Result
Plasmid construction result
The AgaA gene encoding agarase has a length of 1596 bp. As shown in Fig. 1.A, the target fragment of AgaA was successfully amplified by PCR. The target fragment was recovered using a gel extraction technique. Both the plasmid pET-32a(+) and the gel-extracted AgaA target fragment were double-digested with restriction enzymes. Fig. 1.B & C indicate that both the vector plasmid and the target fragment were successfully cut.
The digested products were ligated using T4 DNA ligase, and the ligation products were transformed into competent E. coli TOP10 cells (Fig. 2A). The results of colony PCR, as shown in Fig. 2B, demonstrate that the plasmid was successfully constructed and had been introduced into the cloned strain.
The constructed plasmid AgaA_pET-32a(+) was then subjected to gene sequencing. The sequencing results were aligned with the AgaA gene sequence, confirming that the gene sequence of the successfully constructed expression plasmid was correct, with no mutations occurring during the construction process. The plasmid is now ready for subsequent experiments.
Expression and purification of AgaA result
The AgaA_pET-32a plasmid was transformed into E. coli Rosetta (Fig. 3A), and as shown in Fig. 3B, the plasmid was successfully transferred into the expression strain Rosetta.
We induced expression at 16°C for 18 hours with IPTG at a final concentration of 0.5 mM. Protein expression was detected using SDS-PAGE, and Fig. 4A demonstrates that the target protein was successfully expressed and that the soluble protein can be used for subsequent experiments. The concentration of AgaA before purification was determined using the Bradford assay, resulting in a final protein concentration of 3.09 mg/mL and a total yield of 61.8 mg, which will be used for subsequent production of the new agarooligosaccharide NAOS. The expression strain was then scaled up to 100 mL to express the protein, which was purified using Ni-NTA affinity chromatography (Fig. 4B). The figure indicates that the target protein can be purified using this method, with the wash buffer being chosen as 100 mM imidazole buffer and the elution buffer as 300 mM imidazole buffer.
Enzyme activity detection result
As observed in Fig. 5A, depressions appeared on the LB solid medium where the E. coli Rosetta AgaA_pET-32a(+) strain was cultured, indicating that this expression strain is capable of degrading agar, thereby demonstrating agarase activity. After inducing expression in this strain, the AgaA enzyme was obtained, and further enzyme activity verification is required. A mixture of 200 µl of AgaA and 200 µl of agarose (2%) was incubated in a 55°C metal bath for 30 minutes. The reaction was then terminated by heating at 95°C for 10 minutes. Next, 200 µl of the reaction mixture was transferred to a new 2 mL EP tube, and 200 µl of DNS reagent was added and mixed. The mixture was heated in a 95°C metal bath for 5 minutes (the control group used pre-inactivated AgaA). The color change between the control group and the experimental group was observed. As shown in Fig. 5B, the color of the experimental group significantly darkened, indicating the production of reducing sugars, specifically NAOS, in the reaction. This experimental result confirms that the heterologously expressed AgaA in E. coli has biological activity
Analysis of products of AgaA hydrolyzed agarose
The products of agarose hydrolysis by the AgaA enzyme were analyzed by HPLC. The standard sample was a mixture of neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6). The results showed that the retention times of NA2, NA4 and NA6 were 6.75 min, 12.4 min and 23.5 min, respectively, and the chromatogram of the experimental group and the control group showed that the hydrolyzed products of AgaA enzyme were NA2 and NA4. Previous experiments have shown that the products of AgaA hydrolysis of agarose are NA2, NA4 and NA6, but with the progress of the experiment, NA6 will be gradually hydrolyzed to NA2 and NA4, which proves that the enzyme reaction is relatively thorough, NA6 has been hydrolyzed to NA2 and NA4.
Analysis of oxidation resistance of AgaA hydrolysate
The antioxidant properties of new AgaA oligosaccharides (NAOS) were evaluated by ABTS method. Total antioxidant capacity (T-AOC) test kit was used to determine the T-AOC. The experimental principle is: After oxidation, 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) forms a stable blue-green cationic radical, called ABTS+, soluble in water or acidic ethanol solution, showing a maximum absorbance at 734 nm. When a substance with antioxidant properties is added to the ABTS+ solution, its antioxidant components react with ABTS+, resulting in a decrease in color intensity
As shown in Fig. 6, the antioxidant detection reagent containing the AgaA hydrolysis products exhibited a significant decolorization reaction compared to the control group. This indicates that the NAOS produced by AgaA-mediated hydrolysis of agarose possess notable antioxidant activity.
