Method for preparing fructo-oligosaccharide based on double-enzyme co-expression

Abstract

The invention belongs to the technical field of enzyme engineering and food biology, and particularly relates to a method for preparing fructo-oligosaccharide based on double-enzyme co-expression. According to the invention, a double-enzyme system for co-expressing glucose isomerase and FTase mutants is adopted to catalyze sucrose or a sucrose-containing substrate for reaction. Through an ingenious enzyme engineering strategy, the key inhibition problem in FOS synthesis is solved in principle, raw material pretreatment and process optimization are successfully integrated, a set of efficient, economical and green fructo-oligosaccharide preparation technology is formed, and powerful technical support is provided for production of functional food ingredients and transformation and upgrading of the sugar industry.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and food biotechnology, and specifically relates to a method for preparing fructooligosaccharides based on dual-enzyme co-expression. Background Technology

[0002] The world faces the dual challenges of an aging population and a growing burden of chronic diseases, both of which impact quality of life. Despite advancements in medical technology extending life expectancy, people are increasingly focused on disease prevention through daily practices. Therefore, functional foods with health-promoting effects are gaining attention, such as those that can reduce the risk of chronic diseases and enhance immunity, thereby improving quality of life. Fructose-oligosaccharides (FOS), as a prebiotic, can stimulate the growth of beneficial bacteria and help maintain a balanced gut microbiota. Plants are rich in FOS, but its industrial applications are limited by seasonality. Biocatalysis technology, due to its advantages of mild conditions, high efficiency, and high specificity, has become a hot topic in scientific research and industrial production.

[0003] Studies have shown that the enzymatic production of FOS from sucrose is mainly catalyzed by β-fructofuranosidase (FFase, EC3.2.1.26) and β-D-fructosyltransferase (FTase, EC2.4.1.9). Current research on FTase mainly focuses on resource library expansion, efficient expression, enzyme activity enhancement, and fermentation process optimization. Our previous study found that increased FOS yield is accompanied by glucose accumulation, and glucose has an inhibitory effect on FTase mutants. Disrupting glucose inhibition-related genes can increase FOS yield, which verifies the above results and provides a basis for further yield enhancement. Therefore, we hypothesize that co-expression of glucose isomerase may alleviate glucose inhibition and increase FOS yield.

[0004] Sugarcane, as an important economic crop, plays a crucial role in the global sugar industry. Global sugarcane processing generates over 279 million tons of solid and liquid waste and byproducts (such as bagasse, filter mud, and molasses) annually, which pose risks to the environment and health if not properly managed. In recent years, the trend of waste resource utilization has become increasingly evident, contributing to sustainable development and the goals of a circular bioeconomy. Sugarcane molasses, as a low-cost raw material, has been used to produce various high-value-added biomolecules, such as monosaccharides, D-allulose, L-threonine, and extracellular polysaccharides.

[0005] Guangxi Zhuang Autonomous Region is my country's largest sugarcane producing area and an important research base for the industry. Its abundant resources provide a raw material foundation for converting sugarcane molasses into FOS. Based on this background, this study constructed a dual-enzyme system composed of glucose isomerase (GI) and an FTase mutant, aiming to alleviate glucose inhibition, overcome reaction equilibrium limitations, and utilize inexpensive molasses to produce high-value-added FOS. Through fermentation process optimization, the catalytic efficiency of the co-expression system was maximized, providing a theoretical basis for the high-value utilization of agricultural waste. Summary of the Invention

[0006] The present invention aims to provide a highly efficient method for preparing fructooligosaccharides based on dual-enzyme co-expression, in order to solve the problems of severe glucose inhibition, high raw material cost and low product yield in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing oligofructose based on dual-enzyme co-expression includes the following steps: using a dual-enzyme system co-expressing glucose isomerase and FTase mutant to catalyze the reaction of sucrose or a sucrose-containing substrate, wherein the amino acid sequence of the FTase mutant is shown in SEQ ID NO:1 and its nucleotide sequence is shown in SEQ ID NO:4.

[0009] Furthermore, the glucose isomerase is derived from *Acidothermic cellulose*, and its amino acid sequence is shown in SEQ ID NO:2, and its nucleotide sequence is shown in SEQ ID NO:5.

[0010] Furthermore, the FTase mutant is derived from Aspergillus niger, and its amino acid sequence is obtained by point mutation based on SEQ ID NO:3.

[0011] Furthermore, the molar ratio of glucose isomerase to FTase mutant in the dual-enzyme system is 0.5:1 to 2:1.

[0012] Furthermore, the catalytic reaction conditions are: a temperature of 40-50℃, a pH of 5.0-6.0, and the presence of 0.5-2 mM Ba in the reaction system. ²⁺ As an auxiliary factor, it was carried out in a shaker at 150–250 r / min.

[0013] Furthermore, the sucrose-containing substrate is cane molasses.

[0014] Furthermore, the total enzyme content of glucose isomerase and FTase mutant in the dual-enzyme system is 0.5–2.0 U / mL.

[0015] Furthermore, the catalytic reaction takes 2–24 hours.

[0016] The beneficial effects of this invention are:

[0017] 1. Fundamentally eliminates glucose inhibition, significantly improving catalytic efficiency and product yield.

[0018] This invention, through molecular docking and experimental verification, elucidates for the first time the inhibitory mechanism of glucose on the key enzyme FTase mutant: glucose, a reaction byproduct, competitively occupies the enzyme's substrate / product channel, hindering the entry of sucrose substrate and the release of fructooligosaccharide (FOS) product, thereby disrupting reaction equilibrium. To address this fundamental bottleneck, this invention innovatively constructs a dual-enzyme system co-expressing glucose isomerase (GI) and the FTase mutant. The core principle of this system is that GI can convert glucose, an inhibitor produced during FTase catalysis, into fructose in situ and in real time. This conversion process achieves dual benefits: on the one hand, it directly reduces the concentration of glucose in the reaction system, relieving its competitive inhibition of the FTase mutant activity; on the other hand, the released fructose can participate as an additional substrate in subsequent glycosylation reactions. Experimental data strongly confirms the effectiveness of this strategy; the dual-enzyme system successfully increased the FOS conversion rate significantly from 42.31% in the single-enzyme system to 55.51%, achieving a breakthrough increase in catalytic efficiency.

[0019] 2. It has enabled the high-value utilization of inexpensive raw materials and green and sustainable production.

[0020] This invention combines the aforementioned highly efficient dual-enzyme system with low-cost raw material sugarcane molasses to construct a complete "waste-to-quality" process chain. By optimizing the pretreatment method (adsorption-heat treatment), impurities in the molasses were effectively removed, and high-value-added FOS was successfully produced using it as a substrate. 67.12 g / L of FOS was obtained from 124.48 g / L molasses, achieving a conversion rate of 53.92%. This process not only significantly reduces raw material costs and improves economic feasibility but also provides a practical technical path for the resource utilization of sugar industry by-products, aligning with the development direction of a circular bioeconomy.

[0021] 3. The process is stable and has good prospects for industrial application.

[0022] This invention, through systematic optimization of fermentation conditions (inducer concentration, temperature) and catalytic system (temperature, pH, metal ions), established the optimal culture conditions (25℃, 1 mM IPTG) and optimal reaction parameters (45℃, pH 5.5, Ba) for the dual-enzyme co-expression strain. 2+ (Activation) ensures efficient enzyme expression and stable catalysis. The process conditions are mild and controllable, and the whole-cell catalysis and pure enzyme catalysis schemes provide flexibility for different production scenarios, exhibiting strong applicability.

[0023] In summary, this invention, through a clever enzyme engineering strategy, fundamentally solves the key inhibition problem in FOS synthesis and successfully integrates raw material pretreatment and process optimization, forming a set of efficient, economical, and green fructooligosaccharide preparation technologies, providing strong technical support for the production of functional food ingredients and the transformation and upgrading of the sugar industry. Attached Figure Description

[0024] Figure 1 : Glucose inhibition verification and molecular docking analysis diagram, where (A) glucose inhibition experiment; (B) molecular docking schematic diagram (glucose green, sucrose red, GF2 orange, GF3 yellow);

[0025] Figure 2 FTase 142P-242K SDS-PAGE images of GI expression and purification, where (A) expression and purification of FTase mutant; (B) expression and purification of GI; M: protein marker; lane 1: supernatant; lane 2: purification elution buffer; lanes 3-5: purified enzyme;

[0026] Figure 3 Comparison of FOS conversion rate and sugar composition between single-enzyme and dual-enzyme systems, where (A) FOS conversion rate; (B) sugar composition ratio after catalysis;

[0027] Figure 4 : Enzymatic characteristics analysis diagram of a dual-enzyme system (temperature, pH, metal ions), where (A) temperature; (B) pH; (C) effect of metal ions on enzyme activity. CK is the control without added metal ions;

[0028] Figure 5 The graph shows the effect of fermentation process optimization on FOS conversion rate. Among them, (A) the effect of IPTG concentration on whole-cell catalysis; (B) the effect of temperature on whole-cell catalysis; (C) the effect of IPTG on pure enzyme catalysis; (D) the effect of temperature on pure enzyme catalysis; and (E) a comparison of FOS conversion rates catalyzed by whole-cell and pure enzyme catalysis.

[0029] Figure 6 : Effects of fermentation conditions on cell growth and enzyme expression, where (A1-A2) shows the effect of IPTG; (B1-B2) shows the effect of temperature;

[0030] Figure 7 : Process optimization diagram for molasses to FOS conversion under different pretreatment methods, where (ad) represents the effect of IPTG concentration; (eh) represents the effect of temperature. CK: Untreated; H: Adsorption-heat treatment; P: Calcium phosphate treatment; E: EDTA flocculation treatment;

[0031] Figure 8The effect of pretreatment methods on FOS conversion rate and the comparison of the advantages of the dual-enzyme system are shown in the figure. (A) FOS conversion rate of different pretreatment methods; (B) Comparison of FOS conversion rate of single enzyme and dual-enzyme system under optimal pretreatment (H). Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0033] Example 1

[0034] 1 Introduction

[0035] Fructooligosaccharides (FOS), as an important prebiotic, have broad application prospects in the functional food field. However, traditional preparation methods suffer from problems such as severe glucose inhibition, high raw material costs, and low conversion rates. This invention achieves efficient and low-cost FOS preparation by constructing a dual-enzyme co-expression system.

[0036] 2 Materials and Methods

[0037] 2.1 Strains, Plasmids and Materials

[0038] Escherichia coli DH5α and Rosetta (DE3) were purchased from Sangon Biotech (Shanghai) Co., Ltd. The strains and plasmids used are shown in Tables 1 and 2. Escherichia coli DH5α was used for cloning and plasmid preparation, and was cultured at 37°C in LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, m / v) containing ampicillin (100 μg / mL). Escherichia coli Rosetta (DE3) was used as the expression host.

[0039] Table 1. Strains used in this invention

[0040]

[0041] Table 2. Plasmids used in this invention

[0042]

[0043] 2.2 Gene Cloning and Protein Expression

[0044] The natural sequences of the FTase gene (GenBank accession number KF699529.1) from Aspergillus niger QU10 and the glucose isomerase (GI) gene (GenBank accession number ABK53836.1) from Acidothermus cellulolyticus 11B provide reference sequences and design starting points for this invention.

[0045] Based on the above sequences, we performed codon optimization to adapt them to the E. coli expression system, and introduced nucleotide fragments encoding a C-terminal 6×His tag at the 3' end of each coding sequence, thereby constructing a series of engineered genes suitable for efficient expression and purification:

[0046] The optimized FTase (wild-type) engineered gene carrying a C-terminal 6×His tag has the nucleotide sequence shown in SEQ ID NO:6 and the encoded amino acid sequence (excluding the tag portion) shown in SEQ ID NO:3.

[0047] Using the engineered gene shown in SEQ ID NO:6 as a template, S142P and W242K mutations were introduced through site-directed mutagenesis to obtain an FTase mutant carrying a C-terminal 6×His tag (FTase). 142P-242K The engineered gene has a nucleotide sequence as shown in SEQ ID NO:4 and an encoded amino acid sequence (excluding the tag portion) as shown in SEQ ID NO:1.

[0048] An optimized glucose isomerase (GI) engineered gene carrying a C-terminal 6×His tag has the nucleotide sequence shown in SEQ ID NO:5 and the encoded amino acid sequence (excluding the tag portion) shown in SEQ ID NO:2.

[0049] All of the optimized genes mentioned above were artificially synthesized. The primer sequences used in the experiment are shown in Table 3.

[0050] Plasmids pCold II and pCold sumo were digested with BamHI and HindIII, and then the synthesized His-tagged FTase mutant gene (SEQ ID NO:4) and GI gene (SEQ ID NO:5) were inserted into the vector using a one-step cloning enzyme-mediated homologous recombination technique to construct expression plasmids, named pCold II-FTase. 142P-242K The recombinant plasmids were transformed into *E. coli* Rosetta(DE3) competent cells, and transformants were obtained through antibiotic selection. Sequencing confirmed the successful acquisition of two engineered bacteria, named: one expressing a C-terminal His-tagged FTase...142P-242K The engineered bacteria (SEQ ID NO:1) and the engineered bacteria expressing GI (SEQ ID NO:2) with a His tag at the C-terminus.

[0051] Recombinant bacteria used for protein expression were cultured in fermentation medium until an OD600 of 0.6–0.8 was reached. 1 mmol / L IPTG was added, and the cells were induced at 25°C and 200 r / min for 24 hours. The cells were collected by centrifugation, washed three times with 50 mM phosphate-buffered saline (pH 7.0), and then sonicated in an ice-water bath (300 W, 2-second pulses, 3-second intervals, total duration 10 minutes). Cell debris was removed by centrifugation. The target protein was purified using a Ni Sepharose 6 Fast Flow affinity chromatography column (FTase mutant and GI both fused with a C-terminal 6×His tag). Protein expression was verified by SDS-PAGE.

[0052] Table 3 Primer sequences used in this invention

[0053]

[0054] 2.3 Enzyme activity assay

[0055] The fructosyltransferase activity assay was performed according to the method of Kubota et al., with slight modifications. An appropriate amount of enzyme solution was mixed with 500 g / L sucrose substrate in 0.1 M phosphate buffer (pH 5.5). After the reaction was complete (45°C, 10 minutes), the reaction was terminated by heating in a boiling water bath for 15 minutes.

[0056] A Waters 2595 liquid chromatography system (equipped with a Waters 2414 differential refractive index detector) and an XBridge BEH Amide column (130) were used. The FOS content was determined using a 5 μm, 4.6 × 250 mm spectrophotometer. Chromatographic conditions: column temperature 30℃, mobile phase 75% acetonitrile, flow rate 1.0 mL / min.

[0057] 2.4 Optimization of Reaction Conditions

[0058] The activity of the two-enzyme system was determined by whole-cell reaction. To determine the optimal reaction conditions, the activity of metal ions (1 mMk) was investigated. + Co 2+ Mg 2+ Ca 2+ Cu 2+ Ba 2+ Na + The effects of temperature (30–80 °C) and pH (3.0–8.0) on the activity of the dual-enzyme system (FTase mutant and GI, with a molar ratio controlled at 1:1).

[0059] 2.5 Effects of culture conditions on the activity of the two enzymes

[0060] The recombinant bacteria were inoculated into LB medium containing 100 μg / mL ampicillin and cultured at 37°C and 200 rpm until the OD600 reached 0.6–0.8. A 1% seed culture was transferred to the medium, and different concentrations of IPTG (0.1, 0.2, 0.25, 0.5, 1.0 mmol / L) were added. After induction at 25°C and 200 rpm for 24 hours, cells were collected to determine enzyme activity and cell growth. Alternatively, cells were inducing at different temperatures (15, 20, 25, 28°C) for 24 hours with the addition of 1 mmol / L IPTG, and enzyme activity and growth were also measured.

[0061] 2.6 Molasses Pretreatment

[0062] Adsorption-heat treatment (H): Add 6% activated carbon (m / v), adsorb in a 60℃ water bath for 30 minutes, then add 3% silica (m / v) for 1 hour, and remove impurities by centrifugation and filtration.

[0063] Calcium phosphate treatment (P): Adjust the pH to 3.0 with phosphoric acid, centrifuge and collect the supernatant; adjust the pH to 6.0 with calcium hydroxide, add polyacrylamide and stir for 30 minutes; then adjust the pH to 6.0 with hydrochloric acid, centrifuge and filter.

[0064] EDTA flocculation treatment (E): Add 2 mM EDTA to bind heavy metals at room temperature for 12 hours, then add 1.5% activated carbon (m / v), and centrifuge and filter.

[0065] 2.7 Molecular docking

[0066] Molecular docking and structural visualization analysis were performed using PyMOL (Schrödinger, USA).

[0067] 2.8 Statistical Analysis

[0068] All experiments were independently repeated three times (n=3), and data are expressed as mean ± standard deviation. Statistical analysis and graphing were performed using Prism 10.1.2 (GraphPad, USA) and Origin 9.1 (OriginLab, USA). Significance between means (P<0.05) was determined using the LSD test.

[0069] Results and Discussion

[0070] 3.1 Analysis of glucose inhibition effect

[0071] Following sucrose hydrolysis, the FTase mutant transfers fructose groups to sucrose and releases glucose. Previous studies have shown that high concentrations of glucose may inhibit the activity of the FTase mutant. To verify this hypothesis, different concentrations of glucose were added to the reaction system. Figure 1 As shown in Figure A, the FOS yield gradually decreased with increasing glucose concentration, confirming that glucose has an inhibitory effect on the FTase mutant. To elucidate the mechanism, molecular docking was used to predict glucose binding sites. Figure 1 As shown in B, glucose, sucrose, and FOS (including trisaccharides and tetrasaccharides) share the same binding region in the FTase mutant. Glucose occupies the channel for substrate and product to enter and exit the active site. As glucose is continuously released and accumulates in the channel, it hinders the binding and transport of FOS in the FTase mutant, thereby disrupting the reaction equilibrium.

[0072] 3.2 Construction and expression of the two-enzyme system

[0073] Section 3.1 confirmed the inhibitory effect of glucose on the FTase mutant. To increase FOS production, strains co-expressing glucose isomerase (GI) were constructed to reduce glucose inhibition. The FTase mutant and GI contained 629 and 414 amino acids, respectively, with predicted molecular weights of 68.00 kDa and 65.87 kDa (including the SUMO tag), respectively. SDS-PAGE results showed clear bands at approximately 55–75 kDa, indicating successful expression of both enzymes. Figure 2 A single protein band was obtained by purification using the His tag, which can be used for enzyme activity assay.

[0074] In our previous work, through the rational design of FTases, we obtained mutant FTases with significantly improved performance. 142P-242K (i.e., the mutant with the sequence shown in SEQ ID NO:1), its FOS conversion rate was 42.31%. This embodiment further constructed a two-enzyme system co-expressing glucose isomerase (GI) to relieve glucose inhibition and improve conversion efficiency. Experimental results showed that this two-enzyme system successfully increased the FOS conversion rate to 55.51% (…). Figure 3 A). Comparing the changes in sugar composition during single-enzyme and dual-enzyme catalysis (as shown in Comparative Experiment 1 below), it was found that in the dual-enzyme system, the glucose proportion decreased from 47.16% to 20.66%, while the fructose proportion increased from 10.64% to 26.73%. Figure 3 B). This indicates that after glucose is converted into fructose, it is released from the binding region, keeping the channel open and prompting more sucrose and FOS to enter the active site, thereby driving the catalytic cycle.

[0075] 3.3 Characterization of Enzymatic Properties

[0076] Temperature significantly alters enzyme activity by affecting the three-dimensional conformation of proteins. Testing the activity of a two-enzyme system within the range of 30–80℃ showed that 45℃ was the optimal temperature, and enzyme activity decreased sharply above 55℃. Figure 4 A). Temperature has a dual effect on enzymatic reactions: on the one hand, it accelerates molecular motion and collision probability; on the other hand, excessively high temperatures lead to protein denaturation. pH is also a key factor affecting enzyme activity, altering the enzyme's spatial structure and the affinity between the substrate and the active site. This study determined that the optimal pH for the two-enzyme system is 5.5 (…). Figure 4 B), is in a slightly acidic environment.

[0077] Metal ions, acting as cofactors, can enhance the activity of specific enzymes. This study investigated the effects of various metal ions on two-enzyme systems and found that Na+... + and Ba 2+ It has an activating effect, of which 1 mM Ba 2+ The activation effect is better ( Figure 4 C). The optimal conditions for the FTase mutant were initially determined to be 45℃, pH 6.0, and 1 mM Na. + In comparison, the two-enzyme system exhibits a pH shift of 0.5. When multiple enzymes work synergistically, the overall efficiency depends on the compatibility between the enzymes. If the optimal pH of each enzyme differs, a compromise must be made in the selection of the reaction pH, potentially sacrificing synthetic efficiency. To further optimize the synergistic efficiency of the two enzymes, we investigated the effect of different enzyme molar ratios (see Comparative Experiment 3). The results showed that when the molar ratio of GI to FTase mutant was 1:1, the FOS conversion rate was the highest (55.51%), demonstrating the best synergistic effect.

[0078] 3.4 Fermentation process optimization

[0079] Fermentation conditions such as inducer concentration and temperature affect enzyme quantity and activity, and thus FOS yield. This study investigated the effects of IPTG concentration and fermentation temperature. The results showed that the optimal fermentation conditions were 1 mM IPTG and 25℃ (…). Figure 5 When the IPTG concentration reaches 0.5–1.0 mM, the enzyme expression level is stable, but the OD600 decreases. Figure 6 This indicates that IPTG inhibits cell growth but does not reduce enzyme expression. Appropriate temperature increase promotes cell growth and enzyme synthesis, but excessively rapid synthesis may lead to misfolding and reduced catalytic efficiency. Comparison of the transformation efficiency of the pure enzyme and whole cells revealed that the transformation yield of the pure enzyme was approximately 1.14 times that of the whole cell (…). Figure 5 E). Sucrose needs to be transported into the cell via cell membrane transport proteins for catalysis, while pure enzymes can act directly on the substrate, thus having higher catalytic efficiency under the same conditions.

[0080] 3.5 High-value utilization of sucrose-containing substrates (cane molasses)

[0081] Guangxi Zhuang Autonomous Region, as a core sugar-producing area, boasts abundant sugarcane resources, providing a raw material base for FOS production. However, improper disposal of sugar industry by-products can put pressure on the environment. Sugarcane molasses, rich in sucrose, is an ideal raw material for FOS production. This study evaluated the potential of molasses for FOS production and optimized the process. All pretreated samples were fermented at 25°C and 1 mM IPTG. Figure 7 To screen for the best pretreatment method, we compared the effects of different treatments (see Comparative Experiment 2). Under optimal conditions, the FOS yields of different pretreatment groups were: control group (CK) 41.45%, H treatment group 53.92%, E treatment group 34.77%, and P treatment group 17.13%. Figure 8 A). The H treatment group achieved the highest yield, obtaining 67.12 g / L FOS from 124.48 g / L molasses. Choosing a suitable pretreatment method is crucial for the project's economic viability and sustainability. The H treatment significantly increased the glucose content in molasses, and the dual-enzyme system effectively alleviated the resulting inhibition, thus increasing FOS yield. Previous studies have mostly used pure sucrose as a substrate for FOS production. From an environmental and economic perspective, utilizing inexpensive sucrose sources such as molasses is more advantageous. Although fungi such as Aspergillus exhibit high FTase activity, strains such as Aspergillus niger and Penicillium citrinum have not previously achieved ideal yields. This study achieved a breakthrough through technological optimization and provides a feasible strategy for the high-value utilization of molasses.

[0082] in conclusion

[0083] This study confirmed through experiments and structural analysis that glucose inhibits the FTase mutant and proposed that glucose may occupy the substrate / product channel, thereby hindering the reaction. To this end, a two-enzyme system co-expressing glucose isomerase was constructed to convert glucose to fructose, thus removing the blockage in the enzyme pathway and increasing the FOS conversion rate from 42.31% to 55.51%. The enzymatic properties and fermentation conditions of the two-enzyme system were optimized, and the optimal catalytic conditions were determined to be 45℃, pH 5.5, and 1 mM Ba. 2+ The optimal fermentation conditions were 25℃ and 1 mM IPTG induction. Finally, using pretreated molasses as a substrate, a two-enzyme system was employed to achieve efficient FOS production, increasing the FOS yield from 44.54% to 53.92% after fermentation optimization. This study provides an effective enzyme engineering strategy for enhancing FOS biosynthesis, expands the high-value utilization pathways of inexpensive raw materials, and contributes to promoting the high-quality development and sustainable future of the sugar industry in Guangxi Zhuang Autonomous Region.

[0084] Comparative Experiment 1: Comparison between the single FTase system and the two-enzyme system

[0085] To verify the glucose inhibition effect and compare it with the dual-enzyme system of this invention, the inventors conducted the following experiment:

[0086] 1. Strain construction: An Escherichia coli Rosetta(DE3) strain expressing only the FTase mutant (SEQ ID NO:1) was constructed, see Section 2.2 of Example 1; this strain was used to prepare a single FTase system;

[0087] 2. Reaction System: Two reaction systems were set up: the experimental group was a single FTase system (containing only purified FTase mutant), and the control group was the dual-enzyme system of this invention (containing purified FTase mutant and glucose isomerase (GI), with a molar ratio of 1:1). The reaction conditions were uniformly set as follows: 45℃, pH 5.5 (using 0.1 M phosphate buffer), 1 mM Ba... 2+ (Added in the form of BaCl2), shaken at 150 r / min, substrate was 500 g / L sucrose solution, total enzyme dosage was 1.0 U / mL, reaction time was 24 hours.

[0088] 3. Results Analysis: The 24-hour FOS conversion rate (FOS yield / initial total sucrose × 100%) was calculated. The results showed that the FOS conversion rate of the single FTase system was 42.31%, with a glucose concentration of 47.16% and a fructose concentration of 10.64% in the reaction solution; while the FOS conversion rate of the dual-enzyme system increased to 55.51%, with a significant decrease in glucose concentration to 20.66% and an increase in fructose concentration to 26.73%.

[0089] Conclusion: In a single FTase system, the accumulation of glucose during the reaction causes significant product inhibition, shifting the reaction equilibrium in the reverse direction and limiting FOS conversion. In contrast, the dual-enzyme system of this invention converts glucose to fructose in real time via GI, not only eliminating the inhibition but also providing an additional substrate, thereby significantly improving FOS conversion.

[0090] Comparative Experiment 2: Comparison of the effects of different molasses pretreatment methods on FOS yield

[0091] To compare the effects of different molasses pretreatment methods on FOS yield, the inventors conducted the following experiments:

[0092] 1. Pretreatment methods: Sugarcane molasses is treated using the following methods:

[0093] CK: Untreated, as follows: Take raw molasses and dilute it with deionized water to a total sugar concentration (calculated as sucrose) of 124.48 g / L, without any other treatment;

[0094] H: Adsorption-heat treatment (see Example 1, Section 2.6), specifically as follows: Take the diluted molasses solution, add 6% (w / v) powdered activated carbon, and stir and adsorb in a 60°C water bath for 30 minutes; then add 3% (w / v) diatomaceous earth, and continue stirring and adsorbing for 60 minutes; then centrifuge at 8000 r / min for 15 minutes, collect the supernatant, and filter it through a 0.45 μm filter membrane. The resulting clear liquid is for use.

[0095] P: Calcium phosphate treatment, as follows: Take the diluted molasses solution, adjust the pH to 3.0 with 85% phosphoric acid, stir for 30 minutes and then centrifuge (8000 r / min, 15 minutes); take the supernatant, adjust the pH to 6.0 with 10% calcium hydroxide suspension, add 0.1% polyacrylamide flocculant, stir for 30 minutes; then adjust the pH to 6.0 with 2 M hydrochloric acid, centrifuge and filter, and collect the clear liquid;

[0096] E: EDTA flocculation treatment, as follows: Take the diluted molasses solution, add 2 mM disodium ethylenediaminetetraacetate (EDTA-2Na), stir at room temperature to bind heavy metal ions for 12 hours; then add 1.5% (w / v) activated carbon, stir for 30 minutes, centrifuge and filter, and collect the clear liquid.

[0097] 2. Reaction System: Using the pretreated molasses solution from each group as the substrate (total sugar concentration uniformly converted to 124.48 g / L sucrose equivalent), the dual-enzyme system of this invention (pure enzyme, total enzyme dosage 1.0 U / mL, GI to FTase mutant molar ratio 1:1) was used under optimized conditions (45℃, pH 5.5, 1 mM Ba) 2+ The reaction was carried out at 200 r / min for 24 hours.

[0098] 3. Results Analysis: After the reaction was completed, samples were taken for HPLC analysis to determine the FOS yield and calculate the conversion rate. The results are as follows:

[0099] CK group: FOS conversion rate 41.45%;

[0100] Group H: FOS conversion rate 53.92% (optimal);

[0101] Group P: FOS conversion rate 17.13%;

[0102] Group E: FOS conversion rate 34.77%.

[0103] Conclusion: Adsorption-heat treatment (H) effectively removes pigments, colloids, and some metal ions from molasses, significantly improving substrate purity and enzyme accessibility, thus achieving the highest FOS conversion rate. Other methods either introduce new impurities or result in sugar loss due to harsh treatment conditions, leading to lower conversion rates.

[0104] Comparative Experiment 3: Effect of different enzyme molar ratios on FOS conversion rate

[0105] To determine the optimal synergistic ratio of glucose isomerase (GI) to fructosyltransferase mutant (FTase) in a two-enzyme system, this experiment investigated the effect of different molar ratios on catalytic efficiency.

[0106] 1. Enzyme preparation: GI and FTase mutants were obtained by purification. The concentration of the purified protein was determined using the BCA method, and the molar concentration was calculated based on its molecular weight.

[0107] 2. Reaction System Setup: Three experimental groups were set up, maintaining a consistent total protein molar concentration (10 μM), and adjusting the molar ratio of GI to FTase mutant to 0.5:1, 1:1, and 2:1, respectively. The reaction system consisted of: 500 g / L sucrose solution (dissolved in 0.1 M, pH 5.5 phosphate buffer), 1 mM BaCl2, and enzyme solution in appropriate proportions. The reaction was carried out at 45℃ and 200 r / min for 12 hours.

[0108] 3. Process monitoring and sampling: 0.5 mL samples were taken at 2, 4, 8, and 12 hours of reaction. The sugar composition was analyzed after the reaction was terminated. The FOS conversion rate at 12 hours of reaction was calculated.

[0109] 4. Results:

[0110] When the GI:FTase molar ratio is 0.5:1, the FOS conversion rate is 48.23%. At this point, glucose conversion is incomplete, and there is still some inhibitory effect.

[0111] When the GI:FTase molar ratio is 1:1, the FOS conversion rate reaches its highest value of 55.51%. The production and consumption of glucose and fructose reach a dynamic equilibrium.

[0112] When the GI:FTase molar ratio was 2:1, the FOS conversion rate decreased to 51.67%. This indicates that excess GI may compete for moisture in the reaction system or cause a shift in the reaction pathway.

[0113] Conclusion: When the molar ratio of GI to FTase mutant is 1:1, the two-enzyme system exhibits the best synergistic catalytic efficiency, most effectively relieving glucose inhibition and driving the reaction forward. This ratio was determined to be the optimal process parameter.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing fructooligosaccharides based on dual-enzyme co-expression, characterized in that, Includes the following steps: A dual-enzyme system co-expressing glucose isomerase and FTase mutant is used to catalyze the reaction of sucrose or sucrose-containing substrates; wherein the amino acid sequence of the FTase mutant is shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that, The glucose isomerase is derived from *Acidothermic*; the amino acid sequence of the glucose isomerase is shown in SEQ ID NO:

2.

3. The method according to claim 1, characterized in that, The FTase mutant was derived from Aspergillus niger, and its amino acid sequence was obtained by point mutation based on SEQ ID NO:

3.

4. The method according to claim 1, characterized in that, The molar ratio of glucose isomerase to FTase mutant in the dual-enzyme system is 0.5:1 to 2:

1.

5. The method according to claim 1, characterized in that, The catalytic reaction conditions are: temperature 40-50℃, pH 5.0-6.0, and the presence of 0.5-2 mM Ba in the reaction system. 2+ As a cofactor.

6. The method according to claim 1, characterized in that, The sucrose-containing substrate is sugarcane molasses.

7. The method according to claim 1, characterized in that, The total enzyme amount of glucose isomerase and FTase mutant in the dual-enzyme system is 0.5–2.0 U / mL.

8. The method according to claim 1, characterized in that, The catalytic reaction takes 2–24 hours.

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