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LMT-linker-253E

Biology

LMT Lytic murein transglycosylase (LMT) is an enzyme which is able to degrade murein, a component of cell wall of bacteria. There are two kind of LMTs existing in E.coli: the membrane-binding one and the soluble one. The gene coding LMT homolog is also incorporated in the genome of Vibrio natriegens.

CYP199A4 CYP199A4 is a NADH-dependent cytochrome P450 monooxygenase from Rhodopseudomonas palustris cytochrome P450, a heme-dependent enzyme that is a versatile bio-oxidation catalyst for C–X (e.g., X = H, N, S) bond oxidations (1). CYP199A4 can also function as peroxygenase. The engineered CYP199A4 peroxygenases showed good functional group tolerance and preferential O-demethylation at the meta- or para-position, indicating potential O-demethylation of H- and G-type lignin monomers (1).

Usage and design

CYP199A4 can cause alkaline fracture of conjugated side chains of lignin and other colored substances such as azo dyes through nucleophilic reaction, increasing the hydrophilicity of the reaction products, which can be easily removed in the subsequent washing process to achieve the purpose of bleaching (2). We plan to use the signal peptide LMT to secrete our deinking enzyme, so as to achieve low cost and efficient acquisition of deinking enzyme and achieve the deinking of pulp. Therefore, we built the LMT-linker-CYP199A4 253E protein to test the usability of our system.

Construction

We use pET-28a(+) to construct this circuit. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), and the positive transformants were confirmed by kanamycin, colony PCR, and sequencing.

Deinking Experiments

2024 XMU-China has summarized a set of practical and actionable experimental steps for pulp deinking regarding industrial processes and extensive experimental explorations (see 2024 XMU-China SOP page for details). The experiment can be divided into five processes: pulping, deinking, separation, drying, and measurement, in which our enzymes play a role in the deinking process, and grayscale measurements are used to characterize and evaluate our experimental results.

We screened enzymes through three stages. In the first stage, we used cellulase, laccase, and monooxygenase, among which monooxygenase had the most prominent effect, so we made monooxygenase the focus of our team's research.
1 mL monooxygenase (0.2 mg/mL) were added in the deinking blank system and reacted at 30 °C (the average of the two optimal temperature) for 60 min. After standard operating procedure, read the gray scale value automatically. As shown in (Figure 3), the pulp treated by SfmD-277F showed a slight increase in ΔGray scale value compared to that of wild type. However, the value decreased significantly in the mutant of OleT_JE than that of the wild type. The pulp treated by CYP199A4-253E exhibit the highest ΔGray scale value, which was increase by more than ten-fold compared to the wild type enzyme. As shown in the picture from the microscope (Figure 4), the paper treated with CYP199A4 T253E (Figure 4) has minimal ink residue among these enzymes, demonstrating the highest deinking efficiency.

Based on the results from preliminary experiment, many enzymes exhibit the excellent deinking performance, resulting the saturation of the gray scale value. Thus, each enzyme was diluted from 0.2 mg/mL to 0.05 mg/mL. 1-mL each monooxygenase (0.05 mg/mL) were added in the deinking blank system and reacted at 30 °C (the average of the two optimal temperature) for 60 min. After standard operating procedure, read the gray scale value automatically. As shown in Figure 5, 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 6), the paper treated with CYP199A4 T253A (Figure 6) has minimal ink residue among these enzymes. The paper is relatively white, and the observed effect has reached the 1.06 times of chemical deinking (Figure 6 chemical method).

We have proved that some CYP199A4 mutants showed stronger deinking, and LMT showed a good secretion effect. So, we try to verified 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 7A). 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 7B. As shown in the picture from the microscope, LMT-CYP199A4 T253E in the supernatant showed a perfect deinking effect. 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.

After three stages of screening, we found that CYP199A4 253A had the best deinking efficiency. And we successfully combined the deinking enzyme CYP199A4 253E with the secretion system to get a good deinking effect. Our work provides a new biological idea of environmental protection for the processing and production of recycled paper. It provides an effective reference for the future deinking research team to help them quickly obtain the required deinking enzyme, and further modify it or conduct mechanism research.

Reference

1. P. Zhao, Y. Jiang, Q. Wang, J. Chen, F. Yao, Z. Cong, Crucial gating residues govern the enhancement of peroxygenase activity in an engineered cytochrome P450 O-demethylase. Chemical Science 15, 8062-8070 (2024).
2. Shen Kui-zhong, Application of Hydrogen Peroxide in the Pulp and Paper Industry. (2005).

Sequence and Features