Latest revision as of 12:50, 1 October 2024

INP-Bpul

We modified the existing part accase gene BBa_K863001 using surface display technology, resulting in a new part INP-Bpul (BBa_K5458001). Laccases could bleach indigo dyed denim fabrics to lighter shades. The N-terminal domain of ice crystal nucleoprotein (INP-N) can carry Laccases and anchor on the surface of E. coli cell membranes.

Description

Laccases (EC 1.10.3.2) are a class of copper-containing enzymes that can catalyze the oxidation of various substances while simultaneously reducing oxygen. Many white-rot fungi secrete laccases, and over 60 species from different categories of fungi have been reported to produce these lignin-degrading enzymes(Gianfreda et al., 1999). Laccases remove hydrogen atoms from the hydroxyl groups of ortho- and para-substituted mono- and polyphenolic substrates, as well as aromatic amines, through single-electron abstraction to form free radicals. These free radicals can further undergo depolymerization, repolymerization, demethylation, or quinone formation. Laccases exhibit relatively broad substrate specificity toward electron donors. The addition of redox mediators such as ABTS, HBT, or compounds secreted by lignin-degrading fungi can further expand their substrate range, as these mediators transfer electrons between the enzyme and the substrate(Wei et al., 2023). In our program, laccases could bleach indigo dyed denim fabrics to lighter shades. We started with the existing part laccase gene BBa_K863001 and successfully modified it to create new part INP-Bpul (BBa_K5458001). Considering the activity of laccase and its ability to degrade indigo, we applied a surface display technique to present laccase on the bacterial cell surface(Mo et al., 2022). In our engineered bacterium, we utilized the INP (ice nucleation protein) gene sequence from Pseudomonas syringae KCTC1832 (NCBI Reference Sequence No. AF013159), fusing it with the gene sequence encoding the exogenous functional protein, laccase. The fusion protein is expressed using the pET23b vector and is targeted to specific structures on the cell surface, allowing laccase to be displayed directly on the host cell’s exterior. This surface expression enhances the degradation of indigo by increasing the accessibility and activity of laccase(Li et al., 2021).

Figure 1. Schematic diagram of laccase displayed on E. coli BL21 cell surface for indigo degradation.

Usage and Biology

We selected the laccase gene and introduced an INP truncation sequence upstream of Bpul, which was successfully cloned into the pET23b plasmid (using EcoRI and XhoI cleavage sites). Subsequently, the recombinant plasmid was transformed into E. coli BL21, which was validated by resistance plate screening and sequencing, and finally the engineered strain BL21/INP-Bpul(BBa_K5458001) was obtained.

Figure 2. The gene circuit of INP-Bpul.

Characterization

Surface display of laccase-engineered bacteria and activity in different cellular fractions

Figure 3. Gel image of INP-Bpul.

First, a truncated INP sequence was inserted upstream of thelaccase gene and cloned into the pET23b plasmid. The recombinant plasmid was then transformed into E. coli BL21 to generate the engineered strain BL21/INP-Bpul (BBa_K5458001). The transformation was verified using resistance plate screening and sequencing (Figure 3). To measure laccase activity in the cell membrane and cytoplasm, 100 mL of overnight-cultured engineered strain was collected, and the OD600 was measured. Cells were centrifuged at 10,000 rpm for 1 min and resuspended in 20 mL of BR buffer (pH=5). After sonication to disrupt the cells, centrifugation was performed at 10,000 rpm for 20 min at 4℃. A 5 mL sample of the supernatant was collected as the intracellular fraction. Ultracentrifugation at 39,000 rpm for 1 h was performed to separate cellular components, with the supernatant representing the cytoplasmic fraction and the pellet resuspended in 1 mL of BR buffer (pH=5) representing the cell membrane fraction. Finally, 0.1 mM ABTS was added to 200 μL of each sample, incubated at 37℃, pH=5, and the absorbance at 420 nm was measured using a microplate reader to determine the enzyme activity in both the cell membrane and cytoplasm. Results showed that the cytoplasmic laccase activity in BL21/Bpul was higher than in BL21/INP-Bpul, while the surface-displayed laccase activity in BL21/INP-Bpul was significantly higher on the cell membrane compared to BL21/Bpul. This indicates that surface display enhanced laccase activity on the membrane.

Figure 4. Effect of surface display on laccase activity in engineered bacteria (cellular content VS cell membrane).laccase activity was compared between BL21/Bpul and BL21/INP-Bpul strains across different cell fractions. In the cytoplasmic fraction, BL21/Bpul exhibited significantly higher absorbance compared to BL21/INP-Bpul, indicating stronger activity in cells without surface display. However, in the cell membrane fraction, BL21/INP-Bpul showed significantly higher absorbance than BL21/Bpul, suggesting that surface-displayed laccase has enhanced activity on the cell membrane.

Analysis of indigo degradation by engineered bacteria

This experiment aims to test how laccase-engineered bacteria, using surface display technology, degrade indigo. Live bacteria will be used in the degradation process. The engineered bacteria were first inoculated into 5 mL of M9 medium (Na2HPO4 3.0 g/L, KH2PO4 0.5 g/L, NaCl 1.0 g/L, NH4Cl 1.0 g/L, MgSO4 5.0 mM, CaCl2 0.1 mM, and supplemented with 10 g/L glucose) at a ratio of 1:100. The pH was adjusted using a pH meter (Mettler Toledo). After 12 h, the OD600 was measured and adjusted to OD600 = 1. Then, 1 mL of the bacterial solution was taken, and 1 mM indigo solution was added. Finally, the samples were incubated at 37°C, pH=5, and the absorbance at 620 nm was measured every 2 h. The experimental data were compared with BL21/pET23b and BL21/Bpul. The addition of surface display technology enhanced laccase activity, and BL21/INP-Bpul showed stronger indigo degradation compared to BL21/Bpul without surface display technology.

Figure 5. Degradation effect of BL21/pET23b, BL21/Bpul, and BL21/INP-Bpul on Indigo over time. As shown in Figure 5, BL21/pET23b had no degradation effect on indigo after 8 h, and the curve showed no significant changes. However, both BL21/Bpul and BL21/INP-Bpul showed a decreasing trend in indigo concentration at 8 h, with BL21/INP-Bpul exhibiting a greater reduction in indigo concentration compared to BL21/Bpul.

Bleaching experiment on jeans

In this experiment, we used enzyme powder to bleach a dark denim fabric. First, we sprinkled the enzyme powder directly onto the fabric. Then, using a sponge dipped in water, we scrubbed the upper half of the denim repeatedly. Afterward, the fabric was left to air dry, allowing the bleaching effect to develop. Finally, the results were observed and displayed. Our project demonstrates that using laccase to bleach denim is effective.

Figure 6. Pre-treatment and post-treatment photos of the dark denim bleaching process.

The existing part BBa_K863001 characterization

We modified the existing part accase gene BBa_K863001 using surface display technology, resulting in a new part INP-Bpul (BBa_K5458001).

Laccase activity assay

Figure 7. Gel image of the Bgls and Bpul. The Bgls gene is related to cellulase and will be studied in later experiments.

The experimental procedure was as follows: 100 mL of overnight culture of the engineered strain was collected, and its OD600 was measured. After centrifugation at 10,000 rpm for 1 min, the bacterial cells were resuspended in 20 mL of Britton-Robinson (BR) buffer and then sonicated to disrupt the cells. The supernatant was collected after centrifuging at 10,000 rpm for 20 min at 4℃, yielding the crude enzyme solution. 100 μL of the crude enzyme solution was taken, and the total protein concentration was determined using the Bradford assay, adjusting the concentration to 100 μg/mL. To assess laccase activity, 1 mM ABTS was used as the substrate, and the reaction was incubated at 37℃. The oxidation of ABTS, indicated by an increase in absorbance at 420 nm, was measured using a microplate reader. The results demonstrate that the Bpul gene from Bacillus pumilus was successfully expressed in E. coli BL21 and exhibited strong laccase activity. Compared to the control groups, the engineered strain expressing laccase significantly accelerated the degradation of ABTS, confirming its potential for degrading indigo dye in denim applications.

Figure 8. Absorbance at 420nm for different strains. As shown in the Figure 8, the absorbance values at 420 nm for the wild-type BL21, the BL21/pET23b empty vector, and the BL21/p23b-Bpul strains were compared. Both the wild-type BL21 and the BL21/pET23b strains showed negligible laccase activity, with absorbance values close to zero. In contrast, the BL21/p23b-Bpul strain exhibited a significant increase in absorbance, reaching 2.17, indicating that the expressed laccase effectively catalyzed the oxidation of ABTS.

Laccase catalytic time assay

First, 100 μL of crude enzyme solution was collected, and the total protein concentration was measured using the Bradford protein assay. The protein concentration was adjusted to 100 μg/mL using BR buffer. Then, 1 mM ABTS was added to 200 μL of the enzyme solution. The reaction mixture was incubated at 37℃ in a sealed environment, and the absorbance at 420 nm was measured at different time points: 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min, using a microplate reader.The optimal catalytic time forlaccase is 15 min. This finding is valuable for applications in denim treatment, as it helps optimize the enzyme’s use in degrading indigo dye efficiently within a controlled timeframe.

Figure 9. Absorbance at 420 nm for ABTS degradation by laccase over time. As shown in Figure 9, the absorbance values at 420 nm, reflecting the degradation of ABTS by laccase, varied across the different time intervals. The control group (wild-type E. coli BL21) exhibited absorbance values close to zero throughout the experiment. In contrast, the E. coli BL21-Bpul strain expressing laccase showed a rapid increase in absorbance during the early stages of the reaction. The absorbance reached its peak at approximately 2.05 at 15 min. After 15 min, the absorbance remained stable.

Influence of temperature on laccase activity

100 μL of previously prepared crude enzyme solution was used. The protein concentration was measured using the Bradford protein assay and adjusted to 100 μg/mL. A 100 mM ABTS solution was prepared with distilled water, and 1 mM ABTS was added to 200 μL of the enzyme solution. Laccase activity was tested at different temperatures (25℃, 37℃, 45℃, 55℃, 70℃) using a thermostat water bath. The absorbance at 420 nm was measured over a 30-min period to assess ABTS degradation.The results showed that the optimal temperature for laccase activity is 55℃, where the enzymatic reaction efficiency is highest.

Figure 10. Laccase activity at different temperatures. The results showed that laccase activity increased with rising temperature and reached its peak at 55℃, with an absorbance of 2.93. Beyond 55℃, the activity dropped sharply, with absorbance falling to 1.65 at 70℃.

Influence of pH on laccase activity

The engineered bacteria were resuspended in BR buffer with varying pH values from 4 to 9. After disrupting the cells, the supernatant containing the crude enzyme was collected by centrifugation at 10,000 rpm for 20 min at 4℃. Then, 1 mM ABTS was added to the enzyme solution, and the reactions were incubated at 37℃ for 30 min in a sealed environment. Absorbance at 420 nm was measured using a microplate reader to assess laccase activity.The experimental results indicate that the optimal pH for laccase activity is pH=5, where the enzyme exhibits the highest catalytic efficiency.

Figure 11. Laccase activity at different pH. As shown in Figure 11, the activity of laccase varied across the pH range tested. The enzyme's activity increased as the pH rose from 4 to 5, reaching its peak at pH=5, with an absorbance value of approximately 2.65. Beyond pH=5, the activity decreased rapidly, indicating that the enzyme becomes less efficient as the pH increases toward neutral and alkaline conditions.

Influence of copper irons on laccase activity

This experiment aims to study the effect of copper ions on laccase activity, as copper ions serve as the active center of laccase. By determining the optimal copper ion concentration, we can assess whether adding copper chloride to the final product can enhance the enzyme's catalytic efficiency. A 100 mM CuCl2 solution was prepared, and different concentrations of CuCl2 (0, 0.1, 0.25, 0.5, and 1 mM) were added to the ABTS catalytic reaction system to evaluate the effect of copper ions on laccase activity. The reactions were incubated at 37℃, pH=5, for 30 min, and the absorbance at 420 nm was measured to assess enzyme activity.The results indicate that 0.5 mM is the optimal copper ion concentration to enhance laccase activity. Based on this finding, we can add an appropriate concentration of copper ions to our laccase products to improve enzymatic activity.

Figure 12. Effect of different concentrations of copper ions on laccase activity.The experimental results showed that laccase activity increased as the copper ion concentration increased from 0 mM to 0.5 mM, with the highest activity observed at 0.5 mM. However, when the concentration increased to 1 mM, laccase activity decreased.

Testing the degradation of indigo by laccase

Previous experiments demonstrated laccase activity but did not confirm the enzyme's ability to degrade indigo. Therefore, this experiment aims to test laccase's effectiveness in degrading indigo dye. To improve the solubility of indigo, 10 mM indigo (SM4168, Beyotime) was dissolved in dimethyl sulfoxide (DMSO). A 1 mM indigo solution was added to 200 μL of crude enzyme samples (BR buffer, pH=5). A standard curve was established using different concentrations of indigo (0 mM, 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, and 3 mM). The experiment was performed in a 96-well plate, sealed, and incubated at 37℃ for 2 hours. Each group was done in triplicate, and the optical density was measured at 620 nm, with indigo concentrations calculated based on the standard curve.The experimental results demonstrate that the BL21/Bpul strain, which expresses the laccase gene, significantly degrades indigo more effectively than the BL21 control strain. This confirms that laccase has strong catalytic activity in indigo degradation.

Figure 13. Effect of laccase on indigo degradation. As shown in Figure 13, after 2 hours of incubation, the indigo concentration in the BL21 strain averaged 0.95 mM, while in the BL21/Bpul strain it averaged 0.51 mM. The indigo concentration in the BL21 strain was significantly higher than that in the BL21/Bpul strain, indicating that the BL21/Bpul strain has a stronger ability to degrade indigo.

Potential application directions

The experimental results demonstrated that using surface display technology effectively anchored laccase on the cell membrane, which enhanced its activity and improved its ability to degrade indigo. In the future, surface-displayed engineered bacteria could be applied in dye wastewater treatment and environmental pollution remediation. By enhancing the anchoring of laccase on cell membranes, these engineered bacteria could more efficiently break down harmful dyes, particularly indigo, which is notoriously difficult to treat in industrial wastewater. Moreover, surface display technology could be adapted to other enzymes, improving their efficiency in bioconversion and synthesis reactions across various industrial applications.

References

Mo Y, Lao H I, Au S W, et al. Expression, secretion and functional characterization of three laccases in E. coli[J]. Synthetic and Systems Biotechnology, 2022, 7(1): 474-480.

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.

Li R, Zhou T, Khan A, et al. Feed-additive of bioengineering strain with surface-displayed laccase degrades sulfadiazine in broiler manure and maintains intestinal flora structure[J]. Journal of Hazardous Materials, 2021, 406: 124440.

Gianfreda L, Xu F, Bollag J M. Laccases: a useful group of oxidoreductive enzymes[J]. Bioremediation journal, 1999, 3(1): 1-26.

Sequence and Features