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).

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.

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.

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.

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).

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).

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).

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.

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.

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).

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.

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.

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.

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).

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).

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.

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.

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).

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.

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.

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.

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.