Part:BBa_K5136005

CYP199A4 T253N-His tag

Biology

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). It has been pointed out that position T253 of CYP199A4 can affect the coordination environment of the active center (1). Therefore, we carried out saturation mutagenesis on this site, hoping to screen out enzymes with higher activity.
Partial amino acid sequence of CYP199A4 T253N: …AGLDNTVNGI…

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.

Figure 1 Gene circuit of CYP199A4 T253N-His tag.

Routine Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We obtained the target fragment of 1412 bp.

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136005_pET-28a(+).

The pET-28a (+) vectors containing CYP199A4 variants were transformed into E. coli BL21(DE3), and the cells were cultivated in LB medium containing 50 μg/mL kanamycin. The cultures were grown at 37 °C with vigorous shaking (~200 rpm). When the OD₆₀₀ of the cultures reached 0.8~1.0, the temperature was cooled to 20 °C, and the expression was induced by the addition of IPTG (0.2 mM) and δ-aminolevulinic acid hydrochloride (0.5 mM). Following 16-20 h of expression, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. SDS-PAGE and Coomassie blue staining were used to verify the expression of the target protein (about 45.8 kDa).

Figure 3 SDS-PAGE analysis of CYP199A4 T253N-His tag protein.

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.

Figure 4 Diagram of Pulp Deinking Standard Operating Procedure in Lab.

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) was added in the deinking blank system and reacted at 30 °C (the average of the two optimal temperatures) for 60 min. After the standard operating procedure, read the gray scale value automatically. As shown in (Figure 5), 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 exhibits the highest ΔGray scale value, which was increased by more than ten-fold compared to the wild type enzyme. As shown in the picture from the microscope (Figure 6), the paper treated with CYP199A4 T253E (Figure 6) has minimal ink residue among these enzymes, demonstrating the highest deinking efficiency.

Figure 5 Comparison of the Relative Gray Scale of different monooxygenases and their mutants.

Figure 6 Pulp Recycled Paper under a High-resolution Microscope, the percentage is the value obtained by dividing by the Gray of negative.

Based on the results from the preliminary experiment, many enzymes exhibit excellent deinking performance, resulting in 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) was added in the deinking blank system and reacted at 30 °C (the average of the two optimal temperatures) for 60 min. After the standard operating procedure, read the gray scale value automatically. As shown in Figure 7, 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 8), the paper treated with CYP199A4 T253A (Figure 8) has minimal ink residue among these enzymes. The paper is relatively white, and the observed effect has reached 1.06 times of chemical deinking (Figure 8 chemical method).

Figure 7 Comparison of the Relative Gray Scale of different monooxygenases and their mutants.

Figure 8 Pulp Recycled Paper under a High-resolution Microscope, the percentage is the value obtained by dividing by the Gray of A.

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 9A). 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 9B. 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.

Figure 9 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).

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 150
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 831
  Illegal XhoI site found at 1231
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 150
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 150
  Illegal AgeI site found at 691
  
• 1000
  COMPATIBLE WITH RFC[1000]