With the advancement of biotechnology and increasing demand for effective anti-aging solutions, the cosmetics and skincare industries have seen a improved development toward products that target cellular oxidation. The free radical theory of aging identifies reactive oxygen species (ROS) as major leading factors to cellular aging. In response, catalase (CAT) and superoxide dismutase (SOD) have been widely studied for their role in mitigating oxidative damage. This project aims to develop a novel method to investigate CAT and SOD by enhancing the expression and combined activity of SOD and CAT enzymes using synthetic biology techniques. The genes encoding SOD and CAT were inserted into a plasmid vector and expressed in E.coli. Following bacterial transformation, colonies were screened, and positive clones were identified through PCR amplification and agarose gel electrophoresis. Enzymatic activity assays were conducted to confirm the expression and functions of the enzymes. The invention of this method largely improved and emphasized the effectiveness of the formation of SOD and CAT. The engineered SOD-CAT complex demonstrated a significance antioxidant activity, highlighting the potential for improved efficacy in neutralizing superoxide anions.

1. Amplification and Verification of the targeted Gene and plasmid backbone

First, the nucleotide sequences of sodB (SOD, GeneID: 944953) and katG (CAT, GeneID: 948431) were retrieved from the database and subjected to codon optimization specifically designed for efficient expression in Escherichia coli. Gene synthesis was then performed based on the optimized sequences. Subsequently, the pET28a plasmid vector was selected for cloning, and recombinant constructs were generated using the synthesized gene fragments. The resulting plasmids were further validated through standard molecular biology techniques. The results are presented in Figure 1: We successfully amplified the target genes SOD and CAT, along with the plasmid backbone fragments.

Fig.1 Agarose gel electrophoresis of target genes and linearized vector: SOD, CAT and pET28a plasmid.

2. Transformation using Escherichia coli DH5α, followed by monoclonal verification and DNA sequencing

Following PCR amplification, the target DNA fragments were excised from an agarose gel and purified to obtain the desired gene segments. Subsequently, the SOD and CAT genes were individually cloned into the pET28a plasmid vector via homologous recombination. The resulting recombinant plasmids were transformed into Escherichia coli DH5α competent cells, which were then plated on selective solid medium containing kanamycin and incubated overnight for the identification of positive clones.

Fig. 2 Verification of pET28a-SOD and pET28a-CAT plasmid construction by colony PCR and Sanger sequencing

Individual colonies from the obtained monoclonal cultures were selected for PCR verification. Electrophoresis results, as shown in Figure 2, revealed clear and intense bands corresponding to the expected PCR products, indicating preliminary success in plasmid construction. Positive clones were subsequently expanded through culturing and subjected to Sanger sequencing for further validation. The sequencing results confirmed both the accuracy and integrity of the recombinant plasmid constructs.

Fig .3 Transformation of BL21 competent cells and verification by colony PCR

Subsequently, for the two successfully constructed plasmids, we respectively introduced them into the competent cells of Escherichia coli BL21(DE3), and after screening on solid plates, we confirmed the final positive engineered bacteria through colony verification.

1. Induction and expression of pET28a-SOD and pET28a-CAT

Following successful construction of the expression vector, it was transformed into Escherichia coli BL21 (DE3) competent cells. The target engineered strains, BL21-pET28a-SOD and BL21-pET28a-CAT, were selected through culturing on solid medium and confirmed by colony PCR. These strains were then subjected to large-scale cultivation, followed by induction of protein expression with 1 mM IPTG. After cell lysis via ultrasonic disruption, the recombinant proteins were purified using His-tag affinity chromatography, yielding high-purity SOD (molecular weight approximately 21,289 Da) and CAT (molecular weight approximately 80,054 Da).

Fig. 4 SDS-PAGE gel electrophoresis was used to analyze the expression of SOD and CAT proteins, and nickel column affinity chromatography was employed.

As shown in the SDS-PAGE results in Figure 4, gradient elution with increasing imidazole concentrations (20 mM, 50 mM, 100 mM, and 150 mM) was employed during the purification process to effectively remove contaminating proteins. The target proteins were enriched through three consecutive elutions using 500 mM imidazole. The collected fractions from the 500 mM imidazole elution contained highly purified target proteins, which were subsequently used as samples for enzyme activity assays.

2. BCA assay to measure the concentration of SOD and CAT

Fig. 5 Determination of protein concentration by BCA method

The protein concentrations of the purified target proteins were determined using the BCA assay. A standard curve was constructed to calculate the precise concentrations of SOD and CAT based on sample absorbance values, which served as critical parameters for subsequent enzyme activity assays. The elution fractions from two independent purification batches yielded CAT concentrations of 60.94 μg/mL and 144.25 μg/mL, respectively, while SOD concentrations were determined to be 83.95 μg/mL and 94.07 μg/mL in the corresponding batches.

1. Detection of SOD enzyme activity by WST8 method

Fig. 6 Assay of superoxide dismutase (SOD) enzyme activity

To evaluate SOD enzyme activity individually, the WST-8 assay was utilized owing to its high sensitivity and stability. As shown in Figure 6, Blank1, serving as the positive control, contained a complete enzymatic reaction system capable of generating abundant superoxide anions. In contrast, Blank2, which lacked a functional superoxide anion generation system, produced no detectable signal and thus functioned as the negative control. The target protein treatment group exhibited significant superoxide anion scavenging activity, with clearance efficiency approaching that of the negative control, indicating robust enzymatic activity of the purified SOD. These results establish a solid foundation for the subsequent characterization of dual-enzyme synergistic effects.

2. The activity of CAT enzyme was detected by the hydrogen peroxide removal method.

The activity of CAT enzyme was assessed using a catalase (CAT) activity detection kit. This method indirectly measures CAT enzymatic activity by quantifying the residual hydrogen peroxide and its corresponding chromogenic product in the reaction system. As shown in Figure 7, the Blank group, serving as the negative control, contained no components capable of decomposing hydrogen peroxide, resulting in the highest hydrogen peroxide concentration and consequently the highest absorbance value. In contrast, both purified CAT protein samples demonstrated significant catalytic activity, reducing hydrogen peroxide levels by approximately 50%. These findings provide strong support for subsequent dual-enzyme co-activity assays.

Fig. 7 The determination of catalase activity

As shown in Figure 7, the absorbance of enzymatic products derived from residual hydrogen peroxide was measured at 520 nm to assess hydrogen peroxide levels, clearance capacity, and catalase activity. Compared with the blank control group, the purified CAT protein reduced hydrogen peroxide concentration by approximately 50%. Furthermore, both replicate preparations of purified CAT exhibited consistent enzymatic activity, indicating high batch-to-batch stability of the purified enzyme. This assay confirmed the functional activity of CAT as a single enzyme and provides a basis for its subsequent application in combination with other enzymes.

3. Orthogonal Experiment 1: Superoxide Anion Scavenging (Effects of Temperature, pH, and SOD/CAT Ratio)

Fig. 8 Orthogonal experiments verify the clearance of superoxide anions

A three-factor, three-level orthogonal experiment was conducted to systematically evaluate the effects of SOD/CAT ratio, temperature, and pH on free radical scavenging efficiency. By calculating the mean scavenging efficiency at each factor level and analyzing the corresponding ranges (R), it was determined that temperature exerted the most significant influence (R = 74.38). Specifically, the scavenging efficiency at 25°C was markedly higher than at 37°C and 45°C. At elevated temperature (45°C), a substantial decline in efficiency was observed, with some measurements yielding negative values, indicating thermal inhibition of antioxidant activity. The pH value exhibited the second-largest effect (R = 13.56), with alkaline conditions (pH 8.0) favoring enhanced scavenging performance, whereas pH 7.0 resulted in the lowest efficiency among the tested levels. The SOD/CAT ratio had a comparatively minor impact (R = 11.03). A ratio of 1:1 demonstrated the highest scavenging efficiency and the greatest consistency across replicates, while a ratio of 0.5:1 showed pronounced variability and frequent negative values, suggesting poor stability. A ratio of 2:1 yielded moderate but less optimal performance. Overall, the optimal condition was identified as an SOD/CAT ratio of 1:1, a temperature of 25°C, and a pH of 8.0, under which the system achieved peak scavenging efficacy. These findings provide a robust experimental basis for parameter optimization in the design of efficient antioxidant enzyme systems.

4. Orthogonal Experiment 2: ABTS⁺ Radical Scavenging Activity (Effects of Temperature, pH, and SOD/CAT Ratio)

Fig. 9 Orthogonal experiments verified the clearance of ABTS⁺

Based on a three-factor, three-level orthogonal experimental design, a systematic evaluation was conducted to investigate the effects of SOD/CAT ratio, temperature, and pH on antioxidant capacity, as quantified by the Trolox equivalent antioxidant capacity (TEAC) assay. By calculating the mean TEAC values across different levels of each factor and comparing their respective ranges (R), the results revealed that temperature exerted the most significant influence on antioxidant activity, followed by pH, whereas the SOD/CAT ratio had a relatively minor yet discernible effect. Specifically, the experimental data demonstrated that antioxidant capacity at 25°C was significantly higher than at 37°C and 45°C. Moreover, performance at pH 7.0 and 8.0 was superior to that at pH 6.0, indicating that lower temperatures and neutral-to-alkaline conditions are more favorable for enhancing antioxidant efficacy. With respect to the SOD/CAT ratio, although the highest average TEAC value was observed at a ratio of 2:1, the difference compared to the 1:1 ratio was modest (range ≈ 0.1), suggesting that both ratios contribute to antioxidant activity with only limited variation between them. Overall, the optimal combination of conditions was determined to be an SOD/CAT ratio of 2:1, a temperature of 25°C, and a pH of 7.0, under which the TEAC value reached its maximum of 0.6519. These findings provide a scientific basis for subsequent experimental design and process optimization. Furthermore, the results highlight the critical importance of precise control over temperature and pH, while allowing for flexible selection of the SOD/CAT ratio depending on practical requirements.

Based on the present study, superoxide dismutase (SOD) and catalase (CAT) have been successfully expressed, purified, and functionally validated as active enzymes in vitro. The antioxidant performance of the dual-enzyme system under varying conditions has been systematically assessed through orthogonal experimental design. Future research may be directed toward the following aspects: First, given that superoxide anion scavenging activity is optimal at 25°C, pH 8.0, and an SOD/CAT ratio of 1:1, further investigation into the system’s stability and kinetic behavior across broader temperature and pH ranges is recommended to enhance its applicability under diverse environmental or physiological conditions. Second, since the highest ABTS⁺ radical scavenging efficiency was observed at an SOD/CAT ratio of 2:1, 25°C, and pH 7.0, it is essential to elucidate the underlying synergistic mechanisms between the two enzymes, particularly the minimal performance variation between the 1:1 and 2:1 ratios, which could inform rational enzyme proportion optimization and cost-effective formulation development. Third, in vivo relevance should be evaluated by extending in vitro findings to cellular and animal models to assess the therapeutic efficacy and biosafety of the dual-enzyme system, thereby validating its translational potential. Finally, strategies such as protein engineering or advanced delivery systems (e.g., nanocarriers or targeted vectors) should be explored to improve enzymatic stability, bioavailability, and site-specific targeting, better meeting clinical or industrial requirements for oxidative stress intervention. Collectively, these research directions will provide a solid theoretical foundation and technical support for the rational design and application of dual-enzyme antioxidant systems.