We aim to use enzymes such as cellulase, laccase, catalase, and lipAse in denim processing for environmentally friendly whitening, fraying, and stain removal. The key enzyme laccase (Bpul) enhances indigo dye degradation, a critical element in denim bleaching. Our research explores the optimal conditions for its application and effectiveness in sustainable denim treatment.

The goal is to employ laccase to degrade indigo dye in denim fabric, offering a sustainable alternative to chemical bleaching. After reviewing multiple literature sources, we selected the laccase gene Bpul from Bacillus pumilus for expression in Escherichia coli BL21. This enzyme was chosen for its broad substrate specificity and promising oxidative capabilities.

We first selected and codon-optimized the laccase gene Bpul (BBa_K863001) from Bacillus pumilus to ensure efficient expression in E. coli. To improve expression efficiency, we optimized the gene sequence and sent it to a biotechnology company for synthesis. Next, the synthesized Bpul gene was inserted into the pET23b expression vector via EcoRI and XhoI double digestion. The Bpul gene was precisely positioned at the multiple cloning site (MCS) of the vector, ensuring efficient expression. After constructing the recombinant plasmid, it was transformed into E. coliBL21, and positive clones were confirmed through antibiotic selection. Ultimately, we successfully obtained the engineered strain BL21/pET23b-Bpul, which expresses the laccase.

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. A 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.

Wild-type E. coliand E. coliwith the empty vector showed minimal activity. The engineered strain expressing Bpul exhibited high absorbance at 420 nm (about 2.17), demonstrating significant laccase activity.

The optimal catalytic time was determined to be 15 min. Activity increased sharply until 15 min and plateaued afterward.

The optimal temperature for laccase was 55°C, beyond which activity decreased sharply.

The enzyme exhibited peak activity at pH=5. Activity declined rapidly outside this range.

The experiment investigated the effect of copper ions on laccase activity, as copper ions are essential for laccase activity. By adding different concentrations of CuCl2 (0, 0.1, 0.25, 0.5, and 1 mM) to the reaction system and measuring enzyme activity at 420 nm, it was found that laccase activity increased with copper concentration up to 0.5 mM, where it peaked, but decreased at 1 mM. This suggests that 0.5 mM is the optimal concentration for enhancing laccase activity.

The substrate ABTS showed saturation at 0.5 mM.

Laccase successfully degraded indigo dye, showing significantly lower indigo concentration compared to control strains.

The experiments confirmed that the laccase gene Bpul from Bacillus pumilus can be effectively expressed in E. coliand significantly enhance indigo dye degradation under optimal conditions (55°C, pH=5, 0.5 mM Cu²⁺). The enzyme’s ability to break down indigo dye offers a promising, eco-friendly solution for denim bleaching. Further optimization, such as surface display of laccase on bacterial membranes, could further enhance degradation efficiency and stability.

Surface display technology utilizes genetic engineering to fuse a target proteinwith a specific carrier protein, allowing the fusion protein to be expressed on the surface of host cells. In our project, the goal is to display the laccase on the surface of E. coliand measure the activity of surface-displayed laccase in different cellular fractions.

By employing surface display, the laccase can be anchored directly on the bacterial surface, theoretically enhancing its stability and activity by eliminating the need for substrates to penetrate the cell membrane. This is expected to significantly improve laccase efficiency.

We first introduced a truncated ice nucleation protein (INP) sequence upstream of the Bpul laccase gene, allowing it to be displayed on the cell surface. The INP-Bpul gene(BBa_K5458001) was cloned into the pET23b vector, and the recombinant plasmid was transformed into E. coliBL21. After transformation, we screened for positive clones using antibiotic selection and verified the sequences by sequencing. Finally, the engineered strain BL21/INP-Bpul, capable of surface-displaying laccase, was successfully constructed.

First, 100 ml of overnight culture was collected, and OD600 was measured. Cells were lysed by sonication, and after centrifugation, both cytoplasmic and cell membrane fractions were collected. Laccase activity was tested using ABTS as the substrate. 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.

To further validate the effect of surface-displayed laccase, we performed indigo degradation tests. The engineered strains were inoculated into M9 medium, supplemented with 1 mM indigo, and incubated at 37°C. The absorbance at 620 nm was measured over time to monitor indigo degradation. Data showed that BL21/INP-Bpul had a significantly greater indigo degradation effect compared to BL21/Bpul. BL21/pET23b (empty vector) had no impact on indigo concentration, while BL21/INP-Bpul caused a more rapid decrease in indigo concentration over 8 hours.

This cycle of experiments demonstrated that the surface-displayed laccase significantly enhanced both activity and indigo degradation efficiency. The BL21/INP-Bpul strain exhibited higher laccase activity on the cell membrane, and the indigo degradation efficiency was greatly improved compared to non-displayed laccase. Surface display technology offers an effective approach to enhancing laccase performance, showing great potential for applications in denim dye degradation.

We recognized that laccase activity is closely tied to the oxygen concentration in the solution. To address this, we first engineered a strain to produce catalase, aiming to break down hydrogen peroxide and increase the available oxygen. We then combined catalase and laccase to evaluate whether this combination could enhance laccase activity. Specifically, we focused on improving its effectiveness in indigo dye degradation.

In this step, we first selected and codon-optimized the catalase gene katA (BBa_K5458002) to ensure its efficient expression in E. coliBL21. 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. coliBL21, and positive clones were confirmed via antibiotic selection and sequencing verification. The resulting strain, BL21/pET23b-katA, was successfully obtained.

To verify catalase activity of engineered strains. The engineered strains were cultured overnight in LB medium, then lysed by sonication, and the supernatant was collected as the crude enzyme solution. The reaction with hydrogen peroxide was incubated for 60 minutes, and the residual hydrogen peroxide concentration was measured by detecting absorbance at 415 nm. As shown in Figure 16, 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.

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.

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.

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.

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.

Preparation involved four pumice stones, one pair of average-sized jeans, and a 335ml water bottle. The pumice stones were used to grind the jeans, scraping off the indigo dye to achieve a bleached effect. A large amount of gravel powder was produced during the grinding process. For rinsing, the powder was washed off in a 3.35-liter basin by pouring water ten times using the 335ml water bottle. Then, a water gun was used to rinse the jeans, consuming approximately 1 to 1.5 liters of water (exact amount could not be estimated). In conclusion, it is conservatively estimated that washing a pair of jeans requires at least 4.35 to 5.5 liters of water, and a significant amount of powder is generated during the grinding process.

We successfully produced catalase and confirmed its ability to degrade hydrogen peroxide and release oxygen. By combining crude enzyme solutions of catalase and laccase, we demonstrated that this combination had a significant effect on indigo degradation. However, we found that high concentrations of hydrogen peroxide negatively impacted laccase activity, likely due to interference with the copper active sites of laccase. Additionally, through experiments with our enzyme formulation on denim, we demonstrated that our product effectively achieves a bleaching effect on jeans.These findings provide important guidance for further optimization of the catalase-laccase synergistic system.

In our project, the goal was to degrade cellulose in denim using cellulase, which would result in a fraying effect. We selected the cellulase gene Bgls from Bacillus subtilis (BBa_K1175006). To ensure efficient expression in E. coli, the gene was codon-optimized and synthesized by a biotechnology company. We then measured the cellulase activity of the engineered strain and further optimized the optimal temperature and pH conditions for enzyme activity.

We performed double digestion to insert the synthesized Bgls gene into the pET23b vector, which was then transformed into E. coliBL21. Through antibiotic selection and sequencing, we successfully obtained the engineered strain BL21/pET23b-Bgls, expressing cellulase(Figure 2).

We used carboxymethyl cellulose (CMC) as the substrate to test the cellulase activity of the engineered strain. After sonicating the bacterial cells and collecting the crude enzyme solution, we measured the glucose produced from cellulose degradation. The results showed that the engineered strain BL21/pET23b-Bgls exhibited significantly higher cellulase activity compared to the empty vector control group.

The engineered strain BL21/p23b-Bgls demonstrated a much higher cellulase activity compared to the control strains, indicating its strong ability to degrade cellulose.

We further tested the enzyme's activity under different temperatures and pH conditions. The results demonstrated that BL21/pET23b-Bgls showed the highest activity at 30℃ and pH=5.3, indicating that these are the optimal conditions for cellulose degradation.

Through this cycle of experiments, we validated that the engineered strain BL21/pET23b-Bgls has strong cellulose-degrading capability and identified the optimal conditions for its enzyme activity. This provides a solid foundation for achieving the desired fraying effect on denim.

One of the goals of our project was to use lipAse to remove oil stains from denim. We selected the lipAse gene lipA from Pseudomonas and codon-optimized it for efficient expression in E. coli. We then measured the lipAse activity and optimized the temperature and pH conditions to maximize its effectiveness in stain removal.

The synthesized lipA gene was inserted into the pET23b vector through double digestion, and the recombinant plasmid was transformed into E. coliBL21. After antibiotic selection and sequencing verification, we successfully constructed the engineered strain BL21/pET23b-lipA, which expresses lipAse.

We used p-NPB (p-nitrophenyl butyrate) as the substrate to measure lipAse activity by detecting the absorbance of the product at 405 nm. The results showed that the BL21/pET23b-lipA strain had significantly higher lipAse activity compared to the control group, confirming its strong ability to degrade fats.

Further experiments revealed that lipAse exhibits the highest activity at 30℃ and pH=8.2. These were identified as the optimal conditions for removing oil stains from denim.

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In this experiment, light-colored denim fabric was stained with edible oil to assess the cleaning efficacy of different treatments. Six oil spots, each containing 100 µL of edible oil, were applied to the fabric in a 3×2 grid pattern. The fabric was divided into three treatment groups: untreated, treated with laundry detergent, and treated with enzyme powder. A sponge moistened with water was used to scrub each section continuously to simulate a washing process. After treatment, the fabric was allowed to air dry, and the effectiveness of stain removal for each method was evaluated based on the visible cleaning results.

This cycle of experiments successfully demonstrated the potential of lipAse in denim stain removal and identified the optimal conditions for its activity. By optimizing the expression and reaction environment of lipAse, we provided strong support for enhancing stain removal efficiency. Additionally, experiments on denim demonstrated that our product has an excellent effect in removing oil stains from jeans.

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