Levan sucrase as well as mutant, recombinant expression vector and application thereof

Abstract

The invention discloses levan sucrase as well as a mutant, a recombinant expression vector and application thereof, and belongs to the technical field of bioengineering. The amino acid sequence of the fructan sucrase is as shown in SEQ ID NO.2 or SEQ ID NO.4, and the amino acid sequence of the fructan sucrase mutant is as shown in SEQ ID NO.8. The invention further discloses a preparation method of the fructan sucrase mutant. The recombinant expression vector contains the coding gene of the levan sucrase or the mutant of the levan sucrase. The invention also discloses application of the fructan sucrase and the mutant thereof in hydrolysis of cane sugar or synthesis of kestose. The specific activity of the fructan sucrase is 66.17 U / mg, a fructan sucrase mutant with higher enzyme activity and higher thermal stability is obtained after mutation modification of the fructan sucrase, and the specific activity of the fructan sucrase mutant is 169.37 U / mg. The fructan sucrase and the mutant thereof can be used for hydrolysis of cane sugar and synthesis of kestose, and have a good application prospect.

Description

Technical Field

[0001] This invention relates to a fructan sucrase and its mutants, recombinant expression vectors and applications, belonging to the field of bioengineering technology. Background Technology

[0002] Fructotriose (CAS No.: 470-69-9, Molecular Formula: C) 18 H 32 O 16 Fructose (molecular weight: 504.44) is a non-reducing trisaccharide formed by linking sucrose to fructose via β-1,2-glycosidic bonds. It exists in three isomers, including 1-fructosaccharide, and is the smallest molecular weight and most active component among fructooligosaccharides (FOS). Fructose possesses excellent physiological functions, not only specifically promoting the proliferation of beneficial bacteria such as Bifidobacteria and Clostridium perfringens, maintaining intestinal microecological balance, but also promoting T cell differentiation and interferon-γ secretion, enhancing the body's immune regulation capacity, and simultaneously promoting mineral absorption and improving glucose metabolism. As a functional component integrating nutritional and health benefits, it has broad application prospects in the food, health product, and pharmaceutical fields, becoming a current hot topic in prebiotic research.

[0003] Fructose trisaccharides are naturally found in plants and animals such as onions, Jerusalem artichokes, and honey, but their content is low and extraction is difficult. Industrially, they are mainly produced by converting sucrose through fructosyltransferases. Currently, the biosynthesis of fructosyltransferase primarily relies on sucrose: 1-sucrose 1-fructosyltransferase (1-SST), fructosyltransferase (FTase), and β-fructofuranosidase (FFase). Among these, 1-SST has high substrate specificity and is an ideal tool for synthesizing high-purity fructosyltransferase; however, the extraction of enzymes from natural plant sources is difficult and costly. Although heterologous expression systems (such as *Escherichia coli* and *Pichia pastoris*) have been established, they still face bottlenecks such as low expression levels, poor solubility, and the need for expensive inducers. While fungal-derived FTases, widely used industrially, are lower in cost, they suffer from insufficient catalytic specificity and are prone to continuous transglycosylation reactions, generating byproducts such as fructotetrasaccharides and pentasaccharides, leading to difficulties in separating and purifying the target product. Furthermore, existing enzyme preparations generally suffer from poor thermal and acid-base stability, as well as significant substrate and product inhibition, limiting conversion efficiency at high substrate concentrations. While immobilization technology can improve reusability, it is often accompanied by problems such as high mass transfer resistance and low enzyme activity recovery, making it difficult to achieve large-scale industrial application. Therefore, developing fructooligosaccharide synthases with high specificity, high stability, and low cost has become a key technical challenge that urgently needs to be solved in the field of functional oligosaccharides. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a fructan sucrase and its mutants, recombinant expression vectors and applications, belonging to the field of bioengineering technology.

[0005] This invention is achieved through the following technical solution: A fructan sucrase, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.4.

[0006] The encoding gene for the fructan sucrase has a nucleotide sequence as shown in SEQ ID NO.1 or SEQ ID NO.3.

[0007] The application of fructan sucrase in the hydrolysis of sucrose and in the synthesis of sucrose trisaccharides.

[0008] A fructan sucrase mutant, the amino acid sequence of which is shown in SEQ ID NO.8, differs from the fructan sucrase shown in SEQ ID NO.4 in that the amino acid at position 99 is mutated from valine to isoleucine.

[0009] The coding gene of the fructan sucrase mutant has the nucleotide sequence shown in SEQ ID NO.7. The difference between the fructan sucrase gene shown in SEQ ID NO.3 and the gene at position 295-297 is that the codon gtg for valine is mutated to the codon att for isoleucine.

[0010] The application of the fructan sucrase mutant in sucrose hydrolysis and in the synthesis of sucrose trisaccharides.

[0011] A recombinant expression vector containing the fructan sucrase gene shown in SEQ ID NO.3 or the fructan sucrase mutant gene shown in SEQ ID NO.7.

[0012] A recombinant engineered bacterium whose genome contains the fructan sucrase gene shown in SEQ ID NO.3 or the fructan sucrase mutant gene shown in SEQ ID NO.7, and can express the fructan sucrase shown in SEQ ID NO.4 or the fructan sucrase mutant shown in SEQ ID NO.8.

[0013] Furthermore, the host of the recombinant engineered bacteria is *Escherichia coli* (E. coli). Escherichia coli ).

[0014] Application of the recombinant engineered bacteria in the preparation of fructan sucrase or fructan sucrase mutants.

[0015] This invention has identified a novel fructan sucrase, MLLase, from a specific strain. MLLase exhibits high enzyme activity (66.17 U / mg), but its thermostability is not ideal. This invention further mutates MLLase to obtain a mutant fructan sucrase with enhanced activity and higher thermostability (169.37 U / mg). The fructan sucrase and its mutant of this invention can be used for sucrose hydrolysis and the synthesis of fructosaccharides, showing promising application prospects. This invention also prepares a recombinant expression vector and recombinant engineered bacteria containing the coding gene for fructan sucrase or its mutant, enabling heterologous expression of fructan sucrase or its mutant, thus achieving the industrial production of fructan sucrase or its mutant.

[0016] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0017] Figure 1 Electrophoresis diagrams of the fructan sucrase gene and the fructan sucrase mutant gene, where M is the DL5000 nucleic acid marker, lane 1 is the fructan sucrase gene, and lane 2 is the fructan sucrase mutant gene.

[0018] Figure 2 SDS-PAGE electrophoresis images of fructan sucrase and fructan sucrase mutants, where M is the protein molecular weight standard, lane 1 is the supernatant of the blank control, lane 2 is the crude enzyme solution of Experiment 1, lane 3 is the elution buffer of Experiment 1, lane 4 is the crude enzyme solution of Experiment 3, and lane 5 is the elution buffer of Experiment 3.

[0019] Figure 3 Results of the determination of the optimal temperature for fructan sucrase.

[0020] Figure 4 Results of the determination of the optimal pH for fructan sucrase.

[0021] Figure 5 Results of the determination of the temperature stability of fructan sucrase.

[0022] Figure 6 Results of pH stability determination of fructan sucrase.

[0023] Figure 7 TLC image of the products of fructan hydrolysis of sucrose by sucrase. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0025] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0026] The vector pET-29a used in this embodiment of the invention is from Invitrogen; Escherichia coli Escherichia coli BL21 (DE3) was obtained from Invitrogen; restriction endonucleases were obtained from Takara; and the seamless cloning kit was obtained from Nanjing Novizan Pharmaceutical Co., Ltd.

[0027] This invention is based on a self-isolated strain of microbacterium ( Microbacterium A gene segment was discovered in the genome of sp. B42. After genome draft sequencing and bioinformatics analysis, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO.2. It is predicted that this protein may possess fructan sucrase activity; therefore, this protein is named fructan sucrase MLLSase. This invention uses PCR technology to amplify this gene, performs heterologous expression, and studies its characteristics, as detailed below.

[0028] The nucleotide sequence of the fructan sucrase gene is shown in SEQ ID NO.1, as shown below (direction 5'-3'):

[0029] The amino acid sequence of fructan sucrase MLLSase is shown in SEQ ID NO.2, as follows: .

[0030] The fructan sucrase MLLSase has a signal peptide (containing 37 amino acid residues) at its N-terminus. During cloning, this signal peptide sequence was removed. The nucleotide sequence of the fructan sucrase gene after signal peptide removal is shown in SEQ ID NO.3, as shown below (direction 5'-3'):

[0031] After removing the signal peptide sequence, the amino acid sequence of fructan sucrase MLLSase is shown in SEQ ID NO.4, as follows: ADEIDAVPGHPAPTVHTQQAYDPAADFTARWTRADARQIAAMSDPTAAPRQNSLPEEYTMPTVPQDFPDMSNDQVWVWDSWTLTDETAAQPSFKGWEVVFSLVADRRLGFDDRHTFARLGYFFRK ADVAERPADGGWTYGGLVFPDGASGAIFEDQSFSHQTEWSGSTRIFDGNKLRIFYTAVAFYRNEDGSNRKPYDPRIVQSEGRIFADENGVWLTGFRDQHDMLRADGTYYQTGAQNEFFNFRDPFT FEDPHPGKTFMVFEGNTAAPRGERVCTEEDMGYRTGDPYAETAAEVMDRGAHYQLANVGLAVADNAALTEWTFLPPILSANCVNDQTERPQIYIKDGKYYLFTITHRGTYAAGVDGPEGVYGFV GDGIRSDFQPVNRGSGLALASPSNLNFAAGTPFAPDVNQHPGQFQAYSHYVMPGGLVQSFIDTIGTSDDFVRGGTLAPTVKVDIDGSSVTVDRAYGDNGLGQWADIPSGYAHDVPATADVRPTR.

[0032] Example 1: Cloning of the fructan sucrase gene and construction of a recombinant expression vector PCR amplification of S1, fructan sucrase gene S11. The gene shown in SEQ ID NO.3 was artificially synthesized by the Hunan Branch of Beijing Qingke Biotechnology Co., Ltd.; based on the sequence of the fructan sucrase gene, specific primers were designed. During the cloning process, the signal peptide sequence was removed when designing the primers.

[0033] The nucleotide sequence of upstream primer F1 is shown in SEQ ID NO.5, as shown below (direction 5'-3'): tccatggctgatatcggatccgagatcgacgccgttcc.

[0034] The nucleotide sequence of the downstream primer R1 is shown in SEQ ID NO.6, as shown below (direction 5'-3'): ctcgagtgcggccgcaagcttccgcgtcggacgcac.

[0035] S12. Using the specific primers designed above, and the artificially synthesized gene fragments described above as templates, perform PCR amplification.

[0036] The PCR reaction system consisted of: 1.0 μL total DNA, 1.0 μL each of primers F1 and R1, and PrimeSTAR. ® Max DNAPolymerase 25.0 μL, add ddH2O to bring the total volume of the system to 50.0 μL.

[0037] The PCR amplification conditions were as follows: pre-denaturation at 98℃ for 5 min; denaturation at 98℃ for 30 s; annealing at 60℃ for 30 s; extension at 72℃ for 2 min; a total of 30 cycles were performed; and a final extension at 72℃ for 10 min was performed.

[0038] The amplified product is the fructan sucrase gene, denoted as MLLSase (wild-type). The amplified product was subjected to agarose gel electrophoresis; the electrophoresis image is shown below. Figure 1 As shown.

[0039] S2. Construction and validation of the recombinant vector pET-29a-MLLSase (wild type) S21. The vector pET-29a was double-digested to obtain the double-digested product; the double-digestion system was as follows: Hind III, 1.0 μL; BamH Ⅰ, 1.0 μL; 10×K Buffer, 5.0 μL; pET-29a, 30.0 μL; ddH2O, 13.0 μL.

[0040] S22. The double enzyme digestion products were recovered using a DNA agarose gel extraction kit, and the recovered products were cloned. The cloning system was: 6.0 μL of the MLLSase (wild-type) PCR product obtained in S12, 2× Hieff Clone ® EnzymePremix 10 μL; cloning reaction conditions: react at 50 °C for 30 min, then cool on ice for 2 min to obtain the cloning product.

[0041] S23, The cloned product is converted to [a specific process] via thermal shock. E. coli DH5α competent cells: Add the cloning product to 100 μL of melted [material / solution]. E. coli DH5α competent cells were incubated on ice for 30 min; after the ice bath, they were transferred to a 42°C water bath for 90 s heat shock, followed by cooling on ice for 5 min; after cooling, 800 μL of liquid LB medium was added, and the cells were incubated at 37°C and 180 rpm for 60 min on a shaker.

[0042] S24. After incubation, centrifuge at 600 rpm for 3 min, take 100 μL of competent cells and spread them on LB agar plates containing 50 mg / L kanamycin. Invert the plates and incubate at 37℃ for 13 h.

[0043] S25. Extract multiple single colonies from the cultured plate and inoculate them into test tubes containing 4 mL of liquid LB medium. Incubate at 37°C and 180 rpm for 12 h. After incubation, extract plasmids using a plasmid mini-extraction kit.

[0044] S26. Perform double enzyme digestion on the plasmid extracted in step S25; the double enzyme digestion system is as follows: Hind III, 1.0 μL; BamH Ⅰ, 1.0 μL; 10×K Buffer, 5.0 μL; pET-29a-MLLSase, 30.0 μL; ddH2O, 13.0 μL.

[0045] S27. The obtained recombinant plasmid was verified and sent to Hunan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results showed that the pET-29a vector contained the cloned target fragment, which was 1500 bp in length. The nucleotide sequence was shown in SEQ ID NO.3, and the amino acid sequence of the fructan sucrase it expressed was shown in SEQ ID NO.4.

[0046] Expression and purification of S3, fructan sucrase S31. The plasmid pET-29a- obtained in step S25 is... MLLSase (Wild-type) heat shock transformation to host bacteria E. coli Recombinant engineered bacteria were obtained from BL21(DE3).

[0047] S32. Inoculate a single positive clone into 4 mL of LB medium (containing 50 μg / mL kanamycin) and incubate at 37°C and 180 rpm for 12 h with shaking. After incubation, extract plasmids using a plasmid mini-extraction kit and verify them.

[0048] S33. After verification, inoculate the bacterial culture from S32 into 50 mL of LB medium and culture at 37°C and 180 rpm for 8 h with shaking. After the culture is completed, transfer the culture to 400 mL of LB medium with a 1% inoculation amount and culture at 37°C and 180 rpm for 3.5 h with shaking.

[0049] S34, OD Measurement 600 When the concentration reached 0.4–0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a concentration of 2 mmol / L, and expression was induced at 16°C and 180 rpm for 24 h.

[0050] S35. After induction of expression, the culture medium was centrifuged at 5000 rpm for 0.5 h, and the bacterial cells were collected.

[0051] S36. Resuspend and wash the bacterial cells with PBS buffer (20 mmol / L, pH 6.5), centrifuge at 5000 rpm for 0.5 h, and collect the bacterial cells.

[0052] S37. Under ice bath conditions, add the bacterial cells to PBS buffer (20 mmol / L, pH 6.5) and use an ultrasonic cell disruptor at 400W for 3 seconds, with a 7-second interval.

[0053] S38. After the disruption is complete, centrifuge at 4℃ and 12000rpm for 1 hour. The supernatant is the crude enzyme solution of fructan sucrase. Simultaneously, using the empty vector pET-29a as a blank control, perform the above operation to obtain the supernatant of the blank control.

[0054] S39. Purification of the crude enzyme solution using a Ni-NTA affinity chromatography column: After loading the sample, perform gradient elution with imidazole solutions of varying concentrations, collecting the eluent corresponding to each 100 mmol / L imidazole solution; the eluent is then purified using an Amicon chromatography column. ® Ultra (3kDa MWCO) filter, centrifuged at 5000 rpm for 0.5 h to increase the concentration of the purified product, finally obtaining a purified and concentrated fructan sucrase enzyme solution with a concentration of 1.62 mg / ml.

[0055] Experiment 1 Characterization of fructan sucrase (1) The crude enzyme solution and eluent prepared in Example 1 were subjected to SDS-PAGE electrophoresis. The electrophoresis diagram is shown below. Figure 2 As shown, the molecular weight of fructan sucrase is approximately 58.88 kDa, consistent with the prediction.

[0056] Enzyme activity assay: Prepare a 500 μL reaction system by adding 10 μL of the purified enzyme solution prepared in Example 1 to a PBS buffer (20 mmol / L, pH 6.5) with a sucrose concentration of 100 g / L. Incubate the buffer system at 20°C for 20 min. After the reaction, place the reaction solution in a boiling water bath for 10 min to terminate the reaction. The reducing sugar and glucose generated using the glucose kit (source) in the reaction system were determined using the DNS method. Relative enzyme activity and total enzyme activity were calculated based on the amount of reducing sugar and glucose generated.

[0057] Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of glucose per minute under optimal conditions, which is defined as one unit of enzyme activity (U).

[0058] The specific activity of fructan sucrase MLLSase was determined to be 66.17 U / mg, the Km value was 125.05 mmol / L, and the Vmax value was 520.85 μmol / (L·min).

[0059] (2) Determination of the optimal temperature Prepare a 500 μL reaction system: Add 10 μL of the pure enzyme solution prepared in Example 1 to a PBS buffer (20 mmol / L, pH 6.5) with a sucrose concentration of 100 g / L.

[0060] The above reaction system was subjected to a water bath reaction at different temperatures (5℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃) for 20 min. After the reaction was completed, an equal volume of dinitrosalicylic acid (DNS) was added to terminate the reaction. The absorbance at 540 nm was measured, and three parallel experiments were designed for each temperature. The average value of the results was taken.

[0061] The results showed that the highest absorbance was observed at 20℃, indicating the highest enzyme activity at this temperature. With the enzyme activity at 20℃ set as 100% relative activity, the relative activities at other temperatures were calculated. The optimal temperature for fructan sucrase was determined as follows: Figure 3 As shown, the relative activity is highest at 20℃, and decreases sharply with increasing temperature, reaching less than 5% at 35℃.

[0062] (3) Determination of optimal pH Preparation of a 500 μL buffer system: 10 μL of the pure enzyme solution prepared in Example 1 was added to different pH buffer solutions with a sucrose concentration of 100 g / L. The pH buffer solutions used were divided into five groups: Group 1, citric acid-sodium citrate aqueous solution with a pH of 3.5–5.5; Group 2, sodium acetate aqueous solution with a pH of 5.5–6.5; Group 3, Na2HPO4-NaH2PO4 aqueous solution with a pH of 6.0–8.0; Group 4, Tris-HCl aqueous solution with a pH of 8.0–9.0; and Group 5, Glycine-NaOH aqueous solution with a pH of 9.0–10.0.

[0063] The above buffer system was reacted in a 20℃ water bath for 20 min. After the reaction was completed, an equal volume of DNS was added to terminate the reaction. The absorbance at 540 nm was measured, and three parallel experiments were designed for each pH value. The average value of the results was taken.

[0064] The results showed that the highest absorbance was observed in a Na₂HPO₄-NaH₂PO₄ aqueous solution at pH 6.5, indicating the highest enzyme activity at this pH. Setting this enzyme activity as 100% relative activity, the relative activities in other pH buffer systems were calculated. The optimal pH for fructan sucrase was determined as follows: Figure 4 As shown, the relative activity is low under both acidic and alkaline conditions.

[0065] (4) Determination of temperature stability The pure enzyme solution prepared in Example 1 was incubated at different temperatures (20℃, 30℃, 40℃, 50℃, 60℃) for 1h, 3h, 5h, 7h, 9h, and 24h. No incubation treatment was used as a control.

[0066] To prepare a 500 μL reaction system, add 10 μL of the pure enzyme solution that has been incubated at different temperatures to a PBS buffer (20 mmol / L, pH 6.5) with a sucrose concentration of 100 g / L.

[0067] The above reaction system was reacted in a water bath at 20℃ for 20 min. After the reaction was completed, an equal volume of DNS was added to terminate the reaction. The absorbance at 540 nm was measured. Three parallel experiments were designed for each incubation condition, and the average value of the results was taken.

[0068] The enzyme activity without incubation (i.e., treatment time of 0 h) was set as 100% relative activity. The relative activities after incubation at other temperatures for different times were calculated. The results of the temperature stability determination of fructan sucrase are as follows: Figure 5 As shown, after incubation at 20℃ and 30℃ for 1 to 9 hours, the residual activity was relatively high, exceeding 70% after 9 hours. With increasing incubation temperature, the residual activity decreased sharply: after 5 hours of incubation at 40℃, the residual activity dropped below 50%; after 3 hours of incubation at 50℃, the residual activity dropped below 45%; after 5 hours of incubation at 50℃, the residual activity dropped below 8%; after 1 hour of incubation at 60℃, the residual activity dropped below 30%; and after 3 hours of incubation at 60℃, the residual activity dropped below 10%.

[0069] (5) Determination of pH stability Prepare a 500 μL buffer system: Add 10 μL of the pure enzyme solution prepared in Example 1 to different pH buffers and incubate at 20°C for 24 h. The pH buffers used are divided into five groups: Group 1, citric acid-sodium citrate aqueous solution with pH 5.0–5.5; Group 2, sodium acetate aqueous solution with pH 5.5–6.5; Group 3, Na2HPO4-NaH2PO4 aqueous solution with pH 6.0–8.0; Group 4, Tris-HCl aqueous solution with pH 8.0–9.0; and Group 5, Glycine-NaOH aqueous solution with pH 9.0–10.0.

[0070] Add sucrose to the buffer system to a concentration of 100 g / L and react for 20 min in a 20°C water bath. After the reaction is complete, add an equal volume of DNS to terminate the reaction. Measure the absorbance at 540 nm. Design three parallel experiments for each pH value, and take the average value.

[0071] The enzyme activity after incubation in a Na₂HPO₄-NaH₂PO₄ aqueous solution at pH 6.5 for 0 h was set as 100% relative activity. The relative activities after incubation in each pH buffer system for 24 h were calculated. The pH stability of fructan sucrase was determined as follows: Figure 6 As shown, it exhibits excellent stability in the pH range of 6.5 to 8.0, and the residual activity remains above 90% after 24 hours of incubation.

[0072] Experiment 2: Fructan sucrase hydrolysis of sucrose to synthesize fructotriose and levulin (1) Qualitative analysis of hydrolysis products: Prepare a 500 μL reaction system, add 10 μL of the pure enzyme solution prepared in Example 1 to PBS buffer (20 mmol / L, pH 6.5) with a sucrose concentration of 700 g / L, and react for 20 min in a water bath at 20 °C. After the reaction is complete, boil for 10 min to terminate the reaction. Centrifuge at 12000 rpm for 10 min and collect the supernatant.

[0073] Thin-layer silica gel chromatography was used to analyze the reaction products. A suitable amount of sample was pipetted into a 1 mL EP tube, and the sample was gently spotted onto a labeled TLC silica gel plate using a capillary tube, with the spot diameter controlled within 2 mm. Simultaneously, the sample on the plate was dried using a blower. After spotting, the TLC silica gel plate was placed in a development tank containing a pre-prepared developing solvent of n-butanol:methanol:water (4:2:1, volume ratio). Once the liquid level reached the mark line, the plate was removed, dried, and sprayed with a colorimetric reagent (sulfuric acid:methanol = 1:9, volume ratio). The plate was then placed in a 100℃ oven until color development was achieved.

[0074] TLC images of the products of fructan sucrase hydrolysis of sucrose are shown below. Figure 7As shown in the figure, the left side represents the hydrolysis products, and the right side represents the standard monosaccharides. It is evident that after fructan sucrase hydrolyzes sucrose, the main product is fructotriose.

[0075] (2) HPLC was used to detect sucrose hydrolysis products for quantitative analysis. The separator used was a Waters 2695e, the detector was a 2414 RID, and the liquid chromatography column was a Cosmosilsugar-D column, 4.6 × 250 mm. The test conditions were: mobile phase acetonitrile:water = 70:30 (volume ratio), flow rate 1 mL / min, column temperature 30℃, injection volume 20 μL, and sample run time 15 min. A fructotriose standard was prepared, and a standard curve was plotted to determine the fructotriose content in the sample.

[0076] The experimental results showed that the yield of fructooligosaccharides was 224 g / L, with a yield rate of 32%.

[0077] (3) Quantitative analysis of levofloxacin products: Prepare a 20 mL reaction system, add 600 μg of pure enzyme solution to PBS buffer (20 mmol / L, pH 6.5) with a sucrose concentration of 700 g / L to make the final enzyme concentration 30 μg / mL, and react for 48 h in a water bath at 20 °C. After the reaction is complete, boil for 10 min to terminate the reaction. After cooling, add 20 mL of Sevag reagent (chloroform and n-butanol mixed at a volume ratio of 4:1) to the reaction system and stir thoroughly on a magnetic stirrer for 30 min. After standing and separating the layers, discard the lower organic phase. Repeat this step three times. Place the upper polysaccharide solution in a fume hood and stir magnetically overnight to allow the organic reagent to fully evaporate. Add 4 times the volume of ethanol, stand at 4 °C to precipitate the polysaccharide, and centrifuge at 12000 r / min for 25 min at 4 °C. Discard the supernatant. The polysaccharide precipitate was dissolved in water and transferred to a cellulose ester membrane (CE) dialysis bag with a molecular weight cutoff of 1000 Da. Dialysis was performed with distilled water at 4°C for 48 hours, with the water changed every 8 hours. The dialyzed polysaccharide solution was then frozen at -80°C and freeze-dried under vacuum to obtain a levodiamine sample powder with a yield of 81.1 g / L.

[0078] Experiment 3 Mutational modification of fructan sucrase The results of Experiment 1 show that fructan sucrase has poor thermal stability. To improve the thermal stability of fructan sucrase, this invention attempts to modify it through mutation. Using the online tool of the HotSpo Twizard website to predict sites related to catalytic activity and thermal stability, the valine residue at position 99 (codon gtg) of the fructan sucrase shown in SEQ ID NO.4 was selected and mutated at a specific site to isoleucine (codon att).

[0079] Based on the above-mentioned mutation modification of the amino acid sequence, primers for the mutation site were designed. Using the fructan sucrase gene shown in SEQ ID NO.3 as a template, PCR amplification of the fructan sucrase mutation site was performed to obtain the coding gene of the mutant. Then, the recombinant expression vector of the mutant was constructed according to the method in Example 1, and expressed and purified. The electrophoresis diagram of the fructan sucrase mutant gene is shown below. Figure 1 As shown, the SDS-PAGE electrophoresis image of the fructan sucrase mutant is as follows: Figure 2 As shown.

[0080] The enzyme activity of the fructan sucrase mutant V99I was determined. The results showed that the specific activity of V99I was 169.37 U / mg, which was significantly higher than that of wild-type fructan sucrase. The Km value of V99I was 36.68 mmol / L, and the Vmax value was 519.57 μmol / (L·min). Compared with wild-type fructan sucrase, the Km value was significantly decreased, indicating a significantly improved affinity for the substrate.

[0081] The temperature stability of the mutant pure enzyme solution was determined using the same method as in Experiment 1 (4), except that the mutant pure enzyme solution was incubated at 40℃ for 5 hours. Results: After incubation at 40℃ for 5 hours, the residual activity of wild-type fructan sucrase was 44%, and the residual activity of the fructan sucrase mutant V99I was 73%, showing a significant increase.

[0082] Conclusion: Through mutation modification, this invention obtained a fructan sucrase mutant V99I with stronger enzyme activity and higher thermal stability, which is officially named fructan sucrase MLLase-V99I.

[0083] The amino acid sequence of fructan sucrase MLLase-V99I is shown in SEQ ID NO.8, as follows: ADEIDAVPGHPAPTVHTQQAYDPAADFTARWTRADARQIAAMSDPTAAPRQNSLPEEYTMPTVPQDFPDMSNDQVWVWDSWTLTDETAAQPSFKGWEVIFSLVADRRLGFDDRHTFARLGYFFRK ADVAERPADGGWTYGGLVFPDGASGAIFEDQSFSHQTEWSGSTRIFDGNKLRIFYTAVAFYRNEDGSNRKPYDPRIVQSEGRIFADENGVWLTGFRDQHDMLRADGTYYQTGAQNEFFNFRDPFT FEDPHPGKTFMVFEGNTAAPRGERVCTEEDMGYRTGDPYAETAAEVMDRGAHYQLANVGLAVADNAALTEWTFLPPILSANCVNDQTERPQIYIKDGKYYLFTITHRGTYAAGVDGPEGVYGFV GDGIRSDFQPVNRGSGLALASPSNLNFAAGTPFAPDVNQHPGQFQAYSHYVMPGGLVQSFIDTIGTSDDFVRGGTLAPTVKVDIDGSSVTVDRAYGDNGLGQWADIPSGYAHDVPATADVRPTR.

[0084] The nucleotide sequence of the gene encoding fructan sucrase MLLase-V99I is shown in SEQ ID NO.7, as shown below (direction 5'-3'):

[0085] Experiment 4 Synthesis of fructotriose using fructan sucrase MLLase-V99I Using sucrose as a substrate, fructosaccharide was synthesized under the action of fructan sucrase MLLase-V99I. The product was quantitatively analyzed by HPLC, following the same method as in Experiment 2. The results showed that the yield of fructosaccharide was 203 g / L, with a yield of 29%.

[0086] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A fructan sucrase, characterized in that: The amino acid sequence is shown in SEQ ID NO.2 or SEQ ID NO.

4.

2. The gene encoding fructan sucrase according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.

3.

3. The application of the fructan sucrase of claim 1 in the hydrolysis of sucrose, or in the synthesis of fructosaccharides.

4. A fructan sucrase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

8.

5. The encoding gene of the fructan sucrase mutant according to claim 4, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

7.

6. The application of the fructan sucrase mutant according to claim 4 in the hydrolysis of sucrose, or in the synthesis of sucrose trisaccharides.

7. A recombinant expression vector, characterized in that: Contains the fructan sucrase gene shown in SEQ ID NO.3 or the fructan sucrase mutant gene shown in SEQ ID NO.

7.

8. A recombinant engineered bacterium, characterized in that: The genome of the recombinant engineered bacteria contains the fructan sucrase gene shown in SEQ ID NO.3 or the fructan sucrase mutant gene shown in SEQ ID NO.7, and can express the fructan sucrase shown in SEQ ID NO.4 or the fructan sucrase mutant shown in SEQ ID NO.

8.

9. The recombinant engineered bacteria according to claim 8, characterized in that: The host of the recombinant engineered bacteria is Escherichia coli.

10. The use of the recombinant engineered bacteria according to claim 8 or 9 in the preparation of the fructan sucrase according to claim 1 or the fructan sucrase mutant according to claim 4.

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