Our goal of this research project is obesity management. The reason for obesity is that excess amount of hydrogen sulfide may lead to increased oxidative stress and cell damage, thus reducing the secretion of GLP-1. So we aim to decompose hydrogen sulfide by two enzymes FCSD + SQR. During this experiment, we first transformed competent Escherichia coli BL21 and DH5α cells, then used IPTG to induce protein expression and finally examined the concentration of hydrogen sulfide.

The design of this project reflects the synthetic biotechnology: high-yield expression of key enzymes (FCSD + SQR) in hydrogen sulfide metabolism using E.coli BL21 and DH5α as a prokaryotic host.

Figure 1. Roadmap of Experimental Techniques

1. The PCR amplification of pET28a-SQR and pET28a-FCSD

As shown in the Figure 2, the target genes for SQR (1350 bp) and FCSD (1293 bp) were successfully amplified. The amplified bands align precisely with the expected molecular sizes when compared to the DNA ladder, confirming the specificity and efficiency of the amplification process. These results demonstrate the successful preparation of DNA templates for subsequent cloning experiments.



Figure 2. Target gene PCR amplification diagram

2. Double digestion of pET28a

Following digestion with the restriction enzymes XhoI and BamHI and subsequent PCR amplification at 37 °C for 10 minutes, agarose gel electrophoresis was performed. Comparison with the DNA ladder on the left reveals two distinct bands for the undigested pET28a plasmid, which correspond to its supercoiled and open circular conformations. In contrast, the digested pET28a sample appears as a single band, confirming complete linearization of the plasmid by the restriction enzymes. This result indicates successful enzymatic cleavage and conversion of the supercoiled and open circular forms into a uniform linear DNA fragment.



Figure 3. Double digestion with XhoI and BamHI

3. Recombinant plasmid construction by homologous recombination

The purified PCR product and linearized pET28a backbone were then assembled using homologous recombination. The resulting recombinant plasmid was transformed into E. coli DH5α competent cells. Single colonies were observed on the selection plate, indicating successful transformation. According to the sequencing results, the SQR and FCSD gene was correctly inserted into the pET28a vector without mutations, confirming the successful construction of the pET28a -SQR-His and pET28a -FCSD-His plasmid.



Figure 4. Recombinant Plasmid Identification Map

1. Transform the recombinant plasmid into E.coli BL21(DE3)

For protein expression, the validated recombinant plasmid was transformed into E. coli BL21(DE3) competent cells. The transformed culture was then plated onto LB agar plates supplemented with kanamycin and incubated overnight at 37 °C. As shown in Figure 5A, well-distributed single colonies were observed on the plates, demonstrating successful transformation.Subsequently, several single colonies were randomly selected for preliminary identification of the recombinant plasmid via colony PCR. Agarose gel electrophoresis results (Figure 5B) showed that the amplified DNA band size matched the expected target fragment, confirming that the constructed recombinant plasmid was successfully transformed into E.coli BL21(DE3).



Figure 5. Monoclonal Colony Plate Diagram

2. Protein Expression and Purification

Subsequently, we selected a single monoclonal colony and inoculated it into LB liquid medium. After incubating at 37°C with shaking until the logarithmic growth phase (OD600 ≈ 0.6–0.8), we added IPTG to a final concentration of 0.5 mM. Expression was then induced at 16°C for 16 hours to promote soluble protein formation. After induction, bacterial cells were harvested by centrifugation. Cells were lysed using an ultrasonic cell disruptor, and the supernatant was used. Using the histidine tag (His-tag) at the C-terminus of the SQR and FCSD protein, we purified the protein via nickel affinity chromatography. Four key fractions were collected: crude lysate, flow-through, wash fraction, and elution fraction. Analysis of each fraction by SDS-PAGE gel electrophoresis yielded the results shown in Figure 6. A single, dense protein band appeared at the expected molecular weight in the elution fraction, while this band was faint or absent in the crude lysate, flow-through, and wash fractions. Indicating that the target protein SQR-His and FCSD-His successfully bound specifically to the nickel column and was efficiently eluted. This confirms the successful expression of the recombinant protein in the E.coli system and the effective purification achieved through the nickel affinity chromatography step. The collected material was used to obtain the final product.



Figure 6. SDS-PAGE for Protein Expression Analysis

1. ELISA Test For SQR Enzyme

The assay principle of the ELISA kit relies on the specific binding between the antigen and antibody, followed by the use of an enzyme-labeled antibody or antigen to catalyze a chromogenic reaction of the substrate. The presence and concentration of the target analyte (SQR) are determined based on the intensity of the color developed.

In the microplate layout, the left column was designated for the standard curve. The right column wells were loaded with samples including Crude Enzyme, Flow-through fluid, Washing buffer, and Elution Solution. After incubation with the corresponding ELISA reagents, varying degrees of color development were observed in the wells, reflecting the amount of target protein present in each sample.

In this experiment, we employed the enzyme-linked immunosorbent assay (ELISA) to quantitatively analyze the content of sulfquinone reductase (SQR) in different eluate fractions. The experiment began by pre-coating SQR antibodies onto microplate wells. Standard samples, test samples, and HRP-labeled detection antibodies were sequentially added. After incubation and thorough washing, the TMB substrate was introduced for color development. Under peroxidase catalysis, TMB initially turned blue and then converted to yellow upon acid termination. The color intensity correlated positively with SQR concentration in the samples. Absorbance (OD value) was measured at 450 nm, and SQR concentrations in each sample were calculated using the standard curve.



Figure 7. Assaying the Standard Curve for ELISA

The wells corresponding to the Crude Enzyme, Flow-through fluid, and Washing buffer exhibited extremely faint color (almost colorless), indicating that the content of the SQR enzyme in these samples was very low and insufficient to effectively catalyze the substrate to produce a significant amount of yellow product. In contrast, the well containing the Elution Solution displayed a distinct yellow color, demonstrating a high concentration of SQR enzyme. This sample efficiently catalyzed the chromogenic reaction, yielding a markedly stronger yellow coloration compared to the other three sample types.

Following the visual observation of color development in the microplate wells, the plate was transferred to a microplate reader for quantitative analysis. Absorbance was measured at 275 nm, and enzyme activity was calculated in units of U/L based on the obtained values:

Elution Solution: The enzyme activity peaked at approximately 300 U/L, significantly exceeding that of all other samples. This result indicates that the SQR enzyme retained high catalytic activity in this fraction, efficiently generating product per unit time and corresponding to a strong absorbance signal.

Crude Enzyme, Flow-through, and Washing Buffer: The measured enzyme activity in these samples was nearly 0 U/L, demonstrating that the SQR enzyme exhibited minimal catalytic activity and was unable to effectively facilitate substrate conversion. These quantitative results are consistent with the observed lack of visible color development in the corresponding wells.



Figure 8. ELISA assay showing enzyme concentration

Results showed significantly higher SQR enzyme concentrations in the eluate fraction compared to other fractions, indicating this fraction primarily enriched the target protein. A good linear relationship was observed between standard concentrations and OD450 values, with a high standard curve fit, confirming reliable detection results.

Based on the ELISA results, it can be concluded that a higher concentration of the target enzyme leads to increased production of yellow product and correspondingly deeper color development. Throughout the experimental procedure, the SQR enzyme was effectively enriched and maintained its biological activity following the elution step. The ELISA assay confirmed both the high content and strong activity of the SQR enzyme in the elution solution through two complementary approaches: qualitative color visualization and quantitative activity measurement. These results provide critical support for subsequent functional studies and practical applications of this enzyme.

2. SQR Activity Assay by DUQ Reduction

The assay principle is based on the catalytic activity of the SQR enzyme, which oxidizes sulfide while simultaneously reducing DUQ. The reaction is monitored by measuring the change in absorbance at 275 nm (ΔOD₂₇₅) using a microplate reader. Enzyme activity is calculated according to the formula incorporating the molar extinction coefficient of DUQ. The experiment included multiple control groups-negative control, blank control, and enzyme-inactivated control-with three technical replicates performed for each sample.

As shown in the results, the enzyme activity in the experimental group exhibited a continuous increase over time. In contrast, the blank control, negative control, and enzyme-inactivated groups all showed fluctuations near baseline without a significant upward trend. These findings demonstrate that the SQR enzyme effectively catalyzes the reaction, and that the assay system robustly detects its activity. The control results effectively rule out non-specific interference, confirming the reliability of the experimental outcomes.



Figure 9. spectrophotometric determination of SQR enzyme activity based on DUQ reduction

3. Lead acetate test strip
   1. Construction of E.coli MG1655 for H₂S production with L-cys

   To verify the ability of the enzymes (SQR and FCSD) to decompose hydrogen sulfide, lead acetate test strips were employed. The principle of this method is that sulfur-containing compounds (such as H₂S) generated in the enzyme-catalyzed reaction react with lead acetate, forming a black precipitate of lead sulfide (PbS). The intensity of the color change on the test strip reflects the level of enzyme activity.

   As shown in Figure 10A, samples containing 1.5 mM and 2.0 mM L-cysteine exhibited a noticeably darker color compared to those with lower concentrations, indicating more successful construction of E.coli MG1655 for enhanced hydrogen sulfide production. Figure 10B presents a grayscale value analysis of the test strips for semi-quantitative evaluation, demonstrating a clear correlation between higher L-cysteine concentration and increased production, consistent with a dose-dependent response.

   

   Figure 10. Different L-cysteine derivatives induce varying amounts of H₂S production

   Figure 11 presents a bar chart of OD₆₀₀ values, demonstrating that the absorbance reaches a relatively high level at 1.5 mM L-Cys. Correspondingly, the lead acetate test strip also exhibited the most pronounced color change at this concentration. These results indicate that within the concentration range of 0–2 mM, the amount of hydrogen sulfide produced increases with L-Cys concentration and peaks at 1.5 mM. This suggests that L-Cys influences hydrogen sulfide generation in the system, and an appropriate concentration (1.5 mM under these experimental conditions) can enhance H₂S production, thereby subsequently affecting enzyme-mediated decomposition processes.

   The optimal concentration of L-cysteine for hydrogen sulfide (H₂S) production was determined to be 1.5 mM. At this concentration, a significant increase in H₂S generation was observed, indicating efficient utilization of the supplemented precursor by the enzymatic system. Notably, this enhancement in H₂S yield was achieved without exerting inhibitory effects on bacterial growth, as confirmed by OD₆₀₀ measurements, which showed robust microbial density comparable to controls. These results suggest that 1.5 mM L-cysteine effectively supports the metabolic demand for sulfide synthesis while maintaining cellular viability, establishing this concentration as suitable for subsequent applications requiring maximal H₂S production under non-toxic conditions.

   

   Figure 11. OD600 absorbance value of different concentration of H₂S

   2. Hydrogen sulfide decomposition ability validation

   As the concentrations of SQR and FCSD increased (from 0 to 10 mg/mL), the color change of the lead acetate test strips became progressively more pronounced. This observation indicates that both enzymes are capable of promoting the decomposition of hydrogen sulfide. Furthermore, the results suggest a positive correlation between enzyme concentration and the extent of H₂S decomposition within this concentration range.

   

   Figure 12. Reduction in greyscale of lead acetate test strips with increasing SQR and FCSD concentration

   3. Effect of different proportion of mixed enzymes on inhibiting hydrogen sulfide

   When SQR and FCSD were mixed in different proportions (1:1, 1:2, 1:3, 2:1, 3:1), distinct color changes were observed on the lead acetate test strips. These results indicate that the ratio of the two enzymes influences the efficiency of hydrogen sulfide decomposition, and an appropriately optimized ratio may enhance H₂S degradation capability. Notably, the 1:3 mixture (SQR:FCSD) exhibited the most pronounced color change, suggesting that this ratio yields the highest decomposition activity. These findings imply a potential synergistic or regulatory interaction between SQR and FCSD during hydrogen sulfide decomposition.

   

   Figure 13. Effect of different proportion of mixed enzymes on inhibiting hydrogen sulfide

4. Hardware Result

The experiment covered the variation ranges of humidity (63 - 75) and temperature (35.0 - 38.0). However, regardless of how the temperature and humidity fluctuated, the sensor will block out some interfering data. The sensor's displayed value is positively correlated with the hydrogen sulfide concentration (the higher the concentration, the larger the value), and within the experimental temperature and humidity range, this rule is stable, verifying the reliability of the sensor in detecting the H₂S concentration gradient.



Figure 14. Sensor response positively correlates with H₂S concentration

Compared to the control groups with "no enzyme" and those with "enzyme added," the introduction of either SQR or FCSD resulted in a significant decrease in the hydrogen sulfide sensor readings. This reduction clearly indicates that both enzymes are functionally active in decomposing hydrogen sulfide present in the system, thereby effectively lowering the ambient concentration of H₂S.



Figure 15. Sensor display when SQR or FCSD is added

The SQR enzyme exhibited a more pronounced effect, reducing the sensor reading by approximately 129 units (from 1714 to 1585), demonstrating a stronger capacity for hydrogen sulfide decomposition. In contrast, the FCSD enzyme showed a relatively weaker effect, decreasing the sensor value by only about 58 units (from 1714 to 1656), indicating a lower efficiency in H₂S degradation compared to SQR.

To further investigate the effects of H₂S on intestinal endocrine cell function, we established the following in vitro model: the supernatant from L-cysteine-induced E.coli MG1655 was added to the NCI-H716 cell culture system. This supernatant, rich in H₂S produced by bacterial metabolism, significantly inhibited the synthesis and secretion of glucagon-like peptide-1 (GLP-1) by NCI-H716 cells. Results indicate that H₂S intervention disrupts GLP-1-mediated metabolic regulatory pathways, leading to downregulation of GLP-1 gene expression. This process mimics the potential regulatory role of microbial metabolites in the intestinal microenvironment on host cell endocrine function.

Figure 16. Map of GLP-1 Gene Expression Level Changes

NCI-H716 cells were seeded at a density of 5 × 10⁵ cells per well in a 12-well plate. After the cells adhered and reached 70–80% confluence, they were treated with different drugs: One group received supernatant from the L-cysteine-induced Escherichia coli MG1655 strain (Negative group), another group received supernatant from the L-cysteine-induced strain pretreated with SQR protein (SQR-treated group), with corresponding control groups established. After co-incubation for 4 hours, the supernatant was aspirated, cells were washed with PBS to remove drug residues, and fresh medium was added for continued culture for 24 hours, respectively. Following culture, total cellular RNA was extracted, and the expression level of glucagon-like peptide-1 (GLP-1) mRNA was detected using RT-qPCR.

Table 1 qPCR detection of GLP-1 mRNA expression levels in cells following SQR administration

Quantitative real-time PCR analysis revealed that GLP-1 mRNA expression was significantly upregulated in both the SQR-treated group and the FCSD-treated group compared to the Negative control group (p < 0.05). This indicates that both SQR and FCSD proteins effectively alleviate H₂S-mediated suppression of GLP-1 gene expression by degrading extracellular H₂S present in the bacterial supernatant, leading to the recovery of GLP-1 transcription levels.

Furthermore, the combined administration of SQR and FCSD resulted in a more pronounced upregulation of GLP-1 expression than either enzyme alone, with the efficacy following the order: SQR + FCSD > SQR > FCSD. This suggests a synergistic effect between the two enzymes in enhancing GLP-1 expression, likely due to more efficient and sustained removal of inhibitory H₂S.

Figure 17. The map of GLP-1 gene expression level changes under different treatments

The primary objective of this study was to investigate the potential of using synthetically produced enzymes-sulfide:quinone oxidoreductase (SQR) and FCSD-to degrade hydrogen sulfide (H₂S) as a therapeutic strategy for obesity-related metabolic dysregulation. Our findings demonstrate that both enzymes effectively decompose H₂S in a concentration-dependent manner, with SQR exhibiting significantly higher activity than FCSD. Notably, a mixed ratio of SQR to FCSD at 1:3 resulted in the most pronounced degradation effect, suggesting synergistic interaction between the two enzymes. Real-time monitoring using a specialized H₂S sensor confirmed the notable decrease in H₂S concentration following enzymatic treatment, reinforcing the reliability and quantitative accuracy of our detection system.

Excessive H₂S has been implicated in promoting oxidative stress and cellular damage, thereby suppressing GLP-1 secretion-a key regulator of satiety and lipid metabolism. In this study, we achieved high-yield expression and purification of both SQR and FCSD in E.coli BL21(DE3), and validated their functional efficacy in vitro. The observation that 1.5 mM L-cysteine optimally supported H₂S production without inhibiting bacterial growth highlights a suitable concentration range for future in vivo applications or synthetic consortia design. Furthermore, the enhanced degradation efficiency observed with the SQR-FCSD mixture suggests the potential for engineering multi-enzyme complexes for improved H₂S scavenging.

Despite promising in vitro results, several challenges must be addressed before clinical translation. The stability, immunogenicity, and retention of enzymatic activity within the gastrointestinal environment remain to be thoroughly evaluated. Additionally, potential interference from other sulfur compounds or volatile metabolites in real biological samples warrants further investigation. Future work should focus on in vivo validation using animal models of obesity, as well as the development of efficient enzyme delivery systems to enhance localized H₂S degradation and functional persistence.

In conclusion, this study establishes a robust pipeline for the molecular cloning, expression, purification, and functional characterization of H₂S-degrading enzymes. We provide compelling evidence that SQR and FCSD act synergistically to reduce H₂S levels, supporting their potential application as enzyme-based therapeutics for metabolic disorders associated with H₂S accumulation.