A beta-fructofuranosidase mutant

Abstract

The application provides a mutant D110N / R112I of a beta-furan fructosidase, a gene sequence and an amino acid sequence of the mutant enzyme are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence table respectively. The application also provides a recombinant expression vector and an engineering strain containing the gene sequence of the mutant enzyme. The mutant D110N / R112I can effectively solve the technical problems of insufficient catalytic activity of the existing natural beta-furan fructosidase and low yield of oligomeric lactulose synthesis. Compared with the wild-type enzyme, the yield of oligomeric lactulose synthesis of the mutant is increased by 42.8% under the same catalytic conditions, which significantly improves the catalytic efficiency and product yield, effectively reduces the reaction energy consumption and production cost, and has high industrial technology value and good industrial application prospect in the enzymatic synthesis process of oligomeric lactulose.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a β-fructofuranosidase mutant and its applications. Background Technology

[0002] Lactulose oligosaccharide is a naturally sourced sweetener with a sweetness approximately 30% that of sucrose, making it a good alternative to sucrose. It possesses various physiological functions, including regulating gut microbiota, promoting calcium absorption, and assisting in lowering blood lipids. As a novel functional oligosaccharide, it has attracted widespread attention from the food and pharmaceutical industries.

[0003] Currently, lactulose oligosaccharides are mainly biosynthesized using sucrose and lactose as substrates via β-galactosidase, β-fructofuranosyltransferase, or fructanosucase. Among these, β-fructofuranosyltransferase derived from Arthrobacterium has been studied in greater depth. This enzyme hydrolyzes sucrose to produce fructose groups, which are then transferred to the reducing terminal C1 atom of lactose to generate lactulose oligosaccharides.

[0004] However, natural β-fructofuranosyltransferases suffer from low activity and poor stability during the catalytic synthesis of lactulose oligosaccharides. With the continuous development of technologies such as protein structure simulation, combining molecular docking and molecular dynamics simulations for rational design and targeted modification of enzymes is an effective means to improve their catalytic efficiency and stability. Therefore, key modifications to β-fructofuranosyltransferases to significantly enhance their catalytic activity are crucial for the large-scale production of lactulose oligosaccharides using more mature enzymatic processes in the future. Summary of the Invention

[0005] The purpose of this invention is to provide a β-fructofuranosidase (FTase) mutant that can efficiently catalyze the synthesis of oligo-lactulose.

[0006] The specific technical solution of the present invention is as follows:

[0007] This invention provides a mutant of β-fructofuranosidase (FTase), D110N / R112I, the gene sequence of which is shown in SEQ ID NO:1 and the amino acid sequence of which is shown in SEQ ID NO:2. This mutant was obtained by site-directed mutagenesis of wild-type FTase (Genbank ID: KUM30232.1) derived from Arthrobacter sp. EpRS66, specifically by mutating the 110th amino acid residue of the catalytic active site from aspartic acid (Asp) to asparagine (Asn) and the 112th amino acid residue from arginine (Arg) to isoleucine (Ile).

[0008] Furthermore, the present invention provides a recombinant expression vector comprising the gene sequence shown in SEQ ID NO:1. Preferably, the recombinant expression vector is a pET-28a vector.

[0009] Furthermore, the present invention provides an engineered bacterium containing the above-mentioned recombinant expression vector. Preferably, the engineered bacterium uses *Escherichia coli* as the host cell.

[0010] Furthermore, the present invention provides the application of the aforementioned FTase mutant D110N / R112I in the enzyme-catalyzed synthesis of lactulose oligosaccharides.

[0011] Furthermore, the present invention provides a method for in vitro enzymatic synthesis of lactulose oligosaccharides using the aforementioned FTase mutant D110N / R112I, characterized in that: the mutant D110N / R112I is added to a reaction system containing sucrose and lactose, and the reaction is carried out at pH 7.0-8.0 and 35-45℃ for 3-5 hours. After the reaction is completed, the enzyme is inactivated, and the supernatant is collected by centrifugation to obtain the product containing lactulose oligosaccharides.

[0012] Preferably, in the above-described in vitro enzymatic synthesis of lactulose oligosaccharides, the final concentration of the mutant D110N / R112I in the reaction system is 0.3–0.5 μM; the mass concentration of sucrose in the reaction system is 25–35%; and the mass concentration of lactose in the reaction system is 15–25%.

[0013] More preferably, in the above-described in vitro enzymatic synthesis method for lactulose oligosaccharide, the final concentration of the mutant D110N / R112I in the reaction system is 0.4 μM; the mass concentration of sucrose in the reaction system is 30%; the mass concentration of lactose in the reaction system is 20%; the pH of the reaction system is 7.4; the reaction temperature is 40°C; and the reaction time is 4 h.

[0014] This invention obtains a double mutant of β-fructofuranosidase, D110N / R112I, through site-directed mutagenesis, solving the technical problems of insufficient catalytic activity and low yield of lactulose oligosaccharide synthesis in existing natural β-fructofuranosidases. Compared with the wild-type enzyme, the mutant of this invention increases the yield of lactulose oligosaccharide synthesis by more than 42% under the same catalytic conditions, significantly improving catalytic efficiency and product yield, while effectively reducing reaction energy consumption and production costs. The β-fructofuranosidase mutant D110N / R112I of this invention has high industrial technology value and good industrial application prospects in the enzymatic synthesis process of lactulose oligosaccharide. Attached Figure Description

[0015] Figure 1HPLC identification results of in vitro enzyme-catalyzed reaction products of wild-type FTase and FTase-D110N / R112I mutant. Detailed Implementation

[0016] The method of the present invention is described below through specific implementation schemes. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art.

[0017] Example 1

[0018] Obtaining the FTase-D110N / R112I mutant:

[0019] 1) Construction of wild-type gene recombinant expression vector: The β-fructofuranosidase gene sequence from Arthrobacter sp. EpRS66 was synthesized and ligated into the pET vector to obtain a recombinant expression vector containing the wild-type FTase gene. The pET-28a vector is preferred for this step.

[0020] 2) Site-directed mutagenesis: Site-directed mutagenesis PCR amplification was performed using a wild-type recombinant vector as a template. Mutations were performed on two amino acid sites in the enzyme's amino acid sequence using designed site-directed mutagenesis primers: the 110th amino acid residue in the catalytic active site was mutated from Asp to Asn, and the 112th amino acid residue was mutated from Arg to Ile. The corresponding mutagenesis primers D110N / R112I-F and D110N / R112I-R are shown in Table 1, and their specific nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. The PCR amplification reaction system is shown in Table 2, and the reaction conditions and procedures are shown in Table 3.

[0021] Table 1. Site-directed mutagenesis primers

[0022]

[0023] Table 2 Site-directed mutagenesis PCR reaction system

[0024]

[0025] Table 3 PCR reaction conditions and process

[0026]

[0027] The PCR product after the reaction was digested with 0.35 μL of Dpn I enzyme to remove the template, and purified and recovered after incubation at 37 ℃ for 1 h. The purified and recovered target product was then ligated (cyclized), and the ligation reaction system is shown in Table 4.

[0028] Table 4. Linkage reaction system of Dpn I target product

[0029]

[0030] 3) Transformation and Sequencing Validation: 10 μL of the circularized product was transformed into *E. coli* JM109 competent cells using the heat shock method. After resuscitation and culture, the cells were plated and cultured overnight. Once transformants emerged, they were picked and placed in LB broth containing kanamycin. After culturing at 37 °C and 220 rpm / min for 12 h, the plasmid was extracted for sequencing validation. The plasmid that was validated correctly was the recombinant expression vector containing the FTase-D110N / R112I mutant gene sequence (SEQ ID NO:1).

[0031] 4) Induction of expression: The recombinant expression vector containing the FTase-D110N / R112I mutant gene sequence was transformed into *E. coli* BL21(DE3) competent cells via heat shock. After resuscitation and culture, the cells were plated and cultured overnight. Once transformants appeared, they were picked and placed in 5 mL of LB broth containing 50 μg / mL kanamycin in test tubes. The cells were cultured at 37°C and 220 rpm for 12 h, followed by expansion culture in 1% roller bottles. The bacterial concentration was increased to OD500. 600 After reaching a concentration of 0.6–0.8, IPTG was added to a final concentration of 0.5 mM and the mixture was induced and cultured at 16 °C and 200 rpm / min for 18 h.

[0032] 5) Enzyme extraction and purification: After induction culture, the bacterial culture was centrifuged at 4000 rpm at 4 ℃ to collect the bacterial cells. The bacterial cells were then resuspended in Lysis Buffer. 1% lysozyme and PMSF were added according to the volume of Lysis Buffer used (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, pH=7.4). The cells were stirred and resuspended for 15–20 min with a magnetic stirrer, then sonicated. The sonicated bacterial cells were centrifuged at 12000 rpm at 4 ℃ for 30 min, and the supernatant was collected. The supernatant was then mixed with resin equilibrated with Lysis Buffer and stirred for 40 min. After the supernatant and resin were fully combined, the mixture was passed through a Ni-NTA affinity chromatography column. The resin was then washed with 20 mL Wash Buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, pH=7.4) to elute impurities. Finally, 10 mL Elution was used. The resin was rinsed with a buffer (20 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 500 mM imidazole, pH 7.4) to elute the target protein. Finally, the eluent containing the target protein was replaced with PBS buffer at pH 7.4 to obtain the FTase-D110N / R112I mutant, whose amino acid sequence is shown in SEQ ID NO:2.

[0033] Example 2

[0034] In vitro enzyme-catalyzed reactions of wild-type FTase and FTase-D110N / R112I mutant.

[0035] The wild-type FTase was obtained by following the steps in Example 1 above: using a recombinant expression vector containing the wild-type FTase gene as material, the site-directed mutagenesis step was omitted, and the FTase was obtained directly after transformation, induced expression, and Ni-NTA affinity chromatography purification.

[0036] The following in vitro enzyme-catalyzed reactions were performed using wild-type FTase and the FTase-D110N / R112I mutant as test enzymes:

[0037] The total volume of the in vitro enzyme-catalyzed reaction system was 10 mL, which was made up with PBS buffer at pH 7.4. The final concentration of the enzyme in the system was 0.3–0.5 μM, the sucrose concentration was 30%, and the lactose concentration was 20%. After reacting at 40 °C for 4 h, the enzyme was inactivated by heating in a boiling water bath for 10 min, followed by centrifugation at 12000 rpm for 5 min. The supernatant was used for subsequent high-performance liquid chromatography (HPLC) detection.

[0038] Example 3

[0039] The catalytic reaction products of wild-type FTase and the FTase-D110N / R112I mutant obtained in Example 2 were taken separately, filtered, and then identified by HPLC. The HPLC detection conditions were as follows:

[0040] Chromatograph: Agilent 1260;

[0041] Detector: Differential refractive index detector (Shimadzu 10A);

[0042] Injection volume: 10 μL;

[0043] Column: Caprisil NH2-H 5μm 100Å (250×4.6 mm);

[0044] Column temperature: 40℃;

[0045] Mobile phase: acetonitrile:water = 75:25 (v / v), flow rate: 1 mL / min.

[0046] The results of the identification are as follows Figure 1 As shown: In the chromatograms of the catalytic reaction products of wild-type FTase and mutant D110N / R112I, chromatographic peaks appeared at a retention time of approximately 13.8 min. This retention time is consistent with that of the lactulose oligosaccharide standard, indicating that the catalytic reaction product of both enzymes is lactulose oligosaccharide.

[0047] The quantitative determination results of the products catalyzed by the two enzymes are shown in Table 5. Under the same reaction conditions and time, the yield of wild-type FTase catalyzing the synthesis of lactulose oligosaccharide was 132.8 g / L, while the yield of FTase-D110N / R112I mutant reached 189.6 g / L, which was 42.8% higher than that of wild-type. This indicates that the catalytic efficiency of mutant D110N / R112I enzyme is significantly improved compared with wild-type.

[0048] Table 5. Comparison of yields of lactulose oligosaccharides synthesized by FTase and FTase-D110N / R112I catalysts

[0049]

Claims

1. A β-fructofuranosidase mutant D110N / R112I, characterized in that... The gene sequence is shown in SEQ ID NO:

1.

2. The mutant D110N / R112I as described in claim 1, characterized in that... The amino acid sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that... It contains the gene sequence shown in SEQ ID NO:

1.

4. The recombinant expression vector as described in claim 3, characterized in that: The recombinant expression vector is the pET-28a vector.

5. An engineered bacterium, characterized in that... It includes the recombinant expression vector as described in claim 3.

6. The engineered bacteria as described in claim 5, characterized in that: The engineered bacteria use Escherichia coli as the host cell and contains the recombinant expression vector as described in claim 4.

7. The application of the mutant D110N / R112I as described in claim 1 or 2 in the enzyme-catalyzed synthesis of lactulose oligosaccharides.

8. A method for in vitro enzymatic synthesis of lactulose oligosaccharides using the mutant D110N / R112I as described in claim 1 or 2, characterized in that: The mutant D110N / R112I was added to a reaction system containing sucrose and lactose and reacted at pH 7.0–8.0 and 35–45°C for 3–5 hours. After the reaction was completed, the enzyme was inactivated, and the supernatant was collected by centrifugation to obtain the product containing lactulose oligosaccharides.

9. The method for in vitro enzyme-catalyzed synthesis of lactulose oligosaccharides as described in claim 8, characterized in that: The final concentration of the mutant D110N / R112I in the reaction system is 0.3–0.5 μM; the mass concentration of sucrose in the reaction system is 25–35%; and the mass concentration of lactose in the reaction system is 15–25%.

10. The method for in vitro enzyme-catalyzed synthesis of lactulose oligosaccharides as described in claim 8, characterized in that: The final concentration of the mutant D110N / R112I in the reaction system was 0.4 μM; the mass concentration of sucrose in the reaction system was 30%; the mass concentration of lactose in the reaction system was 20%; the pH of the reaction system was 7.4; the reaction temperature was 40℃; and the reaction time was 4 h.

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