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.

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.

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.

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.

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.

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.

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.

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.

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).

Conclusion

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

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.

Conclusion

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

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

Conclusion

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

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.

Conclusion

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

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.

Conclusion

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

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.

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.

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

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

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.

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).

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.

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.

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.

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.

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.

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℃.

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.

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.

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.

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.

Objective

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

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.

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.

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.

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

Conclusion

The optimal temperature for cellulase activity is 30℃.

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.

Conclusion

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

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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