Part:BBa_K5136020

SfmD WT

Biology

SfmD is a heme-dependent enzyme in the biosynthetic pathway of saframycin A, which is a heme-dependent enzyme that is a versatile bio-oxidation catalyst for C–X (e.g., X = H, N, S) bond oxidations.
SfmD from Streptomyces lavendulae was previously reported as a heme peroxidase with atypical peroxidase activity, capable of hydroxylating 3-methyl-L-tyrosine (3-Me-L-Tyr) or L-tyrosine (L-Tyr) using hydrogen peroxide as the oxidant(1).

Usage

SfmD 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).

Construction

We use pET28a(+) to construct this circuit. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3). The positive transformants were confirmed by kanamycin, colony PCR, and sequencing.

Figure 1 Gene circuit of His tag-SfmD WT.

Routine Characterization

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We obtained the target fragment of 1340 bp.

Figure 2 DNA gel electrophoresis of the colony PCR products of BBa_K5136020_pET-28a(+).

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.5 mM IPTG at 20 °C, the 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 (39.0 kDa).

Figure 3 SDS-PAGE analysis of His tag-SfmD WT 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.Shin I, Davis I, Nieves-Merced K, Wang Y, McHardy S, Liu A. A novel catalytic heme cofactor in SfmD with a single thioether bond and a bis-His ligand set revealed by a de novo crystal structural and spectroscopic study. Chem Sci. 12, 3984-3998 (2021).
2.Kui-zhong, S. Application of Hydrogen Peroxide in the Pulp and Paper Industry. (2005).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 231
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 894
  Illegal NgoMIV site found at 1068
  
• 1000
  COMPATIBLE WITH RFC[1000]