1. Plasmid Construction Results

The gene encoding α-agarase (AgaE) has a length of 2.8 kp, and the gene encoding endo-inulinase (Inulinase) is 1.5 kp in length. As shown in Figs. 1A and 1C, the amplified fragment sizes of AgaE and Inulinase are consistent with the expected sizes, indicating that the two target gene fragments were successfully obtained via PCR. Subsequently, the correctly amplified fragments were purified by gel extraction. The vector plasmid pET-22b(+) and the recovered PCR products were separately subjected to double digestion using EcoRI and XhoI. Results in Figs. 1B, 1D, and 1E demonstrate that both the vector plasmid and the two target fragments were effectively digested. The digested vector plasmid and target fragments were then purified again by gel extraction.

(A) PCR amplification of the AgaE gene fragment. M: DNA marker; 1-3: AgaE PCR products. (B) Enzymatic digestion of the AgaE PCR product. M: DNA marker; 1: Digestion product of the gel-purified AgaE PCR product. (C) PCR amplification of the Inulinase gene fragment. M: DNA marker; 1-2: Inulinase PCR products. (B) Enzymatic digestion of the Inulinas PCR product. M: DNA marker; 1-2: Digestion product of the gel-purified Inulinase PCR product. (E) Enzymatic digestion of the pET-22b (+) vector. M: DNA marker; 1-2: Digestion product of the pET-22b (+) plasmid; 4: Negative control (undigested pET-22b (+) plasmid).

The digested target fragments and the vector plasmid pET-22b(+) were ligated separately using T4 DNA ligase, and the ligation products were transformed into E. coli TOP10 competent cells . After cultivation, the growth results of the transformed strains are shown in Figs. 2A and 2C. Single colonies were picked separately for colony PCR identification. Results in Figs. 2B and 2D confirm that the recombinant plasmids AgaE_pET-22b(+) and Inulinase_pET-22b(+) were successfully constructed and have been smoothly introduced into the cloning strains. The successfully constructed recombinant plasmids AgaE_pET-22b(+) and Inulinase_pET-22b(+) were subjected to gene sequencing respectively. The sequencing results show that both are completely consistent with the original gene sequences, indicating that the recombinant plasmids were constructed correctly and no mutations occurred during the cloning process. The above plasmids can be used for subsequent protein expression experiments or functional studies.

(A) Transformation of AgaE_pET-22b (+) into E. coli TOP10. (B) PCR verification of the cloned strains. M: DNA marker. (C) Transformation of Inulinase_pET-22b (+) into E. coli TOP10. (D) PCR verification of the cloned strains. M: DNA marker.

2. Expression Results of AgaE and Inulinase

The plasmids AgaE_pET-22b(+) and Inulinase_pET-22b(+) were transformed into E. coli Rosetta separately (Fig. 3C). As shown in Figs. 3D and 3B, both plasmids were successfully transferred into the expression strain Rosetta.

(A) E. coli Rosetta AgaE_pET-22b(+) Transformation Results. (B) E. coli Rosetta AgaE_pET-22b(+) PCR Results. M: DNA marker; (C) E. coli Rosetta Inulinase_pET-22b(+) Transformation Results. (D) E. coli Rosetta Inulinase_pET-22b(+) PCR Results. M: DNA marker.

For the expression of AgaE, IPTG with a final concentration of 0.025 mM was used for induction at 20°C for 18 hours. SDS-PAGE analysis (Fig. 4A) indicates that the target protein was successfully expressed, and its soluble fraction meets the requirements for subsequent experiments. The protein concentration of the AgaE enzyme solution before purification was determined to be 2.09 mg/mL using the Bradford method, with a total protein yield of 37.62 mg, which can be used for the subsequent synthesis of agarooligosaccharides (AOS).

For the expression of Inulinase, IPTG with a final concentration of 0.5 mM was used for induction at 20°C for 18 hours. SDS-PAGE results (Fig. 4B) show that the target protein was successfully expressed, and its soluble fraction can be used for subsequent experiments. The protein concentration of the enzyme solution before purification was 3.59 mg/mL as measured by the Bradford method, with a total protein yield of 64.62 mg. This enzyme solution will be used for the preparation of fructooligosaccharides (FOS).

(A) Expression of AgaE_pET-22b(+) protein. M: Protein marker. (B) Expression of Inulinase protein. M: Protein marker.

3. Enzyme Activity Detection Results

To quantitatively evaluate enzyme activity, the 3,5-dinitrosalicylic acid (DNS) colorimetric method was employed in this study. The principle is as follows: under alkaline conditions, DNS reacts with reducing sugars to form a reddish-brown product with characteristic absorption at a specific wavelength, and the absorbance value is positively correlated with the content of reducing sugars. Since the enzymatic hydrolysis products of both AgaE and Inulinase have reducing ends, this method is suitable for the accurate determination of the activities of the two enzymes.

1) Standard Curve Preparation

A standard curve of reducing sugar concentration versus absorbance in the DNS method was prepared using D-fructose standard samples. Fructose standard solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg/mL were prepared separately. A 200 μL aliquot of each solution was placed in a 2 mL centrifuge tube, and an equal volume of DNS solution was added and mixed. The mixed samples were reacted in a boiling water bath for 5 minutes, followed by immediate cooling in an ice bath to terminate the reaction (Fig. 5A). Subsequently, 1 mL of deionized water was added to each tube for dilution, and the absorbance value was measured at a wavelength of 540 nm using a microplate reader. A linear regression equation was established with the fructose concentration as the abscissa and the absorbance (OD value) as the ordinate, and the correlation coefficient was calculated to plot the reducing sugar standard curve. As shown in Fig. 5B, the standard curve equation of the DNS solution is: y = 2.79x + 0.09, with a correlation coefficient R² = 0.9969 > 0.99, indicating a good fitting effect of the standard curve.

(A): Reaction solution of D-fructose solution and DNS reagent; (B): Standard curve.

2) Enzyme Activity Determination

The experimental procedure was as follows: 3 mL of AgaE enzyme solution (or heat-inactivated control enzyme solution) was mixed with 2 mL of 2% agarose solution and reacted at 35°C for 30 minutes; separately, 3 mL of Inulinase enzyme solution (or heat-inactivated control enzyme solution) was mixed with 2 mL of 2% inulin solution and reacted at 55°C for 30 minutes. After the reaction, the samples were heat-treated at 95°C for 10 minutes to terminate the reaction. A 200 μL aliquot of each reaction solution was taken, mixed with an equal volume of DNS reagent, reacted at 95°C for 5 minutes, and then cooled to room temperature to observe the color development.

Compared with the control group, the experimental groups of AgaE and Inulinase showed significantly darker color development, indicating a significant increase in the production of reducing sugars. This confirms that AgaE can effectively degrade agarose to generate agarooligosaccharides (AOS), and Inulinase can efficiently catalyze inulin to produce fructooligosaccharides (FOS), with both enzymes exhibiting the expected biocatalytic activity. The absorbance value (OD₅₄₀) of each group of samples was measured using a microplate reader, and the reducing sugar yield was calculated using the aforementioned standard curve: AOS was 0.81 mg/mL, and FOS was 5.41 mg/mL (Fig. 7).

(A) AgaE enzyme activity assay using the DNS method (left: experimental group; right: control group). (B) Inulinase enzyme activity assay using the DNS method (left: experimental group; right: control group).

4. Product Analysis

High-performance liquid chromatography (HPLC) was used to analyze the products of agarose hydrolysis by AgaE. A mixed standard of agarobiose (A2), agarotetraose (A4), and agarohexaose (A6) was used as a reference, with retention times of 10.26 minutes, 18.97 minutes, and 36.81 minutes, respectively. As shown in Fig. 8A, significant characteristic peaks appeared in the chromatogram of the experimental group at 18.97 minutes and 36.81 minutes, confirming the production of A4 and A6 in the product.

Similarly, HPLC was employed to analyze the products of inulin hydrolysis by Inulinase. A mixed standard of 1-kestose (GF2) and nystose (GF3) was used as a reference, with retention times of 23.49 minutes and 34.88 minutes, respectively. By comparing the chromatograms of the experimental group and the control group (Fig. 8B), obvious new peaks were observed at the corresponding retention times, indicating that the product contained GF2 and GF3.

5. Product Activity Verification

Prebiotics are substances that can be selectively utilized by host microorganisms to confer health benefits. In this experiment, the prebiotic activities of FOS and AOS were systematically verified by evaluating their promoting effects on the in vitro growth of two lactic acid bacteria strains—Lactobacillus acidophilus (derived from pharmaceuticals) and Lactobacillus casei LPC100 (isolated from Weiquan beverages). Under anaerobic conditions, both lactic acid bacteria strains can metabolize specific oligosaccharides as carbon sources. The degree of their proliferation was quantified by measuring the absorbance (OD₆₀₀) of the culture medium at a wavelength of 600 nm; an increase in this value directly reflects an increase in bacterial biomass, which was used to evaluate the prebiotic potential of FOS and AOS.

Single colonies were isolated and purified from L. acidophilus tablets and Weiquan beverages using the spread plate method. After identification, they were confirmed to be Lactobacillus acidophilus and Lactobacillus casei LPC100. Typical single colonies were picked and inoculated into liquid MRS medium, followed by anaerobic culture at 37°C for 24 hours to obtain seed cultures in the logarithmic growth phase. Fresh MRS medium was used, and the following groups were set up for treatment (with 3 replicates per group):

- Control group: 6 mL MRS medium + 700 μL PBS buffer + 100 μL seed culture;

- FOS group: 6 mL MRS medium + 600 μL PBS buffer + 100 μL FOS solution (5.41 mg/mL) + 100 μL seed culture;

- AOS group: 6 mL MRS medium + 100 μL PBS buffer + 600 μL AOS solution (0.81 mg/mL) + 100 μL seed culture;

- FOS+AOS group: 6 mL MRS medium + 50 μL FOS solution + 300 μL AOS solution + 350 μL PBS buffer + 100 μL seed culture.

All groups were cultured in a shaker at 37°C for 24 hours and 48 hours, respectively. After cultivation, 200 μL of the culture medium was transferred to a 96-well plate, and the OD₆₀₀ value was measured using a microplate reader. Uninoculated MRS medium was used as a blank control for zero adjustment. Data showed that compared with the control group, the OD₆₀₀ values of the FOS group, AOS group, and FOS+AOS group were significantly higher after 24 hours and 48 hours of culture (Figs10&12). This indicates that both FOS and AOS can effectively promote the proliferation of Lactobacillus acidophilus and Lactobacillus casei LPC100, demonstrating significant prebiotic activity.