The objective of our project is to explore the potential of enzyme preparations in antioxidant and skin anti-aging products. Therefore, through information research, we confirmed that SOD enzyme and CAT enzyme would be our research subjects. Based on the DBTL (Design-Build-Test-Learn) cycle concept in engineering, this project systematically conducted research on the construction and functional verification of the antioxidant enzyme system. The specific work included:
Abstract：
Taking the advantages of Escherichia coli as the expression host as a priority, we screened the SodB gene encoding superoxide dismutase (SOD) and the KatG gene encoding catalase (CAT). To improve the efficiency of heterologous expression and protein yield, codon optimization was carried out for the Escherichia coli expression system, while considering factors such as the secondary structure of the gene sequence and GC content to ensure the optimal transcription and translation efficiency of the genes in the host.
During the process of protein synthesis, codons play a crucial role in converting genetic information into the amino acid sequence of proteins. Different species often use different codons when translating the same amino acid, and this phenomenon is known as the species-specific preference of codons. Although the specific mechanism for the natural formation of codon preference is not yet fully understood, it has a significant impact on the efficiency of protein expression. Especially in the expression of recombinant proteins, to enhance the expression level, the codon usage habits of the host species are usually optimized for the exogenous gene sequence. This strategy is particularly crucial in heterologous protein expression systems because the target gene comes from different species and must adapt to the translation mechanism of the host to ensure efficient expression. In addition, codon optimization can also improve the cloning efficiency of DNA, for example, by adjusting the CG content and reducing repetitive sequences, to achieve more efficient gene construction. At the same time, codon optimization can enhance the stability of mRNA, improve the efficiency of transcription and translation, and thereby comprehensively promote the expression of the target protein. Based on the original gene sequence of sodB and KatG, we optimized its sequence through an online codon optimization platform. On this basis, we carried out subsequent related work. We chose the pET28a vector for integration construction.
Fig .1 Design and SnapGene diagram of pET28a-sodB and pET28a-KatG
Abstract：
Based on molecular cloning technology, the optimized SodB and KatG genes were respectively cloned into the high-expression vector pET28a, and recombinant plasmids were constructed. Through transformation, the screening and amplification of Escherichia coli engineering strains, a stable and efficient recombinant strain library was established. Subsequently, using the IPTG induction expression system, the expression conditions were optimized to obtain highly pure and fully functional recombinant enzyme proteins, laying the foundation for subsequent functional determination.
The sodB (SOD, GeneID: 944953) and katG (CAT, GeneID:
948431) sequences were retrieved and codon-optimized for efficient expression in Escherichia coli. The genes were
synthesized and cloned into the pET28a vector to generate recombinant plasmids, which were validated using standard
molecular biology techniques. As shown in Figure 2, target genes SOD and CAT, along with the plasmid backbone, were
successfully amplified
.
Fig.2 Agarose gel electrophoresis of target genes and linearized vector: SOD, CAT and pET28a linearized plasmid.
Transformation using Escherichia coli DH5α, followed by monoclonal verification and DNA sequencing
After PCR amplification, the target DNA fragments were carefully excised from an agarose gel and purified to isolate the desired gene segments. The SOD and CAT genes were then gently cloned into the pET28a plasmid vector using homologous recombination—a smooth and precise method that helps ensure accuracy. The resulting recombinant plasmids were introduced into competent Escherichia coli DH5α cells, which were plated on kanamycin-containing selective medium and incubated overnight. This step allowed us to identify positive clones with care and confidence, bringing us one step closer to our goal.
Individual colonies from monoclonal cultures were selected for PCR verification. Electrophoresis results (Fig. 3) showed clear, strong bands matching the expected PCR products, confirming initial plasmid construction success. Positive clones were cultured for expansion and verified by Sanger sequencing, which confirmed the accuracy and integrity of the recombinant plasmids.
Fig. 3 Verification of pET28a-SOD and pET28a-CAT plasmid construction by colony PCR and Sanger sequencing
Building on this foundation, the recombinant plasmid was gently introduced into competent Escherichia coli BL21(DE3) cells. As shown in the Fig. 4, Through careful screening on antibiotic-containing plates, we successfully identified positive clones, which were then confirmed by colony PCR. With confidence in their accuracy, these engineered strains were carried forward for target protein expression and functional validation, marking an important step toward our research goals.
Fig .4 Transformation of BL21 competent cells and verification by colony PCR
Strictly combining biochemical and biophysical methods, a series of in vitro functional evaluations were carried out on the purified SOD and CAT proteins. Through enzymatic activity detection, the scavenging efficiency of superoxide anions and hydrogen peroxide was quantified. Further, orthogonal experiments were conducted to optimize the enzyme ratio, pH, temperature, etc., to systematically characterize the synergistic antioxidant performance of the dual enzyme system under different environmental conditions and establish a reliable functional evaluation system.
For the well-constructed engineered bacterial strains, our next goal is to test and evaluate the production and expression of SOD and CAT enzymes based on the design objectives of the project. We will further conduct tests and research on the effects of single enzyme activity and different environmental factors on the activity of double enzymes.
Firstly, we conducted large-scale cultivation and induction expression of the obtained engineered bacteria and extracted the cell proteins through ultrasonic disruption. At the same time, for the crude protein obtained from the protein supernatant, we used a His-tag purification column to purify the target protein and then analyzed the effect of protein purification through SDS-PAGE electrophoresis. The results are shown in Figure 5. We successfully induced the expression of the constructed BL21-pET28a-SOD and BL21-pET28a-CAT and used gradient imidazole for purifying the impurities and eluting the target protein.
Fig. 5 SDS-PAGE gel electrophoresis was used to analyze the expression of SOD and CAT proteins, and nickel column affinity chromatography was employed.
The concentrations of the purified SOD and CAT
proteins were determined by BCA assay using a standard curve based on absorbance values, providing key parameters for
subsequent enzyme activity assays. The two purification batches yielded CAT concentrations of 60.94 μg/mL and 144.25
μg/mL, and SOD concentrations of 83.95 μg/mL and 94.07 μg/mL.
Fig. 6 BCA assay for the test of protein concentration
SOD activity was evaluated using the WST-8 assay due to its high sensitivity and stability. As shown in Figure 7, Blank1 (positive control) contained a complete system for generating superoxide anions, while Blank2 (negative control), lacking this system, produced no detectable signal. The target protein group showed strong superoxide scavenging activity, with clearance efficiency close to that of the negative control, confirming robust SOD activity. These results support further analysis of dual-enzyme synergy.
Fig. 7 Assay of superoxide dismutase (SOD) enzyme activity
CAT activity was measured using a catalase activity
detection kit, which indirectly quantifies enzyme activity by detecting residual hydrogen peroxide and its chromogenic
product. As shown in Figure 8, the Blank group (negative control) lacked components to decompose hydrogen peroxide,
resulting in the highest absorbance. In contrast, both purified CAT samples reduced hydrogen peroxide levels by
approximately 50%, demonstrating significant catalytic activity. These results support subsequent dual-enzyme
co-activity assays.
Fig. 8 The determination of catalase activity
A three-factor, three-level orthogonal experiment was used to evaluate the effects of SOD/CAT ratio, temperature, and pH on free radical scavenging efficiency. Based on mean efficiency and range analysis (R), temperature had the greatest influence (R = 74.38), with peak performance at 25°C. Efficiency dropped sharply at 45°C, with some negative values indicating thermal inhibition. pH showed the second-largest effect (R = 13.56), with optimal activity at pH 8.0 and lowest at pH 7.0. The SOD/CAT ratio had the smallest impact (R = 11.03); the 1:1 ratio yielded the highest and most consistent efficiency, while 0.5:1 resulted in high variability and frequent negative values, suggesting instability. A 2:1 ratio gave moderate results. The optimal conditions were determined to be 1:1 SOD/CAT ratio, 25°C, and pH 8.0, under which the system achieved maximum scavenging efficacy. These results provide a solid basis for optimizing antioxidant enzyme systems.
Fig. 9 Orthogonal experiments verify the clearance of superoxide anions
This assay used a three-factor, three-level experiment to examine how SOD/CAT ratio, temperature, and pH affect antioxidant ability measured by Trolox equivalent antioxidant capacity (TEAC). Results showed temperature had the biggest impact, followed by pH, while SOD/CAT ratio had a smaller effect. Antioxidant activity was highest at 25°C and better at pH 7.0 and 8.0 than at pH 6.0, indicating cooler and neutral-to-alkaline conditions are best. The 2:1 SOD/CAT ratio gave slightly better results than 1:1, but both worked well. The best combination was SOD/CAT = 2:1, temperature 25°C, and pH 7.0, reaching a TEAC of 0.6519. These results guide future experiments and process control, stressing careful temperature and pH management and flexible SOD/CAT ratios as needed.
Fig. 10 Orthogonal experiments verified the clearance of ABTS+
Based on the experimental data analysis, the intrinsic relationship between gene optimization, expression level, and enzyme activity was deeply analyzed. The sensitivity of process parameters was scientifically summarized, forming a theoretical and technical framework for enzyme preparations in antioxidant and skin anti-aging applications. The research results not only verified the feasibility of the recombinant enzyme system but also provided data support and guidance for subsequent protein engineering modifications, improvement of enzyme catalytic efficiency and stability, and laid a foundation for the development and industrial application of functional enzyme preparations.
