. Proof of Concept .

Deinking

1. Introduction

Our understanding of human practices (see IHP for more details) reveals significant issues in the wastepaper recycling industry, such as high energy consumption and severe pollution. The enzymatic method, with its potential to ensure effective deinking while avoiding the environmental pollution caused by chemical reagents, emerges as a promising solution. However, the limited application of this method due to the low catalytic efficiency of enzymes and the extensive screening workload presents a challenge. To address this, we have developed a rapid screening method based on the gray scale to identify enzymes with the best deinking effect, thereby promoting the industrial application of enzymes. In this section, we present the characterization of the selected enzymes, which could serve as a viable alternative to the chemical method.

2. CYP199A4 T253A can significantly remove ink

0.6 g NaOH, 0.6 g Na₂SiO₃, and 0.01 g/mL H₂O₂ were added to 30 mL pulp with 1% concentration, and the final concentration of monooxygenase CYP199A4 T253A was 1 μM. After the reaction at 30°C for 60 min, limonene was added to the pulp for ink extraction, finally drying by using a Büchner funnel. The recycled paper is imaged after drying, and the gray scale value is read by a written program. The higher the gray scale value, the better the deinking effect is proved, which means the whiter the paper is observed, the less ink remains (see the SOP for details). Based on the screen and analysis, we found that the 253rd residue was the targeted position that contributed to the deinking. So, saturation mutation on the 253 position was carried out to harvest the best mutant. As shown in Figure 1, the gray scale value of some mutants increased by 50% at least, in which 253A shows the best performance in deinking. As shown in the picture from the microscope (Figure 2), the paper treated with CYP199A4 T253A (Figure 2A) has minimal ink residue among these enzymes. The paper is relatively white, and the observed lifting effect has reached 100% of the Negative (Figure 2 Negative). The deinking enhancement effect is defined as

Experimental results showed (Figure 1) that the deinking effect of mutant CYP199A4 T253A was the most prominent, with an average increase of 105%.

Figure 1 Comparison of deinking promotion effects of CYP199A4 series mutants.

Figure 2 Ultra-high resolution microscope imaging of CYP199A4 series mutants.

3. Conclusion

We have successfully established a screening method for enzymes with high deinking efficiency based on the gray scale value changes. Leveraging the monooxygenase CYP199A4 and its mutants reported in the literature, we have identified a monooxygenase with high efficiency in deinking, a significant milestone in the wastepaper recycling process (see Design for more details). This, combined with a rapid screening method, structure-activity analysis, and saturation mutation, has led us to the CYP199A4 mutant T253A (see Part Collection for more details), which exhibits superior deinking performance. In our proposed implementation, these enzymes will be introduced into the industrial pulping environment, and further characterization will focus on the efficiency and stability of the enzyme release system.  

Enzyme Release System

1. Introduction

In practical applications, the high-performance deinking enzymes we have identified must be directly applied to large-scale pulp deinking reactors. However, both protein purification and the inducers-dependent E. coli autolytic system FLSA (FhuD-T7 lysozyme-SsrA mediated autolytic system, see Design for more details) can be costly. In addition, the FLSA system can also lead to the production of a large number of miscellaneous proteins, which can affect the deinking effect of the target enzyme. To address these challenges and ensure the practicality of our research, we have decided to use a cost-effective approach. We will use the signal peptide to directly secret the enzyme outside the cell (see Design for more details), thereby bypassing the protein purification step and avoiding the use of inducers. Combined with constitutive promoters, this approach will significantly reduce the operation steps and costs, making our method more practical for industrial application. In this section, we aim to characterize the effectiveness of releasing target proteins and select the most efficient and stable signal peptide.

2. The LMT has the best secretory efficiency

Eight classic signal peptides, such as LMT, PelB, OmpA, OsmY, YebF, AIgen, TorA, and HlyA, were used to construct the signal peptide-sfGFP fusion protein. The contribution values of secretion, leakage, and cleavage processes to the fluorescence of supernatants were used to characterize the secretion efficiency of signal peptides, exploring their secretion dynamics. As shown in Figure 3, sfGFP secreted by the LMT makes a more significant contribution to the supernatant fluorescence, proving that the LMT exhibits the best secretion efficiency under low metabolic pressure.

Figure 3 Comparative tests of performance of multiple signal peptides. (A) Secretion efficiencies of different groups were calculated as time progressed. (B) The OD₆₀₀, fluorescence intensity of supernatant and normalized culture fluorescence intensity of different groups after induction for 10 hours.

3. Secretion and deinking have concurrently achieved

We have proved that some CYP199A4 mutants showed stronger deinking, and LMT showed a good secretion effect. So, we try to verify the deinking efficiency of CYP199A4 mutants secreted to the supernatant by the LMT. The engineered bacteria were cultured at 25 °C, and the supernatant culture was taken at 12 h, 18 h, 24 h, and 36 h, respectively, using SDS-PAGE to demonstrate that the fusion protein could be successfully secreted into the supernatant. Gray scale value analysis was performed on the bands, proving that the concentration of LMT-CYP199A4 T253E in the culture supernatant gradually increased with time (Figure 4A). At the same time, the supernatant from the culture in 36 hours was used for the pulp deinking experiment (see SOP for more details), and the results are shown in Figure 4B. As shown in the picture from the microscope, LMT-CYP199A4 T253E in the supernatant showed a perfect deinking effect, with an increase effect of 71.7%. The above results showed that LMT signal peptide could secrete CYP199A4 T253E to the extracellular environment continuously, which further exhibits the perfect performance in removing the ink from the pulp.

Figure 4 Characterization of His tag-LMT-CYP199A4 T253E. (A) SDS-PAGE analysis (left) and gray scale value analysis (right) of the supernatant at different times. (B) Deinking characterization of His tag-LMT-CYP199A4 T253E (BBa_K5136047).

4. Conclusion

By characterizing classic signal peptides, we proved that LMT has a high secretion efficiency. It can realize the continuous secretion of CYP199A4 T253E with significant deinking performance. Finally, we proved that the scheme we designed is reasonable and feasible, which paves the way for its application at an industrial scale.

Life Cycle Assessment

1. Introduction

Life Cycle Assessment (LCA) is a comprehensive method used to evaluate the environmental influence of a product or process from inception to disposal (from "cradle" to "grave"), enabling a more complete assessment of the contribution of our projects to the emission reduction of wastepaper recycling industry processes. In this part, we will combine experimental data and related studies to quantify energy consumption, greenhouse gas emissions, and waste generation. By calculating and comparing the carbon emissions of the enzymatic method and chemical method in recycled paper production, we comprehensively evaluate the contribution of our project to emission reduction.

2. Emission reduction contributes to recycled paper production

We calculated the input/output process of recycled paper production without considering the difference in carbon emissions caused by the pretreatment process. We considered the carbon emissions from the production of chemical reagents in the deinking process, the respiratory volume of enzyme-producing bacteria, and the corresponding electricity consumption. The results showed that the carbon emissions were reduced by the enzymatic deinking method by 30.7% compared with the chemical deinking method. The waste treatment process considers the chemical oxygen demand (COD), biochemical oxygen demand (BOD), suspended solids (SS), and other harmful substances in the wastewater. Compared with the chemical method, the carbon emission of the wastewater treated by the enzymatic method is reduced by 54.2%. The final scheme's overall emission reduction optimization degree is 21.1% (Figure 5), accompanied by an overall reduction of 31.9% in electricity consumption and reagents costs, including chemical reagents (see LCA for more details). It is highlighted that the enzymatic method has more advantages in reducing carbon emissions than the classical chemical method, and it can be a more sustainable alternative to wastepaper deinking in the future.

Figure 5 Overall carbon emissions and distribution.

3. Conclusion

Through life cycle evaluation, we have successfully evaluated our project's contribution to reducing the carbon emission of recycled paper production. Therefore, our project is expected to be applied to industrial production in the future to reduce the carbon emission generated by the paper-making process with an efficient, low-energy consumption, and environmentally friendly deinking process.

4. Future

We have successfully demonstrated that the monooxygenase CYP199A4 T253A has a good deinking effect while demonstrating that the LMT has the best secretion efficiency. Using constitutive promoters, we can achieve the requirements of low cost and high efficiency on an industrial scale. We also characterized the combination of LMT and CYP199A4 T253E, proving that LMT-CYP199A4 T253E can achieve secretion-deinking coupling. Due to time constraints, we regret not presenting the secretion-deinking effect of LMT- CYP199A4 T253A (the best variant, see Part Collection) in our wiki, but the deinking result is sufficient to prove the feasibility and efficiency of our project design concept. In the future, we will show our best results in the promotion video or presentation.