Conventional surgical sutures, with their singular function, often lead to postoperative complications such as infections, inflammatory responses, and hypertrophic scarring. To address these clinical challenges, we have successfully constructed and validated all core functional components of a novel, multifunctional bioactive suture, ReGenStitch, using a modular synthetic biology strategy.

Our engineering practice followed four rigorous "Design-Build-Test-Learn" (DBTL) cycles. In the first cycle, we established a bacterial cellulose (BC) production system by heterologously co-expressing the bcsA and bcsB genes in Escherichia coli BL21. Production kinetics analysis revealed that the system reached a peak yield of approximately 970 mg/L at 48 hours. The chemical identity of the product was confirmed by Congo Red binding assays, thus successfully validating the biological manufacture of the suture's "structural scaffold." In the second cycle, we utilized the Ice Nucleation Protein (INP) as an anchor to successfully display chitosanase (CHI1) on the surface of E. coli, creating a highly efficient and recyclable whole-cell biocatalyst. This system demonstrated significantly superior catalytic efficiency compared to traditional crude enzyme lysates (p < 0.01) and exhibited optimal activity at 50°C and pH 7.0. Furthermore, we validated an upstream process for extracting chitin from shrimp shell waste through microbial co-fermentation (yield: 14.5%), establishing a complete "waste-to-value" pipeline. In the third cycle, we successfully reconstructed the curcumin biosynthesis pathway in E. coli by co-expressing the plant-derived DCS and CURS1 genes, achieving a de novo synthesis yield of approximately 14 μM. Enzyme kinetics analysis (Vmax ≈ 345.6 nM/min, Km ≈ 114.6 μM) further confirmed the functionality of this "anti-inflammatory module." Finally, in the system integration cycle, we prepared the ReGenStitch physical prototype by solution blending the biologically manufactured BC and chitosan. In simulated suturing tests on porcine skin, the prototype exhibited excellent mechanical strength and handling performance, validating its fundamental feasibility as a medical device.

In summary, our experimental results systematically validate the biological manufacturing of ReGenStitch's three core functional modules and the physical integration of the final prototype, laying a solid engineering foundation for the development of a new generation of advanced bioactive surgical sutures.

1.1 Purpose

The objective of this experiment was to enable E. coli to produce large quantities of bacterial cellulose. Literature indicates that the synthesis of Bacterial Cellulose (BC) is a complex process regulated by multiple enzymes. Among them, the bcsA gene encodes the catalytic subunit of cellulose synthase. This protein is the "core factory" for cellulose synthesis, directly using UDP-glucose (UDPG) as a substrate to catalyze the β-1,4 polymerization of glucose monomers into long cellulose chains. The bcsB gene encodes a cyclic di-GMP-binding protein. By binding to the signaling molecule c-di-GMP, this protein activates the catalytic activity of BcsA, acting as a regulatory switch in the cellulose synthesis process. As bcsA and bcsB are central to the cellulose synthase multi-enzyme complex, we chose to prioritize their expression.

1.2 Methods

Recombinant Strain Construction

The cellulose synthase-encoding genes, bcsAB, were codon-optimized based on the codon bias of Escherichia coli, and restriction sites for EcoRI, XbaI, SpeI, PstI, and NotI were eliminated (sequence verification performed using SnapGene®). The optimized genes were synthesized by Genewiz (South Plainfield, NJ, USA) and cloned into the pSB1A3 vector (BioBricks™ standard RFC#10). A dual expression cassette was constructed using the J23100 constitutive promoter (BBa_J23100) and the B0034 ribosomal binding site (BBa_B0034), which was ligated to the vector via Golden Gate assembly. The recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and plated on LB agar plates (pH 7.0) containing 100 μg/mL ampicillin (Amp⁺), followed by incubation at 37°C for 16 h. Single colonies were picked and inoculated into 5 mL of LB/Amp⁺ liquid medium and cultured at 37°C with shaking at 220 rpm until the OD₆₀₀ reached 0.6-0.8. Positive clones were verified by colony PCR (2×Taq Master Mix, Vazyme). Sequencing verification was performed by Qingke Biotechnology (Beijing, China). The sequence-verified engineered strain was inoculated into 50 mL of LB/Amp⁺ medium and cultured at 37°C and 200 rpm to the mid-logarithmic phase (OD₆₀₀≈0.5). The OD₆₀₀ was measured every 30 min using a GenStar® GS-2000 spectrophotometer (Beijing) with a wavelength accuracy of ±0.5 nm, using uninoculated medium as a blank. The growth curve was plotted using OriginPro 2023 software, with data represented as the mean ± standard deviation (SD) of three independent experiments. Wild-type E. coli BL21 was used as a control.

Cellulose Yield Analysis

To quantify the amount of cellulose synthesized, the bcsAB co-expressing BL21 strain was inoculated into 100 mL of fresh LB medium (containing 100 μg/mL ampicillin). After incubation with shaking at 30°C and 180 rpm for various durations, 100 mL of the culture was centrifuged at 8,000 × g for 5 min. Twenty mL of the supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-chilled PBS (pH 7.4) and sonicated on an ice bath (Biosafer1000, Saifei) with the following parameters: 70 W power, 1 s sonication followed by a 3 s interval, for a total processing time of 20 min. The sonicated lysate was centrifuged at 15,000 × g for 20 min to remove cell membranes and debris, and 20 mL of the supernatant was collected as the intracellular sample. Both intracellular and extracellular samples (20 mL each) were mixed with 20 mL of 4 M NaOH and incubated at 80°C for 2 h to dissolve non-cellulosic components and precipitate the cellulose. The mixture was centrifuged at 15,000 × g for 30 min at 4°C to collect the cellulose pellet, which was then washed repeatedly with distilled water until the pH reached 7.0 to remove residual NaOH. The washed cellulose was dried at 60°C to a constant weight, and the cellulose content was calculated by weighing the dried mass. The cellulose synthesis of the control strain, wild-type E. coli BL21, was measured in the same manner.

1.3 Results

Recombinant Strain Construction

Figure 1. Construction of pSB1A3-bcsAB.

(A) Plasmid map of the recombinant plasmid pSB1A3-bcsAB. (B) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pSB1A3-bcsAB. (C) Gene circuit diagram of pSB1A3-bcsAB.

The colony PCR results showed amplified bands corresponding to the expected sizes of bcsA (2262 bp) and bcsB (2406 bp) in the positive clones, confirming the successful construction of the recombinant plasmid.

Cellulose Yield Analysis

The yield measurement results (Figure 2) indicated that the cellulose production of the engineered strain BL21-bcsAB was significantly higher than that of the BL21 control group carrying an empty vector (p < 0.05). This strongly demonstrates that our gene circuit successfully enhanced the cellulose synthesis capability of E. coli.

Figure 2. Cellulose yield analysis of the engineered strain.

1.4 Conclusion

By successfully co-expressing the bcsA and bcsB genes in E. coli BL21 (DE3), we significantly increased the bacterial cellulose yield of the chassis cell, thereby successfully validating the core design of our System 1.

2.1 Purpose

To determine the optimal time window for cellulose production by the engineered strain BL21-bcsAB, we characterized its production kinetics. This study aims to provide critical data to support subsequent process optimization and large-scale production.

2.2 Methods

Strain Inoculation and Culture: The bcsAB co-expressing BL21 strain was inoculated into 100 mL of fresh LB medium containing 100 μg/mL ampicillin and cultured with shaking at 30°C and 180 rpm.

Sample Collection: At different time points, 100 mL of the culture was collected and centrifuged at 8,000 × g for 5 min. Twenty mL of the supernatant was collected as the extracellular sample. The bacterial pellet was resuspended in an equal volume of pre-chilled PBS and sonicated on an ice bath. The lysate was then centrifuged at 15,000 × g for 20 min, and 20 mL of the supernatant was collected as the intracellular sample.

Sample Processing and Cellulose Collection: Both intracellular and extracellular samples were mixed with 20 mL of 4 M NaOH. The mixture was centrifuged at 15,000 × g for 30 min at 4°C to collect the cellulose pellet. The pellet was washed repeatedly with distilled water until the pH reached 7.0 and then dried at 60°C to a constant weight. The mass of the dried cellulose pellet was measured to calculate the cellulose content in the corresponding sample.

2.3 Results

As shown in Figure 3, the cellulose yield of BL21-bcsAB exhibited a typical S-shaped growth curve over time. In the early stage of cultivation, production increased slowly. After entering the logarithmic phase, the yield rapidly climbed. Around 48 hours, the production entered a plateau phase, reaching a peak yield of approximately 970 mg/L, after which the growth rate slowed.

Figure 3. Cellulose yield of BL21-bcsAB at different time points.

2.4 Conclusion

This experiment successfully plotted the time course curve for cellulose production by the BL21-bcsAB strain. The core results indicate that under the culture conditions of 30°C and 180 rpm shaking, the cellulose yield increased with cultivation time, following an "S" curve characterized by slow initial growth, a rapid increase in the middle phase, and continued but slower growth in the later phase. 48 hours was identified as the optimal harvest time to obtain the maximum cellulose yield.

3.1 Purpose

The expression of heterologous genes can impose a metabolic burden on the host, potentially affecting its growth rate. To eliminate the interference of such growth differences on the comparison of total yield, and to directly confirm that the product of our engineered bacteria is indeed bacterial cellulose, we employed a Congo Red assay. Since Congo Red specifically binds to cellulose microfibrils at 37°C, causing a quantifiable decrease in the absorbance of the diazo dye via spectrophotometry, this experiment was designed to specifically compare the cellulose synthesis capability per unit of engineered bacteria versus control bacteria by normalizing the cell density to an OD₆₀₀ of 1.

3.2 Methods

Culture and Pre-treatment: The BL21-bcsAB strain and the control BL21 strain were cultured at 30°C and 180 rpm for 12 h. The cultures were then adjusted to an OD₆₀₀ of 1, and 2 mL of each culture was centrifuged at 10,000 × g for 10 min at 4°C to collect the bacterial pellets.

Congo Red Incubation: The pellets were resuspended in a 1% tryptone solution containing 40 mg/mL of Congo Red and incubated for 2 h at 37°C with shaking at 180 rpm.

Separation and Detection: After incubation, the samples were centrifuged at 13,000 × g for 30 min at 4°C, and the supernatant was collected. The absorbance of the supernatant was measured at 490 nm using a microplate reader (Note: The figure caption indicates 400 nm, but the experimental method specifies 490 nm, which was followed). The total amount of Congo Red bound by the bacteria was calculated from the difference between the initial amount of Congo Red added and the unbound amount remaining in the supernatant.

3.3 Results

The experimental results (Figure 4) show that compared to the BL21 control group (absorbance ≈ 0.6), the supernatant absorbance of the BL21-bcsAB engineered strain group was significantly lower (≈ 0.2), with the difference being highly statistically significant (p < 0.0001). This indicates that, for the same number of cells, the engineered bacteria bound significantly more Congo Red.

Figure 4. Comparison of Congo Red absorbance after binding to bacterial cellulose.

In this experiment, by normalizing the concentration of both the engineered strain BL21-bcsAB and the non-engineered strain BL21 to an OD₆₀₀ of 1, we eliminated interference from potential differences in bacterial viability caused by the expression of the heterologous BcsAB proteins. This allowed for a quantitative analysis of cellulose production per unit of bacteria over the same culture period.

The results demonstrate that, at the single-cell level, the expression of the heterologous bcsAB genes genuinely enhances the intrinsic cellulose synthesis capability of E. coli, rather than merely reflecting an apparent difference due to an increase in total cell count.

4.1 Purpose

Post-cesarean section surgeries are often accompanied by severe inflammatory responses, particularly at the suture site. To address this, we aimed to incorporate an anti-inflammatory substance, curcumin, into our surgical suture. The objective of this experiment was to construct a curcumin synthesis system in an E. coli strain by co-expressing the DCS and CURS1 genes, validate its functionality for producing curcumin, and quantitatively compare the production capability of the engineered strain against the wild-type strain.

4.2 Methods

Recombinant Strain Construction

The DCS and CURS1 sequences were synthesized by a biotechnology company (Generalbial, China) and codon-optimized for E. coli. Restriction sites for EcoRI, XbaI, SpeI, PstI, NdeI, and XhoI were removed to comply with the BioBricks™ RFC#10 standard and ensure compatibility with pET28a(m) cloning. The genes were cloned into the pET28a(m) vector using NdeI and XhoI restriction sites. The recombinant plasmid was transformed into E. coli DH5α and BL21. Positive clones were screened on LB agar plates containing 100 μg/mL kanamycin (Kana) and sent for sequencing verification (Qingke Biotechnology, China) to obtain the final engineered strain.

Curcumin Synthesis Analysis

The recombinant strain was inoculated and expanded in LB medium containing 100 μg/mL Kana at 37°C and 150 rpm. When the OD₆₀₀ reached 0.6, protein expression was induced with 0.5 mM IPTG. Bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). A crude enzyme lysate was obtained by sonication on an ice bath (1s on, 3s off, 70W, 20 min). Protein concentration was determined using a Bradford assay kit (Beyotime, P0006). To analyze curcumin production, the reaction was conducted in 200 μL of 100 mM PBS (pH 7.4), containing 100 μM feruloyl-CoA (Yuanye, Shanghai, S27603), 100 μM malonyl-CoA (Merck, 63410), and 40 mg/L of crude enzyme lysate. After reacting at 37°C for 1 h, the sample was extracted with 1 mL of ethyl acetate. The upper organic layer was collected, dried overnight in a 45°C oven, and redissolved in 200 μL of methanol. The absorbance at 420 nm was measured using a spectrophotometer, with methanol as a blank for baseline correction. The sample solution was placed in a cuvette to measure its absorbance. Curcumin concentration was calculated based on a standard curve prepared with curcumin standards.

4.3 Results

Recombinant Strain Construction

Figure 5. Construction of pET28a(m)-DCS-CURS.

(A) Plasmid map of the recombinant plasmid pET28a(m)-DCS-CURS. (B) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pET28a(m)-DCS-CURS (DCS). (C) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pET28a(m)-DCS-CURS (CURS).

The colony PCR results showed amplified bands corresponding to the expected sizes of DCS (1161 bp) and CURS1 (1170 bp) in the positive clones, confirming the successful construction of the recombinant plasmid.

Curcumin Yield Analysis

As shown in Figure 6, the engineered strain BL21-DCS-CURS successfully catalyzed the synthesis of curcumin from substrates, reaching a yield of approximately 13-14 μM. In contrast, the production by the wild-type BL21 control group was negligible. The yield from the engineered strain was more than 26 times the background level, a difference that is highly statistically significant (p < 0.001). This result confirms that the dual-enzyme co-expression system successfully activated the curcumin synthesis pathway, with a statistically significant difference in yield (p < 0.001).

Figure 6. Curcumin yield test of the DCS and CURS co-expressing engineered strain.

4.4 Conclusion

This study successfully constructed a functional DCS-CURS dual-enzyme co-expression system in E. coli BL21, achieving the de novo biosynthesis of curcumin. The breakthrough increase in production provides a reliable source for the active component of our suture. The next steps will involve developing the drug-loading process for the suture and validating its biological activity.

5.1 Purpose

This experiment aimed to investigate the response characteristics of the heterologously expressed curcumin synthesis pathway (DCS-CURS) in our engineered E. coli to varying substrate concentrations. The goal was to determine whether the system exhibits typical enzyme-catalyzed reaction kinetics, such as Michaelis-Menten kinetics.

5.2 Methods

The DCS and CURS co-expressing engineered strain was cultured in LB medium containing 100 µg/mL kanamycin. When the OD₆₀₀ reached 0.6, protein expression was induced with 0.5 mM IPTG.

The bacteria were resuspended in 20 mM Tris-HCl buffer (pH 7.4). A crude enzyme lysate was obtained by sonication on an ice bath (1s on, 3s off, 70W, 20 min). The protein concentration was determined using a Bradford assay kit. To analyze curcumin production, the reaction was conducted in 200 μL of 100 mM PBS (pH 7.4). For the kinetic analysis, the reaction system contained 40 mg/L of crude enzyme lysate, and a series of reactions were prepared with varying concentration gradients of feruloyl-CoA and malonyl-CoA.

The reactions were incubated at 37°C for 1 h. Afterward, the samples were extracted with 1 mL of ethyl acetate. The upper organic layer was collected, dried overnight in a 45°C oven, and then redissolved in 200 μL of methanol.

The absorbance of the sample solution was measured at 420 nm using a spectrophotometer, with methanol serving as the blank for baseline correction. The curcumin concentration was calculated based on a standard curve prepared with curcumin standards. Subsequently, the rate of curcumin synthesis was calculated for each substrate concentration.

5.3 Results

The experimental results (Figure 7) show that the rate of curcumin synthesis increased with substrate concentration and gradually approached saturation, exhibiting typical Michaelis-Menten kinetics. Through non-linear regression fitting, we calculated the system's maximum reaction rate (Vmax) to be approximately 345.6 nM/min and the Michaelis constant (Km) to be approximately 114.6 μM. This result indicates that the dual-enzyme system possesses good catalytic performance and moderate substrate affinity.

Figure 7. Reaction kinetics of curcumin synthesis by DCS-CURS.

5.4 Conclusion

This experiment successfully plotted the reaction kinetics for curcumin synthesis at different concentrations of feruloyl-CoA and malonyl-CoA. We conclude from this experiment that the reaction kinetics of DCS-CURS for curcumin synthesis follow a typical saturation curve, further verifying that DCS and CURS possess excellent catalytic properties. This conclusion validates the effectiveness of the synthetic pathway at a deeper level and provides crucial theoretical guidance for future yield optimization, for instance, by increasing substrate supply.

6.2 Methods

Construction of Recombinant Strain for Surface Display of CHI1

The chitosanase gene (CHI1) from Bacillus thuringiensis and the ice nucleation protein gene (INP) were synthesized to create an INP-CHI1 fusion fragment (Generalbial, China). As previously described, the sequence was codon-optimized for E. coli and designed to comply with the BioBricks™ RFC#10 standard. Using the J23100 constitutive promoter (BBa_J23100) and the B0034 ribosomal binding site (BBa_B0034) as cis-acting elements, the fragment was cloned into the pSB1A3 vector via EcoRI and XbaI restriction sites to generate pSB1A3-INP-CHI1. The pSB1A3-CHI1 plasmid (control) was obtained via inverse PCR using a BKL Kit. The recombinant plasmids were transformed into E. coli DH5α. Positive clones were screened on LB agar plates containing 100 μg/mL ampicillin and verified by sequencing (Qingke Biotechnology, China). The sequence-verified plasmid, pSB1A3-INP-CHI1, was then extracted and transformed into E. coli BL21. Single colonies were picked and cultured in 5 mL of LB liquid medium (containing 100 μg/mL ampicillin) at 37°C and 180 rpm until the OD₆₀₀ reached 0.6. A 1 mL aliquot of the culture was preserved with 25% (v/v) glycerol and stored at -80°C for future use.

Catalytic Activity Test of the Strain

A standard enzyme activity assay was employed to evaluate the catalytic performance of both the BL21-INP-CHI1 engineered strain and the CHI1 crude enzyme lysate. For the assay, 5 mL of PBS-resuspended BL21-INP-CHI1 cells (OD₆₀₀ = 0.6) or an equal volume of CHI1 crude enzyme lysate (obtained by sonicating cells at 70W power, 1s on, 3s off for 20 min, followed by centrifugation at 10,000 × g for 30 min) were incubated with 1 mL of 1% chitosan substrate in a PBS buffer system (pH 7.0) at 37°C for 30 min. The reaction was terminated, and the amount of reducing sugar produced was measured using a Biosharp reducing sugar assay kit. The concentration was quantified against a glucose standard curve (0-100 µg/mL). One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 µmol of reducing sugar per minute at 37°C. Each experiment was performed in triplicate, and the data are presented as mean ± standard deviation.

6.3 Results

Recombinant Strain Construction

Figure 9. Construction of pSB1A3-INP-CHI1.

(A) Construction of pSB1A3-INP-CHI1. (B) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pSB1A3-INP-CHI1 (CHI). (C) Agarose gel electrophoresis of colony PCR products for the recombinant plasmid pSB1A3-INP-CHI1 (INP).

The colony PCR results showed amplified bands corresponding to the expected sizes of CHI1 (2028 bp) and INP (537 bp) in the positive clones, confirming the successful construction of the recombinant plasmid.

Catalytic Activity Test of the Strain

Figure 10. Assessment of chitooligosaccharide production by BL21-INP-CHI1 and CHI1 crude enzyme lysate.

Figure 11. Comparison of enzyme activity between BL21-INP-CHI1 and CHI1 crude enzyme lysate.

The results from both direct product detection (Figure 10) and calculated enzyme activity (Figure 11) consistently demonstrated that the catalytic efficiency of the BL21-INP-CHI1 whole-cell catalyst was significantly higher than that of the CHI1 crude enzyme lysate. This difference was statistically significant (p < 0.01).

Conclusion

In this experiment, we successfully constructed the recombinant strain BL21-INP-CHI1. Furthermore, we demonstrated that the INP-mediated surface display system exhibits a dramatically enhanced capability for producing chitooligosaccharides compared to the traditional strategy of using crude enzyme lysate extracted from disrupted cells.

7.1 Purpose

Effect of Temperature on Enzyme Activity: To determine the enzyme activity profile of the chitosanase-expressing engineered strain BL21-INP-CHI1 at different temperatures, thereby identifying the optimal temperature for maximal enzyme activity. This will guide the efficient cultivation of the engineered strain and enhance the conversion efficiency of chitosanase.

Effect of pH on Enzyme Activity: To investigate the impact of different pH conditions on the activity of the chitosanase produced by BL21-INP-CHI1, thereby determining the optimal pH environment for peak enzyme performance. This will aid in optimizing the culture system for the engineered strain and improving the conversion efficiency of chitosanase.

Figure 12. Characterization of the engineered strain.

7.2 Methods

In the chitosanase activity assay, 1 mL of a PBS suspension of BL21-INP-CHI1 or crude enzyme lysate was mixed with 1 mL of 1% commercial chitosan (Cat. No. C105799, Innochem) and incubated for 30 min at various temperatures: 25, 37, 45, 50, and 65°C. To investigate the effect of pH, the reaction was conducted at 37°C in PBS buffers with pH values of 4, 5, 6, 7, and 8. The total reducing sugar content was measured using a reducing sugar assay kit (Cat. No. BL1789B, Biosharp). One unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 µmol of reducing sugar per minute. Enzyme activity units were calculated based on a glucose standard curve, accounting for the molecular weight of glucose and dilution factors.

7.3 Results

Figure 13. Comparison of enzyme activity of BL21-INP-CHI1 at different culture temperatures.

Figure 14. Comparison of enzyme activity of BL21-INP-CHI1 at different pH conditions.

Within the temperature range of 25 - 65°C, the enzyme activity (U/mL) of BL21-INP-CHI1 first increased and then decreased with rising temperature. The maximum activity was achieved at 50°C. At 25°C, the activity was lowest, while at 37°C, 45°C, and 65°C, the activities were intermediate. Within the pH range of 4 - 8, the enzyme activity (U/mL) also showed a trend of first increasing and then decreasing. The highest activity was observed at pH 7.0. At pH 4, the activity was relatively low, while at pH 5, 6, and 8, the activities were intermediate.

7.4 Conclusion

We successfully determined that the optimal reaction temperature for the BL21-INP-CHI1 whole-cell catalyst is 50°C, and the optimal pH is 7.0. These critical parameters provide a vital scientific basis for subsequent process optimization and industrial application.

8.1 Purpose

To enhance the sustainability of our project, this experiment aimed to develop an environmentally friendly and efficient microbial fermentation method for extracting chitin from shrimp shell waste. This process is intended to provide a green source of raw material for the subsequent production of chitosan and chitooligosaccharides.

8.2 Methods

Bacterial Culture:B. subtilis was cultured from a stab culture in 5 mL of LB medium at 30°C and 180 rpm for 12 h (OD₆₀₀=0.6), then cryopreserved. This stock was used to inoculate 50 mL of LB medium (2% v/v) and cultured for 8 h to prepare for fermentation. Similarly, Acetobacter sp. was cultured in LB medium with 2% ethanol for 24 h (OD₆₀₀=0.6), cryopreserved, and then used to inoculate 50 mL of LB with 4% ethanol (5% v/v) and cultured for 16 h.

Shrimp Shell Pre-treatment: Shrimp shells recovered from restaurant waste were washed, pulverized with a blender, passed through a 60-mesh sieve, and dried at 60°C for 48 h. The fermentation medium was prepared with the following composition: shrimp shell powder 50 g/L, glucose 50 g/L, yeast extract 5 g/L, and KH₂PO₄ 10 g/L. The pH was adjusted to 7.2, and the medium was autoclaved.

Yield Determination: Three groups were established. In the experimental group, 10 mL of B. subtilis culture was added to 100 mL of fermentation medium and fermented at 30°C, 180 rpm for 3 days. Subsequently, ethanol (to a final concentration of 6%) and 10 mL of Acetobacter sp. culture were added, and fermentation continued for another 3 days. In the two control groups, either the B. subtilis or the Acetobacter sp. culture was replaced with an equal volume of PBS. After fermentation, the residue was collected by centrifugation (13,000 × g, 10 min), washed to neutrality with distilled water, and dried at 60°C for 48 h to obtain chitin. The chitin yield was calculated by comparing its mass to the initial mass of the shrimp shells.

Figure 15. Chitin Yield Analysis

8.3 Results

Figure 16. Chitin yield from different bacterial combinations.

As shown in Figure 16, when using only Bacillus subtilis or Acetobacter sp. for treatment, the chitin yield was merely around 1%. However, when the two bacteria worked in synergy, the chitin yield dramatically increased to approximately 14.5%, demonstrating a significant synergistic effect.

8.4 Conclusion

By employing a co-fermentation strategy using Bacillus subtilis and Acetobacter sp. for sequential deproteinization and demineralization of shrimp shells, we have established a highly efficient and environmentally friendly new process for chitin extraction. This successfully achieves the resource utilization of biomass waste.

9.1 Purpose

This experiment was designed to evaluate the performance of our BL21-INP-CHI1 whole-cell catalyst in a practical application scenario. Specifically, we tested its efficiency in hydrolyzing the crude chitosan prepared from shrimp shells (as described above) over different treatment times and compared its performance to that of a traditional crude enzyme lysate.

9.2 Methods

One gram of the extracted chitin was mixed with 10 mL of 10% (v/v) hydrogen peroxide and reacted at 60°C for 2 hours to obtain chitosan. This was then dissolved in 0.1 M acetic acid to prepare a 1% solution. The engineered E. coli BL21-pSB1A3-INP-CHI1 fermentation culture was centrifuged (10,000 × g, 2 min), and the cell pellet was washed twice with PBS (pH 7.0). For the hydrolysis reaction, 1 mL of the bacterial suspension was mixed with 1 mL of the 1% crude chitosan solution. The reactions were carried out at 50°C for different durations. After incubation, the supernatant was collected by centrifugation (13,000 × g, 15 min) and filtered through a 0.22 μm membrane to obtain the chitooligosaccharide sample. A 100 μL aliquot of the sample was used to determine the total reducing sugar content with a reducing sugar assay kit (BL1789B, Biosharp). Enzyme activity was defined as the amount of enzyme required to release 1 µmol of reducing sugar per minute.

Figure 17. Determination of the hydrolysis rate of crude chitosan by the engineered chitosanase bacteria.

9.3 Results

Figure 18. Hydrolysis efficiency of BL21-INP-CHI1 and CHI1 crude enzyme lysate on prepared crude chitosan at different time points.

As shown in Figure 18, at all measured time points, the hydrolysis efficiency of the BL21-INP-CHI1 engineered strain was significantly superior to that of the CHI1 crude enzyme lysate. After 6 hours of reaction, its hydrolysis rate reached nearly 35%, demonstrating rapid and sustained catalytic ability. This confirms its high efficiency in processing authentic biomass-derived raw materials.

9.4 Conclusion

The BL21-INP-CHI1 engineered strain not only performs excellently on ideal substrates but also demonstrates outstanding performance when processing crude substrates extracted from waste. This fully substantiates its application potential as a robust and effective biocatalyst.

10.1 Purpose

The ultimate goal of this project is to create a functional surgical suture that actively promotes wound healing. This experiment aimed to prepare the final product by loading a standard suture material with the biologically active components—curcumin and chitooligosaccharides—produced by our engineered bacteria. Subsequently, we sought to comprehensively evaluate its anti-inflammatory and antibacterial properties and compare its performance against commercially available sutures to validate its enhanced functionality and clinical potential.

10.2 Methods

• Preparation of the Functional Suture: PGA (polyglycolic acid) surgical sutures were used as the base material. A loading solution was prepared by dissolving the previously produced curcumin and chitooligosaccharides in a 75% ethanol solution to final concentrations of 1 mg/mL and 10 mg/mL, respectively. The PGA sutures were immersed in this solution and incubated for 24 hours at room temperature with gentle agitation to ensure uniform coating. After incubation, the sutures were removed and air-dried in a sterile, laminar flow hood for 48 hours to obtain the final functionalized suture. A control suture was prepared by soaking it in 75% ethanol without the active components.
• Anti-inflammatory Activity Assay: RAW 264.7 macrophage cells were seeded in a 24-well plate. An inflammatory response was induced by adding 1 µg/mL lipopolysaccharide (LPS). Segments (1 cm) of our functionalized suture and the control PGA suture were added to the respective wells. A group of cells without LPS or suture served as the negative control, and a group with only LPS served as the positive inflammatory control. After 24 hours of co-culture, the concentration of nitric oxide (NO) in the culture supernatant was measured using a Griess reagent kit to quantify the level of inflammation.
• Antibacterial Activity Assay: The zone of inhibition method was used to assess antibacterial properties. Agar plates were uniformly spread with cultures of Escherichia coli and Staphylococcus aureus. Sterile segments (1 cm) of our functionalized suture and the control PGA suture were placed firmly on the surface of the agar. The plates were incubated at 37°C for 24 hours. The antibacterial activity was evaluated by measuring the diameter of the clear zone of inhibition around each suture segment.

10.3 Results

Figure 19. Anti-inflammatory effect of the functionalized suture on LPS-stimulated macrophages.

As shown in Figure 19, the LPS-stimulated cells co-cultured with the control PGA suture showed high levels of NO production, indicating a strong inflammatory response. In stark contrast, the cells cultured with our curcumin- and chitooligosaccharide-loaded suture exhibited a significant reduction (p < 0.01) in NO levels, bringing them close to the baseline levels of the unstimulated control group. This result powerfully demonstrates the potent anti-inflammatory effect of our functionalized suture.

Figure 20. Antibacterial activity of the functionalized suture against E. coli and S. aureus. (A) Zone of inhibition test plate. (B) Quantification of the inhibition zone diameter.

The results of the zone of inhibition assay are presented in Figure 20. The control PGA suture showed no antibacterial activity against either E. coli or S. aureus. However, our functionalized suture produced clear and significant zones of inhibition for both bacterial species. The average diameter of the inhibition zone was approximately 15 mm for E. coli and 18 mm for S. aureus, confirming its effective, broad-spectrum antibacterial properties.

10.4 Conclusion

The final surgical suture, successfully prepared by loading a PGA base with biologically synthesized curcumin and chitooligosaccharides, demonstrated powerful dual-action functionality. It exhibited potent anti-inflammatory properties by significantly suppressing the inflammatory response in macrophages and displayed broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria. Compared to standard commercial sutures, our functionalized suture shows vastly superior therapeutic capabilities, validating its significant potential as an advanced wound closure device for reducing post-operative complications like infection and excessive inflammation, thereby promoting better and faster healing.

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