iGEM 2024 — Team gec-beijing

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Contents

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[Abstract](#title1_position)

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[System 1 ：Laboratory notebook（Total 41 pages）](#title2_position)

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[System 2 ：Laboratory notebook（Total 17 pages）](#title3_position)

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[System 3 ：Laboratory notebook（Total 17 pages）](#title4_position)

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[Related kit instructions](#title5_position)

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Abstract

At GEC-Beijing, we meticulously documented the daily experiments, including all experimental
protocols, processes, and raw data.

Our team's work primarily focused on the treatment of denim. The experiments were divided
into three systems, each targeting a different enzyme. System 1 focused on the design of
laccase for denim bleaching. System 2 involved cellulase for creating distressed effects,
while System 3 concentrated on lipase for oil stain removal. Through rigorous experimental
design and procedures, we ensured the successful expression of these genes and the progress
of subsequent experiments. These efforts laid a solid foundation for further functional
validation and enzyme activity studies.

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System 1 ：Laboratory notebook（Total 41 pages）

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System 2 ：Laboratory notebook（Total 17 pages）

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

System 3 ：Laboratory notebook（Total 17 pages）

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Related kit instructions

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Hydrogen peroxide content

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Catalase (CAT) activity

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Boxbio-AKSU002M- reducing sugar content test kit

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### Boxbio-AKSU002M- reducing sugar content testkit.pdf

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{% block title %}Engineering{% endblock %}
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Contents

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[Abstract](#title1_position)

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[Cycle 1-1 Construction of laccase-producing strains](#title2_position)

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[Cycle 1-2 Construction of laccase-producing strains with surface display](#title3_position)

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[Cycle 1-3 Synergistic effect of catalase and laccase](#title4_position)

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[Cycle 2 Construction of cellulase-producing strains](#title5_position)

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[Cycle 3 Construction of lipAse-producing strains](#title6_position)

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[Reference：](#title7_position)

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Abstract

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.

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Cycle 1-1 Construction of laccase-producing strains

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Design

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.

![](https://static.igem.wiki/teams/5458/engineering/img2.png)

Figure 1. The gene circuit of Bpul.

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Build

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.

![](https://static.igem.wiki/teams/5458/engineering/img3.png)

Figure 2. Gel image of the Bgls and Bpul. The Bgls gene is related to cellulase and will be studied in later experiments.

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Test

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.

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Laccase Activity Assay:

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.

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Figure 3. Absorbance at 420nm for different strains.

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Catalytic Time Assay

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

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Figure 4. Absorbance at 420 nm for ABTS degradation by laccase over time.

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Temperature Optimization:

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

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Figure 5 Laccase activity at different temperatures.

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pH Sensitivity

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

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Figure 6. Laccase activity at different pH.

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Copper Ion Influence

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.

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Figure 7. Effect of different concentrations of copper ions on laccase activity.

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Substrate Saturation:

The substrate ABTS showed saturation at 0.5 mM.

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Figure 8 Effect of different concentrations of ABTS ions on laccase activity.

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Indigo Degradation

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

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Figure 9. Effect of laccase on indigo degradation.

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Learn

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.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Cycle 1-2 Construction of laccase-producing strains with surface display

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Design

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.

![](https://static.igem.wiki/teams/5458/engineering/img11.png)

Figure 10. The gene circuit of INP-Bpul.

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Build

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.

![](https://static.igem.wiki/teams/5458/engineering/img12.png)

Figure 11. Gel image of INP-Bpul.

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Test

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Laccase Activity in Different Cellular Fractions

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.

![](https://static.igem.wiki/teams/5458/engineering/img13.png)

Figure 12. Absorbance comparison of laccase in cytoplasmic and membrane fractions between BL21/Bpul and BL21/INP-Bpul.

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Indigo Degradation Efficiency

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.

![](https://static.igem.wiki/teams/5458/engineering/img14.png)

Figure 13. Effect of different strains on indigo concentration over 8 hours

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Learn

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.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Cycle 1-3 Synergistic effect of catalase and laccase

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Design

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.

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Figure 14. The gene circuit of katA.

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Build

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.

![](https://static.igem.wiki/teams/5458/engineering/img16.png)

Figure 15. Gel image of katA.

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Test

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Catalase Activity Assay

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.

![](https://static.igem.wiki/teams/5458/engineering/img17.png)

Figure 16. Effect of PBS, BL21/PET23b, and BL21/p23b-katA on oxygen concentration.

![](https://static.igem.wiki/teams/5458/description/write-title3.png)

Oxygen Concentration Measurement

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.

![](https://static.igem.wiki/teams/5458/engineering/img18.png)

Figure 17. Effect of PBS, BL21/PET23b, and BL21/p23b-katA on oxygen concentration

![](https://static.igem.wiki/teams/5458/description/write-title3.png)

Combination of Catalase and Laccase

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.

![](https://static.igem.wiki/teams/5458/engineering/img19.png)

Figure 18. Effect of hydrogen peroxide and catalase on indigo concentration.

![](https://static.igem.wiki/teams/5458/description/write-title3.png)

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.

![](https://static.igem.wiki/teams/5458/engineering/img20.png)

Figure 19. Effect of catalase on indigo degradation.

![](https://static.igem.wiki/teams/5458/description/write-title3.png)

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.

![](https://static.igem.wiki/teams/5458/engineering/img21.png)

Figure 20. Pre-treatment and post-treatment photos of the dark denim bleaching process.

![](https://static.igem.wiki/teams/5458/description/write-title3.png)

Water Consumption for Bleaching Denim Using Pumice

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.

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Learn

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.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Cycle 2 Construction of cellulase-producing strains

![](https://static.igem.wiki/teams/5458/description/write-title2.png)

Design

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.

![](https://static.igem.wiki/teams/5458/engineering/img22.png)

Figure 21. The gene circuit of Bgls

![](https://static.igem.wiki/teams/5458/description/write-title2.png)

Build

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).

![](https://static.igem.wiki/teams/5458/engineering/img23.png)

Figure 2. Gel image of the Bgls and Bpul. The Bpul gene is related to laccase(Fomer experienment).

![](https://static.igem.wiki/teams/5458/description/write-title2.png)

Test

![](https://static.igem.wiki/teams/5458/description/write-title3.png)

Cellulase Activity Assay

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.

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Figure 22. Comparison of glucose production by BL21/pET23b-Bgls and BL21/pET23b over different incubation times.

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

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Figure 23. Cellulase activity of engineered bacterial strains.

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Effect of Temperature and pH on Enzyme Activity

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.

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Figure 24. Effect of temperature on cellulase activity.

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Figure 25. Effect of pH on cellulase activity.

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Learn

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.

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Cycle 3 Construction of lipAse-producing strains

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Design

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.

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Figure 26. The gene circuit of lipA

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Build

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.

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Figure 27. Gel image of lipA

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Test

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lipAse Activity Assay

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.

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Figure 28. Comparison of lipAse activity among BL21, BL21/pET23B, and BL21/p23B-lipA.

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Effect of Temperature and pH on Enzyme Activity:

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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Figure 29. The effect of temperature on lipAse activity.

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Figure 30. The effect of pH on lipAse activity.

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Oil Stain Removal on Light-Colored Denim

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.

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Figure 31 Pre-treatment and post-treatment photos of the oil stain removal from light-colored denim.

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Learn

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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Reference：

[1] Flurkey W H. Laccase[M]//Handbook of food enzymology. CRC Press, 2002: 540-553.

[2] 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.

[3] Xu F. Dioxygen reactivity of laccase: dependence on laccase source, pH, and anion inhibition[J]. Applied biochemistry and biotechnology, 2001, 95: 125-133.

[4] Kiiskinen L L, Viikari L, Kruus K. Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces[J]. Applied Microbiology and Biotechnology, 2002, 59: 198-204.

[5] Guan Z B, Zhang N, Song C M, et al. Molecular cloning, characterization, and dye-decolorizing ability of a temperature-and pH-stable laccase from Bacillus subtilis X1[J]. Applied biochemistry and biotechnology, 2014, 172: 1147-1157.

[6] Fillat U, Roncero M B. Biobleaching of high quality pulps with laccase mediator system: Influence of treatment time and oxygen supply[J]. Biochemical Engineering Journal, 2009, 44(2-3): 193-198.

[7] Ammam M, Fransaer J. Combination of laccase and catalase in construction of H2O2–O2 based biocathode for applications in glucose biofuel cells[J]. Biosensors and Bioelectronics, 2013, 39(1): 274-281.

[8] 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.

[9] Zhao Y, Yang J, Wu Y, et al. Construction of bacterial laccase displayed on the microbial surface for ultrasensitive biosensing of phenolic pollutants with nanohybrids-enhanced performance[J]. Journal of Hazardous Materials, 2023, 452: 131265.

[10] 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.

[11] Wood T M, Bhat K M. Methods for measuring cellulase activities[M]//Methods in enzymology. Academic Press, 1988, 160: 87-112.

[12] Sadhu S, Maiti T K. Cellulase production by bacteria: a review[J]. British Microbiology Research Journal, 2013, 3(3): 235-258.

[13] Sohail M, Siddiqi R, Ahmad A, et al. Cellulase production from Aspergillus niger MS82: effect of temperature and pH[J]. New biotechnology, 2009, 25(6): 437-441.

[14] Jonsson U, Snygg B G. lipAse production and activity as a function of incubation time, pH and temperature of four lipolytic micro‐organisms[J]. Journal of Applied Microbiology, 1974, 37(4): 571-581.

[15] Buthe A, Recker T, Heinemann M, et al. pH-optima in lipAse-catalysed esterification[J]. Biocatalysis and Biotransformation, 2005, 23(5): 307-314.

{% endblock %}

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{% block title %}Experiments{% endblock %}
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Contents

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[Construction of cellulase-producing strain](#title1_position)

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[Testing overexpression of cellulase in engineered strains](#title2_position)

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[Testing cellulase activity](#title3_position)

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[Effect of temperature on cellulase activity](#title4_position)

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[Effect of pH on cellulase activity](#title5_position)

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[Construction of laccase-producing strain](#title6_position)

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[Testing laccase activity](#title7_position)

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[Laccase characterization](#title8_position)

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[Engineering bacteria for surface display of laccase](#title9_position)

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[Analyzing active sites of surface-displayed laccase in engineered strains](#title10_position)

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[Testing the indigo degradation ability of laccase](#title11_position)

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[Testing the indigo degradation effect of engineered strains](#title12_position)

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[Denim bleaching experiment](#title13_position)

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[Construction of catalase overexpression engineered strain](#title14_position)

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[Testing catalase in engineered strains](#title15_position)

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[Combination of laccase and catalase](#title16_position)

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[Catalase promotes indigo biocatalytic activity](#title17_position)

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[Construction of lipase overexpression engineered strain](#title18_position)

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[Testing lipase activity in engineered strains](#title19_position)

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[Analyzing optimal pH and temperature for lipase](#title20_position)

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[Denim oil stain removal experiment](#title21_position)

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[Statistical analysis](#title22_position)

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[Related kit instructions:](#title23_position)

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Construction of cellulase-producing strain

The cellulase gene Bgls from Bacillus subtilis was synthesized and codon-optimized for Escherichia coli , eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). The gene sequence was then cloned into the pET23b plasmid using EcoRI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli  BL21. Positive clones were selected on LB (Luria Bertani) plates containing 50 μg/mL ampicillin and verified by sequencing (Beijing, Qingke), resulting in the engineered strain BL21/p23b-Bgls. The engineered strain was cultured at 37℃ in LB liquid medium for inoculation and expansion. Bacterial growth was monitored by measuring optical density at 600 nm (OD600) using a spectrophotometer (GenStar, China).

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Testing overexpression of cellulase in engineered strains

The engineered strain BL21-pET23b-Bgls was inoculated into LB medium and grown at 37℃ for 3 days. After centrifugation at 13,000 rpm for 5 minutes, 1 mL of supernatant (enzyme solution) was collected. Simultaneously, 1 g of carboxymethyl cellulose sodium (CMC, Sigma-Aldrich) was weighed into a beaker and fully dissolved in 100 mL PBS at 45℃ in a water bath. Then, 1 mL of enzyme solution was mixed with 1 mL of 1% CMC and incubated at 37℃ with shaking (120 rpm) for different times. Glucose standard solutions were prepared, and the amount of glucose produced was measured using a reducing sugar assay kit (Boxbio, AKSU002M) by measuring absorbance at 540 nm. An OD600=1 corresponds to an E. coli  bacterial density of 109/mL. The reducing sugar content produced by the engineered strain (mg/109 cells) was calculated by dividing the measured reducing sugar value by OD600 and the dilution factor.

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Testing cellulase activity

Collect 100 mL of overnight cultured engineered strain and measure OD600. After centrifugation at 10,000 rpm for 1 minute, resuspend in 20 mL PBS (10 mM, pH=7.4). Then, lyse the bacteria using ultrasonic disruption (150 W, 1 s ultrasonication, 3 s intervals, total 20 min). Centrifuge the bacterial lysate at 10,000 rpm for 20 minutes at 4℃ and collect the supernatant as the crude enzyme solution. Take 100 μL of crude enzyme solution and measure the total protein concentration using the Bradford Protein Assay Kit (P0006, Beyotime). Then, mix 1 mL of crude enzyme solution with 1 mL of 1% CMC (dissolved in PBS) and incubate at 37℃ for 30 minutes. Preheat the spectrophotometer (GenStar, China) for 30 minutes, set the wavelength to 540 nm, and zero with distilled water. Measure the amount of glucose produced using the reducing sugar assay kit (Boxbio, AKSU002M). One unit (U) of enzyme activity is defined as producing 1 μmol of product per minute. The specific activity of cellulase is calculated by dividing the product concentration (μmol reducing sugar/min) by the total protein (mg) in the sample.

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Effect of temperature on cellulase activity

The engineered strain BL21/p23b-Bgls was inoculated into fresh LB medium containing 50 μg/mL ampicillin and grown overnight at 37℃. Collect 200 mL of bacterial culture and resuspend in PBS. Use the Biosafer1000 ultrasonic disruptor (150 W, 1 s ultrasonication, 3 s intervals, total 20 min) to lyse the bacteria. Next, centrifuge at 10,000 rpm for 20 minutes and collect the supernatant as the crude enzyme solution. Mix with 1% CMC to analyze the effect of different temperatures (16℃, 25℃, 30℃, 37℃, 45℃) on cellulase activity.

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Effect of pH on cellulase activity

The recombinant strain was inoculated at a 1:100 ratio into fresh LB medium containing 50 μg/mL ampicillin (Amp) and cultured overnight at 37℃. Collect 2 mL of bacterial culture. Centrifuge at 8,000 rpm for 10 minutes to collect the bacterial pellet, and resuspend in 2 mL of sodium acetate buffer (pH=4.6), sodium acetate buffer (pH=3.6), PBS (pH=7.0), or Tris-HCl buffer (pH=9.2). Then, perform ultrasonic disruption on ice using an ultrasonic homogenizer (Biosafer1000, Saifei) (150 W, 1 s ultrasonication, 3 s intervals, total 20 min). Centrifuge at 10,000 rpm for 20 minutes at 4℃, collect the supernatant to obtain the crude enzyme solution. In 1 mL of crude enzyme solution, add 1% CMC and incubate at 37℃ for 30 minutes. Measure the amount of glucose produced using the reducing sugar assay kit (Boxbio, AKSU002M).

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Construction of laccase-producing strain

The laccase gene Bpul from Bacillus pumilus was synthesized and codon-optimized for E. coli , eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). The gene sequence was then cloned into the pET23b plasmid using EcoRI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli  BL21. Positive clones were selected on LB plates containing 50 μg/mL ampicillin and verified by sequencing (Beijing, Qingke), resulting in the engineered strain BL21/p23b-Bpul. The engineered strain was cultured at 37℃ in LB liquid medium for inoculation and expansion. Bacterial growth was monitored by measuring OD600 using a spectrophotometer (GenStar, China).

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Testing laccase activity

Collect 100 mL of overnight cultured engineered strain and measure OD600. After centrifugation at 10,000 rpm for 1 minute, resuspend in 20 mL of Britton-Robinson (BR) buffer (pH=5, R26355, Yuanye). Then, lyse the bacteria using ultrasonic disruption (150 W, 1 s ultrasonication, 3 s intervals, total 20 min). Centrifuge at 10,000 rpm for 20 minutes at 4℃ and collect the supernatant as the crude enzyme solution. Take 100 μL of crude enzyme solution and measure the total protein concentration using the Bradford Protein Assay Kit (P0006, Beyotime). Adjust the protein concentration to 100 μg/mL using BR buffer. ABTS is commonly used to test laccase activity. Prepare a 100 mM ABTS solution (D6132, Macklin) with distilled water. Then, add 1 mM ABTS to 200 μL of crude enzyme solution. Incubate at 37℃, and measure absorbance at 420 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific).

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Laccase characterization

Copper is the active center of laccase. To determine the effect of copper on laccase activity, prepare a 100 mM CuCl2 solution (C804816, Macklin). Then, add different concentrations of CuCl2 (0, 0.1, 0.25, 0.5, 1 mM) to the ABTS catalytic reaction system to analyze the effect of copper on laccase activity. Use a constant temperature shaker or water bath to test the effect of different temperatures (16℃, 25℃, 37℃, 45℃, 55℃, 70℃) on the rate of laccase degradation of ABTS. To test the activity of laccase Bpul at different pH values, resuspend and lyse the bacterial cells using Britton-Robinson buffer at pH=4–9. After centrifugation at 10,000 rpm for 20 minutes (4℃), collect the supernatant, add 1 mM ABTS, seal, and incubate at 37℃ for 30 minutes before measuring absorbance at 420 nm. Additionally, use different concentrations of ABTS to determine the substrate saturation of laccase.

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Engineering bacteria for surface display of laccase

To display laccase on the bacterial surface, an INP truncated sequence was introduced upstream of Bpul. After codon optimization, it was cloned into the pET23b plasmid using EcoRI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli  BL21. Positive clones were selected and verified by sequencing to obtain the engineered strain BL21/INP-Bpul.

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Analyzing active sites of surface-displayed laccase in engineered strains

Collect 100 mL of overnight cultured engineered strain and measure OD600. After centrifugation at 10,000 rpm for 1 minute, resuspend in 20 mL of BR buffer (pH=5, R26355, Yuanye). Then, lyse the bacteria using ultrasonic disruption (150 W, 1 s ultrasonication, 3 s intervals, total 20 min). Centrifuge at 10,000 rpm for 20 minutes at 4℃ and collect 5 mL of supernatant to obtain the cell extract. Then, perform ultracentrifugation at 39,000 rpm for 1 hour; collect 1 mL of supernatant as the cytoplasmic sample and resuspend the pellet in 1 mL of BR buffer (pH=5) as the cell membrane sample. Add 0.1 mM ABTS to 200 μL of each sample. Incubate at 37℃ and measure absorbance at 420 nm using a microplate reader (Multiskan GO, Thermo Fisher Scientific).

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Testing the indigo degradation ability of laccase

Collect 100 mL of overnight cultured engineered strain and measure OD600. After centrifugation at 10,000 rpm for 1 minute, resuspend in 20 mL of BR buffer (pH=5, R26355, Yuanye). Then, lyse the bacteria using ultrasonic disruption (150 W, 1 s ultrasonication, 3 s intervals, total 20 min). Centrifuge at 10,000 rpm for 20 minutes at 4℃ and collect 5 mL of supernatant to obtain the cell extract. Dissolve 10 mM indigo (SM4168, Beyotime) in DMSO. Add 1 mM indigo solution to 200 μL of protein sample (BR buffer, pH=5). Prepare a standard curve using different concentrations of indigo (0 mM, 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, 3 mM). Seal the 96-well plate and incubate at 37℃. Each experimental group was set up in triplicate. After 2 hours, measure optical density at 620 nm.

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Testing the indigo degradation effect of engineered strains

To test the indigo degradation effect of engineered strains, inoculate the engineered strain at a 1:100 ratio 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, 5.0 mM MgSO4, and 0.1 mM CaCl2; supplemented with 10 g/L glucose). Measure pH using a pH meter (Mettler Toledo). After 12 hours, measure OD600 and adjust to OD600=1. Then, take 1 mL of bacterial culture and add 1 mM indigo solution. Incubate at 37℃ and measure absorbance at 620 nm every 2 hours.

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Denim bleaching experiment

To demonstrate the bleaching effect, a dark denim fabric was used. The enzyme preparation powder was evenly sprinkled directly onto the upper portion of the denim to create a gradient effect. Then, a sponge dampened with water was used to continuously scrub the powdered area, facilitating the penetration of the enzyme into the fabric fibers. After thorough scrubbing, the denim was left to air dry completely, allowing the enzyme to react with the fabric. Upon completion of the bleaching process, the denim was examined to assess the extent of fading achieved through the enzyme treatment.

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Construction of catalase overexpression engineered strain

The catalase coding gene katA from E. coli  was synthesized (Genewiz, USA). The gene sequence was then cloned into the pET23b plasmid using EcoRI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli  BL21. Positive clones were selected on LB plates containing 50 μg/mL ampicillin and verified by sequencing (Beijing, Qingke), resulting in the engineered strain BL21/p23b-katA. The engineered strain was cultured at 37℃ in LB liquid medium for inoculation and expansion. Bacterial growth was monitored by measuring OD600 using a spectrophotometer (GenStar, China).

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Testing catalase in engineered strains

The engineered strain carrying katA or the control strain was inoculated into 5 mL LB medium and cultured overnight at 37℃. Measure OD600 and adjust to OD600=1. After centrifugation (10,000 rpm, 1 min), resuspend in PBS (pH=7.4). Lyse the bacteria using ultrasonic disruption (150 W, 1 s ultrasonication, 3 s intervals, total 20 min) with a Biosafer1000 (Saifei). Centrifuge at 10,000 rpm for 20 minutes at 4℃ and collect the supernatant as the crude enzyme solution. Measure total protein concentration using the Bradford Protein Assay Kit (P0006, Beyotime). Adjust the protein concentration to 100 μg/mL using PBS buffer. Take 1 mL of crude enzyme solution and detect katA activity using the Catalase Activity Assay Kit (BC0205, Solarbio) according to the instructions. Dilute 3% hydrogen peroxide solution (H792073, Macklin) with distilled water to 100 mM. Take another 1 mL of crude enzyme solution, add 5 mM hydrogen peroxide, and incubate at 37℃ for 60 minutes. Measure hydrogen peroxide content at 415 nm using the Hydrogen Peroxide Assay Kit (BC3595, Solarbio). Analyze the oxygen concentration in the reaction solution using a dissolved oxygen analyzer (DO9100, Jiyi).

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Combination of laccase and catalase

Since oxygen content can promote laccase activity, overexpressed Bpul and katA engineered strains were cultured overnight in LB medium containing ampicillin (50 μg/mL). The next day, adjust OD600=1 using PBS. After centrifugation to collect bacterial pellets, resuspend in PBS (pH=7.4), and lyse using the Biosafer1000 ultrasonic disruptor (150 W, 1 s ultrasonication, 3 s intervals, total 20 min). Centrifuge at 10,000 rpm for 20 minutes at 4℃ and collect the supernatant as the crude enzyme solution. Measure total protein concentration using the Bradford Protein Assay Kit (P0006, Beyotime). Adjust the protein concentration to 100 μg/mL using PBS buffer. Take 200 μL of Bpul crude enzyme solution, add 200 μL of katA crude enzyme solution, 1 mM indigo solution, and 5 mM hydrogen peroxide. Incubate at 37℃ for 1 hour. Measure absorbance at 620 nm.

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Catalase promotes indigo biocatalytic activity

Inoculate the engineered strain at a 1:100 ratio 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, 5.0 mM MgSO4, and 0.1 mM CaCl2; supplemented with 10 g/L glucose). Measure pH using a pH meter (Mettler Toledo). After 12 hours, measure OD600 and adjust to OD600=1. Then, take 1 mL of bacterial culture, add 1 mM indigo solution, 200 μL of katA crude enzyme solution, and 5 mM hydrogen peroxide. Incubate at 37℃ and measure absorbance at 620 nm after 1 hour.

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Construction of lipase overexpression engineered strain

The lipA gene from Pseudomonas sp. 7323 was synthesized and codon-optimized for E. coli , eliminating EcoRI, XbaI, SpeI, and PstI restriction sites to comply with RFC#10 standards (Genewiz, USA). The gene sequence was then cloned into the pET23b plasmid using EcoRI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli  BL21. Positive clones were selected on LB plates containing 50 μg/mL ampicillin and verified by sequencing (Beijing, Qingke), resulting in the engineered strain BL21/p23b-lipase. The engineered strain was cultured at 37℃ in LB liquid medium for inoculation and expansion. Bacterial growth was monitored by measuring OD600 using a spectrophotometer (GenStar, China).

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Testing lipase activity in engineered strains

The engineered strain BL21/p23b-lipA was inoculated into LB medium and cultured overnight at 37℃. After centrifugation at 10,000 rpm for 1 minute, recombinant E. coli  cells were harvested and resuspended in 25 mM Tris-HCl (pH=8.0). Ultrasonic treatment (150 W, 1 s ultrasonication, 3 s intervals) was performed for 20 minutes to obtain the supernatant containing soluble lipase. Lipase activity was measured spectrophotometrically using p-nitrophenyl butyrate (p-NPB, Sigma) as the substrate. Prepare a 10 mM p-NPB solution (acetonitrile). In 200 μL of crude enzyme solution, add 0.1 mM p-NPB. The hydrolysis reaction was carried out at 37℃. The amount of p-nitrophenol released was determined by measuring the increase in absorbance at 405 nm using a spectrophotometer. One unit of lipase activity is defined as the amount of enzyme that releases 1 μmol of p-nitrophenol per minute.

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Analyzing optimal pH and temperature for lipase

In the reaction system of lipase crude enzyme solution and p-NPB, adjust the ambient temperature to 16℃, 25℃, 30℃, 37℃, and 45℃. After incubating for 30 minutes, test the effect of different temperatures on lipase activity. To test the effect of pH on lipase, resuspend bacterial pellets in citrate buffer (pH=3.0, 5.5), PBS (pH=7.4), or Tris-HCl buffer (pH=8.2, 10.8). After lysis, obtain the crude enzyme solution and add 1 mM p-NPB. Incubate at 37℃ for 1 hour and measure absorbance at 405 nm.

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Denim oil stain removal experiment

On a light-colored denim fabric, 100 μL of edible oil was applied at six points arranged in a 3 × 2 grid. Different treatments were applied: no treatment, laundry detergent, and enzyme preparation. Each treated area was scrubbed continuously using a sponge dampened with water. The fabric was then allowed to air dry, and the cleaning effect was observed to evaluate the efficacy of each treatment.

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Statistical analysis

Data were analyzed and plotted using GraphPad Prism software. Data are presented as mean ± standard deviation (SD). For comparisons among multiple groups, one-way analysis of variance (ANOVA) and Tukey's post hoc test were used for difference analysis. A p-value less than 0.05 was considered statistically significant.

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Related kit instructions:

![](https://static.igem.wiki/teams/5458/description/write-title2.png)

Hydrogen peroxide (H2O2) content detection kit instructions

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### Hydrogen peroxide (H2O2) content detection kit instructions.pdf

size: 0.2MB

[Download](https://static.igem.wiki/teams/5458/experiments/hydrogen-peroxide-h2o2-content-detection-kit-instructions.pdf)

![](https://static.igem.wiki/teams/5458/description/write-title2.png)

Catalase (CAT) activity

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### Catalase (CAT) activity.pdf

size: 0.4MB

[Download](https://static.igem.wiki/teams/5458/experiments/catalase-cat-activity.pdf)

![](https://static.igem.wiki/teams/5458/description/write-title2.png)

Boxbio-AKSU002M- reducing sugar content testkit

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### Boxbio-AKSU002M- reducing sugar content testkit.pdf

size: 0.5MB

[Download](https://static.igem.wiki/teams/5458/experiments/boxbio-aksu002m-reducing-sugar-content-test-kit.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
{% block page\_content %}

{{self.title()}}

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Contents

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[Summary](#title1_position)

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[Laccase](#title2_position)

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[Laccase activity assay](#title3_position)

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[Laccase catalytic time assay](#title4_position)

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[Influence of temperature on laccase activity](#title5_position)

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[Influence of pH on laccase activity](#title6_position)

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[Influence of copper irons on laccase activity](#title7_position)

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[Laccase substrate saturation](#title8_position)

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[Testing the degradation of indigo by laccase](#title9_position)

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[Surface display of laccase-engineered bacteria and activity in different cellular fractions:](#title10_position)

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[Analysis of indigo degradation by engineered bacteria](#title11_position)

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[Catalase](#title12_position)

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[Analysis of catalase activity](#title13_position)

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[Catalase increases dissolved oxygen content](#title14_position)

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[The combination of catalase and laccase promotes indigo degradation](#title15_position)

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[Catalase promotes indigo biocatalyst activity](#title16_position)

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[Bleaching experiment on jeans](#title17_position)

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[Water Consumption for Bleaching Denim Using Pumice](#title18_position)

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[Cellulase](#title19_position)

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[Engineered bacteria degrade cellulose](#title20_position)

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[Cellulase activity assay](#title21_position)

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[Effect of temperature on cellulase activity](#title22_position)

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[Effect of pH on Cellulase Activity](#title23_position)

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[Lipase](#title24_position)

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[Analysis of Lipase Characteristics](#title25_position)

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[The effect of temperature on lipase activity](#title26_position)

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[The effect of pH on lipase activity](#title27_position)

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[Oil Stain Removal on Light-Colored Denim](#title28_position)

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[Reference：](#title29_position)

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Summary

The purpose of our project is to apply cellulase, laccase, catalase and lipase in the processing of jeans to achieve whitening, fraying and stain removal effects. We successfully constructed engineered strains capable of producing cellulase, laccase, catalase, and lipase. The experimental results showed that cellulase, laccase, and lipase were successfully produced, and their optimal reaction conditions were explored. Among them, laccase significantly enhanced indigo dye degradation, and catalase further promoted laccase activity by increasing oxygen concentration. The entire experimental process proved the feasibility and effectiveness of our project, providing biosynthetic support for the environmentally friendly processing of jeans.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Laccase

We first overexpressed the laccase and experimentally verified the activity of the gene. In the experiment, it was found that laccase gene Bpul the highest activity at 55℃, pH=5 and copper ion concentration 0.5 mM, and the optimal treatment time is 15 min. Further studies have shown that laccase reaches saturation at an ABTS concentration of 0.5 mM, and the use of surface display technology can effectively enhance the anchoring of laccase on the cell membrane and the degradation effect of indigo.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Laccase activity assay

**Objective**

This experiment aims to evaluate the enzymatic activity of the Bacillus pumilus-derived laccase gene Bpul expressed in Escherichia coli BL21. Laccase has great potential for applications in denim treatment, as it can oxidatively degrade indigo dye, offering an eco-friendly bleaching and fading effect on denim. By measuring the ability of thelaccase to oxidize the substrate ABTS, this experiment provides foundational data to verify its effectiveness in denim processing and helps to optimize conditions for laccase expression and dye degradation efficiency. The ABTS assay works by detecting the oxidation of ABTS into a green radical cation, which can be quantified by measuring the increase in absorbance at 420 nm, serving as an indicator of laccase activity.

![](https://static.igem.wiki/teams/5458/results/img1.png)

Figure 1. The gene circuit of Bpul.

**Methods**

The Bpul gene (BBa\_K863001) was first cloned into the pET23b vector and overexpressed in E. coli BL21. 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.

![](https://static.igem.wiki/teams/5458/results/img2.png)

Figure 2. Gel image of the Bgls and Bpul. The Bgls gene is related to cellulase and will be studied in later experiments.

**Results**

As shown in the Figure 3, 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.

![](https://static.igem.wiki/teams/5458/results/img3.png)

Figure 3. Absorbance at 420nm for different strains.

**Conclusion**

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.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Laccase catalytic time assay

**Objective**

This experiment aims to determine the optimal catalytic time for the laccase.

**Methods**

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.

**Results**

As shown in Figure 4, 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.

![](https://static.igem.wiki/teams/5458/results/img4.png)

Figure 4. Absorbance at 420 nm for ABTS degradation by laccase over time.

**Conclusion**

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.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Influence of temperature on laccase activity

**Objective**

This experiment aims to investigate the activity of laccase at different temperatures to determine its optimal catalytic temperature.

**Methods**

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.

**Results**

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℃ (Figure 5).

![](https://static.igem.wiki/teams/5458/results/img5.png)

Figure 5 Laccase activity at different temperatures.

**Conclusion**

The optimal temperature for laccase activity is 55℃, where the enzymatic reaction efficiency is highest.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Influence of pH on laccase activity

**Objective**

This experiment aims to determine the effect of different pH values on the activity of Bpul to identify the optimal pH for its catalytic activity.

**Methods**

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.

**Results**

As shown in Figure 6, 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 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.

![](https://static.igem.wiki/teams/5458/results/img6.png)

Figure 6. Laccase activity at different pH.

**Conclusion**

The experimental results indicate that the optimal pH for laccase activity is pH=5, where the enzyme exhibits the highest catalytic efficiency.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Influence of copper irons on laccase activity

**Objective**

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.

**Methods**

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.

**Results**

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 (Figure 7).

![](https://static.igem.wiki/teams/5458/results/img7.png)

Figure 7. Effect of different concentrations of copper ions on laccase activity.

**Conclusion**

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.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Laccase substrate saturation

**Objective**

This experiment aims to determine the substrate saturation concentration of laccase by measuring the effect of different ABTS concentrations on laccase activity.

**Methods**

Four identical ABTS catalytic reaction systems were prepared, with different concentrations of ABTS (0.1 mM, 0.5 mM, 1 mM, 5 mM) added to each system. The reaction conditions were set at pH=5 and 37℃ for 30 min. The substrate saturation concentration was determined by measuring the absorbance at 420 nm to assess the effect of substrate concentration on laccase activity.

**Results**

As shown in Figure 8, the absorbance values varied significantly with different ABTS concentrations. As the substrate concentration increased from 0.1 mM to 0.5 mM, the average absorbance rose from 1.98 to 3.45, indicating an increase in laccase activity with higher substrate concentration. However, when the ABTS concentration increased to 1 mM and 5 mM, the absorbance values were 3.18 and 3.20, respectively, showing little change, suggesting that laccase activity had approached saturation.

![](https://static.igem.wiki/teams/5458/results/img8.png)

Figure 8 Effect of different concentrations of ABTS ions on laccase activity.

**Conclusion**

The experimental results indicate that the substrate saturation concentration for laccase is 0.5 mM. Beyond this concentration, further increases in substrate do not significantly enhance laccase activity, indicating that the enzyme has reached its saturation point.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Testing the degradation of indigo by laccase

**Objective**

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.

**Methods**

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.

**Results**

As shown in Figure 9, 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.

![](https://static.igem.wiki/teams/5458/results/img9.png)

Figure 9. Effect of laccase on indigo degradation.

**Conclusion**

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.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

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

**Objective**

The aim of this experiment is to use surface display technology to anchor laccase on the cell membrane of E. coli and measure its activity at the level of engineered bacteria. Surface display allows the enzyme to be directly localized on the cell surface, improving its stability and activity, thereby enhancing its catalytic efficiency (Figure 10). By comparing laccase activity in the cell membrane and cytoplasm, this experiment evaluates the effect of surface display on laccase activity.

![](https://static.igem.wiki/teams/5458/results/img10.png)

Figure 10. Schematic diagram of laccase displayed on E. coli BL21 cell surface for indigo degradation.

**Methods**

First, a truncated INP (ice nucleation protein) 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 11).

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.

![](https://static.igem.wiki/teams/5458/results/img11.png)

Figure 11. The construction of INP-Bpul. (A) The gene circuit of INP-Bpul. (B) Gel image of INP-Bpul.

**Results**

As shown in Figure 12, 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.

![](https://static.igem.wiki/teams/5458/results/img12.png)

Figure 12. Effect of surface display on laccase activity in engineered bacteria (cellular content VS cell membrane).

**Conclusion**

The experimental results demonstrate that surface display technology successfully anchored laccase to the cell membrane, significantly enhancing its activity on the membrane. These findings are consistent with expectations, confirming that surface-displayed laccase exhibits higher catalytic efficiency on the cell membrane.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Analysis of indigo degradation by engineered bacteria

**Objective**

This experiment aims to test the effect of laccase-engineered bacteria using surface display technology on the decomposition of indigo, with the plan to use live bacteria for the laccase degradation experiment.

**Methods**

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.

**Results**

As shown in Figure 13, 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.

![](https://static.igem.wiki/teams/5458/results/img13.png)

Figure 13. Degradation effect of BL21/pET23b, BL21/Bpul, and BL21/INP-Bpul on Indigo over time.

**Conclusion**

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.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Catalase

To improve the stability of laccase in the presence of oxygen supply in water, we conducted experiments by expressing the catalase gene. The results showed that catalase not only effectively degraded hydrogen peroxide, but also increased the content of dissolved oxygen, significantly promoting the degradation activity of laccase against indigo.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Analysis of catalase activity

**Objective**

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.

![](https://static.igem.wiki/teams/5458/results/img14.png)

Figure 14. The gene circuit of katA.

**Methods**

The katA gene (BBa\_K5458002), encoding catalase, was sourced from Bacillus subtilis and overexpressed in E. coli BL21 using the pET23b plasmid. The engineered strain was inoculated into 5 mL of LB medium and incubated overnight at 37°C. The OD600 was measured and adjusted to 1. The cells 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℃for 60 min. The remaining hydrogen peroxide content was measured at 415 nm using a hydrogen peroxide assay kit (BC3595, Solairebio).

![](https://static.igem.wiki/teams/5458/results/img15.png)

Figure 14. Gel image of katA.

**Results**

As shown in Figure 15, 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.

![](https://static.igem.wiki/teams/5458/results/img16.png)

Figure 15. Effect of PBS, BL21/PET23b, and BL21/p23b-katA on hydrogen peroxide concentration

**Conclusion**

The results demonstrate that the BL21/p23b-katA strain efficiently decomposes hydrogen peroxide, confirming the high activity of catalase.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Catalase increases dissolved oxygen content

**Objective**

This experiment aims to verify whether catalase can increase the oxygen concentration in the solution by decomposing hydrogen peroxide.

**Methods**

The catalase crude enzyme solution was prepared as described in previous experiments. A 1 mL sample of the crude enzyme solution was mixed with 5 mM hydrogen peroxide and incubated at 37℃for 60 min. The oxygen concentration in the reaction solution was measured using a dissolved oxygen analyzer.

**Results**

As shown in Figure 16, after 60 min of incubation at 37°C, the oxygen concentration in the BL21/pET23b sample slightly increased due to the partial decomposition of hydrogen peroxide. In contrast, the oxygen concentration in the BL21/p23b-katA sample significantly increased, indicating that catalase effectively decomposed hydrogen peroxide into oxygen and water, greatly enhancing the dissolved oxygen content.

![](https://static.igem.wiki/teams/5458/results/img17.png)

Figure 16. Effect of PBS, BL21/PET23b, and BL21/p23b-katA on oxygen concentration

**Conclusion**

The experimental results show that catalase can effectively decompose hydrogen peroxide and significantly increase the oxygen concentration in the solution.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

The combination of catalase and laccase promotes indigo degradation

**Objective**

To verify whether oxygen concentration can enhance laccase activity, we combined catalase and laccase to observe the degradation of indigo.

**Methods**

First, engineered strains overexpressing the Bpul and katA genes were cultured overnight in LB medium containing ampicillin (50 μg/ml). The next day, the bacterial suspension was adjusted to OD600 = 1 using PBS, and the bacterial pellet was collected by centrifugation. The pellet was resuspended in PBS (pH=7.4) and sonicated using a Biosafer1000 (150 W, 1 second on, 3 s off, for a total of 20 min). After centrifugation (4℃, 10,000 rpm, for 20 min), the supernatant was collected to obtain the crude enzyme solution. The total protein concentration was measured using the Bradford assay, and the protein concentration was adjusted to 100 μg/ml with PBS. In the experiment, 200 μL of Bpul crude enzyme solution was mixed with 200 μL of katA crude enzyme solution, 1 mM indigo solution, and 5 mM hydrogen peroxide. The mixture was incubated at 37℃ for 1 hour, and absorbance was measured at 620 nm.

**Results**

As shown in Figure 17, after 60 min of incubation at 37°C, the indigo concentration in the katA group remained relatively high, with an average absorbance of approximately 0.99, indicating limited degradation by catalase alone. In the Bpul group, the indigo concentration decreased moderately, with an average absorbance of 0.81, suggesting some degradation by laccase. However, in the Bpul + katA (1:1) group, the indigo concentration significantly decreased, with an average absorbance of 0.45, demonstrating that the combination of catalase and laccase effectively enhanced indigo degradation compared to either enzyme alone.

![](https://static.igem.wiki/teams/5458/results/img18.png)

Figure 17. The combination of catalase and laccase promotes indigo degradation.

**Conclusion**

The combination of catalase and laccase significantly enhanced the degradation of indigo.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Catalase promotes indigo biocatalyst activity

**Objective**

This experiment aims to verify whether catalase can enhance laccase activity by decomposing hydrogen peroxide and increasing the oxygen concentration in the solution. By incorporating surface display technology, a combination of laccase and catalase was used to assess their synergistic effect on indigo degradation at the bacterial level.

**Methods**

The engineered bacteria were inoculated into 5 mL M9 medium at a 1:100 ratio. The initial pH was measured using a pH meter (Mettler Toledo). After 12 hours, the OD600 value was measured and adjusted to OD600 = 1. Then, 1 mL of the bacterial solution was mixed with 1 mM indigo solution, 200 μL of crude catalase solution, and 5 mM hydrogen peroxide. The samples were incubated at 37℃ for 1 hour, and the absorbance was measured at 620 nm.

**Results**

As shown in Figure 18, after 60 min of incubation at 37℃, the indigo concentration in the BL21/INP-Bpul group was 0.76 mM. In comparison, the group with only hydrogen peroxide (BL21/INP-Bpul + H₂O₂) showed a slight increase in indigo concentration to 0.79 mM, but the difference was not significant. This suggests that the addition of hydrogen peroxide alone did not effectively promote indigo degradation and may have inhibited laccase activity. Literature suggests that high concentrations of hydrogen peroxide may inhibit laccase activity by interfering with the copper ions in its active site, weakening its catalytic ability. However, in the group with both hydrogen peroxide and catalase (BL21/INP-Bpul + katA + H₂O₂), the indigo concentration significantly decreased to 0.57 mM, indicating that catalase enhanced the oxygen concentration in the solution, which effectively promoted indigo degradation.

![](https://static.igem.wiki/teams/5458/results/img19.png)

Figure 18. Catalase promotes indigo biocatalyst activity.

**Conclusion**

The results demonstrate that the combination of hydrogen peroxide and catalase can effectively enhance the activity of laccase, leading to improved degradation of indigo.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

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.

![](https://static.igem.wiki/teams/5458/results/img20.png)

Figure 19. Pre-treatment and post-treatment photos of the dark denim bleaching process.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Water Consumption for Bleaching Denim Using Pumice

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.

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Cellulase

In order to achieve the fraying effect of jeans. we constructed an engineering strain expressing the cellulase gene Bgls. The experimental results showed that within 60 min, the strain was able to effectively decompose cellulose, and we found through further experiments that cellulase showed the highest enzyme activity at 37℃ and pH=5.3.

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Engineered bacteria degrade cellulose

**Objective**

The aim of this experiment is to construct a cellulase-producing bacterial strain and evaluate the overexpression of cellulase in the engineered strain (Figure 20).

![](https://static.igem.wiki/teams/5458/results/img21.png)

Figure 20. The gene circuit of Bgls

**Methods**

A cellulase-producing strain was constructed by integrating the cellulase gene Bgls (BBa\_K1175006) from Bacillus subtilis into the pET23b plasmid, which was then transformed into Escherichia coli BL21 (Verification see Figure 2). The engineered strain, BL21/pET23b-Bgls, was inoculated in LB medium and incubated at 37℃ for 3 days. The bacterial cells were then lysed using ultrasonic disruption, followed by centrifugation at 13,000 rpm for 5 min to collect 1 mL of supernatant, which contained the enzyme solution.

To assess cellulase activity, carboxymethyl cellulose (CMC), a water-soluble cellulose analogue, was used as the substrate. One gram of CMC was dissolved in 100 mL of PBS at 45℃. The enzyme solution (1 mL) was mixed with 1 mL of 1% CMC solution and incubated at 37℃ with shaking at 120 rpm for different time periods. Glucose production was measured using a glucose assay kit (Boxbio, AKSU002M) by measuring absorbance at 540 nm (Figure 21). The optical density at 600 nm (OD600) was used to determine the bacterial cell density, with OD600 = 1 corresponding to 1x10^9 cells/mL. The reducing sugar content produced by the engineered strain was calculated in mg/10^9 cells by dividing the measured reducing sugar by the OD600 and dilution factor.

![](https://static.igem.wiki/teams/5458/results/img22.png)

Figure 21. Principle of the reducing sugar assay. Glucose, as a reducing sugar, reacts with 3,5-dinitrosalicylic acid (DNS) in an alkaline solution, producing a red-colored compound with a characteristic absorption peak at 540 nm.

**Results**

The results are shown in Figure 22. The control group (BL21/pET23b) exhibited minimal changes in absorbance over different incubation times (10, 20, 30, and 60 min), indicating a lack of cellulase activity and limited glucose production. In contrast, the engineered strain (BL21/pET23b-Bgls) showed a significant increase in glucose production, with the highest reducing sugar content reaching 2.194 mg/10^9 cells at 60 min, far exceeding that of the control group. This demonstrates successful cellulase expression in the engineered strain, which exhibited strong cellulose-degrading capability.

![](https://static.igem.wiki/teams/5458/results/img23.png)

Figure 22. Comparison of glucose production by BL21/pET23b-Bgls and BL21/pET23b over different incubation times.

**Conclusion**

The experiment demonstrated that the engineered cellulase-producing strain BL21/pET23b-Bgls effectively degraded CMC and produced high concentrations of glucose. This confirms that the strain exhibits strong cellulase activity, significantly enhancing glucose production in a short time frame.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Cellulase activity assay

**Objective**

The purpose of this experiment is to measure the cellulase activity of the engineered bacterial strain.

**Methods**

A 100 mL of overnight culture of the engineered strain was collected, and its OD value was measured. The culture was centrifuged at 10,000 rpm for 1 minute, and the cell pellet was resuspended in 20 mL of PBS buffer (10 mM, pH=7.4). The cells were then disrupted by sonication (150 W, 1 second on, 3 s off, for a total of 20 min). The lysate was centrifuged at 10,000 rpm for 20 min at 4℃, and the supernatant containing the crude enzyme solution was collected.

Next, 100 μL of the crude enzyme solution was used to measure the total protein concentration using the Bradford Protein Assay Kit. To measure cellulase activity, 1 mL of the crude enzyme solution was mixed with 1 mL of 1% CMC dissolved in PBS and incubated at 37℃ for 30 min. The amount of glucose produced was determined by measuring absorbance at 540 nm using a spectrophotometer. Enzyme activity was defined as the amount of product (μmol of reducing sugar) generated per minute, normalized by the total protein concentration (mg) in the sample.

**Results**

The results, shown in Figure 23, present the cellulase activity of the control and experimental strains (BL21, BL21/pET23b, and BL21/p23b-Bgls). The BL21/p23b-Bgls strain exhibited a significantly higher cellulase activity, reaching approximately 12.482 μmol/min/mg protein, far exceeding the activity observed in the control groups.

![](https://static.igem.wiki/teams/5458/results/img24.png)

Figure 23. Cellulase activity of engineered bacterial strains.

**Conclusion**

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

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Effect of temperature on cellulase activity

**Objective**

The objective of this experiment is to study the effect of temperature on cellulase activity and to determine the optimal reaction temperature.

**Methods**

The cellulase activity of the engineered strain was measured at various temperatures (16℃, 25℃, 30℃, 37℃, and 45℃) using a temperature-controlled incubator. The same method described previously was used to assess cellulase activity under each temperature condition.

**Results**

The graph in Figure 24 illustrates the trend in cellulase activity across different temperatures. The cellulase activity increased with temperature up to 30℃, where the highest activity (11.953 μmol/min/mg protein) was observed. Beyond 30℃, the activity declined, with significantly lower activity at both 16℃ and 45℃.

![](https://static.igem.wiki/teams/5458/results/img25.png)

Figure 24. Effect of temperature on cellulase activity.

**Conclusion**

The optimal temperature for cellulase activity is 30℃. The enzyme activity shows a clear peak at this temperature, while activity decreases at both lower and higher temperatures.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Effect of pH on Cellulase Activity

**Objective**

The purpose of this experiment is to investigate the effect of pH on cellulase activity and determine the optimal pH for the enzyme reaction.

**Methods**

The recombinant strain was inoculated at a 1:100 ratio into fresh LB medium containing 50 μg/mL ampicillin and incubated overnight at 37℃. A 2 mL culture was collected and centrifuged at 8,000 rpm for 10 min to collect the bacterial pellet. The pellet was resuspended in buffers with different pH values. Cell disruption was performed using sonication (150 W, 1-second pulse with 3-second intervals, for 20 min) in an ice bath. The lysate was then centrifuged at 10,000 rpm for 20 min, and the supernatant containing the crude enzyme solution was collected.

To measure cellulase activity, 1 mL of the crude enzyme solution was mixed with 1% CMC and incubated at 37℃ for 30 min. The amount of glucose produced was measured using a reducing sugar assay kit.

**Results**

As shown in Figure 25, the cellulase activity varied under different pH conditions (pH=4.2, 5.3, 7.3, 8.5, 11.4). The activity increased with pH and reached its peak at pH=5.3, where the highest activity (16.61 μmol/min/mg protein) was recorded. Beyond this point, the activity decreased as pH increased further.

![](https://static.igem.wiki/teams/5458/results/img26.png)

Figure 25. Effect of pH on cellulase activity.

**Conclusion**

The optimal pH for cellulase activity is 5.3, where the enzyme exhibits the highest activity. Activity decreases at both lower and higher pH values, indicating that the enzyme functions best in slightly acidic conditions.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Lipase

Finally, we explored the use of lipase in removing oil stains from jeans. Experimental results show that the BL21/p23b-LipA strain has significant lipase activity, and the lipase activity is optimal at 30℃ and pH=8.2.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Analysis of Lipase Characteristics

**Objective**

The aim of this experiment is to construct a lipase-producing bacterial strain and evaluate the overexpression of lipase in the engineered strain

![](https://static.igem.wiki/teams/5458/results/img27.png)

Figure 26. The gene circuit of lipA

**Methods**

The lipA gene, derived from Pseudomonas sp. 7323, was synthesized and cloned into the pET23b plasmid, followed by overexpression in the BL21 strain. The soluble lipase in the supernatant was obtained through ultrasonic disruption of the engineered strain. Since lipids are insoluble in water and degrade at a very low rate, p-NPB (butyric acid p-nitrophenyl ester, Sigma) was used as the substrate due to its ester bond and similar properties to lipids. Lipase activity was measured using microplate reader. A 10 mM p-NPB solution (dissolved in acetonitrile) was prepared, and 0.1 mM p-NPB was added to 200 μL of the crude enzyme solution. The reaction was carried out at 37°C, and the hydrolysis rate was determined by measuring the absorbance of the produced p-nitrophenol at 405 nm using a microplate reader.

![](https://static.igem.wiki/teams/5458/results/img28.png)

Figure 27. Gel image of lipA

**Results**

As shown in Figure 28, after 60 min of reaction at 37℃ and pH=7, the absorbance at 405 nm in the BL21/p23B-LipA group was significantly higher than that in the BL21 and BL21/pET23B groups. This indicates that the BL21/p23B-LipA group has the highest degradation efficiency and possesses lipase activity.

![](https://static.igem.wiki/teams/5458/results/img29.png)

Figure 28. Comparison of lipase activity among BL21, BL21/pET23B, and BL21/p23B-LipA.

**Conclusion**

Under the conditions of 37°C, pH=7, and 60 min of reaction time, the BL21/p23B-LipA group showed significantly higher absorbance than the control groups, indicating higher lipase activity and superior degradation efficiency.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

The effect of temperature on lipase activity

**Objective**

The **Objective** of this experiment is to study the effect of temperature on lipase activity and to determine the optimal reaction temperature.

**Methods**

In the lipase crude enzyme solution and p-NPB reaction system, the environmental temperatures were adjusted to 16 ℃, 25℃, 30℃, 37℃, and 45℃. After incubating for 30 min, the effect of different temperatures on lipase activity was tested.

**Results**

Figure 29 shows the trend in lipase activity across different temperatures. Under the conditions of pH=7 and a reaction time of 60 min, the lipase activity increased with temperature up to 30℃, where the highest activity was observed. Beyond 30℃, the activity declined, with significantly lower activity at both 16℃ and 45℃.

![](https://static.igem.wiki/teams/5458/results/img30.png)

Figure 29. The effect of temperature on lipase activity.

**Conclusion**

The optimal temperature for cellulase activity is 30℃.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

The effect of pH on lipase activity

**Objective**

To detect the effect of pH on lipase activity and explore the optimal pH.

**Methods**

To test the influence of pH on lipase, bacterial pellets were resuspended using citrate buffer (pH=3.0, 5.5), PBS (pH=7.4), or Tris-HCl buffer (pH=8.2, 10.8). After cell lysis, a crude enzyme solution was obtained, and 1 mM p-NPB was added. After incubating at 37℃ for 1 hour, the absorbance of the reaction system was measured at 405 nm.

**Results**

Under the conditions of incubation at 37 ℃ for 1 h with the addition of 1 mM p-NPB, the crude enzyme solution at pH=8.2 exhibited the highest absorbance, indicating that lipase has maximum activity under these conditions.

As shown in Figure 30, the lipase activity varied under different pH conditions (pH=3.0, 5.5, 7.4, 8.2, 10.8). The activity increased with pH and reached its peak at pH=8.2. Beyond this point, the activity decreased as pH increased further.

![](https://static.igem.wiki/teams/5458/results/img31.png)

Figure 30. The effect of pH on lipase activity.

**Conclusion**

To achieve optimal reaction results, it is recommended to use the crude enzyme solution at pH=8.2.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

Oil Stain Removal on Light-Colored Denim

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.

![](https://static.igem.wiki/teams/5458/results/img32.png)

Figure 31 Pre-treatment and post-treatment photos of the oil stain removal from light-colored denim.

![](https://static.igem.wiki/teams/5458/description/write-title1.png)

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[2] 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.

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[8] 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.

[9] Zhao Y, Yang J, Wu Y, et al. Construction of bacterial laccase displayed on the microbial surface for ultrasensitive biosensing of phenolic pollutants with nanohybrids-enhanced performance[J]. Journal of Hazardous Materials, 2023, 452: 131265.

[10] 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.

[11] Wood T M, Bhat K M. **Methods** for measuring cellulase activities[M]//**Methods** in enzymology. Academic Press, 1988, 160: 87-112.

[12] Sadhu S, Maiti T K. Cellulase production by bacteria: a review[J]. British Microbiology Research Journal, 2013, 3(3): 235-258.

[13] Sohail M, Siddiqi R, Ahmad A, et al. Cellulase production from Aspergillus niger MS82: effect of temperature and pH[J]. New biotechnology, 2009, 25(6): 437-441.

[14] Jonsson U, Snygg B G. Lipase production and activity as a function of incubation time, pH and temperature of four lipolytic micro‐organisms[J]. Journal of Applied Microbiology, 1974, 37(4): 571-581.

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