Difference between revisions of "Part:BBa K5136020"

Revision as of 20:37, 30 September 2024

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 in Streptomyces lavendulae was previously reported as a heme peroxidase with an atypical peroxidase activity of hydroxylating 3-Me-l-Tyr or l-Tyr using hydrogen peroxide as 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.

Routine Characterization

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

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).

Deinking Experiments

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