The obtained recombinant genetically engineered bacteria were induced and cultured. Bacterial cells containing cellobiose epimerase mutants were isolated from the culture medium. The bacterial cells were broken and centrifuged to obtain crude enzyme solution. The catalytic activity of the mutant enzyme and the original enzyme was compared, and mutants with excellent catalytic performance were screened.

[0020] As a preferred embodiment, the wet bacterial cells are prepared as follows: The recombinant engineered bacteria are inoculated into LB liquid medium containing a final concentration of 50 µg / mL kanamycin and cultured at 37°C and 200 rpm for 10–12 h. Then, at a 3% (v / v) inoculation rate, the bacteria are inoculated into fresh LB liquid medium containing 50 µg / mL kanamycin and cultured at 37°C and 200 rpm until OD (dose-free ratio) is reached. 600 Add 0.6–0.8 g of IPTG to a final concentration of 0.1 mM, induce culture at 30°C for 12 h, centrifuge at 4°C and 10,000 rpm for 10 min, discard the supernatant, collect the precipitate, and obtain wet bacterial cells.

[0021] In a preferred embodiment, the crude enzyme solution is prepared as follows: The wet bacterial cells are suspended in PBS buffer (preferably pH 6.3) at a ratio of 6.0–6.5, and then disrupted using ultrasound to release the intracellular enzyme protein. The disruption program is as follows: disruption time 2 seconds, interval 2 seconds, power 650W, total disruption time 30 minutes under ice bath conditions. The cell disruption solution is centrifuged at 10,000 rpm for 20 minutes, and the supernatant is treated at 70°C for 30 minutes, then centrifuged at 10,000 rpm for 20 minutes. The collected supernatant is the crude cellobiose epimerase.

[0022] This invention involves site-directed mutagenesis of existing cellobiose epimerases, maximizing the catalytic performance of the natural enzyme. The resulting mutant exhibits high lactulose conversion while having a low ipilactose yield. Under optimized conditions, using crude enzyme solution from recombinant bacteria as a catalyst and lactose as a substrate, the conversion rate from lactose to lactulose reaches 70%–73.3%, while the ipilactose yield is only 7–10%, lower than the byproduct ratio allowed by the Chinese Pharmacopoeia. This invention possesses the potential for large-scale application and has extremely high application value in the industrial production of lactulose. Attached Figure Description

[0023] Figure 1 SDS-PAGE of cellobiose epimerase: Lane 1 is the protein molecular weight marker, Lane 2 is the supernatant of E. coli BL21(DE3) after cell wall disruption, Lane 3 is the supernatant of E. coli BL21(DE3)-Pet-28a-CE / E246M, and Lane 4 is the supernatant of E. coli BL21(DE3)-Pet-28a-CE / L227V after cell wall disruption.

[0024] Figure 2 The high-performance liquid chromatography (HPLC) chromatogram shows the detection of lactose to lactulose conversion catalyzed by E. coli BL21(DE3)-Pet-28a-CE / L227V. Detailed Implementation

[0025] The LB liquid culture medium formulation (g / L) in the example is: peptone 20, yeast extract 15, NaCl 10.

[0026] Example 1: Construction of a genetically engineered bacterium carrying cellobiose epimerase

[0027] The gene sequence of cellobiose epimerase from *Rhodothermus marinus* in the gene library was codon-optimized, and then synthesized into a whole gene (nucleotide sequence shown in SEQ ID NO.1). This gene was then transformed into the Nco I and Xho I sites of the vector pET28a(+) to obtain the pET28a(+)-CE plasmid. This plasmid was then transformed into *E. coli* BL21(DE3) to obtain wild-type *E. coli* BL21(DE3) / pET28a-CE. *E. coli* BL21(DE3) / pET28a-CE was plated on LB agar plates containing 100 mM kanamycin and incubated at 37°C for 12 h. Clones were randomly selected from the colonies grown on the plates, and plasmids were extracted for identification by agarose gel electrophoresis and nucleotide sequencing. This yielded a genetically engineered bacterium containing the cellobiose epimerase gene, namely *E. coli* BL21(DE3) / pET28a-CE. The above-mentioned genetically engineered bacteria were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37℃ and 200 rpm for 8 h to obtain seed culture. The seed culture was then inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin at a 3% (v / v) inoculation rate and cultured at 37℃ and 200 rpm to obtain OD. 600 When the concentration reaches 0.6-0.8, add IPTG to the culture medium to a final concentration of 1 mM, induce expression at 28℃ for 12 h, centrifuge at 4℃ and 10000 r / min for 20 min, discard the supernatant, and collect the wet cells for later use.

[0028] Example 2 Construction of mutants

[0029] Based on the gene sequence of the original enzyme, site-directed mutagenesis primers were designed. Using rapid PCR technology and the recombinant vector pET28a-CE as a template, single mutations were introduced at positions 227 and 246, respectively. The primers designed were as follows:

[0030] Forward primer L227V: TGGCTATCCGTCT GTA TGGCACCGTGC (Underlined bases are mutant bases)

[0031] Reverse primer L227V: CAATGGACATTGC CTG ACCGGTAGCCG (underlined bases are mutant bases)

[0032] Forward primer E246M: CATCCATGGCT ATG TACCACCCGGCTAC (underlined bases are mutant bases)

[0033] Reverse primer E246M: ACGCTTAACC GAA GTCCAGCGGTGTAA (underlined bases are mutant bases)

[0034] The PCR reaction system (20 μL) consisted of: 10 μL of 2×Phanta Max Buffer, 0.4 μL of dNTPs, 0.4 μL of forward primer (5 pmol / μL), 0.4 μL of reverse primer (5 pmol / μL), 0.4 μL of template DNA (20 ng / μL), 0.4 μL of Phanta Max Super-Fidelity DNA Polym eraser, and ddH2O added to a final volume of 20 μL.

[0035] PCR amplification conditions were as follows: 95℃ pre-denaturation for 5 min; (95℃ for 15 s, 55℃ for 15 s, 72℃ for 6 min) 30× cycles; 72℃ extension for 10 min; incubation at 16℃. PCR products were verified by 0.9% agarose gel electrophoresis, and the amplified gene fragment matched the size of the target vector was observed. The product was stored at 4℃. 4 μL of the PCR product was added to 100 μL of E. coli BL21(DE3) competent cell suspension (on ice), and incubated on ice for 30 min. The transformation product was then heat-shocked at 42℃ for 90 s, rapidly cooled on ice for 5 min, and 600 μL of LB liquid medium was added to the tube. The tube was incubated at 37℃, 150 rpm for 50 min, centrifuged at 12000 rpm for 1 min, 400 μL of supernatant was discarded, and the bacterial suspension was resuspended. 200 μL of the above resuspension was spread onto an LB solid medium plate containing a final concentration of 50 μg / mL kanamycin resistance. After the bacterial suspension was completely absorbed by the medium, the plate was incubated upside down at 37°C for 12 h. Colonies were then picked and inoculated into 10 mL of LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and incubated at 37°C for 12 h to obtain the respective bacterial suspensions. The bacterial suspensions were sent to a sequencing company for nucleotide sequence analysis, and the sequencing results matched correctly, identifying them as recombinant bacteria E. coli BL21(DE3) / pET28a / RmCE / L227V and BL21(DE3) / pET28a / RmCE / E246M containing the mutant enzyme gene. The nucleotide sequence of the L227 mutant is shown in SEQ ID NO. 2.

[0036] Example 3 Preparation of wet cells of recombinant cellobiose epimerase mutant

[0037] The recombinant *E. coli* containing the gene expressing the cellobiose epimerase mutant obtained in Example 2 was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 200 rpm until OD500. 600When the concentration reaches 0.6–0.8, add IPTG to a final concentration of 0.1 mM, induce culture at 28°C for 12 h, centrifuge at 4°C and 10,000 rpm for 15 min, discard the supernatant, collect the precipitate, and obtain recombinant Escherichia coli wet cells containing the gene expressing the recombinant cellobiose epimerase mutant.

[0038] Example 4 Purification and Enzyme Activity Assay of Recombinant Enzyme

[0039] 1g of wet bacterial cells were resuspended in 20mL of 50mM HEPES (pH 6.5) buffer and sonicated for 30min, with a 2s working time followed by a 2s interval. The lysed mixture was centrifuged at 10000r / min for 10min, and the supernatant was collected as the crude enzyme solution, which was used as the loading solution. Purification was performed using a Nickel-NTA affinity chromatography column (Bio-Scale Mini Profinity IMAC pre-packed column, 40mm long × 12.6mm inner diameter). After purification, the enzyme solution was dialyzed overnight in ultrapure water (pH 6.5) to obtain pure enzyme solution. SDS-PAGE electrophoresis analysis showed that a protein band with a molecular weight of approximately 47kDa was obtained, indicating successful expression of the target protein.

[0040] Take 9 mL of 100 g / L lactose solution, preheat to constant temperature at 75℃, add 1 mL of enzyme solution, mix well, react in a water bath shaker at 75℃ and 200 rpm for 30 min, and terminate the isomerization reaction by boiling water bath for 20 min. Centrifuge at 10000 rpm and 4℃ for 10 min, take the supernatant, filter through a 0.22 µm organic membrane, and detect the lactulose content by HPLC. The results are shown in Table 1.

[0041] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol / L lactulose per minute, which is one unit of enzyme activity, denoted by U.

[0042] Table 1. Activities of cellobiose epimerases and mutant enzymes

[0043]

[0044] Example 5: Preparation of lactulose from lactose catalyzed by a cellobiose epimerase mutant.

[0045] The mutant RmCE / L227V from Example 2 was induced to express recombinant cellobiose epimerase crude enzyme solution according to the methods in Examples 3 and 4. This enzyme solution was then used as a biocatalyst to prepare lactulose through a biotransformation reaction with lactose as a substrate.

[0046] The catalytic system and conditions are as follows: 12 U / g lactose of recombinant cellobiose epimerase was added to 10 mL of the reaction system, resulting in a substrate concentration of 350 g / L lactose. The pH of the reaction solution was adjusted to 6.5 using pH 7.4 PBS buffer. The reaction was carried out at a constant temperature of 200 rpm and 78℃ in a water bath. Samples were taken periodically during the reaction, and the yields of lactose converted to lactulose and ipilactose were determined by HPLC. The results showed that the lactulose yield reached 65.2% and the ipilactose yield reached 13.8% after 6 hours of catalysis.

[0047] Example 6: Preparation of lactulose from lactose catalyzed by a cellobiose epimerase mutant.

[0048] The mutant RmCE / L227V from Example 2 was induced to express recombinant cellobiose epimerase crude enzyme solution according to the methods in Examples 3 and 4. This enzyme solution was then used as a biocatalyst to prepare lactulose through a biotransformation reaction with lactose as a substrate.

[0049] The catalytic system and conditions are as follows: 16 U / g lactose crude enzyme solution of recombinant cellobiose epimerase was added to 10 mL of the reaction system, resulting in a substrate concentration of 350 g / L lactose. The pH of the reaction solution was adjusted to 6.5 using pH 7.4 PBS buffer. The reaction was carried out at a constant temperature of 200 rpm and 78℃ in a water bath. Samples were taken periodically during the reaction, and the yields of lactose converted to lactulose and ipilactose were determined by HPLC. The results showed that the lactulose yield reached 73.3% after 6 hours of catalysis, while the ipilactose yield was only 7.9%.

[0050] Example 7: Preparation of lactulose from lactose catalyzed by a cellobiose epimerase mutant.

[0051] The mutant RmCE / L227V from Example 2 was induced to express recombinant cellobiose epimerase crude enzyme solution according to the methods in Examples 3 and 4. This enzyme solution was then used as a biocatalyst to prepare lactulose through a biotransformation reaction with lactose as a substrate.

[0052] The catalytic system and conditions are as follows: 20 U / g lactose recombinant cellobiose epimerase solution was added to 10 mL of the reaction system, resulting in a substrate concentration of 350 g / L lactose. The pH of the reaction solution was adjusted to 6.5 using pH 7.4 PBS buffer. The reaction was carried out at a constant temperature of 200 rpm and 78℃ in a water bath. Samples were taken periodically during the reaction, and the yields of lactose converted to lactulose and ipilactose were determined by HPLC. The results showed that the lactulose yield reached 70.7% and the ipilactose yield reached 9.5% after 6 hours of catalysis.

[0053] Example 8: Preparation of lactulose from lactose catalyzed by a cellobiose epimerase mutant.

[0054] The mutant RmCE / E246M from Example 2 was induced to express recombinant cellobiose epimerase crude enzyme solution according to the methods in Examples 3 and 4. This enzyme solution was then used as a biocatalyst to prepare lactulose through a biotransformation reaction with lactose as a substrate.

[0055] The catalytic system and conditions are as follows: 20 U / g lactose of recombinant cellobiose epimerase solution was added to 10 mL of the reaction system, resulting in a substrate concentration of 100 g / L lactose. The pH of the reaction solution was adjusted to 6.5 using pH 7.4 PBS buffer. The reaction was carried out at a constant temperature of 200 rpm and 78℃ in a water bath. Samples were taken periodically during the reaction, and the yields of lactose converted to lactulose and ipilactose were determined by HPLC. The results showed that the lactulose yield reached 67.4% and the ipilactose yield reached 12.8% after 6 hours of catalysis.

Claims

1. A cellobiose epimerase mutant, characterized in that, The mutant was obtained by mutating leucine at position 227 of cellobiose epimerase to valine. The nucleotide sequence of the cellobiose epimerase is shown in SEQ ID NO.

1.

2. An expression vector containing the nucleotide sequence of the mutant of claim 1.

3. Recombinant engineered bacteria containing the expression vector of claim 2.

4. The recombinant engineered bacteria according to claim 3, characterized in that, The recombinant engineered bacteria were constructed using Escherichia coli as the host bacterium.

5. The application of the cellobiose epimerase mutant of claim 1 in the fermentation of lactulose.

6. The application according to claim 5, characterized in that, The cellobiose epimerase mutant was used as a catalyst and lactose as a substrate to carry out the conversion reaction at 75–80 °C.

7. The application according to claim 6, characterized in that, The cellobiose epimerase mutant was used as a catalyst in the form of pure enzyme or crude enzyme solution. The crude enzyme solution is prepared as follows: a recombinant engineered bacterium is constructed using the cellobiose epimerase mutant as the target gene. After induction culture, the recombinant engineered bacterium is collected as wet cells. The wet cells are then resuspended, sonicated, and the supernatant is collected, which is the crude enzyme solution.

8. The application according to claim 6, characterized in that, The concentration of the substrate lactose is 100–400 g / L.

9. The application according to claim 6, characterized in that, The enzyme dosage is 16-25 U / g lactose.

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