Heterologous expression and structural characterization of polyamide 4-degrading enzyme from a soil bacterium

Polyamide 4 (PA4) is a bio-based plastic with thermal stability, excellent mechanical properties, and good biodegradability in various environments. To understand the biodegradation of PA4 under natural environments, PA4-degrading microorganisms and enzymes have been investigated. Although our previous research identified the amino acid sequence and predicted the three-dimensional (3D) structure of a PA4-degrading enzyme from a marine environment (Nyl4A _pa ), those of an enzyme from terrestrial environments have remained unidentified. In this study, we identified the PA4-degrading enzyme gene (nyl4A_px ) from the PA4-degrading soil bacterium Pseudoxanthomonas sp. TN-N1. In addition, nyl4A_px was successfully expressed in Escherichia coli BL21(DE3) and Brevibacillus choshinensis HPD31-SP3. The PA4-degrading activity of the enzyme secreted by recombinant B. choshinensis HPD31-SP3 reached 68.8 Δ655 nm/h/100 mL broth, representing a 2.4-fold increase compared with that produced by recombinant E. coli BL21(DE3). Based on a homology search using the amino acid sequence and predicted 3D structure of the enzyme, Nyl4A _px was predicted to be composed of a substrate-binding domain, a middle domain, and a catalytic domain. Among these domains, the substrate-binding and catalytic domains of Nyl4A _px are sequentially and structurally similar to those of Nyl4A _pa . Furthermore, putative homologs of Nyl4A _px and Nyl4A _pa were found in marine-associated environmental metagenomes through BLAST searches. To our knowledge, this is the first report describing the structural properties of a PA4-degrading enzyme from a soil bacterium.