Construction and application of a beta-(2,6) fructosaccharase mutant

Abstract

The application discloses construction and application of a beta-(2,6) fructosaccharose sucrase mutant, and belongs to the technical field of enzyme genetic engineering. The beta-(2,6) fructosaccharose sucrase from a microorganism Pseudomonas orientalis is used as a parent to construct a single-point mutant enzyme R91T. The mutant R91T has obviously improved the ability of catalyzing sucrose polymerization, and the conversion rate of 20% sucrose and 20% lactose into oligomeric lactulose reaches 40% within 1 hour. Meanwhile, the mutant can convert 30% sucrose into high-molecular-weight fructan, and the conversion rate reaches 49%. The finding has important research value for industrialized preparation of oligomeric lactulose and industrialized application of the beta-(2,6) fructosaccharose sucrase.

Description

Technical Field

[0001] This invention relates to the construction and application of a β-(2,6)fructan sucrase mutant, belonging to the field of enzyme genetic engineering technology. Background Technology

[0002] Lactosucrose, a novel trisaccharide composed of fructose, glucose, and galactosyl groups, can be synthesized enzymatically from sucrose and lactose through transglycosylation. Because it cannot be digested and utilized by the human body but can promote the proliferation of bifidobacteria in the gut, it can be considered a prebiotic. Currently, lactosucrose is mainly produced enzymatically, using sucrose and lactose as substrates, undergoing a transglycosylation reaction under the action of enzymes. The transglycosylation reactions in the synthesis of lactosucrose include two types: first, through enzymatic transfructation, the fructose groups produced from sucrose decomposition are transferred to the C1 hydroxyl group of the glucose moiety in lactose; suitable enzymes include β-(2,6)fructansulanase and β-fructofuranosidase from various microbial sources; second, through enzymatic transgalactosylation, the galactosyl groups produced from lactose decomposition are transferred to the C4 hydroxyl group of the glucose moiety in sucrose; suitable enzymes are mainly β-galactosidase. Although β-fructofuranosidases are widely found in nature, only Arthrobacter sp. K-1 β-fructofuranosidase has been found to convert sucrose and lactose into lactulose oligosaccharides. However, the synthesis of lactulose oligosaccharides using β-galactosidase is accompanied by various side reactions. Therefore, β-(2,6)fructan sucrase is considered a valuable tool enzyme for the synthesis of lactulose oligosaccharides.

[0003] β-(2,6)fructan sucrase (EC 2.4.1.10) is a member of the glycosyltransferase family and is widely found in microorganisms. Due to its hydrolytic, fructose-transfer, and polymerization functions, β-(2,6)fructan sucrase plays an important role in the biosynthesis of functional food ingredients such as fructans and oligosaccharides. Many microbial β-(2,6)fructan sucrase enzymes have been shown to be capable of synthesizing lactulose oligosaccharides, such as Bacillus natto, Bacillus subtilis ATCC 6501, Paenibacillus polymyxa IFO 3020, Sterigmatomyces elviae ATCC18894, and Pseudomonas aurantiaca. The β-(2,6)fructan sucrase from Leuconostoc mesenteroides B-512FMC exhibited the highest capacity for synthesizing oligo-lactulose, achieving an equilibrium conversion of 41.5% after 1 hour of reaction using 27% (w / v) sucrose and 27% (w / v) lactose as substrates. However, this LS-derived enzyme has a narrow product spectrum, only capable of generating low-polymerization fructans and lactulose, and there are currently few reports on its synthesis of high-molecular-weight fructans. Compared to oligo-lactulose, high-molecular-weight β-(2,6)fructans cannot be directly digested by the human body, but possess antioxidant, antitumor, and anti-inflammatory properties, and have been widely used in food, pharmaceutical, and materials fields.

[0004] Currently, β-(2,6)fructan sucrase is an important enzyme for synthesizing lactulose oligosaccharides (LOS), and its ability to synthesize LOS is related to its glycosylation ability. The LOS content is limited by enzymes from different sources, making a stable industrial production process impossible. Furthermore, no β-(2,6)fructan sucrase has yet been discovered that can both improve LOS conversion rate and produce a broad product spectrum. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention utilizes site-directed mutagenesis to molecularly modify β-(2,6)fructan sucrase (Psor-LS) from the microorganism P. orientalis, providing a mutant for the efficient preparation of lactulose oligosaccharides. This discovery has significant practical implications for the industrial production of lactulose oligosaccharides.

[0006] The first objective of this invention is to provide a β-(2,6)fructan sucrase mutant, the amino acid sequence of which is shown in SEQ ID NO.4.

[0007] In one embodiment, the mutant is based on β-(2,6)fructan sucrase with the amino acid sequence shown in SEQ ID NO.2, by replacing arginine at position 91 with threonine.

[0008] A second objective of this invention is to provide a gene encoding the β-(2,6)fructan sucrase mutant.

[0009] In one embodiment, the sequence of the gene is shown in SEQ ID NO.3.

[0010] A third objective of this invention is to provide an expression vector carrying the gene.

[0011] In one embodiment, the carrier includes, but is not limited to, pET series carriers.

[0012] In one embodiment, the carrier comprises pET-22b(+).

[0013] A fourth objective of this invention is to provide a genetically engineered bacterium expressing the β-(2,6)fructan sucrase mutant.

[0014] In one embodiment, the genetically engineered bacteria uses Escherichia coli as the host and pET-22b(+) as the vector.

[0015] The fifth object of the present invention is to provide a method for producing lactulose oligosaccharide, wherein the method uses the β-(2,6) fructan sucrase mutant or genetically engineered bacteria containing the β-(2,6) fructan sucrase mutant as a catalyst and sucrose and lactose as substrates to prepare lactulose oligosaccharide.

[0016] In one embodiment, the preparation uses sodium phosphate buffer as the buffer system.

[0017] In one embodiment, the substrate of the method is sucrose and lactose, and the final concentration ratio of lactose to sucrose is (0.5-2):1.

[0018] In one embodiment, the amount of β-(2,6)fructan sucrase mutant added to the method is 5-15 U / mL.

[0019] In one embodiment, the production conditions of the method are pH 4-7, temperature 50-70°C, and reaction time 0.5-3h.

[0020] The sixth objective of this invention is to provide a method for preparing high molecular weight fructans, using the mutant as a catalyst and sucrose as a substrate for the reaction.

[0021] The present invention also provides the application of the mutant, the gene, the expression vector, the genetically engineered bacteria, or the method for preparing lactulose oligosaccharides in the pharmaceutical and food industries.

[0022] Beneficial effects:

[0023] The single-point mutant enzyme R91T significantly improved polymerization ability, enhancing the ability of β-(2,6)fructan sucrase to synthesize oligo-lactulose. Using sucrose and lactose as substrates, at 65℃ and pH 6.5 for 1 h, the conversion rate of R91T to produce oligo-lactulose reached 40%, which is 1.3 times that of the parent enzyme. The space-time conversion rate of the R91T-catalyzed reaction was 16 g / (L·h), which is 2.6 times that of the parent enzyme.

[0024] The mutant enzyme R91T enhances the ability to catalyze the production of high molecular weight fructans. At 65°C and pH 6.5 for 6 hours, the mutant enzyme R91T can convert sucrose into high molecular weight β-(2,6) fructans with a conversion rate of 49%, which is 1.6 times that of the parent enzyme. Attached Figure Description

[0025] Figure 1 Comparison of the original enzyme and the mutant R91T in catalyzing the synthesis of lactulose oligosaccharides from sucrose and lactose. Detailed Implementation

[0026] The β-(2,6)fructan sucrase (Psor-LS) gene involved in this invention is derived from the microorganism Pseudomonas orientalis, with the NCBI database number WP_124433094.1. Its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2.

[0027] The enzyme activity assay system consisted of 1 mL of sucrose containing 30% (w / v) sucrose and 10 μg of purified enzyme. All enzyme activity assays were performed at the optimal temperature (65°C) and pH (6.5) of the LS reaction, with a reaction time of 15 min. The reaction was terminated by placing the reaction solution in a boiling water bath for 10 min. Total enzyme activity and hydrolytic enzyme activity were calculated using the amounts of glucose and fructose produced in the reaction system, respectively. Total enzyme activity was defined as the amount of enzyme required to produce 1 μmol of glucose per 1 min, and hydrolytic enzyme activity was defined as the amount of enzyme required to produce 1 μmol of fructose per unit time. Transfructase activity was calculated by subtracting hydrolytic enzyme activity from total enzyme activity. (In the following embodiments, enzyme activity refers to total enzyme activity.)

[0028] Protein detection method: Protein concentration was determined by Coomassie brilliant blue assay.

[0029] Enzyme activity calculation method (U / mg) = Enzyme activity (U) / Enzyme protein mass (mg)

[0030] Molecular weight determination method: The molecular weight of polysaccharides was determined by high-performance gel filtration chromatography (HPGFC). A differential detector and a gel column (7.8 × 300 mm) were used. The mobile phase was 0.1 N sodium nitrate solution, and the flow rate and detection temperature were 0.5 mL / min and 40 °C, respectively. The standard reference for molecular weight was dextran T-2000 (molecular weight 2 × 10⁻⁶). 6 Da), Dextranamide T-300 (3.0×10) 5 Da), dextran T-150 (1.4×10) 5 Da), Dextran T-10 (9.7×10) 3 Da) and dextran T-5 (2.7×10) 3 Da).

[0031] Example 1: Three-dimensional structural analysis of β-(2,6)fructan sucrase and construction of mutant plasmids

[0032] 1. Construction of plasmids.

[0033] (1) Construction of the original plasmid pET-22b(+)-Psor-LS

[0034] The β-(2,6) fructan sucrase gene (Psor-LS) with the nucleotide sequence shown in SEQ ID NO.1 was synthesized and inserted into the Nde I and Xho I restriction enzyme sites of the vector pET-22b(+). Sequencing verification was performed to obtain the original plasmid pET-22b(+)-Psor-LS.

[0035] (2) Construction of mutant plasmid pET-22b(+)-R91T

[0036] Mutation primers were designed for site-directed mutagenesis (Table 2). Using the original plasmid pET-22b(+)-Psor-LS carrying the Psor-LS gene as a template, a single-point mutation was performed, replacing the 91st arginine residue of β-(2,6)fructan sucrase (Psor-LS) with a threonine residue, resulting in the mutant plasmid pET-22b(+)-R91T. The mutation was verified by PCR and template digestion. The product of template digestion was then sequenced. The nucleotide sequence of the mutant enzyme is shown in SEQ ID NO. 3, and the amino acid sequence is shown in SEQ ID NO. 4.

[0037] Table 1 PCR reaction system

[0038]

[0039] PCR reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 3 min 40 s, 32 cycles, stored at 4℃.

[0040] Table 2 Primer sequence list

[0041]

[0042] Table 3 Template digestion reaction system

[0043]

[0044] Reaction conditions: 37℃, reaction time 90 min.

[0045] Example 2: Construction of engineered strains and expression and purification of mutant enzymes

[0046] (1) Construction of engineered strains.

[0047] The mutant plasmid pET-22b(+)-R91T and the original plasmid pET-22b(+)-Psor-LS obtained in Example 1 were transformed into E. coli BL21(DE3) competent cells, respectively. The cells were plated on LB solid medium containing 100 μg / mL ampicillin and cultured at 37°C for 12 h to obtain plates containing recombinant engineered bacteria E. coli BL21 / pET-22b(+)-R91T and E. coli BL21 / pET-22b(+)-Psor-LS, respectively.

[0048] (2) Expression of mutant enzymes.

[0049] Single colonies from the plates in step (1) were picked and transferred to 4 mL of LB liquid medium containing 50 μg / mL ampicillin. The culture was incubated at 37°C for 12 h to obtain a seed culture. This seed culture was then transferred to 200 mL of LB liquid medium containing 50 μg / mL ampicillin and incubated at 37°C for 2–3 h until OD (Organic Oxygen Demand) was reached. 600 The expression level was set at 0.6–0.8. IPTG was added to a final concentration of 1 mmol / L to induce protein expression, and the mixture was incubated at 28°C for 6–8 h to obtain the fermentation broth. The fermentation broth was then centrifuged at 4°C and 8000 rpm for 10 min, and the cells were collected. The expression level of the mutant enzyme was comparable to that of the original enzyme, and its fermentation enzyme activity reached 400 U / mL.

[0050] (3) Purification of mutant enzymes.

[0051] Add 20 mL of disruption buffer (50 mmol / L phosphate buffer, 200 mmol / L NaCl, pH 7.0) to the bacterial cells obtained in step (2), fully resuspend the bacterial cells, and then perform ultrasonic disruption. After disruption, centrifuge at 4℃ and 8000 rpm for 15 min, and collect the supernatant, which is the crude enzyme solution.

[0052] The crude enzyme solution was purified using a nickel ion affinity chromatography column. First, the column was equilibrated with equilibration buffer (50 mmol / L phosphate buffer, 500 mmol / L NaCl, pH 7.0). Then, the obtained crude enzyme solution was added to the column. Next, contaminating proteins were washed with a buffer containing a low concentration of imidazole (50 mmol / L phosphate buffer, 500 mmol / L NaCl, 50 mmol / L imidazole, pH 7.0). Finally, elution was performed with a buffer containing a high concentration of imidazole (50 mmol / L phosphate buffer, 500 mmol / L NaCl, 500 mmol / L imidazole, pH 7.0) to obtain the mutant enzyme R91T and the parent enzyme Psor-LS.

[0053] (4) The enzyme activity of β-(2,6)fructan sucrase was determined under optimal temperature and pH conditions.

[0054] Under reaction conditions of pH 6.5 and 65℃, using sucrose at a final concentration of 300 g / L as the substrate, 10 μg of the mutant enzyme R91T obtained in step (3) was added, and the reaction was carried out for 15 min. The enzyme was then inactivated by boiling for 10 min. The specific enzyme activity of the original enzyme was 1219 U / mg. The final measured specific enzyme activity of the mutant enzyme was 790 U / mg, retaining most of its activity.

[0055] Example 3: Reaction process of producing lactulose oligosaccharides using mutant enzyme R91T

[0056] The conversion rate of lactulose oligosaccharides (LOS) was obtained using the mass conservation method. The mass of LOS = substrate mass - remaining substrate mass - mass of glucose and fructose produced; the conversion rate is the mass of LOS divided by the total mass of sucrose and lactose. The mass of each component was obtained using high-performance liquid chromatography (HPLC). The conversion rate of LOS produced by R91T reached 40%, significantly higher than that of the original enzyme. The production conditions were: pH 6.5 phosphate buffer, 65℃, final concentrations of 200 g / L sucrose and 200 g / L lactose, enzyme dosage of 10 U / mL, reaction time of 1 h, and a reaction system of 50 mL. The final conversion rate of sucrose and lactose to LOS by the mutant R91T was 40%, significantly higher than the equilibrium conversion ratio of 30% for the original enzyme. Furthermore, the mutant R91T reached equilibrium within 1 h, with a space-time conversion rate of 16 g / (L·h), while the original enzyme was 6 g / (L·h).

[0057] Example 4: Reaction process of producing high molecular weight fructan from mutant enzyme R91T

[0058] The conversion rate of high molecular weight fructan was obtained by the mass conservation method. The mass of high molecular weight fructan = substrate mass - remaining substrate mass - mass of glucose and fructose produced; the conversion rate is the mass of high molecular weight fructan divided by the total mass of sucrose. The amounts of each component were obtained by high performance liquid chromatography. Product spectrum comparison: Using 300 g / L sucrose as substrate, 10 U / mL of the mutant enzyme R91T obtained in Example 2 and the parent enzyme were added to 1 mL of the reaction system (pH 6.5), and the reaction was carried out at 65 °C for 6 h. After the reaction, the mixture was boiled for 10 min. The mutant enzyme R91T accumulated 147 g / L of high molecular weight β-(2,6) fructan, with an average molecular weight of 1.2 × 10⁻⁶. 6 The conversion rate was 49%; while the parent enzyme reaction system accumulated 87 g / L of high molecular weight β-(2,6) fructan, with an average molecular weight of 1.3 × 10⁻⁶. 6 The conversion rate was 29%.

[0059] Comparative Example 1:

[0060] The specific implementation method is as described in Example 3, except that the final concentration of lactose is fixed at 200 g / L, and the substrate sucrose / lactose concentration ratio is changed to 0.5:1 and 2:1, respectively. The conversion rate of lactulose oligosaccharide production was tested, and the results showed that the conversion rates of lactulose oligosaccharide were 26% and 17%, respectively.

[0061] Comparative Example 2:

[0062] The specific implementation method is as described in Example 3, except that the amount of enzyme added was changed. The conversion rate of producing oligo-lactulose was detected at enzyme addition amounts of 5 U / mL and 15 U / mL, respectively. The results showed that the conversion rates of oligo-lactulose were 37% and 35%, respectively.

[0063] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A kind β -(2,6) fructan sucrase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

4.

2. The gene encoding the mutant of claim 1.

3. An expression vector carrying the gene of claim 2, characterized in that, Including the pET series of vectors.

4. Genetically engineered bacteria expressing the mutant of claim 1.

5. The genetically engineered bacterium according to claim 4, characterized in that, Including E. coli.

6. A method for preparing lactulose oligosaccharides, characterized in that, The reaction is carried out using the mutant described in claim 1 as a catalyst and sucrose and lactose as substrates.

7. The method according to claim 6, characterized in that, The catalyst concentration is 5-15 U / mL, and the substrate is sucrose and lactose, with a final concentration ratio of lactose to sucrose of (0.5~2):

1.

8. The method according to claim 7, characterized in that, React at pH 4-7 and 50-70℃ for 0.5-3 h.

9. A method for preparing high molecular weight fructans, characterized in that, The reaction was carried out using the mutant described in claim 1 as a catalyst and sucrose as a substrate.

10. The use of the mutant of claim 1, or the gene of claim 2, or the expression vector of claim 3, or the genetically engineered bacteria of claim 4 or 5, or the method of any one of claims 6 to 9, in the production of lactulose oligosaccharides or high molecular weight fructans in the fields of pharmaceutical production and food processing.

Images