Molecular Cloning and Biochemical Characterization of a Novel Disulfide Bond-Reductase from Feather-Degrading Stenotrophomonas sp. Yang-5

Abstract

Disulfide bond-reductase (DSBR) exerts an initial role in the enzymatic degradation of feather waste. However, despite its significance, there are currently very few reports on the heterologous expression and enzymatic properties of this type of enzyme. This study focuses on a novel DSBR derived from the strain Stenotrophomonas sp. Yang-5 (Yang-5-DSBR), which was previously found to efficiently degrade feathers. The target band of the DSBR was cloned by PCR, with a size of 687 bp. Domain prediction revealed that the enzyme consists of two distinct parts: N-terminal domain similar to glutathione-S-transferase, spanning amino acids from 1 to 92, and C-terminal domain spanning amino acids from 110 to 204. Subsequently, recombinant expression was carried out using a prokaryotic expression host, followed by nickel column purification of the protein. SDS-PAGE analysis showed that the size of the target protein was approximately 26 kDa, which was in line with our expectations. Enzymatic property assays indicated that the optimal reaction temperature and pH for the Yang-5-DSBR were 35 °C and pH 7.0, respectively. It maintained over 80% enzymatic activity at pH levels ranging from 6.0 to 8.0 and at 40 °C, demonstrating moderate tolerance with significant potential for further improvement. Notably, most metal ions and chemicals inhibited the activity of Yang-5-DSBR, but among them, Al³⁺, Mg²⁺ and Mn²⁺ at a concentration of 0.025 mM could enhance enzymatic activity by 20–50%. Additionally, the presence of 2.5% reducing reagent DTT increased enzymatic activity by 20%, akin to enzymes with Cys as a catalytic key amino acid, such as keratinase and nitrilase. Substrate specificity studies revealed that Yang-5-DSBR had a preference for GSSG and soluble keratin but exhibited lower activity towards other feather substrates. Kinetic parameter assays determined the V_max and K_M of DSBR to be 7.4 and 6.7 mg/mL, respectively. Overall, these insights enhance understanding of DSBR’s role in feather degradation and facilitate its potential applications in biochemical processes and industry.