Difference between revisions of "Part:BBa K5458002"

Latest revision as of 04:05, 30 September 2024

katA

Catalase is an enzyme that decomposes hydrogen peroxide into water and oxygen, protecting cells from oxidative damage by reactive oxygen species. It can be used in conjunction with laccases to enhance oxidation reactions.

Description

To further enhance laccase activity, literature suggests that the combination of catalase and laccase can effectively boost enzymatic efficiency. Based on this, we aimed to construct a bacterial strain capable of producing catalase to explore its potential in improving laccase activity. This experiment focuses on measuring the catalase activity of the engineered strain to assess its ability to decompose hydrogen peroxide.

Usage and Biology

We first selected and codon-optimized the catalase gene katA (BBa_K5458002) to ensure its efficient expression in E. coli BL21. The optimized gene sequence was synthesized by a biotechnology company. Next, we performed EcoRI and XhoI double digestion to insert the katA gene into the pET23b expression vector. After constructing the recombinant plasmid, it was transformed into E. coli BL21, and positive clones were confirmed via antibiotic selection and sequencing verification. The resulting strain, BL21/pET23b-katA, was successfully obtained.

Figure 1. The gene circuit of katA.

Characterization

Analysis of catalase activity

The engineered strains were centrifuged at 10,000 rpm for 1 min and resuspended in PBS (pH=7.4), followed by sonication to lyse the cells. After centrifugation at 10,000 rpm for 20 min at 4°C, the crude enzyme was collected. The total protein concentration was determined using the Bradford assay and adjusted to 100 μg/mL with PBS. A 3% hydrogen peroxide solution was diluted to 100 mM using distilled water. Then, 1 mL of the crude enzyme solution was incubated with 5 mM hydrogen peroxide at 37°C for 60 min. The remaining hydrogen peroxide content was measured at 415 nm using a hydrogen peroxide assay kit (BC3595, Solairebio).

Figure 2. Gel image of katA.

As shown in Figure 3, after 60 min of incubation at 37°C, partial decomposition of hydrogen peroxide was observed in the BL21/pET23b sample due to its inherent instability. However, in the BL21/p23b-katA sample, the hydrogen peroxide concentration significantly decreased, indicating efficient catalase activity.

Figure 3. Effect of PBS, BL21/PET23b, and BL21/p23b-katA on hydrogen peroxide concentration.

Catalase increases dissolved oxygen content

To determine whether catalase increased oxygen concentration in the solution, we added hydrogen peroxide to the catalase solution and incubated it for 60 minutes. After the reaction, the oxygen concentration in the solution was measured using a dissolved oxygen analyzer. The results demonstrated that catalase significantly increased the oxygen concentration.

Figure 4. Effect of PBS, BL21/PET23b, and BL21/p23b-katA on oxygen concentration.

The combination of catalase and laccase promotes indigo degradation

We then tested whether the combination of catalase and laccase could improve the degradation of indigo. Laccase and catalase crude enzyme solutions were mixed with 1 mM indigo and 5 mM hydrogen peroxide, and the reaction was incubated at 37℃ for 1 hour. The absorbance at 620 nm was measured to determine the indigo degradation efficiency. The results showed that adding hydrogen peroxide alone did not significantly enhance laccase activity and, in fact, led to a reduction in indigo degradation efficiency due to high concentrations of hydrogen peroxide interfering with the laccase’s active site. The combination of catalase and laccase effectively enhanced indigo degradation compared to either enzyme alone.

Figure 5. The combination of catalase and laccase promotes indigo degradation.

Catalase promotes indigo biocatalyst activity

To further investigate the role of catalase in promoting laccase activity, we performed indigo degradation tests in engineered strains. Engineered strains were cultured in M9 medium for 12 hours, and then indigo, catalase, and hydrogen peroxide were added. After incubating for 1 hour, the absorbance at 620 nm was measured. The results demonstrate that the combination of hydrogen peroxide and catalase can effectively enhance the activity of laccase, leading to improved degradation of indigo.

Figure 6. Catalase promotes indigo biocatalyst activity.

Potential application directions

Active hydrogen peroxide enzyme effectively degrades hydrogen peroxide, hydrogen peroxide enzyme effectively increases the solubility of oxygen, hydrogen peroxide enzyme and hydrogen peroxide promote the degradation of indigo, and indigo biocatalyst activity. Therefore, fture teams can enhance the efficiency of breaking down indigo by using catalase.

References

Wei J, Yang L, Feng W. Efficient oxidation of 5-hydroxymethylfurfural to 2, 5-furandicarboxylic acid by a two-enzyme system: Combination of a bacterial laccase with catalase[J]. Enzyme and Microbial Technology, 2023, 162: 110144.

Sequence and Features